Proteolytic fragmentation of bovine heart heavy meromyosin

Tada, Michihiko; Bailin, Gary; Bárány, Kate; Bárány, Michael

doi:10.1021/bi00840a029

Cited by 46 publications

(8 citation statements)

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“…Because the total protein extracted was ~30.5% MHC and ~25.1% actin, 1,008 μg myofibrillar protein should consist of 307 μg MHC and 253 μg actin. When the MHC and actin bands are cut from the gel and hydrolyzed, the MHC should yield 31 μg leucine and the actin should yield 20 μg leucine, with the assumption that MHC and actin are 10 and 7.8% leucine by weight, respectively (12,22). We have found these amounts of leucine to be adequate for about five MHC and three actin determinations of [ 13 C]leucine by GC-C-IRMS.…”

Section: Resultsmentioning

confidence: 89%

Isolation of human skeletal muscle myosin heavy chain and actin for measurement of fractional synthesis rates

Hasten

Morris

Ramanadham

et al. 1998

American Journal of Physiology-Endocrinology and Metabolism

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Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), we have developed a simple method to isolate myosin heavy chain (MHC) and actin from small (60-80 mg) human skeletal muscle samples for the determination of their fractional synthesis rates. The amounts of MHC and actin isolated are adequate for the quantification of [ 13 C]leucine abundance by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Fractional synthesis rates of mixed muscle protein (MMP), MHC, and actin were determined in six healthy young subjects (27 ± 1 yr) after they received a 14-h intravenous infusion (prime = 7.58 μmol/kg body wt, constant infusion = 7.58 μmol·kg body wt −1 ·h −1 ) of [1-13 C]leucine. The fractional synthesis rates of MMP, MHC, and actin were found to be 0.0468 ± 0.0048, 0.0376 ± 0.0033, and 0.0754 ± 0.0078%/h, respectively. Overall, the synthesis rate of MHC was 20% lower (P = 0.012), and the synthesis rate of actin was 61% higher (P = 0.060, not significant) than the MMP synthesis rate. The isolation of these proteins for isotope abundance analysis by GC-C-IRMS provides important information about the synthesis rates of these specific contractile proteins, as opposed to the more general information provided by the determination of MMP synthesis rates.Keywords muscle protein synthesis; amino acid metabolism; protein metabolism; stable isotope tracers; mass spectrometry MOST STUDIES that have used stable isotope tracer methodology to determine the fractional synthesis rate of human muscle protein have reported the rate of "mixed" muscle protein (MMP) synthesis (14,15,23,24,26,27). A problem with the measurement of MMP synthesis is that it reflects the average rate of synthesis of several muscle proteins in the sample (i.e., contractile, enzymatic, mitochondrial). Changes in the synthesis rate of Copyright © 1998 the American Physiological Society Address for reprint requests: K. E. Yarasheski, Washington Univ. School of Medicine, 660 S. Euclid Ave., Box 8127, St. Louis, MO 63110.. NIH Public Access Author ManuscriptAm J Physiol. Author manuscript; available in PMC 2014 August 21.Published in final edited form as: Am J Physiol. 1998 December ; 275(6 0 1): E1092-E1099. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript individual contractile proteins could be confounded by corresponding or opposing changes in the synthesis rates of other proteins in the MMP sample. In particular, Rooyackers et al. (19) found that the rate of mitochondrial protein synthesis was ~95% higher than that of MMP in young subjects. Therefore, isolating specific contractile proteins from human muscle samples obtained during a stable isotopically labeled amino acid (e.g., [1-13 C]leucine) administration protocol, and measuring the amount of labeled amino acid incorporated into these specific contractile proteins by use of gas isotope ratio mass spectrometry (IRMS) would provide a more refined approach to in vivo studies of muscle contractile protein metabolism.Recent effort...

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Section: Resultsmentioning

confidence: 89%

Isolation of human skeletal muscle myosin heavy chain and actin for measurement of fractional synthesis rates

Hasten

Morris

Ramanadham

et al. 1998

American Journal of Physiology-Endocrinology and Metabolism

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“…Concentrations of cardiac myosin and myosin-Si were determined from absorption at 280 nm using extinction coefficients of 0.53 and 0.64, respectively (14). The concentration of rabbit skeletal muscle actin was determined using the micro-Biuret procedure (15) standardized with bovine serum albumin.…”

Section: Methodsmentioning

confidence: 99%

ADP dissociation from actomyosin subfragment 1 is sufficiently slow to limit the unloaded shortening velocity in vertebrate muscle.

Siemankowski¹,

Wiseman²,

White³

1985

Proc. Natl. Acad. Sci. U.S.A.

