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1.
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. 相似文献
2.
M Maeshima 《European journal of biochemistry》1991,196(1):11-17
The effects of divalent cations, especially Ca2+ and Mg2+, on the proton-translocating inorganic pyrophosphatase purified from mung bean vacuoles were investigated to compare the enzyme with other pyrophosphatases. The pyrophosphatase was irreversibly inactivated by incubation in the absence of Mg2+. The removal of Mg2+ from the enzyme increased susceptibility to proteolysis by trypsin. Vacuolar pyrophosphatase required free Mg2+ as an essential cofactor (K0.5 = 42 microM). Binding of Mg2+ stabilizes and activates the enzyme. The formation of MgPPi is also an important role of magnesium ion. Apparent Km of the enzyme for MgPPi was about 130 microM. CaCl2 decreased the enzyme activity to less than 60% at 40 microM, and the inhibition was reversed by EGTA. Pyrophosphatase activity was measured under different conditions of Mg2+ and Ca2+ concentrations at pH 7.2. The rate of inhibition depended on the concentration of CaPPi, and the approximate Ki for CaPPi was 17 microM. A high concentration of free Ca2+ did not inhibit the enzyme at a low concentration of CaPPi. It appears that for Ca2+, at least, the inhibitory form is the Ca2(+)-PPi complex. Cd2+, Co2+ and Cu2+ also inhibited the enzyme. The antibody against the vacuolar pyrophosphatase did not react with rat liver mitochondrial or yeast cytosolic pyrophosphatases. Also, the antibody to the yeast enzyme did not react with the vacuolar enzyme. Thus, the catalytic properties of the vacuolar pyrophosphatase, such as Mg2+ requirement and sensitivity to Ca2+, are common to the other pyrophosphatases, but the vacuolar enzyme differs from them in subunit mass and immunoreactivity. 相似文献
3.
AVP2, a sequence-divergent, K(+)-insensitive H(+)-translocating inorganic pyrophosphatase from Arabidopsis 总被引:1,自引:0,他引:1
Plant vacuolar H(+)-translocating inorganic pyrophosphatases (V-PPases; EC 3.6.1.1) have been considered to constitute a family of functionally and structurally monotonous intrinsic membrane proteins. Typified by AVP1 (V. Sarafian, Y. Kim, R.J. Poole, P.A. Rea [1992] Proc Natl Acad Sci USA 89: 1775-1779) from Arabidopsis, all characterized plant V-PPases share greater than 84% sequence identity and catalyze K(+)-stimulated H(+) translocation. Here we describe the molecular and biochemical characterization of AVP2 (accession no. AF182813), a sequence-divergent (36% identical) K(+)-insensitive, Ca(2+)-hypersensitive V-PPase active in both inorganic pyrophosphate hydrolysis and H(+) translocation. The differences between AVP2 and AVP1 provide the first indication that plant V-PPases from the same organism fall into two distinct categories. Phylogenetic analyses of these and other V-PPase sequences extend this principle by showing that AVP2, rather than being an isoform of AVP1, is but one representative of a novel category of AVP2-like (type II) V-PPases that coexist with AVP1-like (type I) V-PPases not only in plants, but also in apicomplexan protists such as the malarial parasite Plasmodium falciparum. 相似文献
4.
5.
Structure, function and regulation of the plant vacuolar H(+)-translocating ATPase 总被引:20,自引:0,他引:20
Ratajczak R 《Biochimica et biophysica acta》2000,1465(1-2):17-36
6.
The influence of phosphate (Pi) deprivation on the vacuolar H(+)-translocating pyrophosphatase (PPiase) and ATPase in tonoplast vesicles from Brassica napus suspension cells was assessed. Pi starvation significantly elevated the ratios of PPi-:ATP-dependent H(+) translocation rate and H(+)-PPiase:H(+)-ATPase hydrolytic activities. These increases were reversed 36 h following resupply of 2.5 mM Pi to the Pi-starved cells. Immunoblotting indicated that Pi starvation also induced a two-fold increase in the amount of H(+)-PPiase protein, whereas the amount of H(+)-ATPase remained unchanged. It is proposed that H(+)-PPiase facilitates the conservation of limited ATP pools, and Pi recycling during Pi stress. 相似文献
7.
