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Binding of phosphorylase a and b to skeletal muscle thin filament proteins
ARCHIVESOFBIOCHEMISTRYANDBIOPHYSICS Vol. 245, No. 2, March, pp. 404-410,1986
Binding of Phosphorylase a and b to Skeletal Muscle Thin Filament Proteins’ RAINER
MARQUETANT,
JOHN P. MANFREDI,
AND EDWARD
W. HOLMES’
Howard Hughes Medical Institute Laboratories and Departments of Medicine and Biochemistry, Duke University Medical Center, hrham, North Carolina 27710 Received May 15,1985, and in revised form November 6,1985
Phosphorylase plays an important role in energy generation during muscle contraction. We have demonstrated that purified rabbit skeletal muscle phosphorylase a and phosphorylase b bind to rabbit muscle F-actin, F-actin-tropomyosin, F-actin-tropomyosintroponin, and myofibrils. Neither phosphorylase a nor phosphorylase b binds to myosin. Phosphorylase a and b bind to F-actin with S,,s values of 1.5 X 10e6 and 2.1 X lop6 M, respectively. At saturation, 0.035 mol of phosphorylase a and b is bound for every seven G-actin monomers in the F-actin polymer. Using the F-actin-tropomyosin-troponin complex as opposed to F-actin as a binding target, there are five- and threefold increases in the maximal binding capacity for phosphorylase a and phosphorylase b, respectively, without a significant change in the S,, value for either form of the enzyme. A similar stoichiometry and affinity of phosphorylase binding are observed when myofibrils are used as the binding target. Ca2+ ions and AMP increase the maximal binding capacity for phosphorylase a to myofibrils while ATP decreases the B,,,. Our study suggests that in skeletal muscle, phosphorylase a and phosphorylase b may interact with the thin filament, and that this binding to thin filament proteins may be controlled by during changes in sarcoplasmic concentration of Cazf and ligands of phosphorylase muscle contraction. 0 I986 Academic press. I~~.
Interconvertible forms of phosphorylase (EC 2.4.1.1) have been isolated from skeletal muscle, as well as liver. Phosphorylase b, the less active form of the enzyme and the predominant species in resting muscle, is phosphorylated by phosphorylase kinase to phosphorylase a, the more active form of the enzyme and the predominant species found in stimulated muscle. Following activation of phosphorylase kinase and conversion of phosphorylase b to a, the activated enzyme removes a terminal glucose from glycogen and the glucose l-phosphate generated in this reaction enters the gly-
1Supported by ROl AM-12413 and F32 AM-07097 from the National Institutes of Health. ’ To whom correspondence should be addressed. 0003-9861/86 $3.00 Copyright All rigbta
Q 1986 by Academic Press. Inc. of reproduction in any form reserved.
colytic cascade leading to energy production in the myocyte. Danforth and Helmreich (1) showed that glycogenolysis increases almost immediately upon activation of muscle contraction. The increase in glycogenolysis and glycolysis in response to small, and in some cases unmeasurable, changes in myocyte content of ligands for the highly regulated enzymes in this cascade has led investigators to question whether some of the enzymes in these pathways are compartmentalized in the myocyte. Such compartmentalization could enable these enzymes to respond rapidly to small, local changes in ligand or substrate concentrations (2-4). In support of this hypothesis several enzymes of the glycolytic pathway, i.e., aldolase, pyruvate kinase, and lactate dehydrogenase, 404
INTERACTION
OF PHOSPHORYLASE
have been shown to bind to thin filament proteins (5-9). Phosphorylase binds to glycogen granules (2,10, ll), and these protein granules have been localized in direct proximity to the myofibril(2,12). Histochemical studies (10) indicate that phosphorylase is Iocalized in the same striated pattern as the glycolytic enzymes (5-9) which bind to thin filament proteins. These findings admit the possibility that phosphorylase may bind to thin filament or other myofibrillar proteins, as well as glycogen granules. Prior studies (6, 13) have not examined phosphorylase binding to contractile proteins in great detail, and the results of these studies are somewhat contradictory. Arnold et al. found (6) relatively little binding of phosphorylase to F-a&in in comparison to that observed for aldolase, pyruvate kinase, and lactate dehydrogenase. Another report described the copurification of phosphorylase with M-line proteins (13). However, the same investigators questioned the significance of phosphorylase binding to the M-line region since their experiments with phosphorylase antibodies localized this enzyme to the Z-line region in the sarcomere. Because several of the above studies indicate that phosphorylase is localized to distinct regions of the sarcomere in skeletal muscle, the present study was undertaken to determine through direct binding experiments whether phosphorylase a and b bind to myofibrillar proteins. Our results demonstrate that both forms of phosphorylase bind to native thin filament proteins, as well as myofibrils, but this enzyme does not bind appreciably to myosin. MATERIALS
AND
METHODS
Chemicals. Dithiothreitol was purchased from Bachem and EDTA was obtained from Mallinckrodt. Reagents for polyacrylamide gel electrophoresis and staining were purchased from Bio-Rad. All other chemicals were obtained from Sigma Chemical Compaw. Preparation of proteins. Actin was prepared as described by Drabikowski and Gergely (14) with the inclusion of an additional depolymerization-polymerization step. Tropomyosin, a gift of Dr. W. Longley,
WITH
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405
was prepared by the method of Bailey (15), tropomyosin-troponin (native tropomyosin) by the method of Eisenberg and Kielley (16), myosin by the method of Perry (l?), and myofibrils according to Perry and Corsi (18). Phosphorylase a and b were purchased from Sigma Chemical Company. The preparations were r95% pure as assessed by SDS-PAGE.3 Protein concentrations were determined spectrophotometrically from the absorbance at 280 nm using a 0.1%/280 = 1.1 for actin, 0.24 for tropomyosin, 0.38 for tropomyosin-troponin, 0.7 for myofibrils, 0.53 for myosin, and 1.3 for phosphorylase. To determine the molar concentration the following molecular weights were assumed: actin-42,000, tropomyosin-‘70,000, native tropomyosin-138,000, myosin-470,000, and phosphorylase monomer-97,400. The actin content of myofibrils was assumed to be 22% of the total protein (19). Actin-tropomyosin and a&in-native tropomyosin complexes were assembled by polymerizing actin in the presence of an excess of the regulatory proteins according to Ebashi and Ebashi (20). The mixtures were incubated at room temperature for 45 min in the presence of 120 mM KCl, 5 mM MgCl, and 0.1 mM ATP, pH 7.0. The reconstituted thin filaments were collected by centrifugation at 100,OOOgfor 1 h at 25°C using an airfuge (Beckman). The pellet was resuspended in binding buffer (see below), allowed to swell, and gently homogenized. Binding experiments. Phosphorylase a and b, F-actin, F-actin-tropomyosin, and F-actin-native tropomyosin were dialyzed against 1000 vol of 10 mM imidazole-HCI, 120 mM KU, 1 mM dithiothreitol, 1 mM MgCl, and 1 mM EGTA, pH 7.0 (binding buffer). Myofibrils were introduced into the binding buffer by three cycles of sedimentation and resuspension in the appropriate buffer. After dialysis, phosphorylase a and b were centrifuged in a Beckman airfuge at 100,OOOg for 10 min to remove any sedimentable material which may have formed during dialysis. Protein determinations were then made. All binding experiments with phosphorylase and thin filament proteins were conducted in the above buffer. NaF was included where indicated. To assess binding of phosphorylase to myosin a slightly different protocol was utilized. Myosin was solubilized in 10 mM imidazole-HCl, 500 mM KCI, 1 mM dithiothreitol, 1 mM MgC12, and 1 mM EDTA, pH 7.0, and just prior to mixing with phosphorylase it was diluted IO-fold with the same buffer containing no KC1 to achieve complete precipitation
a Abbreviations used: EGTA, ethylene glycol bis(Baminoethyl ether)-N,N,N’,N’-tetraacetic acid; SDSPAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Pi, inorganic phosphate; DTT, dithiothreitol.
