OF BIOCHEMISTKY AND BIOPHYSICS Vol. 189, No. 1, July, pp. 63-75, 1978



of Detergent-Dispersed GEORGE






of Chemistry, Canada Received



The University T2N IN4




DUNHAM of Calgary,



21, 1977

Adenylate cyclase from rabbit ventricle was solubilized in 30 to 50% yield by the nonionic detergent Lubrol PX. The detergent, when present in the assay at concentrations above 0.05%, rapidly inactivated the enzyme in assays conducted above 26’C; assays were valid only when conducted below this temperature. The solubilized enzyme was eluted from diethylaminoethyl (DEAE)-Bio-Gel A (DEAE-agarose) with 100 mM NaCl in a yield of 25% and was free of detergent. Several properties of the solubilized detergent-free enzyme were similar to properties of the native membrane-bound species. The K,, for substrate was 0.1 mM., the K,, for Mg2+ was 2.5 mM, and ATP in excess of Mg”’ was inhibitory. The enzyme was activated by F-- and guanyl-5’-yl imidodiphosphate [Gpp(NH)p] in a timeand temperature-dependent manner, and activation by the latter was persistent. Activation by F- and Gpp(NH)p reduced the K,, for Mg”+. Activation by Gpp(NH)p was increased by Mg’+; the apparent K. for activation was 0.1 PM. Multiple binding sites for Gpp(NH)p were present: one class with a & value of 0.11 pM was probably associated with activation of the enzyme. The soluble enzyme was insensitive to catecholamines, in both the presence and the absence of Gpp(NH)p. Sensitivity to catecholamines was not restored by the addition of phospholipids, particularly phosphatidyl inositol, in either the presence or the absence of Gpp(NH)p, and this phospholipid did not increase the sensitivity of the membrane-bound enzyme to epinephrine. Catecholamine binding sites were present, and their association with adenylate cyclase was seemingly not affected by phospholipids.

Adenylate cyclase [ATP pyrophosphatelyase (cyclizing); EC is bound to membranes, particularly plasma membranes, in eukaryotic tissues. Following the initial studies of Sutherland et al. (l), nonionic detergents have been used to solubilize the enzyme from heart (2,3), brain (4-8), liver (g-11), testis (12, 13), kidney (14, 15), adrenal cortex (161, and erythrocytes (17). A characteristic feature of the solubilized enzyme is loss of hormonal responsiveness, presumably due to uncoupling of the hormone receptor from the catalytic unit. However, the liver enzyme solubilized with Triton X-305 retained sensitivity to glucagon and epinephrine (lo), a preparation of the brain enzyme solubilized with Lubrol PX was stimulated by catecholamines (8), and the renal cortex enzyme solubilized with this detergent responded to calcitonin (14). Levey -found that catecholamine responsiveness of the cat heart enzyme solu-

bilized with Lubrol PX was dramatically restored by the addition of phosphatidylinositol (18, 19); glucagon sensitivity was similarly restored by phosphatidylserine (19,20). Solubilization of the catecholamine binding site (receptor) in frog erythrocytes was effected with digitonin (but not with other detergents) (21) and resolved from adenylate cyclase by gel filtration (22). Glucagon binding sites were present in preparations of cat heart adenylate cyclase solubilized with Lubrol PX (19, 23, 24). Since the observations of Rodbell et al. (25) that glucagon stimulation of the liver enzyme was enhanced by GTP, it has been established that a requisite for hormonal activation of the mammalian enzyme is binding of this nucleotide to a specific site. Most studies on the role of guanine nucleotides have employed the more stable GTP analog, Gpp(NH)p2 (26). Guanine nucleotide ’ Abbreviations dodiphosphate; aminoethyl.

’ This work was supported by the Medical Research Council of Canada and the Alberta Heart Foundation.

used: Gpp(NH)p, CAMP, cyclic AMP;

guanyl-5’-yl imiDEAE, diethyl-

63 0003-9861/78/1891-0063$02.00/O Copyright 0 1978 by Academic

Press, Inc.



converts the enzyme to an activated state; the hormone increases the rate of active state formation (27-36). The detergent-solubilized enzyme retains sensitivity to Gpp(NH)p in liver (ll), adrenal cortex (16), pigeon erythrocytes (17), dog heart (32,33), and renal medulla (37). Gpp(NH)p binding sites have been partially resolved from the catalytic unit of adenylate cyclase in adrenal cortex (16) and erythrocytes (17) by gel filtration. We have been engaged in studies designed to examine the properties of solubilized myocardial adenylate cyclase with the hope of ultimately purifying the enzyme; our findings to date are described herein. MATERIALS