8.
On the subunit structure of yeast inorganic pyrophosphatase 总被引:1,自引:0,他引:1
R L Heinrikson R Sterner C Noyes B S Cooperman R H Bruckmann 《The Journal of biological chemistry》1973,248(7):2521-2528
9.
A. Teplyakov G. Obmolova K. S. Wilson K. Ishii H. Kaji T. Samejima I. Kuranova 《Protein science : a publication of the Protein Society》1994,3(7):1098-1107
The 3-dimensional structure of inorganic pyrophosphatase from Thermus thermophilus (T-PPase) has been determined by X-ray diffraction at 2.0 A resolution and refined to R = 15.3%. The structure consists of an antiparallel closed beta-sheet and 2 alpha-helices and resembles that of the yeast enzyme in spite of the large difference in size (174 and 286 residues, respectively), little sequence similarity beyond the active center (about 20%), and different oligomeric organization (hexameric and dimeric, respectively). The similarity of the polypeptide folding in the 2 PPases provides a very strong argument in favor of an evolutionary relationship between the yeast and bacterial enzymes. The same Greek-key topology of the 5-stranded beta-barrel was found in the OB-fold proteins, the bacteriophage gene-5 DNA-binding protein, toxic-shock syndrome toxin-1, and the major cold-shock protein of Bacillus subtilis. Moreover, all known nucleotide-binding sites in these proteins are located on the same side of the beta-barrel as the active center in T-PPase. Analysis of the active center of T-PPase revealed 17 residues of potential functional importance, 16 of which are strictly conserved in all sequences of soluble PPases. Their possible role in the catalytic mechanism is discussed on the basis of the present crystal structure and with respect to site-directed mutagenesis studies on the Escherichia coli enzyme. The observed oligomeric organization of T-PPase allows us to suggest a possible mechanism for the allosteric regulation of hexameric PPases. 相似文献
10.
Y Sakai-Tomita C Moritani H Kanazawa M Tsuda T Tsuchiya 《Journal of bacteriology》1992,174(21):6743-6751
Cells of Vibrio parahaemolyticus grown in the presence of glucose showed reduced (by about 40%) oxidative phosphorylation. With this observation as a basis, we examined the effect of glucose on the level of H(+)-translocating ATPase. The addition of glucose to the growth medium reduced the specific activity and the amount of the H(+)-translocating ATPase in membrane vesicles of V. parahaemolyticus. These reductions were reversed by adding cyclic AMP (cAMP) to the growth medium. We cloned some parts of the unc genes encoding subunits of the H(+)-translocating ATPase of V. parahaemolyticus by means of the polymerase chain reaction. Using an amplified DNA fragment, we carried out Northern (RNA) blot analysis and found that glucose reduced the mRNA level of the H(+)-translocating ATPase gene by about 40% and that cAMP restored it. We determined the DNA sequence of the unc promoter region of V. parahaemolyticus and found a consensus sequence for the cAMP receptor protein-cAMP-binding site. Such a sequence was also found in the promoter region of the unc operon of Vibrio alginolyticus but not in its counterpart in Escherichia coli. We observed a similar reduction in the level of ATPase due to glucose in V. alginolyticus. In E. coli, however, reductions in the ATPase and the unc mRNA levels were not observed. Thus, the unc operon is controlled by cAMP-regulated catabolite repression in V. parahaemolyticus and V. alginolyticus but not in E. coli. Catabolite repression of the unc operon in V. parahaemolyticus is not severe. 相似文献
11.
12.
P A Rea Y Kim V Sarafian R J Poole J M Davies D Sanders 《Trends in biochemical sciences》1992,17(9):348-353
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. 相似文献
13.
14.
Molecular cloning and characterization of a novel H+-translocating pyrophosphatase gene in Zea mays.
