Proteolysis of smooth muscle myosin by Staphylococcus aureus protease: preparation of heavy meromyosin and subfragment 1 with intact 20 000-dalton light chains.
The proteolysis of gizzard myosin by Staphylococcus aureus protease produces both heavy meromyosin and subfragment 1 in which the 20 000-dalton light chains are intact, and conditions are suggested for the preparation of each. Cleavage of the myosin heavy chain to produce subfragment 1 is dependent on the myosin conformation. Proteolysis of myosin in the 10S conformation yields predominantly heavy meromyosin, and myosin in the 6S conformation yields mostly subfragment 1 and some heavy meromyosin. Two sites are influenced by myosin conformation, and these are located at approximately 68 000 and 94 000 daltons from the N-terminus of the myosin heavy chain. The latter site is thought to be located at the subfragment 1-subfragment 2 junction, and cleavage at this site results in the production of subfragment 1. The time courses of phosphorylation of both heavy meromyosin and subfragment 1 can be fit by a single exponential. The actin-activated Mg2+-ATPase activity of heavy meromyosin is markedly activated by phosphorylation of the 20 000-dalton light chains. From the actin dependence of Mg2+-ATPase activity the following values are obtained: for phosphorylated heavy meromyosin, Vmax approximately 5.6 s-1 and Ka (the apparent dissociation constant for actin) approximately 2 mg/mL; for dephosphorylated heavy meromyosin, Vmax approximately 0.2 s-1 and Ka approximately 7 mg/mL. The actin-activated ATPase activity of subfragment 1 is not influenced by phosphorylation, and Vmax and Ka for both the phosphorylated and dephosphorylated forms are 0.4 s-1 and 5 mg/mL, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Ikebe
M
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Hartshorne
D J
DJ
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002850:Chromatography, Gel; D010450:Endopeptidases / Q000378:metabolism*; D008666:Metalloendopeptidases; D008970:Molecular Weight; D009130:Muscle, Smooth / Q000201:enzymology*; D015879:Myosin Subfragments / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D011189:Potassium Chloride / Q000494:pharmacology; D013997:Time Factors; D014422:Turkeys
D015879:Myosin Subfragments; D010446:Peptide Fragments; D011189:Potassium Chloride; D010450:Endopeptidases; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase; D009218:Myosins; D000252:Calcium-Transporting ATPases