Ca2+-dependent protein phosphorylation associated with microsomal fraction of rat pancreas.
Microsomes isolated from cat pancreas were incubated with [gamma-32P]ATP in the presence or absence of Ca2+. Following fractionation of phosphoproteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis a single microsomal protein with an apparent molecular mass of 77,000 dalton (77K) was found to be phosphorylated in a Ca2+-dependent mechanism. Maximal phosphate incorporation into the 77K protein was observed at 10(-6) mol/l [Ca2+] and was 4-fold higher than in the absence of Ca2+. The 77K phosphoprotein showed characteristic of a stable phosphoester rather than an acyl phosphate. Measurable phosphate incorporation into the 77K protein was noted 5 s following addition of [gamma-32P]ATP and reached maximum at 9-10th min. The lack of effect of exogenous cyclic AMP, cyclic AMP-dependent protein kinase, calmodulin, the calmodulin antagonist trifluoperazine, leupeptin and the suppression of phosphorylation by some phospholipid-interacting drugs suggested that the 77K protein is a substrate for cyclic AMP- and calmodulin-independent, Ca2+-activated phospholipid-sensitive kinase activity. Centrifugation of the pancreatic homogenate in a ficoll-sucrose density gradient indicated that both the 77K protein and enzyme were associated in a fraction enriched in rough endoplasmic reticulum.
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002415:Cats; D005260:Female; D007700:Kinetics; D008297:Male; D008861:Microsomes / Q000378:metabolism*; D008970:Molecular Weight; D010179:Pancreas / Q000378:metabolism*; D010750:Phosphoproteins / Q000302:isolation & purification*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism*
D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D011506:Proteins; D002118:Calcium