AT P















 ) basn24ala, thr64ala, glnl0aala






 L.





 glnl0eala
 bthr6ala


 basn24ala
 2 min wt,

Figure 5-3. ATP-driven energization of membrane vesicles prepared from b subunit
 membrane domain mutants. Cell membrane vesicles were prepared by
 differential centrifugation (see Materials and Methods). Membrane protein
 (200 Gig) was suspended in 3 mL of assay buffer (50 mM MOPS, 10 mM
 MgCl2, pH 7.3). The fluorescent dye ACMA was added to a final
 concentration of 1 mM and fluorescence was recorded with excitation at 410
 nm and emission at 490 nm. ATP was added as indicated to a final
 concentration of 1 mM. The samples for each trace have been labeled
 according to the mutation, so the strains used are as follows: Ab, membranes
 from strain KM2/pBR322; bwt, KM2/pKAM14; basn24ala, thr64ala, glnl04ala,
 KM2/pAWH24; basn24ala, KM2/pTAM43; bthr64ala, KM2/pTAM44;
 bginlonala, KM2/pTAM45.