Adenylate Cyclase in Skeletal Muscle

Adenylate cyclase was studied in plasma membranes prepared from rabbit skeletal muscle. Such preparations represented an increase in specific activity of loto ZO-fold over the whole homogenate with a yield of activity of approximately 30%. Various parameters, such as phase contrast microscopy, “marker” enzyme activities, and chemical composition, suggested that the preparation constituted sarcolemma in a high degree of purity. The K, for substrate (MgATP) was 0.3 to 0.5 II~M. The K, for Mg2+ was 3 to 5 mm ‘Binding of Mg2+ to a second site resulted in increased reactivity of the catalytic site for substrate. Stimulation by fluoride resulted from an increase in maximal velocity; the K, for Mg2+ and the Km for substrate were not appreciably altered. The effect of Fwas markedly temperature-sensitive and partially irreversible. Fluoride-stimulated activity was particularly sensitive to inhibition by pyrophosphate and this inhibition was competitive with respect to ATP (Kit 0.45 m). Catecholamines stimulated the enzyme in a typical fl adrenergic fashion. The prominent kinetic effect of epinephrine was (like F-) to increase reaction velocity without affecting atlinity for Mg2+ or ATP. The regulation of adenylate cyclase in skeletal muscle may be classified as a “V” allosteric system since metal ions, F-, and epinephrine all result in increased maximal velocity of the enzyme reaction.

Much evidence exists that adenylate cyclase is located primarily, but not exclusively, in plasma membranes of cells of most tissues. The enzyme has not been extensively studied in skeletal muscle although it was shown to be present in this tissue some years ago (1). The formation of cyclic adenosine 3',5'monophosphate by a microsomal fraction of rabbit skeletal muscle was reported by Seraydarian binowitz et al. (3) have reported that the enzyme present in ho mogenates of this tissue was distributed largely in mitochondrial and microsomal fractions.
Methods have been available for some time for the isolation of skeletal muscle sarcolemma (4, 5), but these preparations have not been examined for adenylate cyclase activity.
In previous studies (6,7) we have examined the nature of fluoride and catecholamine stimulation of the enzyme in particulate preparations of cardiac tissue. The present communication describes the isolation of plasma membrane preparations from rabbit skeletal muscle containing a good yield of adenylate cyclase. Kinetic properties of the enzyme are examined especially with respect to fluoride and neurohormonal stimulation. Solutions of labeled ATP were diluted with unlabeled nucleotide to the desired specific activity and with water to the desired concentrations.
Prostaglandins E, Ez, F,,, Fear, and AZ were supplied through the courtesy of Dr. John Pike of The Upjohn Company.

Methods
Isolation of Plasma Membranes-Plasma membranes were isolated from rabbit skeletal muscle by a modification of the method of Kono and Colowick (4). All procedures were carried out at 4". Fresh leg muscle from rabbits in 5-g portions was Skeletal Muscle Adenylate Cyclase Vol. 247, Xo. 9 minced with scissors and homogenized for 15 s in 5 volumes of 50 mM CaClz in a Sorvall Omnimixer at maximal velocity.
The homogenate was passed through a coarse nylon sieve (pore size 1 mm2) and centrifuged for 10 min at 2,000 x g. The precipitate was washed twice by suspending to the original volume in 10 mM Tris-HCI, pH 8, and centrifuging at 2,000 x g for 10 min.
Washing effectively removed soluble protein and superficially bound calcium. Preparations of washed particles were suspended in 5 volumes of 10 mM Tris-HCl, pH 8 (based on initial tissue weight), and lithium bromide (4 M solution) was added to produce a final concentration of 0.4 M. The solution was stirred magnetically for 4 to 5 hours. The viscous suspension was diluted with 100 ml of 10 KIM Tris-HCl, pH 8, and centtifuged for 10 min at 2,000 X g. Dilution in this manner was necessary to effect sedimentation of the partially extracted membranes. The sediment was again washed by suspending in 10 mM Tris-HCl, pH 8, and centrifuging at 2,000 x g for 10 min. The lithium bromide-extracted particles were suspended in 4 volumes of 25% (w/w) potassium bromide (based on initial tissue weight) and centrifuged at 25,000 x g for 30 min. Under these conditions any unextracted muscle fibers remained in the supernatant. The pellet was washed by suspending in 10 mM Tris-HCl, pH 8, and centrifuging at 2,000 x g for 10 min. The resulting pellet was carefully suspended in 1 ml of 10 mM Tris-HCl, pH 8, per g of initial tissue weight.
