P36, a Dictyostelium discoideum protein whose phosphorylation is stimulated by GDP, is homologous to the alpha-subunit of succinyl-CoA synthetase.
Previously, we reported the phosphorylation of a 36 kDa protein, p36, in crude membranes from the amoeba Dictyostelium discoideum (Anschutz, A.L., Howlett, A. and Klein, C. (1989) Proc. Natl. Acad. Sci. USA 86, 3665-3668). Here, we report the purification and identification of p36. The protein was purified approximately 35-40-fold with a yield of 8-10%. This material was then separated on 10% SDS-polyacrylamide gels and the band corresponding to p36 was isolated. Partial peptide sequencing of this band revealed p36 to be homologous to the alpha-subunit of succinyl-CoA synthetase. This identification of the protein was supported by the results of phosphorylation studies which examined the effects of substrates of succinyl-CoA synthetase on p36 phosphorylation. In crude sample preparations, p36 could be phosphorylated by both ATP or GTP and in either case, its phosphorylation was stimulated by low concentrations of GDP. Partially purified p36 retained its ability to be phosphorylated with GTP while exhibiting little or no phosphorylation with ATP. GDP still enhanced the rate of p36 phosphorylation with GTP. Therefore, the stimulation of p36 phosphorylation by GDP is not due to substrate conversion and is best explained by a regulatory mechanism.
Anschutz
A L
AL
E.A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, MO 63104.
Um
H D
HD
Siegel
N R
NR
Veron
M
M
Klein
C
C
D000293:Adolescent; D000478:Alkylation; D000595:Amino Acid Sequence; D000818:Animals; D006224:Cricetinae; D004023:Dictyostelium / Q000737:chemistry*; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000494:pharmacology*; D006801:Humans; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010766:Phosphorylation / Q000187:drug effects; D012689:Sequence Homology, Nucleic Acid; D013389:Succinate-CoA Ligases / Q000737:chemistry*; D014357:Trypsin
D005656:Fungal Proteins; D006153:Guanosine Diphosphate; D014357:Trypsin; D013389:Succinate-CoA Ligases