Stoichiometry of labeling of myosin's proteolytic fragments by a purine disulfide analog of adenosine triphosphate.

A site-specific analog of ATP, 6,6'-dithiobis (inosinyl imidodiphosphate (S2P-PNP), inactivates the ATPase activities of myosin's proteolytic fragments, heavy meromyosin (HMM) and subfragment one (SF1), by formation of mixed disulfides between the 6 position of the purine ring and certain key cysteines. The stoichiometry of the reaction was determined by quantitatively displacing the thiopurine nucleotides from the labeled enzymes with sodium[14-C]cyanide. The thiocyanatoenzyme formed regained 25 percent of the original activity showing that the cysteines modified were not essential for catalysis. The rate of uptake of label paralleled the rate of inactivation. HMM was completely inactivated when 4 mol of thiopurine nucleotide was bound. SF1 made by a papain digestion of myosin incorporarted 2 mol of thiopurine nucleotide when completely inactivated. Having adenylyl imidodiphosphate, areversible competitive inhibitor of myosin's ATPase, present during the inactivation of HMM by S2P-PNP demonstrated that only one cysteine per head needed to be blocked to inactivate the enzyme. Moreover, SF1 made by a trypsin digest of HMM was completely inactivated when only 1.1 mol of the thiopurine nucleotide bound again indicating that blocking only a single cysteine per head was sufficient to cause inactivation. This sulfhydryl is thought to be at an ATP binding site distinct from the ATPase site. The properties of this second ATP binding site are consistent with it being an ATP regulatory site.     

Subunit location of sulfhydryl groups of myosin labeled with a purine disulfide analog of adenosine triphosphate.

A purine disulfide analog of ATP, 6,6'-dithiobis(inosinyl imidodiphosphate), forms mixed disulfides with cysteine residues at what are believed to be ATP regulatory sites of myosin. Blocking these sites causes inactivation of the ATPase activity at the active sites. Two cysteine residues per head are specifically modifed by this disulfide analog. The thiopurine nucleotides can be stoichiometrically displaced from myosin by [14-C]cyanide to give a more stable thiocyanato derivative of the enzyme. [14-C]Thiocyanatomyosin (3.7 14-CN/myosin) was dissociated in 4 M urea and the individual subunits were isolated. The heavy chains each had 0.78 14-CN bound per 200,000 molecular weight unit. The light chain with molecular weight of 20,700 had 1.00 14-CN bound and the 16,500 molecular weight light chain had 0.65 14-CN bound. The two 19,000 molecular weight light chains were not labeled. The two labeled light chains have only a single cysteine which is stoichiometrically modified. These two light chains show a high degree of homology and presumably perform identical functions in myosin. Their specific modification by the purine disulfide analog and their other known properties suggest that they contribute directly to the ATP regulatory sites and may, in fact, function as regulatory subunits.     

Reaction of cardiac myosin with a purine disulfide analog of adenosine triphosphate. I. Kinetics of inactivation and binding of adenylyl imidodiphosphate.