For those experiments which involved precise determination of adenylate cyclase in crude homogenates, the tissue was homogenized in 5 volumes of 10 mM Tris-HCl, pH 8, rather than 50 mM CaC12. Washed particles were then prepared as described above. It was necessary to avoid the presence of Ca2+ in such preparations because this cation is a strong inhibitqr of adenylate cyclase.
Adenylate Cyclase Assay---The assay for adenylate cyclase was essentially that previously described (6) except that the pH was 8.5 and theophylline was omitted. In addition, 1 mM EGTA was present in the assay of crude homogenates and washed particle preparations.
Assays were carried out at 37" for 20 min and were terminated by placing the tubes in a boiling water bath for 3 min. Cyclic [14C]AMP was isolated chromatographically and quantitatively determined by scintillation spectrometry (6). In experiments in which substrate concentrations greater than 0.3 mM were required, [ol-32P]ATP was used as substrate instead of [I%]ATP.
Following termination of the reaction by boiling, 50 ~1 of 0.25 M ZnS04 were added to each tube, followed by 50 ~1 of 0.25 M Ba(OH)%.
Precipitates were removed by centrifugation at 8,000 x g, and 150 ~1 of each clear supernatant were subjected to chromatography in the usual way (6). Bariumzinc precipitation removed more than 95% of the ATP, ADP, and 5'-AMP present; recovery of cyclic AMP ranged from 97 to 100% as determined by the addition of known amounts of the 8-'%-labeled compound to control tubes. Assay of Other Enzymes--The assay for Mgzf-dependent Na+, K+-stimulated ATPase contained 50 mM Tris-HCl, pH 7.5, 5 mM ATP, 5 mM MgC12, 100 mM NaCl, 20 mM KCl, and appropriate quantities of membrane protein (0.20 to 0.30 mg) in a total volume of 1 ml. The tubes were incubated for 10 min at 37". Ouabain when present was 0.4 mM.
The reaction was terminated by the addition of 1 ml of cold 127, trichloroacetic acid, and, following centrifugation, an aliquot (1.0 ml) of the supernatant was assayed for inorganic phosphate by the method of Taussky and Shorr (8). Mg+dependent ATPase was measured under similar conditions with the omission of NaCl and KCl. Caz+dependent ATPase was determined by incubating appropriate quantities (0.20 to 0.30 mg) of membrane protein with 50 mM Tris-HCl, pH 7.0, 5 mM ATP, and 5 mM CaClz in a final volume of 1 ml for 10 to 20 min at 37". Phosphate was determined as described above. 5'-Nucleotidase was assayed by incubat;ng muscle protein with 8 mM 5'-AMP, and 18 rnbt MgS04, in a final volume of 250 ~1 for 10 min at 37". Reactions were terminated by the addition of 1 ml of cold 3% trichloroacetic acid. After centrifugation, inorganic phosphate was determined in the supernatant by the Fiske and SubbaRow method (9). Acid phosphatase was estimated by determining inorganic phosphate released (9) following the incubation of muscle protein with 25 mM flglycerol phosphate buffered at pH 5.0 with 50 mM acetate in a final volume of 200 ~1 for 20 min at 37". Pyrophosphatase u-as assayed according to the method of Nordlie and Arion (10) ; acid maltase by the method of Hers and van Hoof (11) ; cytochrome c oxidase as described by Cooperstein and Lazarow (12) and acetyl cholinesterase by the spectrophotometric method of Ellman et al. (13).
