Eur. J. Biochem. 81, 129-139 (1977)
The Hysteretic Properties of Glycogen Synthase I Henrik SQLLING and Viggo ESMANN Department of Medicine, Marselisborg Hospital, Aarhus (Received June 29, 1977)
Glycogen-free synthase I from human polymorphonuclear leukocytes is activated by its own substrate, glycogen, in a slow, time-dependent proces (hysteretic activation). This lag in response to addition of glycogen depends on the concentration of glycogen, pH and temperature. At pH 7.4 and at a temperature of 30 "C, the half-time of activation, tl:2, decreases from 89 min at 0.004 mg/ml glycogen to 6 min at 25 mg/ml. The activation is accelerated by increasing temperature and pH, but is not influenced by enzyme concentration, glucose 6-phosphate, UDP, high ionic strength, EDTA, mercaptoethanol, glucose, sucrose or amylase limit dextrin. The K,,, for UDP-glucose (0.024 mM) and the activity ratio were unchanged during the activation process. The activation can be described by tit = uf (vo - or) eCkrwhere ut, uf and uo are velocities at times t , 0 and co and k is a complex rate constant. Evidence from ultracentrifugation and kinetic studies is presented to substantiate the hypothesis that the underlying mechanism is a simple bimolecular process :enzyme glycogeneenzyme-glycogen complex, with the dissociation constant K, = 0.003 mg/ml. The hysteretic activation may become rate-limiting during experiments in vitro with synthase. The possibility of a physiological role in glycogen metabolism, perhaps in the form of a concerted hysteresis with H + is discussed.
+
+
The activity of glycogen synthase, the rate-limiting UDPenzyme of glycogen synthesis (glycogen, glucose+glycogen,+ I UDP), is controlled by covalent modification and by metabolite (allosteric) control [l]. In human polymorphonuclear leukocytes, the control is exerted at three levels with regard to degree of phosphorylation and value of kinetic constants. The kinetic constants for substrates and modifiers of the fully phosphorylated enzyme form (D for dependent on glucose-6-P for activity) are of such magnitude that this form of the enzyme can be considered inactive under intracellular conditions [2]. A recently discovered partially phosphorylated enzyme form (R for rheostatic) has been found to account for glycogen synthesis under several physiological conditions, the kinetic constants being of a magnitude compatible with control exerted by the intracellular concentration of glucose-6-P 13 - 51. The third non-phosphorylated enzyme form, synthase 1, must from preliminary evidence [6] be considered fully active under all conditions. It can be formed in leukocytes under different experimental conditions, in particular when the inhibitory effect of glycogen on the D to 1 conversion is relieved during starvation [7], and/or when the activity of phosphorylase a reaches a critical, low level [S], suggesting the relief of an inhibitory effect on the R to I conversion [4].
+
Enzyme. Glycogen synthase (EC 2.4.1.11).
+
When we attempted to detail the kinetics of synthase I we became aware that glycogen, besides being both substrate and product in the enzymatic reaction and inhibitor of the D to I conversion, also confers stability on synthase I against inactivation, as described in the preceding paper [9]. In addition, glycogen induces a slow, time-dependent activation of initially glycogen-freesynthase I. Since this phenomenon, which also has been found by others [lo-131, sometimes may become rate-limiting, at least in vitro, it became clear that the kinetic behavior of synthase I is more complicated than initially expected. In this communication, we introduce the hysteretic enzyme concept [14] to describe the activation of synthase I by glycogen and discuss the physiological significance as well as the possible mechanisms behind the phenomenon. A preliminary account of this work has been given 1151. A slow increase or decrease in enzyme activity induced by a rapid change in the concentration of a modifier or a substrate is called a hysteretic process by Frieden 1141, who showed that regardless of the underlying mechanism, the change in enzyme activity can, in most cases, be described by ut = vf + (210 - vr) e-kr (1) which transforms to
130
where ot is the velocity at time t , u0 is the velocity at t = 0, uf is the velocity at t = co,and k is a complex rate constant which depends on the concentration of the modifier, the temperature, pH, and the underlying mechanism. Hysteretic changes in enzyme activity may be caused by isomerization or polymeriLation/ depolymerization of the enzyme and by the dissociation or association of a tightly bound ligand. The same phenomenon has also been treated theoretically by others [16,17] and it is now realized that hysteresis is a feature of many regulatory enzymes [18].
MATERIALS AND METHODS In addition to the materials described in the preceding paper 191, glycogen from leukocytes (a gift from Dr L. Plesner) and glycogen from oyster and rabbit liver glycogen type TI and V (Buedinger glycogen), purchased from Sigma Chemical Co. (St Louis, Mo.) were used. The glycogens were purified as described [9]. x-Amylase limit dextrin was produced by incubating 100 mg of rabbit liver glycogen in 10 ml 50 mM Tris-HCl (pH 7.4), 5 mM EDTA (buffer A) with 1000 units of diisopropylphospliorofluoridate-treated hog pancreas amylase (Sigma) at 37 ' C for 48 h. After addition of 1 ml 307; KOH to destroy the amylase, the preparation was gel-filtered on Sephadex G-50. Glycogen-free and glycogen-containing synthase I from human polymorphonuclear leukocytes were purified as described [9] to a specific activity of 7 - 11 U j mg protein. The glycogen-free enzyme contaiiied more than 5 U enzymelmg of residual glycogen and was essentially inactive in the absence of added primer. The glycogen-containing enzyme contained approximately 0.2 U enzyme/mg glycogen and was fully active even in the absence of added glycogen. Incubations of synthase were performed in buffer with 50 mM Tris-HC1 (pH 7.4), 5 mM EDTA and 14 mM 2-mercaptoethanol (buffer B), usually at 30 "C. The stability of the cnzyine during prolonged incubations in this buffer has been investigated in the preceding paper [9]. Glycogen-containing synthase activity was assayed with the filter paper method of Thomas et al. [lo]. The activity ratio is the ratio between activities measured in the presence of either 10 mM Na2S04 (I form) or 6.7 mM glucose-6-P (total activity). Before assay, glycogen-free enzyme was activated by incubation for 2 h, at 30 C with 20 mg/ml glycogen and 10 mM glucose-6-P. Glucose-6-P was removed by gel filtration before determination of activity ratio. Assay of synthase by fluorimetric measurement of UDP was slightly rnodificd from Passoneau et al. [19] using an Aminco-Bowman spectrofluorimeter (SPF 125). Glycogen and protein were determined as previously described [9].
Hysteresis of Glycogen Synthase I
RESULTS
In our initial experiments a remarkable difference was observed between progress curves obtained with glycogen-free and glycogen-containing synthase I. When glycogen-containing enzyme is assayed in the presence of sulfate (10 mM) or glucose-6-P (6.7 mM) perfect linear progress curves arc observed for 30 60 min with the concentration of enzyme used (Fig. 1A). After this time substrate depletionjproduct inhibition becomes notable. In the absence of sulfate or glucose-6-P, the progress curve is not linear. The deviation from linearity is more pronounced at low (0.2 mM) than at high (4 mM) UDP-glucose concentrations and is not influenced by the addition of glycogen. The decrease in velocity is caused by product inhibition in the absence of the activator, but a partial inactivation of the unprotected enzyme during the 60-min assay period at 30 "C cannot be excluded as contributory [9]. When the glycogen-free enzyme was assayed, sigmoidal progress curves were obtained (Fig. 1 B). The first upward concave part is suggestive of an activation of the enzyme during the assay period, while the last part probably represents substrate depletionjproduct inhibition as for the glycogen-containing enzyme. In the presence of Na2S04 or glucose6-P, the decrease in activity is first apparent after 60 min. In Fig. 1C an enzyme preparation was incubated with glycogen and glucose-6-P for various periods before a time course was run. From this experiment it is quite clear that the glycogen-free enzyme is slowly activated during the incubation. l n the absence of glucose-6-P or in the presence of inactivating substances like Mg2+ [9] during incubation or assay, underestimation of the degree of activation will take place. It is also evident from Fig. 1C that an apparently linear progress curve does not prove that an enzyme is fully activated as the relative change in the concentration of the activated form is too small to be detected during a short assay period. To further elucidate the nature of this activation process, a number of experiments were carried out. Fig. 2 shows that glycogen-free synthase I may be incubated for 48 h in the presence of mercaptoethanol and glucose-6-P without undergoing activation, whereas incubation of the enzyme in the presence of 5 mg glycogenjml resulted in a gradual activation, which in this experiment became maximal after 5 - 6 h and then remained constant. When the glycogen-frce enzyme was assayed without glycogen in thc assay mixture, essentially no activity was observed. When glycogen was present in the assay mixture a constant relatively low activity was measured corresponding to the activation induced by glycogen during the 120-s assay period. The same additional activity was observed during the
131
H. S$lling and V. Esmann
Time (min)
Fig. 1. Progress curvesfor glycogen-confaining andglycogen-free synfhase I . (A) A glycogcn-containing cnzyme (4 mU/ml) was incubated with 4 mM UDP-glucose (spec. act. 170 counts min nmol- ') and 6.6 mglml glycogen in buffer B at 30 "C without (A) or with 6.7 mM glucose-6-P (0)or 10 mM NazS04 (A). At time indicated aliquots were removed and spotted on squares of filter paper and procedured as described by Thomas [lo]. (B) A glycogen-free enzyme (8 mU/ml) was incubated exactly as in (A), except for the spec. act. of 1JDP-glucosebeing 275 counts min-' nmol-'. The curvcs obtained in the presence of glucose-6-P and Na2S04 were indistinguishable and only one is shown ( 7 ) .Control (v).(C) A glycogen-free enzyme (2 mU/ml) was incubated in buffcr B at 30 "C with 10 mM glucosc-6-P and 0.2 mg/nd glycogen. At the indicated intervals, progress curves over a 13-min period were obtained after transferring 600 p1 of the incubation mixture to 200 111 0.36 mM UUP-glucose (spec. act. 3000 counts min-' nmol-') preheated to 30 ' C
rapid initial phase of the activation, and waned with the progress of activation, when synthase I incubatcd in the presence of glycogen was assayed with assay mixture containing glycogen. The glycogen-free enzyme remained potentially completely active during the 24 h of incubation without glycogen, as it could be activated to the same extent, and at the same rate, upon subsequent incubation with glycogen. Clearly, the activation process cannot be a simple reactivation of a reversibly inactivated protein, but must be a specific property of the enzyme, which is induced by its one substrate, glycogen. In control experiments it was ascertained that the activity ratio was higher than 0.93 during the whole activation process. It is essential to realize that enzyme preparations having a higher glycogen content than about 1 mg residual glycogen per 5 U enzyme, for example preparations with an activity of 1 - 2 U/mg glycogen, contain enough glycogen to have demonstrable enzymatic activity when assayed in the absence of added, exogeneous, glycogen. In such cases incubation of the enzyme preparation in the presence of mercaptoethanol and glucose-6-P will lead to the erroneous impression of an activating effect of glucose-6-P similar to that of glycogen. The activation of a glycogen-free enzyme can also be accomplished by the endogenous glycogen of a glycogen-containing enzyme (Fig. 3 ) , although the enzyme activities are not completely additive. The native glycogen thus has unoccupied sites for the addition of enzyme. It is obvious that if glycogen-free and glycogen-containing enzyme occur simultaneously in
