Studies on an endogenous substrate of wheat germ protein kinase.
An endogenous phosphoryl acceptor has been purified 138-fold from wheat germ extracts. This protein, termed T-substrate, is far more effective than casein or phosvitin as a phosphoryl acceptor for a wheat germ kinase recently purified by our laboratory. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the T-substrate preparation is not homogeneous. The T-substrate, which migrates at a Mr = 48,000, constitutes about 90% of the total protein stain on the gel and is the only protein component which is phosphorylated by the wheat germ kinase. The hydrodynamic properties of the T-substrate have been determined by gel filtration and glycerol density gradient centrifugation. The protein exhibits a Stokes radius of 65.5 A, a sedimentation coefficient of 3.85 S, a frictional ratio of 2.11, and a molecular weight of approximately 104,000. The results suggest that the wheat germ T-substrate is a dimer. The protein exhibits a greater substrate specificity for the wheat germ kinase than for the cyclic AMP-dependent and several independent protein kinases isolated from rabbit skeletal muscle and human erythrocytes. The T-substrate can be maximally phosphorylated by the wheat germ kinase to the extent of about 8 mol of phosphate/48,000 g of protein. Complete dephosphorylation of the phospho-T-substrate occurred upon treatment with phosphatases. The phosphorylated amino acid was identified by high voltage electrophoresis of an acid hydrolysate of 32P-T-substrate. The results indicate that phosphorylation occurs mainly on the seryl residues of T-substrate.
D000818:Animals; D047388:Casein Kinases; D004912:Erythrocytes / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology; D010750:Phosphoproteins; D010766:Phosphorylation; D010940:Plant Proteins / Q000302:isolation & purification*; D010944:Plants / Q000201:enzymology*; D011494:Protein Kinases / Q000378:metabolism*; D011817:Rabbits; D013379:Substrate Specificity; D014908:Triticum / Q000201:enzymology
D010750:Phosphoproteins; D010940:Plant Proteins; C034921:T-substrate protein, Triticum aestivum; D011494:Protein Kinases; D047388:Casein Kinases