[12] Myosin active-site trapping with vanadate ion

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Acknowledgments We wish to thank Mary Sheldon, William Perkins, Ross Dalbey, Cathy Knoeber, Dr. Philip Buzby,and Dr. Kay Nakamayefor makingunpublished results availableand for many useful discussions. We are also indebted to them and to Dr. Alan G. Weeds and Gary J. Pielak for helpfulcommentson this manuscript. This researchwas supported by grants from the MuscularDystrophyAssociation,the National Institutes of Health (AM-05195),and the American Heart Association.

[12] M y o s i n A c t i v e - S i t e T r a p p i n g w i t h V a n a d a t e I o n

By CHARLES C. GOODNO In the ideal world it would be possible to study the actomyosin ATPase by slowing it to the point where each chemical intermediate could be examined at leisure. Although the ideal is unattainable, it is possible to obtain stable complexes of myosin that mimic transient intermediates of ATP hydrolysis. One such model complex, which resembles the central myosin-products intermediate (M • Pr), is formed by the binding of ADP and vanadate ion to the myosin active site. 1"1aThis chapter is intended as an introduction to the properties and prospective applications of the complex, as well as a discussion of techniques for its synthesis and analysis. The stable complex is best understood in the context of the properties of vanadate ion (i.e., orthovanadate, VOW-), which is a tetrahedral oxyanion of pentavalent vanadium. 2 By virtue of its geometry, size, and charge, vanadate (abbreviated Vi) is an analog of phosphate ion. The functional aspects of this analogy have been recognized in studies of several phosphatases and phosphotransferases. 3-5 In contrast to its readily reversible interaction with other enzymes, vanadate forms with myosin a complex that appears to be uniquely stable. Vanadate alone forms a weak reversible complex with myosin, much like phosphate. Yet when ADP is included, a stable, enzymically inactive Abbreviations: Vl, vanadate ion (degree o f protonation unspecified); M, single active site o f the m y o s i n ATPase; M • Pr, m y o s i n - p r o d u c t s central complex; M • A D P • Vi, reversible t e r n a r y c o m p l e x of m y o s i n ; M t • A D P • Vl, stable ternary c o m p l e x (trapped intermediate); P~, p h o s p h a t e ion; PP~, p y r o p h o s p h a t e ion; A M P P N P , adenylyl imidodiphosphate; PAR, 4-(2-pyridylazo)resorcinol; SDS, sodium dodecyl sulfate. la C. C. G o o d n o , Proc. Natl. Acad. Sci. U.S.A. 76, 2620 (1979). 2 M. T. Pope and B. W. Dale, Q. Rev. Chem. Soc. 22, 527 (1968). 3 E. G. D e M a s t e r and R. A. Mitchell, Biochemistry 12, 3616 (1973). 4 V. L o p e z , T. Stevens, a n d R. N. Lindquist, Arch. Biochem. Biophys. 175, 31 (1976). 5 L. J o s e p h s o n a n d L. C. Cantley, Jr., Biochemistry 16, 4572 (1977).

METHODS IN ENZYMOLOGY, VOL. 85

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complex is formed, which can be isolated free of unbound vanadate and ADP. This complex has a stoichiometry of one ADP and one Vi per myosin active site and a half-life of approximately 3 days at 25°. Although this stability is unaffected by dialysis, chelating agents, and ATP, denaturation of the myosin produces a rapid release of ADP and Vi (<3 min). Thus, the complex appears to be held together by strong secondary forces rather than covalent bonds. The formation of this inactive complex, symbolized by Mt • ADP. V~ (the dagger representing the demise of ATPase activity), is highly specific. If the reaction is carried to various degrees of completion, there is a linear relationship (with a slope of 1.0) between the percentage incorporation of V~ and the percentage of inactivation of the ATPase. 6 This result is consistent with the interpretation that a single vanadate ion inactivates a single myosin ATPase site. The relationship between V~ incorporation and ATPase inactivation remains constant as the concentration of V~ is varied over a range of 4- to 1000-fold excess over myosin active sites. Similar results are obtained when the concentration of ADP is varied. The use of excess ADP and Vi, therefore, does not lead to incorporation at additional sites. The stable incorporation of Vi is nucleotide-specific in that neither AMP, ATP, AMPPNP, nor PPi will substitute for ADP. ATP actually protects myosin from modification in the presence of V~ and ADP, causing the rate of V~ incorporation to drop to 3% of its usual value. Since roughly 3% of the steady-state complexes of the myosin ATPase are in the form of M . ADP, 7 which can react directly with V~, this finding suggests that M • ATP and M • Pr complexes are completely protective. These observations, in concert with kinetic studies (see below), lead to the conclusion that V~ and ADP function together as a binary active-site directed reagent for myosin, with the unusual feature that the labeling is apparently not covalent. F o r m a t i o n o f M+ •A D P

