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, respectivelyThe 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
