H+-translocating ATPase 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 response to inhibitors, inactivation by cold treatment in the presence of MgATP, and subunit composition judged by antibody cross-reactivity.
Moriyama
Y
Y
Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110.
Nelson
N
N
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002837:Chromaffin Granules / Q000201:enzymology; D003080:Cold Temperature; D006056:Golgi Apparatus / Q000201:enzymology*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D008099:Liver / Q000648:ultrastructure*; D018942:Macrolides; D008970:Molecular Weight; D009566:Nitrates / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D014617:Vacuoles / Q000201:enzymology*
D000900:Anti-Bacterial Agents; D018942:Macrolides; D009566:Nitrates; C057620:bafilomycin A; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; C031618:sodium nitrate; D006180:Proton-Translocating ATPases