enhanced chemiluminescence. The V5-subunit antibody incubation was performed as

described by the manufacturer followed by a secondary antibody incubation with

horseradish peroxidase-linked sheep anti-mouse antibody (1:10,000). Signals were

visualized on high performance chemiluminescence film using a Kodak X-Omat.

Assays of F1Fo ATP Synthase Activity

 Growth on a minimal succinate medium was used as an initial, in vivo, assay for

enzyme viability. ATP hydrolysis activity was assayed by the acid molybdate method

(146). Membranes were assayed in buffer (50 mM Tris-HC1, 1 mM MgCl2, pH 9.1) to

determine the linearity with respect to time and enzyme concentration. Membrane

energization was detected by the fluorescence quenching of 9-amino-6-chloro-2-

methoxyacridine (ACMA) (271).

 Results

Construction and Growth Characteristics of Mutants

 To investigate the function of individual b subunits in a F1Fo ATP synthase enzyme

complex, plasmids expressing defective b subunits with either a histidine or a V5 epitope-

tag were generated by site-directed mutagenesis. A total of five plasmids were

constructed expressing the bwt-his, bAl534end-his, bwt-vs, barg364ile-V5, Or barg364glu-V5 Subunits

(Table 3-1). The epitope tags were needed to facilitate enzyme purification and subunit

detection on a Western blot, respectively. In order to express two different b subunits in

the same cell, we used the two-plasmid expression system described previously in

Chapter 2(195). Enzyme complexes incorporated with a histidine or V5 epitope-tagged b

subunit homodimer have been studied previously and were shown to result in a functional

enzyme complex (195). E. coli strain KM2 (Ab) was used as the host because the

uncF(b) gene has been deleted, eliminating any background b subunit.