phosphorus-radioisotopes and acetyl-phosphate

Sarcoplasmic reticulum Ca(2+)-ATPase is an ion pump whose catalytic cycle includes the transient formation of an acyl phosphate at Asp(351), and fluorescein isothiocyanate is a covalent inhibitor of ATP binding to this pump, known to specifically derivatize Lys(515) in the nucleotide-binding site. It was previously found that an unusually stable, phosphorylated form of fluorescein-ATPase, with low fluorescence, is obtained following Ca (2+) loading with acetyl phosphate as energy source and then chelation with EGTA of Ca(2+) on the cytosolic side. Here we show that the phospho-linkage in this low fluorescent species is stable at alkaline pH, unlike the acyl phosphate at Asp(351). Moreover, the low fluorescence and stable phosphoryl group track together in primary and secondary tryptic subfragments, separated by SDS-PAGE after denaturation. Finally, normal fluorescence and absorbance are recovered upon treatment with alkaline phosphatase after extensive trypsinolysis. We conclude that the low fluorescent species is the result of the phosphoryl group being transferred from Asp (351) to the fluorescein moiety during pump reversal, yielding fluorescein monophosphate tethered to Ca(2+)-ATPase.

Topics: Alkaline Phosphatase; Animals; Aspartic Acid; Catalysis; Enzyme Stability; Fluorescein; Hydrogen-Ion Concentration; Lysine; Organophosphates; Peptide Fragments; Phosphorus Radioisotopes; Phosphorylation; Rabbits; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Spectrometry, Fluorescence; Substrate Specificity; Trypsin

Intrinsic GTPase activity of GTP-binding proteins plays the vital role in regulating the downstream activation pathway. We examined the GTP and ATP hydrolyzing (NTPase) abilities of various bacterial and human GTP-binding proteins under different metabolic conditions. Two metabolic components, acetate and 3-phosphoglyceric acid (3-PG), have shown significant stimulatory action on NTPase activity of G-protein preparations. Acetyl phosphate and 2,3-bisphosphoglyceric acid (2,3-BPG) blocked these stimulations. From gel filtration analyses, we have determined two fractions containing metabolite-inducible NTPase activities which are independent of GTP-binding protein enzymatic actions. Therefore, one should be cautious when NTPase activity is examined in a buffer containing acetate often used for NTPase assay.

Topics: Acetates; Adenosine Triphosphate; Buffers; Diphosphoglyceric Acids; Escherichia coli; Escherichia coli Proteins; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Organophosphates; Organophosphorus Compounds; Phosphorus Radioisotopes; ras Proteins; RNA-Binding Proteins

Clostridial glycine reductase consists of proteins A, B, and C and catalyzes the reaction glycine + Pi + 2e(-)----acetyl phosphate + NH4+. Evidence was previously obtained that is consistent with the involvement of an acyl enzyme intermediate in this reaction. We now demonstrate that protein C catalyzes exchange of [32P]Pi into acetyl phosphate, providing additional support for an acetyl enzyme intermediate on protein C. Furthermore, we have isolated acetyl protein C and shown that it is qualitatively catalytically competent. Acetyl protein C can be obtained through the forward reaction from protein C and Se-(carboxymethyl)selenocysteine-protein A, which is generated by the reaction of glycine with proteins A and B [Arkowitz, R. A., & Abeles, R. H. (1990) J. Am. Chem. Soc. 112, 870-872]. Acetyl protein C can also be generated through the reverse reaction by the addition of acetyl phosphate to protein C. Both procedures lead to the same acetyl enzyme. The acetyl enzyme reacts with Pi to give acetyl phosphate. When [14C]acetyl protein C is denaturated with TCA and redissolved with urea, radioactivity remained associated with the protein. At pH 11.5 radioactivity was released with t1/2 = 57 min, comparable to the hydrolysis rate of thioesters. Exposure of 4 N neutralized NH2OH resulted in the complete release of radioactivity. Treatment with KBH4 removes all the radioactivity associated with protein C, resulting in the formation of [14C]ethanol. We conclude that a thiol group on protein C is acetylated. Proteins A and C together catalyze the exchange of tritium atoms from [3H]H2O into acetyl phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acid Oxidoreductases; Carbon Radioisotopes; Clostridium; Kinetics; Models, Chemical; Multienzyme Complexes; Organophosphates; Phosphates; Phosphorus Radioisotopes; Protein Binding; Radioisotope Dilution Technique; Tritium

A simple, rapid, and inexpensive method is described for the synthesis of gamma-32P-labeled ribo- or deoxyribonucleoside triphosphates. The procedure involves chemical synthesis of [32P]acetylphosphate and subsequent phosphorylation of nucleoside diphosphates using acetate kinase (EC 2.7.2.1) and a final purification step. The entire procedure is performed 8 h or less.

Topics: Acetate Kinase; Charcoal; Chromatography, DEAE-Cellulose; Nucleotides; Organophosphates; Phosphorus Radioisotopes