Thermoinactivation analysis of vacuolar H(+)-pyrophosphatase

Vacuolar H(+)-translocating pyrophosphatase (H(+)-PPase; EC 3.6.1.1) catalyzes both the hydrolysis of PP(i) and the electrogenic translocation of proton from the cytosol to the lumen of the vacuole. Vacuolar H(+)-PPase, purified from etiolated hypocotyls of mung bean (Vigna radiata L.), is a homodimer with a molecular mass of 145 kDa. To investigate the relationship between structure and function of this H(+)-translocating enzyme, thermoinactivation analysis was employed. Thermoinactivation studies suggested that vacuolar H(+)-PPase consists of two distinct states upon heat treatment and exhibited different transition temperatures in the presence and absence of ligands (substrate and inhibitors). Substrate protection of H(+)-PPase stabilizes enzyme structure by increasing activation energy from 54.9 to 70.2 kJ/mol. We believe that the conformation of this enzyme was altered in the presence of substrate to protect against the thermoinactivation. In contrast, the modification of H(+)-PPase by inhibitor (fluorescein 5'-isothiocyanate; FITC) augmented the inactivation by heat treatment. The native, substrate-bound, and FITC-labeled vacuolar H(+)-PPases possess probably distinct conformation and show different modes of susceptibility to thermoinactivation. Our results also indicate that the structure of one subunit of this homodimer exerts long distance effect on the other, suggesting a specific subunit-subunit interaction in vacuolar H(+)-PPase. A working model was proposed to interpret the relationship of the structure and function of vacuolar H(+)-PPase.     

Roles of histidine residues in plant vacuolar H(+)-pyrophosphatase

Vacuolar proton pumping pyrophosphatase (H(+)-PPase; EC 3.6.1.1) plays a pivotal role in electrogenic translocation of protons from cytosol to the vacuolar lumen at the expense of PP(i) hydrolysis. Alignment analysis on amino acid sequence demonstrates that vacuolar H(+)-PPase of mung bean contains six highly conserved histidine residues. Previous evidence indicated possible involvement of histidine residue(s) in enzymatic activity and H(+)-translocation of vacuolar H(+)-PPase as determined by using histidine specific modifier, diethylpyrocarbonate [J. Protein Chem. 21 (2002) 51]. In this study, we further attempted to identify the roles of histidine residues in mung bean vacuolar H(+)-PPase by site-directed mutagenesis. A line of mutants with histidine residues singly replaced by alanine was constructed, over-expressed in Saccharomyces cerevisiae, and then used to determine their enzymatic activities and proton translocations. Among the mutants scrutinized, only the mutation of H716 significantly decreased the enzymatic activity, the proton transport, and the coupling ratio of vacuolar H(+)-PPase. The enzymatic activity of H716A is relatively resistant to inhibition by diethylpyrocarbonate as compared to wild-type and other mutants, indicating that H716 is probably the target residue for the attack by this modifier. The mutation at H716 of V-PPase shifted the optimum pH value but not the T(1/2) (pretreatment temperature at which half enzymatic activity is observed) for PP(i) hydrolytic activity. Mutation of histidine residues obviously induced conformational changes of vacuolar H(+)-PPase as determined by immunoblotting analysis after limited trypsin digestion. Furthermore, mutation of these histidine residues modified the inhibitory effects of F(-) and Na(+), but not that of Ca(2+). Single substitution of H704, H716 and H758 by alanine partially released the effect of K(+) stimulation, indicating possible location of K(+) binding in the vicinity of domains surrounding these residues.     

Diethylpyrocarbonate inhibition of vacuolar H+-pyrophosphatase possibly involves a histidine residue