Cyclic 3', 5'.nucleotide phosphodiesterase was measured by incubating cyclic [3H]AMP (5 mM, 2 PCi per pmole) with 25 mM Tris-HCl, pH 7.5, 2.5 mM MgS04, and protein in a final volume of 200 ~1 for 20 min at 30". Reactions were terminated by immersing the tubes in a boiling water bath, and, following removal of denatured protein by centrifugation, 150 ~1 of the supernatant fluid were chromatographed, with 1 M ammonium acetate-95% ethanol (15:35) as the developing solvent (6). Areas of the chromatogram corresponding to 5'-AMP (identified by marker) were cut out and counted by scintillation spectrometry. Under the conditions of the assay there was no detectable conversion of 5'-AMP to adenosine. Cyclic 2', 3'-nucleotide phosphodiesterase was assayed by the method of Drummond et al. (14). Measurement of Calcium Binding-Membrane protein (0.20 to 0.30 mg per ml) was incubated in a medium (final volume 2 ml) containing 50 mM Tris-maleate, pH 6.0, 5 mM MgC12, 5 mM ATP, and 0.1 mM 4SCaC12 (4000 to 6000 cpm per nmole) at 37". Reactions were terminated by filtering the mixtures through Millipore filters (0.45 ~1, 25 mm). The filters were carefully washed with 5 ml of the above buffer, dried, and radioactivity was determined by scintillation spectrometry. Appropriate controls were run containing no ATP, no MgC12, or no protein.
In the absence of membrane protein, retention of radioactivity on the filters was negligible.
Calcium binding was calculated from the specific activity of added 45CaC12 and the activity retained by the membrane protein.

Chemical
Assays-Cholesterol was determined by the method of Zak et al. (15). Lipid phosphorus was determined by the procedure described by Bartlett (16); phospholipid was estimated by multiplying the lipid phosphorus value by 25. Sialic acid was determined by the method of Warren (17) following hydrolysis of membrane protein by the procedure of Svennerholm (18). Fluoride was estimated with the Amadac-F reagent (19). Protein determinations were made by the method of Lowry et al. (20).  (6) to test the effectiveness of the phosphoenol pyruvate-pyruvate kinase regenerating system used. This solvent system separates ADP from ATP more effectively than the 1 K{ ammonium acetate-95% ethanol (15:35) solvent system (6). In the absence of the regenerating system, incubating standard amounts of membrane protein (75 pg) in the assay led to the destruction of up to 75% of the ATP, the radioactivity being distributed between ADP and 5'-AMP. The inclusion of 8 mM F-did not prevent the disappearance of ATP, but decreased the amount of 5'.AMP (with an increasing proportion of radioactivity in ,4DP). addition of the phosphoenol pyruvate-pyruvate kinasc regenerating system effectively maintained more than 90% of the ATP.

Preliminary-
Assay conditions under which the regenerating system may fail to conserve substrate have been discussed previously (6, 7). Recovery of cyclic AMP in the reaction mixtures was also examined.
In a mock assay, cyclic [WC]AMP (10,902 dpm) was added to the incubation mixture and the tubes were incubated with the omission of ATP, and with the addition of unlabeled cyclic AMP or theophylline to prevent destruction of the labeled material. Table I, Experiment 1, shows that when cyclic [%W]AMP was incubated in the presence of membmne protein (95 pug), at least 95% was destroyed.
Radioactivity on t,he chromatogram was recovered in the area corresponding to 5'-AMP (4,740 dpm) and adenosine (6,287 dpm), a reflection of the presence of both cyclic 3', 5'-nucleotide phosphodiesterase and 5'-nucleotidase.
Recovery of the labeled cyclic AMP was increased to greater than 95y0 by the addition of unlabeled cyclic nucleotide (0.5 mM and 2 mM) ( Table I). The addition of theophylline (1.3 mM and 13.4 mM) also prevented the destruction of the cyclic [8-W]AMP, but was less effective than unlabeled cyclic nucleotide.
Addition of unlabeled cyclic AMP to the reaction mixture greatly increased the synthesis of labeled cyclic nucleotide from [U-14C]ATP (Table I, Experiment II). Addition In a separate experiment, it xas shown that increasing amounts of theophylline in the presence of 2 mM cyclic AMP, indeed, led to a concentrationdependent decrease in adenylate cyclase activity; 50% inhibition was produced with 20 mM theophylline.