[a-‘“P]ATP (tetratriethylammonium salt, 15-20 Ci/mmol), [@HIcAMP (ammonium salt, 30 Ci/mmol), levo-[propyl-2,3-“Hldihydroalprenolol (50 Ci/mmol), [:‘H]acetic anhydride (50 Ci/mol), Aquasol, and Omnifluor were obtained from New England Nuclear, Montreal; /3,y-imido-[&“H]guanosine 5’-triphosphate (ammonium salt, 5 Ci/mmol) was from Amersham/Searle Corp., Arlington Heights, Illinois; and DEAE-Bio-Gel A (DEAE-agarose) (100-200 mesh) was purchased from Bio-Rad Laboratories, Richmond, California. Phosphoenolpyruvate (trisodium salt), pyruvate kinase (rabbit skeletal muscle type II), L-Ophosphatidyl-L-serine (bovine brain), L-a-phosphatidylethanolamine (ovine brain), and l.-a-phosphatidylcholine were obtained from Sigma Chemical Co., St. Louis; phosphatidylinositol and guanyl-5’-yl imidodiphosphate (sodium) from PL Biochemicals, Inc., Milwaukee; and sphingomyelin from Pierce Chemical Products. (-)-Propanolol was obtained through the courtesy of Dr. D. J. Marshall, Ayerst Research Laboratories, Montreal. Lubrol PX was obtained from ICI Products Group, Montreal; a sample of detergent was labeled by reaction with [:‘H]acetic anhydride by the method of Gaylor and Delwiche (38). Solubilization of adenylate cyclase. Hearts were removed from white female rabbits (2.5 to 3 kg) anesthetized with sodium pentobarbital and were perfused with warm, oxygenated Krebs-Ringer bicarbonate to remove blood. All succeeding operations were performed at 4°C. Ventricular muscle, approximately 4 g per heart, freed of fat, atria, large vessels, and chorda tendenae, was homogenized in 5 vol of 0.25 M sucrose, 20 mM Tris-HCl, 1 mM dithiothreitol (pH 7.5) for two 15-s intervals in a Polytron PT 10 homogenizer at a rheostat setting of 4, followed by 2 s at maximal velocity. The homogenate was filtered through a 475. pm nylon mesh under light suction, brought to 10 vol with homogenizing medium, and centrifuged at 25,OOOg for 10 min. The pellet was suspended in 2 vol of medium (based on original tissue weight), brought to



1% with Lubrol PX (prepared as a 50% solution in ethanol), dispersed with a Polytron for 30 sat a setting of 3, and sedimented at 38.009g for 20 min. Following reextraction of the pellet in an identical manner, the combined supernatants were centrifuged at 100,000g for 1 h. The resulting 100,000g supernatant fluid was stored at -80°C. DEAE-&o-Gel A chromatography. Twenty milliliters of the 100,009g supernatant fraction (4 mg of protein/ml) were added to a DEAE-Bio-Gel A column (2 x 20 cm) previously equilibrated with 0.25 M sucrose, 1 mM dithiothreitol, 20 mM Tris-HCI (pH 7.5). The column was then eluted (flow rate, 1 ml/min) with 125 ml of this buffer and, subsequently, with similar volumes of buffer containing 40 mM NaCl, 100 mM NaCl, and 100 mM NaCl plus 0.2% Lubrol PX. The protein eluted with 100 mM NaCl was concentrated fivefold by ultrafiltration (Amicon Diaflo PM 10) under 40 psi pressure. The resulting solution was stored at -80°C. Solubilization of Gpp(NHp-activated enzyme. A 25,000g pellet fraction was suspended in 10 vol of homogenizing medium (based on initial tissue weight) and incubated with 10 mM MgSO,, 10 aM [8-“HIGpp(NH)p (1000 dpm/pmol), and 50 pM epinephrine at 26°C for 30 min. The particles were sedimented at 38,OOOg and washed three times with 10 vol of the original buffer by sedimentation. The final pellet was suspended in 2 vol of buffer and extracted with Lubrol PX as described above. The resulting 100,OOOg supernatant fraction, which contained 57 pmol of bound [:‘H]Gpp(NH)p/mg of protein, was subjected to DEAE-Bio-Gel A chromatography as described for the nonactivated enzyme. Adenylate cyclase assay. The method used was that of Salomon et al. (39), with minor modifications. The assay system contained 40 mM Tris-HCl, pH 7.5, 8 mM theophylline, 8 mM MgSO,, 5.5 mM KCl, 20 mM phosphoenolpyruvate, 170 pg/ml of pyruvate kinase, 1 mM CAMP, 0.5 mM [a-“‘P]ATP (40 dpm/pmol), and extract (20 to 400 pg of protein) in a final vol of 150 ~1. Tubes were made up in an ice bath and equilibrated at 20°C (unless otherwise indicated) for 1 min before initiating the reaction by the addition of ATP; the incubation time was 10 min. The reaction was terminated by the addition of 0.1 ml of “stop” solution, and following the addition of [“HIcAMP (5 x IO” cpm) for recovery determinations, [“‘PICAMP was quantitated as described by the above-mentioned authors (39). All assays were performed in duplicate. Reaction velocity was proportional to time and protein concentration under all conditions, unless otherwise stated. Specific activity is defined as picomoles of CAMP formed per minute-’ milligram-‘. Protein was determined by the method of Lowry et al. (40). To measure activation with Gpp(NH)p, tubes (in duplicate) containing the complete assay medium, except ATP (30 to 300 pg of protein), were incubated at 26°C with 0.1 mM Gpp(NH)p for 30 min in the case of the washed particle and 100,OOOg supernatant frac-



tions and for 2 h in the case of the solubilized detergent-free preparation. To measure F activation, tubes were similarly prepared to contain 8 mM NaF and were incubated for 30 min at 26°C. Tubes were then placed in ice and equilibrated at 20°C for 1 min. and the adenylate cycl.ase assay was initiated by the addition of ATP. Binding of f’h’/Gpp(NH)p. Binding of Gpp(NH)p in soluble preparations was quantitated by the polyethylene glycol precipitation technique (41), as adapted for guanine nucleotide by Glossmann (16) and Pfeuffer and Helmreich (17). The assay (performed in duplicate) contained 20 mM Tris-HCl (pH 7.5), 0.5 PM [8-“HJGpp(NH)p (1 X IO” dpm/pmol), and 5 mM MgSO, (unless otherwise stated) in a final vol of 100 ~1. After a 2-min equilibration at 26”C, the reaction was started by the addition of extract (IO to 30 pg of protein) and incubations were carried out for 40 min. Nonspecific binding was determined in tubes containing a IOOO-fold excess of unlabeled Gpp(NH)p. Controls were also carried without protein to measure binding to the filters. Bound Gpp(NH)p was precipitated by adding 50 ~1 of 0.3% human y-globulin and 1 ml of 5% polyethylene glycol 6000, and the tubes were kept on ice for 20 min. The mixtures were then filtered through a microfilter (Millipore HAWP024, 0.45 pm) and washed with 5 ml of 5% polyethylene glycol6000. The filters were dried and dissolved in 1 ml of ethylene glycol monometh,yl ether, and radioactivity was determined in 16 ml of toluene-ethylene glycol monomethyl ether (3:l) cocktail containing 4 g of Omnifluor/liter. “Nonspecific” binding ranged from 0 to 10% of the total disintegrations pe; minute bound. All values were corrected for binding to the filters (usually 2% of the binding to protein). Binding was proportional to protein over the range used. Detergent or NaCl at the concentrations present did not interfere with binding or precipitation of bound counts. Binding of (-)-f’H/dihydroalprenolol. Binding of c’H]dihydroalprenolol to particulate preparations was determined in a medium containing 50 mM Tris-HCl (pH 7.5), extract (300-600 pg of protein), and 10 nM (-)-[“Hldihydroalprenolol (6 x 10” dpm/pmol), as described by Mukherjee et al. (42). Following equilibration of the samples (in duplicate) for 2 min at 3O”C, the reaction was initiated by the addition of the antagonist and the tubes were incubated for 10 min. Nonspecific binding was determined in tubes which contained, in addition, 10 pM (-)-propanolol. Tubes were also carried containing no extract to correct for binding to the filters. Reactions were terminated by the addition of 1.85 ml of 50 mM Tris-HCl (pH 7.5), and the mixtures were filtered through glass-fiber filters (Gelman Type A/E). The filters were washed with 20 ml of buffer and dried, and radioactivity was determined in 7 ml of toluene containing 4 g/liter of Omnifluor. Nonspecllfic binding amounted to 36-48%> of the total radioactivity bound. To quantitate (-)[“HJdihydroalprenolol binding to the soluble deter-




gent-free preparation, the method of Caron and Lefkowitz (21) was used. The sample (200-300 pg of protein) was incubated in 50 mM Tris-HCl with 40 nM (-j-[,‘H]dihydroalprenolol (7 x IO” dpm/pmol) in a final vol of 150 ~1 for 90 min at 4’. Nonspecific binding was measured in the presence of 40 FM (-)-propanolol and amounted to 40% of the total binding after correction for blank values (protein absent). Following dilution of the incubation mixture with 150 ~1 of buffer, the solutions were applied to 0.6 x 10.5-cm Sephadex G-50 columns, 0.5 ml of buffer was added, and the eluate was discarded. Then 1.2 ml of buffer was added, this effluent was collected in scintillation vials, and radioactivity was determined using 8 ml of Aquasol. Assay of samples containing 1% Lubrol PX, i.e., the 100,000~ supernatant fraction, could not be performed because of high blank values. RESULTS

Effects of Detergent on Adenylate


In early studies in which the enzyme was assayed at 37”C, the apparent yield of solubilized enzyme was extremely low, less than 10% based on the starting activity. It was found that Lubrol PX added directly to the assay at final concentrations of 0.1 to 0.2% [equivalent to that carried into the assay from extracts containing 0.5 or 1% (8.3 or 16.6 mM)] caused a marked decrease in both basal and F--stimulated activity (Fig. 1A). F- did not prevent this decrease when present in concentrations as high as 20 mM (Fig. 1B). The data in Fig. 1 were obtained using whole homogenates; the enzyme in washed particle preparations was equally sensitive. Lubrol WX and Triton X-100 were also inhibitory to the rabbit heart enzyme. The damaging effect of Lubrol PX was also seen with homogenates and washed particle preparations from rat, guinea pig, dog, cat, and pigeon hearts. To investigate the inhibitory effect further, the enzyme was assayed in the presence and absence of Lubrol PX at several temperatures (Fig. 2A). In the absence of detergent, the activity in the crude homogenate increased with temperature up to 32”C, but declined above this temperature, reflecting the lability of the native enzyme at physiological temperatures (43). In the presence of F-, the activity increased with temperature up to 37°C. The activity in the homogenate containing 0.5% Lubrol PX (0.1% concentration in the assay) declined precipitously above 27”C, and this was not pre-







03 1%)

FIG. 1. Effect of Lubrol PX on rabbit heart adenylate cyclase. protein) was assayed at 37°C. (A) Lubrol concentration was varied: in the presence of 8 mM NaF. (B) NaF concentration was varied: (D) PX in assay. Values are means of two experiments with different directly to the assay without prior incubation


Crude homogenate (400 pg of (0) basal activity; (0) activity no detergent; (0) 0.3% Lubrol preparations. NaF was added

100 -*--------o

: :$ :, ’‘\ : ‘\ b‘\ b. -. ‘\ a... -m--- ----a ‘\ *-------* k I E




/ 4


1 6

I 8

I 10

4 li


AT 37”

