Comp. Biochem. Physiol.Vol. 102B,No. 1, pp. 77-82, 1992 Printed in Great Britain
0305-0491/92 $5.00+ 0.00 © 1992PergamonPress Ltd
THE EFFECTS OF AESTIVATION ON THE CATALYTIC AND REGULATORY PROPERTIES OF PYRUVATE KINASE FROM HELIX ASPERSA JEREMYH. A. FIELDS Department of Biology, University of San Diego, Alcala Park, San Diego, CA 92110, U.S.A. (Tel: 619 260-4729) (Received 16 September 1991)
Abstract--1. Pyruvate kinase was partially purified from the foot, mantle, and digestive gland of active and aestivating snails. 2. At pH 7.0 the apparent Km values for phosphoenolpyruvate (PEP) were 0.064 mmol/l for the enzyme from foot and 0.071 mmol/1 for the enzyme from mantle; those for ADP were 0.35 mmol/1 for the foot enzyme and 0.33 mmol/l for the mantle enzyme. 3. Both enzymes were ihibited by alanine, and this could be reversed by fructose 1,6-bisphosphate (FBP), although FBP alone was a weak activator. 4. Decreasing the pH to 6.5 markedly increased the inhibition by alanine and reduced the response to FBP. 5. The enzymes from these tissues of aestivating snails showed a small decrease in their affinity for PEP and a small increase in the effectiveness of alanine as an inhibitor. 6. These changes are indicative of a down-regulation of this enzyme which is consistent with the observations in other species during metabolic depression. 7. In contrast the enzyme from the digestive gland of active animals showed sigmoidal saturation kinetics for PEP with a S0.5of 1.2 mmol/1, but had a markedly higher affinity for PEP, S0.5= 0.20 mmol/1 during aestivation. This may be indicative of other metabolic changes occurring in the digestive gland.
INTRODUCTION Terrestrial snails commonly aestivate in order to survive dry periods. This metabolic state is characterized by withdrawal in to the shell, secretion of an epiphragm in some species, and a metabolic rate reduced below that of normal, quiescent animals (Machin, 1971; Herreid, 1977; Riddle, 1977; Vorhaben et al., 1984; Barnhart and McMahon, 1987). The organisms' energy needs during this period are met by the catabolism of carbohydrate reserves and some protein (Horne, 1973, 1979; Heeg, 1977; Swami and Reddy, 1978; Umezurike and Iheanacho, 1983; Cedefio-Leon, 1984). Some changes in the activity of some catabolic enzymes have been reported, notably decreases in alanine and aspartate aminotransferases, cytochrome oxidase and glutamate dehydrogenase, but the activities of glycolytic or Krebs cycle enzymes do not change to an extent that would explain the reduction in metabolic rate (Raghupathiramireddy, 1967; Vorhaben et al., 1984). Recent studies on the physiology of aestivation in the snail Otala lactea have shown that the depression in metabolic rate is correlated with an elevated level of carbon dioxide in the blood which produces a decrease in the intracellular pH (Barnhart and McMahon, 1988). The effects of pH on metabolic rate has been studied in a few model systems, such as the dormant embryos of Artemia (Busa, 1982; Busa and Crowe, 1983). This reduction in metabolic rate is probably mediated through the effects of pH on key regulatory enzymes that are very sensitive to changes in pH (Busa, 1982; Busa and Nuccatelli, 1984). This may constitute one mechanism for these observed
effects in O. lactea. On the other hand, studies have shown that intertidal invertebrates also depress their metabolic rate when subjected to anoxic stress (Ebberink et al., 1979; Shick et al., 1983). These organisms phosphorylate the glycolytic enzymes phosphofructokinase (EC 2.7.1.11) and pyruvate kinase (EC 2.7.1.40) which reduces their affinity for substrates and activators while enhancing the effects of inhibitors (Storey, 1985; Storey and Storey, 1990). This causes a slowdown of glycolysis and crossover plots are consistent with an inhibition of these enzymes during anoxia (Ebberink and de Zwann, 1980). If this mechanism is a general one for the reduction of metabolic rates, then one should expect to find evidence for phosphorylation of regulatory enzymes during aestivation in terrestrial gastroods and other organisms that show metabolic depression during times of environmental stress. Pyruvate kinase has been found to be phosphorylated during aestivation in O. lactea (Whitwam and Storey, 1990). This is consistent with an earlier study on changes in glycolyric intermediates which indicated that pyruvate kinase was one of rate limiting steps in the glycolytic pathway during aestivation (Churchill and Storey, 1989). Helix aspersa will also aestivate under dry conditions but not for periods as long as O. lactea (Barnhart, personal communication), thus it is of interest whether similar changes in catalytic and regulatory properties of pyruvate kinase are found during aestivation. This study reports the results of an investigation of the kinetic properties of pyruvate kinase from the foot, mantle collar, and digestive gland of active and aestivating H. aspersa. 77
78
JEREMYH. A. FIELDS MATERIALS AND METHODS
All biochemicals and coupling enzymes were purchased from Sigma Chemical Co. (St. Louis, MO); all other standard chemicals were reagent grade. Snails were collected locally and stored in plastic boxes lined with moist paper towels until used for the experiments. They were fed daily on a mixture of lettuce, potato, carrot and cornflour. Snails would remain active as long as food was available and they were kept under moist conditions. Aestivation was induced by removing all food and paper towels, and the snails were allowed to aestivate for at least six weeks before any tissues were used in the experiments.
Partial purification of pyruvate kinasefrom mantle and foot musele The mantle or foot was rapidly excised from 4 snails, then immediately homogenized in 10 volumes of ice cold 100 mM Tris, 1.5 mmol/l EDTA, 1.5 mmol/l EGTA, 25 mmol/1 NaF, 0.1 mmol/l phenylmethylsulfonylflouride, and 7mmol/l 2mercaptoethanol, adjusted to pH 7.5 with 10mol/l HCI (buffer A). The homogenate was centrifuged at 30,000 g for 20 min at 4°C. The supernatant was decanted and treated with 0.209 g/ml of solid (NH4)2SO4. This solution was stirred for 1 hr at 4°C, then centrifuged at 23,000g for 10min. The supernatant was then treated with a further 0.221 g/ml of solid (NH4)2SO4, stirred and centrifuged as described above. The pellet was dissolved in a small volume of 5mmol/1 KH2PO4, I mM EDTA, 10mmol/l NaF, 7 mmol/1 2-mercaptoethanol adjusted to pH 6.5 with 1 mol/1 KOH (buffer B), dialyzed for 2 hr against 21. of the same buffer, and then applied to a 5 × 1 cm column of cellulose phosphate that had been equilibrated with buffer B. Pyruvate kinase was eluted from this column with a 0-1 mol/l gradient of KCI in buffer B. This enzyme preparation was substantially free of other enzymes that might interfere with the measurements of the catalytic and regulatory properties, and was used without further purification.
