Column centrifugation generates an intersubunit disulfide bridge in Escherichia coli F1-ATPase

Abstract

Passage of F1-ATPase through a centrifuge column [Penefsky, H. S. (1979) Methods Enzymol. 56, 527-539] caused formation of a product with a relative molecular mass of 72,000 as determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The product was identified as cross-linked .alpha. and .delta. subunits by using Western blots and subunit-specific monoclonal antibodies. The cross-link was reversed by 50 mM dithiothreitol implying that it was a disulfide bridge. Formation of the cross-link was inhibited by 2 mM EDTA and was stimulated in some buffers by the addition of 10 .mu.M CuCl2. Time course experiments indicated that the majority of the cross-link formed while the enzyme was passing through the column. Thus the cross-link induced by column centrifugation arose from the rapid, heavy-metal-ion-catalysed oxidation of two sulfhydryl groups, one on the .alpha. subunit and one on the .delta. subunit, to a disulfide. These results demonstrate that care must be exercised when running proteins through centrifuge columns as potentially deleterious disulfide formation can result. An anti-.beta. monoclonal antibody was capable of immunoprecipitating the entire enzyme including the cross-linked subunits, implying that the cross-linked .alpha. and .delta. subunits were still a part of F1. The formation of the cross-link affected neither the hydrolytic activity of the enzyme nor its susceptibility to inhibition by .epsilon. subunit. The cross-linked enzyme was unable to bind to F1-depleted membranes in experiments in which soluble F1 and membranes were separated by centrifugation. Column centrifugation did not generate the cross-link on membrane-bound enzyme. These results indicate that the .alpha.- .delta. cross-link results in a loss of binding affinity between F1 and F0.