FIG. 2. Effect of temperature on enzyme activity in the presence and absence of Lubrol PX. (A) Crude homogenate (400 pg of protein) (O,O), homogenate containing 0.5% Lubrol PX (M, Cl), and supernatant obtained by centrifuging the Lubrol-treated preparation at 38,OOOg (150 8g of protein) @,A) were assayed under standard conditions, except that the temperature was varied. Closed symbols indicate basal activity; open symbols indicate that 8 mM NaF was present. Where present, detergent carried into the assay gave a final concentration of 0.1% (B) The three preparations were incubated in the complete assay system (minus ATP) at 37°C for the time periods indicated. When present, NaF was 8 mM. The tubes were then placed on ice and equilibrated at 24°C for 2 min, and the reaction (which was conducted at 24°C) was started by the addition of ATP. Symbols are as in A. Values are means obtained from two separate preparations.

vented by F-. Similarly, enzyme activity in the 38,OOOgsupernatant prepared from a detergent-treated homogenate declined above 24’C, and F--stimulated activity in this preparation declined above 27°C. It is apparent from Fig. 2A that, at temperatures below 27”C, the detergent slightly stimulated basal activity (compare n with 0).

The rate of inactivation of the enzyme at 37°C was examined by incubating the above preparations in the assay system in the presence and absence of F- for various time periods prior to the addition of ATP. The assay was then conducted at 24°C. Loss of activity in the presence of Lubrol occurred with extreme rapidity at 37°C






(Fig. 2B) and was only slightly slowed by F-. Inactivated basal enzyme could not be stimulated by either F- or Gpp(NH)p (data not shown). Marked loss of activity in the native enzyme in the absence of detergent also occurred under these conditions; in this case, F had a pronounced stabilizing effect (compare 0 with 0). Thus the destructive action of Lubrol PX could be largely prevented simply by conducting the assay at 20 or 26°C. Solubilization and DEAE-Bio-Gel A Chromatography of Adenylate Cyclase When extracted with Lubrol PX as descri.bed under Materials and Methods, the recovery of enzyme in the 100,OOOg supernatant fraction varied within the range of 30 to 50% of the starting homogenate (assay conducted at 2O’C) in at least 20 preparations. Basal specific activity of the initial homogenate ‘was in the range of 20 to 35 pm01 min-’ mg-‘, and that of the 100,OOOg supernatant, 70 to 100 pm01 min-’ mg-‘. The detergent-dispersed enzyme readily passed through a 0.2~pm filter and did not sediment at 150,000g for 90 min. The preparation rapid1.y lost activity at 4 and -2O”C, but retained activity for at least 3 weeks at -80°C provided thawing and freezing were avoided. When this fraction was subjected to chromatography on DEAE-agarose (Fig. 3A), an active fraction was eluted with buffer contai:ning 100 mM NaCl (peak III). Protein in this peak was concentrated by ultrafiltration and is referred to as solubilized detergent-free enzyme; it was stored at -80°C. Recovery of protein in this fraction was 15%; recovery of activity ranged from 15 to 25’6, with specific activities (after ultrafiltration) ranging from 70 to 120 pm01 min-’ mg-‘. Thus very little purification was achieved by the chromatographic procedure; however, the enzyme was obtained essentially free of detergent. When [“H]Lubrol was used, all radioactivity (8.5 x lo” dpm) was recovered in the washout peak (Fig. 3A, peak I); radioactivity in peak III was not above background. This does not preclude the possibility that detergent still remained associated with the enzyme or with some other constituent of the fraction; it only meanls that the detergent concentra-




FIN. 3. DEAE-Bio-Gel A chromatography of 100. 000~ supernatant fractions. (A) The 100,000~ fraction (80 mg of protein) was chromatographed as described under Materials and Methods. Peak I, washout; peak II, eluted with 40 mM NaCl (started at arrow A); peak III, eluted with 100 mM NaCl (started at arrow B); peak IV, eluted with 100 mM NaCI, 0.2% Lubrol PX (started at arrow C). To facilitate assay procedures, tubes were pooled so that volumes varied between 8 and 25 ml; thus elution is expressed as total volume, not fraction number. Three separate experiments are represented: One contained [“H]Lubrol, 8.1 x IO” dpm. The recovery of radioactivity was 100%. Duplicate experiments were performed to measure the binding of [“H]Gpp(NH)p and adenylate cyclase. The values for protein and adenylate cyclase are representative of 12 separate column fractionations. (A) Protein; (0) [“H]Lubrol PX; (0) adenylate cyclase; and (Cl) bound Gpp(NH)p. (B) Fifteen milliliters of a solubilized preparation (3.6 mg of protein/ml) previously activated with [“H]Gpp(NH)p (bound nucleotide, 3073 pmol) was chromatographed as in A. (A) Protein; (0) adenylate cyclase; and (A) bound [‘H]Gpp(NH)p (determined as described under Materials and Methods).

