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 (M_r = 16,400, 15,700, and 15,500) that remained tightly bound to the membrane. A water-soluble fragment (M_r 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.