The catalytic cycle of the vacuolar H(+)-ATPase. Comparison of proton transport in kidney- and osteoclast-derived vesicles.
To characterize the catalytic cycle involved in the initial rate of proton transport by the vacuolar ATPases (V-ATPase), we have analyzed and compared the catalytic parameters of V-ATPase-dependent proton transport in vesicles from chicken kidney or purified osteoclasts. The relationship of acidification to ATP obeyed simple Michaelis-Menten kinetics with a single Km for ATP of 62 microM in the kidney and 191 microM in the osteoclast. The activity was dependent on the presence of Mg2+, with a single Km of 258 microM MgCl2 in the kidney and 504 microM MgCl2 in the osteoclast. In both preparations, ADP competed with ATP and inhibited the proton transport (Ki = 37 microM in kidney and 17 microM in osteoclast). Phosphate was found to be a noncompetitive inhibitor of ATP, with a calculated Ki of about 10.5 and 5.5 mM, respectively. In both preparations, the catalytic mechanism determining the V-ATPase-mediated proton transport, fits the model of a "uni-bi-ordered release" mechanism. According to this model, ATP is the single substrate, and P(i) and ADP are the two products where phosphate, being noncompetitive, is released first and ADP, being competitive, second. The findings of an elevated Km for ATP and Mg2+ and a decrease in the Ki of ADP and phosphate in the osteoclast relative to the kidney preparation suggests that the V-ATPases present in these two tissues may differ.
David
P
P
Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510.
Baron
R
R
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002645:Chickens; D005260:Female; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology* / Q000648:ultrastructure; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D008861:Microsomes / Q000201:enzymology; D010010:Osteoclasts / Q000201:enzymology* / Q000648:ultrastructure; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D014617:Vacuoles / Q000201:enzymology*
D015636:Magnesium Chloride; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases