Abstracts of papers (1999)

Last Update: 07/04/2000

Abstracts of papers (1999)

[1999-1] Antognoni, F. et al., J. Biol. Chem. 274, 1942-1948 (1999)

The mechanism of inhibition of spermidine uptake by polyamines in Escherichia coli has been studied using the spermidine uptake operon, which consists of the potA, -B, -C, and -D genes. Transcriptional activity of the operon was inhibited by the PotD protein, a spermidine binding protein usually existing in the periplasm, and the inhibition was increased by spermidine. Transcription was not affected by bovine serum albumin, PotA or PotF, suggesting that the effects of PotD are specific. It was found that PotD bound to regions -258 to -209 nucleotides upstream and +66 to +135 nucleotides downstream from the ATG initiation codon of the potA gene. Binding of PotD to the latter was stimulated by spermidine. The 50% inhibition of transcription in the presence of 8 mM spermidine was observed in the approximate molar ratio of 1:500 of template DNA and PotD compared to the activity in the absence of PotD and spermidine. Spermidine uptake activity, PotABCD mRNA synthesis, and expression of a lacZ gene fused downstream of the potA gene containing the PotD binding sites in E. coli DH5[alpha] were inhibited significantly by overexpression of the PotD protein. When excess amounts of PotD were synthesized, significant amounts of PotD precursor existed in spheroplast. Our results indicate that PotD precursor also inhibits the spermidine transport activity. Amino acid residues involved in the interaction of PotD with the spermidine uptake operon were determined using mutated PotD, and Thr35 and Ser83 of PotD were found to be important for this interaction. These results strongly suggest that transcription of the spermidine transport operon is inhibited by PotD (or PotD precursor) through its binding to two regions close to the transcriptional initiation site of the operon.

[1999-2] Tomitori, H. et al., J. Biol. Chem. 274, 3265-3267 (1999)

Properties of a membrane protein encoded by YLL028w were examined using yeast cells transformed with the gene. The transformed cells became resistant to polyamine toxicity, and the resistance was overcome by bafilomycin A1, an inhibitor of vacuolar H+-ATPase. Although spermine uptake activity of the transformed cells was almost the same as that of wild type cells, the uptake activity of vacuolar membrane vesicles from the transformed cells was higher than that from wild type cells. The transformed cells became resistant to MGBG [methylglyoxal bis(guanylhydrazone)] and paraquat, but not Ni2+ and Co2+, suggesting that the protein encoded by YLL028w is a transport protein specific for polyamines. When the YLL028w gene was disrupted by inserting the HIS3 gene, the cells became sensitive to polyamines, and spermine uptake activity of the vacuolar membrane vesicles decreased significantly. The accumulated spermine in YLL028w gene-disrupted cells decreased greatly compared with that in wild type cells. The results indicate that a membrane protein encoded by YLL028w (TPO1) is a polyamine transport protein on the vacuolar membrane.

[1999-3] Iwata, S. et al., Oncogene 18, 165-172 (1999)

Cell proliferation and transformation induced by growth factor stimulation or by carcinogens, viruses, or oncogenes are characterized by an associated increase in polyamine levels, which is mediated by increased polyamine biosynthesis and enhanced uptake of polyamines. Polyamine biosynthesis is catalyzed particularly, in the level of ornithine decarboxylase (ODC). The elevation of cellular polyamine levels on the other hand accelerates the induction of ornithine decarboxylase antizyme (antizyme), which is involved not only in ODC-degradation, but in the negative regulation of polyamine transport. Taking advantage of these characteristics of antizyme, the potential of antizyme as a factor having anti-cell growth and anti-tumor activity was investigated. We show that antizyme can induce cell death associated with a rapid decline of intracellular polyamine contents. The possible anti-tumor activities of ectopically expressed antizyme were teted in p21H-ras (Val 12)-transformed NIH3T3 cells and several human malignant cell lines including a line with loss of p53 expression, and they were shown to be as sensitive as nontransformed NIH3T3 cells in vitro. The in vivo anti-tumor activity was also tested using nude mice inoculated with H-ras transformed NIH3T3 cells that had been transfected with inducible antizyme expression vector and the results showed that antizyme expression in vivo blocks tumor formation in these mice. These results suggest that ectopic antizyme expression is of possible therapeutic benefit in the treatment of cancer, which is mediated by inactivation and intracellular polyamine depletion.