" Vi

During the formation of the M? • ADP • V~ complex, ADP and Vi are incorporated into the complex at identical rates, within experimental error. For fixed concentrations of ADP and Vi, the kinetics are pseudo-first order. When the concentrations are varied, the rate of formation of the complex is a hyperbolic function of both ADP and Vi. This hyperbolic dependence is characteristic of active-site-directed reagents, which bind at the enzyme active site prior to the modification reaction. The maximum 6 C. C. G o o d n o and E. W. Taylor, in preparation. 6a C. C. G o o d n o a n d E. W. Taylor, Proc. Natl. Acad. Sci. U.S.A. 79, 21 (1982). 7 D. R. T r e n t h a m , J. F. Eccleston, and C. R. B a g s h a w , Q. Rev. Biophys. 9, 217 (1976).

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M. ADP

k5

M

M.ADP.V 1 ~ k-5

-- Mt.ADP.V i

M-V i FIG. 1. Mechanism of formation of the stable vanadate complex. Rapid preequilibration leads to a reversible ternary complex (M • ADP • Vi), which slowly isomerizes to the stable complex. The isomerization (step 5) is the rate-limiting step in both forward and reverse directions. (plateau) rate o f formation o f the stable complex is approximately 0.01 sec -1 (pH 7 or 8.5, ionic strength 0.1, 25°). Half-saturation o f the rate occurs with 400 /zM V~ and less than 10 /xM ADP. T h e s e findings are consistent with the formation o f a reversible ternary complex o f myosin, ADP, and V~, followed by a slow isomerization step leading to the stable complex (trapped intermediate), as illustrated in Fig. 1. Although the Mt • A D P . V~ complex is exceedingly stable, its formation is slowly and spontaneously reversible, with a first-order rate constant o f approximately 2.5 x 10 -6 sec -1 (pH 8.5, ionic strength 0.1, 25°). The release of V~ and r e c o v e r y o f ATPase activity o c c u r at the same rate, indicating the genuine reversibility of the binding. Thus, the half-life of the complex is approximately 3 days. Combination o f the forward and reverse rate constants for the isomerization step gives an equilibrium constant of 4 x 10a in the forward direction. Since the tight binding o f Vl is the result o f a coupled equilibrium (binding followed by isomerization), the overall binding constant for V~ is given by the product of the two individual binding constants, which is approximately 107 M -1. It is noteworthy that this binding constant is about 100,000-fold higher than the binding constant o f myosin for P~, yet it is similar to the binding constant o f P~ in the M • Pr intermediate in ATP hydrolysis. Interaction o f M't • A D P • V t with A c t i n

It is well known that actin exerts its activating effect on the myosin ATPase by accelerating the otherwise slow product release step. s One would expect, then, that a genuine trapped intermediate of the myosinproducts type would interact with actin in such a way as to displace the products. Figure 2 shows that actin indeed displaces Vi from the M t • ADP • V~ complex.ta Moreover, increasing concentrations o f actin produce higher 8 R. W. Lymn and E. W. Taylor, Biochemistry 10, 4617 (197i).

VANADATE TRAPPING O F MYOSIN

[12]

1 19

100

80

2 '-'

u.I

60

u.I ..J

,,, tr

40

>20 V ,

--V

Y

,

,

I

2

3

4

5

TIME

(HRI

F=o. 2. Kinetics of actin-mediated vanadate displacement from M~ • ADP • Vt. The displacement of Vi from the complex by various concentrations of actin was determined colorimetrically. Conditions: 10/xM Mt - ADP • Vi, 90 mM NaC1, 20 mM Tris, 5 mM MgCl2, pH 8.5, 25°. Actin concentrations: ~7, 0; rq, l0/.tM; ©, 25/zM; O, 50 tzM; •, 75 #M.

rates of Vi displacement, even in the range of actin concentrations that lead to 100% displacement of Vi at equilibrium. Thus, actin not only shifts the equilibrium for the binding of Vi, it also catalyzes the removal of V~ from the myosin active site in much the same fashion as it catalyzes the removal of Pi. The role of actin is corroborated by the observation that ATPase activity is recovered at the same rate as V~ displacement (Fig. 3), with full displacement of Vl leading to complete recovery of ATPase activity. The binding of actin to MS • ADP • V~ is ionic-strength dependent, as it is with the M • Pr complex. Under the most favorable conditions examined to date (ionic strength 0.03, pH 7.0), the rate of V~ release is a hyperbolic function of actin concentration, with an apparent binding constant of approximately 104 M -~ and a limiting rate constant in the vicinity of 0.5-1.0 sec -~. This rate is approximately 5% of the rate of actinmediated release of products from the M • Pr complex. Since the rate of spontaneous release of V~ under these conditions is approximately 5 × 10-6 sec -~, actin is able to accelerate the process by a factor of about 100,000. Thus, actin binds to the complex and catalyzes a reaction analogous to product-displacement from the myosin ATPase site. Conversely, studies 6a have shown that ADP and V~ together have a

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I00

80 r~ rid <

uJ 60

.J W R.