tion was vanishingly low and did not interfere with the enzyme assay. A second peak of activity comprising 5 to 8% of the applied activity was recovered by further elution of the column with 100 mM NaCl containing 0.2% Lubrol (Fig. 3A, peak IV). No further activity could be recovered from the column, by elution with either 400 mM NaCl or 400 mM NaCl with 1% Lubrol. Thus the comparative low yield seems to result from loss of activity during the fractionation pro-



cedure. The binding of Gpp(NH)p to protein in each peak was also measured. It can be seen (Fig. 3A) that significant binding of the nucleotide occurred in peaks I and II (30% of the total) and that these fractions were devoid of adenylate cyclase. Maximal binding occurred in peak III (46% of total), with 23% in peak IV. The specific activity of binding in fractions comprising this latter peak (which ranged from 50 to 120 pmol mg-*) was higher, however, than that in peak III (range, 35-60 pmol mg-I). In other experiments, the enzyme preparation previously activated with [“H]Gpp(NH)p was subjected to chromatography (see Materials and Methods). Enzyme eluted in precisely the same manner (Fig. 3B) as the nonactivated preparation. The specific activity of the 100,OOOg supernatant applied was 930 pmol min-’ mg-‘; the highest specific activity recovered in peak III was 3100 pm01 min-’ mg-‘; the recovery of activity was 66% of that applied. An additional 15% was recovered in peak IV. In one experiment, the 100,OOOg supernatant fraction applied contained bound [“H]Gpp(NH)p (3.1 x lo” dpm). Following chromatography, significant amounts of radioactivity appeared in peaks I and II (12 and 14% of the total, respectively); 32% was present in peak III, and 12% in peak IV. Thus, again [“HIGpp(NH)p was bound to fractions devoid of adenylate cyclase. Adenylate cyclase recovered in peak III remained in the fully activated form, as evidenced by the excellent yield and by the fact that it could not be further activated by incubation with





+ t/' N b x


5 :






FIG. 4. Effect of temperature on assay of solubilized detergent-free enzyme (24 pg of protein). Factivation was affected by prior incubation of the enzyme with 8 mM NaF (see Materials and Methods). Lubrol PX, where present, was added immediately before the addition of ATP, and the final concentration was 0.1%. (0,O) No detergent; (A& detergent present. Open symbols indicate basal activity: closed symbols indicate F-stimulated activity.


Properties of the Detergent-Free Enzyme The solubilized detergent-free enzyme (concentrated peak III) was highly labile; preparations, however, could be stored at -80°C for 1 month. At 4°C aggregation took place, so that activity did not pass through a 0.2~pm filter unless 0.1% Lubrol was present. When assayed at various temperatures (Fig. 4), activity did not increase proportionally above 24°C; thus all assays of this fraction were conducted at 20°C. The enzyme was activated and markedly stabilized by F-. Lubrol PX added to the assay at a concentration of 0.1% severely



Frc. 5. Time and temperature dependence of Fand Gpp(NH)p activation. Detergent-free enzyme (30 pg of protein; duplicate samples) was incubated in the complete assay system (without ATP), with (---) and without (---) 8 mM NaF (A) and 0.1 mM Gpp(NH)p (B), at 15°C (O), 20°C (a), 26’C (Cl), 32°C (O), and 37°C (A), for various time periods prior to assay. The inset in B (upper left) provides data on the activation of the 100,000g supernatant fraction by Gpp(NH)p.



reduced basal activity above 20°C and decreased F--stimulated activity above 24°C. The enzyme was activated by F- (Fig. 5A) and by Gpp(NH)p (Fig. 5B) in a time- and temperature-dependent manner. The rate of F- activation was maximal at 26”C, and maximal activation was achieved in 20 min at this temperature. Activation did not proceed to peak values at 32 and 37”C, primarily because ba.sal activity deteriorated rapidly at these temperatures (see Fig. 5A, the controls). The rate of F- activation of the enzyme at 20 and 26°C was identical to that of the washed particle fractions (data not shown). Activation by Gpp(NH)p proceeded very ~slowly at 20 and 26°C (Fig. 5B); the maximal rate occurred at 30 and 37°C. The activated enzyme, however, was not stable at 37°C. Activation of the enzyme in the 100,OOOg fraction occurred much more rapidly at 20 and 26°C (Fig. 5B, inset), reaching a maximum in 15 min at 20°C and 10 min at 26’C. Enzyme in the washed-particle fraction and in peak IV (which contained 0.2% detergent) was activated at rates analogous to that of the 100,OOOg supernatant fraction. The reason for the slower rate of activation of the solubilized detergent-free enzyme is not understood. It was probably not due to removal of the Gpp(NH)p binding site during chromatography, since peak III contained the highest percentage of Gpp(NH)p binding sites (Fig. 3). It was probably not due to the absence of detergent, since the washed particle fraction activated as rapidly as the two fractions containing detergent (lOO,OOOg supernatant and peak IV). The K,,, of the detergent-free enzyme for substrate was 0.1 mM; activation by F- or Gpp(NH)p d.id not affect affinity (Fig. 6). In an experiment in which MgS04 was reduced to 4 mM and ATP varied up to 6 mM, the nucleotide produced inhibition at concentrations in excess of Mg”+ (Fig. 6D-F). The solubilized detergent-free enzyme was stimulated by Mg”+ at concentrations greater than that required for the binding of substrate (MgATP) (Fig. 7B), the K, for this divalent cation being about 2.5 mM. In this regard, it was similar to the enzyme in the washed-particle fraction (Fig. 7A) except that the K, for Mg2+ in the latter










ATP (mM)

FIG. 6. Effect of ATP on basal, F -activated, and Gpp(NH)p-activated enzyme. Enzyme was incubated (duplicate samples; 30 pg of protein) in the complete assay system (without ATP) with 0.1 mM Gpp(NH)p for 2 h at 26°C (A and D), with 8 mM NaF for 30 min at 26°C (B and E), and without the addition of either at 4°C (C and F). The MgSO, concentration in A, B, and C was 8 mM; the concentration in D, E, and F was 4 mM. Following incubation, the tubes were placed in ice and equilibrated to 20°C and ATP at various concentrations was added to start the reaction, which was conducted for 10 min at 20°C.

fraction was somewhat higher. Activation by F- and Gpp(NH)p was accompanied by a reduction in the Mg”’ concentration required for half-maximal velocity (Fig. 7B); this reduction was not as marked as that in the washed-particle fraction (Fig. 7A). The effect of Mg”+ on Gpp(NH)p activation was also examined. Two types of experiments were performed. In the first, incubated with buffer, enzyme was Gpp(NH)p, and varying concentrations of Mg2+. The preparations were then assayed (Fig. 8) before (curve A) and after (curve B) dialysis. In the second experiment, activation was carried out in the adenylate cyclase assay medium (without ATP) with varying concentrations of Mg2+ (curve C). In each case, Mg”’ greatly increased the total activation. Significant activation oc-






FIG. 7. Effect of Mg’+ on enzyme activity. Basal (0), F--activated (A), and Gpp(NH)p-activated enzyme (O), in washed-particle (A) and in solubilized detergent-free (B) preparations, was assayed with varying concentrations of Mg’+. (A) Washed particles were prepared from an homogenate in 20 mM Tris-HCl, 1 mM dithiothreitol, 0.25 M sucrose, 1 mM EDTA (pH 7.5). After centrifugation, the pellet was washed once in 10 vol of this medium, then washed twice in 10 vol of medium lacking EDTA, and finally suspended in 10 vol (based on original tissue weight) of the latter medium. An aliquot (3 ml, 7.63 mg of protein) was incubated with 8 mM NaF, 10 mM MgS04 for 30 min at 26°C; another aliquot was similarly incubated with 0.1 mM Gpp(NH)p, 10 mM MgSO,. Each mixture was centrifuged for 20 min a< 38,OOOg, washed three times by centrifugation in medium lacking EDTA, and finally suspended in the original volume of this medium. Duplicate aliquots were then assayed (100 ag of protein) at 20°C (incubation time, 10 min). (B) An aliquot of solubilized detergent-free enzyme (3 ml, 2.19 mg of protein) was incubated with 8 mM NaF, 5 mM MgS04 for 30 min at 26°C; a similar aliquot was incubated with 0.1 mM Gpp(NH)p, 8 mM MgS04 for 120 min at 26°C. The two samples and a similar sample of the starting fraction were dialyzed separately against 100 ml of 20 mM Tris-HCl, 1 mM dithiothreitol, 0.25 M sucrose, 100 mM NaCl for 5 h, with the buffer changed three times. The samples were then assayed (40 ng of protein) at 20°C.

curred in the absence of added Mg’+, however, as evidenced by the zero Mg”+ values being higher than the basal activity, particularly in the first experiment. Activation of the particulate enzyme (washed-particle fraction) by Gpp(NH)p was similarly affected by Mg”+ (data not shown). A series of experiments was carried out to examine the relationship between Gpp(NH)p-facilitated activation of adenylate cyclase and binding of the nucleotide to protein. The time dependence of












FIG. 8. Effect of Mg”+ on Gpp(NH)p activation. Two experimental procedures were used. In curves A and B, l-ml aliquots of a solubilized detergent-free preparation (1.2 mg of protein) were incubated with 0.01 mM Gpp(NH)p and varying concentrations of MgSO, for 30 min at 30°C. Appropriate aliquots were removed for assay (curve B). The remainder of each was dialyzed for 5 h against three lOO-ml portions of the usual buffer, and assays were performed (curve A) at 20°C for 10 min (30 pg of protein), with a final MgS04 concentration of 8 mM. In the second procedure (curve C), enzyme (3Oag of protein per tube) was incubated in the complete assay mixture (without ATP) with 0.10 mM Gpp(NH)p and varying concentrations of MgSO, for 120 min at 26”C, as described under Materials and Methods. Tubes were then placed in ice, the MgSO, concentration in each tube was adjusted to a final concentration of 10 mM, and after I-min equilibration at 2O”C, incubation was initiated by the addition of ATP.

Gpp(NH)p activation is shown in Fig. 5B. In Fig. 9A, data are presented showing the effect of Gpp(NH)p concentration on activation of the enzyme in the solubilized detergent-free preparation. Half-maximal activation occurred at 0.1 PM (mean of three experiments); maximal activation occurred at 0.01 mM under the conditions used (120 min at 26°C). An apparent K, of 0.1 PM was also established for activation of the enzyme in the 100,OOOg fraction and in peak IV at 26’C (data not shown). The time dependence of Gpp(NH)p binding is shown in Fig. 9B. Binding at 26’C occurred slowly, a maximum being approached at 80 min. Binding at 37°C (0) did not reach the same



maximum as at 26°C possibly reflecting the lability of the binding sites. Binding in the 100,OOOg supernatant fraction (A) (0.054% Lubrol carried into the assay) proceeded more rapidly than in the detergentfree preparation. It may be significant that activation of adenylate cyclase in this fraction also occurred much more rapidly at 26°C (Fig. 5B, inset). In Fig. 9C, data on Gpp(NH)p binding to the solubilized deter-

