Sodium-calcium exchange in membrane vesicles from aortic myocytes: stimulation by endogenous proteolysis masks inactivation during vesicle preparation.
Plasma membrane vesicles were purified from rat aortic myocytes by centrifugation in a discontinuous sucrose gradient. Vesicles were prepared in the presence or absence of five proteinase inhibitors (aprotinin, benzamidine, leupeptin, pepstatin A and phenylmethylsulfonyl fluoride). The proteinase inhibitors decreased the Vmax by 3.4-fold and had no effect on the Km for Ca2+ of Na+ gradient-dependent 45Ca2+ influx. The proteinase inhibitors had no direct effect on exchange activity, and they had no effect on membrane purity as indicated by 5'-nucleotidase activity. Removing the proteinase inhibitors or adding trypsin or chymotrypsin increased exchange activity approx. 2-fold. The Vmax of exchange activity in intact aortic myocytes is approx. 10-fold higher than the Vmax in plasma membrane vesicles prepared in the presence of proteinase inhibitors. Exchange activity in plasma membrane vesicles is only a sixtieth of the expected value, because the vesicles have approx. 7-fold higher 5'-nucleotidase activity and approx. 6-fold higher specific exchange activity than the crude homogenate. The large loss of exchange activity may be caused by a change in a regulatory domain of the exchanger because endogenous proteolysis restores some of the activity lost during vesicle preparation.
Lyu
R M
RM
Department of Pharmacology, School of Medicine, University of Alabama, Birmingham 35294.
Reeves
J P
JP
Smith
J B
JB
D015720:5'-Nucleotidase / Q000378:metabolism; D000818:Animals; D001011:Aorta; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism*; D002478:Cells, Cultured / Q000378:metabolism; D007700:Kinetics; D009131:Muscle, Smooth, Vascular / Q000201:enzymology / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D019831:Sodium-Calcium Exchanger; D014357:Trypsin / Q000494:pharmacology
D002352:Carrier Proteins; D011480:Protease Inhibitors; D019831:Sodium-Calcium Exchanger; D015720:5'-Nucleotidase; D014357:Trypsin