Ca2+-dependent membrane bound protein fraction from rabbit gastric mucosa contains a protein whose histidyl residue is phosphorylated.
We found an autophosphorylated protein with a molecular weight of 40 kDa (p40) in the crude annexin fraction of rabbit gastric mucosa, i.e., the materials released by EGTA from the membrane fraction obtained in the presence of Ca2+. This protein was enriched in chief cells in the gastric glands, and also found in the heart and the liver by Western blotting. The protein bound to phenyl-Sepharose in the presence of Ca2+ and showed extremely basic nature. The phosphorylation site of p40 was considered to be histidyl residue based on the stability to the various agents, the synthesizing activity of ATP from ADP, and the results of phosphoamino acid analysis. The autophosphorylation of p40 was augmented several tenth fold by GDP, Ras, myelin basic protein, or H1 histone at micromolar range. The phosphorylated form was rapidly dephosphorylated in the presence of cold ATP, succinate, and CoA, suggesting that p40 has succinyl-CoA synthetase activity. In fact, a peptide fragment from p40 showed a striking homology with the alpha subunits of succinyl-CoA synthetases from Escherichia coli, Dictyostelium discoideum, and rat liver. These results suggest that p40 is extramitochondrial alpha subunit of succinyl-CoA synthetase or its homologue.
Urushidani
T
T
Department of Toxicology and Pharmacology, Faculty of Pharmaceutical Sciences, The University of Tokyo, Hongo, Japan. urushi@mol.f.u-tokyo.ac.jp
Nagao
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T
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D005753:Gastric Mucosa / Q000737:chemistry*; D006639:Histidine / Q000031:analogs & derivatives* / Q000032:analysis; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification*; D010766:Phosphorylation; D011817:Rabbits; D012697:Serine Endopeptidases; D013385:Succinate Dehydrogenase / Q000032:analysis
D008565:Membrane Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D006639:Histidine; D013385:Succinate Dehydrogenase; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D002118:Calcium; C005436:phosphohistidine