Inhibition of adenylate cyclase activity by theophylline has also been observed in the toad bladder (21) and rat erythrocyte (22). In t.he standard assay, therefore, we have employed unlabeled cyclic nucleotide as the sole means of preserving the labeled product.
Under conditions finally adopted (theophylline absent, 2 mM cyclic AMP present), adenylate cyclase activity was proportional to time and to protein content when measured in the presence or absence of 8 mM F- (Fig. I). The pH dependence was examined from pH 6.0 to 10.5 with the use of combinations of P-glycerol phosphate, Tris-HCl, and 2-amino-2-mcthyl-l , 3-propancdiol (each at 40 mM) as buffers. The pH optimum both in the presence and absence of F-was 8.5, somewhat higher than that previously found for the cardiac enzyme (6).

Adenylate
Cyclase in Skeletal Muscle of Several Organisms-Enzyme activity in whole homogenates of skeletal muscle from several species is shown in Table II. Activity in back leg muscle of rabbit and guinea pig was greater than in rat leg muscle, particularly in the presence of F-.
Mammalian skeletal muscles were more act,ive than frog gastrocnemius.
Pigeon breast muscle was much more active than chicken breast muscle. The low activity in frog muscle when compared with mammalian tissues and the differences between breast muscle from chicken and pigeon suggest that adenylate cyclase may be richer in red fibers adapted for oxtdative metabolism than in white muscle fibers. Because of ready availability and high activity, rabbit hind leg muscle was selected for all subsequent studies.

Sedimentation of Adenylate Cyclase and Preparation
of Plasma Membranes-When homogenates were prepared with a blade homogenizer, it was found that 807; of the enzyme activity was present in the particulate fraction prepared by sedimenting at 2,000 x g and washing with dilute buffer ( Most of the activity remaining in the 2,000 x g supernatant could be sedimented at 37,000 x g. Such high yields of activity in particles sedimenting at low gravitational forces suggested that adenylate cyclase might reside in the plasma membrane. Accordingly, attempts were made to isolate sarcolemma from this tissue. Procedures based on the method of McCollester (5) resulted in large losses of activity. Modification of the procedure of Kono and Colowick (4) (see under "Methods"), however, yielded a plasma membrane fraction with a high yield of adenylate cyclase activity (Table III, Fraction B). Extraction of the washed particles with LiBr resulted in an increase in specific activity of about 2-fold with a 78vc yield of activity.
Centrifugation in KBr (density 1.21) produced a membrane pellet with a yield of 37yc of the total activity and an increase in specific ac- Portions of hind leg muscle from rat, guinea pig, and rabbit, the gastrocnemius from frog, and breast muscle from pigeon and chicken were homogenized in 5 volumes of 10 mM Tris-HCl, pH 8.0, for 10 s in a Sorvall Omnimixer.
The homogenates were strained through a coarse nylon sieve (pore size 1 mm2  Rabbit leg muscle (5 g) was fractionated as described under "Methods." In Fraction A, the tissue was homogenized in 10 mM Tris-HCl, pH 8, rather than in 50 rnl\l CaClz as in Fraction B.
This was necessary in order to obtain a reliable value for activity in the crude homogenate since Ca*+ inhibits the enzyme. Standard assay conditions were used; the substrate was 0.3 mM [U-14C]-ATP and the assay mixture contained 1 mM EGTA and 8 mM F-. tivity of adenylate cyclase of about 1Bfold. Purification of activity measured in the absence of F-paralleled that measured in the presence of this anion (8 mM). The enzyme present in membranes was highly unstable. Storage of membrane preparations for 18 hours at -4' or even at -80" led to 50cs loss of activity. From 50 to 80% of the activity survived lyophilization of membrane preparations; lyophilized powders, however, lost 5O(r, of their activity during storage at -18" for 1 week. Because of this, all experiments were performed with freshly prepared membranes. Membrane Properties--A number of criteria were esamined to ascertain the relative purity of the plasma membrane preparation. At each step of the procedure, fractions were routinely examined by phase contrast microscopy. Photomicrographs of a washed particle and final membrane preparation are shown in Fig. 2. Cross striations are clearly visible in washed particle preparations (Plate A), whereas the membranes (Plate B) contain no cross striations and appear as empty, transparent, sac like structures analogous to those described by Kono and Colowick (4) and McCollester (5).
