The H(+)-ATPase of the plasma membrane from yeast. Kinetics of ATP hydrolysis in native membranes, isolated and reconstituted enzymes.
The H(+)-ATPase of the plasma membrane from Saccharomyces cerevisiae has been isolated, purified and reconstituted into asolectin liposomes. The kinetics of ATP hydrolysis have been compared for the H(+)-ATPase in the plasma membrane, in a protein/lipid/detergent micelle (isolated enzyme) and in asolectin proteoliposomes (reconstituted enzyme). In all three cases the kinetics of ATP hydrolysis can be described by Michaelis-Menten kinetics with Km = 0.2 mM MgATP (plasma membranes), Km = 2.4 mM MgATP (isolated enzyme) and Km = 0.2 mM MgATP (reconstituted enzyme). However, the maximal turnover decreases only by a factor of two during isolation of the enzyme and does not change during reconstitution; the activation of the H(+)-ATPase by free Mg2+ is also only slightly influenced by the detergent. The dissociation constant of the enzyme-Mg2+ complex Ka, does not alter during isolation and the dissociation constant of the enzyme-substrate complex, Ks, increases from Ks = 30 microM (plasma membranes) to Ks = 90 microM (isolated enzyme). ATP binding to the H(+)-ATPase ('single turnover' conditions) for the isolated and the reconstituted enzyme resulted in both cases in a second-order rate constant k1 = 2.6 x 10(4) M-1.s-1. From these observations it is concluded that the detergent used (Zwittergent TM 3-14) interacts reversibly with the H(+)-ATPase and that practically all H(+)-ATPase molecules are reconstituted into the liposomes with the ATP-binding site being directed to the outside of the vesicle.
Wach
A
A
Institut für Biochemie und Molekularbiologie, Freie Universität Berlin.
Ahlers
J
J
Gräber
P
P
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology*; D007700:Kinetics; D008081:Liposomes / Q000302:isolation & purification; D010713:Phosphatidylcholines; D010743:Phospholipids / Q000302:isolation & purification; D006180:Proton-Translocating ATPases / Q000032:analysis*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D008081:Liposomes; D010713:Phosphatidylcholines; D010743:Phospholipids; C024990:asolectin; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases