Characterization of the high and low affinity components of the renal Ca2(+)-Mg2+ ATPase.
The purpose of this study was to characterize the interrelationship between free calcium (Ca2+) and magnesium (Mg2+) in the Ca2+ ATPase enzyme cycle of kidney membranes. Experiments were performed with basolateral membranes from rat renal cortex and microdissected proximal and distal tubules from mice. Results were similar in the three types of preparations. We first investigated the effect of ATP concentration on Ca2(+)- and Mg2(+)-dependent ATP hydrolysis. With 0.2 microM Ca2+, the enzyme activity, as a function of ATP concentration, showed two saturable components: a high affinity component with a Km of 33 microM ATP and a low affinity component with a Km of 0.63 mM ATP. These components may represent either two distinct sites of ATP binding or two forms of the same site. For the sake of simplicity, it was assumed that the two components correspond to a high affinity and a low affinity substrate site. At the high affinity site (ATP = 50 microM), the Ca2+ dependence of ATP hydrolysis followed a single Michaelis-Menten kinetics with Km for Ca2+ of 0.08 microM. The addition of 1 mM Mg2+ resulted in a relatively constant increase in ATP hydrolysis at all Ca2+ concentrations, indicating that the effects of the two cations were additive. With high ATP concentration (ATP = 3 mM), Ca2+ also induced an ATP hydrolysis according to a saturable process, with a Km for Ca2+ of 0.2 microM. In contrast with what occurred with low concentrations of ATP, addition of millimolar Mg2+ completely curtailed the sensitivity of the enzyme to Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)
Brunette
M G
MG
Research Centre, Maisonneuve-Rosemont Hospital, Montreal, Que., Canada.
Mailloux
J
J
Chan
M
M
Ramachandran
C
C
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002147:Calmodulin / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D006868:Hydrolysis; D007668:Kidney / Q000201:enzymology* / Q000648:ultrastructure; D007672:Kidney Cortex / Q000201:enzymology; D007686:Kidney Tubules, Distal / Q000201:enzymology / Q000648:ultrastructure; D007687:Kidney Tubules, Proximal / Q000201:enzymology / Q000648:ultrastructure; D007700:Kinetics; D007811:Lanthanum / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D051379:Mice; D051381:Rats; D011919:Rats, Inbred Strains; D014638:Vanadates / Q000494:pharmacology
D002147:Calmodulin; D014638:Vanadates; D007811:Lanthanum; D000255:Adenosine Triphosphate; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002118:Calcium