Phosphorylated intermediate of (Ca2+ + K+)-stimulated Mg2+-dependent transport ATPase in endoplasmic reticulum from rat pancreatic acinar cells.
Formation and decomposition of the phosphorylated intermediate of endoplasmic reticulum (Ca2+ + Mg2+)-ATPase from pancreatic acinar cells have been studied using lithium dodecyl sulfate- and tetradecyltrimethylammonium bromide-polyacrylamide gel electrophoresis. Incorporation of 32P from [gamma-32P]ATP is Ca2+-dependent (approximate Km for free [Ca2+] = 2-3 X 10(-8) mol/liter). Formation of the 100-kDa phosphoprotein is rapid, reaching maximal 32Pi incorporation within 1 s at room temperature. At 4 degrees C, phosphorylation is slower and dephosphorylation is drastically decreased. For dephosphorylation, Mg2+ and monovalent cations such as K+ or Na+ are necessary. Vanadate inhibits both 32P incorporation and 32P liberation dose dependently (Km = 3 X 10(-6) mol/liter), whereas mitochondrial inhibitors and ouabain have no effect. The phosphoprotein is stable at pH 2 and destabilizes with increasing pH being completely decomposed at pH 9. Reduction of 32P incorporation in the presence of high concentrations of cold ATP and hydroxylamine suggests formation of acylphosphate present in the ATPase intermediate. The characteristics of Ca2+, cation, and pH dependencies of the ATPase activity are similar to those previously described for MgATP-dependent Ca2+ transport into rough endoplasmic reticulum from pancreatic acinar cells (Bayerdörffer, E., Streb, H., Eckhardt, L., Haase, W., and Schulz, I. (1984) J. Membr. Biol. 81, 69-82). The data suggest that the 100-kDa phosphoprotein as described in this study is the intermediate of this Ca2+ transport ATPase.
Imamura
K
K
Schulz
I
I
D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase; D002118:Calcium / Q000494:pharmacology; D002414:Cations, Monovalent; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008970:Molecular Weight; D010179:Pancreas / Q000166:cytology / Q000201:enzymology*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011485:Protein Binding; D051381:Rats; D011919:Rats, Inbred Strains; D014638:Vanadates; D014639:Vanadium / Q000494:pharmacology
D002414:Cations, Monovalent; D010761:Phosphorus Radioisotopes; D014639:Vanadium; D014638:Vanadates; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase; C046459:calcium potassium-stimulated magnesium adenosine triphosphatase; D008274:Magnesium; D002118:Calcium