The tonoplast proton-translocating ATPase from the crassulacean acid metabolism plant Kalanchoë daigremontiana

A rapid procedure was developed for the purification and reconstitution into proteoliposomes of the tonoplast H⁺-translocating ATPase from the Crassulacean acid metabolism (CAM) plant Kalanchoe daigremontiana. It involved the fractionation of crude tonoplast membranes with the detergent Triton X-114, resolubilization of the ATPase with octyl glucoside in the presence of an optimized lipid mixture and formation of liposomes on removal of detergent by gel filtration. The enzyme could be further purified by sedimentation through glycerol gradients. It contained polypeptides of apparent molecular mass 72, 57, 48, 42, 39, 33 and 16 kDa; the smallest of these was labelled by [¹⁴C]-dicyclohexylcarbodiimide. There was no evidence for the presence of any large subunits. In these proteoliposomes, ATP hydrolysis and H⁺-translocation were measured independently, by a coupled enzyme assay and by quenching of the fluorescence of a permeant weak base, respectively The kinetic properties of the reconstituted plant ATPase were studied in detail. Rate equations derived from theoretical models of the enzyme's behaviour were fitted to experimental data by weighted non-linear regression, using a computer program that calculated the kinetic parameters that accorded to the optimal fit. The dependence of the rate of H⁺- translocation on the concentration of MgATP was well fitted by the Michaelis equation, with a Kᵐ value about 30 μM. ATP could be replaced by dATP, ITP, GTP, UTP or CTP and Mg²⁺ by Mn²+ or Ca²⁺; kinetic parameters for these substrates were determined. In contrast hydrolysis of MgATP showed complex kinetics which suggested either negative cooperativity between nucleotide-binding sites, or the presence of two non-interacting catalytic sites. Both the hydrolytic and the H⁺-translocating activities of the proteoliposomes were inhibited by nitrate, though not in parallel, the latter activity being more sensitive. Both activities were inhibited in parallel by bafilomycin A₁, which did not produce complete inhibition; the bafilomycininsensitive component had complex ATPase kinetics similar to those of the uninhibited enzyme. ADP behaved as an allosteric inhibitor of the ATPase, inducing apparent cooperativity in saturation with MgATP, together with a reduction in Vᵐᵃˣ . By fitting the experimental data to the concerted allosteric model proposed by Monod et al. (1965) a theoretical model was proposed in which the complex kinetics were due to the interaction between two substrate binding sites and a single inhibitor-binding site. Antibodies raised against specific subunits of the tonoplast ATPase were shown to cross-react with V-type ATPases from different species. Immunoblotting of the plant antibodies against the V-type ATPase purified from several bovine tissues has suggested the existence of isoforms of this particular enzyme.