Phosphorylase kinase from bovine stomach smooth muscle: a Ca2(+)-dependent protein kinase associated with an actin-like molecule.
Phosphorylase kinase was purified (110-fold) from bovine stomach smooth muscle by a procedure involving DEAE-cellulose chromatography, ammonium sulfate fractionation and glycerol density ultracentrifugation. On sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) the final enzyme preparation shows a single protein band of 43 kDa. The purified protein exhibits a close similarity with bovine aortic actin, as revealed by amino acid analysis and sequencing of a tryptic decapeptide fragment, although it differs widely from actin in several respects. In our effort to separate phosphorylase kinase activity from the 43 kDa protein we used a variety of chromatographic procedures, but in all cases the catalytic activity (when eluted) was accompanied by the 43 kDa protein band. Bovine stomach phosphorylase kinase exhibits an apparent molecular mass of 950 kDa, it shows a low Vmax value for phosphorylase b (85 nmol.min-1.mg-1), a pH 6.8/8.2 activity ratio of 0.23, it has an absolute requirement for Ca2+ and it is activated 1.8-fold by Ca2+/calmodulin. Furthermore, the protein kinase activity is neither inhibited by antibodies against rabbit skeletal muscle phosphorylase kinase nor activated by protein phosphorylation. These results suggest that bovine stomach phosphorylase kinase is tightly bound to an aggregate of actin-like molecules.
Zevgolis
V G
VG
Institute of Biological Research, National Hellenic Research Foundation, Athens, Greece.
Sotiroudis
T G
TG
Evangelopoulos
A E
AE
D000199:Actins / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta / Q000201:enzymology; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology; D002417:Cattle; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000201:enzymology*; D009131:Muscle, Smooth, Vascular / Q000201:enzymology; D010764:Phosphorylase Kinase / Q000302:isolation & purification*; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification*; D013270:Stomach / Q000201:enzymology
D000199:Actins; D002147:Calmodulin; D011494:Protein Kinases; D010764:Phosphorylase Kinase; D002118:Calcium