406
MARQUETANT,
MANFREDI,
of the myosin. In the above experiments, increasing amounts of phosphorylase a and b (0.85-3.5 PM) were incubated with F-actin, F-actin-tropomyosin, F-actinnative tropomyosin, myofibrils, and myosin. The Factin concentration (10 NM) was kept constant in all experiments. The effect of Caz+ and ligands on binding of phosphorylase a and b was examined using myofibrils as the binding target. Only B-, or maximal binding capacity, was evaluated in these studies; a saturating concentration of phosphorylase a or b (3.5 PM) and a fixed concentration of myofibrils (2 mg/ml) were used in all studies. In experiments examining the effect of Ca’+ on phosphorylase binding to myofibrils, CaClz was added to give a final concentration of 100 *M in the binding buffer; EGTA was omitted. To examine the effect of various ligands, 10 mM ATP, 0.5 mM AMP, or 10 mM KPi was added to the binding buffer. All binding experiments were performed in a reaction volume of 100 ~1. After incubation for 10 min at 25”C, the reaction mixture was centrifuged at 100,OOOg(30 psi) in a Beckman airfuge for either 20 min (F-actin, F-actin-tropomyosin, F-actin-tropomyosin-troponin) or 1 min (myosin and myofibrils). Aliquots of the supernatants were mixed with 1 vol of SDS sample buffer, boiled for 2 min, and analyzed by SDS-PAGE. The pellets of F-a&in, F-actin-tropoand F-actin-tropomyosin-troponin plus myosin, phosphorylase were resuspended in 100 pl of binding buffer; in the case of myosin or myofibrils the pellets were resuspended in 100 pl of 0.1 M NaOH. Aliquots of the resuspended pellets were prepared as described above and analyzed on SDS-PAGE. Control experiments demonstrated that all of the F-actin, F-actin-tropomyosin, F-actin-tropomyosintroponin, myofibrils, and myosin were recovered in the pellets, while none of the phosphorylase sedimented in the absence of these contractile proteins under the experimental conditions described above. SDS-Polyacrylumide gel electropkoresis. Gel electrophoresis was performed on 10% polyacrylamide slab gels in the presence of SDS using the system described by Laemmli (21). The gels were stained with Coomassie brilliant blue. Densitometry. Densitometry analyses were performed using a soft laser scanning densitometer (BioRad Instruments). The zero gain signal was adjusted to a blank area on the gel, and the maximal signal gain was adjusted to peaks of interest. Control experiments with phosphorylase a and b, as well as actin, demonstrated a linear relationship between the amount of the proteins applied to the gel and the magnitude of the signal obtained in the densitometry tracings. All experiments were performed with concentrations of proteins in this linear range. Application of known amounts of actin and phosphorylase served as internal standards on each gel. These stan-
AND
HOLMES
dards were used to calculate the relative molar ratios of actin and phosphorylase in the supernatant and pellet samples obtained from the binding studies. The data represent the averages of three scans taken from each of the bands of interest in the gel. Calculation of binding umstanta Scatchard analyses indicated that plots of bound vs free/bound were nonlinear. Therefore, the data were analyzed by the logarithmic form of the Hill equation, where log b/(B,, - b) versus log S was plotted and s$, and 71’were determined graphically, (B,, = amount of phosphorylase bound at saturating concentrations, b = amount of phosphorylase bound at nonsaturating concentrations, and S = total phosphorylase concentration in the reaction mixture.) Statistical analyses. An analysis of variance was used. RESULTS
As indicated in Fig. 1 phosphorylase a and phosphorylase b exhibit saturable binding to F-actin. The affinity of phosphorylase a for F-actin is slightly greater (SO, = 1.5 PM) than the affinity of phosphorylase b for F-actin (S,, = 2.1 PM) (Table I). No difference in the maximal binding capacity for these two forms of phosphorylase was found. In contrast to F-actin, the major component of the thick filament, myosin, does not bind phosphorylase a or phosphorylase b (data not shown). The F-actin-tropomyosin complex prepared as described by Ebashi and Ebashi (20) contains 1 mol of tropomyosin per 7 mol of G-actin. The B,,, (mol of native phosphorylase per seven G-actin monomers) of the F-actin-tropomyosin complex is 0.62 for phosphorylase a, a value which is 1’7times greater than that observed with F-actin alone (Table I). The B,, for phosphorylase b binding to the F-actin-tropomyosin complex is also greater than that observed for F-actin alone (0.17 vs 0.035 mol of native phosphorylase per seven Gactin monomers), but the magnitude of the increase in B,, is not as great as observed for phosphorylase a (Fig. 1). The affinities of phosphorylase a and phosphorylase b for the F-actin-tropomyosin complex are not very different from those for F-actin alone (Table I). The reconstituted thin filament contains 1 mol of tropomyosin and 1 mol of the tro-
INTERACTION
OF PHOSPHORYLASE
IA
I
011 05
IO
15
20
TOTAL PHOSPHORYLASE
25
30
35
[/Al
FIG. 1. Binding of phosphorylase a (A) and phosphorylase b (B) to thin filament proteins. Increasing amounts of phosphorylase a and phosphorylase b (0.85-3.5 PM assuming native molecular weights of 390,000 and 195,000, respectively) were incubated (pH 7.0,25”C, 10 min) with F-actin-tropomyosin, F-actintropomyosin-troponin, myofibrils, and F-actin. The F-actin concentration (10 PM) was constant in all experiments, and this in turn dictated the amount of thin filament associated proteins or myofibrils added to the reaction mixture (see Materials and Methods). The amount of bound phosphorylase was quantitated by precipitation with subsequent gel electrophoresis as specified under Materials and Methods. The data shown represent the means of three experiments.
