High performance liquid chromatography purification and structural characterization of the subunits of rabbit muscle phosphorylase kinase.
A rapid reverse-phase high performance liquid chromatography (HPLC) method is presented for isolating the alpha, beta, gamma, and delta subunits of rabbit muscle phosphorylase kinase. The HPLC separation allows micropreparative purification of all the subunits with 66-88% recoveries. Relative molecular weights of the subunits as determined by sodium dodecyl sulfate gel electrophoresis in 4, 5, 7, and 10% acrylamide are alpha 132,000, alpha' 127,000, beta 113,000 and gamma 43,000. Amino acid compositions are reported for the HPLC purified subunits. alpha contains about 2 mol of endogenous phosphate/mol of protein and beta, gamma, and delta each contain about 1 mol of phosphate/mol of protein. Despite the identity of delta and calmodulin, essentially no protein-bound phosphate was found associated with bovine brain calmodulin. Holophosphorylase kinase contains about 20 mol of endogenous phosphate/mol of protein. The first NH2-terminal sequence analyses of the alpha, beta, and gamma subunits were determined by Tarr manual Edman degradation. Within the NH2-terminal 23 residues of gamma ( TRDAALPGSHSTHGFYENYESKE . . . ) there are six identities and one conservative interchange with the catalytic subunit of bovine cAMP-dependent protein kinase. The first 17 residues of the NH2-terminal sequence of alpha ( MRSRSNSGVRLDSYARL . . . ) exhibit six identities and one conservative interchange with the transforming protein from the Rous sarcoma virus (Schmidt- Rupin strain) provided a single gap is inserted in the src gene product. Further structural information is required to evaluate the significance of these sequence similarities. The beta subunit has a blocked NH2 terminus.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D046911:Macromolecular Substances; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D010750:Phosphoproteins / Q000032:analysis; D010764:Phosphorylase Kinase / Q000302:isolation & purification*; D011817:Rabbits
D000596:Amino Acids; D046911:Macromolecular Substances; D010750:Phosphoproteins; D010764:Phosphorylase Kinase