Removal of the epsilon subunit from Escherichia coli F1-ATPase using monoclonal anti-epsilon antibody affinity chromatography.
The usefulness of two monoclonal antibodies, epsilon-1 and epsilon-4, which recognize the epsilon subunit of Escherichia coli F1-ATPase, for removing that subunit from ATPase was assessed. The epsilon subunit is a tightly bound, but dissociable, inhibitor of the ATPase. epsilon-1 binds epsilon with 10-fold higher affinity than epsilon-4. epsilon-1 recognizes a site on epsilon which is hidden by the quaternary structure of ATPase, while epsilon-4 can recognize epsilon when it is part of ATPase. Each antibody was purified and coupled to Sepharose to generate affinity columns. Solutions of ATPase in a buffer which was designed to reduce the affinity of epsilon for the enzyme were pumped through the columns and the degree of epsilon depletion was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by Western blotting. Neither column retained ATPase significantly. At low ATPase concentrations and low flow rates, the epsilon-1 column was more efficient than the epsilon-4 column, removing in excess of 95% of the epsilon in a single passage compared with 93% removal by the epsilon-4 column. At higher protein concentrations or flow rates, however, the performance of the epsilon-1 column was substantially poorer, while that of the epsilon-4 column was much less affected. Very little epsilon emerged from the epsilon-4 column before most of the measured epsilon-binding capacity was filled. A second passage through the epsilon-4 column reduced residual epsilon to less than 2% of that which was originally present. Pure, active epsilon was eluted from either column by 1 M NH4OH, pH 11.(ABSTRACT TRUNCATED AT 250 WORDS)
D000911:Antibodies, Monoclonal; D002846:Chromatography, Affinity / Q000379:methods; D000939:Epitopes / Q000276:immunology; D004926:Escherichia coli / Q000201:enzymology*; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000276:immunology / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D006180:Proton-Translocating ATPases