An Essential Arginyl Residue in the Tonoplast Pyrophosphatase from Etiolated Mung Bean Seedlings

Abstract

Tonoplast membrane of etiolated mung bean (Vinga radiata.L.) seedlings contained HW-translocating pyrophosphatase (PPase).Modification of tonoplast vesicles and partially purified PPase from etiolated mung bean seedlings with arginine-specific reagents, phenylglyoxal (PGO) and 2,3-butanedione (BD), resulted in a marked decline in HW-translocating PPase activity. The halfmaximal inhibition was brought about by 20 millimolar PGO and 50 millimolar BD for membrane bound and 1.5 millimolar PGO and 5.0 millimolar BD for soluble PPase, respectively. The substrate, Mg2 -pyrophosphate, provided partial protection against inactivation by these reagents. Loss of activity of partially purified PPase followed pseudo-first order kinetics. The double logarithm plots of pseudo-first order rate constant versus reagent concentrations gave slopes of 0.88 (PGO) and 0.90 (BD), respectively, suggesting that the inactivation may possibly result from reaction of at least one arginyl residue at the active site of H+-translocating PPase.

The Role of Magnesium, Pyrophosphate, and Their Complexes as Substrates and Activators of the Vacuolar H+-Pumping Inorganic Pyrophosphatase (Studies Using Ligand Protection from Covalent Inhibitors)

“…This study supports earlier findings that Cys, Arg, and carboxyl residues may be present in the vicinity of domains involved in the binding of substrate and/or free Mg2+ (Britten et al, 1989;Kuo and Pan, 1990;Romero and Celis, 1992;Zhen et al, 1994). The location of the residues sensitive to PGO and EDAC remains to be established, but substrate-protectable NEM inhibition is due to binding to Cys"' , which is located on a cytoplasmic loop between membrane-spanning helices X and XI (Sarafian et al, 1992a; Zhen et al, 1994).…”

“…In this paper we have determined the binding constants for Mg,PPi and Mg2+ by making use of the observation that the H+-PPase is sensitive to inhibitors that react preferentially with certain amino acids and that inhibition is less when mixtures of Mg2+ and PPi are present in the incubation medium (Britten et al, 1989;Kuo and Pan, 1990;Baykov et al, 1993;Zhen et al, 1994). The results support the conclusion that Mg,PPi is the substrate and that there is a high-affinity Mgz+ binding site (Leigh et al, 1992;Baykov et al, 1993).…”

Proton pumping inorganic pyrophosphatase of endoplasmic reticulum-enriched vesicles from etiolated mung bean seedlings

“…The fluorescence quenching of ER-enriched vesicles and tonoplast were initiated by adding PP i . The ionophore gramicidin (2 mg/mL) was added at the end of each assay (Maeshima and Yoshida, 1989;Kuo and Pan, 1990).…”

Localization of a carboxylic residue possibly involved in the inhibition of vacuolar H+-pyrophosphatase by N,N′-dicyclohexylcarbodi-imide

“…from site-directed mutagenesis studies and the analysis of alignments for all the sequenced soluble PPases [20] and those from vacuolar H + -PPases [13][14][15][16][17][18]29]. The structure-function relationship of vacuolar H + -PPase has been studied extensively by conventional chemical modification and\or site-directed mutagenesis [10,19,[30][31][32]. It has been shown that vacuolar H + -PPase contains an arginine residue and a tyrosine residue crucial to enzymic activity [10,30].…”

“…The structure-function relationship of vacuolar H + -PPase has been studied extensively by conventional chemical modification and\or site-directed mutagenesis [10,19,[30][31][32]. It has been shown that vacuolar H + -PPase contains an arginine residue and a tyrosine residue crucial to enzymic activity [10,30]. A cytosolically oriented substrate-protectable cysteine residue was further shown to be involved in the inhibition of enzymic activity by Nethylmaleimide, but not to participate in the catalytic reaction [31,32].…”