Partial purification of pyruvate kinase from digestive gland The digestive gland was rapidly excised from 4 snails, then immediately homogenized in 5 vol of ice cold buffer A. The homogenate was centrifuged at 30,000g for 20min. The supernatant was decanted and applied to a cohimn of Cibacron Blue 3GA-agarose equilibrated with buffer A. Pyruvate kinase was then eluted from the column by a gradient of 0-I mol/l of KCI in buffer A. The enzyme did not bind to cellulose phosphate, nor DEAESephadex, nor CM-cellulose, and was somewhat unstable; therefore it was used for kinetic studies without further purification. Table 1. Apparent kinetic parameters (mmol/l+ SEM, N = 3) of pyruvate kinase from foot of Helix aspersa Active Aestivating pH 7.0 Igm(pEp) 0.068 _+0.008 0.097 + 0.011 2Km(ADP) 0.35 _+0.06 0.29 +_0.01 % 0.00050 + 0.000,3 0.00096 +_ 0.00045 ,.'" 2.3 +0.63 I . , + I.O 4150%(ATP) 3.8 + 1.1 4.8 4- 1.9 pH 6.5 IKm(pEv~ 0.11 _+0.021 0.098 ___0.024 2Km(ADP) 0.44 _ 0.07 0.37 + 0.03 3/(a(FBP) 0.0012 ----.0.0005 0.0018 _+0.0005 415o,/.(aa~ni,c) 0.93 + 0.29 0.49 + 0.18 4Is0%IATPl 2.1 + 1.1 1.2 + 0.40 Standard conditions were 100mmol/l KCI, 10mmol/1 MgSO4, 0.1 mmol/I NADH, 0.2 unit LDH, 50 mmol/imidazoleat stated pH, 25°C. Other conditions: ~2mmol/l ADP; 22mmol/1 PEP; 30.06 mmol/I PEP, 2.0 mmol/l ADP, 5.0 mmol/l alanine; 40.06 mmol/l PEP, 2.0 mmol/I ADP. 50%
(alanine)
--
140
~c-
,_, 120 7 --= 2o loo ~_ e, 80 E 7 ._c 60 E 4O E
~
Z3
L
F
_
_
_
i
L
i
-
8
20 0 0.0
i
i
[PEP]
i
J
i
i
1'0 (mmol.l-')
i
i
1'5
i
k
i
2'0
Fig. 1. Saturation kinetics for phosphoenolpyruvate of pyruvate kinase from the foot of active Helix aspersa showing the effects of alanine and fructose 1,6-bisphosphate. (O O) control; (O O) 5mmol/l alanine; (A . . . . A) 0.05 mmol/1 fructose 1,6-bisphosphate; (& . . . . A) 0.05 mmol/l fructose 1,6-bisphosphate plus 5mmol/1 alanine. Other conditions: 2 mmol/l ADP, I00 mmol/1 KC1, I0 mmol/l MgSO4, 0.1 mmol/l NADH, 0.2 unit LDH.
Assay of pyruvate kinase Pyruvate kinase activity was determined by a coupled enzyme assay with lactate dehydrogenase (EC 1.1.1.27). The rate of oxidation of NADH was measured with a recording spectrophotometer (B/icher and Pfleiderer, 1955). Standard assay conditions were 2mmol/1 ADP, 2mmol/l phosphoenolpyruvate, 100mmol/l KCI, 10mmol/l MgSO4, 0.I mmol/1 NADH, 0.5 unit of lactate dehydrogenase (EC 1.I.1.27) in 50 mmol/l imidazole adjusted to pH 7.0 with 10 mol/1 HC1. All assays were conducted at 25°C.
Measurement of protein concentration The protein content of all samples was determined by Coomassie Blue binding (Bradford, 1976). Bovine serum albumin was used as a standard.
Calculation of enzyme kinetic parameters and statistical analysis Michaelis-Menten constants were calculated by the method of weighted least-squares regression (Wilkinson, 1961) when the enzymes displayed simple hyperbolic kinetics. If the kinetic data were sigmoidal in nature, they were fitted to the Hill equation by an iterative method giving the S0.5 and Hill (nil) values. All data were replicated 3 times. They are reported as means ___SEM. They were analysed by Student's t-test (Sokal and Rohlf, 1981). RESULTS
Pyruvate kinase from foot and mantle These enzymes were found to be very similar. Both showed hyperbolic saturation kinetics for their substrates p h o s p h o e n o l p y r u v a t e (PEP) and A D P (Table 1). Alanine inhibited b o t h enzymes, changing the P E P saturation kinetics to a slightly sigmoidal form (Fig. 1). In the presence o f 5 mmol/l alanine the enzyme from the foot o f active animals had a S0.s(pBp) o f 0.25 + 0 . 0 5 m m o l / l (N = 3) and n M o f 1.2 + 0.04; the data for the enzyme from aestivating animals were S0.5(pBp)= 0.38 + 0.10 mmol/I (N = 3) and n a = 1 . 4 + 0 . 0 4 . The inhibition by alanine
Changes in kinetics of pyruvate kinase from Helix
was readily reversed by fructose 1,6-bisphosphate (FBP) (Fig. 1). A concentration of 0.05mmol/i was sufficient to reduce the Km(vee) to 0.049 ± 0.003 mmol/l (N ffi 3) for the foot enzyme from active animals, and 0.046 ± 0.001 mmol/l (N ffi 3) for the enzyme from the foot of aestivating animals. The activation by FBP was not as prominent in the absence of alanine (Fig. 1), but 0.05 mmol/I FBP did reduce the Km(ee.p ) of the enzyme from active animals to 0.047 ± 0.003 mmol/l (N = 3), and 0.037 ±0.005mmol/l (N = 3) for the enzyme from aestivating animals. The enzyme from the mantle collar responded in similar fashion, the data being essentially similar to that of the enzyme from the foot. ATP was also found to be an inhibitor of these enzymes, but the saturation kinetics of PEP remained hyperbolic. Comparing the properties of pyruvate kinase from the foot and mantle of aestivating and active animals showed a small decrease in affinity for phosphoenolpyruvate (PEP) (P < 0.05) and the activator FBP (P < 0.05). There was no significant difference in the Kin(AVe), nor in the concentration of ATP or alanine required to produce 50% inhibition in the enzymes from active and aestivating animals. The effects of reducing the pH to 6.5 were found to be at least as dramatic than the changes in properties of the enzyme in the active and aestivating animals (Tables 1 and 2). For the enzyme from the foot and mantle the affinity for phosphoenolpyruvate was decreased at pH 6.5, the inhibition by ATP and alanine was more pronounced (Iso./. was decreased), and the affinity for ADP was also decreased (P < 0.05 for all comparisons).
Pyruvate kinase fiom digestive gland In contrast to the properties of the enzyme from the mantle collar and foot, those of the enzyme from the digestive gland were markedly different. The enzyme from the digestive gland of active animals showed sigmoidal saturation kinetics with a low affinity for PEP whereas the enzyme from aestivating snails showed hyperbolic saturation kinetics and a higher affinity for PEP (Fig. 2, Table 3). Both enzymes were inhibited by alanine; at a concentration of 5 mmol/I the S0.s value for PEP was increased to 4.3 ± 0.4 mmol/l (N = 3) (nil ffi 1.9 ± 0.3) for the enTable 2. Apparent kinetic parameters (mmol/I ± SEM, N = 3) of pyruvate kinase from mantle collar of Helix aspersa Active
3Ka(FBP) 4150%(iluine)
4l~(^Tp )
IK~(eEe)
5.0 I
e-
•~
O 4.0
O.