ponin complex per seven G-actin monomers. The preparation of reconstituted thin filaments used in the experiments reported here confers calcium regulability to actin activation of myosin ATPase (data not shown). Both forms of the enzyme bind to reconstituted thin filaments with similar affinities (L&, = 2.4 and 2.0 PM for phosphorylase a and phosphorylase b, respectively), values which are similar to those observed for binding of the two enzyme forms to the F-actin-tropomyosin complex (Table I). In contrast, the maximal binding
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capacity of the F-actin-tropomyosin-troponin complex for phosphorylase a is about one-fourth of the maximal binding capacity of the F-actin-tropomyosin complex (0.18 vs 0.62 mol of native phosphorylase per seven G-a&in monomers). The Bmaxfor phosphorylase b, however, is similar for the two complexes of thin filament protein (Table I). Both enzyme forms bind to myofibrils with affinities similar to those observed with thin filament proteins (SO.b= 2.0 and 2.4 PM for phosphorylase a and phosphorylase b, respectively). The maximal binding capacity of the myofibril is 0.18 for phosphorylase a and 0.10 for phosphorylase b (moles of native phosphorylase per seven G-a&in monomers). These values are similar to those obtained for the reconstituted thin filament, i.e., the F-actin-tropomyosin-troponin complex (Table I). In other experiments not shown, NaF was included at concentrations of 5, 10, and 20 mM to inhibit possible phosphatase activity (22) in the myofibrillar preparations, but this precaution did not influence the binding data obtained with either phosphorylase preparation. Since the B,,for phosphorylase a is reduced in the presence of the troponin complex and the conformation of thin filament proteins is changed consequent to Ca2+ binding, the influence of Ca2+ on B,,, for phosphorylase a and phosphorylase b binding to myofibrils was assessed. As shown in Fig. 2, the maximal binding capacity of myofibrils for phosphorylase a increased by 33% when 100 ~.LM Ca2’ was added to and EGTA omitted from the buffer used in the binding studies, from 0.18 f 0.008 to 0.24 f 0.015 mol of native phosphorylase per seven G-a&in monomers (P < 0.05). The B,,, for phosphorylase b on the other hand decreased slightly but significantly from 0.10 + 0.032 to 0.08 + 0.007 mol of native phosphorylase per seven Gactin monomers in buffer containing 100 PM Ca2+ (P < 0.05). Similar results were obtained when 200 PM Ca2+ was included in the binding buffer. Since AMP, ATP, and Pi are potential regulators of phosphorylase catalytic ac-
MARQUETANT,
MANFREDI, TABLE
AND
HOLMES
I
BINDING PARAMETERS FOR THE INTERACTION OF PHOSPHORYLASE a AND PHOSPHOR~LASE b WITH MYOFIBRILLAR PROTEINS Phosphorylase Contractile
protein
F-actin F-actin-tropomyosin F-actin-tropomyosintroponin Myofibrils
a
Phosphorylase
b
B mu
n
ST.6(PM)
B mu
sJ.6(PM)
1.5 z!z0.09 1.5 k 0.25
0.035 + 0.006 0.62 + 0.09
2.1 + 0.13 2.0 + 0.41
0.035 + 0.01 0.17 + 0.02
4 3
2.4 k 0.33 2.0 + 0.28
0.18 + 0.016 0.18 + 0.008
2.0 k 0.18 2.4 + 0.18
0.12 f 0.036 0.10 + 0.032
3 4
Note. Increasing amounts of phosphorylase a and phosphorylase b were incubated with thin filament proteins as described under Materials and Methods and in the legend of Fig. 1. Indicated are the amounts of phosphorylase bound at saturating concentrations (B,,,,). B,, data are expressed as moles of native phosphorylase bound per 7 mol of G-actin monomer. The dissociation constants (&J were analyzed by the logarithmic form of the Hill equation, assuming native molecular weights of 390,000 for phosphorylase and 195,000 for phosphorylase b.
tivity, the influence of these ligands on the maximal binding capacity of myofibrils for + HPHOSPHORYLASE B phosphorylase a and b was also evaluated. * p co.05 As demonstrated in Fig. 2,10 mM ATP de** p (0.01 creased the maximal binding capacity for phosphorylase a and phosphorylase b by approximately 25% (P < O.(N), whereas 0.5 mM AMP increased the B,, of phosphorylase a by 30% (P < 0.01) and phosphorylase b by 10%. Ten millimolar inorganic phosphate had no effect on B,,, for phosphorylase a or phosphorylase b binding to ATP AMP KPi myofibrils, demonstrating that these nuFIG. 2. Effect of Ca2+ and ligands on the binding of cleotide effects are not nonspecific effects phosphorylase a and phosphorylase b to myofibrils. of phosphate ions. Phosphorylase a or phosphorylase b at saturating
q
PHOSPHORYLASE
A
I
concentrations of 3.5 PM was incubated (pH 7.0,25”C, 10 min) with isolated myofibrils (2 mg/ml) in the presence of Ca’+ (100 pM, with the omission of EGTA) or in the presence of various ligands (ATP, 10 mbi; AMP, 0.5 mrd; KPi, 10 mM). The amount of bound phosphorylase was quantitated as described in the legend to Fig. 1 and under Materials and Methods. 100% phosphorylase bound (control) refers to the amount bound in the presence of 10 mb! imidazoleHCl, 126 mM KCI, 1 mM DTT, 1 mM MgCl,, 1 mM EGTA, pH 7.0. The effects of Car+, ATP, AMP, and Pi are presented as percentages of this control B,,,,. The data shown represent the means of four experiments (&SE). Statistical comparisons were done by an analysis of variance.
DISCUSSION
Prior studies have demonstrated that phosphorylase is bound to glycogen granules in skeletal muscle (2, 10, 11). In the present study evidence is presented which indicates that both phosphorylase a and b are also capable of binding to F-actin, Factin-tropomyosin, F-actin-tropomyosintroponin, and myofibrils. The binding to thin filament proteins is saturable and demonstrable at what are reported to be physiological salt and hydrogen ion concentrations. Although the affinity of phos-
INTERACTION
OF PHOSPHORYLASE
phorylase for thin filament proteins is not as great as that reported for binding of some other enzymes to contractile proteins, the affinity of this enzyme for thin filament proteins, as estimated from SO,values (1.5 to 2.4 PM), is substantially below the calculated concentration of phosphorylase in sarcoplasmic water, i.e., 9 to 20 j&M, depending on the state of aggregation of the enzyme. (Phosphorylase concentrations were estimated based on the reported value of 3.0 mg of phosphorylase per gram of skeletal muscle [lo].) Coupled with the histochemical studies which indicate that the distribution of phosphorylase follows the same striated pattern as a number of glycolytic enzymes which bind to thin filament proteins (23), the results of the present study suggest that this enzyme may also be bound to thin filament proteins in myocytes. This conclusion is not necessarily in disagreement with prior studies (2,10,11) demonstrating that phosphorylase binds to glycogen granules, since these granules are also concentrated in the region of the thin filament in the sarcomere. One might speculate that phosphorylase binds to both thin filament proteins and glycogen granules thereby serving to anchor these granules in the region of the myocyte where a number of glycolytic enzymes are concentrated. Maximal binding of phosphorylase to thin filament proteins is observed with the F-actin-tropomyosin complex, and at saturation approximately 1.2 mol of phosphorylase a is bound per 14 G-a&in monomers, a value similar to that obtained for aldolase (9). Addition of the troponin complex, either as reconstituted thin filaments or as native myofibrils, reduces the maximal number of binding sites, especially for phosphorylase a. This reduction in binding capacity in the presence of the troponin complex could be the result of simple steric inhibition or it may reflect a mechanism for controlling phosphorylase binding. Although we have not excluded steric effects, experiments with variable Ca2+concentration indicate that the maximal number of binding sites for phosphorylase on myofibrils can be modulated by changes in the concentration of this cation. The opposing
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409
effects of Ca2+on the B maxof the two forms of phosphorylase provides a potential mechanism for increasing binding of phosphorylase a, the more catalytically active form of the enzyme, and decreasing the binding of phosphorylase b, the less active form, during muscle contraction. Because of the influence of the troponin complex on the Bmaxfor phosphorylase, we assume that the effect of changing Ca2+concentrations is mediated through effects of this cation on the troponin complex. Another potential mechanism for regulating phosphorylase binding to thin filament proteins is suggested by the experiments with ATP and AMP. These ligands which control the distribution of the enzyme between the R and T conformations have opposing effects on the maximal binding capacity. ATP shifts the enzyme to the T conformation and decreases B,,, for both forms of the enzyme, while AMP shifts the enzyme to the R conformation and increases B,,,, providing a mechanism for increasing phosphorylase binding during intense muscle contraction through changes in energy charge in the myocyte. In summary, results of the present study demonstrate that phosphorylase binds to thin filament proteins at what are reported to be physiological ionic strengths and hydrogen ion concentrations, and the affinity of this binding is an order of magnitude less than the intracellular concentration of this protein. Data are presented which suggest that phosphorylase binding to thin filament proteins may be controlled through changes in conformation of the thin filament mediated by Ca2+ and/or changes in the conformation of phosphorylase mediated by the ligands ATP and AMP. ACKNOWLEDGMENTS The authors thank Dr. W. Longley of the Anatomy Department at Duke University for the supply of tropomyosin. We are grateful to Dr. R. H. Strasser for helpful suggestions in the preparation of the manuscript.We greatly appreciate the expert technical assistance of Jeana Meade. We thank Carolyn Mills for tireless secretarial help in preparation of this manuscript.
410
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MANFREDI,
REFERENCES 1. DANFORTH, W. H., AND HELMREICH, E. (1964) .I Bid Ch 239,3133-3138. 2. MEYER, F., HEILMEYER, L. M. G., JR., HASCHKE, R. H., AND FISCHER, E. H. (1970) J. Biol Chew. 245,6642-6648. 3. HASCHKE, R. H., HEILMEYER, L. M. G., JR., MEYER, F., AND FISCHER, E. H. (1970) J. Biol. Chem 245,6657-6663. 4. HEILMEYER, L. M. G., JR., MEYER, F., HASCHKE, R. H., AND FISCHER, E. H. (1970) J. Biol. Chew. 245,6649-6656. 5. ARNOLD, H., AND PETTE, D. (1968) Eur. J Biockem 6,163-171. 6. ARNOLD, H., HENNING, R., AND PEITE, D. (1971) Eur. J. Bioch~m. 22,121-126. 7. CLARKE, F. M., AND MASTERS, C. J. (1974) Biochim Biophys. Acta 139,785-733. 8. CLARKE, F. M., AND MASTERS, C. J. (1974) Biochim Biophys. Actu 358,193-207. 9. WALSH, T. P., WINZOR, D. J., CLARKE, F. M., MASTERS, C. J., AND MORTON, D. J. (1980) Biochem J. 186,89-g% 10. FISCHER, E. H., AND KREBS, E. G. (1958) J. Bill Chem 231,65-71.
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11. LELOIR, L. F., AND GOLDEMBERG, S. H. (1960) J. BioL Chem. 235,919-923. 12. ENTMAN, M. L., BORNET, E. P., VAN WINKLE, M. A., GOLDSTEIN, A., AND SCHWARTZ, A. (1977) J. Mol. Cell Cardiol 9,515-528. 13. TRINICK, J., AND LOWEY, S. (1977) J. Md Biol 113, 343-368. 14. DRABIKOWSKI, W., AND GERGELY, J. (1963) J. Biol Chem. 238,640-643. 15. BAILEY, K. (1948) Biochem .I 43,271. 16. EISENBERG, E., AND KIELLEY, W. W. (1974) .I Biol Chem. 249,4742. 17. PERRY, S. V. (1955) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 2, p. 582, Academic Press, New York. 18. PERRY, W. V., AND CORSI, A. (1958) Biochem J. 68,5. 19. YATES, L. D., AND GREASER, M. L. (1983) J. Mel BioL 168,123-141. 20. EBASHI, S., AND EBASHI, F. (1964) J. Biochem (Tokyo) 55,604. 21. LAEMMLI, U. K. (1970) Nature (London) 227,680685. 22. TALLANT, E. A., AND CHEUNG, W. Y. (1984) Arch B&hem Biophys. 232,269-279. 23. SIGEL, P., AND PEITE, D. (1969) J. H&o&em Cyto&em 17,225-237.
Binding of Phosphorylase a and b to Skeletal Muscle Thin Filament Proteins’ RAINER
MARQUETANT,
JOHN P. MANFREDI,
AND EDWARD
W. HOLMES’
Howard Hughes Medical Institute Laboratories and Departments of Medicine and Biochemistry, Duke University Medical Center, hrham, North Carolina 27710 Received May 15,1985, and in revised form November 6,1985
Phosphorylase plays an important role in energy generation during muscle contraction. We have demonstrated that purified rabbit skeletal muscle phosphorylase a and phosphorylase b bind to rabbit muscle F-actin, F-actin-tropomyosin, F-actin-tropomyosintroponin, and myofibrils. Neither phosphorylase a nor phosphorylase b binds to myosin. Phosphorylase a and b bind to F-actin with S,,s values of 1.5 X 10e6 and 2.1 X lop6 M, respectively. At saturation, 0.035 mol of phosphorylase a and b is bound for every seven G-actin monomers in the F-actin polymer. Using the F-actin-tropomyosin-troponin complex as opposed to F-actin as a binding target, there are five- and threefold increases in the maximal binding capacity for phosphorylase a and phosphorylase b, respectively, without a significant change in the S,, value for either form of the enzyme. A similar stoichiometry and affinity of phosphorylase binding are observed when myofibrils are used as the binding target. Ca2+ ions and AMP increase the maximal binding capacity for phosphorylase a to myofibrils while ATP decreases the B,,,. Our study suggests that in skeletal muscle, phosphorylase a and phosphorylase b may interact with the thin filament, and that this binding to thin filament proteins may be controlled by during changes in sarcoplasmic concentration of Cazf and ligands of phosphorylase muscle contraction. 0 I986 Academic press. I~~.
Interconvertible forms of phosphorylase (EC 2.4.1.1) have been isolated from skeletal muscle, as well as liver. Phosphorylase b, the less active form of the enzyme and the predominant species in resting muscle, is phosphorylated by phosphorylase kinase to phosphorylase a, the more active form of the enzyme and the predominant species found in stimulated muscle. Following activation of phosphorylase kinase and conversion of phosphorylase b to a, the activated enzyme removes a terminal glucose from glycogen and the glucose l-phosphate generated in this reaction enters the gly-
1Supported by ROl AM-12413 and F32 AM-07097 from the National Institutes of Health. ’ To whom correspondence should be addressed. 0003-9861/86 $3.00 Copyright All rigbta
Q 1986 by Academic Press. Inc. of reproduction in any form reserved.
colytic cascade leading to energy production in the myocyte. Danforth and Helmreich (1) showed that glycogenolysis increases almost immediately upon activation of muscle contraction. The increase in glycogenolysis and glycolysis in response to small, and in some cases unmeasurable, changes in myocyte content of ligands for the highly regulated enzymes in this cascade has led investigators to question whether some of the enzymes in these pathways are compartmentalized in the myocyte. Such compartmentalization could enable these enzymes to respond rapidly to small, local changes in ligand or substrate concentrations (2-4). In support of this hypothesis several enzymes of the glycolytic pathway, i.e., aldolase, pyruvate kinase, and lactate dehydrogenase, 404
INTERACTION
OF PHOSPHORYLASE
have been shown to bind to thin filament proteins (5-9). Phosphorylase binds to glycogen granules (2,10, ll), and these protein granules have been localized in direct proximity to the myofibril(2,12). Histochemical studies (10) indicate that phosphorylase is Iocalized in the same striated pattern as the glycolytic enzymes (5-9) which bind to thin filament proteins. These findings admit the possibility that phosphorylase may bind to thin filament or other myofibrillar proteins, as well as glycogen granules. Prior studies (6, 13) have not examined phosphorylase binding to contractile proteins in great detail, and the results of these studies are somewhat contradictory. Arnold et al. found (6) relatively little binding of phosphorylase to F-a&in in comparison to that observed for aldolase, pyruvate kinase, and lactate dehydrogenase. Another report described the copurification of phosphorylase with M-line proteins (13). However, the same investigators questioned the significance of phosphorylase binding to the M-line region since their experiments with phosphorylase antibodies localized this enzyme to the Z-line region in the sarcomere. Because several of the above studies indicate that phosphorylase is localized to distinct regions of the sarcomere in skeletal muscle, the present study was undertaken to determine through direct binding experiments whether phosphorylase a and b bind to myofibrillar proteins. Our results demonstrate that both forms of phosphorylase bind to native thin filament proteins, as well as myofibrils, but this enzyme does not bind appreciably to myosin. MATERIALS
AND
METHODS
Chemicals. Dithiothreitol was purchased from Bachem and EDTA was obtained from Mallinckrodt. Reagents for polyacrylamide gel electrophoresis and staining were purchased from Bio-Rad. All other chemicals were obtained from Sigma Chemical Compaw. Preparation of proteins. Actin was prepared as described by Drabikowski and Gergely (14) with the inclusion of an additional depolymerization-polymerization step. Tropomyosin, a gift of Dr. W. Longley,
