Kinetic characterization of plasma membrane ATPase from Saccharomyces cerevisiae.
1. Plasma membrane preparations have been isolated from spheroplasts of Saccharomyces cerevisiae, strain R XII, via lysis and subsequent differential centrifugation. These preparations are almost devoid of mitochondrial contamination. 2. The plasma membrane ATPase is fairly stable when refrigerated, but loses activity at 8 degrees C and above. Below pH 5.6 the ATPase is irreversibly inactivated. The enzyme also splits GTP and ITP, although to a lesser extent. 3. Mg2+-ions are essential as part of the reactive substrate, MgATP, and furthermore they activate the ATPase. Optimal conditions depend on substrate concentration. When the concentration of free Mg2+ ions exceeds about 0.1 mM, competitive inhibition occurs. 4. In the range of pH 5.6-9.2 two functional groups dissociate. One, with pKb = 8.1 +/- 0.1 participated in substrate binding and another one with pKb' = 8.1 +/- 0.1 is involved in substrate splitting. 5. The experiments with group-specific inhibitors suggest that an alpha-amino group and a sulfhydryl residue are involved in substrate binding and conversion. Furthermore, imidazole, tryptophan and carboxyl residues may be important for the catalytic process.
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000201:enzymology; D004355:Drug Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008433:Mathematics; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity
D000251:Adenosine Triphosphatases; D008274:Magnesium