Correlation of enzymatic properties and conformation of bovine erythrocyte myosin.
Myosin was purified from bovine erythrocytes by chromatography on DEAE-cellulose, Sepharose CL-4B, hydroxylapatite, and DEAE-5PW. The yield was about 200 micrograms/L of packed cells. From SDS-polyacrylamide gels, the purity was estimated to be greater than 95%. The bovine erythrocyte myosin is composed of heavy chains of 200 kDa and light chains of 20 and 17 kDa, in a molar stoichiometry of 1. Myosin was also purified from human erythrocytes by the same method. The molecular weights of two light chains were 26K and 19.5K which confirmed the earlier reports [Fowler, V. M., Davis, J. Q., & Bennet, V. (1985) J. Cell Biol. 100, 47-55; Wong, A. J., Kiehart, D. P., & Pollard, T.D. (1985) J. Biol. Chem. 260, 46-49]. Phosphorylation by gizzard myosin light chain kinase, to a level of 1 mol of phosphate/mol of 20-kDa light chain, increased actin-activated ATPase, and the extent of activation was dependent on the MgCl2 concentration. Both Ca2+-ATPase and Mg2+-ATPase activities were dependent on KCl concentration and markedly decreased below 0.3 M KCl. Mg2+-ATPase of phosphorylated myosin, while more resistant to decreasing ionic strength, was also decreased below 0.2 M KCl. These results are similar to those obtained with smooth muscle myosin and suggest that the 10S-6S transition occurs. In confirmation of this, gel filtration, viscosity, and electron microscopy (rotary shadowing) show that erythrocyte myosin forms extended and folded conformations in high and low salt, respectively. It is proposed that each conformation is characterized by distinct enzymatic properties.(ABSTRACT TRUNCATED AT 250 WORDS)
Higashihara
M
M
Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106.
Hartshorne
D J
DJ
Craig
R
R
Ikebe
M
M
D000251:Adenosine Triphosphatases / Q000097:blood* / Q000302:isolation & purification; D000818:Animals; D002417:Cattle; D002845:Chromatography; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D004912:Erythrocytes / Q000201:enzymology*; D006882:Hydroxyapatites; D007700:Kinetics; D008970:Molecular Weight; D009218:Myosins / Q000097:blood* / Q000302:isolation & purification; D011487:Protein Conformation
D006882:Hydroxyapatites; D017886:Durapatite; D000251:Adenosine Triphosphatases; D009218:Myosins