The membrane preparation contained a Mg2+-dependent ATPase activity (Table IV, Addition A) which was further stimulated by the addition of 100 mu Na+ and 20 rnivr Kf (Table IV, Addition B). This Na+, Ktstimulated ?\Ig?+-1TPase was partially inhibited by the addition of 0.4 rnnf ouabain. The specific activity of this enzyme was greater than that observed by Peter (23) for rat sarcolemma, but less than that present in hamster skeletal muscle sarcolemma (24). The membrane preparation also possessed Ca&-stimulated ATPase activity which did not require Mg* (  (Table V) ; binding was enhanced by the addition of 2 mat phosphate. 5'-Nucleotidase is commonly used as a plasma membrane marker, particularly in liver.
Most of this activity in rabbit skeletal muscle was soluble and activity could not be detected in the membrane preparation by the assay used (see under "Methods").
However, this activity was present as evidenced by the appearance of adenosine on paper chromatograms of the adenylate cyclase assay. The activity of acetyl cholinesterase in the membrane fraction was 37 nmoles of acetylcholine hydrolyzed per min per mg, an increase in specific activity of 6-fold from the whole homogenate with a yield of about 10%. Similar activities from rabbit muscle sarcolemma have been reported by Ferdman et al. (25). Cytochrome c oxidase, acid maltase, acid phosphatase, and pyrophosphatase could not be detected in the membrane fraction, suggesting minimal contamination by mitochondria and lysosomes. The activity of cyclic 3', 5'-nucleotide phosphodiesterase was determined to be 0.3 nmole per min per mg of protein in a yield of 1 to 3% of the activity of the whole homogenate. Cyclic 2', 3'-nucleotide phosphodiesterase, a membrane-bound enzyme f6und predominantly in nerve tissue (14), was present in the membrane fractions in activities of 1.8 nmoles per min per mg (cyclic adenosine 2',3'-monophosphate used as substrate). It has been suggested (26) that a property of plasma membranes from various sources is a high molar ratio of cholesterol to phospholipid.
The analysis of three separate membrane preparations for cholesterol, phospholipid, and sialic acid is shown in  (7), metal ions stimulated skeletal muscle adenylate cyclase when present at concentrations in excess of ATP. The effect of Mg2+, Mn2+, and Co2f in the presence of 0.3 rnN ATP is shown in Fig. 3   from most mammalian sources. The skeletal muscle membrane enzyme was stimulated lo-to 20-fold by this anion.
The effect of F-on the enzyme at two temperatures (27' and 37") is shown in Fig. 5A. Maximal activity Kas achieved at 12 mM; halfmaximal stimulation occurred at 4 mM. The effect of temperature from 12-43" on basal and F--stimulated enzyme activity is presented in Fig. 5B in the form of an hrrhenius plot. There is a linear relationship between activity and t'emperature, except at 43", where presumably inactivation occurs. Energies of activation calculated from the slopes are 7.8 kcal per mole for basal activity and 17.4 kcal per mole for F--stimulated activity. Fluoride stimulation of adenylate cyclase from brain (28), parotid gland (29), and adrenal (30, 31) has been shown to be irreversible.
To examine t'his possibility in skeletal muscle, membrane preparations were incubated with Mg2+, with I?-, and with Mg2+ plus F-, for 30 min at 4". Following dialysis, samples were assayed in the presence of 9 mM Mg2+ (Table VII).
Dialysis of control preparations (previously incubated with no additions) showed that the enzyme did not lose activity during this procedure.
Preparations previously incubated with Mg2+, followed by dialysis, were totally inactive unless this cation was present in the assay, indicating the complete reversibility of Mg2+ stimulation.
