Heterogeneous Na+ sensitivity of Na+,K(+)-ATPase isoenzymes in whole brain membranes.
The Na+ sensitivity of whole brain membrane Na+,K(+)-ATPase isoenzymes was studied using the differential inhibitory effect of ouabain (alpha 1, low affinity for ouabain; alpha 2, high affinity; and alpha 3, very high affinity). At 100 mM Na+, we found that the proportion of isoforms with low, high, and very high ouabain affinity was 21, 38, and 41%, respectively. Using two ouabain concentrations (10(-5) and 10(-7) M), we were able to discriminate Na+ sensitivity of Na+,K(+)-ATPase isoenzymes using nonlinear regression. The ouabain low-affinity isoform, alpha 1, exhibited high Na+ sensitivity [Ka of 3.88 +/- 0.25 mM Na+ and a Hill coefficient (n) of 1.98 +/- 0.13]; the ouabain high-affinity isoform, alpha 2, had two Na+ sensitivities, a high (Ka of 4.98 +/- 0.2 mM Na+ and n of 1.34 +/- 0.10) and a low (Ka of 28 +/- 0.5 mM Na+ and an n of 1.92 +/- 0.18) Na+ sensitivity activated above a threshold (22 +/- 0.3 mM Na+); and the ouabain very-high-affinity isoform, alpha 3, was resolved by two processes and appears to have two Na+ sensitivities (apparent Ka values of 3.5 and 20 mM Na+). We show that Na+ dependence in the absence of ouabain is the result of at least of five Na+ reactivities. This molecular functional characteristic of isoenzymes in membranes could explain the diversity of physiological roles attributed to isoenzymes.
Gerbi
A
A
INSERM U. 26, Hôpital Fernand Widal, Paris, France.
Debray
M
M
Maixent
J M
JM
Chanez
C
C
Bourre
J M
JM
D000818:Animals; D001921:Brain / Q000201:enzymology*; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008566:Membranes / Q000201:enzymology; D010042:Ouabain / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D012044:Regression Analysis; D012964:Sodium / Q000494:pharmacology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D007527:Isoenzymes; D010042:Ouabain; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase