Cumulative subject index volumes 160–165

Abstract

Plant vacuolar vesicles contain a novel H+-translocating pyrophosphatase (H+-PPase, EC 3.6.1.1). Modification of tonoplast vesicles and purified vacuolar H+-PPase from etiolated mung bean seedlings with tetranitromethane (TNM) resulted in a progressive decline in H+-translocating pyrophosphatase activity. The half-maximal inhibition was brought about by 0.6, 1.0, and 0.8 mM TNM for purified and membrane-bound H+-PPases, and its associated proton translocation, respectively. The maximal inhibition of vacuolar H+-PPase by TNM occurred at a pH value above 8. Loss of activity of purified H+-pyrophosphatase followed pseudo-first order rate kinetics, yielding a first-order rate constant (k2) of 0.039 s−1 and a steady-state dissociation constant of inactivation (Ki) of 0.02 mM. Covalent modification of vacuolar H+-PPase by TNM increased Km value of the enzyme for its substrate without a significant effect on Vmax. Double logarithmic plots of the pseudo-first order rate constant (kobs) versus TNM concentration exhibited a slope of 0.88, suggesting that at least one tyrosine residue was involved in the inactivation of H+-PPase enzymatic activity. Further spectrophotometric measurements of the nitrated H+-pyrophosphatase indicated that TNM could modify approximately two tyrosine residues/subunit of the enzyme. However, Tsou's analysis revealed that only one of those modified tyrosine residues directly participated in the inhibition of enzymatic activity of vacuolar H+-PPase. The physiological substrate, i.e., dimagnesium pyrophosphate, provided substantial protection against inactivation by TNM. Moreover, NEM pretreatment of the enzyme decreased the number of subsequent nitration of vacuolar H+-PPase. Taken together, we suggest that vacuolar H+-pyrophosphatase contains a substrate-protectable tyrosine residue conferring to the inhibition of its activity and this tyrosine residue may be located in a domain sensitive to the modification of Cys-634 by NEM.