Phosphorylation of a low molecular weight polypeptide in beef heart Na plus, K plus-ATPase preparations.

Sodium dodecyl sulfate-polyacrylamide gel profiles of a NaI-treated beef heart Na⁺,K⁺-ATPase preparation revealed the presence of two protein kinase substrates of low molecular weight, whereas a more purified citrate beef heart Na⁺,K⁺-ATPase preparation contained one low molecular weight polypeptide substrate. This enzyme preparation was phosphorylated in the presence of protein kinase, and phosphorylation was inhibited by protein kinase inhibitor. The phosphorylated product was identified as a phosphoester. Half maximal stimulation of protein kinase-catalyzed phosphorylation occurred at approximately 9 × 10^?8m cyclic AMP. The low molecular weight (11,700) protein kinase substrate present in the heart preparations was eluted from polyacrylamide slab gels. The polypeptide fraction was reelectrophoresed and the polypeptide was removed from the gels, hydrolyzed, and analyzed for amino acid content. This polypeptide was different from other low molecular weight protein kinase substrates including troponin components, myosin light chains, and histones and is most likely of plasma membrane origin.