906
Biochemistry 1982, 21, 906-9 15
Potentiated State of the Tropomyosin Actin Filament and Nucleotide-Containing Myosin Subfragment 1+ John M. Murray, Mary K. Knox, Cynthia E. Trueblood, and Annemarie Weber*
ABSTRACT: The main purpose of this study was to determine whether potentiation of acto-S-1 ATPase activity (activity higher than that obtained with tropomyosin-free actin) could be caused by nucleotide-containing acto-S- 1 complexes. In addition, we wanted to know whether these complexes also have a positive cooperative effect on their own apparent binding constant under conditions where nucleotide-free acto-S- 1 complexes cause potentiation of ATPase activity. Using calcium-saturated troponin-tropomyosin actin filaments, we observed potentiation of ATPase activity in the presence of 5 .O mM magnesium 5’-adenylyl imidodiphosphate (MgAMPPNP) and calculated that the ability of acto-S- 1AMPPNP complexes to cause potentiation must have been very similar to that of nucleotide-free acto-S-1 complexes. In extension of earlier studies, potentiated acto-S- 1 ATPase activity was characterized by an increase in V ,and, as observed
before, a lowering of the apparent KM for subfragment 1 (S-1). Under conditions similar to those that produce the potentiation of acto-S-1 ATPase activity, the apparent actin binding constant of nucleotide-free s-1 was increased about 3-5-fold while the apparent binding constant of AMPPNP to actin-bound S-1 was reduced to (2.5-10) X lo2 M-’ compared to that of about (1-5) X lo3 M-’ for S-1 bound to tropomyosin-free actin. Under the same conditions, the apparent binding constant of S-1-AMPPNP to actin was not increased. We suggest that a potentiated state of the tropomyosin actin filament is produced by the cooperative action of acto-S- 1 or acto& 1-AMPPNP complexes. The potentiated state is characterized by an increase in the V,,, of the acto-S- 1 ATPase activity, increased binding constants for S-1 and S-1ADP, and increased binding of tropomyosin to actin.
Topomyosin is a constituent of all muscle thin filaments and is bound to some of the actin filaments of many nonmuscle cells. It plays an essential role in the regulation of those muscles in which the transition between contraction and relaxation depends on a change of state of the thin filament (Ebashi et al., 1969; Huxley, 1972; Haselgrove, 1972; Parry & Squire, 1973). Its role has not been understood in muscles where this regulation is accomplished by the myosin molecule directly or possibly indirectly through myosin phosphorylation. Therefore, it is of interest that tropomyosin is capable of modifying the behavior of actin filaments that do not contain troponin (Murray et al., 1975, 1980a,b; Bremel et al., 1972). Tropomyosin, in the absence of troponin, modifies the activation of myosin ATPase by actin in two opposite ways: either by reducing this activation or by enhancing it. These two tropomyosin effects are called inhibition or potentiation of actomyosin ATPase. Inhibition occurs when the S-1 (subfragment 1)’ to actin ratio is low, i.e., about one s-1per 30 actin molecules (Murray et al., 1975, 1980a,b). Under these conditions, the presence of tropomyosin on the actin filament reduces the acto-S-1 ATPase activity by half (Eaton et al., 1975; Murray et al., 1975, 1980a,b). Increasing the S-1 to actin ratio changes tropomyosin inhibition to tropomyosin-dependent potentiation of acto-S- 1 ATPase activity, so that it is severalfold faster than the ATPase activity with tropomyosin-free actin. In other words, potentiation of the cofactor activity of actin requires both the presence of tropomyosin and a sufficiently high degree of saturation of the actin filaments with S-1. What may con-
stitute a sufficiently high degree of saturation with S-1 will be discussed later. Of the various intermediate acto-S-1 complexes that occur during acto-S-1 ATPase activity [for a review, see Taylor (1979) and Adelstein & Eisenberg (1980)], only rigor complexes (nucleotide-free acto-S- 1 complexes) have been shown unequivocally to cause potentiated ATPase activity (Bremel et al., 1972). Although rigor complexes are postulated to be an intermediate during bridge cycling (Eisenberg & Greene, 1980), their steady-state concentration is expected to be low at physiological ATP concentrations. A number of years ago (Bremel et al., 1972), we described experiments which seemed to indicate that the nucleotide-containing acto-S-1 complexes which prevail at saturating ATP concentrations (possibly acto-S- 1-ADP-P) also cause potentiation. Later, however, we learned that this interpretation of the experiments is not unambiguous. Even in the presence of saturating ATP levels, rigor complexes cannot be ruled out as the cause of potentiation unless the existence of ATP-resistant rigor complexes can be excluded. ATP-resistant rigor complexes are formed with S-1 whose ATP binding site is nonfunctional so that ATP can no longer dissociate the rigor complex (Pemrick & Weber, 1976). We have shown previously that such myosin is produced during treatment with sulfhydryl reagents (Barany & Barany, 1959; Pemrick & Weber, 1976), and we have evidence suggesting that each S-1 preparation contains this S-1 as a contaminant, possibly due to sulfhydryl oxidation by air (Murray et al., 1981). Therefore, it is difficult to be sure to what extent potentiation at saturating ATP concentrations is due to these rigor complexes rather than to one of the nucleotide-containing acto-S- 1 intermediates. To minimize the ambiguities intro-
’
From the Medical Research Council, Cambridge, England, and the Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104. Received April 16, 1981; revised manuscript received August 21, 1981. This work was supported by a research grant from the National Institutes of Health (HL 15-692). * Correspondence should be addressed to this author at the Department of Biochemistry and Biophysics, University of Pennsylvania.
0006-2960/82/0421-0906$01.25/0
Abbreviations: AMPPNP, 5’-adenylyl imidodiphosphate; EGTA, ethylene glycol bis(@-aminoethyl ether)-N,N,N’,N’-tetraacetic acid; [3H]NEM, N-[3H]ethylmaleimide; S-1, myosin subfragment 1; S-IADP.P, the fluorescent S-1 complex with both ADP and P that is the first product of ATP hydrolysis; NaDodS04, sodium dodecyl sulfate.
0 1982 American Chemical Society
POTENTIATED STATE OF MUSCLE PROTEINS
duced by ATP-resistant rigor complexes, we determined whether S-1 complexed with the ATP analogue 5’-adenylyl imidodiphosphate (AMPPNP) can cause potentiation. We present experiments showing that acto& 1 complexes containing the nucleotide AMPPNP are just as competent to cause potentiation of ATPase activity as are rigor complexes. We characterized the potentiated state with respect to the binding of S-1. Measurements of binding can provide an estimate of how great an energy barrier separates the potentiated from the nonpotentiated state of the actin filament (we describe the actin filament as potentiated when actomyosin ATPase is potentiated). We compared S-1 binding to troponin-tropomyosin actin in the potentiated state with S-1 binding to tropomyosin-free pure actin and to troponin-tropomyosin actin in the nonpotentiated state. A method which permits one to obtain actin binding constants for nucleotidefree S-1 and S-1-AMPPNP is the titration of acto-S-1 with increasing concentrations of AMPPNP (Greene & Eisenberg, 1978; Murray et al., 1981). Transition to the potentiated state increased binding of nucleotide-freeS-1 4-6-fold. By contrast, we did not measure an increase in the binding constant for S- 1-AMPPNP. Materials and Methods Protein Preparations. All proteins were derived from the hind leg and back muscles of rabbits. Myosin subfragment 1 (S-1) was prepared according to Lowey et al. (1969) with the modifications described previously (Murray et al., 1981). The main change in the preparation consisted of selecting the S-1 fraction that precipitated as a discrete fraction between 50 and 55% ammonium sulfate saturation and rejecting the fraction that precipitated between 60 and 65% saturation. The 50-55% fraction had a greater actin-activated ATPase activity than the 60-65s fraction (50-70% higher) and was less digested than the later fraction. However, there was not a really distinct difference in gel bands between the two fractions such as the appearance or disappearance of a band on NaDodSO, gels. Actin was prepared according to Spudich & Watt (1971) with the modifications described recently (Murray et al., 1981). It was homogeneous on overloaded NaDodSO, gels except for an occasional small band just in front of actin but not completely separated from it. We are not sure whether this occasional, very minor contamination is a proteolytic actin fragment. Troponin and tropomyosin were prepared as a complex by extraction of a lyophilized Ebashi powder (Ebashi & Ebashi, 1964) with 1 M KCl, 2 mM MgClz, and 10 mM Tris, pH 7.4, followed by centrifugation at 1OOOOOg to remove contaminating actin. Troponin-tropomyosin actin filaments were prepared according to Bremel and colleagues [ 1972; see also Murray et al. (1981)l. In a number of preparations, the troponin-tropomyosin actin filaments contained an unidentified contaminant from the Ebashi extract that caused increasing depolymerization of the troponin-tropomyosin actin with time. Protein concentrations were measured with the Lowry reaction (Lowry et al., 1951) by using bovine serum albumin as the color standard and calculating the protein concentrations from tables for myosin and actin constructed after determination of the protein concentrations by Kjeldahl. Zonal NaDodSO, gel electrophoresis was carried out with disc gels according to Laemmli (1 970) with fast green as a stain as described by Potter (1974). Protein Modification. For a few experiments, myosin was labeled before proteolysis with N-[3H]ethylmaleimide ( [3H]NEM) as recently described (Murray et al., 1981). In
