Calcium-activated, phospholipid-dependent protein kinase in pancreatic acinar cells.
In the present study we partially purified calcium-activated, phospholipid-dependent protein kinase (protein kinase C) from pancreatic acinar cells of the guinea pig using diethylaminoethylcellulose and Sephadex G-150 chromatography and characterized the dependence of the enzyme on calcium, phospholipids, diacylglycerol (diolein), and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). The enriched preparation of protein kinase C contained no cyclic nucleotide-dependent or calcium-dependent, calmodulin-dependent protein kinase activity. The values of Km for H1-histone and ATP were 0.74 +/- 0.22 and 13.1 +/- 3.2 microM, respectively. Pancreatic protein kinase C demonstrated an absolute requirement for calcium and phospholipid for its activation, and diolein or TPA increased the affinity of the enzyme for calcium by 10-fold. With phosphatidylserine the calcium concentration that caused a half-maximal activation (Ka) was 74 +/- 17 microM, whereas with phosphatidylserine and diolein or TPA the Ka for calcium was 7.9 +/- 1.6 or 6.8 +/- 1.3 microM, respectively. Adding phosphatidylethanolamine and phosphatidylserine decreased the Ka for calcium to 2.0 +/- 0.9 microM with diolein and to 0.7 +/- 0.4 microM with TPA. Activation of protein kinase C by TPA and diolein was identical with calcium concentrations greater than 1 microM, but at low calcium concentrations (less than 1 microM) in the presence of phospholipids, maximally effective concentrations of diolein caused only 55% of the activation seen with TPA. In addition to TPA, other phorbol esters such as phorbol dibutyrate and phorbol diacetate, but not phorbol itself, activated protein kinase C.(ABSTRACT TRUNCATED AT 250 WORDS)
Noguchi
M
M
Adachi
H
H
Gardner
J D
JD
Jensen
R T
RT
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004075:Diglycerides / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006168:Guinea Pigs; D007515:Islets of Langerhans / Q000201:enzymology*; D008297:Male; D010703:Phorbol Esters / Q000494:pharmacology; D010743:Phospholipids / Q000378:metabolism; D011493:Protein Kinase C; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D013268:Stimulation, Chemical
D004075:Diglycerides; D010703:Phorbol Esters; D010743:Phospholipids; D011494:Protein Kinases; D011493:Protein Kinase C; D002118:Calcium