Bovine cardiac myosin ATPase activity was rapidly inactivated by the purine disulfide analog of ATP,6,6'-dithiobis(inosinyl imidodiphosphate). Kinetic investigations showed that this analog acted as a site-specific reagent at 0 degrees with a Ki of 130 muM and a half-life of 8.2 min at saturating inhibitor concentrations. Concentrations (50 to 500 muM) of ATP, adenyl-5'-yl imidodiphosphate (AMP-PNP), or ADP that saturated the active site caused an enhancement in the rate of inactivation, indicating the purine disulfide analog was not reacting at the active site. Under these conditions saturation kinetic data were still observed with Ki values remaining unchanged (120 muM) but with the half-life of inactivation decreasing to 6.0 min (ATP) and 4.6 min (AMP-PNP) at saturating inhibitor concentrations. At concentrations greater than 0.5 mM ATP, AMP-PNP, or ADP there was a decrease in the rate of inactivation, implying protection by these nucleotides. However, saturation kinetics of inactivation could no longer be demonstrated, implying a change in the mechanism of inactivation. A comparison of the inactivation of the Mg2+, Ca2+, and EDTA-ATPase activities of cardiac myosin after modification by the purine disulfide analog showed that the Mg2+- and Ca2+ATPase activities plateaued at approximately 60% and 40%, respectively, while the EDTA-ATPase activity continued to decrease to below 10%. This evidence supports the suggestion that the purine disulfide analog was not reacting at the active site. Equilibrium dialysis experiments were used to measure the binding of [8-3H]AMP-PNP to native cardiac myosin, the thiopurine nucleotide-modified myosin, and the derivative formed by displacing the thiopurine nucleotide by cyanide (thiocyanato-myosin). Native myosin bound a total of 2.1 mol of AMP-PNP with a binding constant of 6.0 X 10(6) M-1. There was a 15 to 40% decrease in the number of AMP-PNP binding sites in the enzyme derivatives, but the active sites appeared not to be blocked since the association constants remained essentially unchanged (KA=3.9 X 10(6) M-1 for thiopurine nucleotide-myosin and 12.0 X 10(6) M-1 for thiocyanato-myosin). The kinetic studies and the binding experiments indicate that the purine disulfide analog reacts at a specific site other than the active site but do not offer support to earlier suggestions from skeletal myosin studies that this site is a possible ATP control site.     

The covalent modification of myosin's proteolytic fragments by a purine disulfide analog of adenosine triphosphate. Reaction at a binding site other than the active site.

A purine disulfide analog of ATP, 6,6'-dithiobis(inosinyl imidodiphosphate), forms mixed disulfide bonds between the 6 thiol group on the purine ring and certain key cysteines on myosin, heavy meromyosin, and subfragment one. The EDTA ATPase activities of myosin and heavy meromyosin were completely inactivated when 4 mol of thiopurine nucleotide was bound. When similarly inactivated, subfragment one, depending on its method of preparation, incorporated either 1 or 2 mol of thiopurine nucleotide. Modification of a single cysteine on subfragment one resulted in an inhibition of both the Ca2+ and the EDTA ATPase activities, but the latter always to a greater extent. Modification of two cysteines per head of heavy meromyosin had the same effect suggesting that the active sites were not blocked by the thiopurine nucleotides. Direct evidence for this suggestion was provided by equilibrium dialysis experiments. Heavy meromyosin and subfragment one bound 1.9 and 0.8 mol of [8-3H]adenylyl imidodiphosphate per mol of enzyme, respectively, with an average dissociation constant of 5 X 10(-7) M. Heavy meromyosin with four thiopurine nucleotides bound or subfragment one with two thiopurine nucleotides bound retained 65-80% of these tight adenylyl imidodiphosphate binding sites confirming the above suggestion. Thus previous work assuming reaction of thiopurine nucleotide analogs at the active site of myosin must be reevaluated. Ultracentrifugation studies showed that heavy meromyosin which had incorporated four thiopurine nucleotides did not bind to F-actin while subfragment one with one thiopurine nucleotide bound interacted only very weakly with F-actin. Thus reaction of 6,6'-dithiobis(inosinyl imidodiphosphate) at nucleotide binding sites other than the active sites reduces the rate of ATP hydrolysis and inhibits actin binding. It is suggested that these second sites may function as regulatory sites on myosin.     

Substructure of the myosin molecule: IV. Interactions of myosin and its subfragments with adenosine triphosphate and F-actin

The enzymic activity of several single-headed subfragments of myosin (HMM S-1 and single-headed HMM) has been compared to the double-headed derivative of myosin (HMM) both in the presence and absence of aetin. Under the assay conditions of our experiments, we find that HMM hydrolyses ATP at approximately twice the rate of any single-headed species. These results suggest a relatively independent functional role for each of the two heads of the myosin molecule.An attempt has been made to determine the stoichiometry of association between subfragments and actin, either in the absence of nucleotide or during the hydrolysis of ATP. It was originally thought that a comparison of the maximum turnover rate of HMM at infinite concentrations of actin with the maximum rate at infinite concentrations of enzyme (but with a fixed amount of actin) would yield the combining ratio of actin to HMM. However, the considerable variation of ATP turnover rates with the conditions of the experiment has made it impossible to reach any firm conclusions regarding stoichiometry. A more direct approach to the question of stoichiometry is possible in the absence of ATP. By reacting varying amounts of F-actin with a given concentration of subfragment and centrifuging the resulting complex, it is possible to determine the unbound concentration of subfragment in the supernatant. These data provide sufficient information to construct a Scatchard plot and show that twice as many moles of actin are bound by HMM as by HMM S-1. Furthermore, the association constant of actin for HMM is several orders of magnitude higher than that for the single-headed species.In connection with the question of why myosin has two “heads”, we have examined the ability of single-headed molecules to undergo the phenomenon of “superprecipitation”. We find that single-headed myosin (the preparation of which was discussed in the preceding paper) is able to superprecipitate in much the same manner as native myosin.We conclude from these studies that each head of the myosin molecule is able to function in a relatively independent fashion. These studies do not, of course, exclude the possibility of more subtle interactions between the heads of myosin which our techniques are not able to detect.     

The magnesium ion-dependent adenosine triphosphatase of myosin. Two-step processes of adenosine triphosphate association and adenosine diphosphate dissociation

The kinetics of protein-fluorescence change when rabbit skeletal myosin subfragment 1 is mixed with ATP or adenosine 5'-(3-thiotriphosphate) in the presence of Mg(2+) are incompatible with a simple bimolecular association process. A substrate-induced conformation change with DeltaG(0)<-24kJ.mol(-1) (i.e. DeltaG(0) could be more negative) at pH8 and 21 degrees C is proposed as the additional step in the binding of ATP. The postulated binding mechanism is M+ATPright harpoon over left harpoonM.ATPright harpoon over left harpoonM*.ATP, where the association constant for the first step, K(1), is 4.5x10(3)m(-1) at I 0.14m and the rate of isomerization is 400s(-1). In the presence of Mg(2+), ADP binds in a similar fashion to ATP, the rate of the conformation change also being 400s(-1), but with DeltaG(0) for that process being -14kJ.mol(-1). The effect of increasing ionic strength is to decrease K(1), the kinetics of the conformation change being essentially unaltered. Alternative schemes involving a two-step binding process for ATP to subfragment 1 are possible. These are not excluded by the experimental results, although they are perhaps less likely because they imply uncharacteristically slow bimolecular association rate constants.     

Direct photoaffinity labeling of gizzard myosin with vanadate-trapped adenosine diphosphate

The active-site topology of smooth muscle myosin has been investigated by direct photoaffinity-labeling studies with [3H]ADP. Addition of vanadate (Vi) and Co2+ enabled [3H]ADP to be stably trapped at the active site (t1/2 greater than 5 days at 0 degrees C). The extraordinary stability of the myosin.Co2+.[3H]ADP.Vi complex allowed it to be purified free of excess [3H]ADP before irradiation began and ensured that only active-site residues became labeled. Following UV irradiation, approximately 10% of the trapped [3H]ADP became covalently attached at the active site. All of the [3H]ADP incorporated into the 200-kDa heavy chain, confirming earlier results using untrapped [alpha-32P]ATP [Maruta, H., & Korn, E. (1981) J. Biol. Chem. 256, 499-502]. After extensive trypsin digestion of labeled subfragment 1, HPLC separation methods combined with alkaline phosphatase treatment allowed two labeled peptides to be isolated. Sequence analysis of both labeled peptides indicated that Glu-185 was the labeled residue. Since Glu-185 has been previously identified as a residue at the active site of smooth myosin using [3H]UDP as a photolabel [Garabedian, T. E., & Yount, R. G. (1990) J. Biol. Chem. 265, 22547-22553], these results provide further evidence that Glu-185, located immediately adjacent to the glycine-rich loop, is located in the purine binding pocket of the active site of smooth muscle myosin.     

Radioactive labeling and location of specific thiol groups in myosin from fast, slow and cardiac muscles.

1. Based on incorporation of radioactively labeled N-ethylmaleimide, the readily reactive thiol groups of isolated myosin (EC 3.6.1.3) from fast, slow and cardiac muscles could be classified into 3 types. All 3 myosins contain 2 thiol-1, 2 thiol-2 and a variable number of thiol-3 groups per molecule. Both thiol-1 and thiol-2 groups which are essential for functioning of the K+-stimulated ATPase, are located in the heavy chains in all 3 myosin types. 2. The variation in the incorporation pattern of N-ethylmaleimide over the 3 thiol group classes under steady-state conditions of Mg(2+) - ATP hydrolysis allowed different conformations of some reaction intermediates to be characterized. In all 3 types of myosin the hydrolytic cycle of Mg(2+) - ATP was found to be controlled by the same step at 25 degrees C. In all three cases, this rate-limiting step is changed in the same way by lowereing temperature. 3. Using the chemically determined molecular weights for myosin light chains, their stoichiometry was found on the basis of sodium dodecyl sulfate electrophoresis to be 1.2 : 2.1 : 0.8 for light chain-1: light chain-2:light chain-3 per molecule of fast myosin, 2.0 : 1.9 for light chain-1:light chain-2 per molecule of slow myosin and 1.9 : 1.9 for light chain-1:light chain-2 per molecule of cardiac myosin. This qualitative difference in light subunit composition between the fast and the two types of slow myosin is not reflected in the small variations of the characteristics exhibited by the isolated myosins, but rather seems to be connected with their respective myofibrillar ATPase activities.     

Disorder induced in nonoverlap myosin cross-bridges by loss of adenosine triphosphate.

Adenosine triphosphate-dependent changes in myosin filament structure have been directly observed in whole muscle by electron microscopy of thin sections of rapidly frozen, demembranated frog sartorius specimens. In the presence of ATP the thick filaments show an ordered, helical array of cross-bridges except in the bare zone. In the absence of ATP they show two distinct appearances: in the region of overlap with actin, there is an ordered, rigorlike array of cross-bridges between the thick and thin filaments, whereas in the nonoverlap region (H-zone) the myosin heads move away from the thick filament backbone and lose their helical order. This result suggests that the presence of ATP is necessary for maintenance of the helical array of cross-bridges characteristic of the relaxed state. The primary effect of ATP removal on the myosin heads appears to be weaken their binding to the thick filament backbone; released heads that are close to an actin filament subsequently form a new actin-based, ordered array.     

Action of the adenosine triphosphate analog, adenylyl imidodiphosphate in mitochondria.

Adenylyl imidodiphosphate (AMP-PNP), and analog of adenosine triphosphate (ATP), is a potent competitive inhibitor of mitochondrial ATPase activity. It inhibits both the soluble oligomycin-insensitive ATPase (K_i = 9.2 × 10^?7 M) and the bound oligomycin-sensitive APTase (K_i = 1.3 × 10^?6 M). ATPase activity of inside-out submitochondrial preparations are more sensitive to AMP-PNP in the presence of an uncoupler (K_i = 2.0 × 10^?7 M). Mitochondrial ATP-dependent reactions (reversed electron transfer and potassium uptake) do not proceed if ATP is replaced with AMP-PNP; however, the analog does affect these systems. Oxidative phosphorylation of whole mitochondria and submitochondrial preparations were unaffected by AMP-PNP.     

Quantitative labeling of the regulatory subunit of type II cAMP-dependent protein kinase from bovine heart by a photoaffinity analog.

Several methods were compared for estimating the amount of regulatory subunit of an 800-fold purified Type II cAMP-dependent protein kinase from bovine heart. These methods included a reversable binding assay using either cAMP, or 8-N3-[32P]cAMP, photoaffinity labeling with 8-N3-[32P]cAMP, and autophosphorylation of the regulatory subunit of the enzyme. Although the regulatory subunit had a slightly lower affinity for 8-N3-cAMP than for cAMP, the total amount of regulatory subunit could be determined by each of the procedures examined. The results indicate that the photoaffinity analog 8-N3-[32P]cAMP is able to label quantitatively all cAMP-binding sites of the regulatory subunit of this cAMP-dependent protein kinase.