T
&o
-~
2.0
1.0
0.0
0.0
1.0
2.0
[PEP] (retool.l-')
0.071 + 0.002 0.33 + 0.004 0.00076 + 0.000003
0.097 + 0.02 0.26 ~ 0.005 0.00091 + 0.00018 I.I :[:0.06 5.6 =[:1.0
1.7 _+ 0.07 5.3 + 1.0
4.0
zyme from active animals, and 2.3 _+0.4 mmol/1 (N f3) ( n H = 1.2+0.03) for the enzyme from aestivating animals• Fructose 1,6-bisphosphate (0.05 retool/l) reversed the inhibition by alanine, decreasing the S0.S(pEp) to 0•29 + 0.03 mmol/1 (N = 3) (n H = 1.5 _+0.2) for the enzyme from the digestive gland of active animals, and 0.16+0.02mmol/l (N = 3) (nil----1.0) for the enzyme from aestivating animals. In the absence of alanine, fructose 1,6bisphosphate activated the enzyme from active and aestivating snails (Table 3), reducing the S05(pEp) to 0.11±0.02mmol/1 ( N - - 3 ) (nH=l.3_+0.2) for the enzyme from active animals and 0•068 + 0.007 mmol/I (N = 3) (nH = 1.1 ± 0.1) for the enzyme from aestivating snails. The concentration of alanine required for 50% inhibition was not markedly different in the two forms of the enzyme, nor was it altered by decreasing the pH to 6.5. On the other hand, the 15O./ofor ATP was markedly lower for the enzyme from active animals, and for both forms it was significantly lower at pH 6.5 (Table 3) (P < 0.05 for both comparisons). It is also notable that the affinity for FBP was clearly Table 3. Apparent kinetic parameters (mmol/l + SEM, N = 3) of pyruvate kinase from the digestive gland of Helix aspersa Active
Aestivating pH 7.0
IS0.s(pep~ ntt 2Krn(AVP }
3K,~rsp ) 3150,/o(at.ni~)
3150%(ATI,)
p H 6.5
0.12 4- 0.04 0.12 __. 0 . 0 0 6 2KmcADP) 0.,44 __ 0.01 0.40 + 0.007 3Ko(Fse) 0.0019 + 0.0003 0.0018 + 0 . 0 0 0 3 44150%(a]anine) 0.52 + 0.11 0.54 _+0.04 I5o%|^TP~ 0.96 + 0.11 I. I + 0. !0 Standard conditions were 100mmol/I KCI, 10retool MgSO4, 0.1 mmol/I NADH, 0.2 unit LDH, 50 mmol/imidazole at stated pH, 25°C. Other conditions: ]2.0 mmol/I ADP; 22.0mmol/I PEP; 30.06mmol/I PEP, 2.0mmol/l ADP, 5.0mmol/I alaninc; 40.06 mmol/l PEP, 2.0 mmol/I ADP.
3.0
Fig. 2. Saturation kinetics for phosphoenolpyruvate kinase from the digestive gland of active (O) and aestivating (0) Helix aspersa. Conditions: 2 mmol/1 ADP, 100 mmol/l KCI, 10mmol/l MgSO4, 0.1 mmol/l NADH, 0.2 unit LDH.
Aestivating pH 7.0
~Km(pEe) 2Km(ADP)
79
1.2 + 2.0 + 0.23 + 0.0013 +
0.28 +_0.04 1.1 _+0.1 0.28 + 0.01 0.00066 _+0.00002
0.09 0.1 0.04 0.00002
0.54 + 0.03 2.0 _+ 0.5
0.78 + 0.20 7.6 + 1.5 p H 6.5
IS0.~PEp ) /'1H 2Km(AOP)
0.36 + 0.04 1.3 + 0.2 1.8 ± 0.3 1.3 + 0.2 0.27 _+0.06 0.33 + 0.03 3K,(vse) 0.0032 +_0.0006 0.0013 _+ 0.0006 3 3150~(al.,i,~) 0.51 + 0.02 1.0 _+ 0.6 3.6 + 0.6 ls0~|^Tr? 1.2 +0.2 Standard conditions were 100mmol/I KCI, 10mmol MgSO4, 0.1 mmol/I NADH, 0.2 unit LDH, 50 mmol/imidazole at stated pH, 25°C. Other conditions: J2.0 mmol/l ADP; 22.0mmol/l PEP; 30.6 mmol/I PEP, 2.0 mmol/I ADP.
80
JEREMY H. A. FIELDS
higher in the enzyme from aestivating snails than in that from active snails.
of the enzyme through phosphorylation or dephosphorylation. The phosphorylation of rate-limiting glycolytic enzymes, especially pyruvate kinase, in response to anoxic stress has been extensively studied DISCUSSION in intertidal molluscs. In these organisms pyruvate The results of this study have shown that there are kinase and phosphofructokinase are converted to less differences in the kinetic properties ofpyruvate kinase active forms by phosphorylation as part of the stratfrom the foot, mantle and digestive gland of active egy of reducing metabolic rate to cope with long-term and aestivating H. aspersa. These kinetic differences anoxic stress (Storey and Storey, 1990). The hallpoint towards an enzyme that is slightly less active in marks of the changes are a reduction in affinity for the foot and mantle collar of aestivating snails than one or more substrates, an increase in sensitivity to in the active ones. It has a lower affinity for PEP, and inhibitors, and a decrease in sensitivity to activators is less responsive to the activator FBP. The differ- (Storey, 1985; Storey and Storey, 1990). For pyruvate ences in the properties of the enzyme from the kinase, in terms of absolute magnitude, the greatest digestive gland were found to be reversed as com- change in the S0.5 (5-10-fold) for PEP has been pared to the enzyme from foot and mantle collar, i.e. observed in the gastropods Busycotypus canaliculait was the enzyme from aestivating animals that had turn and Concholepas concholepas (Plaxton and a higher affinity for PEP, the activator FBP, and a Storey, 1984a,b, 1985; Carvajal et al., 1990); that in lower 15oo/ofor the inhibitor ATP. This would suggest the limpet Patella caerulea, the bivalves Mytilus that the enzyme from the digestive gland in aestivat- edulis, Venus gallina, and Scapharca inaequivalvis was ing snails would be more active than that from the lower (1.3-3-fold) (Holwerda et al., 1983; Hakim et al., 1985; Michaelidis et al., 1988). The changes digestive gland of active snails. The changes in the kinetic properties of pyruvate observed in the foot and mantle of aestivating kinase from the tissues of H. aspersa are consistent H. aspersa (this study) and O. lactea (Whitwam and with the enzyme being covalently modified, perhaps Storey, 1990) are small, but comparable to those through phosphorylation. Indeed, the data obtained observed in the limpet P. caerulea (Michaelidis et al., for H. aspersa are very similar to those obtained for 1988). Thus it would appear that conversion of key O. lactea (Whitwam and Storey, 1990) except for the enzymes, as exemplified by pyruvate kinase, to less observation of activation by FBP in the enzyme from active forms is a characteristic response to long-term environmental stress amongst the Mollusca, at least foot and mantle collar of active and aestivating snails, in some tissues. and also from the digestive gland of aestivating snails. The above argument clearly does not apply to the Whitwam and Storey (1990) also showed that the changes in the properties of pyruvate kinase could be changes observed in pyruvate kinase from the digesmimicked by treatment of the enzymes from active tive gland of terrestrial gastropods during aestivation. snails with stimulators of protein kinase, or by treat- The data from O. lactea (Whitwam and Storey, 1990) ment of the enzyme from aestivating snails with and H. aspersa (this study) indicate that the enzyme alkaline phosphatase. They concluded that the kinetic should be more active during aestivation, having a changes were brought about by covalent modification lower S0.5 for PEP, a lower Ka for FBP, and a higher 15oo/. for alanine. On the other hand, this process is occurring during a time when metabolism is de60 pressed. It is noteworthy that the catalytic properties ~ • r . . . . of pyruvate kinase from the digestive gland are f 4x / characteristic of those of gluconeogenic tissues, the i z~ ~ " A ~ cmammalian liver being the archetype for comparison / / I (Scrutton and Utter, 1968; Engstrom, 1979). It has "6 been assumed that gluconeogenesis occurs in the ~'r'" 4.0 0"~ / digestive gland of gastropods (Livingstone and de Zwaan, 1983). If this is a major site of gluconeogenT / esis, then the change in the properties of pyruvate kinase may reflect a switch in metabolism, from being gluconeogenic to non-gluconeogenic. It has been -o 2.0 '[-2~A shown that blood glucose in gastropods declines during aestivation, and that glycogen stores in tissues are also consumed (Swami and Reddy, 1973; Home, 1973; Heeg, 1977; Riddle, 1977; Cedefio-Leon, 1984). It may well be that the digestive gland must switch from being primarily gluconeogenic in the active °'°°c !°pEP][ (mmol.l-2'°1) 3.~--4.o phase to primarily glycolytic in the aestivating phase Fig. 3. Saturation kinetics for phosphoenolpyruvate of in order to supply the necessary metabolic energy to pyruvate kinase from the digestive gland of active Helix the cell. This may be the explanation for the effects aspersa showing the effects of alanine and fructose 1,6-bis- observed in the digestive gland being opposite to phosphate. (0 O) control; (Q 0 ) 5mmol/l ala- those found in the foot and mantle collar. nine; (/X .... /X) 0.05mmol/l fructose 1,6-bisphosphate; Studies of gas exchange in active and aestivating (A .... A) 0.05mmol/l fructose 1,6-bisphosphate plus O. lactea have shown that the rate of oxygen con5mmol/l alanine. Other conditions: 2mmol/l ADP, 100 mmol/1 KC1, 10 mmol/l MgSO4, 0.1 mmol/l NADH, 0.2 sumption is inversely proportional to the concentration of carbon dioxide (Barnhart and McMahon, unit LDH.
