PREPARATION AND FUNCTIONAL-CHARACTERIZATION OF A CATALYTICALLY ACTIVEFRAGMENT OF PHOSPHORYLASE-KINASE

Limited proteolysis of rabbit muscle phosphorylase kinase catalyzed by chymotrypsin generates a 33kD product whose kinase activity is indepe ndent of both calcium and pH over the range of 6.8 to 8.3 (Malencik, D .A. & Fischer, E.H. Calcium and Cell Function III: 161-188, 1982). Thi s active preparation consists of three related species containing resi dues 1-290, 1-296, and 1-298 of the 44.7kD gamma-subunit of phosphoryl ase kinase (Harris, W.R., Malencik, D.A., Johnson, C.M., Carr, S.A., R oberts, G.D., Byles, C.E., Anderson, S.R., Heilmeyer, L.M.G., Fischer, E.H. & Crabb, J.W. J. Biol. Chem. 265: 11740-11745, 1991). Good recov eries of catalytic activity-with varying degrees of calcium dependence - result upon the digestion of phosphorylase kinase with assorted pro teases. However, especially high yields of the chymotryptic fragment a re obtainable, with purification on an Ultrogel-34 column and a DEAE S epharose CL-6B column giving 23% of the maximum possible protein. Phys ical characterization shows that the 33 kD chymotryptic fragment is gl obular, with s(20,w) = 2.9S, and that it has an isoelectric point of 5 .3. Our continuous catalytic assay, based on differences in the bindin g of the fluorescent dye 1-anilinonaphthalene-8-sulfonate by phosphory lase a and b, shows that, on a molar basis, the activity of the fragme nt is 2.8 fold greater than that of phosphorylase kinase (Malencik, D. A., Zhao, Z. and Anderson, S.R. Biochem. Biophys. Res. Comm. 174: 344- 350, 1991). The active fragment also undergoes autophosphorylation. In cubation with Mg[gamma-P-32] ATP results in the reaction of 0.7 mol P- 32/mol fragment. When the catalytic subunit of the cAMP-dependent prot ein kinase is also present, the amount of P-32 incorporated increases to 1.1 mol/mol. In the former case, phosphorylation occurs primarily a t Ser(30) while in the latter an additional reaction takes place at Se r(81). The phosphopeptides correspond to sequences occurring in the ga mma-subunit of phosphorylase kinase.