Membrane samples that had been previously incubated with F-, or F-plus Mg zf, showed an &fold stimulation over basal activity when assayed after dialysis in the absence of Ii".
This corresponded to about 40%, of the F--stimulated activity of the dialyzed control.
Analysis for F-by the Amadac-F reagent (which can detect as little as 5 to 10 /lg of I?) indicated F-had been completely removed by dialysis.
Identical results were obtained when the prior incubations were carried out for 10 min at 25". The results suggest that F-stimulation of the skeletal muscle enzyme is at least partially irreversible. Ephedrine (0.1 mM) was inactive. Stimulation due to epinephrine was blocked by propranolol (0.1 mM); propranolol had no effect on basal activity.
Preliminary incubation of the enzyme in reaction mixtures lacking only substrate for various times (10 to 30 min) with insulin in concentrations varying from 1 milliunit per ml to 10 units per ml did not alter either basal or epinephrine-stimulated activity. Nature of Neurohormone and F-Stimulation-In our studies with the cardiac enzyme (6, 7), it was shown that the prominent action of F-and epinephrine was to increase Ti,,,, of the Mgz+bound enzyme. The action of F-(4 and 12 mM) and epinephrine (0.1 mM) on Mgz+ saturation of the skeletal muscle enzyme is shown in Fig. 7. Both F- (Fig. 7A) and epinephrine (B) increased reaction velocity at all MgZf concentrations.
The predominant kinetic effect was to increase V,,,; there was no appreciable effect on the K, for Mg2+. The effect of F-(12 mM) and epinephrine (0.01 mM) on BTP saturation in the presence of two fixed Mg2+ concentrations (2.0 and 9.0 mM) is seen in Fig. 8. Under these conditions, both F-(a) and epinephrine (B) stimulated the enzyme at all ATP concentrations.
As previously observed (Fig. 4)) increasing ATP concentrations were inhibitory. This inhibition was not reversed by F-or epinephrine; it was reversed by increasing the XI@+ concentration to 9 mM. The action of both I? and epinephrine was to increase V,,,. The affinity of the enzyme for ATP was not altered substantially by these agents. This is most clearly seen with 9 mM Mg2+ where The substrate was [oI-~~P]ATP, and 250 rg of membrane protein were used. A, effect of ATP concentration on enzyme velocity with two fixed Mgz+ concentrations (Mgz+ concentration, mM, indicated by the numbers on each curve) in the absence (-) and presence (---) of 12 mM I?. B, same as A, except that the assay was performed in the absence (--) and presence (---) of epinephrine, 0.01 mM.
ATP was not inhibitory.
We conclude from these experiments that the kinetic nature of F-and epinephrine stimulation of the skeletal muscle enzyme is essentially identical with that of the cardiac enzyme (6, 7).
Effect of Pyrophosphate-Pyrophosphate is a product of the adenylate cyclase reaction (33, 34). Addition of pyrophosphate at concentrations of 0.1 to 2 mM to the assay resulted in inhibition of basal, epinephrine-stimulated, and F--stimulated adenylate cyclase activity (Fig. 9). The F--stimulated activity was much more sensitive to pyrophosphate inhibition than basal or epinephrine-stimulated activities, and was observed at F-concentrations varying from 2 to 16 mM. Pyrophosphate (2 mM) has been shown to inhibit adenylate cyclase from Escherichia coli (35), and a differential response to this agent was observed for glucagon and F--stimulated adenylate cyclase in plasma membranes from liver ( has also found the enzyme present in particulate fractions sedimenting at 18,800 x g. The difference between these results and ours may arise from the longer periods of homogenization employed (3, 44) which could result in disruption of the sarco-lemma1 membrane, yielding particulate fragments sedimenting at higher gravitational forces. Our results indicate that a considerable portion of adenylate cyclase in skeletal muscle resides in the plasma membrane.
,4 number of methods exist for the preparation of skeletal muscle sarcolemma (4,5,23,24,45,46). The procedure we have employed is a modification of the method of Kono and Colowick (4) and yields plasma membranes with an excellent yield of adenylate cyclase in a relatively short time interval.
