Functional effects and cross-reactivity of antibody to purified subunit b (uncF protein) of Escherichia coli proton-ATPase.
Subunit b (uncF protein) of the proton-ATPase (F1F0) of Escherichia coli was purified from membranes of strain AN1460 (unc+). Antibody to purified subunit b was raised in rabbits. It reacted with F1-depleted membranes and blocked F1 binding. Bound antibody had no effect on proton transport through F0. F1-Depleted membranes competed with purified subunit b for antibody in an enzyme-linked immunosorbent assay. F1-Depleted membranes which had been pretreated with trypsin or preincubated with saturating amounts of soluble F1 competed poorly with purified subunit b for antibody. The antibody to subunit b was used to further evaluate the trypsin-cleavage data previously reported [D. S. Perlin, D. N. Cox, and A. E. Senior (1983) J. Biol. Chem. 258, 9793-9800]. The results indicated that trypsin proteolysis of F1-depleted membranes resulted in the transient appearance of three fragments of subunit b (Mr = 16,400, 15,700, and 15,500) that remained tightly bound to the membrane. A water-soluble fragment (Mr 14,800), previously thought to be derived from subunit b, was not detected by the antibody. The antibody to subunit b did not cross-react with any subunit of mitochondrial, chloroplast, or other bacterial proton-ATPase, or with the proton-ATPase of clathrin-coated vesicles, plant microsomal membranes, or Neurospora crassa plasma membranes.
Perlin
D S
DS
Senior
A E
AE
D000818:Animals; D000918:Antibody Specificity; D001667:Binding, Competitive; D002462:Cell Membrane / Q000201:enzymology; D003101:Collodion; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D009154:Mutation; D010446:Peptide Fragments / Q000276:immunology; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000276:immunology*; D011817:Rabbits; D014357:Trypsin
D010446:Peptide Fragments; D003101:Collodion; D014357:Trypsin; D006180:Proton-Translocating ATPases