H⁺-transloaction ATP in Golgi apparatus. Characterization as vacuolar H⁺-ATPase and its subunit structures

Golgi apparatus was prepared from rat liver, and enzymatic properties and the subunit structure of the H⁺ATPase were characterized. GTP (and also ITP) was found to drive H⁺-transport with about 20% of the initial velocity as that of ATP. Bafilomycin, a specific inhibitor for vacuolar H⁺-ATPase, inhibited the activity at 2.5 nM. The H⁺-ATPase was completely inhibited in the cold in the presence of MgATP (5 mM) and NaNO₃ (0.1 M). The cold inactivation of the H⁺-ATPase resulted in release of a set of polypeptides from Golgi membrane, with molecular masses almost identical to that of the hydrophilic sector of chromaffin granule H⁺-ATPase (72, 57, 41, 34, and 33 kDa). Three of these polypeptides (72, 57, and 34 kDa), cross-reacted with antibodies against the corresponding subunits of the chromaffin granule H⁺-ATPase. A counterpart of the 39-kDa hydrophobic component of chromaffin granule H⁺-ATPase was identified in the membrane, but no 115-kDa component was found. Hence, the Golgi H⁺-ATPase, shows typical features of vacuolar H⁺-ATPase, in relatively low substrate specificity, its repsonse to inhibitors, inactivation by cold treatment in the presence of MgATP, and subunit composition judged by antibody cross-reactivity.