Prolactin kinase activity in bovine anterior pituitary sub-cellular fractions.
Bovine anterior pituitary cells phosphorylate prolactin (PRL). We describe the phosphorylation of endogenous and exogenous bPRL in highly enriched subcellular fractions of bovine anterior pituitary using [gamma-32P]-ATP. 32P-labeling of endogenous and exogenous bPRL occurred in all subcellular membrane fractions, but most significantly in the fraction enriched for secretory granules. Zn2+ (0.8 mM), Cu2+ (0.8 mM), and Mn2+ (9.8 mM) increased bPRL phosphorylation by 268, 214, and 154%, respectively, relative to basal phosphorylation with no added cations. Neither Mg2+ (10 mM) nor Ca2+ (0.9 mM) increased bPRL phosphorylation above basal levels. Phosphorylation was dependent on the concentration of Zn2+ with an apparent Km of 570 microM. bPRL phosphorylation occurred over a wide pH range of 5.9-8.3, with the greatest activity at pH of 6.7 or greater. Phosphorylation of bPRL was time-dependent. The apparent Kms of the bPRL kinase for exogenous bPRL and ATP were 15.3 and 267 microM, respectively. bPRL incorporation of 32P was unaffected by the presence of calcium and calmodulin, cAMP, phosphotidylserine and diolein, or spermine. From these results we conclude that in vitro phosphorylation of bPRL occurs under physiological conditions that would be found in pituitary cells.
Wicks
J R
JR
Department of Veterinary Biosciences, The Ohio State University, Columbus 43212, USA.
Brooks
C L
CL
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology; D002412:Cations / Q000494:pharmacology; D002417:Cattle; D002458:Cell Fractionation; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D000242:Cyclic AMP / Q000494:pharmacology; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D004075:Diglycerides / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D010710:Phosphates / Q000378:metabolism; D010718:Phosphatidylserines / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D011388:Prolactin / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D013096:Spermine / Q000494:pharmacology
D002147:Calmodulin; D002412:Cations; D004075:Diglycerides; D008670:Metals; D010710:Phosphates; D010718:Phosphatidylserines; D013096:Spermine; D000255:Adenosine Triphosphate; D011388:Prolactin; D000242:Cyclic AMP; D011494:Protein Kinases; D002118:Calcium; C013965:diolein