Preparation and functional characterization of a catalytically active fragment of phosphorylase kinase.
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 gamma-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, with S20,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 phosphorylase a and b, 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[gamma-P32] ATP results in the reaction of 0.7 mol 32P/mol fragment. When the catalytic subunit of the cAMP-dependent protein kinase is also present, the amount of 32P 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 gamma-subunit of phosphorylase kinase.
Malencik
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DA
Department of Biochemistry and Biophysics, Oregon State University, Corvallis 97331-7305.
Zhao
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Z
Anderson
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SR
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009132:Muscles; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification; D010764:Phosphorylase Kinase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D014461:Ultracentrifugation
D046911:Macromolecular Substances; D010446:Peptide Fragments; D010748:Phosphopeptides; D010764:Phosphorylase Kinase; D010450:Endopeptidases; D002918:Chymotrypsin