[1999-4] Arikado, E. et al., Eur. J. Biochem. 259, 262-268 (1999)

The amount of F1Fo-ATPase in Enterococcus hirae (formerly Streptococcus faecalis) increases when the cytoplasmic pH is lowered below 7.6, and protons are extruded to maintain the cytoplasmic pH at around 7.6. In the present study, we found that the transcriptional activity of the F1F0-ATPase operon was not regulated by pH. The synthesis of F1 subunits was increased 1.65 +/- 0.12-fold by the acidification of medium from pH 8.1 to pH 5.3. Western-blot analysis showed that there were F1 subunits in the cytoplasm, and the number of alpha plus beta subunits in the cytoplasm was 50% of the total number of the subunits in cells growing at pH 8.0. This decreased to 22% after shifting the medium pH to 5.3, with a concomitant 5.1-fold increase in the level of membrane-bound F1F0-ATPase. The cytoplasmic F1 subunits were shown to be degraded, and F0 subunits not assembled into the intact F1F0 complex were suggested to be digested. These data suggest that regulation of the enzyme level of F1F0-ATPase by the intracellular pH takes place mainly at the step of enzyme assembly from its subunits.

[1999-5] Nishimura, K. et al., Biochem. Biophys. Res. Commun. 257, 835-838 (1999)

Saccharomyces cerevisiae became less sensitive to nickel by a defect of the SPT7 gene encoding a transcription factor. Initial rate of nickel uptake by whole cells of a SPT7-negative mutant FY963 accumulated nickel about 1.7-fold of the value of FY61 when cultured in medium containing 0.1 mM NiCl2; most of which was sequestered into vacuoles. The pH gradient-driven nickel uptake by vacuolar membrane vesicles was not altered in FY963, but the amount of polyphosphate in vacuoles was highly elevated. Involvement of Spt7p in nickel detoxification through regulation of vacuolar polyphosphate level in S. cerevisiae was discussed.

[1999-6] Murata, T. et al., J. Bioichem. 125, 414-421 (1999)

We report here the large-scale purification of vacuolar (V0V1)-type Na+-ATPase from Enterococcus hirae achieved using column anion-exchange and gel filtration chromatographies; 32 mg of purified enzyme comprising nine suunits, A, B, C, D, E, F, G, I, and K, was obtained from 20 liter culture. This amount is 500-fold larger than that reported in the previous paper [Murata, T., Takase, K., Yamato, I., Igarashi, K., and Kakinuma, Y. (1997) J. Biol. Chem. 272, 24885-24890]. The purified enzyme shows a high specific activity of ATP hydrolysis (35.7 micromol Pi released/min/mg protein). ATP-driven [22]Na+ uptake by reconstituted V0V1-proteoliposomes exhibited an apparent Ki value for Na+ of 40 [micro]M, which is near the Km value (20 [microM) for Na+ of the ATP hydrolytic activity. Denatured gel electro-phoresis revealed that six subunits, A, B, C, D, E, and F, are releasable as the V1 subunit from the V0V1 complex by incubation with ethylenediaminetetraacetic acid; subunit G was not identified. The remaining V0-liposomes containing I and K sununits catalyzed Na+ uptake in response to potassium diffusion potential (DY, inside negative): the Ki value for Na+ of this reaction was estimated to be about 2 mM. Inhibition by N, N'-dicyclohexylcarbodiimide (DCCD) of the Na+-ATPase activity and DY-driven Na+-uptake by the V0-liposomes was prevented by the presence of Na+, suggesting that the Na+ binding site overlaps with the DCCD-reactive site.

