Abstract
The properties of Mg2+-ATPase in the vacuole of Saccharomyces cerevisiae were studied, using purified intact vacuoles and right-side-out vacuolar membrane vesicles prepared by the method of Y. Ohsumi and Y. Anraku ((1981) J. Biol. Chem. 256, 2079). The enzyme requires Mg2+ ion but not Ca2+ in. Cu2+ and Zn2+ ions inhibit the activity. The optimal pH is at pH 7.0. The enzyme hydrolyzes ATP, GTP, UTP, and CTP in this order and the Km value for ATP was determined as 0.2 mM. It does not hydrolyze ADP, adenosyl-5'-yl imidodiphosphate, or p-nitrophenyl phosphate. ADP does not inhibit hydrolysis of ATP by the enzyme. The activities of intact vacuoles and of vacuolar membrane vesicles were stimulated 3- and 1.5-fold, respectively, by the protonophore uncoupler 3,5-di-tert-butyl-4-hydroxybenzilidenemalononitrile and the K+/H+ antiporter ionophore nigericin. Sodium azide at a concentration exerting an uncoupler effect also stimulated the activity. The activity was sensitive to the ATPase inhibitor N,N'-dicyclohexylcarbodiimide, but not to sodium vanadate. The ATP-dependent formation of an electrochemical potential difference of protons, measured by the flow-dialysis method, was determined as 180 mV, with contribution of 1.7 pH units, interior acid, and of a membrane potential of 75 mV. It is concluded that the Mg2+-ATPase of vacuoles is a new marker enzyme for these organelles and is a N,N'-dicyclohexylcarbodiimide-sensitive, H+-translocating ATPase whose catalytic site is exposed to the cytoplasm.
Highlights
Amersham. 4-Methylumbelliferyl-a-~-mannopyranosidwe as obtained from Koch-Light Laboratories, England
The fist half of this paper describes biochemical criteria for the preparations of intact vacuoles and vacuolar membrane
H'-translocating ATPase of S. cerevisiae Vacuole vesicles according to themarker concept of cellular organelles of de Duve [26]
Summary
We established a procedure for preparing rightet a!. [7] and 1 unit of enzyme was defined as the amount liberating 1 pmol of inorganic phosphate/min under the standard conditions side-out vacuolar membrane vesicleosf high purityfrom cells described above. T h i s paper describes the properties of vacuolar membrane- mixture (2.5 m l ) , which contained 50 mM Tricine/NaOH, pH 7.5, 5 bound M8+-ATPase and its characteristic functionas a H'- mM MgCIz, 2p~ quinacrine, and 180 pgof protein of membrane translocating ATPase.The Mg2+-dependent, ATPase of the vacuoles was fountdo be a new of these organelles and t o differ from mitoch. H+-translocating specific marker ondrialATPase vesicles,was incubated in a quartz cuvette in a Hitachi-MPF-4 spectrofluorometer at 25 "C for 2 to 5 min To this mixture, ATP (0.5 mM) was added tostart quenching. The internal water space of vacuolar membrane vesicles under standard assay conditions was determined as 5.2 d/mg of membrane protein by the inulin method [17]
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