TABLE 1.

ATP hydrolysis activities of membranes containing F0F1 and purified F1 before and after induction of the γ-β cross-link by oxidizing reagent

Conditions of assays are as described under “Experimental Procedures.”

Mutation	F0F1 expressiona	ATP hydrolysisa
			Isolated F1
		Membranesb	Untreatedc	After induction of γ-β cross-linkc,d	After treatment with 20 mm DTTc	Untreated Kme	Untreated kcat/Kme
	%	μmol Pi/min/mg	s−1	s−1	s−1	μm	s−1m1
Wild type	12 ± 3	8.0 ± 0.9	95 ± 3	NDf	NDf	45	5.9 × 105
βE381C	20 ± 4	8.0 ± 1.9	120 ± 4	4.0 ± 0.9g	130 ± 2	42	1.0 × 106
βD380C	15 ± 2	5.0 ± 0.9	84 ± 2	1.2 ± 0.02g	77 ± 3	37	1.1 × 106

^aExpression of F₀F₁ on membranes was determined by quantitative immunoblot as outlined under “Experimental Procedures,” and values were expressed as % of total membrane protein, n = 4.

^bMembrane assays contained 1 μm carbonyl cyanide 3-chlorophenylhydrazone.

^cATPase activities, under saturating ATP conditions, were measured in 50 mm HEPES-KOH, 10 mm ATP, 5 mm MgSO₄, pH 7.5, at 30 °C, with an ATP-regenerating system of 5 mm phospho(enol)pyruvate and 50 μg/ml pyruvate kinase.

^dThe γ-β cross-link was induced by treatment with 50 μm CuCl₂ for βE381C-F₁ and by treatment with 25 μm DTNB for βD380C-F₁.

^eValues for K_m and k_cat were determined by measuring ATPase activity as a function of Mg·ATP concentration under conditions maintaining a low free Mg²⁺ concentration of 50 μm. The buffer consisted of 25 mm TES-KOH, 0.2 mm EDTA, and varying amounts of Na₂-ATP (25 μm to 10 mm) and MgCl₂ (0.269 mm to 4.57 mm), pH 7.5, at 25 °C, with an ATP-regenerating system of 2 mm phospho(enol)pyruvate, 50 μg/ml pyruvate kinase.

^fND means experiment was not repeated here. Duncan et al. (27) have already shown there is no effect of either DTNB or DTT on wild-type activity.

^gActivity was fully inhibitable by 1 mm sodium azide, indicating the extent of uncross-linked enzyme.