[1999-7] Kakinuma, Y. et al., Biosci. Biotechnol. Biochem. 63, 875-878 (1999)

An energy-dependent K+/H+ antiport system is found in Enterococcus hirae ATCC9790 cultured in a standard complex medium (Y. Kakinuma, and K. Igarashi, J. Biol. Chem. 263: 14166-14170, 1998). We have now found that the activity of this antiport system was totally missing in cells cultured in a defined medium. In this defined medium, E. hirae did not grow well at pH near 9, but grew normally at pH below 7.5. This antiport system is important at high pH but dispensable at lower pH for ion homeostasis of this bacterium.

[1999-8] Kakinuma, Y. et al., J. Bioenerg. Biomembr. 31, 7-14 (1999)

A Na+-translocating ATPase was discovered in a gram-positive bacterium Enterococcus hirae. Our biochemical and molecular biological studies revealed that this Na+-ATPase belongs to the vacuolar-type enzyme. Purified Na+-ATPase consisted of nine subunits: NtpA, B, C, D, E, F, G, I, and K; reconstituted proteoliposomes showed ATP-driven electrogenic Na+ translocation. All these subunits were encoded by the ntp operon: ntpFIKECGABDHJ. The deduced amino acid sequences of the major subunits, A, B, and K (16 kDa proteolipid), were highly similar to those of A, B, and proteolipid subunits of vacuolar ATPases, although the similarities of other subunits were moderate. The ntpJ gene encoded a K+ transporter independent of the Na+-ATPase. Expression of this operon, encoding two transport systems for Na+ and K+ ions, was regulated at transcriptional level by intracellular Na+ as the signal. Two related cation pumps, vacuolar Na+-ATPase and F0F1, H+-ATPase, coexist in this bacterium.

[1999-9] Takase, K. et al., Biosci. Biotechnol. Biochem. 63, 1125-1129 (1999)

Enterococcus hirae vacuolar ATPase catalyzes translocation of Na+ or Li+ coupled with ATP hydrolysis. It is suggested that the glutamic acid residue (Glu139) or NtpK proteolipid subunit of this multisubunit enzyme is the binding site of these ions for translocation. Here we established a complementation system for the ntpK gene with its deletion mutant, and found that the ATPase activity disappeared upon replacement of Glu139 by aspartic acid. The side-chain length of this acidic residue of NtpK is thus important for this ATPase reaction.

[1999-10] Masuko, T. et al., Mol. Pharmacol. 55, 957-969 (1999)

There are complex interactions between spermine, protons, and ifenprodil at N-methyl-D-aspartate receptors. Spermine stimulation may involve relief of protien inhibition, whereas ifenprodil inhibition may involve an increase in proton inhibition. We studied mutations at acidic residues in the NR1 subunit using voltage-clamp recording of NR1/NR2B receptors expressed in Xenopus oocytes. Mutations at residues near the site of teh exon-5 insert, including E181 and E185, reduced spermine stimulation and proton inhibition. Mutation NR1 (D130N) reduced sensitivity to ifenprodil by more than 500-fold, but had little effect on sensitivity to spermine and pH. Mutations at six other residues in this region of the NR1 subunit reduced the potency and, in some cases, the maximum effect of ifenprodil. These mutants did not affect sensitivity to pH, glutamate, glycine, or other hallmark properties of N-methyl-D-aspartate channels such as Mg2+ block and Ba2+ permeability. Residues in this region presumably form part of the ifenprodil-binding site. To model this region of NR1 we compared the predicted secondary structure of NR1 (residues 19-400) with the known structures of 1,400 proteins. This region of NR1 is most similar to bacterial leucine/isoleucine/valine binding protein, a globular amino acid binding protein containing two lobes, similar to the downstream S1-S2 region of glutamate receptors. We propose that the tertiary structure of NR1 (22-375) is similar to leucine/isoleucine/valine binding protein, containing two "regulatory" domains, which we term R1 and R2. This region, which contains the binding sites for spermine and ifenprodil, may influence the downstream S1 and S2 domains that constitute the glycine binding pocket.

[1999-11] Ikegami, M. et al., FEBS Lett. 454, 67-70 (1999)

The Enterococcus hirae ntp operon encodes both a vacuolar ATPase, which transports Na+ as well as Li+, and the KtrII K+ transporter. A plasmid, in which the chloramphenicol acetyltransferase gene (CAT) was placed downstream of the ntp promoter, was introduced into a mutant totally defective in Na+ extrusion. The CAT activity of this transformant was increased preferentially by addition of NaCl, but not by LiCl, in the media or by elevating the medium pH, correlating well with the increase in amounts of the ATPase subunits ovserved by Western blotting. The physiological significance of these responses of the ntp promoter is discussed.