>40 o

g. 2o F-

I

I

i

I

2

4 TIME

I

I

6

(HR.)

FIG. 3. Correlation of vanadate release arid ATPase recovery. 10 p.M M? - ADP. Vl was mixed with 17 ~ M actin (final concentrations). Samples were withdrawn at intervals for measurement of ATPase activity and free V~. ©, ATPase activity; A, free Vi. Conditions: 90 mM NaCi, 20 mM Tris, 5 m M MgC12, pH 8.5, 25°.

dissociating effect on the actomyosin complex under conditions where neither species separately has any effect. These results suggest that ADP and Vi may have a relaxing effect similar to that of ATP. Preliminary studies with frog semitendinosus have shown that when Vl is added to a muscle fiber in the presence of endogenously generated ADP (in the approach to rigor), relaxation is obtained under conditions where ADP and Vi have no effect separately, a Stiffness is simultaneously reduced to a low level, indicating the almost total dissociation of myosin cross-bridges. These effects are completely reversed by washing out the ADP and Vi. Methods

Vanadate Analysis Stock solutions of 100 mM Vi are conveniently prepared by dissolving Na3VO4 or V205 in H~O and adjusting the solution to approximately pH 10 with 6 N HC1 or 10 N NaOH, respectively. Since Vi exists in a slow, pH-dependent equilibrium with various polymeric vanadate species, z this adjustment perturbs the equilibrium. Resulting polymeric species A. Magid and C. C. Goodno, Biophys. J. 37, 107A (1982).

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VANADATETRAPPINGOF MYOSIN

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(orange-yellow) are destroyed by boiling the solution until colorless, followed by rechecking the pH of the cooled solution. Several cycles of adjustment and boiling may be required. Stock solutions can be stored for several months in a stoppered bottle away from reducing agents. Solutions are discarded when they turn noticeably yellow. The final concentration of Vi is checked by measuring the absorbance at 265 nm (E2e5 = 2925 M -1 c m -1) o n an aliquot of stock solution diluted with H~O. A solution (2 mM) of the metal-indicator dye 4-(2-pyridylazo)resorcinol (PAR), is prepared by dissolving the dye powder in HzO, followed by filtration to remove any particulate matter. A stock solution of 1.0 M imidazole buffer, pH 6.0, is also prepared. Vanadate analysis is carried out by the addition of 0.1 ml of 1.0 M imidazole to a 1.0-ml sample of vanadate, followed by addition of 0.1 ml of 2 mM PAR, then mixing. After 5 min of color development at room temperature, the absorbance is measured at 540 nm in a 1-cm semimicro cuvette. The resulting red color is stable for several hours. Calibration curves are prepared over the range of 0-20 t~M V~, the region in which Beer's law is obeyed.

Preparation of Vanadate-Trapped Myosin For ease in chromatographic separations at low ionic strength, either subfragment-1 or heavy meromyosin is generally used, although whole myosin readily undergoes modification. AtypicalpreparationofMt • ADP • Vi is carried out with a myosin site concentration of 20 ~ M in 0.09 M NaCI, 5 mM MgCI~, and 20 mM Tris, pH 8.5. (Ionic strength is kept low and pH high to facilitate separation of M~ • ADP • Vi by ion-exchange chromatography. Preparation can be carried out at pH 7 with equal ease, although subsequent separation is more complex.) A small aliquot of stock ADP (100 raM) is added to produce a final ADP concentration of approximately 0.2 raM, and the solution is incubated for 5 min in a water bath at 25° to hydrolyze any ATP present in the ADP. Stock Vi (100 mM) is then added to a final concentration of 1 mM, and the modification reaction is allowed to proceed for 5 rain. Separation of Mt • ADP • Vi from excess ADP and Vi is readily performed by ion-exchange chromatography. Up to 5 ml of the reaction solution are applied to a 0.5 × 2 cm column of washed Dowex l-X8 resin (200-400 mesh), which has been fully equilibrated with 0.09 M NaC1, 5 mM MgCI2, and 20 mM Tris, pH 8.5. (Pasteur pipettes make convenient columns.) The reaction mixture is allowed to percolate into the column under gravity flow (approximately 0.2 ml/min), and the resulting effluent is collected. The column is then washed with 2-5 ml of the column buffer, again under gravity flow, and the effluent is collected in the same vessel.

