Calmodulin-dependent protein kinase in acini from lactating rat mammary tissue: subcellular locale, characterization, and solubilization.
Acini isolated from lactating rat mammary tissue were used as the starting material to determine the subcellular location and characteristics of a calcium and calmodulin-dependent protein kinase. The kinase activity phosphorylated a 53,600-Da endogenous protein, required Mg2+, and was stimulated only by the simultaneous presence of calcium and calmodulin. Fractionation by differential and sucrose gradient centrifugation demonstrated the enzyme activity in acinar homogenates to be largely particulate; yet the activity did not co-fractionate with markers for nuclei, secretory vesicles, endoplasmic reticulum, mitochondria, lysozymes, Golgi or plasma membranes. The addition of dephosphorylated K-casein to these preparations resulted in a calcium and calmodulin-dependent phosphorylation of the exogenous substrate. A combination of differential centrifugation and equilibrium sucrose density gradient centrifugation purified the kinase 15-fold and revealed a density for the kinase activity between 1.33 and 1.27 g/cm3, suggesting that the kinase was associated with a particle composed largely or entirely of protein. Gel chromatography on Sephacryl S-1000 also purified the activity significantly, and provided a molecular weight of approximately 10(6). In both procedures, the enzymatic activity and principal endogenous protein substrate were enriched indicating that the kinase was associated with the 53,600-Da substrate. Sodium dodecyl sulfate-gel electrophoresis of the fractions enriched in kinase activity by either gel-exclusion chromatography or equilibrium density gradient centrifugation revealed a discrete set of proteins common to both preparations. These included proteins with molecular weights of approximately 32, 35, 54, 70, 94, 100 and 103 K. The calmodulin-dependent protein kinase of mammary acini may be associated in a large complex with these protein species or may represent a polymer of one or several of the proteins. Despite no apparent association with the common phospholipid membranous organelles, the kinase activity was solubilized by treatment with a mixture of phospholipases C and D. After phospholipase treatment and chromatography on Sephacryl S-1000, calcium and calmodulin-dependent phosphorylation was no longer detectable, indicating separation of enzyme and endogenous substrate. Phospholipase treatment of the kinase preparation may be useful in future studies as a method to solubilize the activity.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007774:Lactation; D008274:Magnesium / Q000494:pharmacology; D008321:Mammary Glands, Animal / Q000201:enzymology*; D008970:Molecular Weight; D010739:Phospholipase D / Q000378:metabolism; D011247:Pregnancy; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility; D013347:Subcellular Fractions / Q000201:enzymology; D014268:Trifluoperazine / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism
D002147:Calmodulin; D014268:Trifluoperazine; D011494:Protein Kinases; D010738:Type C Phospholipases; D010739:Phospholipase D; D008274:Magnesium; D002118:Calcium