A cDNA encoding a putative H+-translocating pyrophosphatase (H+-PPase) has been cloned from Zea mays by suppression subtractive hybridization (SSH) coupled with in silico cloning approach. The isolated 2974 bp full-length cDNA named ZmGPP contains a single 2400 bp open reading frame encoding a putative protein of 799 amino acids. The predicted protein has 16 transmembrane domains and is significantly similar to Golgi apparatus resident type-II H+-PPase from Arabidopsis thaliana. DNA gel blotting analysis shows that ZmGPP is a low-copy gene. Organ expression pattern analysis reveals that ZmGPPexpressed highly in leaf and tassel, followed by in stem, root, and ear. The Real-time RT-PCR assays showed that the expression of ZmGPP was up-regulated both in shoots and roots of maize seedlings under dehydration, cold and high salt stresses. Those results suggest that the ZmGPP product may play an important role in abiotic stress tolerance of Z. mays. 相似文献
15.
Na(+)-coupled ATP synthesis in a mutant of Vibrio parahaemolyticus lacking H(+)-translocating ATPase activity. 总被引:1,自引:0,他引:1
Y Sakai-Tomita M Tsuda T Tsuchiya 《Biochemical and biophysical research communications》1991,179(1):224-228
An H(+)-translocating ATPase-defective mutant of Vibrio parahaemolyticus YS-1 grew well on lactate as a sole source of carbon at pH 8.5 under aerobic conditions, but not under anaerobic conditions. Both wild type cells and the mutant cells could grow on lactate at pH 8.5 even in the presence of an H+ conductor, carbonylcyanide m-chlorophenylhydrazone (CCCP), but not at pH 7.5. Oxidative phosphorylation resistant to CCCP in the mutant occurred at pH 8.5. These findings suggest the existence of Na(+)-coupled oxidative phosphorylation which is functional at alkaline pHs in V. parahaemolyticus. In fact, we observed ATP synthesis driven by an artificially imposed Na+ gradient in YS-1 cells, which was resistant to CCCP. 相似文献
16.
The earliest known H+-proton-pumping inorganic pyrophosphatase, the integrally membrane-bound H+-proton-pumping inorganic pyrophosphate synthase from Rhodospirillum rubrum, is still the only alternative to H+-ATP synthase in biological electron transport phosphorylation. Cloning of several higher plant vacuolar H+-proton-pumping inorganic pyrophosphatase genes has led to the recognition that the corresponding proteins form a family of extremely similar proton-pumping enzymes. The bacterial H+-proton-pumping inorganic pyrophosphate synthase and two algal vacuolar H+-proton-pumping inorganic pyrophosphatases are homologous with this family, as deduced from their cloned genes. The prokaryotic and algal homologues differ more than the H+-proton-pumping inorganic pyrophosphatases from higher plants, facilitating recognition of functionally significant entities. Primary structures of H+-proton-pumping inorganic pyrophosphatases are reviewed and compared with H+-ATPases and soluble proton-pumping inorganic pyrophosphatases. 相似文献
17.
Carboxamidomethylation of yeast inorganic pyrophosphatase 总被引:2,自引:0,他引:2
18.
The activity of inorganic pyrophosphatase and pyrophosphate content were studied in developing and germinating cotton seeds. It was shown that the content of pyrophosphate in germinating seeds reached its maximum value after two days of their development, and the activity of inorganic pyrophosphatase, one day after the beginning of seed bud formation. The low pyrophosphatase activity of dormant seeds increased during their germination under open-ground conditions, reaching its maximum on day 6–7. Properties of partly purified pyrophosphatase from three-day-old cotton seedlings grown under laboratory conditions were studied. 相似文献
19.
The complete amino acid sequence of the inorganic pyrophosphatase from thermophilic bacterium PS-3 was determined by automated Edman analysis of the intact protein and of peptides derived from digests obtained with lysylendopeptidase, Staphylococcus aureus strain V8 protease, and arginylendopeptidase. The monomer peptide chain comprises 164 amino acid residues and has a calculated molecular weight of 18,792. The sequence is identical at about 46% of the amino acid positions with that of the Escherichia coli enzymes. 相似文献
20.
The activity of inorganic pyrophosphatase and pyrophosphate content were studied in developing and germinating cotton seeds. It was shown that the content of pyrophosphate in germinating seeds reached its maximum value after two days of their development, and the activity of inorganic pyrophosphatase, one day after the beginning of seed bud formation. The low pyrophosphatase activity of dormant seeds increased during their germination under open-ground conditions, reaching its maximum on day 6-7. Properties of partly purified pyrophosphatase from three-day-old cotton seedlings grown under laboratory conditions were studied. 相似文献