FIG. 9. Characteristics of Gpp(NH)p binding and activation of adenylate cyclase. (A) Detergent-free enzyme (duplicate tubes; 30 pg of protein) was incubated in the complete assay system (without ATP) for 120 min at 26°C with varying concentrations of Gpp(NH)p, before assay at 20°C. (B) Detergent-free enzyme (duplicate tubes; 18 pg of protein) was incubated (see Materials and Methods) with 0.5 pM ]‘H]Gpp(NH)p (1 x IO4 dpm/pmol) at 26’C in the absence (0) and presence (0) of 0.054% Lubrol PX, and at 37°C with no detergent (a), for the times indicated. Binding in the 100,OOOg supernatant fraction (A) was also measured at 26°C (18 pg of protein); detergent concentration in the assay was 0.054%. (C) Scatchard analysis of ]‘H]Gpp(NH)p binding in solubilized detergent-free prmeparation (0,O) and in 100,OOOg supernatant fraction (A,A). The concentration of [“HIGpp(NH)p (9186 dpm/pmol) was varied between 2.5 nM and 4.4 pM, using 18 pg of protein per tube. Incubation was for 40 min at 2fi”C. Open symbols indicate no MgZ+; closed symbols indicate 5 mM Mg”. (D) Specific [“H]Gpp(NH)p binding to the detergent-free preparation (0) and the 100,OOOg supernatant fraction (a) (18 and 22 pg of protein, respectively), using 0.5 pM [“H]Gpp(NH)p (1 x lo4 dpm/pmol) with the MgSO, concentration shown. Incubations were for 40 min at ZVC.




gent-free preparation and the 100,OOOg supernatant fraction are presented as a Scatchard analysis, where binding was measured in the absence and presence of Mg”‘. The data reveal two populations of binding sites in the former preparation, a high-affinity site with Ku = 0.11 PM and a loweraffinity site with KU = 0.61 PM (means of three separate experiments). The 100,OOOg supernatant actually displayed three populations of binding sites with apparent Ku values of 0.03, 0.11, and 0.62 PM (means of three experiments). Binding of Gpp( NH) p did not require Mg’+; in fact, the cation decreased the amount of nucleotide bound but did not alter the affinity. Figure 9D reveals that Mg” decreased binding but did not eliminate it completely. The solubilized enzyme in both the lOO,OOOg supernatant fraction and the detergent-free preparation was completely unresponsive to catecholamines. In view of Levey’s observations (18-20)) it was considered pertinent to examine the effect of phospholipids for possible restoration of hormonal sensitivity. Levey also reported that phosphatidylinositol increased the sensitivity of the particulate cat heart enzyme to norepinephrine by a factor of 10 (19). In our hands, phosphatidylinositol had a slight stimulatory action on adenylate cyclase in crude homogenates of rabbit heart, but the sensitivity to epinephrine remained unchanged (Fig. lOA). Phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, and a combination of all five added to the assay had no effect on adenylate cyclase in the solubilized detergent-free preparation, and epinephrine produced no stimulation when the assay was supplemented with phosphatidylinositol or phosphatidylcholine (Fig. 10B); glucagon and histamine remained inactive in the presence of phosphatidyl serine. In additional experiments, phosphatidylinositol did not alter the rate of activation of the detergent-free preparation by Gpp(NH)p; epinephrine in the presence or absence of phospholipids had no effect on the rate of Gpp(NH)p activation. Sanders et al. (44) reported a factor in the 10,OOOg supernatant fractions of rat heart homogenates that in-



A t


5 E ‘0 a P


9 6



bit preparations, using [“HJdihydroalpren0101 (see Materials and Methods). Specific binding in the crude homogenate amounted to 0.07 pmol mg-‘; nonspecific binding amounted to 36% of the total bound radioactivity. In the washed-particle preparation, specific binding was 0.10 pmol mg-‘; the yield based on the crude homogenate was 45%. Specific binding in the solubilized detergent-free preparation was 0.04 pmol binding in this fraction mg-‘; nonspecific amounted to 40% of the total bound radioactivity (values are means of three experiments with different preparations), Dihydroalprenolol binding in the 100,OOOg supernatant fraction could not be measured because the detergent interfered with development of the Sephadex columns, the blanks becoming unreliably high. I>ISCUSSION

FIG. 10. Lack of effect of phospholipids on adenylate cyclase. (A) Fresh whole homogenate (390 pg of protein; duplicate tubes) was assayed in the absence (0) and presence (0) of phosphatidylinositol, 74 pg/ml in the assay, with varying epinephrine concentrations. Incubation was for 10 min at 26°C. The values are means of two separate experiments. (B) Phospholipids dispersed in buffer by sonication (30 s) were added to solubilized detergent-free enzyme (20 pg of protein). All assay components [epinephrine (EPI), glucagon (GLUC), and histamine (HIST)] were then added, and the reaction was started by the addition of ATP; reaction was for 10 min at 26°C. The data are representative of four experiments with different enzyme preparations.

creased epinephrine stimulation in a lOO,OOOgpellet fraction. We have confirmed the presence of this factor in 100,OOOg supernatant fractions prepared from fresh homogenates of rabbit heart. The factor increased epinephrine stimulation in a washed-particle fraction in a concentrationdependent manner over the range of 40 to 200 pg of protein. However, when added to the solubilized detergent-free preparation, the factor was without effect and did not produce sensitivity to epinephrine, in either the presence or the absence of phosphatidylinositol or other phospholipids. Thus we were entirely unable to restore catecholamine sensitivity of the solubilized enzyme, whether detergent was present or not. We examined catecholamine binding in the rab-

