Isolation, characterization, and kinetics of mitochrondrial F₃-ATPASE from crayfish (Orconectes nais) gills.

Abstract

A soluble Fl-ATPase was isolated from the mitochondria of crayfish (Orconectes nais) gill tissue. The maximal mitochondrial disruption rate (95%) was obtained by sonicating for 4 min at pH 8.6. A purification of 15-fold was estimated. The F1-ATPase preparation was stable at 4°C to -70°C when kept in 20% glycerol. The pH optimum for soluble F1-ATPase (7.0-7.2) was slightly lower and more narrow than for membrane-bound enzyme (7.0-7.8). Both membrane-bound and soluble F1-ATPase were stimulated by HC03 -, S042-, and CI-i the highest stimulated activity for all was obtained at 35 mM. The activity was in the order HC03 -> SO42-> C1-. In addition, the apparent Ka for soluble enzyme was 10.1, 10.7, and 11.1 mM, respectively. Oligomycin and DCCD inhibited the membrane-bound F1-ATPase with I50 of 0.019 ug/ml and 2.2 uM, respectively, but were ineffective in inhibiting the soluble enzyme. Both were similarly sensitive to DIDS and vanadate. Soluble ATPase was significantly more sensitive to pCMB and N03 than the membrane-bound enzyme. In addition, soluble Fl-ATPase was slightly more sensitive to azide and NBD-Cl than membrane-bound enzyme. The differences between soluble and membrane-bound enzyme in the general properties as well as inhibitor and modulator sensitivities suggest conformational change transmission between Fe and Fl sectors, and slight conformational differences between soluble Fl and membrane-bound Fl' Kinetic studies on the membrane-bound Fl-ATPase indicated that the MgATP complex was the true substrate for the ATPase activity and had a ~ value of 0.327 mM. Free ATP was a competitive inhibitor (Ki = 0.77 mM), and free Mg2 + was a mixed inhibitor (Ki = 0.81 mM, Kit = 5.89 mM). However, free ATP also acted as an activator. Lineweaver-Burk plots for MgATP hydrolysis at high free Mg2 + concentration exhibited apparent negative cooperativity. This was not the case for high free ATP level. These results suggest that, although free ATP inhibited the enzyme by binding to catalytic sites, it stimulated the ATPase activity by binding to noncatalytic sites.