Vacuolar proton pumping pyrophosphatase (H⁺-PPase; EC 3.6.1.1) plays a pivotal role in electrogenic translocation of protons from cytosol to the vacuolar lumen at the expense of PP_i hydrolysis. A histidine-specific modifier, diethylpyrocarbonate (DEPC), could substantially inhibit enzymic activity and H⁺-translocation of vacuolar H⁺-PPase in a concentration-dependent manner. Absorbance of vacuolar H⁺-PPase at 240 nm was increased upon incubation with DEPC, demonstrating that an N-carbethoxyhistidine moiety was probably formed. On the other hand, hydroxylamine, a reagent that can deacylate N-carbethoxyhistidine, could reverse the absorption change at 240 nm and partially restore PP_i hydrolysis activity as well. The pK _a of modified residues of the enzyme was determined to be 6.4, a value close to that of histidine. Thus, we speculate that inhibition of vacuolar H⁺-PPase by DEPC possibly could be attributed to the modification of histidyl residues on the enzyme. Furthermore, inhibition of vacuolar H⁺-PPase by DEPC follows pseudo-first-order rate kinetics. A reaction order of 0.85 was calculated from a double logarithmic plot of the apparent reaction constant against DEPC concentration, suggesting that the modification of one single histidine residue on the enzyme suffices to inhibit vacuolar H⁺-PPase. Inhibition of vacuolar H⁺-PPase by DEPC changes V _max but not K _m values. Moreover, DEPC inhibition of vacuolar H⁺-PPase could be substantially protected against by its physiological substrate, Mg²⁺-PP_i. These results indicated that DEPC specifically competes with the substrate at the active site and the DEPC-labeled histidine residue might locate in or near the catalytic domain of the enzyme. Besides, pretreatment of the enzyme with N-ethylmaleimide decreased the degree of subsequent labeling of H⁺-PPase by DEPC. Taken together, we suggest that vacuolar H⁺-PPase likely contains a substrate-protectable histidine residue contributing to the inhibition of its activity by DEPC, and this histidine residue may located in a domain sensitive to the modification of Cys-629 by NEM.     

Isolation and characterisation of the major outer membrane protein of Erwinia carotovora

Treatment of the tonoplast H(+)-ATPase from mung bean seedlings (Vigna radiata L.) with histidine-specific modifier, diethyl pyrocarbonate (DEP), caused a marked loss of the ATP hydrolysis activity and the proton translocation in a concentration-dependent manner. The reaction order of inhibition was calculated to be 0.98, suggesting that at least one histidine residue of vacuolar H(+)-ATPase was modified by DEP. The absorbance of the vacuolar H(+)-ATPase at 240 nm was progressively increased after incubation with DEP, suggesting that N-carbethoxyhistidine had been formed. Hydroxylamine, which could break N-carbethoxyhistidine, reversed the absorbance change and partially restored the enzymic activity. The pK(a) of modified residues of vacuolar H(+)-ATPase was kinetically determined to be 6.73, a value close to that of histidine. Thus, it is assuredly concluded that histidine residues of the vacuolar H(+)-ATPase were modified by DEP. Kinetic analysis showed that V(max) but not K(m) of vacuolar H(+)-ATPase was decreased by DEP. This result is interpreted as that the residual activity after DEP inhibition was primarily due to the unmodified enzyme molecules. Moreover, simultaneous presence of DEP and DCCD (N,N'-dicyclohexyl-carbodiimide), an inhibitor modified at proteolipid subunit of vacuolar H(+)-ATPase, did not induce synergistic inhibition, indicating their independent effects. The stoichiometry studies further demonstrate that only one out of four histidine residues modified was involved in the inhibition of vacuolar H(+)-ATPase by DEP. Mg(2+)-ATP, the physiological substrate of vacuolar H(+)-ATPase, but not its analogs, exerted preferentially partial protection against DEP, indicating that the histidine residue involved in the inhibition of enzymatic activity may locate at/or near the active site and directly participate in the binding of the substrate.     

Vacuolar H(+)-pyrophosphatase

The H(+)-translocating inorganic pyrophosphatase (H(+)-PPase) is a unique, electrogenic proton pump distributed among most land plants, but only some alga, protozoa, bacteria, and archaebacteria. This enzyme is a fine model for research on the coupling mechanism between the pyrophosphate hydrolysis and the active proton transport, since the enzyme consists of a single polypeptide with a calculated molecular mass of 71-80 kDa and its substrate is also simple. Cloning of the H(+)-PPase genes from several organisms has revealed the conserved regions that may be the catalytic site and/or participate in the enzymatic function. The primary sequences are reviewed with reference to biochemical properties of the enzyme, such as the requirement of Mg(2)(+) and K(+). In plant cells, H(+)-PPase coexists with H(+)-ATPase in a single vacuolar membrane. The physiological significance and the regulation of the gene expression of H(+)-PPase are also reviewed.     

