An immunoreactive 8-azido ATP-labeled protein common to the lysosomal and chromaffin granule membrane.
The H+-ATPases of eukaryotic cell organelles, including the rat liver lysosomal (tritosomal) H+-ATPase and the bovine chromaffin granule ATPase, exhibit similarities in function, substrate requirements, and inhibitor responses. We have explored the possibility that these pumps also exhibit immunological similarities, and that common determinants may be present on polypeptides important to function, such as ATP binding. Toward this end, antibodies were produced in rabbits against a highly purified, detergent-solubilized and fractionated chromaffin granule proton pump preparation. This antibody reacted with a 70-80 kDa protein of the lysosomal membrane on Western blots. We have previously shown that photolysis with 8-azido-ATP inhibits lysosomal N-ethylmaleimide-sensitive, vanadate-, ouabain- and oligomycin-insensitive ATP hydrolysis and H+ transport, with concomitant labeling of a 70-80 kDa membrane protein, amongst others. Here, we report that the photolysis with 8-azido-ATP also leads to inhibition of chromaffin granule H+ pump function and pump-related ATP hydrolysis, with concomitant N-ethylmaleimide-sensitive, ATP-protectable, 8-azido-[alpha-32P]ATP labeling. The anti-chromaffin granule antibody reacts with an approx. 70 kDa protein of the chromaffin granule and the lysosome. This raises the possibility that the 70 kDa 8-azido-ATP-reactive, immunologically similar proteins may play a similar role in pump function such as ATP binding and/or hydrolysis in these organelles.
Cuppoletti
J
J
Department of Physiology and Biophysics, University of Cincinnati College of Medicine, OH 45267-0576.
Strasser
J E
JE
Dean
G E
GE
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D001386:Azides / Q000378:metabolism*; D002417:Cattle; D002837:Chromaffin Granules / Q000032:analysis*; D002838:Chromaffin System / Q000032:analysis*; D007425:Intracellular Membranes / Q000032:analysis; D008247:Lysosomes / Q000032:analysis*; D010782:Photolysis; D011506:Proteins / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism
D001386:Azides; D011506:Proteins; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase