Partial characterization of protein kinase-catalyzed phosphorylation of low molecular weight proteins in purified preparations of pigeon heart sarcolemma and sarcoplasmic reticulum |
| |
Authors: | Horst Will Tatyana S Levchenko Dmitri O Levitsky Vladimir N Smirnov Albert Wollenberger |
| |
Institution: | 1. Division of Cellular and Molecular Cardiology, Central Institute of Heart and Circulatory Regulation Research, Academy of Sciences of the D.D.R., 115 Berlin-Buch, Germany;2. Laboratory of Cardiac Metabolism, All-Union Research Center of Cardiology, Academy of Medical Sciences of the U.S.S.R., Moscow, U.S.S.R. |
| |
Abstract: | Pigeon heart microsomes contain three minor size protein kinase substrates of minimal molecular weights of 22 000, 15 000, and 11 500, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When the microsomes were partially loaded with calcium oxalate and subjected to rate zonal and isopynic centrifugations in sucrose density gradient columns, the 22 000 and the 15 000 dalton proteins settled in the heaviest fraction, which was composed mainly of vesicles of sarcoplasmic reticular membranes; the 11 500 dalton protein was concentrated in the lightest fractions, which consisted chiefly of vesicles of sarcolemmal origin. During incubation of the membrane fractions with Mgγ-32P]ATP significant amounts of 32P were incorporated into all these proteins. Incorporation of 32P into the 15 000 dalton protein was moderately and 32P incorporation into the 22 000 dalton protein was markedly enhanced in the presence of exogenous soluble cyclic AMP-dependent protein kinase and cyclic AMP. The phosphorylation of the three proteins was virtually unaffected by CA2+ concentrations up to 0.1 mM and by ethyleneglycol-bis(β-aminoethylether)-N,N′-tetraacetic acid in the absence of added Ca2+.Phosphorylation of the 22 000 and the 11 500 dalton proteins occurred mainly at serine residues. In the 15 000 dalton protein threonine residues were the main site of endogenous phosphorylation. Nearly equal amounts of 32P]-phosphate were incorporated into threonine and serine residues of this protein when phosphorylation was supported by exogenous cyclic AMP-dependent protein kinase and cyclic AMP.The 15 000 dalton protein could be removed from its membrane attachment by extraction with an acidic chloroform/methanol mixture. This step opens the way for the purification of this membrane-bound protein kinase substrate. |
| |
Keywords: | cyclic AMP adenosine 3′ 5′-monophosphate EGTA N′-tetraacetic acid |
本文献已被 ScienceDirect 等数据库收录! |
|