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ADP and V~ that are not bound to myosin are quantitatively retained by the column (99.9%), while 95% recovery of the heavy meromyosin or subfragment-1 is obtained in the effluent. The pooled washings, which contain the purified Mt • ADP • Vi complex, are then stored at 4°; and the column is regenerated with 1 N NaOH. Although Dowex 1 treatment may seem harsh, it produces no observable change in the SDS-gel electrophoresis pattern of the protein and less than a 1% change in the ATPase specific activity.

Analysis of Vanadate-Trapped Myosin The vanadate content of the isolated complex can be determined directly by treating the complex with 1% sodium dodecyl sulfate to denature the protein, followed by standard colorimetric analysis of the vanadate that is released. However, frequently 5-10% of the bound vanadate is bound in nonspecific (i.e., reversible) fashion, leading to an overestimate of the tightly bound vanadate. For this reason, we prefer a differential method in which vanadate is measured both before and after the addition of SDS. In this procedure, calibration curves are established for Vi in the presence and in the absence of SDS. A 1-ml sample of Mt • ADP • V~ is used, and V~ analysis is carried out according to the standard assay procedure, with sample and reagents added directly to the spectrophotometer cuvette. After the absorbance at 540 nm is read, a 63-/~1 aliquot of 20% SDS is added with mixing, and an additional 5 min is allowed for color development from the released V~. The absorbance is again read; appropriate blank readings are made with an equivalent amount of protein (without V0; and the concentration of free V~ before and after denaturation is calculated. Tightly bound Vi (i.e., trapped) is given by the difference between these values. Typical reactions using fresh heavy meromyosin or subfragment-1 give 85-95% modification, as judged by both Vi incorporation and ATPase inactivation. Aging of the protein leads to a decline in V~ incorporation and inactivation, particularly for subfragment-1. Preliminary studies e have shown that alkylation of myosin sulfhydryl groups drastically inhibits the incorporation of V~. Thus, myosin must remain intact to ensure optimal modification.

Applications of Vanadate Labeling The combination of ADP and V~ produces an affinity label that is completely selective for one site per head of the myosin molecule, and the preponderance of evidence suggests that this site is identical with the ATPase active site. Moreover, currently available data suggest that

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myosin is the only enzyme that is stably labeled by the combination of ADP and Vi. These properties make ADP/Vi a suitable reagent for active-site titrations of myosin, as well as the presumptive identification of myosin in heterogeneous systems. Using the colorimetric analysis for Vi, one can determine nanomole quantities of myosin sites. With [3H]ADP or other suitable radioactive label in ADP or Vi, the sensitivity can be extended by several orders of magnitude. If desired, the myosin can be recovered unharmed by treatment with actin, which releases the label and restores full ATPase activity. The Mt • ADP 'V~ complex has a number of possible applications in the investigation of energy transduction by myosin, since it resembles M - P r , the central intermediate in the hydrolysis of MgATP. r'8 The 20,000-fold longer lifetime of Mt • ADP • V~ makes it suitable for examination by physicochemical methods that are too slow to be used for the actual intermediate. This long lifetime may also afford a unique opportunity to circumvent the diffusion limitation of muscle fibers by saturating the myosin cross-bridges in a well-defined chemical state (i.e., Mt • ADP • Vi). With blockage of actin by low calcium, this state should have a lifetime in the cross-bridge comparable to its lifetime in isolated myosin. Thus, it may be possible to study the mechanical properties of muscle fibers whose myosin cross-bridges are in a chemically well-defined state, leading to a correlation of chemical and mechanical events involved in muscle contraction.

[13] Assays f o r M y o s i n By THOMAS D. POLLARD The first prerequisite in the purification of any protein is reliable simple assays. Fortunately, the distinctive ATPase activities of all the myosins and, in most cases, the unique high molecular weight of the myosin heavy chains and the characteristic shape of the intact myosin molecules make this a simple matter.

ATPase Assays In most cases myosins have the unique property of having low ATPase activity in Mg 2+ and high activity in Ca ~+ and in EDTA, providing that high concentrations of K ÷ are present. The high ionic strength also prevents any actin in crude fractions from activating the MgATPase. Most other ATPases are more active in Mg 2+ than in Ca ~÷ and are inactive in

METHODS IN ENZYMOLOGY, VOL. 85

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