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was prepared by the method of Bailey (15), tropomyosin-troponin (native tropomyosin) by the method of Eisenberg and Kielley (16), myosin by the method of Perry (l?), and myofibrils according to Perry and Corsi (18). Phosphorylase a and b were purchased from Sigma Chemical Company. The preparations were r95% pure as assessed by SDS-PAGE.3 Protein concentrations were determined spectrophotometrically from the absorbance at 280 nm using a 0.1%/280 = 1.1 for actin, 0.24 for tropomyosin, 0.38 for tropomyosin-troponin, 0.7 for myofibrils, 0.53 for myosin, and 1.3 for phosphorylase. To determine the molar concentration the following molecular weights were assumed: actin-42,000, tropomyosin-‘70,000, native tropomyosin-138,000, myosin-470,000, and phosphorylase monomer-97,400. The actin content of myofibrils was assumed to be 22% of the total protein (19). Actin-tropomyosin and a&in-native tropomyosin complexes were assembled by polymerizing actin in the presence of an excess of the regulatory proteins according to Ebashi and Ebashi (20). The mixtures were incubated at room temperature for 45 min in the presence of 120 mM KCl, 5 mM MgCl, and 0.1 mM ATP, pH 7.0. The reconstituted thin filaments were collected by centrifugation at 100,OOOgfor 1 h at 25°C using an airfuge (Beckman). The pellet was resuspended in binding buffer (see below), allowed to swell, and gently homogenized. Binding experiments. Phosphorylase a and b, F-actin, F-actin-tropomyosin, and F-actin-native tropomyosin were dialyzed against 1000 vol of 10 mM imidazole-HCI, 120 mM KU, 1 mM dithiothreitol, 1 mM MgCl, and 1 mM EGTA, pH 7.0 (binding buffer). Myofibrils were introduced into the binding buffer by three cycles of sedimentation and resuspension in the appropriate buffer. After dialysis, phosphorylase a and b were centrifuged in a Beckman airfuge at 100,OOOg for 10 min to remove any sedimentable material which may have formed during dialysis. Protein determinations were then made. All binding experiments with phosphorylase and thin filament proteins were conducted in the above buffer. NaF was included where indicated. To assess binding of phosphorylase to myosin a slightly different protocol was utilized. Myosin was solubilized in 10 mM imidazole-HCl, 500 mM KCI, 1 mM dithiothreitol, 1 mM MgC12, and 1 mM EDTA, pH 7.0, and just prior to mixing with phosphorylase it was diluted IO-fold with the same buffer containing no KC1 to achieve complete precipitation
a Abbreviations used: EGTA, ethylene glycol bis(Baminoethyl ether)-N,N,N’,N’-tetraacetic acid; SDSPAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Pi, inorganic phosphate; DTT, dithiothreitol.
406
MARQUETANT,
MANFREDI,
of the myosin. In the above experiments, increasing amounts of phosphorylase a and b (0.85-3.5 PM) were incubated with F-actin, F-actin-tropomyosin, F-actinnative tropomyosin, myofibrils, and myosin. The Factin concentration (10 NM) was kept constant in all experiments. The effect of Caz+ and ligands on binding of phosphorylase a and b was examined using myofibrils as the binding target. Only B-, or maximal binding capacity, was evaluated in these studies; a saturating concentration of phosphorylase a or b (3.5 PM) and a fixed concentration of myofibrils (2 mg/ml) were used in all studies. In experiments examining the effect of Ca’+ on phosphorylase binding to myofibrils, CaClz was added to give a final concentration of 100 *M in the binding buffer; EGTA was omitted. To examine the effect of various ligands, 10 mM ATP, 0.5 mM AMP, or 10 mM KPi was added to the binding buffer. All binding experiments were performed in a reaction volume of 100 ~1. After incubation for 10 min at 25”C, the reaction mixture was centrifuged at 100,OOOg(30 psi) in a Beckman airfuge for either 20 min (F-actin, F-actin-tropomyosin, F-actin-tropomyosin-troponin) or 1 min (myosin and myofibrils). Aliquots of the supernatants were mixed with 1 vol of SDS sample buffer, boiled for 2 min, and analyzed by SDS-PAGE. The pellets of F-a&in, F-actin-tropoand F-actin-tropomyosin-troponin plus myosin, phosphorylase were resuspended in 100 pl of binding buffer; in the case of myosin or myofibrils the pellets were resuspended in 100 pl of 0.1 M NaOH. Aliquots of the resuspended pellets were prepared as described above and analyzed on SDS-PAGE. Control experiments demonstrated that all of the F-actin, F-actin-tropomyosin, F-actin-tropomyosintroponin, myofibrils, and myosin were recovered in the pellets, while none of the phosphorylase sedimented in the absence of these contractile proteins under the experimental conditions described above. SDS-Polyacrylumide gel electropkoresis. Gel electrophoresis was performed on 10% polyacrylamide slab gels in the presence of SDS using the system described by Laemmli (21). The gels were stained with Coomassie brilliant blue. Densitometry. Densitometry analyses were performed using a soft laser scanning densitometer (BioRad Instruments). The zero gain signal was adjusted to a blank area on the gel, and the maximal signal gain was adjusted to peaks of interest. Control experiments with phosphorylase a and b, as well as actin, demonstrated a linear relationship between the amount of the proteins applied to the gel and the magnitude of the signal obtained in the densitometry tracings. All experiments were performed with concentrations of proteins in this linear range. Application of known amounts of actin and phosphorylase served as internal standards on each gel. These stan-
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dards were used to calculate the relative molar ratios of actin and phosphorylase in the supernatant and pellet samples obtained from the binding studies. The data represent the averages of three scans taken from each of the bands of interest in the gel. Calculation of binding umstanta Scatchard analyses indicated that plots of bound vs free/bound were nonlinear. Therefore, the data were analyzed by the logarithmic form of the Hill equation, where log b/(B,, - b) versus log S was plotted and s$, and 71’were determined graphically, (B,, = amount of phosphorylase bound at saturating concentrations, b = amount of phosphorylase bound at nonsaturating concentrations, and S = total phosphorylase concentration in the reaction mixture.) Statistical analyses. An analysis of variance was used. RESULTS
As indicated in Fig. 1 phosphorylase a and phosphorylase b exhibit saturable binding to F-actin. The affinity of phosphorylase a for F-actin is slightly greater (SO, = 1.5 PM) than the affinity of phosphorylase b for F-actin (S,, = 2.1 PM) (Table I). No difference in the maximal binding capacity for these two forms of phosphorylase was found. In contrast to F-actin, the major component of the thick filament, myosin, does not bind phosphorylase a or phosphorylase b (data not shown). The F-actin-tropomyosin complex prepared as described by Ebashi and Ebashi (20) contains 1 mol of tropomyosin per 7 mol of G-actin. The B,,, (mol of native phosphorylase per seven G-actin monomers) of the F-actin-tropomyosin complex is 0.62 for phosphorylase a, a value which is 1’7times greater than that observed with F-actin alone (Table I). The B,, for phosphorylase b binding to the F-actin-tropomyosin complex is also greater than that observed for F-actin alone (0.17 vs 0.035 mol of native phosphorylase per seven Gactin monomers), but the magnitude of the increase in B,, is not as great as observed for phosphorylase a (Fig. 1). The affinities of phosphorylase a and phosphorylase b for the F-actin-tropomyosin complex are not very different from those for F-actin alone (Table I). The reconstituted thin filament contains 1 mol of tropomyosin and 1 mol of the tro-
INTERACTION
OF PHOSPHORYLASE
IA
I
011 05
IO
15
20
TOTAL PHOSPHORYLASE
25
30
35
[/Al
FIG. 1. Binding of phosphorylase a (A) and phosphorylase b (B) to thin filament proteins. Increasing amounts of phosphorylase a and phosphorylase b (0.85-3.5 PM assuming native molecular weights of 390,000 and 195,000, respectively) were incubated (pH 7.0,25”C, 10 min) with F-actin-tropomyosin, F-actintropomyosin-troponin, myofibrils, and F-actin. The F-actin concentration (10 PM) was constant in all experiments, and this in turn dictated the amount of thin filament associated proteins or myofibrils added to the reaction mixture (see Materials and Methods). The amount of bound phosphorylase was quantitated by precipitation with subsequent gel electrophoresis as specified under Materials and Methods. The data shown represent the means of three experiments.