" ^ 1
U
I
J
0
2
3
J
U
" 4 5 Time (h)
G
M
A
@-cr--l* 24
48
,Fig. 2. Incubation ofgl,ycogen,free ,syniliatc I with and willlour glycogen. Glycogen-free synthase 1 (3 mUiml) was incubated for 48 h at 30 "C in buffer B supplcmentcd with 10 m M glucose-6-P and with (filled symbols) or without (open symbols) 5 niglml glycogen (type 111). At time indicated, enzyme activity was assaycd by transferring a 90-pl aliquot to preheated (30 "C) disposable tubes containing 30 pI of a mixture of 0.36 mM UDP-glucose (8000 counts min-' nmol-I), 10 mM glucose-6-P and either 100 mg;ml glycogcn (squares) or no glycogen (circlcs). After 120 s the assay was stopped by spotting 100 pl of the reaction mixturc on filter paper. After 24 h 950 pl of the incubation mixture without glycogen was transferred to a new tube containing SO pl of a glycogen solution (100 mgiml) and the incubation was continued for another 24 h (A). For reasons of prcsentation this incubation is drawn as starting at zero time. The slightly lower final vclocity obtained is explained by the dilution of'the enzyme. Only the results obtained with glycogcn in the assay mixture are presented. Synthase activity is measured as the rate of incorporation of glucosyl units
132
Hysteresis of Glycogen Synthase I
I
I 1
2
3
24
Time ( h )
Fig. 3. Activufion of a mi\-rure of glycogijn-jwe undglycogen-containing synthase I . In (A) 1 mU/ml of a glycogen-containing enzyme (6 pg glycogen/mU enzyme) was incubated at 30 "C in buffer B with 10 mM glucose-6-k' for 24 h. .Addition of 250 pgjml glycogen had no influence on the activity. In (B) 3 rnU/ml of a glycogen-free enzyme was activated by incubation with 250 pg/ml glycogen. In (C) both enzymes were incubated together without additional glycogen. At intervals the enzyme activity was assayed as in Fig. 2, but over 240 s and without glycogen in the assay mixture
vivo, the total enzyme content would be underestimated in an ordinary enzyme assay. However, glycogen-free synthase I has not yet been found in leukocytes and not been extensively looked for in other tissues, but has been demonstrated in muscle immediately after a tetanic contraction [2O,211. When the glycogen concentration is varied (Fig. 4) in the incubation, it is easily observed that the rate of activation depends on the glycogen concentration. For concentrations above 100 pg/ml glycogen a common final velocity (of) is obtained in all cases. At lower concentrations, the final velocity was lower and did not increase or decrease even after 72 h of incubation. It was ascertained that the glycogen concentration in the incubation medium was unchanged during these long incubations. It should be noted that the enzyme activity is measured at the concentration of glycogen present during the activation period. When, in parallel assays, the activity was measured with 2% glycogen in the assay mixture only such additional activity was measured in the low-glycogen incubations, as corresponded to the additional activation occurring during the 120-s assay period. No activity which might be ascribed to a kinetic effect of unbound glycogen was observed. Without added glycogen, the enzyme had essentially no catalytic activity. Glycogen from oyster, glycogen type I1 or type V from rabbit liver and glycogen from leukocytes were found to activate synthase in a similar way. Soluble starch and amylase limit dextrin had no effect. The structure of glycogen, for example caused by (undetected) contaminating enzymes, was apparently
not changed during the long incubation periods so as to make the glycogen an increasingly better substrate. When the same glycogen (I00 pgiml) was used for two successive activations of glycogen-free enzyme (2 m u / ml), precisely the same activation curve is observed (not shown). The enzyme used for the first activation period was killed either by heating to 90 "C for 2 min or by freezing at -20 "C in the presence of 50 mM mercaptoethanol. It is unlikely that both these procedures should transform the supposedly changed structure of glycogen to the structure it had before incubation. The demonstration of an activating effect of glycogen on synthase I ultimately depends on the assay of enzyme activity, which is carried out by measuring the incorporation of [14C]glucosefrom labelled UDPglucose into glycogen retained on filter paper (the Thomas assay [lo]). The (kinetically) unlikely claim could be made that the low enzyme activities measured during the activation period were due to the preferential glucosylation of low-molecular-weight primers not retained on filter paper, as for example dithiothreitol or low-molecular-weight dextrins remaining after the amylase treatment of synthase to remove glycogen. The activation of a glycogen-free enzyme by glycogen could, however, also be demonstrated when followed by the liberation of UDP from UDP-glucose (results not shown). Also, the observed activation could be thought due to a change in the binding constants for the substrate UDP-glucose or the activator glucose-6-P. In a following paper we will show that synthase I, in analogy with the D enzyme [2], at saturating glycogen concentrations follows the formula u=
V
KB ' 1+B
(3)
Kn is the apparent K , for UDP-glucose, K i is 24 pM for UDP-glucose for both the I (cf. below) and D enzymes, and Kc is the activation constant for glucose-6-P, which for the D enzyme is 1.7 mM [2] and for the I enzyme 0.017 mM (unpublished results). If, for example, an observed three-fold activation of enzyme by glycogen should instead be accounted for by a change in the kinetic constants, it can be calculated that KB must change by a factor of nine during the activation process, which should be easily discernible. As shown in Fig. 5, KB (24 pM) is cssentially constant during the whole activation process. As mentioned, the activity ratio has also been found to be unchanged and greater than 0.93 during the activation period (cf: Fig. 1 Bj, thus excluding any conversions between an apparent D form and synthase I as a cause of activation.
133
H. Sglling and V. Esmann
-11.5 E .c
.-E
b I
1
e
'
1
-
-
2
4
0
8
24
Time ( h )
Fig.4. Activation of glycogen synthase I by dijJerent concan/ru/ionsoj glycogen. Glycogen-free synthase 1 (2 mU/ml) was incubated for 24 h at 30 "C in buffer B supplemented with 10 mM glucose-6-P and rabbit liver type I11 glycogen (in mg/ml): 25 (+), 10 (v).2 (v),0.2 (A), 0.04 (A), 0.01 (O), 0.005 (m), 0.002 (O), and zero (0).At intervals the enzyme activity was assayed as in Fig. 2, but over 150 s and without glycogen in the assay mixture
10
20 30 1/ [ UDP -glucose] ( m M-' )
40
Fig. 5. Estimation of the KJor UDP-glucose duringmtivation. 8 mU/ml glycogen-free synthase 1 was incubated at 30 "C in buffer B with 10 mM glucose-6-P and 500 pg/ml glycogen. At the indicated times aliquots were removed and assayed with UDP-glucose (16000 counts min-' nmol-I). For each UDP-glucose concentration a time course was run over 10 min to allow estimation of the initial velocity. Except for the 3-min curve, these progress curves were apparently perfectly linear (cJ: Fig. 1 C)
The rate of activation, but not of, of synthase I by glycogen is dependent on temperature in the interval 20-40 "C (Fig.6). At 4 "C and 14 "C, the rate of activation as well as of were not measurably different from that obtained at 20 "C, which most probably is due to the inevitable activation occurring during the equilibration and assay periods at 30 "C. Attempts to demonstrate a temperature-dependent change in M , of glycogen-free synthase have failed [9]. pH also heavily influences the activation (Fig. 7), although less in the physiological range than at alkaline pH. It is observed that at pH 9.0 the enzyme is unstable after 24-h incubation (see also 191).
The rate of activation was independent of the addition of 0.01 - 10 mM glucose-6-P or 10 mM Na2S04, but in the absence of one of these ligands a 5 - 30 % lower vf was obtained due to inactivation of the enzyme (cj: Fig. 1 and the preceding paper [9]). Similarly, the rate of activation was not affected by varying the concentration of mercaptoethanol from 0- 50 mM or omitting EDTA from the buffer. Also the addition of UDP (10- 100 pM), NaF (50 mM): MgClz (10 mM), ATP (1 - 5 mM), glucose (100 mM), sucrose (25 %), glycerol (25 %), amylase limit dextrin, serum albumin ( 5 mgiml), or varying the .ionic strength by addition of 0.3 - 3 M NaCl were without effect on the activa-
134
Hysteresis of Glycogen Synthase I
0
#
0
I
'
1
2
3
4
8
12
24
Time ( h )
Fig. 6. Influence oftemperature on activalion vj'qvnthaseI b y glyc'ogen. 2 m U /nil of a glycogen-free enzyme was incubated in buffer B with 10 mM glucose-6-P and 2.5 mg/ml glycogen at different temperatures: 40 "C (m), 35 "C (A), 30 ' C (A), 25 'C (O), and 20 "C (0).Incubations were also performed at 14 "C and 4 'C, but these results were identical to those obtained at 20 'C and are omitted from the figure. bnzyme activity was measured at the times indicated. 6O-pl aliquots were transferred to empty test tubes and equilibrated at 30 "C for 45 s. Then the reaction was started by adding 40 pl of 0.3 mM UDP-glucose + 10 mM glucose-6-P (preheated to 30 T).Assay was terminated after 180 s
0
1
2 Tine ( h )
3
4
24
Fig. 7. Influence q f p H on uctivation. 3 mC/ml of glycogen-free enzyme was incubated at 30 "fwith 200 pg/ml ofglycogen. Buffer B contained 10 mM of glncose-6-P, and the pH was adjusted as indicated prior to incubation: pH 9.0 (V).pII 8.6 (V), pH 8.2 (A),pH 7.8 (A), pH 7.4 ( 0 ) and pH 7.0 (0).120-s assays were performed during incubation as described above
tion process. Also the addition of soya bean trypsin inhibitor or phenylmetylsulfonyl fluoride had no influence. For the enzyme glycogen phosphorylase a it has been found [22] that a decrease in specific activity with increasing enzyme concentrations signified a conformational change from dimer to tetramer. For synthase I, albeit in a much lower concentration range, perfectly linear plots of z'f versus enzyme concentration (2.6- 105 mujml) were found, provided the enzyme was suitably protected by glucose-6-P against inactivation. Also, the rate constant of activation was
independent of enzyme concentration. These results are in keeping with our inability to demonstrate changes in M , by incubating the glycogen-free enzyme at different temperatures or with or without glucose6-P [9]. Whether the activation took place after formation of the enzyme-glycogen complex was examined by utilizing a technic developed by Metzger and Helmreich [23]. The enzyme was slowly activated by incubating with a low concentration of high-molecularweight glycogen. which can be sedimented in the ultracentrifuge (Fig.8). It was found that the amount of
H. S$lling and V. Esmann
135
found. however. that when a concentrated glycogencontaining enzyme was rapidly diluted to form a suspension of 0.5 mU enzyme/ml with as little as 2 pg/ml of endogeneous glycogen, no change in activity was observed over a subsequent 24-h incubation with or without added exogeneous glycogen. We must therefore conclude that the formation of the enzyme-glycogen complex is a practically irreversible reaction at most physiological glycogen concentrations. However, in some experiments with an enzyme preparation partly freed of glycogen, and thus not totally inactive in the absence of added primer, such inactivation was observed. The degree of inactivation increased with the degree of dilution and was reversible as judged by full activation upon addition of glycogen.