Changes in kinetics of pyruvate kinase from Helix 1988, 1989). Furthermore, the aestivating state is characterized by hypercapnia and a decrease in intracellular pH (Barnhart and McMahon, 1989). Pyruvate kinase from H. aspersa might be less active at a lower pH. The enzyme from foot and mantle collar of active snails had a higher Km for PEP, a higher Ka for FBP, and lower 15o,/. for ATP. The inhibition by ATP may well be significant at this pH if the snails are maintaining concentrations of ATP that are close to those of an active animal. Decreases i n intracellular pH have been shown to reduce metabolic rate in frog skeletal muscle, mammalian erythrocytes, rat hepatocytes, and embryos of Artemia (Tomoda et al., 1977; Busa and Crowe, 1982; Busa et al., 1983; Fidelman et al., 1982; Kashiwagura et al., 1984). Ellington (1985) showed that a reduction in intracellular pH in perfused hearts of Busycon contrarium caused a negative crossover between fructose 6phosphate and fructose 1,6-bisphosphate, indicating that flux through phosphofructokinase was inhibited. Churchill and Storey (1989) found negative crossovers in tissues of asetivating snails at the level of phosphofructokinase and pyruvate kinase, clearly indicating that both of these enzymes are rate-limiting steps in glycolysis during aestivation. It is plausible that changes in intracellular pH also play a role in reducing metabolism in aestivating snails, as suggested by Barnhart (1989) in addition to covalent modifications of regulatory enzymes (Storey and Storey, 1990). The reduction in flux through pyruvate kinase in the foot might reflect both conditions; a reduction in intracellular pH because of hypercapnia, and covalent modification. The digestive gland needs further study, as it is metabolically more complex than the foot or mantle collar. Acknowledgements--This study was funded by Faculty Research Grants during 1986-1989 from the College of Arts and Sciences, University of San Diego. The technical assistance of Mr. T. C. Matthews and Ms. L. Alvarez is greatly appreciated. REFERENCES
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Busa W. B., Crowe J. H. and Nuccitelli R. (1982) Intracellular pH and the metabolic status of dormant and developing Artemia enbryos. Arehs Bioehem. Biophys. 216,
711-718. Busa W. B. and Nuccitelli R. (1984) Metabolic regulation via intracelhilar pH. Am. J. Physiol. 246, R409-R438. Carvajal N., Gonzalez R. and Kessi E. (1990) Tissue-specific regulation of pyruvate kinase during environmental anoxia in Concholepas concholepas (Gastropoda: Muricidae). J. exp. Zool. 255, 280-285. Cedefio-Leon A. (1984) Carbohydrate reserves during aestivation of Pomacea urceus (Mueller) (Gastropoda, Prosobranchia). Comp. Biochem. Physiol. 78A, 553-557. Churchill T. A. and Storey K. B. (1989) Intermediary energy metabolism during dormancy and anoxia in the land snail Otala lactea. Physiol. Zool. 62, 1015-1030. Ebberink R. H. M. and Zwaan A. de (1980) Control of glycolysis in the posterior adductor muscle of the sea mussel Mytilus edulis. J. comp. Physiol. 137, 165-171. Ebberink R. H. M., Zurburg W. and Zandee D. I. (1979) The energy demand of the posterior adductor muscle of Mytilus edulis in air. Mar. Biol. Lett. 1, 23-31. Ellington W. R. (1985) Metabolic impact of experimental reductions of intracellular pH in molluscan cardiac muscle. Molec. Physiol. 7, 155-164. Engstrom L. (1978) The regulation of liver pyruvate kinase by phosphorylation-dephosphorylation. In Current Topics in Cellular Regulation Vol. 13. (Edited by Horecker B. L. and Stadtman E. R.), pp. 29-51. Academic Press, New York. Fidelman M. L., Seeholzer S. H., Walsh K. B. and Moore R. D. (1982) IntraceUular pH mediates action of insulin in frog skeletal muscle. Am. J. Physiol. 242, C87-C93. Hakim G., Carpene E., Cortesi P. and Isani G. (1985) Regulation by phosphorylation-dephosphorylation of pyruvate kinase in Venus gallina and Scapharca inaequivalvis. Comp. Biochem. Physiol. 80B, 109-112. Heeg J. (1977) Oxygen consumption and the use of metabolic reserves during starvation and aestivation in Bulinus (Physopsis) africanus (Pulmonata: Planorbidae). Malacologia 16, 549-560. Home F. R. (1973) The utilization of foodstuffs and urea production by a land snail during aestivation. Biol. Bull. 144, 321-330. Home F. R. (1979) Comparative aspects of estivating metabolism in the gastropod, Marisa. Comp. Biochem. Physiol. 64A, 309-31I. Kashiwagura T., Deutsch C. J., Taylor J., Erecinska M. and Wilson D. F. (1984) Dependence of gluconeogenesis,urea synthesis, and energy metabolism of hepatocytes on intracellular pH. J. biol. Chem. 259, 237-243. Livingstone D. R. and Zwaan A. de (1983) Carbohydrate metabolism of gastropods. In (K. M. Wilbur, Editor) The Mollusca, Vol. I. Metabolic Biochemistry and Molecular Biomechanics. (Edited by Hochachka P. W.), pp. 177-242. Academic Press, New York. Machin J. (1971) Structural adaptation for reducing water loss in three species of terrestrial snail. J. Zool, Lond. 152, 55-65. Michaelidis B., Gaitanaki C. and Beis I. S. (1988) Modification of pyruvate kinase from the foot muscle of Patella caerula (L) during anaerobiosis. J. exp. Zool. 248, 264-271. Plaxton W. C. and Storey K. B. (1984a) Purification and properties of aerobic and anoxic forms of pyruvate kinase from red muscle tissue of the channelled whelk, Busycotypus canaliculatum. Eur. J. Biochem. 143, 257-265. Plaxton W. C. and Storey K. B. (1984b) Phosphorylation in vivo of red muscle pyruvate kinase from the channelled whelk, Busycotypus canaliculatum, in response to anoxic stress. Eur. J. Biochem. 143, 267-272. Plaxton W. C. and Storey K. B. (1985) Tissue specific isozymes of pyruvate kinase in the channelled whelk
82
JEREMYH. A. FIELDS
Busycotypus canaliuculatum: enzyme modification in response to anoxia. 3". comp. PhysioL B 155, 291-296. Raghupathiramireddy S. (1967) Respiratory enzymes during aestivation of the Indian apple snail Pila globosa. Life Sci. 6, 341-345. Riddle W. A. (1977) Comparative respiratory physiology of a desert snail, Rabdotus schiedeanus, and a garden snail, Helix aspersa. Comp. Biochem. Physiol. 56A, 369-373. Scrutton M. C. and Utter M. F. (1968) The regulation of giycolysis and gluconeogenesis in animal tissues. A. Rev. Biochem. 37, 249-302. Shick J. M., Zwaan A. de and Bont A. M. T. de (1983) Anoxic metabolic rate in the mussel Mytilus edulis L. estimated by simultaneous direct calorimetry and biochemical analysis. Physiol. Zool. 56, 56~3. Sokal R. R. and Rohlf F. J. (1981) Biometry (2nd edn) 859 pp. W. H. Freeman, San Francisco, CA. Storey K. B. (1985) A re-evaluation of the Pasteur effect: new mechanisms in anaerobic metabolism. Molec. Physiol. 8, 439-461. Storey K. B. and Storey J. M. (1990) Metabolic rate