413

318

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The rate constant for dissociation of ADP from actomyosin subfragment 1 (Si) has been measured in this laboratory and elsewhere for a variety of vertebrate muscle types. We have made the following observations: (i) In solution, the dissociation of ADP from actomyosin-Sl limits the rate of dissociation of actomyosin-Sl-ADP by ATP and, presumably, also limits the rate of crossbridge detachment in contracting muscle. (it) For muscle types in which the rate of ADP dissociation from actomyosin-Sl is slow enough to measure using stopped-flow methods, the rate constants are nearly the same as the theoretical value for the minimum allowable rate constant for dissociation of an attached crossbridge. Therefore, ADP dissociation is sufficiently slow to be the molecular step that limits the maximum shortening velocity of these muscles. (i) Variation with muscle type of the rate constant for ADP dissociation may be a general phylogenetic mechanism for regulating shortening velocity.that one or more of the rate constants of the ATP hydrolysis mechanism limits the muscle contraction rate. For a molecular step to limit the shortening velocity of muscle, it must have the following attributes:(i) The rate constant for such a step must be consistent with both the maximum working length of an individual crossbridge and the rate of axial displacement of the thick and thin filaments observed in contracting muscle. The minimum allowable rate constant, kmin, for the conversion of an attached crossbridge state in muscle (corresponding to the AM states in the top line of Eq. 1) to other attached or detached states (corresponding to the M states in the bottom line of Eq. 1) can be estimated from the maximum rate of contraction (the unloaded shortening velocity) and the distance over which a crossbridge can remain attached, using Eq. 2, Muscle contraction is thought to occur as the result of a cyclic association and dissociation of crossbridges formed between myosin molecules in the thick filaments and F-actin molecules in the thin filament, involving concomitant hydrolysis of ATP. This cycle can lead to relative sliding of the actin and myosin filaments and result in the production of work. A rationale for studying the kinetic mechanism of ATP hydrolysis by actomyosin-Sl in solution is that it may directly relate to the observed physiological properties of muscle. A condensed version of the kinetic mechanism, consistent with recent observations (1, 2), is shown in Eq. 1. The second-order reactions of nucleotide and phosphate binding have been shown to be two-step reactions (3-5), but they are condensed here to single steps:where M represents myosin subfragment-1 (Si) and AM represents actomyosin S1. Different types of vertebrate muscles have shortening velocities that vary by almost two orders of magnitude. Barany showed that the steady-state rate of hydrolysis of MgATP by actomyosin is correlated with muscle shortening velocity (6).However, it has not been demonstrated that the rate of ATP hydrolysis directly limits shortening ve...

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“…The myosin and heavy meromyosin were purified by column chromatography [4,5]. Cardiac myosin was prepared [6] from fresh bovine hearts, and purified in the same way as skeletal myosin by ion-exchange chromatography on cellulose A50. Subfragment 1 was prepared by papain hydrolysis, followed by chromatography on a DEAEcellulose (DE52) column, following Taylor and Weeds [7].…”

Section: Methodsmentioning

confidence: 99%

Interaction of ADP with Skeletal and Cardiac Myosin and Their Active Fragments Observed by Proton Release

Kardami

Bruin

Gratzer

1979

European Journal of Biochemistry

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The technique of proton release measurement has been used to explore the binding of ADP to skeletal and cardiac myosins and their active fragments in a variety of conditions. It has proved possible to obtain binding profiles on intact myosin in the filamentous, undissolved form in physiological solvent conditions. Binding constants are given. At higher ionic strength (0.5 M potassium chloride) the binding profile of magnesium-ADP is compatible with the presence of two types of site, differing from one another both in respect of affinity and the number of protons released per site. Studies with cardiac myosin reveal no such indications of heterogeneity, and are consistent with the presence of a single population of thermodynamically indistinguishable sites. In the absence of divalent cations, in solutions containing potassium ions and EDTA, ADP binds with absorption rather than liberation of protons. The pH profile of proton absorption at saturation can be fitted in terms of an ionising group with an unperturbed pK of 9.4, and at least one of lower pK (5.9). The dissociation constant (pH 8 at 5 "C) is about 8 pM, and the affinity for uncomplexed ADP is thus only slightly weaker than that for magnesium-ADP.In a previous paper [l] it was shown that the interaction of rabbit skeletal heavy meromyosin with its natural ligand, magnesium-ADP, leads to the liberation of protons through the perturbation of ionising groups in or near the binding sites. This effect was used to obtain binding profiles and associated equilibrium constants, and it was shown that the method affords arguably the most precise means of studying these interactions. Here follows a more detailed exploration of the scope of the method. Binding curves have been determined for magnesium-ADP to intact myosin in physiological solvent conditions, showing that the method is applicable to undissolved protein and that the binding behaviour of heavy meromyosin approximates closely to that of the undegraded parent molecule. Binding data have also been obtained for cardiac myosin and its subfragment 1. Results obtained at high ionic strength (0.5 M) indicate that the population of binding sites present in a preparation of purified skeletal (but not apparently cardiac) myosin may be heterogeneous. Finally, a study of the binding of ADP in the presence of potassium as the only supporting cation is presented. In the absence of the divalent metal ion, protons are absorbed rather than Enzyme. ATPase (EC 3.6.1.3).liberated. The binding constant has been measured, as has the pH dependence of proton absorption. This leads to an assignment of the pK values of the ionising groups perturbed by attachment of the ligand. MATERIALS AND METHODSRabbit skeletal muscle myosin was prepared by the method of Perry [2] and heavy meromyosin by chymotryptic hydrolysis [3]. The myosin and heavy meromyosin were purified by column chromatography [4,5]. Cardiac myosin was prepared [6] from fresh bovine hearts, and purified in the same way as skeletal myosin by ion-exchange chromatography...

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Proteolytic fragmentation of bovine heart heavy meromyosin

Cited by 46 publications

References 20 publications

Isolation of human skeletal muscle myosin heavy chain and actin for measurement of fractional synthesis rates

Isolation of human skeletal muscle myosin heavy chain and actin for measurement of fractional synthesis rates

ADP dissociation from actomyosin subfragment 1 is sufficiently slow to limit the unloaded shortening velocity in vertebrate muscle.

Interaction of ADP with Skeletal and Cardiac Myosin and Their Active Fragments Observed by Proton Release

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