( A ) Complete incubation
3
DISCUSSION
I
/ E,,
OO
1
2 Time ( h )
3
24
Fig. 8. Ce?ilrifugation experitmw~scturrirg cicrivntion. (A) 4 in Ulml of glycogen-free synthase I was incubated in buffer B with 10 mM glucose-6-P and either (11) without (0)or (1) with (0) 250 pg!ml high-molecular-weight glycogen (type V). At intervals. the enzyme activity was assayed as descri bed in Fig. 2. Also, at intervals indicated in (B) and (C), 1-in1 portions of the reaction mixtures were transferred to small ultracentrifugation tubes, rapidly cooled to 2 'C. and spun at 40000 rev./min for 45 min at 2 "C in an MSE 65 highspeed ullracenlrifuge. A carefully taken sample (100 pl) of the resulting supernatant (B) was allowed 10 be fully activated by addition of 40 i t 1 of a glycogen solution (ef. Methods) before assay of enzyme activity. Indicated by and a1 times zero and 24 11 arc the enzyme activity of a fully activated aliquot of the incubation mixture, which had not been centrifuged. Thc pellets (C) were resuspended in 1 in1 buffer B with 10 mM glucosc-6-P and assayed. Control experiments showed that the enzyme in the pellet fraction was not further activated when incubated with 20 mg/nil glycogen
enzyme remaining in the supernatant slowly decreased during incubation and always required activation for full activity. Conversely, the sedimentable enzyme slowly increased during incubation, and once bound to glycogen exhibited full activity. Considering the time scales involved and the minimal further activatioh occurring during the centrifugation period, this result clearly shows that the hysteretic activation is paralleled by the formation of an enzyme-glycogen complex. The activation of synthase I by glycogen might theoretically be reversed by a suciria drop in glycogen concentration. This should resulL i n a drop in enzyme activity and in the appearance of an inactive enzyme species, which subsequently could be activated. We
The slow activation of glycogen synthase I has been reported by others [lo-121, but a detailed investigation has not previously been carried out. Usually, the enzyme has been activated by incubation for 10-30 min with 5 - 10 mg/ml glycogen at 30 "C and the process has been thought completed on the evidence or linear progress curves. As demonstrated in Fig. 4, the activation process requires one hour at pH 7.4 even at 25 mg/ml glycogen and, as shown in Fig. 1, short-term linear progress curves are not to be relied upon in this respect. It has been reported [l 11 that glucose-6-P accelerates the activation by glycogen (concerted hysteresis). We have in the preceding paper shown that glucose-6-P or Na2S04protects synthase I against inactivation and the present results exclude any effect of glucose-6-P (or Na2S04) on the activation process proper. The activation can be elicited by glycogen from different sources and is not caused by changing properties of the glycogen during incubation. It remains to be investigated whether the activation depends on the detailed structure of glycogen, for example the length of outer chains. It is noted that synthase I purified by methods not involving amylase treatment also exhibit activation by glycogen [ll - 131, as does synthase D [ll,121 (and own control experiments). It seems justified to name the slow (slow compared to the time required to measure enzyme activity) activation of synthase as a hysteretic process since it is elicited solely by addition of glycogen and cannot be caused by a non-specific renaturation. There are six obvious reactions, which will give rise to this behaviour (c$ introduction). a) A slow isomerization of inactive to active enzyme ( E S E ' ) followed by a rapid binding to glycoG e E ' G). E and E' signifies different congen (E' formations of enzyme.
+
136
Hysteresis of Glycogen Synthase I
b) A slow dissociation of tetramer to dimer enzyme followed by rapid binding to glycogen to give the active enzyme. c) Initial rapid binding of enzyme to glycogen followed by a slow isomerization process (E + G$EG+E' G). d) A slow association of a modifier to yield an enzyme-modifier complex that rapidly binds to glycogen to become active. e) A slow dissociation of a modifier to yield an enzyme that rapidly associates with glycogen. f ) A slow bimolecular reaction between enzyme and glycogen without any of the above mechanisms. Mechanism (a) is excluded by the fact that the observed rate constant, k , for the formation of active enzyme is a linear function of the glycogen concentration at 30 "C (see Fig. 10 below) and at 4 "C (not shown), whereas mechanism (a) (and b and c) predicts k as a hyperbolic function of glycogen concentration [34]. A change in molecular weight compatible with a tetramer to dimer conversion has never been observed with the leukocyte enzyme [9]. If the equilibrium between tetramer (inactive) and dimer (active) enzyme is not totally in favour of the former, one should also observe an initial burst phenomenon, which we have never seen with a totally glycogen-free enzyme. Also, varying the enzyme concentration should have caused changes in the rate of activation. This evidence, although negative, speaks against mechanism (b). Mechanism (c) is also ruled out by the results shown in Fig. 8, which demonstrates that the enzyme is fully active as soon as it is bound to glycogen. Mechanism (d) is eliminated, since no substance other than glycogen (and H+) seems to influence the activation process. Finally, mechanism (e) is excluded by the experiment illustrated in Fig. 2, which shows that prolonged incubation does not result in any change in the rate of activation, and also by the fact that the rate of activation was unaffected by enzyme concentration. A possible candidate for mechanism (e) could have been amylase limit dextrin remaining after digestion of glycogen, but this substance was found positively not to influence activation. The data are, however, compatible with the curprising, but simple solution, mechanism (0,that the hysteresis phenomenon is caused by the slow binding of synthase to its one substrate, glycogen, in the bimolecular reaction :
E+G+EG (4) with the dissociation constant K , = k - l / k l . Most of the experiments have been conducted with a high ratio of glycogen to enzyme. Assuming molecular weights of glycogen and synthase I of 5 x lo6 and 360000, respectively, and a maximal specific activity of synthase I of 36 U/mg [24], the molar ratio in- experiments conducted with 1 pg/ml ~~
~
glycogen and 1 mU enzyme/ml would, however, only be 2.6. Due to the uniform structure of glycogen and the difference in size between the two molecules, the number of possible binding sites on each glycogen molecule, even in this case, is far in excess of those on the enzyme. We are thus dealing with a pseudo-firstorder reaction, which explains why the activation follows Eqn (l), the formula for a first-order reaction. What we observe during the activation process is thus the appearance of a tightly associated enzymeglycogen complex. The appearance of this catalytically active complex is monitored by the short (1 20 - 150-s) assays according to Eqn ( 5 ) : ~t
=
k, [EG]
(5)
where k, is a complex constant. During the short assay period a possible further activation of the enzyme due to consumption of enzyme-glycogen complex, EG, and subsequent displacement of Eqn (4) will be negligible and ut is thus a measure of the concentration of EG. In order to relate ut to [EG], when ut is measured at non-saturating concentrations of UDP-glucose, it is necessary to assume that the Michaelis constants of the catalytic reaction are also dissociation constants, which has been found true for synthase D of human polymorphonuclear leukocytes [ 2 ] . This also appears to be true for synthase I according to the results of Fig. 5, which demonstrates a common intercept on the horizontal axis, when UDP-glucose is varied at constant but increasing concentrations of EG, as formed during the course of the association between synthase and glycogen. Under the present experimental conditions k, is therefore the rate constant for the rate-limiting conversion of the ternary enzyme-glycogen-UDP-glucose complex to products, divided by 1 + K B / B(cf: Eqn 3). It was demonstrated in Fig. 4 that uf, the velocity measured when reaction (4) had reached equilibrium
1
'5
25
200
100
l/[Glycogen]
(mlerng')
Fig. 9. Klotz plot of corresponding values o j glycogen concentration and VJ as obtained from Fig. 4 . For symbols see Fig. 4. K, in this experiment was 6.25 pg/ml (determined by the weighted least-squares procedure of Wilkinson [35])
131
H. S$lling and V. Esmann
Fig. 10. The dutu of Fig. 4 replottedaccording
10
Eqn ( 2 J . Symbols as in Fig.4. The following values for k and t , ,Z = In 2/kmay be calculated
Glycogen concn.
k
fl/Z
mg/ml
min-l
min
25 10 2 0.2 0.04
0.115
6
0.056 0.021 0.0106
12
0.0079
33 65 88
The insert shows k as a function of the glycogen concentration [GI. In the interval 0.2-25 mg/ml glycogen k = 0.010 having k = 0.017 0.0040 [GI parison the insert also shows the result of another experiment (&--o)
+
depended on the concentration of glycogen. Formation of EG follows the equation
where [Elo is the total enzyme concentration and [GIo is the initial concentration of glycogen. At equilibrium, the exponential term can be ignored and
- -
If vf (sat) is vf at saturating glycogen concentrations, then Q(sat)
- uf
[GI = K,
(8)
Of
which, using the data of Fig. 4, is illustrated in doublereciprocal form (Klotz plot [33]) in Fig. 9, from which K, is easily obtained. In five experiments with glycogen concentrations ranging between 0.001 and 0.1 mg/ml, K, was estimated to 0.0033 f: 0.0012 (S.E.), which means that formation of the enzyme-glycogen complex is strongly favoured, a fact that is common knowledge to those who have
+ 0.0045 [GI. For com-
attempted to separate (completely) synthase I from glycogen without the help of glycogenolytic enzymes. The binding of glycogen to synthase I as expressed by Eqn (4) is to be considered apart from interactions of the terminal glycosyl unit with the catalytic site. The present investigation does not contradict the rapid equilibrium assumption [21, but the presence of hysteresis prevents the determination of a dissociation constant (K,) for glycogen at the active site. The slow binding of glycogen to the enzyme is a prerequisite for any catalytic activity and the amount of active enzyme will therefore not be constant, but vary with the amount of added glycogen as stated by Eqns (2) and (6). The data from Fig.4. showing the influence of glycogen on enzyme activation, are replotted in Fig. 10 according to Eqn (2). It is observed that within the interval 0.04- 25 mg/ml glcyogen, Eqn (2) is strictly obeyed, which supports the assumptions underlying Eqn (4). For the lowest glycogen concentrations (0.001-0.010 mg/ml), the determination of k is highly sensitive to small variations in ut and vf and has usually been indistinguishable from that obtained with 0.04 mg/rnl glycogen.
