Phosphorylation of low molecular weight proteins in purified preparations of rat heart sarcolemma and sarcoplasmic reticulum.
A rat heart sarcolemmal preparation could be obtained in which both 5'-nucleotidase and adenylate cyclase were enriched approx. 9-fold by subjecting a homogenate to a discontinuous sucrose gradient, without the use of a high salt extraction. After incubation of this fraction with Mg[gamma-32P]ATP, the majority of 32P incorporated was present in 24 000- and 9000-dalton protein components. Only when a heart cytosol fraction or a purified cyclic AMP-dependent protein kinase was added, was enhancement of 32P-incorporaton found by addition of cyclic AMP. The 9000- and 24 000-dalton proteins appeared to be interconvertible. The degree of conversion could be affected by changing the temperature during solubilizaion of the membranes in SDS prior to electrophoresis. This suggested that the 24 000-dalton protein does not correspond to phospholamban, first identified by others in canine heart sarcoplasmic reticulum. Moreover, it could be excluded that the 24 000-dalton protein was derived from contaminating myofibrillar troponin I. When the sarcolemmal fraction was preincubated with Ca2+, Mg2+, ATP and oxalate, contaminating sarcoplasmic reticulum vesicles, loaded with calcium oxalate, settled to a greater density in the sucrose gradient. Membrane constituents other than those with enzymatic activity were monitored to confirm the separation between sarcolemmal and sarcoplasmic reticulum membranes: Coomassie blue staining material, sialic acid, cholesterol and phospholipid. The 24 000- and 9000-dalton proteins were equally distributed among the sarolemmal and sarcoplasmic reticulum fractions present in the sucrose gradient. However, the rate of 32P-incorporation in the presence of heart cytosol fraction was much slowr in the sarcoplasmic reticulum than in the sarcolemmal fraction.
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D000242:Cyclic AMP / Q000494:pharmacology; D004789:Enzyme Activation; D006168:Guinea Pigs; D008970:Molecular Weight; D009206:Myocardium / Q000378:metabolism*; D009708:Nucleotidases / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D012508:Sarcolemma / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
D011506:Proteins; D000242:Cyclic AMP; D011494:Protein Kinases; D009708:Nucleotidases; D000262:Adenylyl Cyclases