Acylspermidine derivatives isolated from a soft coral,Sinularia sp,inhibit plant vacuolar H(+)-pyrophosphatase

H(+)-pyrophosphatase (H(+)-PPase), which pumps H(+) across membranes coupled with PP(i) hydrolysis, is found in most plants, and some parasitic protists, eubacteria and archaebacteria. We assayed a number of extracts derived from 145 marine invertebrates as to their inhibitory effect on plant vacuolar H(+)-PPase. Acylspermidine derivatives [RCONH(CH(2))(3)N(CH(3))(CH(2))(4)N(CH(3))(2)] from a soft coral (Sinularia sp.) inhibited the PPi-hydrolysis activity of purified H(+)-PPase and the PP(i)-dependent H(+) pump activity (half inhibition concentration, 1 micro M) of vacuolar membranes of mung bean. The apparent K(i) was determined to be 0.9 micro M. Acylspermidines did not affect the activity of vacuolar H(+)-ATPase, plasma membrane H(+)-ATPase, mitochondrial ATPase or cytosolic PPase. Acylspermidines inhibited the acidification of vacuoles in protoplasts, as found on monitoring by the acridine orange fluorescent method. These results indicate that acylspermidine derivatives represent new inhibitors of H(+)-PPase with relatively high specificity.     

Identification of essential lysines involved in substrate binding of vacuolar H+-pyrophosphatase

H+-translocating pyrophosphatase (H+-PPase; EC 3.6.1.1) drives proton transport against an electrochemical potential gradient by hydrolyzing pyrophosphate (PPi) and is found in various endomembranes of higher plants, bacteria, and some protists. H+-PPase contains seven highly conserved lysines. We examined the functional roles of these lysines, which are, for the most part, found in the cytosolic regions of mung bean H+-PPase by site-directed mutagenesis. Construction of mutants that each had a cytosolic and highly conserved lysine substituted with an alanine resulted in dramatic drops in the PPi hydrolytic activity. The effects caused by ions on the activities of WT and mutant H+-PPases suggest that Lys-730 may be in close proximity to the Mg2+-binding site, and the great resistance of the K694A and K695A mutants to fluoride inhibition suggests that these lysines are present in the active site. The modifier fluorescein 5'-isothiocyanate (FITC) labeled a lysine at the H+-PPase active site but did not inhibit the hydrolytic activities of K250A, K250N, K250T, and K250S, which suggested that Lys-250 is essential for substrate binding and may be involved in proton translocation. Analysis of tryptic digests indicated that Lys-711 and Lys-717 help maintain the conformation of the active site. Proteolytic evidence also demonstrated that Lys-250 is the primary target of trypsin and confirmed its crucial role in H+-PPase hydrolysis.     

A lysine substitute for K+. A460K mutation eliminates K+ dependence in H+-pyrophosphatase of Carboxydothermus hydrogenoformans

The H(+) proton-translocating inorganic pyrophosphatase (H(+)-PPase) family is composed of two phylogenetically distinct types of enzymes: K(+)-dependent and K(+)-independent. However, to date, the sequence criteria governing this dichotomy have remained unknown. In this study, we describe the heterologous expression and functional characterization of H(+)-PPase from the thermophilic bacterium Carboxydothermus hydrogenoformans. Both PP(i)-hydrolyzing and PP(i)-energized H(+) translocation activities of the recombinant enzyme in Escherichia coli inner membrane vesicles are strictly K(+)-dependent. Here we deduce the K(+) requirement of all available H(+)-PPase sequences based on the K(+) dependence of C. hydrogenoformans H(+)-PPase in conjunction with phylogenetic analyses. Our data reveal that K(+)-independent H(+)-PPases possess conserved Lys and Thr that are absent in K(+)-dependent H(+)-PPases. We further demonstrate that a A460K substitution in C. hydrogenoformans H(+)-PPase is sufficient to confer K(+) independence to both PP(i) hydrolysis and PP(i)-energized H(+) translocation. In contrast, a A463T mutation does not affect the K(+) dependence of H(+)-PPase.     

