Reversible dissociation of dog cardiac myosin regulatory light chain 2 and its influence on ATP hydrolysis.
The regulatory light chains of dog heart myosin were removed by digestion with myopathic hamster neutral protease. The heavy chains were also cleaved to an extent of 15%, but a homogeneous, rod-free LC2-deficient myosin was obtained by ion-exchange chromatography. A similar approach was used to prepare LC2-deficient heavy meromyosin. Neither Ca2+- nor K+-EDTA-activated ATPases were affected by LC2 removal. The Lineweaver-Burk plots for actin-activated ATPase in 25 mM KCl were biphasic giving a Vmax of 1.54 s-1 for control and LC2-recombined myosins and 1.08 s-1 for LC2-deficient myosin at low actin concentrations. At high actin concentrations, the Vmax for control and recombined myosins was 2.33 s-1 and 1.39 s-1 for LC2-deficient myosin. Increasing the KCl concentration in the reaction mixtures resulted in more linear plots without suppressing the 35-45% decrease in Vmax that accompanied LC2 removal. The results from assays with control and LC2-deficient heavy meromyosin performed in the absence of KCl, paralleled those obtained with myosin. The latter was also assayed in the presence of equimolar concentrations of C-protein in 50 mM KCl: C-protein induced a significant increase in the actin-activated ATPase of both control and LC2-recombined myosins, with no effect on LC2-deficient myosin. The Vmax for actin-activation in the presence of C-protein was 2.38 s-1, 0.83 s-1, and 1.71 s-1 for control, LC2-deficient, and recombined myosins, respectively. The enhancement of actin-activation in both the control and LC2-recombined myosins represents a possible role for C-protein in a LC2-mediated potentiation of actomyosin ATPase.
Margossian
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D000251:Adenosine Triphosphatases / Q000032:analysis*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins; D006224:Cricetinae; D004285:Dogs; D004492:Edetic Acid / Q000494:pharmacology; D010450:Endopeptidases / Q000494:pharmacology; D006868:Hydrolysis; D007700:Kinetics; D009124:Muscle Proteins / Q000494:pharmacology; D009206:Myocardium / Q000201:enzymology*; D015879:Myosin Subfragments; D009218:Myosins / Q000032:analysis*; D015260:Neprilysin; D000099163:Myosin Binding Protein C
D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate; D002352:Carrier Proteins; D004492:Edetic Acid; D010450:Endopeptidases; D009124:Muscle Proteins; D015879:Myosin Subfragments; D009218:Myosins; D015260:Neprilysin; D000099163:Myosin Binding Protein C