ponin complex per seven G-actin monomers. The preparation of reconstituted thin filaments used in the experiments reported here confers calcium regulability to actin activation of myosin ATPase (data not shown). Both forms of the enzyme bind to reconstituted thin filaments with similar affinities (L&, = 2.4 and 2.0 PM for phosphorylase a and phosphorylase b, respectively), values which are similar to those observed for binding of the two enzyme forms to the F-actin-tropomyosin complex (Table I). In contrast, the maximal binding
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407
capacity of the F-actin-tropomyosin-troponin complex for phosphorylase a is about one-fourth of the maximal binding capacity of the F-actin-tropomyosin complex (0.18 vs 0.62 mol of native phosphorylase per seven G-a&in monomers). The Bmaxfor phosphorylase b, however, is similar for the two complexes of thin filament protein (Table I). Both enzyme forms bind to myofibrils with affinities similar to those observed with thin filament proteins (SO.b= 2.0 and 2.4 PM for phosphorylase a and phosphorylase b, respectively). The maximal binding capacity of the myofibril is 0.18 for phosphorylase a and 0.10 for phosphorylase b (moles of native phosphorylase per seven G-a&in monomers). These values are similar to those obtained for the reconstituted thin filament, i.e., the F-actin-tropomyosin-troponin complex (Table I). In other experiments not shown, NaF was included at concentrations of 5, 10, and 20 mM to inhibit possible phosphatase activity (22) in the myofibrillar preparations, but this precaution did not influence the binding data obtained with either phosphorylase preparation. Since the B,,for phosphorylase a is reduced in the presence of the troponin complex and the conformation of thin filament proteins is changed consequent to Ca2+ binding, the influence of Ca2+ on B,,, for phosphorylase a and phosphorylase b binding to myofibrils was assessed. As shown in Fig. 2, the maximal binding capacity of myofibrils for phosphorylase a increased by 33% when 100 ~.LM Ca2’ was added to and EGTA omitted from the buffer used in the binding studies, from 0.18 f 0.008 to 0.24 f 0.015 mol of native phosphorylase per seven G-a&in monomers (P < 0.05). The B,,, for phosphorylase b on the other hand decreased slightly but significantly from 0.10 + 0.032 to 0.08 + 0.007 mol of native phosphorylase per seven Gactin monomers in buffer containing 100 PM Ca2+ (P < 0.05). Similar results were obtained when 200 PM Ca2+ was included in the binding buffer. Since AMP, ATP, and Pi are potential regulators of phosphorylase catalytic ac-
MARQUETANT,
MANFREDI, TABLE
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I
BINDING PARAMETERS FOR THE INTERACTION OF PHOSPHORYLASE a AND PHOSPHOR~LASE b WITH MYOFIBRILLAR PROTEINS Phosphorylase Contractile
protein
F-actin F-actin-tropomyosin F-actin-tropomyosintroponin Myofibrils
a
Phosphorylase
b
B mu
n
ST.6(PM)
B mu
sJ.6(PM)
1.5 z!z0.09 1.5 k 0.25
0.035 + 0.006 0.62 + 0.09
2.1 + 0.13 2.0 + 0.41
0.035 + 0.01 0.17 + 0.02
4 3
2.4 k 0.33 2.0 + 0.28
0.18 + 0.016 0.18 + 0.008
2.0 k 0.18 2.4 + 0.18
0.12 f 0.036 0.10 + 0.032
3 4
Note. Increasing amounts of phosphorylase a and phosphorylase b were incubated with thin filament proteins as described under Materials and Methods and in the legend of Fig. 1. Indicated are the amounts of phosphorylase bound at saturating concentrations (B,,,,). B,, data are expressed as moles of native phosphorylase bound per 7 mol of G-actin monomer. The dissociation constants (&J were analyzed by the logarithmic form of the Hill equation, assuming native molecular weights of 390,000 for phosphorylase and 195,000 for phosphorylase b.
tivity, the influence of these ligands on the maximal binding capacity of myofibrils for + HPHOSPHORYLASE B phosphorylase a and b was also evaluated. * p co.05 As demonstrated in Fig. 2,10 mM ATP de** p (0.01 creased the maximal binding capacity for phosphorylase a and phosphorylase b by approximately 25% (P < O.(N), whereas 0.5 mM AMP increased the B,, of phosphorylase a by 30% (P < 0.01) and phosphorylase b by 10%. Ten millimolar inorganic phosphate had no effect on B,,, for phosphorylase a or phosphorylase b binding to ATP AMP KPi myofibrils, demonstrating that these nuFIG. 2. Effect of Ca2+ and ligands on the binding of cleotide effects are not nonspecific effects phosphorylase a and phosphorylase b to myofibrils. of phosphate ions. Phosphorylase a or phosphorylase b at saturating
q
PHOSPHORYLASE
A
I
concentrations of 3.5 PM was incubated (pH 7.0,25”C, 10 min) with isolated myofibrils (2 mg/ml) in the presence of Ca’+ (100 pM, with the omission of EGTA) or in the presence of various ligands (ATP, 10 mbi; AMP, 0.5 mrd; KPi, 10 mM). The amount of bound phosphorylase was quantitated as described in the legend to Fig. 1 and under Materials and Methods. 100% phosphorylase bound (control) refers to the amount bound in the presence of 10 mb! imidazoleHCl, 126 mM KCI, 1 mM DTT, 1 mM MgCl,, 1 mM EGTA, pH 7.0. The effects of Car+, ATP, AMP, and Pi are presented as percentages of this control B,,,,. The data shown represent the means of four experiments (&SE). Statistical comparisons were done by an analysis of variance.