Studies with the solubilized enzyme were greatly complicated by the strong inhibitory effects of Lubrol PX. Assays could not be conducted above 26°C because of the extreme rapidity with which the enzyme was inactivated when detergent carried into the assay amounted to 0.1%. Inactivation appeared to result from thermal denaturation, since enzyme incubated in the presence of 0.1% detergent could not be activated by either F- or Gpp(NH)p. The effect of nonionic detergents on the enzyme from other sources has not been systematically examined, except in the study by Johnson and Sutherland (5). They found that basal activity in rat cerebellum and cerebrum was markedly increased by Lubrol PX at concentrations between 0.01 and 0.3% in the assay (conducted at 37’C); F- seemed to decrease Lubrol stimulation. Middlemiss and Franklin (7) also observed stimulation of the brain enzyme by this detergent, and in separate studies we have confirmed this. Perkins and Moore (4) noted stimulation of the brain enzyme by Triton X-100; both Lubrol PX and Triton X-100 increased basal activity of the testis enzyme (13). It can be inferred from other studies that Lubrol decreased enzyme activity in liver (9, ll), frog erythrocytes (45), and turkey erythrocytes (46). Digitonin has also been reported to inactivate the enzyme from



liver plasma membranes (47, 48). Fortunately, in our studies Lubrol PX did not decrease enzyme activity when the assay was conducted below 26°C. Recovery of the activity in peak III from DEAE-Bio-Gel A chromatography was only 25% of that applied; recovery of the Gpp(NH)p-activated enzyme was substantially higher (66%) in peak III. In view of the persistence of Gpp(NH)p effects, purification of the activated enzyme may be preferred. In the case of both th.e nonactivated and the Gpp(NH)p-activated preparations, a second peak of a.ctivity was eluted by further development of the column with 100 mM NaCl containing 0.2% Lubrol (peak IV). Properties of the enzyme in this fraction seemed very similar to those of the enzyme in peak III, except that it was activated much more rapidly by Gpp(NH)p. We do not know whether it constitutes a separate enzyme species. Properties of the solubilized detergentfree enzyme were similar in several respects to those of the native membrane-bound activity. The K, for substrate (MgATP) was 0.1 mM; the V was increased by Mg’+, the apparent K, for this cation being 2.5 IIIM; and activity was inhibited by ATP in excess of Mg’+. These properties are quite analogous to those of the membrane-bound cardiac enzyme (49). The enzyme was stimulated by F- and Gpp(NH)p in a time- and temperature-dependent manner; both ligands not only activated but also stabilized the enzyme, and Gpp(NH)p activation was persistent. ‘Thus the activated enzyme could be dialyzed and chromatographed without reversal of the activated state. This pattern of a.ctivation is similar to that of the membrane-bound enzyme from a variety of sources (29-31, 33), including rabbit heart (43) as well as the solubilized canine heart enz:yme Activation by (33). Gpp(NH)p did not alter the Km for substrate, which was similar to that of the membrane-bound enzyme from rabbit heart (43), frog erythrocytes, and the solubilized canine heart enzyme (33). Activation by both. F- and Gpp(NH)p reduced the apparent K,, for Mg” in a manner analogous to the enzyme in the washed-particle fraction. It has been reported that




Gpp(NH)p activation of the membranebound (34) and solubilized (33) canine heart enzyme did not decrease the Mg2+ required for half-maximal activity. Previous studies indicate that Gpp(NH)p activates as an unchelated species (16, 17, 30, 31, 32, 36, 41). We found that in the solubilized detergent-free enzyme, Mg2+ significantly increased maximal activation by the nucleotide, although there was not an absolute requirement for cation. The particulate enzyme behaved similarly. Alvarez and Bruno (50) have made similar observations with the particulate guinea pig heart enzyme. The solubilized detergent-free enzyme possessed two classes of binding sites for Gpp(NH)p, and the 100,OOOg supernatant fraction actually contained three. Recently, Narayanan and Sulakhe have reported three classes of binding sites in guinea pig heart plasma membranes (51) with KD values reasonably close to those we have found. Possibly, the site with a KD value of 0.11 pM is associated with activation of adenylate cyclase, since the apparent K, for activation was 0.1 pM. In agreement with the results of the above-mentioned authors, binding did not require Mg’+; in fact, the cation strongly reduced binding. In the guinea pig heart preparation (51), Mg”+ at concentrations as low as 0.05 mM obliterated binding at the highest-affinity site. The presence of multiple binding sites and the fact that binding sites were eluted from the chromatographic column which contained no adenylate cyclase suggest that a significant portion of Gpp(NH)p bound is not associated with activation of the enzyme. Welton et al. (52) reported that only 1% of the Gpp(NH)p binding sites in liver plasma membranes is associated with activation of adenylate cyclase. The solubilized enzyme, in both the 100,OOOg supernatant fraction and the detergent-free preparation, was completely unresponsive to catecholamines in the absence or presence of Gpp(NH)p. Under a variety of conditions, the addition of phospholipids did not restore catecholamine sensitivity. Our findings thus differ from those of Levey, who found that catecholamine sensitivity of the solubilized cat heart enzyme was restored by phosphatidylino-




sitol (18, 19) and glucagon and histamine sensitivity by phosphatidylserine (19, 20). Levey also found that phosphatidylinositol increased the sensitivity of the particulate cat heart enzyme to norepinephrine (19). This phospholipid increased basal activity slightly in a crude homogenate of rabbit heart, but did not increase sensitivity to epinephrine (Fig. 10A). The detergent-free enzyme bound [“Hldihydroalprenolol, indicating the presence of catecholamine binding sites. Thus, it seems that such sites were dissociated from adenylate cyclase and that phospholipids did not bring about association.

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Properties of detergent-dispersed myocardial adenylate cyclase.

OF BIOCHEMISTKY AND BIOPHYSICS Vol. 189, No. 1, July, pp. 63-75, 1978 ARCHIVES Properties of Detergent-Dispersed GEORGE Biochemistry Group, Myoc...
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