A vacuolar ATPase and pyrophosphatase in Acetabularia acetabulum.

Vacuole-rich fractions were isolated from Acetabularia acetabulum by Ficoll step gradient centrifugation. The tonoplast-rich vesicles showed ATP-dependent and pyrophosphate-dependent H(+)-transport activities. ATP-dependent H(+)-transport and ATPase activity were both inhibited by the addition of a specific inhibitor of vacuolar ATPase, bafilomycin B1. A 66 kDa polypeptide present in the preparation cross-reacted with the anti-IgG fractions against the alpha and beta subunits of Halobacterium halobium ATPase and with the antibody against the A subunit (68 kDa subunit) of mung bean vacuolar ATPase. A 56 kDa polypeptide present in the vacuole preparation showed cross-reactivity with the antibody against the B subunit (57 kDa) of mung bean vacuolar ATPase but not with the anti-beta subunit of H. halobium ATPase. A 73 kDa polypeptide cross-reacted with the antibody against inorganic pyrophosphatase of mung bean vacuoles. These results suggest that vacuolar membrane of A. acetabulum equipped energy transducing systems similar to those found in other plant vacuoles.     

Heterogeneity of the vacuolar pyrophosphatase protein fromChenopodium rubrum

Activities of the tonoplast ATPase (V-ATPase EC 3.6.1.3) and PPase (V-PPase EC 3.6.1.1) provide the proton gradient driving the accumulation of various metabolites, organic and inorganic ions in the plant vacuole. We used anion exchange chromatography, liquid-phase isoelectric focusing (IEF), and continuous-elution native polyacrylamide gel electrophoresis (preparative PAGE) to enrich the V-PPase from solubilized tonoplast proteins from suspension cultured cells of Chenopodium rubrum L.The fractions were identified by their enzymatic activity, sensitivity towards the specific PPase inhibitor aminomethylenediphosphonate, apparent molecular weight, and immunological reactivity with an antibody raised against mung bean V-PPase. All these different methods used for the separation of solubilized tonoplast proteins revealed the existence of two physically separable V-PPase proteins exhibiting substrate specific enzymatic activity and 66 kDa apparent molecular weight after sodium dodecyl sulfate(SDS)-PAGE. The isoelectric points of the active V-PPase forms were 5.05 and 5.48 (V-ATPase 6.1). On the basis of the observation of high recoveries of enzymatic activity after different preparations we suggest that the V-PPase proteins separated may represent physiologically occurring forms of the enzyme which cannot be distinguished by SDS-PAGE and Western blot.     

Heterogeneity of the vacuolar pyrophosphatase protein fromChenopodium rubrum

Summary Activities of the tonoplast ATPase (V-ATPase EC 3.6.1.3) and PPase (V-PPase EC 3.6.1.1) provide the proton gradient driving the accumulation of various metabolites, organic and inorganic ions in the plant vacuole. We used anion exchange chromatography, liquid-phase isoelectric focusing (IEF), and continuous-elution native polyacrylamide gel electrophoresis (preparative PAGE) to enrich the V-PPase from solubilized tonoplast proteins from suspension cultured cells ofChenopodium rubrum L.The fractions were identified by their enzymatic activity, sensitivity towards the specific PPase inhibitor aminomethylenediphosphonate, apparent molecular weight, and immunological reactivity with an antibody raised against mung bean V-PPase. All these different methods used for the separation of solubilized tonoplast proteins revealed the existence of two physically separable V-PPase proteins exhibiting substrate specific enzymatic activity and 66 kDa apparent molecular weight after sodium dodecyl sulfate(SDS)-PAGE. The isoelectric points of the active V-PPase forms were 5.05 and 5.48 (V-ATPase 6.1). On the basis of the observation of high recoveries of enzymatic activity after different preparations we suggest that the V-PPase proteins separated may represent physiologically occurring forms of the enzyme which cannot be distinguished by SDS-PAGE and Western blot.     

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.     

Intracellular PPi concentration is not directly dependent on amount of inorganic pyrophosphatase in Escherichia coli K-12 cells.

No correlation was observed between the level of inorganic pyrophosphatase (PPase) and the intracellular concentration of PPi in Escherichia coli cells. In exponentially growing cells the intracellular PPi concentration was in every case 1.5 nmol/mg (dry weight) or about 0.5 mM, even though the amount of PPase was varied from 15 to 2,600% of the control amount by mutation or by using a multicopy plasmid with an inserted gene (ppa) encoding PPase. The PPi concentration could, however, be increased or decreased from the control level under some stressful conditions.     

The influence of pretreatment and enzyme loading on the effectiveness of batch and fed-batch hydrolysis of corn stover

To try to improve hydrolysis yields at elevated solids loadings, a comparison was made between batch and fed-batch addition of fresh substrate at the initial and later phases of hydrolysis. Both ethanol (EPCS) and steam-pretreated corn stover (SPCS) substrates were tested at low (5 FPU) and high (60 FPU) loadings of cellulase per gram of cellulose. The fed-batch addition of fresh substrate resulted in a slight decrease in hydrolysis yields when compared with the corresponding batch reactions. A 72-h hydrolysis of the SPCS substrate resulted in a hydrolysis yield of 66% compared with 51% for the EPCS substrate. When the enzyme adsorption and substrate characteristics were assessed during batch and fed-batch hydrolysis, it appeared that the irreversible binding of cellulases to the more recalcitrant original substrate limited their access to the freshly added substrate. After 72-h hydrolysis of the SPCS substrate at low enzyme loadings, ～40-50% of the added cellulases were desorbed into solution, whereas only 20% of the added enzyme was released from the EPCS substrate. Both simultaneous and sequential treatments with xylanases and cellulases resulted in an up to a 20% increase in hydrolysis yields for both substrates at low enzyme loading. Simons' stain measurements indicated that xylanase treatment increased cellulose access, thus facilitating cellulose hydrolysis.     

Two pathways of pyrophosphate hydrolysis and synthesis by yeast inorganic pyrophosphatase.

Initial rates of pyrophosphate hydrolysis and synthesis by baker's yeast inorganic pyrophosphatase and equilibrium amounts of enzyme-bound and free pyrophosphate were measured over wide ranges of Mg2+ and respective substrate concentrations. Computer analysis of these data, in conjunction with those on phosphate/water oxygen exchange [Kasho, V. N. & Baykov, A. A. (1989) Biochem. Biophys. Res. Comm. 161, 475-480], yielded values of the equilibrium constants for Mg2+ binding to free enzyme and central complexes and values of the forward and reverse rate constants for the four reaction steps, namely, PPi binding/release, PPi hydrolysis/synthesis and two Pi binding/release steps. All catalytic steps were found to proceed through two parallel pathways, involving 3 or 4 Mg2+/PPi or 2 Pi bound. Product release is the slowest catalytic event in both hydrolysis and synthesis of pyrophosphate, at least, for the four-metal pathway. In the hydrolytic reaction, magnesium pyrophosphate binding is faster for the four-metal pathway, dissociation of the second Pi is faster for the three-metal pathway, while PPi hydrolysis and the release of the first Pi may proceed with similar rates. Release of pyrophosphate formed on the enzyme is faster for the three-metal pathway. Both pathways are expected to operate in vivo, and their relative contributions will vary with changes in the Mg2+ concentration, thus providing a means for pyrophosphatase-activity regulation.     

Formation of nuclear HSF1 granules varies depending on stress stimuli

In concert with the stress-induced activation of human heat shock factor 1 (HSF1), the factor becomes inducibly phosphorylated and accumulates into nuclear granules. To date, these processes are not fully understood. Here, we show that although stress caused by the proteasome inhibitors MG132 and clasto-lactacystine beta-lactone induces the expression of Hsp70, the formation of HSF1 granules is affected differently in comparison to heat shock. Furthermore, proteasome inhibition increases serine phosphorylation on HSF1, but to a lesser extent than heat stress. Our results suggest that, depending on the type of stress stimulus, the multiple events associated with HSF1 activation might be affected differently.     

Purification and properties of vacuolar membrane proton-translocating inorganic pyrophosphatase from mung bean

Inorganic pyrophosphatase was purified from the vacuolar membrane of mung bean hypocotyl tissue by solubilization with lysophosphatidylcholine and QAE-Toyopearl chromatography. The molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 73,000 daltons. Among the amino-terminal first 30 amino acids are 25 nonpolar hydrophobic residues. For maximum activity, the purified pyrophosphatase required 1 mM Mg2+ and 50 mM K+. The enzyme reaction was stimulated by exogenous phospholipid in the presence of detergent. Excess pyrophosphate as well as excess magnesium inhibited the pyrophosphatase. The enzyme reaction was strongly inhibited by ATP, GTP, and CTP at 2 mM, and the inhibition was reversed by increasing the Mg2+ concentration. An antibody preparation raised in a rabbit against the purified enzyme inhibited both the reactions of pyrophosphate hydrolysis of the purified preparation and the pyrophosphate-dependent H+ translocation in the tonoplast vesicles. N,N'-Dicyclohexylcarbodiimide became bound to the purified pyrophosphatase and inhibited the reaction of pyrophosphate hydrolysis. It is concluded that the 73-kDa protein in vacuolar membrane functions as an H+-translocating inorganic pyrophosphatase.     