DISCUSSION
Prior studies have demonstrated that phosphorylase is bound to glycogen granules in skeletal muscle (2, 10, 11). In the present study evidence is presented which indicates that both phosphorylase a and b are also capable of binding to F-actin, Factin-tropomyosin, F-actin-tropomyosintroponin, and myofibrils. The binding to thin filament proteins is saturable and demonstrable at what are reported to be physiological salt and hydrogen ion concentrations. Although the affinity of phos-
INTERACTION
OF PHOSPHORYLASE
phorylase for thin filament proteins is not as great as that reported for binding of some other enzymes to contractile proteins, the affinity of this enzyme for thin filament proteins, as estimated from SO,values (1.5 to 2.4 PM), is substantially below the calculated concentration of phosphorylase in sarcoplasmic water, i.e., 9 to 20 j&M, depending on the state of aggregation of the enzyme. (Phosphorylase concentrations were estimated based on the reported value of 3.0 mg of phosphorylase per gram of skeletal muscle [lo].) Coupled with the histochemical studies which indicate that the distribution of phosphorylase follows the same striated pattern as a number of glycolytic enzymes which bind to thin filament proteins (23), the results of the present study suggest that this enzyme may also be bound to thin filament proteins in myocytes. This conclusion is not necessarily in disagreement with prior studies (2,10,11) demonstrating that phosphorylase binds to glycogen granules, since these granules are also concentrated in the region of the thin filament in the sarcomere. One might speculate that phosphorylase binds to both thin filament proteins and glycogen granules thereby serving to anchor these granules in the region of the myocyte where a number of glycolytic enzymes are concentrated. Maximal binding of phosphorylase to thin filament proteins is observed with the F-actin-tropomyosin complex, and at saturation approximately 1.2 mol of phosphorylase a is bound per 14 G-a&in monomers, a value similar to that obtained for aldolase (9). Addition of the troponin complex, either as reconstituted thin filaments or as native myofibrils, reduces the maximal number of binding sites, especially for phosphorylase a. This reduction in binding capacity in the presence of the troponin complex could be the result of simple steric inhibition or it may reflect a mechanism for controlling phosphorylase binding. Although we have not excluded steric effects, experiments with variable Ca2+concentration indicate that the maximal number of binding sites for phosphorylase on myofibrils can be modulated by changes in the concentration of this cation. The opposing
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409
effects of Ca2+on the B maxof the two forms of phosphorylase provides a potential mechanism for increasing binding of phosphorylase a, the more catalytically active form of the enzyme, and decreasing the binding of phosphorylase b, the less active form, during muscle contraction. Because of the influence of the troponin complex on the Bmaxfor phosphorylase, we assume that the effect of changing Ca2+concentrations is mediated through effects of this cation on the troponin complex. Another potential mechanism for regulating phosphorylase binding to thin filament proteins is suggested by the experiments with ATP and AMP. These ligands which control the distribution of the enzyme between the R and T conformations have opposing effects on the maximal binding capacity. ATP shifts the enzyme to the T conformation and decreases B,,, for both forms of the enzyme, while AMP shifts the enzyme to the R conformation and increases B,,,, providing a mechanism for increasing phosphorylase binding during intense muscle contraction through changes in energy charge in the myocyte. In summary, results of the present study demonstrate that phosphorylase binds to thin filament proteins at what are reported to be physiological ionic strengths and hydrogen ion concentrations, and the affinity of this binding is an order of magnitude less than the intracellular concentration of this protein. Data are presented which suggest that phosphorylase binding to thin filament proteins may be controlled through changes in conformation of the thin filament mediated by Ca2+ and/or changes in the conformation of phosphorylase mediated by the ligands ATP and AMP. ACKNOWLEDGMENTS The authors thank Dr. W. Longley of the Anatomy Department at Duke University for the supply of tropomyosin. We are grateful to Dr. R. H. Strasser for helpful suggestions in the preparation of the manuscript.We greatly appreciate the expert technical assistance of Jeana Meade. We thank Carolyn Mills for tireless secretarial help in preparation of this manuscript.
410
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11. LELOIR, L. F., AND GOLDEMBERG, S. H. (1960) J. BioL Chem. 235,919-923. 12. ENTMAN, M. L., BORNET, E. P., VAN WINKLE, M. A., GOLDSTEIN, A., AND SCHWARTZ, A. (1977) J. Mol. Cell Cardiol 9,515-528. 13. TRINICK, J., AND LOWEY, S. (1977) J. Md Biol 113, 343-368. 14. DRABIKOWSKI, W., AND GERGELY, J. (1963) J. Biol Chem. 238,640-643. 15. BAILEY, K. (1948) Biochem .I 43,271. 16. EISENBERG, E., AND KIELLEY, W. W. (1974) .I Biol Chem. 249,4742. 17. PERRY, S. V. (1955) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 2, p. 582, Academic Press, New York. 18. PERRY, W. V., AND CORSI, A. (1958) Biochem J. 68,5. 19. YATES, L. D., AND GREASER, M. L. (1983) J. Mel BioL 168,123-141. 20. EBASHI, S., AND EBASHI, F. (1964) J. Biochem (Tokyo) 55,604. 21. LAEMMLI, U. K. (1970) Nature (London) 227,680685. 22. TALLANT, E. A., AND CHEUNG, W. Y. (1984) Arch B&hem Biophys. 232,269-279. 23. SIGEL, P., AND PEITE, D. (1969) J. H&o&em Cyto&em 17,225-237.