Isolation and characterization of a new Ca2+/calmodulin-dependent protein kinase from isoproterenol-stimulated proliferating rat parotid acinar cells.
A new Ca2+/calmodulin-dependent serine kinase was isolated from rat parotid gland acinar cells following chronic treatment with the beta-agonist isoproterenol. A single-step purification was performed on a calmodulin-agarose affinity column, following solubilization with Triton X-100. Among various substrates tested, bovine galactosyltransferase was the preferred substrate of the kinase, followed by glycogen synthetase greater than histone greater than phosphodiesterase greater than phenylalanine hydroxylase greater than phosphorylase b greater than bovine serum albumin. In comparison, a spleen preparation of Ca2+/calmodulin-dependent kinase did not show galactosyltransferase to be the preferred substrate. Thus, the enzyme would appear to be similar to the human galactosyltransferase-associated kinase. The kinase activity was saturable with 100 microM Ca2+ and 2 microM calmodulin. The molecular mass determined by nondenaturing and sodium dodecyl sulfate polyacrylamide gel electrophoreses was 75 kDa with a pI of 4.3. The Vmax was 3500 mumol/(min.mg protein) with a Km of 1.6 microM for the transferase substrate. Leukotriene C and prostaglandin E2 were found to be specific noncompetitive inhibitors of the rat galactosyltransferase-associated kinase.
Purushotham
K R
KR
Department of Oral Biology, University of Florida, Gainesville 32610.
Bologna
J
J
Nakagawa
Y
Y
Humphreys-Beher
M G
MG
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002846:Chromatography, Affinity; D015232:Dinoprostone / Q000494:pharmacology; D004586:Electrophoresis; D005700:Galactosyltransferases / Q000378:metabolism*; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D015289:Leukotrienes / Q000494:pharmacology; D008297:Male; D010306:Parotid Gland / Q000187:drug effects / Q000201:enzymology*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D051381:Rats; D011919:Rats, Inbred Strains
D002147:Calmodulin; D015289:Leukotrienes; D047428:Protein Kinase Inhibitors; D005700:Galactosyltransferases; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D015232:Dinoprostone; D007545:Isoproterenol; D002118:Calcium