Patch clamp analysis of a H+ pump heterologously expressed in giant yeast vacuoles

Despite the usefulness of the patch-clamp technique, its application to ion pumps and transporters in biomembranes is limited. We developed a novel method for determining the activity of a proton-pumping pyrophosphatase (H(+)-PPase) made of a single protein. We heterologously highly expressed the enzyme in Saccharomyces cerevisiae, prepared giant vacuoles from the cells, and measured a PPi-dependent electrical current of 18 pA (10.5 fA/ micro m(2)) using the patch-clamp technique in the whole-vacuole recording mode. We determined the inhibitor sensitivity and affinity for substrate (K(m), 4.6 micro M). The enzyme number in a giant vacuole (4.2 x 10(6)) and the molecular activity of the expressed H(+)-PPase (14 s(-1)) were determined. An uncoupling-type H(+)-PPase mutant, of which the 263rd glutamate residue was replaced by aspartate, and of which H(+) pump activity was not detected with the fluorescence quenching method, showed a weak current with a high K(m). The high accuracy, effectiveness and applicability of the method for exogenously expressed ion transporters were also discussed.     

H+-pyrophosphatase of Rhodospirillum rubrum. High yield expression in Escherichia coli and identification of the Cys residues responsible for inactivation my mersalyl

H(+)-translocating pyrophosphatase (H(+)-PPase) of the photosynthetic bacterium Rhodospirillum rubrum was expressed in Escherichia coli C43(DE3) cells. Recombinant H(+)-PPase was observed in inner membrane vesicles, where it catalyzed both PP(i) hydrolysis coupled with H(+) transport into the vesicles and PP(i) synthesis. The hydrolytic activity of H(+)-PPase in E. coli vesicles was eight times greater than that in R. rubrum chromatophores but exhibited similar sensitivity to the H(+)-PPase inhibitor, aminomethylenediphosphonate, and insensitivity to the soluble PPase inhibitor, fluoride. Using this expression system, we showed that substitution of Cys(185), Cys(222), or Cys(573) with aliphatic residues had no effect on the activity of H(+)-PPase but decreased its sensitivity to the sulfhydryl modifying reagent, mersalyl. H(+)-PPase lacking all three Cys residues was completely resistant to the effects of mersalyl. Mg(2+) and MgPP(i) protected Cys(185) and Cys(573) from modification by this agent but not Cys(222). Phylogenetic analyses of 23 nonredundant H(+)-PPase sequences led to classification into two subfamilies. One subfamily invariably contains Cys(222) and includes all known K(+)-independent H(+)-PPases, whereas the other incorporates a conserved Cys(573) but lacks Cys(222) and includes all known K(+)-dependent H(+)-PPases. These data suggest a specific link between the incidence of Cys at positions 222 and 573 and the K(+) dependence of H(+)-PPase.     

Immunological cross-reactivity between proton-pumping inorganic pyrophosphatases of widely phylogenic separated species.

Immunological cross-reactivity among three types of inorganic pyrophosphatases, that is, the proton pumping inorganic pyrophosphate synthase (H(+)-PPi synthase) and the soluble inorganic pyrophosphatase, both from Rhodospirillum rubrum, and the vacuolar membrane inorganic pyrophosphatase (H(+)-PPase) from mung bean (Vigna radiata), were examined by means of immunoblot analyses. Antibodies raised against the mung bean H(+)-PPase cross-reacted with the H(+)-PPi synthase from R. rubrum but not with the soluble PPase from R. rubrum. N,N'-dicyclohexylcarbodiimide (DCCD), which inhibits both synthesis and hydrolysis of PPi catalysed by purified and chromatophore H(+)-PPi synthase, binds to the enzyme as shown by fluorography of [14C]DCCD labelling. These results suggest that the R. rubrum H(+)-PPase share close structural similarities with the vacuolar H(+)-PPase from Mung bean.     

