Phosphorylation of proteins in rat ovarian plasma membranes by [gamma-32P]GTP: evidence for the formation of a high energy phosphoprotein.
Incubation of rat ovarian plasma membranes with [gamma-32P]guanosine 5'-triphosphate (GTP) in the presence of an adenosine triphosphate (ATP)-trapping system results in the labeling of a single protein, Mr 33,000 +/- 3000 designated 'a' (Amir-Zaltsman, Y., Ezra, E., Walker, M., Lindner, H. R. and Salomon, Y. (1980) FEBS Lett. 122, 166-170). Based on competition with other nucleotides it is concluded that protein 'a' is preferentially phosphorylated by [gamma-32P]GTP (Km = 0.28 microM). Phosphorylation of protein 'a' does not occur at pH less than 5 and progressively increases to plateau levels at pH 7-9. Phosphorylation of protein 'a' is absolutely dependent on the presence of divalent cations 1 mM Mg2+, Ca2+, or Cd2+. At higher concentrations, 5-20 mM, Mg2+ or in the presence of 1 mM Mn2+ ions other proteins are also phosphorylated. While vanadate ions selectively prevent the labeling of protein 'a', molybdate ions were found to inhibit phosphorylation of all the membrane proteins including protein 'a'. In contrast to molybdate ions, vanadate ions were found to accelerate the dephosphorylation of phosphoprotein 'a'. We suggest that phosphoprotein 'a' is a high energy protein intermediate in which the phosphate is present as a phosphoramidate for the following reasons: (i) Guanosine diphosphate (GDP) but not guanosine 5'-O-(2-thiodiphosphate) selectively accelerated the dephosphorylation of phosphoprotein 'a' but only in the presence of Mg2+ ions. (ii) The phosphoprotein intermediate is hydrolyzed in the presence of hydroxylamine. (iii) Phosphoprotein 'a' is labile in the presence of 1 N HCl but stable in 1 N NaOH at 37 degrees C. (iv) Phosphoprotein 'a' is heat labile. Phosphoprotein 'a' is readily digested by several proteolytic enzymes and a single cleavage peptide is generated upon treatment with Staphylococcus aureus V8 protease. The properties of protein 'a' were compared and found different from another phosphoprotein Mr 90,000 +/- 1000, designated 'b' that was selected arbitrarily. We propose that protein 'a' is a GTP requiring enzyme intermediate, of yet unidentified function.
Amir-Zaltsman
Y
Y
Department of Hormone Research, Weizmann Institute of Science, Rehovot, Israel.
Salomon
Y
Y
D000818:Animals; D002462:Cell Membrane / Q000032:analysis / Q000378:metabolism; D005260:Female; D006160:Guanosine Triphosphate / Q000032:analysis / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D006898:Hydroxylamines / Q000494:pharmacology; D008274:Magnesium / Q000502:physiology; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism*; D009711:Nucleotides / Q000494:pharmacology; D010053:Ovary / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure*; D010750:Phosphoproteins / Q000032:analysis / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D051381:Rats
D006898:Hydroxylamines; D008565:Membrane Proteins; D009711:Nucleotides; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D006160:Guanosine Triphosphate; D008274:Magnesium