(146). Membranes were assayed in buffer (50 mM Tris-HC1, 1 mM MgCl2, pH9.1) to

determine the linearity with respect to time and enzyme concentration.

Immunoblot Analysis

 Proteins were loaded on a 15% tris-glycine SDS gel and transferred onto

nitrocellulose by electroblot. The b subunit antibody incubation was performed

essentially as described previously, using a 1:25,000 dilution of the anti-b subunit

antibodies. Secondary antibody incubation was performed with horseradish peroxidase-

linked donkey anti-rabbit antibody (1:50,000), and the antibody was detected by

enhanced chemiluminescence. Signals were visualized on high performance

chemiluminescence film using a Kodak X-Omat.

 Results

Construction and Growth Characteristics of Mutants

 Single cysteines, which will provide reactive thiols for site-specific chemical

modification, were strategically placed within the F1Fo ATP synthase enzyme complex.

Several cysteine mutations were generated in the unc operon in a multi-step construction

scheme to ultimately result in 12 plasmids encoding different combinations of six

different amino acid substitutions as well as b subunits of altered length (Table 4-2)

(Figure 4-3).

 A previous analysis of F1Fo ATP synthase complexes with b subunits shortened and

lengthened by 11 amino acids found the mutants to be essentially wild-type when

expressed in the presence of 40 C1M IPTG (193, 194). Functional F1Fo ATP synthase

complexes have been studied with many different cysteine mutations;