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Membranes and Bioenergetics

Anion-Sensitive Mg²⁺ ATP-Dependent Proton Pumping in Microsomal Membranes from Phycomyces blakesleeanus Bgff

Botanisches Institut der Johann Wolfgang Goethe Universität, Siesmayerstrasse 70, D-6000-Frankfurt, Federal Republic of Germany

A light microsomal membrane fraction (collected on 28% weight/volume sucrose) from young mycelia of Phycomyces blakesleeanus Bgff. stationary cultures exhibited Mg²⁺ ATP-dependent proton pumping as monitored with the dye neutral red or by accumulation of [¹⁴C]methylamine. The substrate kinetics for ATP (K_m(ATP) 1.1 millimolar in the presence of 2 millimolar Mg²⁺), the competitive inhibition by ADP (K_i(ADP) 0.8 millimolar), the anion sensitivity (stimulation by Cl^–, inhibition by NO₃^– and SO₄^2–), the reversal of acidification by Ca²⁺, the inhibition by diethylstilbestrol and dicyclohexylcarbodiimide, as well as the absence of any inhibition by azide, molybdate, and vanadate strongly suggest a tonoplast-type ATPase driven proton pumping. The same membrane fraction showed no pyrophosphate driven H⁺-translocation. Electron microscopic examination of the fungal mycelium demonstrated the presence of vacuoles of different sizes and other nonidentified vesicles but no typical Golgi stacks.