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R G Yount

Publications and source records attributed to R G Yount.

At least 19 recordsLinked to original sources
PubMed1992

Stability and photochemical properties of vanadate-trapped nucleotide complexes of gizzard myosin in the 6S and 10S conformations: identification of an active-site serine.

The properties of divalent metal.ADP.vanadate (V(i)) complexes of the 6S extended and 10S folded conformations of gizzard myosin before and after UV irradiation have been studied. The half-lives of both 6S and 10S myosin.MgADP.V(i) complexes in the dark at 0 degrees C are on the order of 2 weeks. Brief irradiation with UV light, however, photomodified the enzyme as suggested by changes in the NH(4+)-, K(+)-, and Ca(2+)-ATPase activities, and destabilized the complexes. The 6S complex, when irradiated, released ADP and V(i) rapidly (t1/2 less than or equal to 1 min) as has been observed in comparable experiments with skeletal myosin subfragment 1 (S1) [Grammer et al. (1988) Biochemistry 27, 8408-8415]. The irradiated 10S complex released approximately 20% of the ADP and V(i) rapidly (t1/2 less than or equal to 1 min), but the remainder stayed trapped, possibly as the vanadyl (VO2+).ADP complex, for much longer times (t1/2 approximately 8 h). The site of photomodification was sought by reducing both photomodified 6S and 10S myosin with NaB3H4. Amino acid composition analyses identified [3H]serine as the only labeled residue(s), suggesting that the hydroxymethyl group of serine had been oxidized to an aldehyde as shown previously for photomodified skeletal myosin S1 [Cremo et al. (1989) J. Biol. Chem. 264, 6608-6611]. The 29-kDa NH2-terminal tryptic peptide from the heavy chain was found to contain essentially all of the [3H]serine. Preparations of 6S and 10S [3H]myosin were digested exhaustively with trypsin. An identical [3H]peptide was purified from each preparation and its sequence determined to be Glu169-Asp-Gln-Ser-Ile-Leu-(Cys)-Thr-Gly-[3H]Ser-Gly-Ala-Gly-Ly s183.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗
PubMed1992

Photochemical mapping of the active site of myosin.

The active sites of myosin from skeletal, smooth and scallop muscle have been partly characterized by use of a series of photoreactive analogues of ATP. Specific labelling was attained by trapping these analogues in their diphosphate forms at the active sites by either cross-linking two reactive thiols (skeletal myosin) or by formation of stable vanadate-metal ion transition state-like complexes (smooth muscle and scallop myosin). By use of this approach combined with appropriate chemistry, several key residues in all three myosins have been identified which bind at or near the adenine ring, the ribose ring and to the gamma-phosphate of ATP. This information should aid in the solution of the crystal structure of the heads of myosin and in defining a detailed structure of the ATP binding site.

Adenosine Triphosphate↗
PubMed1991

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.

Adenosine Diphosphate↗
PubMed1991

Mechanics of glycerinated muscle fibers using nonnucleoside triphosphate substrates.

We have investigated the ability of the photoaffinity, nonnucleotide ATP analogues, 2-[(4-azido-2-nitrophenyl) amino] ethyl triphosphate (NANTP) and 2-[(4-azido-2-nitrophenyl) amino] propyl triphosphate (PrNANTP), to support active contraction in glycerinated rabbit psoas fibers. At millimolar concentrations, in the absence of calcium, both analogues relaxed fibers. In the presence of calcium, MgNANTP produced isometric tension and stiffness that were one-half to two-thirds the values obtained in MgATP. Maximum shortening velocity and the calcium-activated, myofibrillar catalyzed rate of hydrolysis were approximately the same for MgNANTP as for MgATP. With MgNANTP as the substrate, increasing concentrations of the diphosphate analogue, MgNANDP, inhibited shortening velocity but did not change isometric tension. The addition of increased concentrations of orthophosphate (P) decreased tension while shortening velocity increased. Thus, the effects of the hydrolysis products of NANTP were quite similar to those observed previously for ADP and P in the presence of MgATP. Taken together, these observations show that MgNANTP binds to, and functions in the active site of myosin in a manner quite analogous to MgATP. Thus, the aryl azido group should serve as a valid photoaffinity label for the purine portion of the active site. In contrast, MgPrNANTP, which differs from MgNANTP only in an extra CH2 spacer between the nitrophenyl ring and the triphosphate moiety did not support isometric tension or active shortening in the presence of calcium. Fiber stiffness increased in the presence of calcium and MgPrNANTP, with a calcium-activated, myofibrillar MgPrNANTPase which was about half that obtained with MgATP. Thus, in the presence of MgPrNANTP, cross-bridges appeared to be cycling through states that were attached to actin, but not producing force.

Adenosine Triphosphate↗
PubMed1990

Direct photoaffinity labeling of gizzard myosin with [3H]uridine diphosphate places Glu185 of the heavy chain at the active site.

