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

A light microsomal membrane fraction (collected on 28% weight/volume sucrose) from young mycelia of Phycomyces blakesleeanus Bgff. stationary cultures exhibited Mg(2+) ATP-dependent proton pumping as monitored with the dye neutral red or by accumulation of [(14)C]methylamine. The substrate kinetics for ATP (K(m(ATP)) 1.1 millimolar in the presence of 2 millimolar Mg(2+)), the competitive inhibition by ADP (K(i(ADP)) 0.8 millimolar), the anion sensitivity (stimulation by Cl(-), inhibition by NO(3) (-) and SO(4) (2-)), the reversal of acidification by Ca(2+), 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.