The inhibitor of liver plasma membrane (Ca2+-Mg2+)-ATPase. Purification and identification as a mediator of glucagon action.
Rat liver plasma membranes contain (Ca2+-Mg2+)-ATPase sensitive to inhibition by both glucagon and Mg2+. We have previously shown that Mg2+ inhibition is mediated by a 30,000-dalton inhibitor, originally identified as a membrane-bound protein. In fact, this inhibitor is also present in the 100,000 X g supernatant of the total liver homogenate. Its purification was achieved from this fraction by a combination of ammonium sulfate washing, gel filtration, and cationic exchange chromatography. N-Ethylmaleimide (NEM) treatment caused the inactivation of the purified inhibitor, which suggested that this protein possesses at least one NEM-sensitive sulfhydryl group essential for its activity. Treatment of the liver plasma membranes with NEM resulted in a 2- and 5-fold decrease in the affinity of the (Ca2+-Mg2+)-ATPase for glucagon and Mg2+, respectively, while the basal enzyme activity remained unchanged. This effect of NEM was concentration-, pH-, and time-dependent, optimal conditions being obtained by a 60-min treatment of plasma membranes with 50 mM NEM, at pH 7 and at 4 degrees C. The presence of 0.5 mM Mg2+ during NEM treatment of the plasma membranes prevented NEM inactivation. Reconstitution experiments showed that addition of the purified inhibitor to NEM-treated plasma membranes restored the inhibitions of the (Ca2+-Mg2+)-ATPase by both magnesium and glucagon. It is proposed that the (Ca2+-Mg2+)-ATPase inhibitor not only confers its sensitivity of the liver (Ca2+-Mg2+)-ATPase to Mg2+, but also mediates the inhibition of this system by glucagon.
Lotersztajn
S
S
Mallat
A
A
Pavoine
C
C
Pecker
F
F
D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D002462:Cell Membrane / Q000201:enzymology; D003080:Cold Temperature; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide / Q000494:pharmacology; D005260:Female; D005934:Glucagon / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D051381:Rats; D013997:Time Factors
D005934:Glucagon; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D005033:Ethylmaleimide