The active site of chicken gizzard myosin was labeled by direct photoaffinity labeling with [3H]UDP. [3H] UDP was stably trapped at the active site by addition of vanadate (Vi) and Co2+. The extraordinary stability of the myosin.Co2+.[3H]UDP.Vi complex (t1/2 greater than 5 days at 0 degrees C) allowed it to be purified free of extraneous [3H]UDP before irradiation began. Upon UV irradiation, greater than 60% of the trapped [3H]UDP was photoincorporated into the active site. Only the 200-kDa heavy chain was labeled, confirming earlier results (Maruta, H., and Korn, E. (1981) J. Biol. Chem. 256, 499-502) using [3H]UTP. Extensive tryptic digestion of photolabeled myosin subfragment 1 followed by high performance liquid chromatography separations and removal of nucleotide phosphates by treatment with alkaline phosphatase allowed two labeled peptides to be isolated. Sequencing of the labeled peptides and radioactive counting showed that Glu185 was the residue labeled. Since UDP is a "zero-length" cross-linker, Glu185 is located at the purine-binding pocket of the active site of smooth myosin and adjacent to the glycine-rich loop which binds the polyphosphate portion of ATP. This Glu residue is conserved in smooth and nonmuscle myosins and is the same residue identified previously by [3H]UTP photolabeling in Acanthamoeba myosin II (Atkinson, M. A., Robinson, E. A., Appella, E., and Korn, E. D. (1986) J. Biol. Chem. 261, 1844-1848).

Affinity Labels↗
PubMed1990

Photolabeling of the 6 and 10 S conformations of gizzard myosin with 3'(2')-O-(4-Benzoyl)benzoyl-ATP. Proline 324 is near the active site.

3'(2')-O-(4-Benzoyl)benzoyl-ATP (Bz2ATP) was used as a photoaffinity label of the ATP binding site of unphosphorylated chicken gizzard myosin. Specific photolabeling of the active site of 6 S myosin was assured by forming a stable myosin.Co(II)Bz2ADP.orthovanadate complex (termed trapping) prior to irradiation. Co2+ was used in place of Mg2+ to prevent the known photoreaction of vanadate with myosin which destabilizes the trapped complex. [3H] Bz2ADP.Pi was also stably trapped on gizzard myosin by forming the 10 S folded conformation of the protein in the presence of [3H]Bz2ATP and Mg2+. Irradiation of 6 S myosin containing orthovanadate trapped [3H] Bz2ADP or 10 S trapped [3H]Bz2ADP.Pi gave 32 and 30% covalent incorporation, respectively. The 50-kDa and precursor 68-kDa tryptic peptides of the subfragment-1 heavy chain derived from both forms of myosin were found to contain essentially all of the covalently attached [3H]Bz2ADP. Parallel experiments with untrapped [3H]Bz2ADP showed extensive nonspecific labeling of all of the major tryptic peptides and the light chains. Eight labeled peptides, isolated from 6 and 10 S photolabeled myosin, contained the sequence G319-H-V-P-I-X-A-Q326, where X corresponds to labeled proline 324. [14C]Bz2ADP was previously shown to label serine 324 in skeletal subfragment-1 (Mahmood, R., Elzinga, M., and Yount, R. G. (1989) Biochemistry 28, 3989-3995), which corresponds to alanine 325 in the gizzard sequence. Thus, this region of the 50-kDa tryptic fragment, near the nucleotide binding site, in both skeletal and smooth muscle myosins, must fold in essentially the same manner.

Adenosine Triphosphate↗
PubMed1990

Interaction of myosin subfragment 1 with fluorescent ribose-modified nucleotides. A comparison of vanadate trapping and SH1-SH2 cross-linking.

The environment near the ribose binding site of skeletal myosin subfragment 1 (S1) was investigated by use of two adenosine 5'-diphosphate analogues with fluorescent groups attached at the 2'- and 3'-hydroxyls of the ribose ring. We have compared steady-state and time-resolved fluorescent properties of the reversibly bound S1-nucleotide complexes and the complexes generated by N,N'-p-phenylenedimaleimide (pPDM) thiol cross-linking or vanadate (Vi) trapping. A new fluorescent probe, 2'(3')-O-[N-[2-[[[5-(dimethylamino)naphthyl]sulfonyl] amino]ethyl]carbamoyl]adenosine 5'-diphosphate (DEDA-ADP), which contains a base-stable carbamoyl linkage between the ribose ring and the fluorescent dansyl group, was synthesized and characterized. For comparison, we performed parallel experiments with 2'(3')-O-(N-methylanthraniloyl)adenosine 5'-diphosphate (MANT-ADP) [Hiratsuka, T. (1983) Biochim. Biophys. Acta 742, 496-508]. Solute quenching studies indicated that both analogues bound reversibly to a single cleft or pocket near the ribose binding site. However, steady-state polarization measurements indicated that the probes were not rigidly bound to the protein. The quantum yields of both fluorophores were higher for the complexes formed after trapping with pPDM or Vi than for the reversibly bound complexes. Both DEDA-ADP and MANT-ADP, respectively, had nearly homogeneous lifetimes free in solution (3.65 and 4.65 ns), reversibly bound to S1 (12.8 and 8.6 ns), and trapped on S1 by pPDM (12.7 and 8.7 ns) or Vi (12.8 and 8.6 ns). In contrast to the quantum yields, the lifetimes were not increased upon trapping, compared to those of the reversibly bound states. These results suggested that static quenching in the reversibly bound complex was relieved upon trapping. Taken together, the results suggest that there was a conformational change near the ribose binding site upon trapping by either pPDM or Vi. On the basis of the quantum yield, lifetime, polarization, and solute accessibility studies, we could not detect differences between the S1-pPDM-nucleotide analog complex and the S1-Vi-nucleotide analogue complex for either analogue. Thus, previously observed differences with the adenine modified nucleotide analogue 1,N6-ethenoadenosine diphosphate (epsilon ADP) could not be detected with these ribose-modified probes, indicating that structural differences may be localized to the adenine binding site and not transmitted to the region near the ribose ring.