138
Hyslercsis of Glycogen Synthase I
1
0
4
3
2
Time ( h )
Fig. 11. The data of' Fig. 6 replotted accodina to E4n (2). The symbols are as in Fig. 6. The following values [or k and t l i ~(In 2/k) may be calculated. At 20 'C and 25 "C thc first value of k corresponds to the first hour of incubation, the second value to the snbsequent 3 h (c5 text) Temp.
k
(112
T
min-'
min
40 35 30 25 (4-14)-20
0.096 0.029 0.014 0.0096 -0.0069 0.0075 -0.0060
24 50 72- 100 92-116
7
The insert shows the Arrhenius plot of log k as a function of T - ' . The stipled line connccts values o f log k obtained during the last 3 h ofincubation at 20 "C and 25 "C. From the now straight line between 0.003246 and 0.003356 (35 'C to 25 " C ) an activation energy of 25200 caljmol (306 kJ:mol) may be calculated
Within the interval 0.04- 25 mg/ml glycogen, k increased from 0.0078 to 0.115 min-'. For saturating glycogen concentrations ( =. 0.1 mg/ml, c$ above), k appears to be a linear function of [GI, as shown in the insert of Fig. 10 and as predicted by Eqn (6). The regression line allows an estimate of k and the time necessary to activate synthase I at a particular glycogen concentration (at the temperature and pH indicated). It is not warranted to identify the rate constant in the expression fork in Eqn (6) with the empirical constants of the regression line in Fig. 9 (qfi above). The activation of synthase I by glycogen depended markedly on temperature (Fig. 6) and the results are plotted according to Eqn (2) in Fig. 11. A plot of loglok versus T-' (Fig. 11, insert) is non-linear when based on values of k calculated from activities measured during the first hour of incubation. This is presumably due to the error introduced by additional enzyme-glycogen complex formed during the period of equilibration and assay at 30 C , which will result in too high estimates of k . The relative error will decrease with time and when values of k from the later part of the activation period are used instead, a linear relation between
log k and T-' from 25-35 "C is observed allowing the calculation of an activation energy of 25400 cal: mol (106 kJ/mol) for formation of the enzyme-glycogen complex (Eqn 4). This is about twice as high as the activation energy (13 SO0 cal/mol or 56 kJ/mol) found in the preceding paper [9] for the glycogen-containing enzyme (Eqn 5). The influence of pH on the activation of synthase is quite surprising. Further studies along this line might yield information on the composition and localization of the binding sites on enzyme and glycogen. Although the influence of pH is most pronounced at nonphysiologically high values, it is tempting to speculate on a physiological influence of H+ on hysteresis (concerted hysteresis [ 141). The progress curves in Fig. 1 C may be considered to be parts of one long progress curve not influenced by substrate depletion or product inhibition. To describe such progress curves Frieden has derived the equation
P, =
l?ft
-
~
1 ( Z' r - v0) (1 k
-
e-k'),
139
H. SClling and V. Esmann
where Pt is product concentration at time t and the other symbols as above. When ePk t< 1 the progress curve becomes linear and intersects the abscissa at t = ( u f - z:o)/kz?f.The estimation of this point is usual when lag periods are investigated in pre-steadystate kinetics. In a very slow process it is, howcver, difficult to judge when a progress curve is linear even if substrate depletion and product inhibition can be prevented. If 210 = 0, prolongation of the apparently linear portion in the upper curve in Fig. 1 B gives us t = k - ’ = 4mi n and k = 0.25 min-l. tl12 is now calculated to 2.8 rnin. The true value of l l i 2 ,however, is about 17 min (c$ Fig. 10). The physiological significance of hystercsis in glycogen metabolism is difficult to evaluate yet, but it could be, as stated by Frieden [14], that very rapid changes in the concentration of key metabolites are avoided. If concerted hysteresis exists, a very powerful feed-back regulation may result. It is interesling to note that phosphorylase [14] and UDPG-pyrophosphorylase [32] also exhibit hysteretic properties. Until now, hysteresis has only been obsewed in vitva. Since it may be a rate-limiting step, hysteresis deserves consideration in experiments where synthase I and D are used as substrates or products in the study of protein kinases and phosphatases. In such experiments total activity appears to increase in tissue homogenates during incubation [25- 301. Since very high concentrations of glycogen is usually involved in the assay of synthase and this assay is carried out for 10 min or more, a hysteretic activation is easily underestimated. Also in experiments with whole organisms changes in total activity of synthase have been observed [28,31], which may not totally be explained by de nova synthesis of enzyme. The authors are greatly indebted to Mrs Jonna Guldberg for the extreme care exercised during the present expcrimcnts and to Liselotte and Igor W. Plesner for enlightening discussions. Financial support has been given to VE by the Danish Research Council (grants 51211517-2588-3663-5288.6723 and 151169).
REFERENCES 1. Stalmans, W. & Hers, H. G. (1973) in The Eniynzes 3rd edn (Boyer, P. D.; ed.) vol. 9, pp. 309- 361. Academic Press. New York and London. 2. Plesner, L., Plesner, 1. W. & Esmann. V. (1976) Mol. Ce//.Biorhem. 12,45- 61,
H. Splling and V. Esmann, Medicinsk Afdeling, Marselisborg Hospital, Skanderborgvej 9, DK-8000 Arhus C, Denmark
3. Saugmann, P. (1977) Biochem. Biuphys. Res. Commun. 74, 1511 -1519. 4. Saugmann. P. & hsmann, V . (1977) Biochem. Biophyh. Res. Commuu. 74, 1520 - 3 527. 5. Saugmann, P. (1977) Abstr. Commun. 11th Meet. Fed. Eur. Biuchem. Soc. A 1-4-006. 6. S$lling, H. & Esmann, V. (1974) Abstr. Commun. 9th Meet. Fed. Eur. Biochem. Soc., p. 83. 7. Wang, P., Plesncr, L. & Esmann, V. (1972) Eur. J . Biochen~.27, 297 - 300. 8. W-ang,P., Bantle, G. & S$rensen, N.B. (1977) Biochim. Bioph>ls.Aciu, 496, 436 - 447. 9. SQlling,H. & Esmann, V. (1977) EUF.,J, Biochem. 81,119-128. 10. Thomas, J. A., Schlender, K . K. & Larner, J. (1968) Anal. Biuchem. 25,486-499. 11. Rottenberg. D. A,, Passoneau, J. V. & Lust, W. D. (1972) Biochen?. Biopliys. Res. Cornmun. 48, 1192- 1198. 12. Passoneau, J. V. & Schwarti, J. P. (1975) J . Biol. Chrm. 250, 2287 -- 2292. 13. Nimmo, H. G., Proud, C. G . & Cohen, P. (1976) Eur. J . Biochem. 68, 21 -30. 14. Friedcn, C. (1970) J . Biol. Chem. 245, 5788-5799. 15. SQlling, H. & Esmann. V. (1976) Proc. I U t h Int. (’ongr. Biochem., p. 41 5. 16. Rabin, B. R. (1967) Biochem. J . 102, 22c-23c. 17. Ainslie, G. R. Jr, Shill. J. P. & Neet, K . E. (1972) J . Biol. Chem. 747.7088 - 7096. 18. Shill, J. P. & Neet, K. E. (1975)J. Bid. Chem. 250,2259-2268. 19. Passoneau, J. V. & Rottenberg, D. A. (1973) Anal. Biochem. 51, 528 - 541. 20. Staneloni, R. J. & Piras, R. (1971) Biochem. Bioplzys. Res. Commun. 42,237 -244. 21. Piras, R. & Staneloni, R. (1970) Fed. Proc. 29, 676. 22. Wang, J . H. tk Gravcs, D. J. (1964) Biochemistry, 3, 14371445. 23. Metzger, B., Helmreich, E. & Glaser. L. (1967) Biochemistry, 6,994-1001. 24. Takeda, Y., Brewer, H. €3. & Larner, J. (1975) J . Bid. Chem. 250, 8943 8950. 25. Rosell-Perez, M. &Lamer, J. (1962) Biochemisfvyy:1, 769- 772. 26. Hizukuri, S. & Larner, J. (1963) Biochim. Biophys. Acta, 72, 525 527. 27. Gold, A. H. & Segal, H. L. (1967) Arch. Biochem. Biophys. 220, 359 - 364. 28. Vardanis. A. (1969) Arch. Biochem. Biophys. 130,413 ~ ~ 4 2 1 . 29. Sato. K., Abe, N. & Tsuiki, S. (1969) Hiochim. Bzopl~ys.Acta, 268.638 645. 30. Rosell-Perez, M. (1972) Itul. J . Biochem. 21, 34- 69. 31. Stciner, D. F. &King, J. (1964)J. Biol. Chem. 239,1292- 1298. 32. Fitzgerald. D. K., Chen, S. & Ebner, K. E. (1969) Biochim. Biop h y . Acta, 178, 491 498. 33. Segel, I. H. (1975) Enzyyme Kinetics, pp. 218-220, John Wiley and Sons. New York. y, V. (1975) J. Bid. Chem. 34. Strickland, S., Palmer, G. & M 250,4048 4052. 3s. Wilkinson, G. N. (1961) Biochem. J . 80, 324-332. -
-
-
~
-
The Hysteretic Properties of Glycogen Synthase I Henrik SQLLING and Viggo ESMANN Department of Medicine, Marselisborg Hospital, Aarhus (Received June 29, 1977)
Glycogen-free synthase I from human polymorphonuclear leukocytes is activated by its own substrate, glycogen, in a slow, time-dependent proces (hysteretic activation). This lag in response to addition of glycogen depends on the concentration of glycogen, pH and temperature. At pH 7.4 and at a temperature of 30 "C, the half-time of activation, tl:2, decreases from 89 min at 0.004 mg/ml glycogen to 6 min at 25 mg/ml. The activation is accelerated by increasing temperature and pH, but is not influenced by enzyme concentration, glucose 6-phosphate, UDP, high ionic strength, EDTA, mercaptoethanol, glucose, sucrose or amylase limit dextrin. The K,,, for UDP-glucose (0.024 mM) and the activity ratio were unchanged during the activation process. The activation can be described by tit = uf (vo - or) eCkrwhere ut, uf and uo are velocities at times t , 0 and co and k is a complex rate constant. Evidence from ultracentrifugation and kinetic studies is presented to substantiate the hypothesis that the underlying mechanism is a simple bimolecular process :enzyme glycogeneenzyme-glycogen complex, with the dissociation constant K, = 0.003 mg/ml. The hysteretic activation may become rate-limiting during experiments in vitro with synthase. The possibility of a physiological role in glycogen metabolism, perhaps in the form of a concerted hysteresis with H + is discussed.
+
+
The activity of glycogen synthase, the rate-limiting UDPenzyme of glycogen synthesis (glycogen, glucose+glycogen,+ I UDP), is controlled by covalent modification and by metabolite (allosteric) control [l]. In human polymorphonuclear leukocytes, the control is exerted at three levels with regard to degree of phosphorylation and value of kinetic constants. The kinetic constants for substrates and modifiers of the fully phosphorylated enzyme form (D for dependent on glucose-6-P for activity) are of such magnitude that this form of the enzyme can be considered inactive under intracellular conditions [2]. A recently discovered partially phosphorylated enzyme form (R for rheostatic) has been found to account for glycogen synthesis under several physiological conditions, the kinetic constants being of a magnitude compatible with control exerted by the intracellular concentration of glucose-6-P 13 - 51. The third non-phosphorylated enzyme form, synthase 1, must from preliminary evidence [6] be considered fully active under all conditions. It can be formed in leukocytes under different experimental conditions, in particular when the inhibitory effect of glycogen on the D to 1 conversion is relieved during starvation [7], and/or when the activity of phosphorylase a reaches a critical, low level [S], suggesting the relief of an inhibitory effect on the R to I conversion [4].
+
Enzyme. Glycogen synthase (EC 2.4.1.11).
+
When we attempted to detail the kinetics of synthase I we became aware that glycogen, besides being both substrate and product in the enzymatic reaction and inhibitor of the D to I conversion, also confers stability on synthase I against inactivation, as described in the preceding paper [9]. In addition, glycogen induces a slow, time-dependent activation of initially glycogen-freesynthase I. Since this phenomenon, which also has been found by others [lo-131, sometimes may become rate-limiting, at least in vitro, it became clear that the kinetic behavior of synthase I is more complicated than initially expected. In this communication, we introduce the hysteretic enzyme concept [14] to describe the activation of synthase I by glycogen and discuss the physiological significance as well as the possible mechanisms behind the phenomenon. A preliminary account of this work has been given 1151. A slow increase or decrease in enzyme activity induced by a rapid change in the concentration of a modifier or a substrate is called a hysteretic process by Frieden 1141, who showed that regardless of the underlying mechanism, the change in enzyme activity can, in most cases, be described by ut = vf + (210 - vr) e-kr (1) which transforms to
130
where ot is the velocity at time t , u0 is the velocity at t = 0, uf is the velocity at t = co,and k is a complex rate constant which depends on the concentration of the modifier, the temperature, pH, and the underlying mechanism. Hysteretic changes in enzyme activity may be caused by isomerization or polymeriLation/ depolymerization of the enzyme and by the dissociation or association of a tightly bound ligand. The same phenomenon has also been treated theoretically by others [16,17] and it is now realized that hysteresis is a feature of many regulatory enzymes [18].
MATERIALS AND METHODS In addition to the materials described in the preceding paper 191, glycogen from leukocytes (a gift from Dr L. Plesner) and glycogen from oyster and rabbit liver glycogen type TI and V (Buedinger glycogen), purchased from Sigma Chemical Co. (St Louis, Mo.) were used. The glycogens were purified as described [9]. x-Amylase limit dextrin was produced by incubating 100 mg of rabbit liver glycogen in 10 ml 50 mM Tris-HCl (pH 7.4), 5 mM EDTA (buffer A) with 1000 units of diisopropylphospliorofluoridate-treated hog pancreas amylase (Sigma) at 37 ' C for 48 h. After addition of 1 ml 307; KOH to destroy the amylase, the preparation was gel-filtered on Sephadex G-50. Glycogen-free and glycogen-containing synthase I from human polymorphonuclear leukocytes were purified as described [9] to a specific activity of 7 - 11 U j mg protein. The glycogen-free enzyme contaiiied more than 5 U enzymelmg of residual glycogen and was essentially inactive in the absence of added primer. The glycogen-containing enzyme contained approximately 0.2 U enzyme/mg glycogen and was fully active even in the absence of added glycogen. Incubations of synthase were performed in buffer with 50 mM Tris-HC1 (pH 7.4), 5 mM EDTA and 14 mM 2-mercaptoethanol (buffer B), usually at 30 "C. The stability of the cnzyine during prolonged incubations in this buffer has been investigated in the preceding paper [9]. Glycogen-containing synthase activity was assayed with the filter paper method of Thomas et al. [lo]. The activity ratio is the ratio between activities measured in the presence of either 10 mM Na2S04 (I form) or 6.7 mM glucose-6-P (total activity). Before assay, glycogen-free enzyme was activated by incubation for 2 h, at 30 C with 20 mg/ml glycogen and 10 mM glucose-6-P. Glucose-6-P was removed by gel filtration before determination of activity ratio. Assay of synthase by fluorimetric measurement of UDP was slightly rnodificd from Passoneau et al. [19] using an Aminco-Bowman spectrofluorimeter (SPF 125). Glycogen and protein were determined as previously described [9].
Hysteresis of Glycogen Synthase I
RESULTS
In our initial experiments a remarkable difference was observed between progress curves obtained with glycogen-free and glycogen-containing synthase I. When glycogen-containing enzyme is assayed in the presence of sulfate (10 mM) or glucose-6-P (6.7 mM) perfect linear progress curves arc observed for 30 60 min with the concentration of enzyme used (Fig. 1A). After this time substrate depletionjproduct inhibition becomes notable. In the absence of sulfate or glucose-6-P, the progress curve is not linear. The deviation from linearity is more pronounced at low (0.2 mM) than at high (4 mM) UDP-glucose concentrations and is not influenced by the addition of glycogen. The decrease in velocity is caused by product inhibition in the absence of the activator, but a partial inactivation of the unprotected enzyme during the 60-min assay period at 30 "C cannot be excluded as contributory [9]. When the glycogen-free enzyme was assayed, sigmoidal progress curves were obtained (Fig. 1 B). The first upward concave part is suggestive of an activation of the enzyme during the assay period, while the last part probably represents substrate depletionjproduct inhibition as for the glycogen-containing enzyme. In the presence of Na2S04 or glucose6-P, the decrease in activity is first apparent after 60 min. In Fig. 1C an enzyme preparation was incubated with glycogen and glucose-6-P for various periods before a time course was run. From this experiment it is quite clear that the glycogen-free enzyme is slowly activated during the incubation. l n the absence of glucose-6-P or in the presence of inactivating substances like Mg2+ [9] during incubation or assay, underestimation of the degree of activation will take place. It is also evident from Fig. 1C that an apparently linear progress curve does not prove that an enzyme is fully activated as the relative change in the concentration of the activated form is too small to be detected during a short assay period. To further elucidate the nature of this activation process, a number of experiments were carried out. Fig. 2 shows that glycogen-free synthase I may be incubated for 48 h in the presence of mercaptoethanol and glucose-6-P without undergoing activation, whereas incubation of the enzyme in the presence of 5 mg glycogenjml resulted in a gradual activation, which in this experiment became maximal after 5 - 6 h and then remained constant. When the glycogen-frce enzyme was assayed without glycogen in thc assay mixture, essentially no activity was observed. When glycogen was present in the assay mixture a constant relatively low activity was measured corresponding to the activation induced by glycogen during the 120-s assay period. The same additional activity was observed during the
131
H. S$lling and V. Esmann
Time (min)
Fig. 1. Progress curvesfor glycogen-confaining andglycogen-free synfhase I . (A) A glycogcn-containing cnzyme (4 mU/ml) was incubated with 4 mM UDP-glucose (spec. act. 170 counts min nmol- ') and 6.6 mglml glycogen in buffer B at 30 "C without (A) or with 6.7 mM glucose-6-P (0)or 10 mM NazS04 (A). At time indicated aliquots were removed and spotted on squares of filter paper and procedured as described by Thomas [lo]. (B) A glycogen-free enzyme (8 mU/ml) was incubated exactly as in (A), except for the spec. act. of 1JDP-glucosebeing 275 counts min-' nmol-'. The curvcs obtained in the presence of glucose-6-P and Na2S04 were indistinguishable and only one is shown ( 7 ) .Control (v).(C) A glycogen-free enzyme (2 mU/ml) was incubated in buffcr B at 30 "C with 10 mM glucosc-6-P and 0.2 mg/nd glycogen. At the indicated intervals, progress curves over a 13-min period were obtained after transferring 600 p1 of the incubation mixture to 200 111 0.36 mM UUP-glucose (spec. act. 3000 counts min-' nmol-') preheated to 30 ' C
rapid initial phase of the activation, and waned with the progress of activation, when synthase I incubatcd in the presence of glycogen was assayed with assay mixture containing glycogen. The glycogen-free enzyme remained potentially completely active during the 24 h of incubation without glycogen, as it could be activated to the same extent, and at the same rate, upon subsequent incubation with glycogen. Clearly, the activation process cannot be a simple reactivation of a reversibly inactivated protein, but must be a specific property of the enzyme, which is induced by its one substrate, glycogen. In control experiments it was ascertained that the activity ratio was higher than 0.93 during the whole activation process. It is essential to realize that enzyme preparations having a higher glycogen content than about 1 mg residual glycogen per 5 U enzyme, for example preparations with an activity of 1 - 2 U/mg glycogen, contain enough glycogen to have demonstrable enzymatic activity when assayed in the absence of added, exogeneous, glycogen. In such cases incubation of the enzyme preparation in the presence of mercaptoethanol and glucose-6-P will lead to the erroneous impression of an activating effect of glucose-6-P similar to that of glycogen. The activation of a glycogen-free enzyme can also be accomplished by the endogenous glycogen of a glycogen-containing enzyme (Fig. 3 ) , although the enzyme activities are not completely additive. The native glycogen thus has unoccupied sites for the addition of enzyme. It is obvious that if glycogen-free and glycogen-containing enzyme occur simultaneously in
" ^ 1
U
I
J
0
2
3
J
U
" 4 5 Time (h)
G
M
A
@-cr--l* 24
48
,Fig. 2. Incubation ofgl,ycogen,free ,syniliatc I with and willlour glycogen. Glycogen-free synthase 1 (3 mUiml) was incubated for 48 h at 30 "C in buffer B supplcmentcd with 10 m M glucose-6-P and with (filled symbols) or without (open symbols) 5 niglml glycogen (type 111). At time indicated, enzyme activity was assaycd by transferring a 90-pl aliquot to preheated (30 "C) disposable tubes containing 30 pI of a mixture of 0.36 mM UDP-glucose (8000 counts min-' nmol-I), 10 mM glucose-6-P and either 100 mg;ml glycogcn (squares) or no glycogen (circlcs). After 120 s the assay was stopped by spotting 100 pl of the reaction mixturc on filter paper. After 24 h 950 pl of the incubation mixture without glycogen was transferred to a new tube containing SO pl of a glycogen solution (100 mgiml) and the incubation was continued for another 24 h (A). For reasons of prcsentation this incubation is drawn as starting at zero time. The slightly lower final vclocity obtained is explained by the dilution of'the enzyme. Only the results obtained with glycogcn in the assay mixture are presented. Synthase activity is measured as the rate of incorporation of glucosyl units
132
Hysteresis of Glycogen Synthase I
I
I 1
2
3
24
Time ( h )
Fig. 3. Activufion of a mi\-rure of glycogijn-jwe undglycogen-containing synthase I . In (A) 1 mU/ml of a glycogen-containing enzyme (6 pg glycogen/mU enzyme) was incubated at 30 "C in buffer B with 10 mM glucose-6-k' for 24 h. .Addition of 250 pgjml glycogen had no influence on the activity. In (B) 3 rnU/ml of a glycogen-free enzyme was activated by incubation with 250 pg/ml glycogen. In (C) both enzymes were incubated together without additional glycogen. At intervals the enzyme activity was assayed as in Fig. 2, but over 240 s and without glycogen in the assay mixture
vivo, the total enzyme content would be underestimated in an ordinary enzyme assay. However, glycogen-free synthase I has not yet been found in leukocytes and not been extensively looked for in other tissues, but has been demonstrated in muscle immediately after a tetanic contraction [2O,211. When the glycogen concentration is varied (Fig. 4) in the incubation, it is easily observed that the rate of activation depends on the glycogen concentration. For concentrations above 100 pg/ml glycogen a common final velocity (of) is obtained in all cases. At lower concentrations, the final velocity was lower and did not increase or decrease even after 72 h of incubation. It was ascertained that the glycogen concentration in the incubation medium was unchanged during these long incubations. It should be noted that the enzyme activity is measured at the concentration of glycogen present during the activation period. When, in parallel assays, the activity was measured with 2% glycogen in the assay mixture only such additional activity was measured in the low-glycogen incubations, as corresponded to the additional activation occurring during the 120-s assay period. No activity which might be ascribed to a kinetic effect of unbound glycogen was observed. Without added glycogen, the enzyme had essentially no catalytic activity. Glycogen from oyster, glycogen type I1 or type V from rabbit liver and glycogen from leukocytes were found to activate synthase in a similar way. Soluble starch and amylase limit dextrin had no effect. The structure of glycogen, for example caused by (undetected) contaminating enzymes, was apparently
not changed during the long incubation periods so as to make the glycogen an increasingly better substrate. When the same glycogen (I00 pgiml) was used for two successive activations of glycogen-free enzyme (2 m u / ml), precisely the same activation curve is observed (not shown). The enzyme used for the first activation period was killed either by heating to 90 "C for 2 min or by freezing at -20 "C in the presence of 50 mM mercaptoethanol. It is unlikely that both these procedures should transform the supposedly changed structure of glycogen to the structure it had before incubation. The demonstration of an activating effect of glycogen on synthase I ultimately depends on the assay of enzyme activity, which is carried out by measuring the incorporation of [14C]glucosefrom labelled UDPglucose into glycogen retained on filter paper (the Thomas assay [lo]). The (kinetically) unlikely claim could be made that the low enzyme activities measured during the activation period were due to the preferential glucosylation of low-molecular-weight primers not retained on filter paper, as for example dithiothreitol or low-molecular-weight dextrins remaining after the amylase treatment of synthase to remove glycogen. The activation of a glycogen-free enzyme by glycogen could, however, also be demonstrated when followed by the liberation of UDP from UDP-glucose (results not shown). Also, the observed activation could be thought due to a change in the binding constants for the substrate UDP-glucose or the activator glucose-6-P. In a following paper we will show that synthase I, in analogy with the D enzyme [2], at saturating glycogen concentrations follows the formula u=
V
KB ' 1+B
(3)
Kn is the apparent K , for UDP-glucose, K i is 24 pM for UDP-glucose for both the I (cf. below) and D enzymes, and Kc is the activation constant for glucose-6-P, which for the D enzyme is 1.7 mM [2] and for the I enzyme 0.017 mM (unpublished results). If, for example, an observed three-fold activation of enzyme by glycogen should instead be accounted for by a change in the kinetic constants, it can be calculated that KB must change by a factor of nine during the activation process, which should be easily discernible. As shown in Fig. 5, KB (24 pM) is cssentially constant during the whole activation process. As mentioned, the activity ratio has also been found to be unchanged and greater than 0.93 during the activation period (cf: Fig. 1 Bj, thus excluding any conversions between an apparent D form and synthase I as a cause of activation.
133
H. Sglling and V. Esmann
-11.5 E .c
.-E
b I
1
e
'
1
-
-
2
4
0
8
24
Time ( h )
Fig.4. Activation of glycogen synthase I by dijJerent concan/ru/ionsoj glycogen. Glycogen-free synthase 1 (2 mU/ml) was incubated for 24 h at 30 "C in buffer B supplemented with 10 mM glucose-6-P and rabbit liver type I11 glycogen (in mg/ml): 25 (+), 10 (v).2 (v),0.2 (A), 0.04 (A), 0.01 (O), 0.005 (m), 0.002 (O), and zero (0).At intervals the enzyme activity was assayed as in Fig. 2, but over 150 s and without glycogen in the assay mixture
10
20 30 1/ [ UDP -glucose] ( m M-' )
40
Fig. 5. Estimation of the KJor UDP-glucose duringmtivation. 8 mU/ml glycogen-free synthase 1 was incubated at 30 "C in buffer B with 10 mM glucose-6-P and 500 pg/ml glycogen. At the indicated times aliquots were removed and assayed with UDP-glucose (16000 counts min-' nmol-I). For each UDP-glucose concentration a time course was run over 10 min to allow estimation of the initial velocity. Except for the 3-min curve, these progress curves were apparently perfectly linear (cJ: Fig. 1 C)
The rate of activation, but not of, of synthase I by glycogen is dependent on temperature in the interval 20-40 "C (Fig.6). At 4 "C and 14 "C, the rate of activation as well as of were not measurably different from that obtained at 20 "C, which most probably is due to the inevitable activation occurring during the equilibration and assay periods at 30 "C. Attempts to demonstrate a temperature-dependent change in M , of glycogen-free synthase have failed [9]. pH also heavily influences the activation (Fig. 7), although less in the physiological range than at alkaline pH. It is observed that at pH 9.0 the enzyme is unstable after 24-h incubation (see also 191).
The rate of activation was independent of the addition of 0.01 - 10 mM glucose-6-P or 10 mM Na2S04, but in the absence of one of these ligands a 5 - 30 % lower vf was obtained due to inactivation of the enzyme (cj: Fig. 1 and the preceding paper [9]). Similarly, the rate of activation was not affected by varying the concentration of mercaptoethanol from 0- 50 mM or omitting EDTA from the buffer. Also the addition of UDP (10- 100 pM), NaF (50 mM): MgClz (10 mM), ATP (1 - 5 mM), glucose (100 mM), sucrose (25 %), glycerol (25 %), amylase limit dextrin, serum albumin ( 5 mgiml), or varying the .ionic strength by addition of 0.3 - 3 M NaCl were without effect on the activa-
134
Hysteresis of Glycogen Synthase I
0
#
0
I
'
1
2
3
4
8
12
24
Time ( h )
Fig. 6. Influence oftemperature on activalion vj'qvnthaseI b y glyc'ogen. 2 m U /nil of a glycogen-free enzyme was incubated in buffer B with 10 mM glucose-6-P and 2.5 mg/ml glycogen at different temperatures: 40 "C (m), 35 "C (A), 30 ' C (A), 25 'C (O), and 20 "C (0).Incubations were also performed at 14 "C and 4 'C, but these results were identical to those obtained at 20 'C and are omitted from the figure. bnzyme activity was measured at the times indicated. 6O-pl aliquots were transferred to empty test tubes and equilibrated at 30 "C for 45 s. Then the reaction was started by adding 40 pl of 0.3 mM UDP-glucose + 10 mM glucose-6-P (preheated to 30 T).Assay was terminated after 180 s
0
1
2 Tine ( h )
3
4
24
Fig. 7. Influence q f p H on uctivation. 3 mC/ml of glycogen-free enzyme was incubated at 30 "fwith 200 pg/ml ofglycogen. Buffer B contained 10 mM of glncose-6-P, and the pH was adjusted as indicated prior to incubation: pH 9.0 (V).pII 8.6 (V), pH 8.2 (A),pH 7.8 (A), pH 7.4 ( 0 ) and pH 7.0 (0).120-s assays were performed during incubation as described above
tion process. Also the addition of soya bean trypsin inhibitor or phenylmetylsulfonyl fluoride had no influence. For the enzyme glycogen phosphorylase a it has been found [22] that a decrease in specific activity with increasing enzyme concentrations signified a conformational change from dimer to tetramer. For synthase I, albeit in a much lower concentration range, perfectly linear plots of z'f versus enzyme concentration (2.6- 105 mujml) were found, provided the enzyme was suitably protected by glucose-6-P against inactivation. Also, the rate constant of activation was
independent of enzyme concentration. These results are in keeping with our inability to demonstrate changes in M , by incubating the glycogen-free enzyme at different temperatures or with or without glucose6-P [9]. Whether the activation took place after formation of the enzyme-glycogen complex was examined by utilizing a technic developed by Metzger and Helmreich [23]. The enzyme was slowly activated by incubating with a low concentration of high-molecularweight glycogen. which can be sedimented in the ultracentrifuge (Fig.8). It was found that the amount of
H. S$lling and V. Esmann
135
found. however. that when a concentrated glycogencontaining enzyme was rapidly diluted to form a suspension of 0.5 mU enzyme/ml with as little as 2 pg/ml of endogeneous glycogen, no change in activity was observed over a subsequent 24-h incubation with or without added exogeneous glycogen. We must therefore conclude that the formation of the enzyme-glycogen complex is a practically irreversible reaction at most physiological glycogen concentrations. However, in some experiments with an enzyme preparation partly freed of glycogen, and thus not totally inactive in the absence of added primer, such inactivation was observed. The degree of inactivation increased with the degree of dilution and was reversible as judged by full activation upon addition of glycogen.
( A ) Complete incubation
3
DISCUSSION
I
/ E,,
OO
1
2 Time ( h )
3
24
Fig. 8. Ce?ilrifugation experitmw~scturrirg cicrivntion. (A) 4 in Ulml of glycogen-free synthase I was incubated in buffer B with 10 mM glucose-6-P and either (11) without (0)or (1) with (0) 250 pg!ml high-molecular-weight glycogen (type V). At intervals. the enzyme activity was assayed as descri bed in Fig. 2. Also, at intervals indicated in (B) and (C), 1-in1 portions of the reaction mixtures were transferred to small ultracentrifugation tubes, rapidly cooled to 2 'C. and spun at 40000 rev./min for 45 min at 2 "C in an MSE 65 highspeed ullracenlrifuge. A carefully taken sample (100 pl) of the resulting supernatant (B) was allowed 10 be fully activated by addition of 40 i t 1 of a glycogen solution (ef. Methods) before assay of enzyme activity. Indicated by and a1 times zero and 24 11 arc the enzyme activity of a fully activated aliquot of the incubation mixture, which had not been centrifuged. Thc pellets (C) were resuspended in 1 in1 buffer B with 10 mM glucosc-6-P and assayed. Control experiments showed that the enzyme in the pellet fraction was not further activated when incubated with 20 mg/nil glycogen
enzyme remaining in the supernatant slowly decreased during incubation and always required activation for full activity. Conversely, the sedimentable enzyme slowly increased during incubation, and once bound to glycogen exhibited full activity. Considering the time scales involved and the minimal further activatioh occurring during the centrifugation period, this result clearly shows that the hysteretic activation is paralleled by the formation of an enzyme-glycogen complex. The activation of synthase I by glycogen might theoretically be reversed by a suciria drop in glycogen concentration. This should resulL i n a drop in enzyme activity and in the appearance of an inactive enzyme species, which subsequently could be activated. We
The slow activation of glycogen synthase I has been reported by others [lo-121, but a detailed investigation has not previously been carried out. Usually, the enzyme has been activated by incubation for 10-30 min with 5 - 10 mg/ml glycogen at 30 "C and the process has been thought completed on the evidence or linear progress curves. As demonstrated in Fig. 4, the activation process requires one hour at pH 7.4 even at 25 mg/ml glycogen and, as shown in Fig. 1, short-term linear progress curves are not to be relied upon in this respect. It has been reported [l 11 that glucose-6-P accelerates the activation by glycogen (concerted hysteresis). We have in the preceding paper shown that glucose-6-P or Na2S04protects synthase I against inactivation and the present results exclude any effect of glucose-6-P (or Na2S04) on the activation process proper. The activation can be elicited by glycogen from different sources and is not caused by changing properties of the glycogen during incubation. It remains to be investigated whether the activation depends on the detailed structure of glycogen, for example the length of outer chains. It is noted that synthase I purified by methods not involving amylase treatment also exhibit activation by glycogen [ll - 131, as does synthase D [ll,121 (and own control experiments). It seems justified to name the slow (slow compared to the time required to measure enzyme activity) activation of synthase as a hysteretic process since it is elicited solely by addition of glycogen and cannot be caused by a non-specific renaturation. There are six obvious reactions, which will give rise to this behaviour (c$ introduction). a) A slow isomerization of inactive to active enzyme ( E S E ' ) followed by a rapid binding to glycoG e E ' G). E and E' signifies different congen (E' formations of enzyme.
+
136
Hysteresis of Glycogen Synthase I
b) A slow dissociation of tetramer to dimer enzyme followed by rapid binding to glycogen to give the active enzyme. c) Initial rapid binding of enzyme to glycogen followed by a slow isomerization process (E + G$EG+E' G). d) A slow association of a modifier to yield an enzyme-modifier complex that rapidly binds to glycogen to become active. e) A slow dissociation of a modifier to yield an enzyme that rapidly associates with glycogen. f ) A slow bimolecular reaction between enzyme and glycogen without any of the above mechanisms. Mechanism (a) is excluded by the fact that the observed rate constant, k , for the formation of active enzyme is a linear function of the glycogen concentration at 30 "C (see Fig. 10 below) and at 4 "C (not shown), whereas mechanism (a) (and b and c) predicts k as a hyperbolic function of glycogen concentration [34]. A change in molecular weight compatible with a tetramer to dimer conversion has never been observed with the leukocyte enzyme [9]. If the equilibrium between tetramer (inactive) and dimer (active) enzyme is not totally in favour of the former, one should also observe an initial burst phenomenon, which we have never seen with a totally glycogen-free enzyme. Also, varying the enzyme concentration should have caused changes in the rate of activation. This evidence, although negative, speaks against mechanism (b). Mechanism (c) is also ruled out by the results shown in Fig. 8, which demonstrates that the enzyme is fully active as soon as it is bound to glycogen. Mechanism (d) is eliminated, since no substance other than glycogen (and H+) seems to influence the activation process. Finally, mechanism (e) is excluded by the experiment illustrated in Fig. 2, which shows that prolonged incubation does not result in any change in the rate of activation, and also by the fact that the rate of activation was unaffected by enzyme concentration. A possible candidate for mechanism (e) could have been amylase limit dextrin remaining after digestion of glycogen, but this substance was found positively not to influence activation. The data are, however, compatible with the curprising, but simple solution, mechanism (0,that the hysteresis phenomenon is caused by the slow binding of synthase to its one substrate, glycogen, in the bimolecular reaction :
E+G+EG (4) with the dissociation constant K , = k - l / k l . Most of the experiments have been conducted with a high ratio of glycogen to enzyme. Assuming molecular weights of glycogen and synthase I of 5 x lo6 and 360000, respectively, and a maximal specific activity of synthase I of 36 U/mg [24], the molar ratio in- experiments conducted with 1 pg/ml ~~
~
glycogen and 1 mU enzyme/ml would, however, only be 2.6. Due to the uniform structure of glycogen and the difference in size between the two molecules, the number of possible binding sites on each glycogen molecule, even in this case, is far in excess of those on the enzyme. We are thus dealing with a pseudo-firstorder reaction, which explains why the activation follows Eqn (l), the formula for a first-order reaction. What we observe during the activation process is thus the appearance of a tightly associated enzymeglycogen complex. The appearance of this catalytically active complex is monitored by the short (1 20 - 150-s) assays according to Eqn ( 5 ) : ~t
=
k, [EG]
(5)
where k, is a complex constant. During the short assay period a possible further activation of the enzyme due to consumption of enzyme-glycogen complex, EG, and subsequent displacement of Eqn (4) will be negligible and ut is thus a measure of the concentration of EG. In order to relate ut to [EG], when ut is measured at non-saturating concentrations of UDP-glucose, it is necessary to assume that the Michaelis constants of the catalytic reaction are also dissociation constants, which has been found true for synthase D of human polymorphonuclear leukocytes [ 2 ] . This also appears to be true for synthase I according to the results of Fig. 5, which demonstrates a common intercept on the horizontal axis, when UDP-glucose is varied at constant but increasing concentrations of EG, as formed during the course of the association between synthase and glycogen. Under the present experimental conditions k, is therefore the rate constant for the rate-limiting conversion of the ternary enzyme-glycogen-UDP-glucose complex to products, divided by 1 + K B / B(cf: Eqn 3). It was demonstrated in Fig. 4 that uf, the velocity measured when reaction (4) had reached equilibrium
1
'5
25
200
100
l/[Glycogen]
(mlerng')
Fig. 9. Klotz plot of corresponding values o j glycogen concentration and VJ as obtained from Fig. 4 . For symbols see Fig. 4. K, in this experiment was 6.25 pg/ml (determined by the weighted least-squares procedure of Wilkinson [35])
131
H. S$lling and V. Esmann
Fig. 10. The dutu of Fig. 4 replottedaccording
10
Eqn ( 2 J . Symbols as in Fig.4. The following values for k and t , ,Z = In 2/kmay be calculated
Glycogen concn.
k
fl/Z
mg/ml
min-l
min
25 10 2 0.2 0.04
0.115
6
0.056 0.021 0.0106
12
0.0079
33 65 88
The insert shows k as a function of the glycogen concentration [GI. In the interval 0.2-25 mg/ml glycogen k = 0.010 having k = 0.017 0.0040 [GI parison the insert also shows the result of another experiment (&--o)
+
depended on the concentration of glycogen. Formation of EG follows the equation
where [Elo is the total enzyme concentration and [GIo is the initial concentration of glycogen. At equilibrium, the exponential term can be ignored and
- -
If vf (sat) is vf at saturating glycogen concentrations, then Q(sat)
- uf
[GI = K,
(8)
Of
which, using the data of Fig. 4, is illustrated in doublereciprocal form (Klotz plot [33]) in Fig. 9, from which K, is easily obtained. In five experiments with glycogen concentrations ranging between 0.001 and 0.1 mg/ml, K, was estimated to 0.0033 f: 0.0012 (S.E.), which means that formation of the enzyme-glycogen complex is strongly favoured, a fact that is common knowledge to those who have
+ 0.0045 [GI. For com-
attempted to separate (completely) synthase I from glycogen without the help of glycogenolytic enzymes. The binding of glycogen to synthase I as expressed by Eqn (4) is to be considered apart from interactions of the terminal glycosyl unit with the catalytic site. The present investigation does not contradict the rapid equilibrium assumption [21, but the presence of hysteresis prevents the determination of a dissociation constant (K,) for glycogen at the active site. The slow binding of glycogen to the enzyme is a prerequisite for any catalytic activity and the amount of active enzyme will therefore not be constant, but vary with the amount of added glycogen as stated by Eqns (2) and (6). The data from Fig.4. showing the influence of glycogen on enzyme activation, are replotted in Fig. 10 according to Eqn (2). It is observed that within the interval 0.04- 25 mg/ml glcyogen, Eqn (2) is strictly obeyed, which supports the assumptions underlying Eqn (4). For the lowest glycogen concentrations (0.001-0.010 mg/ml), the determination of k is highly sensitive to small variations in ut and vf and has usually been indistinguishable from that obtained with 0.04 mg/rnl glycogen.