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907
experiments using native S-1, actin was labeled at cysteine-373 with [3H]NEM (Porter & Weber, 1979). Reagents. AMPPNP was obtained from Boehringer and was routinely checked for purity before use both by thin-layer chromatography and by enzymatic measurements for a quantitative measurement of the contaminating ADP and ATP concentrations. The enzymatic measurements were performed with Boehringer kits containing the pyruvate kinase lactic dehydrogenase system and the phosphoglycerokinase phosphoglyceraldehyde dehydrogenase + triose isomerase glycerophosphate dehydrogenase system for measurement of contaminating ADP and ATP concentrations, respectively. Contamination with ADP ATP varied from 1 to 3 mmol/mol of AMPPNP during the S- 1 binding experiments and was 1 mmol/mol of AMPPNP during the ATPase activity measurements. ATPase Activity Determination. (1) ATPase Activity as a Function of Increasing S-1 Concentrations. For these experiments, S-1, present in a stock solution of 40 mg/mL, was weighed into every assay tube before adding the actin with the incubation mixture containing (in final concentrations) 10 mM imidazole, pH 7.0, 1.0 mM MgC12, and 1.0 mg/mL creatine kinase. The assay was started by the addition of a mixture of MgATP and creatine phosphate to give final concentrations of 1.0 and 5.0 mM, respectively. The assay was terminated with p-mercuribenzoate (final concentration 50 mM) and analyzed for creatine as previously described (Maruyama & Weber, 1972). ( 2 ) ATPase Activity as a Function of Increasing ATP Concentrations. For these experiments with constant S-1 and actin concentrations, the proteins were mixed with imidazole buffer and creatine kinase as described above and 5.0 mM MgCl, in excess over MgAMPPNP concentration (as indicated in the legends), and the assay was started by the addition of creatine phosphate (5.0 mM final concentration) + MgATP in the appropriate concentration. Each ATP mixture contained in addition KC1 to bring all assays to the same ionic strength (see legends). It is important to avoid mixing AMPPNP and the complete phosphorylating system during temperature equilibration of the enzyme because of the contaminating ADP + ATP in the AMPPNP. Titration of Acto-S-1 with AMPPNP. Dissociation of acto-S-1 by AMPPNP was measured by separating the actin-bound from the free S-1 by a brief high-speed centrifugation. [3H]NEM-labeled actin and native S-1 were mixed in the appropriate proportions with 5 mM MgCl, in excess over MgAMPPNP concentration at pH 7.0 (10 mM imidazole buffer) and the appropriate concentration of KCl to maintain the same ionic strength for all concentrations of MgAMPPNP. Denatured S-1, defined as that which remained in the supernatant after centrifugation with 10 FM actin in the absence of MgAMPPNP, was subtracted from each value. In the experiments with [3H]NEM-labeled S-1, native actin was used, and dissociated S-1 was determined by measuring the radioactivity in the supernatants. In the experiments where native S-1 was used, dissociated S-1 was determined by the Lowry reaction (Lowry et al., 195 1). Nonpolymerized [jH]NEMlabeled actin, present in the supernatant, was subtracted from the Lowry protein values. A detailed protocol, control experiments, and calculations of the data have been described (Murray et al., 1981). Modelling of the ATPase Data. The ATPase activity of S-1 in the presence of tropomyosin-free actin is satisfactorily accounted for under steady-state conditions by the TaylorTonomura-Trentham reaction scheme [cf. Trentham et al.
+
+
+ +
B I o c H E M I sT R Y
908
M U R R A Y ET A L .
Table I: Reactions Included in the Model of Steady-State ATPase Activity of Tropomyosin Actin S-1 a kforward
ref
kreverSe
ref
5 x lo6 1.0 X lo6 (1.8 X lo6) 70 0.1
d,e d
1X 0.5 (K,lk+)
Ag
i,e
30 o
reactants -products S S
+ T-ST
+N
+-$
SN
ST t--.f ST* ST* c---i S + ADP + Pi
d
nonpotentiated
I7
i, e
.f potentiated
reactants cf products A + S +-+AS
kforward ref /