Expression of functional Streptomyces coelicolor H+-pyrophosphatase and characterization of its molecular properties

H(+)-translocating pyrophosphatases (H(+)-PPases) are proton pumps that are found in many organisms, including plants, bacteria and protozoa. Streptomyces coelicolor is a soil bacterium that produces several useful antibiotics. Here we investigated the properties of the H(+)-PPase of S. coelicolor by expressing a synthetic DNA encoding the amino-acid sequence of the H(+)-PPase in Escherichia coli. The H(+)-PPase from E. coli membranes was active at a relatively high pH, stable up to 50 degrees C, and sensitive to N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide and acylspermidine. Enzyme activity increased by 60% in the presence of 120 mM K(+), which was less than the stimulation observed with plant vacuolar H(+)-PPases (type I). Substitutions of Lys-507 in the Gly-Gln-x-x-(Ala/Lys)-Ala motif, which is thought to determine the K(+) requirement of H(+)-PPases, did not alter its K(+) dependence, suggesting that other residues control this feature of the S. coelicolor enzyme. The H(+)-PPase was detected during early growth and was present mainly on the plasma membrane and to a lesser extent on intracellular membranous structures.     

Vacuolar H(+)-translocating pyrophosphatases: a new category of ion translocase.

The membrane surrounding the central vacuole of plant cells contains an H(+)-translocating ATPase (H(+)-ATPase) and an H(+)-translocating inorganic pyrophosphatase (H(+)-PPase). Both enzymes are abundant and ubiquitous in plants but the H(+)-PPase is unusual in its exclusive use of inorganic pyrophosphate (PPi) as an energy source. The lack of sequence identity between the vacuolar H(+)-PPase and any other characterized ion pump implies a different evolutionary origin for this translocase. The existence of the vacuolar H(+)-PPase, in conjunction with increasing recognition of PPi as a key metabolite in plant systems, necessitates reconsideration of ATP as the primary energy source for membrane transport in plant cells.     

Reconstitution of transport function of vacuolar H(+)-translocating inorganic pyrophosphatase.

A procedure for reconstitution of the transport function of the vacuolar H(+)-translocating inorganic pyrophosphatase (H(+)-PPase; EC 3.6.1.1) prepared from etiolated hypocotyls of Vigna radiata (mung bean) is described. The method entails sequential extraction of isolated vacuolar membrane (tonoplast) vesicles with deoxycholate and CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate), combination of CHAPS-solubilized protein with phospholipid-cholesterol mixtures, dialysis, and glycerol density gradient centrifugation. The final proteoliposome preparation is 9-fold enriched for PPase activity and active in pyrophosphate (PPi)-energized electrogenic H(+)-translocation. Since both PPi hydrolysis and PPi-dependent H(+)-translocation by the proteoliposomes are indistinguishable from the corresponding activities of native tonoplast vesicles, the functional integrity of the H(+)-PPase appears to be conserved during solubilization and reconstitution. The high transport capacity and amenability of the reconstituted enzyme to both radiometric membrane filtration and fluorimetric H(+)-translocation assays, on the other hand, demonstrate its applicability to a broad range of transport studies. SDS-polyacrylamide gel electrophoresis of the proteoliposomes reveals selective enrichment of the M(r) 66,000, substrate-binding subunit of the H(+)-PPase and two additional polypeptides of M(r) 21,000 and 20,000. Although the M(r) 21,000 and 20,000 polypeptides have not been described previously, all attempts to reconstitute H(+)-PPase lacking these components were unsuccessful. It is therefore tentatively proposed that the M(r) 21,000 and 20,000 polypeptides, as well as the M(r) 66,000 subunit, are required for the productive reconstitution of PPi-dependent H(+)-translocation.     

Fluorescein isothiocyanate specifically modifies lysine 338 of cytochrome P-450scc and inhibits adrenodoxin binding

Treatment of cytochrome P-450scc with fluorescein isothiocyanate (FITC) resulted in covalent labeling with 1.0 +/- 0.1 eq of FITC. Reverse-phase high performance liquid chromatography of tryptic and chymotryptic digests of the labeled protein revealed that a single FITC-labeled peptide accounted for 75% of the label. This peptide was found to be specifically labeled at lysine 338 by amino acid sequencing. The modification of lysine 338 with FITC resulted in 85 +/- 15% inhibition of adrenodoxin binding to cytochrome P-450scc. In a complementary experiment it was found that if a complex between adrenodoxin and native cytochrome P-450scc was formed in the presence of cholesterol and then treated with FITC, there was almost no labeling of lysine 338. The modification of lysine 338 by FITC was not inhibited by 22(R)-hydroxycholesterol, the first intermediate in the side chain cleavage reaction which binds to the active site 300 times more tightly than cholesterol itself. These experiments suggest that lysine 338 is located at the binding site for adrenodoxin and electrostatically interacts with one of the carboxylate groups on adrenodoxin that has been implicated in binding. The fluorescence emission of the FITC label on cytochrome P-450scc was only 14% as large as that of an equivalent concentration of FITC-labeled bovine serum albumin, suggesting that it was quenched by Forster energy transfer to the heme group.     

