Pyrophosphate:protein phosphotransferase: a membrane-bound enzyme of endoplasmic reticulum.
Recently, we demonstrated that highly purified rat liver microsomal membrane was capable of selectively phosphorylating two intrinsic membrane polypeptides (M(r) 145,000 and M(r) 130,000) and that the course of the reaction was kinetically divided into two distinct stages [Lam, K. S. & Kasper, C. B. (1980) J. Biol. Chem. 255, 259-266]. Evidence was also presented that strongly suggested that a phosphoryl donor other than ATP was involved in the second stage of phosphorylation. In the present study, we demonstrate that incubation of microsomal membrane with [gamma-(32)P]ATP produces a prominent (32)P-labeled compound detectable by thin-layer chromatography on polyethyleneimine-impregnated cellulose. DEAE-cellulose fractionation of detergent-solubilized microsomal membrane generated a protein fraction that could convert in excess of 90% of the [gamma-(32)P]ATP into this newly (32)P-labeled unknown compound (I approximately P) without the formation of significant levels of (32)P(i). When [alpha-(32)P]ATP was used, I approximately P was unlabeled. Enzymically synthesized I approximately P was purified and determined to be pyrophosphate by using (31)P NMR spectroscopy. [(32)P]Pyrophosphate, synthesized chemically or enzymically, was capable of selectively phosphorylating the M(r) 145,000 and M(r) 130,000 polypeptides. Time course studies utilizing pyrophosphate as the phosphate source showed only one phase of phosphorylation that was strongly inhibited by micromolar levels of ATP as well as by NaF (5 mM). These studies further establish that pyrophosphate is the phosphoryl donor involved in the second stage of phosphorylation.
D000818:Animals; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D008970:Molecular Weight; D010766:Phosphorylation; D010770:Phosphotransferases / Q000302:isolation & purification / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats
D008565:Membrane Proteins; D010770:Phosphotransferases; D011494:Protein Kinases; C024910:pyrophosphate-protein phosphotransferase