[74] Determination of the Stoichiometry and Arrangement of α and β Subunits in F₁-ATPase Using Monoclonal Antibodies in Immunoelectron Microscopy

This chapter discusses the methods used for the determination of the stoichiometry and arrangement of α and β subunits in F₁-ATPase using monoclonal antibodies in immunoelectron microscopy. The methods include (1) staining intensities of bands after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (2) metabolic labeling with radioactive precursors, (3) cross-linking, (4) chemical labeling of sulfhydryl groups, (5) titration of the subunits using radioactively labeled antibodies and evaluation of the binding with a Scatchard plot, and (6) reconstitution experiments. These methods are based on the molecular weight of the enzyme, statistical behavior of the enzyme-reagent interactions, and a high homogeneity of the enzyme preparation. Monoclonal antibodies are used to label a specific subunit only once and the number of antibodies bound per molecule F₁-ATPase is determined by electron microscopy. The DNA sequence data of the Escherichia coli ATP synthase operon exclude repetitive sequences within one subunit or sufficiently homologous sequences in different subunits, and thus, one bound antibody corresponds to one subunit.