Arbuscular mycorrhizal fungi induce differential activation of the plasma membrane and vacuolar H+ pumps in maize roots

Roots undergo multiple changes as a consequence of arbuscular mycorrhizal (AM) interactions. One of the major alterations expected is the induction of membrane transport systems, including proton pumps. In this work, we investigated the changes in the activities of vacuolar and plasma membrane (PM) H(+) pumps from maize roots (Zea mays L.) in response to colonization by two species of AM fungi, Gigaspora margarita and Glomus clarum. Both the vacuolar and PM H(+)-ATPase activities were inhibited, while a concomitant strong stimulation of the vacuolar H(+)-PPase was found in the early stages of root colonization by G. clarum (30 days after inoculation), localized in the younger root regions. In contrast, roots colonized by G. margarita exhibited only stimulation of these enzymatic activities, suggesting a species-specific phenomenon. However, when the root surface H(+) effluxes were recorded using a noninvasive vibrating probe technique, a striking activation of the PM H(+)-ATPases was revealed specifically in the elongation zone of roots colonized with G. clarum. The data provide evidences for a coordinated regulation of the H(+) pumps, which depicts a mechanism underlying an activation of the root H(+)-PPase activity as an adaptative response to the energetic changes faced by the host root during the early stages of the AM interaction.     

Characterization of isolated acidocalcisomes of Trypanosoma cruzi

The acidocalcisome is an acidic calcium store in trypanosomatids with a vacuolar-type proton-pumping pyrophosphatase (V-H(+)-PPase) located in its membrane. In this paper, we describe a new method using iodixanol density gradients for purification of the acidocalcisome from Trypanosoma cruzi epimastigotes. Pyrophosphatase assays indicated that the isolated organelle was at least 60-fold purified compared with the large organelle (10,000 x g) fraction. Assays for other organelles generally indicated no enrichment in the acidocalcisome fraction; glycosomes were concentrated 5-fold. Vanadate-sensitive ATP-driven Ca(2+) uptake (Ca(2+)-ATPase) activity was detectable in the isolated acidocalcisome, but ionophore experiments indicated that it was not acidic. However, when pyrophosphate was added, the organelle acidified, and the rate of Ca(2+) uptake increased. Use of the indicator Oxonol VI showed that V-H(+)-PPase activity generated a membrane potential. Use of sulfate or nitrate in place of chloride in the assay buffer did not affect V-H(+)-PPase activity, but there was less activity with gluconate. Organelle acidification was countered by the chloride/proton symport cycloprogidiosin. No vacuolar H(+)-ATPase activity was detectable in isolated acidocalcisomes. However, immunoblots showed the presence of at least a membrane-bound V-H(+)-ATPase subunit, while experiments employing permeabilized epimastigotes suggested that vacuolar H(+)-ATPase and V-H(+)-PPase activities are present in the same Ca(2+)-containing compartment.     

Elucidating the role of conserved glutamates in H+-pyrophosphatase of Rhodospirillum rubrum

