Ca 2+-dependent protein phosphorylation associated with microsomal fraction of rat pancreas

• Published in: International journal of biochemistry Vol. 18; no. 8; pp. 753 - 761
• Main Authors: Shisheva, Assia C., Imamura, Koichiro
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 1986
• ISSN: 0020-711X
• DOI: 10.1016/0020-711X(86)90399-X

1. 1. Microsomes isolated from cat pancreas were incubated with [γ- 32P]ATP in the presence or absence of Ca 2+. Following fractionation of phosphoproteins by sodium dodecyl sulfate-polyacrylamide absence of Ca 2+. 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 Ca 2+-dependent mechanism. 2. 2. Maximal phosphate incorporation into the 77K protein was observed at 10 −6mol/l[Ca 2+ and was 4-fold higher than in the absence of Ca 2+. 3. 3. The 77K phosphoprotein showed characteristic of a stable phosphoester rather than an acyl phosphate. 4. 4. Measurable phosphate incorporation into the 77K protein was noted 5 s following addition of [γ- 32P]ATP and reached maximum at 9–10th min. 5. 5. 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, Ca 2+-activated phospholipid-sensitive kinase activity. 6. 6. Centrifugation of the pancreatic homogenate in a ficoll-sucrose densitu gradient indicated that both the 77K protein and enzyme were associated in a fraction enriched in rough endoplasmic reticulum.