Separation at neutral pH value of 32P-labelled proteins in a membrane preparation from ox brain. Partial characterization of component of the sodium-plus-potassium ion-activited adenosine triphosphatase.
1. Ox brain microsomal fractions were labelled with [(32)P]ATP in the presence of Na(+) and the reaction was stopped with sodium dodecyl sulphate. The Na(+)-dependent bound phosphate was isolated on Sephadex G-25 and by acetone precipitation. The bound phosphate isolated under these neutral conditions was labile to hydroxylamine and gave the same pH profile of hydrolysis as that isolated by precipitation with strong acids. 2. When membrane protein was labelled with [(32)P]ATP, solubilized with sodium dodecyl sulphate and fractionated on Sepharose 6B, the Na(+)-dependent label emerged in a peak corresponding to protein of molecular weight 570000-580000. On fractionation of this protein peak on polyacrylamide gels containing detergent and urea, the Na(+)-dependent label occurred in a single band corresponding to a protein of molecular weight 102000. 3. Fractionation on Sepharose 6B of protein labelled with [(32)P]ATP in the absence of Na(+) revealed three labelled peaks, one of which corresponded in position to the Na(+)-dependent label. Electrophoresis of this peak material on polyacrylamide gels showed that most of the label occurred in two fast-running bands. Cyclic AMP stimulated the labelling in these two bands, but had no effect on the labelling of the band corresponding in position to the Na(+)-dependent label. 4. Di-isopropyl [(32)P]phosphorofluoridate also labelled the band corresponding to the Na(+)-dependent label on gel electrophoresis. The labelling of this band by the reagent was inhibited by 50-60% by 3mm-ATP, but there was no evidence to suggest that the group labelled is normally phosphorylated by ATP.
D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate; D000643:Ammonium Chloride; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000166:cytology / Q000201:enzymology*; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D000242:Cyclic AMP; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D006898:Hydroxylamines; D007531:Isoflurophate; D008566:Membranes / Q000201:enzymology; D008861:Microsomes / Q000201:enzymology; D008970:Molecular Weight; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D011188:Potassium; D011485:Protein Binding; D012964:Sodium; D012995:Solubility
D006898:Hydroxylamines; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D000643:Ammonium Chloride; D007531:Isoflurophate; D000255:Adenosine Triphosphate; D012964:Sodium; D000242:Cyclic AMP; D000251:Adenosine Triphosphatases; D011188:Potassium