H(+)-pyrophosphatase (H(+)-PPase) catalyzes pyrophosphate-driven proton transport against the electrochemical potential gradient in various biological membranes. All 50 of the known H(+)-PPase amino acid sequences contain four invariant glutamate residues. In this study, we use site-directed mutagenesis in conjunction with functional studies to determine the roles of the glutamate residues Glu(197), Glu(202), Glu(550), and Glu(649) in the H(+)-PPase of Rhodospirillum rubrum (R-PPase). All residues were replaced with Asp and Ala. The resulting eight variant R-PPases were expressed in Escherichia coli and isolated as inner membrane vesicles. All substitutions, except E202A, generated enzymes capable of PP(i) hydrolysis and PP(i)-energized proton translocation, indicating that the negative charge of Glu(202) is essential for R-PPase function. The hydrolytic activities of all other PPase variants were impaired at low Mg(2+) concentrations but were only slightly affected at high Mg(2+) concentrations, signifying that catalysis proceeds through a three-metal pathway in contrast to wild-type R-PPase, which employs both two- and three-metal pathways. Substitution of Glu(197), Glu(202), and Glu(649) resulted in decreased binding affinity for the substrate analogues aminomethylenediphosphonate and methylenediphosphonate, indicating that these residues are involved in substrate binding as ligands for bridging metal ions. Following the substitutions of Glu(550) and Glu(649), R-PPase was more susceptible to inactivation by the sulfhydryl reagent mersalyl, highlighting a role of these residues in maintaining enzyme tertiary structure. None of the substitutions affected the coupling of PP(i) hydrolysis to proton transport.     

The polyphosphate bodies of Chlamydomonas reinhardtii possess a proton-pumping pyrophosphatase and are similar to acidocalcisomes

Acidocalcisomes are acidic calcium storage compartments described initially in trypanosomatid and apicomplexan parasites. In this work, we describe organelles with properties similar to acidocalcisomes in the green alga Chlamydomonas reinhardtii. Nigericin and NH(4)Cl released (45)Ca(2+) from preloaded permeabilized cells, suggesting the incorporation of a significant amount of this cation into an acidic compartment. X-ray microanalysis of the electron-dense vacuoles or polyphosphate bodies of C. reinhardtii showed large amounts of phosphorus, magnesium, calcium, and zinc. Immunofluorescence microscopy, using antisera raised against a peptide sequence of the vacuolar type proton pyrophosphatase (H(+)-PPase) of Arabidopsis thaliana which is conserved in the C. reinhardtii enzyme, indicated localization in the plasma membrane, in intracellular vacuoles, and the contractile vacuole where it colocalized with the vacuolar proton ATPase (V-H(+)-ATPase). Purification of the electron-dense vacuoles using iodixanol density gradients indicated a preferential localization of the H(+)-PPase and the V-H(+)-ATPase activities in addition to high concentrations of PP(i) and short and long chain polyphosphate, but lack of markers for mitochondria and chloroplasts. In isolated electron-dense vacuoles, PP(i)-driven proton translocation was stimulated by potassium ions and inhibited by the PP(i) analog aminomethylenediphosphonate. Potassium fluoride, imidodiphosphate, N,N'-dicyclohexylcarbodiimide, and N-ethylmaleimide also inhibited PP(i) hydrolysis in the isolated organelles in a dose-dependent manner. These results indicate that the electron-dense vacuoles of C. reinhardtii are very similar to acidocalcisomes with regard to their chemical composition and the presence of proton pumps. Polyphosphate was also localized to the contractile vacuole by 4',6-diamidino-2-phenylindole staining, suggesting, with the immunochemical data, a link between these organelles and the acidocalcisomes.     

Modification of gastric (H+ + K+)-ATPase with pyridoxal 5'-phosphate

Pig gastric membrane vesicles enriched in (H+ + K+)-ATPase were covalently modified with pyridoxal 5'-phosphate (PLP). The modification resulted in inhibition of K+-dependent ATP hydrolysis, formation of phosphoenzyme and ATP-driven H+-uptake catalyzed by (H+ + K+)-ATPase. ATP, ADP, and adenyl-5'-yl imidodiphosphate were protective ligands, whereas Mg2+ and K+ were not. Specific PLP-binding of about 4.5 nmol/mg membrane protein was necessary for complete inhibition of the enzyme activity, indicating that the stoichiometry of PLP-binding to the enzyme was about 1:1. Limited proteolysis of the enzyme modified with [3H]PLP by trypsin suggests that PLP specifically modifies the lysine residue located in the 16-kDa fragment of the enzyme cleaved by trypsin. These results suggested that PLP binds to a specific lysine residue in the nucleotide-binding site or a region in its vicinity and inhibits the substrate binding or phosphorylation step of (H+ + K+)-ATPase.