depression and biochemical adaptation in anaerobiosis, hibernation and aestivation. Q. Rev. Biol. 65, 145-174. Swami K. S. and Reddy Y. S. (1978) Adaptive changes to survival during aestivation in the gastropod snail, Pila globosa. J. Sci. Ind. Res., India 37, 144-157. Tomoda A. S., Tsuda-Hirota S. and Minakami S. (1977) Glycolysis of red cells suspended in solutions of impermeable solutes. Intracellular pH and glycolysis. J. biol. Chem. 81, 697-701. Umezurike G. M. and Iheanacho E. N. (1983) Metabolic adaptations in aestivating giant African snail (Achatina achatina). Comp. Biochem. Physiol. 74B, 493-498. Vorhaben J. E., Klotz A. V. and Campbell J. W. (1984) Activity and oxidative metabolism of the land snail Helix aspersa. Physiol. Zool. 57, 357-365. Wilkinson G. N. (1961) Statistical estimation in enzyme kinetics. Biochem. J. 80, 324-332. Whitwam R. E. and Storey K. B. (1990) Pyruvate kinase from the land snail Otala lactea: regulation by reversible phosphorylation during estivation and anoxia. J. exp. Biol. 154, 321-337.
0305-0491/92 $5.00+ 0.00 © 1992PergamonPress Ltd
THE EFFECTS OF AESTIVATION ON THE CATALYTIC AND REGULATORY PROPERTIES OF PYRUVATE KINASE FROM HELIX ASPERSA JEREMYH. A. FIELDS Department of Biology, University of San Diego, Alcala Park, San Diego, CA 92110, U.S.A. (Tel: 619 260-4729) (Received 16 September 1991)
Abstract--1. Pyruvate kinase was partially purified from the foot, mantle, and digestive gland of active and aestivating snails. 2. At pH 7.0 the apparent Km values for phosphoenolpyruvate (PEP) were 0.064 mmol/l for the enzyme from foot and 0.071 mmol/1 for the enzyme from mantle; those for ADP were 0.35 mmol/1 for the foot enzyme and 0.33 mmol/l for the mantle enzyme. 3. Both enzymes were ihibited by alanine, and this could be reversed by fructose 1,6-bisphosphate (FBP), although FBP alone was a weak activator. 4. Decreasing the pH to 6.5 markedly increased the inhibition by alanine and reduced the response to FBP. 5. The enzymes from these tissues of aestivating snails showed a small decrease in their affinity for PEP and a small increase in the effectiveness of alanine as an inhibitor. 6. These changes are indicative of a down-regulation of this enzyme which is consistent with the observations in other species during metabolic depression. 7. In contrast the enzyme from the digestive gland of active animals showed sigmoidal saturation kinetics for PEP with a S0.5of 1.2 mmol/1, but had a markedly higher affinity for PEP, S0.5= 0.20 mmol/1 during aestivation. This may be indicative of other metabolic changes occurring in the digestive gland.
INTRODUCTION Terrestrial snails commonly aestivate in order to survive dry periods. This metabolic state is characterized by withdrawal in to the shell, secretion of an epiphragm in some species, and a metabolic rate reduced below that of normal, quiescent animals (Machin, 1971; Herreid, 1977; Riddle, 1977; Vorhaben et al., 1984; Barnhart and McMahon, 1987). The organisms' energy needs during this period are met by the catabolism of carbohydrate reserves and some protein (Horne, 1973, 1979; Heeg, 1977; Swami and Reddy, 1978; Umezurike and Iheanacho, 1983; Cedefio-Leon, 1984). Some changes in the activity of some catabolic enzymes have been reported, notably decreases in alanine and aspartate aminotransferases, cytochrome oxidase and glutamate dehydrogenase, but the activities of glycolytic or Krebs cycle enzymes do not change to an extent that would explain the reduction in metabolic rate (Raghupathiramireddy, 1967; Vorhaben et al., 1984). Recent studies on the physiology of aestivation in the snail Otala lactea have shown that the depression in metabolic rate is correlated with an elevated level of carbon dioxide in the blood which produces a decrease in the intracellular pH (Barnhart and McMahon, 1988). The effects of pH on metabolic rate has been studied in a few model systems, such as the dormant embryos of Artemia (Busa, 1982; Busa and Crowe, 1983). This reduction in metabolic rate is probably mediated through the effects of pH on key regulatory enzymes that are very sensitive to changes in pH (Busa, 1982; Busa and Nuccatelli, 1984). This may constitute one mechanism for these observed
effects in O. lactea. On the other hand, studies have shown that intertidal invertebrates also depress their metabolic rate when subjected to anoxic stress (Ebberink et al., 1979; Shick et al., 1983). These organisms phosphorylate the glycolytic enzymes phosphofructokinase (EC 2.7.1.11) and pyruvate kinase (EC 2.7.1.40) which reduces their affinity for substrates and activators while enhancing the effects of inhibitors (Storey, 1985; Storey and Storey, 1990). This causes a slowdown of glycolysis and crossover plots are consistent with an inhibition of these enzymes during anoxia (Ebberink and de Zwann, 1980). If this mechanism is a general one for the reduction of metabolic rates, then one should expect to find evidence for phosphorylation of regulatory enzymes during aestivation in terrestrial gastroods and other organisms that show metabolic depression during times of environmental stress. Pyruvate kinase has been found to be phosphorylated during aestivation in O. lactea (Whitwam and Storey, 1990). This is consistent with an earlier study on changes in glycolyric intermediates which indicated that pyruvate kinase was one of rate limiting steps in the glycolytic pathway during aestivation (Churchill and Storey, 1989). Helix aspersa will also aestivate under dry conditions but not for periods as long as O. lactea (Barnhart, personal communication), thus it is of interest whether similar changes in catalytic and regulatory properties of pyruvate kinase are found during aestivation. This study reports the results of an investigation of the kinetic properties of pyruvate kinase from the foot, mantle collar, and digestive gland of active and aestivating H. aspersa. 77
78
JEREMYH. A. FIELDS MATERIALS AND METHODS
All biochemicals and coupling enzymes were purchased from Sigma Chemical Co. (St. Louis, MO); all other standard chemicals were reagent grade. Snails were collected locally and stored in plastic boxes lined with moist paper towels until used for the experiments. They were fed daily on a mixture of lettuce, potato, carrot and cornflour. Snails would remain active as long as food was available and they were kept under moist conditions. Aestivation was induced by removing all food and paper towels, and the snails were allowed to aestivate for at least six weeks before any tissues were used in the experiments.
