, Volume 127-128, Issue 1, pp 31-43

Preparation and functional characterization of a catalytically active fragment of phosphorylase kinase

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Abstract

Limited proteolysis of rabbit muscle phosphorylase kinase catalyzed by chymotrypsin generates a 33 kD product whose kinase activity is independent 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). This active preparation consists of three related species containing residues 1–290, 1–296, and 1–298 of the 44.7 kD γ-subunit of phosphorylase kinase (Harris, W.R., Malencik, D.A., Johnson, C.M., Carr, S.A., Roberts, 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 recoveries of catalytic activity — with varying degrees of calcium dependence — result upon the digestion of phosphorylase kinase with assorted proteases. However, especially high yields of the chymotryptic fragment are obtainable, with purification on an Ultrogel-34 column and a DEAE Sepharose CL-6B column giving 23% of the maximum possible protein.

Physical characterization shows that the 33 kD chymotryptic fragment is globular, withs 20,w=2.9S, and that it has an isoelectric point of 5.3. Our continuous catalytic assay, based on differences in the binding of the fluorescent dye 1-anilinonaphthalene-8-sulfonate by phosphorylasea andb, shows that, on a molar basis, the activity of the fragment 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. Incubation with Mg[γ-P32] ATP results in the reaction of 0.7 mol32P/mol fragment. When the catalytic subunit of the cAMP-dependent protein kinase is also present, the amount of32P incorporated increases to 1.1 mol/mol. In the former case, phosphorylation occurs primarily at Ser30 while in the latter an additional reaction takes place at Ser81. The phosphopeptides correspond to sequences occurring in the γ-subunit of phosphorylase kinase.

The expected two to three fold increases in fluorescence intensity occur when assays are conducted with solutions containing 20 mM Tris chloride and no glycerophosphate.