138
Hyslercsis of Glycogen Synthase I
1
0
4
3
2
Time ( h )
Fig. 11. The data of' Fig. 6 replotted accodina to E4n (2). The symbols are as in Fig. 6. The following values [or k and t l i ~(In 2/k) may be calculated. At 20 'C and 25 "C thc first value of k corresponds to the first hour of incubation, the second value to the snbsequent 3 h (c5 text) Temp.
k
(112
T
min-'
min
40 35 30 25 (4-14)-20
0.096 0.029 0.014 0.0096 -0.0069 0.0075 -0.0060
24 50 72- 100 92-116
7
The insert shows the Arrhenius plot of log k as a function of T - ' . The stipled line connccts values o f log k obtained during the last 3 h ofincubation at 20 "C and 25 "C. From the now straight line between 0.003246 and 0.003356 (35 'C to 25 " C ) an activation energy of 25200 caljmol (306 kJ:mol) may be calculated
Within the interval 0.04- 25 mg/ml glycogen, k increased from 0.0078 to 0.115 min-'. For saturating glycogen concentrations ( =. 0.1 mg/ml, c$ above), k appears to be a linear function of [GI, as shown in the insert of Fig. 10 and as predicted by Eqn (6). The regression line allows an estimate of k and the time necessary to activate synthase I at a particular glycogen concentration (at the temperature and pH indicated). It is not warranted to identify the rate constant in the expression fork in Eqn (6) with the empirical constants of the regression line in Fig. 9 (qfi above). The activation of synthase I by glycogen depended markedly on temperature (Fig. 6) and the results are plotted according to Eqn (2) in Fig. 11. A plot of loglok versus T-' (Fig. 11, insert) is non-linear when based on values of k calculated from activities measured during the first hour of incubation. This is presumably due to the error introduced by additional enzyme-glycogen complex formed during the period of equilibration and assay at 30 C , which will result in too high estimates of k . The relative error will decrease with time and when values of k from the later part of the activation period are used instead, a linear relation between
log k and T-' from 25-35 "C is observed allowing the calculation of an activation energy of 25400 cal: mol (106 kJ/mol) for formation of the enzyme-glycogen complex (Eqn 4). This is about twice as high as the activation energy (13 SO0 cal/mol or 56 kJ/mol) found in the preceding paper [9] for the glycogen-containing enzyme (Eqn 5). The influence of pH on the activation of synthase is quite surprising. Further studies along this line might yield information on the composition and localization of the binding sites on enzyme and glycogen. Although the influence of pH is most pronounced at nonphysiologically high values, it is tempting to speculate on a physiological influence of H+ on hysteresis (concerted hysteresis [ 141). The progress curves in Fig. 1 C may be considered to be parts of one long progress curve not influenced by substrate depletion or product inhibition. To describe such progress curves Frieden has derived the equation
P, =
l?ft
-
~
1 ( Z' r - v0) (1 k
-
e-k'),
139
H. SClling and V. Esmann
where Pt is product concentration at time t and the other symbols as above. When ePk t< 1 the progress curve becomes linear and intersects the abscissa at t = ( u f - z:o)/kz?f.The estimation of this point is usual when lag periods are investigated in pre-steadystate kinetics. In a very slow process it is, howcver, difficult to judge when a progress curve is linear even if substrate depletion and product inhibition can be prevented. If 210 = 0, prolongation of the apparently linear portion in the upper curve in Fig. 1 B gives us t = k - ’ = 4mi n and k = 0.25 min-l. tl12 is now calculated to 2.8 rnin. The true value of l l i 2 ,however, is about 17 min (c$ Fig. 10). The physiological significance of hystercsis in glycogen metabolism is difficult to evaluate yet, but it could be, as stated by Frieden [14], that very rapid changes in the concentration of key metabolites are avoided. If concerted hysteresis exists, a very powerful feed-back regulation may result. It is interesling to note that phosphorylase [14] and UDPG-pyrophosphorylase [32] also exhibit hysteretic properties. Until now, hysteresis has only been obsewed in vitva. Since it may be a rate-limiting step, hysteresis deserves consideration in experiments where synthase I and D are used as substrates or products in the study of protein kinases and phosphatases. In such experiments total activity appears to increase in tissue homogenates during incubation [25- 301. Since very high concentrations of glycogen is usually involved in the assay of synthase and this assay is carried out for 10 min or more, a hysteretic activation is easily underestimated. Also in experiments with whole organisms changes in total activity of synthase have been observed [28,31], which may not totally be explained by de nova synthesis of enzyme. The authors are greatly indebted to Mrs Jonna Guldberg for the extreme care exercised during the present expcrimcnts and to Liselotte and Igor W. Plesner for enlightening discussions. Financial support has been given to VE by the Danish Research Council (grants 51211517-2588-3663-5288.6723 and 151169).
REFERENCES 1. Stalmans, W. & Hers, H. G. (1973) in The Eniynzes 3rd edn (Boyer, P. D.; ed.) vol. 9, pp. 309- 361. Academic Press. New York and London. 2. Plesner, L., Plesner, 1. W. & Esmann. V. (1976) Mol. Ce//.Biorhem. 12,45- 61,
H. Splling and V. Esmann, Medicinsk Afdeling, Marselisborg Hospital, Skanderborgvej 9, DK-8000 Arhus C, Denmark
3. Saugmann, P. (1977) Biochem. Biuphys. Res. Commun. 74, 1511 -1519. 4. Saugmann. P. & hsmann, V . (1977) Biochem. Biophyh. Res. Commuu. 74, 1520 - 3 527. 5. Saugmann, P. (1977) Abstr. Commun. 11th Meet. Fed. Eur. Biuchem. Soc. A 1-4-006. 6. S$lling, H. & Esmann, V. (1974) Abstr. Commun. 9th Meet. Fed. Eur. Biochem. Soc., p. 83. 7. Wang, P., Plesncr, L. & Esmann, V. (1972) Eur. J . Biochen~.27, 297 - 300. 8. W-ang,P., Bantle, G. & S$rensen, N.B. (1977) Biochim. Bioph>ls.Aciu, 496, 436 - 447. 9. SQlling,H. & Esmann, V. (1977) EUF.,J, Biochem. 81,119-128. 10. Thomas, J. A., Schlender, K . K. & Larner, J. (1968) Anal. Biuchem. 25,486-499. 11. Rottenberg. D. A,, Passoneau, J. V. & Lust, W. D. (1972) Biochen?. Biopliys. Res. Cornmun. 48, 1192- 1198. 12. Passoneau, J. V. & Schwarti, J. P. (1975) J . Biol. Chrm. 250, 2287 -- 2292. 13. Nimmo, H. G., Proud, C. G . & Cohen, P. (1976) Eur. J . Biochem. 68, 21 -30. 14. Friedcn, C. (1970) J . Biol. Chem. 245, 5788-5799. 15. SQlling, H. & Esmann. V. (1976) Proc. I U t h Int. (’ongr. Biochem., p. 41 5. 16. Rabin, B. R. (1967) Biochem. J . 102, 22c-23c. 17. Ainslie, G. R. Jr, Shill. J. P. & Neet, K . E. (1972) J . Biol. Chem. 747.7088 - 7096. 18. Shill, J. P. & Neet, K. E. (1975)J. Bid. Chem. 250,2259-2268. 19. Passoneau, J. V. & Rottenberg, D. A. (1973) Anal. Biochem. 51, 528 - 541. 20. Staneloni, R. J. & Piras, R. (1971) Biochem. Bioplzys. Res. Commun. 42,237 -244. 21. Piras, R. & Staneloni, R. (1970) Fed. Proc. 29, 676. 22. Wang, J . H. tk Gravcs, D. J. (1964) Biochemistry, 3, 14371445. 23. Metzger, B., Helmreich, E. & Glaser. L. (1967) Biochemistry, 6,994-1001. 24. Takeda, Y., Brewer, H. €3. & Larner, J. (1975) J . Bid. Chem. 250, 8943 8950. 25. Rosell-Perez, M. &Lamer, J. (1962) Biochemisfvyy:1, 769- 772. 26. Hizukuri, S. & Larner, J. (1963) Biochim. Biophys. Acta, 72, 525 527. 27. Gold, A. H. & Segal, H. L. (1967) Arch. Biochem. Biophys. 220, 359 - 364. 28. Vardanis. A. (1969) Arch. Biochem. Biophys. 130,413 ~ ~ 4 2 1 . 29. Sato. K., Abe, N. & Tsuiki, S. (1969) Hiochim. Bzopl~ys.Acta, 268.638 645. 30. Rosell-Perez, M. (1972) Itul. J . Biochem. 21, 34- 69. 31. Stciner, D. F. &King, J. (1964)J. Biol. Chem. 239,1292- 1298. 32. Fitzgerald. D. K., Chen, S. & Ebner, K. E. (1969) Biochim. Biop h y . Acta, 178, 491 498. 33. Segel, I. H. (1975) Enzyyme Kinetics, pp. 218-220, John Wiley and Sons. New York. y, V. (1975) J. Bid. Chem. 34. Strickland, S., Palmer, G. & M 250,4048 4052. 3s. Wilkinson, G. N. (1961) Biochem. J . 80, 324-332. -
-
-
~
-