Partial purification of pyruvate kinasefrom mantle and foot musele The mantle or foot was rapidly excised from 4 snails, then immediately homogenized in 10 volumes of ice cold 100 mM Tris, 1.5 mmol/l EDTA, 1.5 mmol/l EGTA, 25 mmol/1 NaF, 0.1 mmol/l phenylmethylsulfonylflouride, and 7mmol/l 2mercaptoethanol, adjusted to pH 7.5 with 10mol/l HCI (buffer A). The homogenate was centrifuged at 30,000 g for 20 min at 4°C. The supernatant was decanted and treated with 0.209 g/ml of solid (NH4)2SO4. This solution was stirred for 1 hr at 4°C, then centrifuged at 23,000g for 10min. The supernatant was then treated with a further 0.221 g/ml of solid (NH4)2SO4, stirred and centrifuged as described above. The pellet was dissolved in a small volume of 5mmol/1 KH2PO4, I mM EDTA, 10mmol/l NaF, 7 mmol/1 2-mercaptoethanol adjusted to pH 6.5 with 1 mol/1 KOH (buffer B), dialyzed for 2 hr against 21. of the same buffer, and then applied to a 5 × 1 cm column of cellulose phosphate that had been equilibrated with buffer B. Pyruvate kinase was eluted from this column with a 0-1 mol/l gradient of KCI in buffer B. This enzyme preparation was substantially free of other enzymes that might interfere with the measurements of the catalytic and regulatory properties, and was used without further purification.
Partial purification of pyruvate kinase from digestive gland The digestive gland was rapidly excised from 4 snails, then immediately homogenized in 5 vol of ice cold buffer A. The homogenate was centrifuged at 30,000g for 20min. The supernatant was decanted and applied to a cohimn of Cibacron Blue 3GA-agarose equilibrated with buffer A. Pyruvate kinase was then eluted from the column by a gradient of 0-I mol/l of KCI in buffer A. The enzyme did not bind to cellulose phosphate, nor DEAESephadex, nor CM-cellulose, and was somewhat unstable; therefore it was used for kinetic studies without further purification. Table 1. Apparent kinetic parameters (mmol/l+ SEM, N = 3) of pyruvate kinase from foot of Helix aspersa Active Aestivating pH 7.0 Igm(pEp) 0.068 _+0.008 0.097 + 0.011 2Km(ADP) 0.35 _+0.06 0.29 +_0.01 % 0.00050 + 0.000,3 0.00096 +_ 0.00045 ,.'" 2.3 +0.63 I . , + I.O 4150%(ATP) 3.8 + 1.1 4.8 4- 1.9 pH 6.5 IKm(pEv~ 0.11 _+0.021 0.098 ___0.024 2Km(ADP) 0.44 _ 0.07 0.37 + 0.03 3/(a(FBP) 0.0012 ----.0.0005 0.0018 _+0.0005 415o,/.(aa~ni,c) 0.93 + 0.29 0.49 + 0.18 4Is0%IATPl 2.1 + 1.1 1.2 + 0.40 Standard conditions were 100mmol/l KCI, 10mmol/1 MgSO4, 0.1 mmol/I NADH, 0.2 unit LDH, 50 mmol/imidazoleat stated pH, 25°C. Other conditions: ~2mmol/l ADP; 22mmol/1 PEP; 30.06 mmol/I PEP, 2.0 mmol/l ADP, 5.0 mmol/l alanine; 40.06 mmol/l PEP, 2.0 mmol/I ADP. 50%
(alanine)
--
140
~c-
,_, 120 7 --= 2o loo ~_ e, 80 E 7 ._c 60 E 4O E
~
Z3
L
F
_
_
_
i
L
i
-
8
20 0 0.0
i
i
[PEP]
i
J
i
i
1'0 (mmol.l-')
i
i
1'5
i
k
i
2'0
Fig. 1. Saturation kinetics for phosphoenolpyruvate of pyruvate kinase from the foot of active Helix aspersa showing the effects of alanine and fructose 1,6-bisphosphate. (O O) control; (O O) 5mmol/l alanine; (A . . . . A) 0.05 mmol/1 fructose 1,6-bisphosphate; (& . . . . A) 0.05 mmol/l fructose 1,6-bisphosphate plus 5mmol/1 alanine. Other conditions: 2 mmol/l ADP, I00 mmol/1 KC1, I0 mmol/l MgSO4, 0.1 mmol/l NADH, 0.2 unit LDH.
Assay of pyruvate kinase Pyruvate kinase activity was determined by a coupled enzyme assay with lactate dehydrogenase (EC 1.1.1.27). The rate of oxidation of NADH was measured with a recording spectrophotometer (B/icher and Pfleiderer, 1955). Standard assay conditions were 2mmol/1 ADP, 2mmol/l phosphoenolpyruvate, 100mmol/l KCI, 10mmol/l MgSO4, 0.I mmol/1 NADH, 0.5 unit of lactate dehydrogenase (EC 1.I.1.27) in 50 mmol/l imidazole adjusted to pH 7.0 with 10 mol/1 HC1. All assays were conducted at 25°C.
Measurement of protein concentration The protein content of all samples was determined by Coomassie Blue binding (Bradford, 1976). Bovine serum albumin was used as a standard.