Evidence for the role of vacuolar soluble pyrophosphatase and inorganic polyphosphate in Trypanosoma cruzi persistence

Trypanosoma cruzi infection leads to development of a chronic disease but the mechanisms that the parasite utilizes to establish a persistent infection despite activation of a potent immune response by the host are currently unknown. Unusual characteristics of T. cruzi are that it possesses cellular levels of pyrophosphate (PP_i) at least 10 times higher than those of ATP and molar levels of inorganic polyphosphate (polyP) within acidocalcisomes. We characterized an inorganic soluble EF‐hand containing pyrophosphatase from T. cruzi (TcVSP) that, depending on the pH and cofactors, can hydrolyse either pyrophosphate (PP_i) or polyphosphate (polyP). The enzyme is localized to both acidocalcisomes and cytosol. Overexpression of TcVSP (TcVSP‐OE) resulted in a significant decrease in cytosolic PP_i, and short and long‐chain polyP levels. Additionally, the TcVSP‐OE parasites showed a significant growth defect in fibroblasts, less responsiveness to hyperosmotic stress, and reduced persistence in tissues of mice, suggesting that PP_i and polyP are essential for the parasite to resist the stressful conditions in the host and to maintain a persistent infection.     

Effect of ferric tartrate/sodium hydroxide solvent pretreatment on enzyme hydrolysis of cellulose in corn residue

Lignocellulose containing 62% cellulose was prepared from corn residue by dilute acid hydrolysis using 5% H(2)SO(4) at 90 degrees C. The lignocellulose was then treated with a cellulose solvent consisting of a ferric sodium tartrate complex in 1.5N sodium hydroxide at levels ranging from 4:1 to 12:1 (solvent volume: corn residue lignocellulose) or a 1.5N sodium hydroxide solution alone. Subsequent hydrolysis with cellulase enzymes from Trichoderma reesei gave cellulose conversions which were two to three times higher than untreated lignocellulose (30%) and approached 90% conversion after 24 h in the best cases. It was found that increasing cellulase enzyme levels from 3.74 lU/g lignocellulose to 7.71 lU/g lignocellulose increased cellulose conversion by 50% at all pretreatment conditions, while an increase from 7.71 to 10.1 lU/g gave only an additional 5-10% increase. Pretreatment with sodium hydroxide resulted in 5-25% lower conversions than observed for cellulose treated with the solvent, depending on enzyme levels and treatment levels. At high enzyme levels, sodium hydroxide pretreatment is almost as effective in enhancing cellulose conversion after 24 h as is pretreatment using the cellulose solvent.     

Determinism of bacterial metacommunity dynamics in the southern East China Sea varies depending on hydrography

Spatial variation of communities composition (metacommunities) results from multiple assembly mechanisms, including environmental filtering and dispersal; however, whether and why the relative importance of the assembly mechanisms in shaping bacterial metacommunity changes through time in marine pelagic systems remains poorly studied. Here, we applied the elements of metacommunity structure framework and the variation partitioning framework to examine whether temporal variation of hydrographic conditions influences bacterioplankton metacommunity dynamics in the southern East China Sea (ECS). The spatiotemporal variation of bacterial communities composition was revealed using 454 pyrosequencing of 16S rDNA. In addition to the whole bacterial community, we analyzed four dominant taxonomic groups (Cyanobacteria, Alphaproteobacteria, Gammaproteobacteria, and Actinobacteria) separately. Our analyses indicate that, considering the whole community level, the determinism of metacommunity structure varied among seasons. When the degree of connectivity was low (December), the metacommunity exhibited random distribution and was explained mainly by the environmental component. However, Clementsian metacommunity was found at intermediate connectivity (May), during which the environmental and spatial predictors were both significant. When connectivity was high (August), a random distribution pattern was found and no significant effect of environmental filtering or dispersal limitation was detected. Nevertheless, when considering different taxonomic groups, the differences in metacommunity dynamics among groups were found. Our results suggest that the driving forces of metacommunity dynamics varied depending on hydrography, as the degrees of environmental heterogeneity and connectivity among habitat patches were determined by circulation pattern. Moreover, mechanisms varied among different taxonomic groups, suggesting that differential dispersal capacity among taxonomic groups should be integrated into community assembly studies.