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Query: EC:3.6.3.1 (Mg2+-ATPase)
 1,484 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mechanism of transport of basic amino acids into vacuoles of cells of the yeast Saccharomyces cerevisiae was investigated in vitro. Right-side-out vacuolar membrane vesicles were prepared from purified vacuoles. Arginine was taken up effectively by the vesicles only in the presence of ATP, not in the presence of ADP or AMP-adenosyl-5'-yl imidodiphosphate. It was exchangeable and was released completely by a protonophore, 3,5-di-tert-butyl-4-hydroxybenzilidenemalononitrile (SF6847). The transport required Mg2+ ion but was inhibited by Cu2+, Ca2+, or Zn2+ ions. The transport activity was sensitive to the ATPase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD), but not to oligomycin or sodium vanadate. SF6847 or nigericin blocked arginine uptake completely, but valinomycin had no effect. ATP-dependent formation of a delta pH across the membrane vesicles was shown by quenching of 9-aminoacridine fluorescence. These results indicate that DCCD-sensitive, Mg2+-ATPase of vacuolar membranes is essential as an energy-donating system for the active transport, and that an electrochemical potential difference of protons is a driving force of this basic amino acid transport. Arginine transport showed saturation kinetics with a Km value of 0.6 mM and the mechanism was well explained by an H+/arginine antiport.
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PMID:Active transport of basic amino acids driven by a proton motive force in vacuolar membrane vesicles of Saccharomyces cerevisiae. 645 Jul 64

This study describes investigations of the importance of intraacrosomal pH in the hamster sperm acrosome reaction (AR). Washed cauda epididymal sperm were capacitated in vitro in a medium containing 2 mM Ca2+, 144 mM Na+, and 3 mM K+. Such sperm underwent a significant increase in the number of AR within 10 min after the addition of the Mg2+-ATPase (adenosine triphosphatase) inhibitors DCCD (20 microM) or NBD-Cl (10 microM) or the proton ionophore FCCP (6 micrograms/ml) at 3.5 hr of incubation or after addition of HN4Cl (3 mM) at 4 hr of incubation. Addition of the mitochondrial electron transport inhibitor rotenone (2.5 microM) at 3.5 hr or of NaCl (3 mM) or KCl (3 mM) at 4 hr did not stimulate AR over control levels, suggesting that the stimulation of AR by the other compounds was not directly due to depletion of acrosomal adenosine triphosphate (ATP) or alteration of the acrosomal transmembrane potential. The AR also was not stimulated by either DCCD or FCCP added prior to 3 hr of incubation of sperm, whereas both compounds were increasingly effective at stimulating AR with increasing length of preincubation of sperm before the addition of the test compounds. The intraacrosomal pH of sperm incubated in low [K+] (0.6-0.9 mM) for 3.5 hr rose by at least one pH unit (as measured with the fluorescent dye 9-aminoacridine) within 15-30 min after raising extracellular [K+] to 4.2-4.5 mM. The pH rise occurred even in the presence of the Ca2+-chelator EGTA (2 mM). Either FCCP (8 micrograms/ml) or DCCD (20 microM), but not rotenone (2.5 microM), plus K+ (3.6 mM), raised the intraacrosomal pH of sperm incubated for 3 hr in low [K+] within 10 min after addition. No pH rise occurred in the absence of additional K+. These results demonstrate that the intraacrosomal pH of the hamster sperm becomes more alkaline in a process not requiring high concentrations of external Ca2+, but requiring K+. The results of this and previous studies lead us to suggest here that the intraacrosomal pH rise may be mediated via a change in K+ and H+ permeability of sperm head membranes, which allows K+ influx and H+ efflux, and via inhibition of an acrosomal Mg2+-ATPase proton pump. We propose that the permeability changes and the consequent alkalinization of the acrosomal interior are important steps in late capacitation and/or the mammalian AR.
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PMID:Correlation of increased intraacrosomal pH with the hamster sperm acrosome reaction. 661 70


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