Animals↗
PubMed1979

Active site trapping of nucleotides by crosslinking two sulfhydryls in myosin subfragment 1.

Studies with reagents that crosslink two thiol groups have shown that it is possible to trap nucleotides at the active site of myosin chymotryptic subfragment 1. Subfragment 1 incorporates nearly stoichiometric quantities of [14C]ATP or [14C]ADP in a manner that depends linearly on the extent of inactivation by either N,N'-p-phenylenedimaleimide or Co(II)phenanthroline/[Co(III)(phenanthroline)2CO3]+ complexes. The incorporated radioactive nucleotide is retained after gel filtration, even when the enzyme derivatives are stored in the presence of EDTA or nonradioactive nucleotides (t 1/2 approximately 5 days). The nucleotide incorporated is not covalently bound because HClO4 denaturation allows immediate release of bound nucleotide. The nucleotide retained is ADP because the gamma-phosphate of [gamma-32P]ATP is lost after trapping. Subfragment 1 inactivated as above does not bind the competitive inhibitor adenosine 5'-[beta, gamma-imido]triphosphate, indicating that the active site is blocked. It is proposed that a jawlike nucleotide cleft closes on MgADP or MgATP, which can be locked shut by crosslinking two thiol groups by reaction with N,N'-p-phenylenedimaleimide or cobalt phenanthroline complexes.

Adenosine Diphosphate↗
PubMed1977

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.

Adenosine Triphosphatases↗
PubMed1977

Reaction of cardiac myosin with a purine disulfide analog of adenosine triphosphate. II. Stoichiometry and subunit location of labeling.

The reaction of bovine cardiac myosin with the site-specific purine disulfide analog of ATP, 6,6'-dithiobis (inosinyl imidodiphosphate), was studied to determine the stoichiometry of labeling and subunit location of the reactive cysteines. The analog inactivates myosin by forming a mixed disulfide between the thiopurine nucleotide and certain key cysteines. The thiopurine nucleotide was displaced quantitatively by 14CN to form the more stable thiocyanato-enzyme derivatives. In cardiac myosin, the reactive cysteines could be categorized into three classes, nonessential, critical, and noncritical. The modification of the critical cysteines (two per myosin) inactivated the EDTA and Ca2+ ATPase activities, the latter to a lesser extent. The nonessential cysteines (two to three per myosin) and the noncritical cysteines (two per myosin), differentiated by their rates of reaction, had no effect on the ATPase activities after modification. Thiocyanato-modified myosin was analyzed by sodium dodecyl sulfate gel electrophoresis to determine the distribution of 14CN in the subunits. The critical cysteines were found on the 21,000-dalton light chain (LC1) and the noncritical cysteines on the heavy chains. More specifically, the critical cysteine modified in cardiac LC1 (determined from the products after cyclization and chain cleavage at the thiocyanatoalanyl residues) was shown to be the thiol residue whose surrounding amino acid sequence is homologous to that of the single cysteine of the skeletal myosin alkali light chains, confirming the likely similar structure and function of these light chains in the two different muscle types.

Adenosine Triphosphatases↗
PubMed1977

Observations on the kinetics, subunit composition, and sulfhydryl reactivity of myosin from Physarum polycephalum.

A highly purified preparation of myosin from Physarum polycephalum has been shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis to contain heavy chains and only one molecular weight class of light chains, of approx. 15 000 daltons. Kinetic investigations of the Ca2+-ATPase and Mg2+-ATPase (ATP phosphohydrolases, EC 3.6.1.3) at pH 8.0 gave Km and V values of 17.3 muM and 1.25 mumol Pi/min per mg, and 2.4 muM and 0.12 mumol Pi/min per mg, respectively. Adenylyl imidodiphosphate, a beta-gamma-imido ATP analog, inhibited the ATPase activity of Physarum myosin competitively with Ki values equal to 350 and 12 muM in the presence of Ca2+ and Mg2+, respectively. The ATPase activity of Physarum myosin was inhibited at a very low rate (t1/2 = 24 h) by the ATP analog, 6,6'-dithiobis(inosinyl imidodiphosphate), with concentrations of inhibitor previously shown to inactivate (t1/2 approximately 10 min) skeletal and cardiac myosins rapidly by reacting with key cysteines.

Actomyosin↗
PubMed1975

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.

Actins↗
PubMed1975

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.

Adenosine Triphosphatases↗
PubMed1975

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.

Adenosine Triphosphatases↗