the assay. Tubes of stop buffer were required for each time point of each sample,

phosphate standard curve, and "ATP only" control. Prior to the beginning of the time

course, two measures are taken to subtract background phosphate. First, a zero time

point was taken by removing 43 5 C1L of the reaction buffer, containing the membranes

before the addition of ATP, and adding it to 2 mL of stop buffer. Also, to account for

spontaneous hydrolysis, ATP was incubated in 4 mL of reaction buffer with no

membrane protein and then 435 C1L was removed and added to stop buffer. The time

course was started by the addition of 80 CIL ATP (0.15 M in 25 mM Tris-HC1, pH 7.5) to

the reaction buffer and additional time points were taken at 2, 5, 7, 10 and 12 minutes. A

phosphate standard curve was generated by preparing duplicates of 1 mL reaction buffer

containing 0, 0.02, 0.1, 0.2, 0.4 and 0.6 Clmol phosphate (KH2PO4), and then adding 435

CIL of each to 2 mL stop buffer. Once the time course and the standard curve were

completed, all of the samples in stop buffer were removed from the ice and allowed to

come to room temperature. The inorganic phosphate concentration was determined by

adding 100 CIL of Eikonogen solution (1 M NaHSO3, 0.1 M Na2SO3, 0.01 M 4-amino-3-

hydroxy-1 -naphthalenesulfonic acid), diluted 1:10, and incubating at room temperature

for 30 minutes to allow color development. The absorbance of each time point at 700 nm

was recorded with an LKB Biochrom Ultrospec II spectrophotometer and the amount of

free phosphate in each sample was determined using linear regression. The rate of ATP

hydrolysis was determined and the apparent specific activity was expressed as Clmol

Pi/min/mg membrane protein.

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

chloro-2-methoxyacridine (ACMA) (271). First, F1Fo ATP synthase-mediated ATP-