Calculation of enzyme kinetic parameters and statistical analysis Michaelis-Menten constants were calculated by the method of weighted least-squares regression (Wilkinson, 1961) when the enzymes displayed simple hyperbolic kinetics. If the kinetic data were sigmoidal in nature, they were fitted to the Hill equation by an iterative method giving the S0.5 and Hill (nil) values. All data were replicated 3 times. They are reported as means ___SEM. They were analysed by Student's t-test (Sokal and Rohlf, 1981). RESULTS
Pyruvate kinase from foot and mantle These enzymes were found to be very similar. Both showed hyperbolic saturation kinetics for their substrates p h o s p h o e n o l p y r u v a t e (PEP) and A D P (Table 1). Alanine inhibited b o t h enzymes, changing the P E P saturation kinetics to a slightly sigmoidal form (Fig. 1). In the presence o f 5 mmol/l alanine the enzyme from the foot o f active animals had a S0.s(pBp) o f 0.25 + 0 . 0 5 m m o l / l (N = 3) and n M o f 1.2 + 0.04; the data for the enzyme from aestivating animals were S0.5(pBp)= 0.38 + 0.10 mmol/I (N = 3) and n a = 1 . 4 + 0 . 0 4 . The inhibition by alanine
Changes in kinetics of pyruvate kinase from Helix
was readily reversed by fructose 1,6-bisphosphate (FBP) (Fig. 1). A concentration of 0.05mmol/i was sufficient to reduce the Km(vee) to 0.049 ± 0.003 mmol/l (N ffi 3) for the foot enzyme from active animals, and 0.046 ± 0.001 mmol/l (N ffi 3) for the enzyme from the foot of aestivating animals. The activation by FBP was not as prominent in the absence of alanine (Fig. 1), but 0.05 mmol/I FBP did reduce the Km(ee.p ) of the enzyme from active animals to 0.047 ± 0.003 mmol/l (N = 3), and 0.037 ±0.005mmol/l (N = 3) for the enzyme from aestivating animals. The enzyme from the mantle collar responded in similar fashion, the data being essentially similar to that of the enzyme from the foot. ATP was also found to be an inhibitor of these enzymes, but the saturation kinetics of PEP remained hyperbolic. Comparing the properties of pyruvate kinase from the foot and mantle of aestivating and active animals showed a small decrease in affinity for phosphoenolpyruvate (PEP) (P < 0.05) and the activator FBP (P < 0.05). There was no significant difference in the Kin(AVe), nor in the concentration of ATP or alanine required to produce 50% inhibition in the enzymes from active and aestivating animals. The effects of reducing the pH to 6.5 were found to be at least as dramatic than the changes in properties of the enzyme in the active and aestivating animals (Tables 1 and 2). For the enzyme from the foot and mantle the affinity for phosphoenolpyruvate was decreased at pH 6.5, the inhibition by ATP and alanine was more pronounced (Iso./. was decreased), and the affinity for ADP was also decreased (P < 0.05 for all comparisons).
Pyruvate kinase fiom digestive gland In contrast to the properties of the enzyme from the mantle collar and foot, those of the enzyme from the digestive gland were markedly different. The enzyme from the digestive gland of active animals showed sigmoidal saturation kinetics with a low affinity for PEP whereas the enzyme from aestivating snails showed hyperbolic saturation kinetics and a higher affinity for PEP (Fig. 2, Table 3). Both enzymes were inhibited by alanine; at a concentration of 5 mmol/I the S0.s value for PEP was increased to 4.3 ± 0.4 mmol/l (N = 3) (nil ffi 1.9 ± 0.3) for the enTable 2. Apparent kinetic parameters (mmol/I ± SEM, N = 3) of pyruvate kinase from mantle collar of Helix aspersa Active
3Ka(FBP) 4150%(iluine)
4l~(^Tp )
IK~(eEe)
5.0 I
e-
•~
O 4.0
O.
T
&o
-~
2.0
1.0
0.0
0.0
1.0
2.0
[PEP] (retool.l-')
0.071 + 0.002 0.33 + 0.004 0.00076 + 0.000003
0.097 + 0.02 0.26 ~ 0.005 0.00091 + 0.00018 I.I :[:0.06 5.6 =[:1.0
1.7 _+ 0.07 5.3 + 1.0
4.0
zyme from active animals, and 2.3 _+0.4 mmol/1 (N f3) ( n H = 1.2+0.03) for the enzyme from aestivating animals• Fructose 1,6-bisphosphate (0.05 retool/l) reversed the inhibition by alanine, decreasing the S0.S(pEp) to 0•29 + 0.03 mmol/1 (N = 3) (n H = 1.5 _+0.2) for the enzyme from the digestive gland of active animals, and 0.16+0.02mmol/l (N = 3) (nil----1.0) for the enzyme from aestivating animals. In the absence of alanine, fructose 1,6bisphosphate activated the enzyme from active and aestivating snails (Table 3), reducing the S05(pEp) to 0.11±0.02mmol/1 ( N - - 3 ) (nH=l.3_+0.2) for the enzyme from active animals and 0•068 + 0.007 mmol/I (N = 3) (nH = 1.1 ± 0.1) for the enzyme from aestivating snails. The concentration of alanine required for 50% inhibition was not markedly different in the two forms of the enzyme, nor was it altered by decreasing the pH to 6.5. On the other hand, the 15O./ofor ATP was markedly lower for the enzyme from active animals, and for both forms it was significantly lower at pH 6.5 (Table 3) (P < 0.05 for both comparisons). It is also notable that the affinity for FBP was clearly Table 3. Apparent kinetic parameters (mmol/l + SEM, N = 3) of pyruvate kinase from the digestive gland of Helix aspersa Active
Aestivating pH 7.0
IS0.s(pep~ ntt 2Krn(AVP }
3K,~rsp ) 3150,/o(at.ni~)
3150%(ATI,)
p H 6.5
0.12 4- 0.04 0.12 __. 0 . 0 0 6 2KmcADP) 0.,44 __ 0.01 0.40 + 0.007 3Ko(Fse) 0.0019 + 0.0003 0.0018 + 0 . 0 0 0 3 44150%(a]anine) 0.52 + 0.11 0.54 _+0.04 I5o%|^TP~ 0.96 + 0.11 I. I + 0. !0 Standard conditions were 100mmol/I KCI, 10retool MgSO4, 0.1 mmol/I NADH, 0.2 unit LDH, 50 mmol/imidazole at stated pH, 25°C. Other conditions: ]2.0 mmol/I ADP; 22.0mmol/I PEP; 30.06mmol/I PEP, 2.0mmol/l ADP, 5.0mmol/I alaninc; 40.06 mmol/l PEP, 2.0 mmol/I ADP.
3.0
Fig. 2. Saturation kinetics for phosphoenolpyruvate kinase from the digestive gland of active (O) and aestivating (0) Helix aspersa. Conditions: 2 mmol/1 ADP, 100 mmol/l KCI, 10mmol/l MgSO4, 0.1 mmol/l NADH, 0.2 unit LDH.
Aestivating pH 7.0
~Km(pEe) 2Km(ADP)
79
1.2 + 2.0 + 0.23 + 0.0013 +
0.28 +_0.04 1.1 _+0.1 0.28 + 0.01 0.00066 _+0.00002
0.09 0.1 0.04 0.00002
0.54 + 0.03 2.0 _+ 0.5
0.78 + 0.20 7.6 + 1.5 p H 6.5
IS0.~PEp ) /'1H 2Km(AOP)
0.36 + 0.04 1.3 + 0.2 1.8 ± 0.3 1.3 + 0.2 0.27 _+0.06 0.33 + 0.03 3K,(vse) 0.0032 +_0.0006 0.0013 _+ 0.0006 3 3150~(al.,i,~) 0.51 + 0.02 1.0 _+ 0.6 3.6 + 0.6 ls0~|^Tr? 1.2 +0.2 Standard conditions were 100mmol/I KCI, 10mmol MgSO4, 0.1 mmol/I NADH, 0.2 unit LDH, 50 mmol/imidazole at stated pH, 25°C. Other conditions: J2.0 mmol/l ADP; 22.0mmol/l PEP; 30.6 mmol/I PEP, 2.0 mmol/I ADP.
80
JEREMY H. A. FIELDS
higher in the enzyme from aestivating snails than in that from active snails.
of the enzyme through phosphorylation or dephosphorylation. The phosphorylation of rate-limiting glycolytic enzymes, especially pyruvate kinase, in response to anoxic stress has been extensively studied DISCUSSION in intertidal molluscs. In these organisms pyruvate The results of this study have shown that there are kinase and phosphofructokinase are converted to less differences in the kinetic properties ofpyruvate kinase active forms by phosphorylation as part of the stratfrom the foot, mantle and digestive gland of active egy of reducing metabolic rate to cope with long-term and aestivating H. aspersa. These kinetic differences anoxic stress (Storey and Storey, 1990). The hallpoint towards an enzyme that is slightly less active in marks of the changes are a reduction in affinity for the foot and mantle collar of aestivating snails than one or more substrates, an increase in sensitivity to in the active ones. It has a lower affinity for PEP, and inhibitors, and a decrease in sensitivity to activators is less responsive to the activator FBP. The differ- (Storey, 1985; Storey and Storey, 1990). For pyruvate ences in the properties of the enzyme from the kinase, in terms of absolute magnitude, the greatest digestive gland were found to be reversed as com- change in the S0.5 (5-10-fold) for PEP has been pared to the enzyme from foot and mantle collar, i.e. observed in the gastropods Busycotypus canaliculait was the enzyme from aestivating animals that had turn and Concholepas concholepas (Plaxton and a higher affinity for PEP, the activator FBP, and a Storey, 1984a,b, 1985; Carvajal et al., 1990); that in lower 15oo/ofor the inhibitor ATP. This would suggest the limpet Patella caerulea, the bivalves Mytilus that the enzyme from the digestive gland in aestivat- edulis, Venus gallina, and Scapharca inaequivalvis was ing snails would be more active than that from the lower (1.3-3-fold) (Holwerda et al., 1983; Hakim et al., 1985; Michaelidis et al., 1988). The changes digestive gland of active snails. The changes in the kinetic properties of pyruvate observed in the foot and mantle of aestivating kinase from the tissues of H. aspersa are consistent H. aspersa (this study) and O. lactea (Whitwam and with the enzyme being covalently modified, perhaps Storey, 1990) are small, but comparable to those through phosphorylation. Indeed, the data obtained observed in the limpet P. caerulea (Michaelidis et al., for H. aspersa are very similar to those obtained for 1988). Thus it would appear that conversion of key O. lactea (Whitwam and Storey, 1990) except for the enzymes, as exemplified by pyruvate kinase, to less observation of activation by FBP in the enzyme from active forms is a characteristic response to long-term environmental stress amongst the Mollusca, at least foot and mantle collar of active and aestivating snails, in some tissues. and also from the digestive gland of aestivating snails. The above argument clearly does not apply to the Whitwam and Storey (1990) also showed that the changes in the properties of pyruvate kinase could be changes observed in pyruvate kinase from the digesmimicked by treatment of the enzymes from active tive gland of terrestrial gastropods during aestivation. snails with stimulators of protein kinase, or by treat- The data from O. lactea (Whitwam and Storey, 1990) ment of the enzyme from aestivating snails with and H. aspersa (this study) indicate that the enzyme alkaline phosphatase. They concluded that the kinetic should be more active during aestivation, having a changes were brought about by covalent modification lower S0.5 for PEP, a lower Ka for FBP, and a higher 15oo/. for alanine. On the other hand, this process is occurring during a time when metabolism is de60 pressed. It is noteworthy that the catalytic properties ~ • r . . . . of pyruvate kinase from the digestive gland are f 4x / characteristic of those of gluconeogenic tissues, the i z~ ~ " A ~ cmammalian liver being the archetype for comparison / / I (Scrutton and Utter, 1968; Engstrom, 1979). It has "6 been assumed that gluconeogenesis occurs in the ~'r'" 4.0 0"~ / digestive gland of gastropods (Livingstone and de Zwaan, 1983). If this is a major site of gluconeogenT / esis, then the change in the properties of pyruvate kinase may reflect a switch in metabolism, from being gluconeogenic to non-gluconeogenic. It has been -o 2.0 '[-2~A shown that blood glucose in gastropods declines during aestivation, and that glycogen stores in tissues are also consumed (Swami and Reddy, 1973; Home, 1973; Heeg, 1977; Riddle, 1977; Cedefio-Leon, 1984). It may well be that the digestive gland must switch from being primarily gluconeogenic in the active °'°°c !°pEP][ (mmol.l-2'°1) 3.~--4.o phase to primarily glycolytic in the aestivating phase Fig. 3. Saturation kinetics for phosphoenolpyruvate of in order to supply the necessary metabolic energy to pyruvate kinase from the digestive gland of active Helix the cell. This may be the explanation for the effects aspersa showing the effects of alanine and fructose 1,6-bis- observed in the digestive gland being opposite to phosphate. (0 O) control; (Q 0 ) 5mmol/l ala- those found in the foot and mantle collar. nine; (/X .... /X) 0.05mmol/l fructose 1,6-bisphosphate; Studies of gas exchange in active and aestivating (A .... A) 0.05mmol/l fructose 1,6-bisphosphate plus O. lactea have shown that the rate of oxygen con5mmol/l alanine. Other conditions: 2mmol/l ADP, 100 mmol/1 KC1, 10 mmol/l MgSO4, 0.1 mmol/l NADH, 0.2 sumption is inversely proportional to the concentration of carbon dioxide (Barnhart and McMahon, unit LDH.
Changes in kinetics of pyruvate kinase from Helix 1988, 1989). Furthermore, the aestivating state is characterized by hypercapnia and a decrease in intracellular pH (Barnhart and McMahon, 1989). Pyruvate kinase from H. aspersa might be less active at a lower pH. The enzyme from foot and mantle collar of active snails had a higher Km for PEP, a higher Ka for FBP, and lower 15o,/. for ATP. The inhibition by ATP may well be significant at this pH if the snails are maintaining concentrations of ATP that are close to those of an active animal. Decreases i n intracellular pH have been shown to reduce metabolic rate in frog skeletal muscle, mammalian erythrocytes, rat hepatocytes, and embryos of Artemia (Tomoda et al., 1977; Busa and Crowe, 1982; Busa et al., 1983; Fidelman et al., 1982; Kashiwagura et al., 1984). Ellington (1985) showed that a reduction in intracellular pH in perfused hearts of Busycon contrarium caused a negative crossover between fructose 6phosphate and fructose 1,6-bisphosphate, indicating that flux through phosphofructokinase was inhibited. Churchill and Storey (1989) found negative crossovers in tissues of asetivating snails at the level of phosphofructokinase and pyruvate kinase, clearly indicating that both of these enzymes are rate-limiting steps in glycolysis during aestivation. It is plausible that changes in intracellular pH also play a role in reducing metabolism in aestivating snails, as suggested by Barnhart (1989) in addition to covalent modifications of regulatory enzymes (Storey and Storey, 1990). The reduction in flux through pyruvate kinase in the foot might reflect both conditions; a reduction in intracellular pH because of hypercapnia, and covalent modification. The digestive gland needs further study, as it is metabolically more complex than the foot or mantle collar. Acknowledgements--This study was funded by Faculty Research Grants during 1986-1989 from the College of Arts and Sciences, University of San Diego. The technical assistance of Mr. T. C. Matthews and Ms. L. Alvarez is greatly appreciated. REFERENCES
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