Analysis of the Substrate Binding Site and Carboxyl Terminal Region of Vacuolar H+-Pyrophosphatase of Mung Bean with Peptide Antibodies

Abstract

Vacuolar H+-translocating inorganic pyrophosphatase is a single-protein enzyme and uses a simple substance as an energy donor. Functional domains of the enzyme were investigated by using antibodies specific to peptides corresponding to the putative substrate-binding site (DVGADLVGKVE) in the hydrophilic loop and the carboxyl terminal part. The antibody to the former peptide clearly reacted with the pyrophosphatases of different plant species, and strongly inhibited the hydrolytic activity of the purified enzymes and the proton pumping activity of membrane vesicles. These results indicate that the sequence functions as an actual substrate-binding site and is a common motif. The antibody to the carboxyl terminal part reacted only to the mung bean enzyme, suppressing its hydrolytic and proton pumping activities. The results suggest that the carboxyl terminus is exposed to the cytosol and is close to the catalytic site. H+-Pyrophosphatase hydrolyzed triphosphate and tetraphosphate at low rates. Phytic acid, myo-inositol hexaphosphate, inhibited the enzyme even in the presence of Mg2+. The concentration for 50% inhibition was 0.15 mM. The inhibition of H+-PPase by dicyclohexyldiimide was partly reversed by Mg2+. The catalytic site and the membrane topology of the enzyme are discussed.

The Organization of High-Affinity Ammonium Uptake in Arabidopsis Roots Depends on the Spatial Arrangement and Biochemical Properties of AMT1-Type Transporters

“…Enrichment of plasma membrane proteins in the upper phase was verified by protein gel blot analysis using an antibody against the plasma membrane ATPase AHA2 (DeWitt et al, 1996), while enrichment of endosomal membrane proteins in the lower fraction was confirmed by detection of DET3, a subunit of the vacuolar ATPase (Schumacher et al, 1999) and of the vacuolar pyrophosphatase (VPPase; Takasu et al, 1997). Probing of these fractions with the AMT1;2 antibody showed that in both cases, AMT1;2 was enriched in the upper phase (Figure 3).…”

“…Blots were developed using the ECL Advance Western Blotting Detection Kit (Amersham) according to the manufacturer's protocol. Primary antibodies and the secondary antibody (peroxidase-linked anti-rabbit IgG; Amersham) were diluted in blocking solution at the following concentrations and combinations: anti-AMT1;1 at 1:400 with secondary antibody at 1:25,000, anti-AMT1;2 at 1:10,000, anti-AMT1;3 at 1:5000, anti-AMT2;1 at 1:4000, anti-AHA2 at 1:10,000 (DeWitt et al, 1996), anti-VPPase (Takasu et al, 1997) at 1;2000, and anti-DET3 at 1:20,000 (Schumacher et al, 1999) with secondary antibody at 1:10,000. MagicMark Western Standard (Invitrogen) was used as molecular weight marker.…”

Identification and analysis of proton‐translocating pyrophosphatases in the methanogenic archaeon Methanosarcina mazei

“…A multiple alignment of PPase sequences from Eukarya (Arabidopsis, Beta, Acetabularia and Plasmodium), Bacteria (Rhodospirillum, Streptomyces and Thermotoga) and Archaea (Pyrobaculum, M. mazei Mvp1 and Mvp2) was performed (not shown) and conserved residues are shown in white on a black background in Figure 5. The greatest similarities between the Mvp2 sequence and the sequences of other proton-translocating PPases were detected within the hydrophilic loops III and VI with cytoplasmic orientation and the C-terminal tail, all of which are probably part of the substrate-binding and hydrolysis domain or contribute to it (Takasu et al 1997). Moreover, there are highly conserved Gly residues in Helices 3, 4, 5, 9, 13 and 15 that could be responsible for tight localization of the corresponding helices.…”

“…Moreover, antibody binding to the C-terminus inhibited PPi hydrolysis. Therefore, it was supposed that the C-terminus is close to the catalytic site of the cytoplasmic loop III (Takasu et al 1997). Considering the fundamental correspondence of the putative topology of Mvp1 and Mvp2 to that of other V-type PPases, a basic uniformity of secondary structure is most likely (Zhen et al 1997).…”

Functional expression of mung bean Ca2+/H+ antiporter in yeast and its intracellular localization in the hypocotyl and tobacco cells

“…SDS/PAGE was performed in 12% gels. Immunoblotting was carried out with a poly(vinylidene difluoride) membrane (Millipore) by the standard procedure [19]. Protein A conjugated to horseradish peroxidase was used to detect the IgG on the immunoblot.…”