This is important because of the lability of the enzyme to storage even at 4". A number of criteria were employed to examine the purity of the membrane preparation.
Phase contrast microscopy revealed empty, transparent, sac like structures similar to those previously reported (4,5,45,46). The lipid composition was similar to that reported by Fiehn et al. (27), and the high molar ratio of cholesterol to phospholipid is characteristic of plasma membranes (26). The absence of cytochrome c oxidase, acid maltase, acid phosphatase, and pyrophosphatase activities indicates little or no contamination by mitochondria or lysosomes.
The presence of Mg2+ATPase, Na+, K+-stimulated Mgz+ATPase, and Ca2+*4TPase (Table IV) is also consis- phate (0.2 and 1.0 mM) on ATP concentration is shown in Fig. 10. From the double reciprocal plots, it is apparent that pyrophosphate inhibits competitively with respect to ATP.
A Ki of 0.45 m&x was calculated.
Membrane protein (290 rg) was assayed with increasing [olJ*P]ATP in the presence of fixed concentrations of pyrophosphate (concentration, mM, indicated by the numbers on each curve). Concentration of Mg2+ was 9 mM: of F-, 12 mM. tent with a sarcolemmal origin (23-25).
It should be noted that the Ca*+ATPase present did not require Mg2+ for its activity. Sarcolemma from cardiac muscle have been shown to possess a Caz+ATPase (47, 48) with properties similar to those we find for the skeletal muscle membranes.
The presence of Na+,K+stimulated Mg2+ATPase activity in frog skeletal muscle membranes has been used to distinguish plasma membrane fragments from sarcoplasmic reticulum (49). We2 have observed that skeletal muscle sarcoplasmic reticulum Mg2+ATPase can Issue of May 10, 1972 D. L. Severson, G. I. Drummond, and P. V. Sulalche 2957 be further stimulated either by Na+ or K+ (but not as Na+ + KQtimulated) and that such stimulation is insensitive to ouabain; the activity in the membrane preparations described here is inhibited by the glycoside (Table IV).
The skeletal muscle preparation possessed the ability to bind calcium ions in an ATP-dependent manner; binding was further enhanced by the addition of phosphate (Table V). Calcium has also been reported to bind to plasma membranes from bullfrog skeletal muscle (50). Microsomal contamination is not likely to account for our results since although heavy microsomes were present in the first 2000 x g residue, they were effectively removed by the series of washes and low speed centrifugation steps employed.
In addition, extraction of sarcoplasmic reticulum fragments with 0.4 M LiBr under identical conditions greatly reduced their ability to sequester calcium.
This has also been observed for cardiac sarcoplasmic reticulum (51). Several investigators have failed to observe energy-linked calcium binding to skeletal muscle membrane fractions.
This could possibly be due to lability of the calcium-binding mechanism, to high salt concentrations, and to long periods of extraction.
Recently we have observed2 that sarcolemmal membranes prepared by the method of McCollester (5) bound very small amounts of calcium, whereas those prepared by a modification of the method of Rosenthal,Edelman,and Schwartz (45) bound this cation in amounts comparable to our preparation.
Perhaps this is a general property of cell membranes.
All of the criteria cited above lead us to conclude that the membrane fraction consists of plasma membranes in a high degree of purity.
Because of the excellent yield of adenylate cyclase and the ease and speed of isolation, we feel this preparation offers considerable promise for examination of hormonal binding and for further purification and especially solubilization studies. The kinetic properties of adenylate cyclase in skeletal muscle are qualitatively similar to those previously reported for the myocardial enzyme (6, 7). The K, for ATP was about 0.3 mM; increasing concentrations of ATP were inhibitory. ATP inhibition was reversed by Mg2+, making it likely that ATP inhibition results from a competition between enzyme and nucleotide for free metal ions as has been proposed for the cardiac enzyme (7). Magnesium seems able to bind to the enzyme in addition to that involved at the catalytic site since enzyme activity was increased by Mg2+ concentrations greatly in excess of ATP (Figs. 3 and 4). The K, for Mg2+ was 3 to 5 InM.
The consequence of Mg2+ binding to an apparent second site was enhanced reactivity of the catalytic site for substrate.
Fluoride profoundly stimulated skeletal muscle adenylate cyclase. Stimulation occurred at all Mg2f concentrations (Fig. 4A). The K, for Mg2+ was not appreciably altered by the anion, nor was the affinity for substrate.
Temperature had a much greater effect on F-stimulated than on basal activity. This was reflected in an increase in energy of activation in the presence of F- (Fig. 5B). The precise mechanism by which I? stimulates adenylate cyclase will have to be elucidated to explain this effect; conformational changes or changes in the rate-limiting event in the catalytic reaction may be relevant.
Pyrophosphate caused much greater inhibition of F--stimulated activity than either basal or epinephrine-stimulated activity. This may also be related to the mechanism of F-action. Inhibition by pyrophosphate in the presence of F-was competitive with respect to ATP (Ki, 0.45 mrvr), suggesting that the release of products in the adenylate cyclase reaction is ordered, with cyclic AMP being released from the enzyme before pyrophosphate. Adenylate cyclase in skeletal muscle is stimulated by catecholamines, and the order of potency was the same as that observed for stimulation of glycogenolysis in rat diaphragm (55). Inhibition of epinephrine stimulation with propranolol is consistent with an action on @ adrenergic receptors.
Insulin had no effect on either basal or epinephrine-stimulated adenylate cyclase activity.
These results are in accord with the findings of Craig et al. (56) that the activation of muscle glycogen synthetase by insulin was not associated with changes in tissue levels of cyclic AMP.
The kinetic nature of epinephrine stimulation was similar to that of F-.
Epinephrine increased reaction velocity at all Mg2+ and ATP concentrations (Figs. 7B and 8). The K, for Mg2+ and the K, for substrate were not appreciably altered; the primary action of epinephrine was to increase V,.,, i.e. to increase catalytic reactivity.
Increased reaction velocity at all ATP and Mg2+ concentrations has also been observed for glucagon and F-with liver plasma membranes (41) and for thyroidstimulating hormone and F-on thyroid plasma membranes (43). Adenylate cyclase in skeletal muscle may exist as a free enzyme or in a magnesium-bound form, both forms having equal affinity for substrate, with the magnesium-bound enzyme having greater catalytic reactivity.
Similar alterations in conformation may occur from interaction with F or epinephrine with resultant increased maximal velocity without alterations in the affinity for Mg2+ or for substrate.
Thus the regulation of adenylate cyclase in skeletal muscle seems in accord with that of a "V" allosteric enzyme, using the terminology of Monod, Wyman, and Changeux (57). The same mechanism for regulation has been applied to protein kinase from skeletal muscle (58).
Results obtained on hormonal regulation derived from in vitro studies of adenylate cyclase must be interpreted with caution. For example, responses to catecholamines require concentrations much higher than those which are effective physiologically and the skeletal muscle membrane preparation ( Fig. 6) is no exception.
In this regard, it is pertinent to refer to the findings of Stull and Mayer (59) in which the formation of phosphorylase a in rabbit gracilis muscle in response to low doses of isopropylnorepinephrine was dissociated from increases in cyclic AMP levels and the activation of phosphorylase b kinase. Higher doses of the catecholamine resulted in phosphorylase activation which was correlated with increased levels of cyclic AMP.
The examination of adenylate cyclase regulation in membrane preparations may, therefore, not provide final definitive answers concerning regulatory mechanisms in intact cells and tissues. The present studies also suggest that adenylate cyclase activity is higher in red skeletal muscle fibers than in white fibers. The former are equipped for oxidative metabolism and the oxidation of lipids.
In accord with this, it has been found that exerciseinduced lipolysis required normal hormonal mechanisms, whereas exercise-induced glycogenolysis was not influenced by adrenodemedullectomy or adrenergic blockade (60), i.e. glycogenolysis occurred in animals devoid of hormonal regulation.
Our understanding of the role of the adenylate cyclase system in regulating energy supply to tissues, although expanding rapidly, is not yet complete.