Open AccessPyrophosphate:protein phosphotransferase: A membrane-bound enzyme of endoplasmic reticulum
Author(s) -
Kit S. Lam,
Charles B. Kasper
Publication year - 1980
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.77.4.1927
Subject(s) - pyrophosphate , microsome , endoplasmic reticulum , phosphorylation , chemistry , membrane , biochemistry , enzyme , nuclear magnetic resonance spectroscopy , phosphotransferase , phosphate , chromatography , stereochemistry
Recently, we demonstrated that highly purified rat liver microsomal membrane was capable of selectively phosphorylating two intrinsic membrane polypeptides (M r 145,000 andM 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 [γ-32 P]ATP produces a prominent32 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 [γ-32 P]ATP into this newly32 P-labeled unknown compound (I∼P ) without the formation of significant levels of32 Pi . When [α-32 P]ATP was used, I∼P was unlabeled. Enzymically synthesized I∼P was purified and determined to be pyrophosphate by using31 P NMR spectroscopy. [32 P]Pyrophosphate, synthesized chemically or enzymically, was capable of selectively phosphorylating theM r 145,000 andM 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.