[1999-12] Kawano, M. et al., FEMS Microbio. Lett. 176, 449-453 (1999)

We isolated a mutant, JEMK1, which did not grow at pH 6.0 in medium containing 0.5 mM KCl, derived from a Enterococcus hirae mutant deficient in the KtrII K+ uptake system. This mutant showed an impairment in the proton potential-dependent K+ uptake, the activity of the KtrII K+ uptake system, and did not grow well in K+-poor media in a wide pH range from 6 to 10, suggesting that KtrI and KtrII are the main systems for potassium accumulation of this bacterium.

[1999-13] Kakinuma, Y. and Igarashi, K., J. Bacteriol. 181, 4103-4105 (1999)

A K+/H+ antiporter regulates cytoplasmic pH in Enterococcus hirae growing at alkaline pH. Mutants defective in this antiport activity were alkaline pH sensitive. One mutant, Pop1, lacked both K+/methylamine exchange at pH 9.5 and concomitant acidification of cytoplasmic pH. Pop1 grew well at pHs below 8 but did not at pHs above 9, conditions under which cytoplasmic pH was not fully acidified.

[1999-14] Masuko, T et al., J. Pharmacol. Exp. Ther. 290, 1026-1033 (1999)

The effects of aminoglycoside antibiotics on N-methyl-D-aspartate (NMDA) receptors were studied using voltage-clamp recording of recombinant NMDA receptors expressed in Xenopus oocytes. A number of aminoglycosides were found to potentiate macroscopic currents at heteromeric NR1A/NR2B receptors, but not at NR1A/NR2A, NR1A/NR2C, NR1A/NR2D or NR1B/NR2B receptors. The degree of potentiation had a rank order neomycin B > paromomycin > gentamicin C > geneticin > kanamycin A > streptomycin. Potentiation was not seen with kasugamycin and spectinomycin. The degree of stimulation paralleled the number of the amino groups in the aminoglycosides. The stimulatory effects of aminoglycosides were more pronounced at subsaturating concentrations of glycine and at acidic pH, similar to the stimulatory effects of spermine. We measured the effects of aminoglycosides at mutant NMDA receptors to determine which amino acid residues in NMDA receptor subunits are involved in stimulation. Mutations that reduced or abolished spermine stimulation also reduced stimulation by aminoglycosides. Several aminoglycosides produced a weak voltage-dependent block of NMDA receptors, but the degree of inhibition did not appear to correlate with the number of amino groups in the molecule. The results suggest that aminoglycosides having more than three amino groups have stimulatory effects that are mediated through the spermine-binding site on NMDA receptors.

[1999-15] Yoshida, M. et al., J. Biol. Chem. 274, 22723-22728 (1999)

We previously suggested that the degree of polyamine stimulation of oligopeptide-binding protein (OppA) synthesis is dependent on the secondary structure and position of the Shine-Dalgarno (SD) sequence of OppA mRNA. To study the structural change of OppA mRNA induced by polyamines and polyaine stimulation of initiaion complex formation, four different 130-mer OppA mRNAs containing the initiation region were synthesized in vitro. The structural change of these mRNAs induced by polyamines was examined by measuring their sensitivity to RNase T1, specific for single-stranded RNA, and RNase V1, which recognizes double-stranded or stacked RNA. in parallel, the effect of spermidine on mRNA-dependenet fMet-tRNA binding to ribosomes was examined. Our results indicate that the secondary structure of the SD sequence and initiation codon AUG is important for the efficiency of initiation complex formation and that spermidine relaxes the structure of the SD sequence and the initiaion codon AUG. The existence of a GC-rich double-stranded region close to the SD sequence is important for spermidine stimulation of fMet-tRNA binding to ribosomes. Spermidine apparently binds to this GC-rich stem and causes a structural change of the SD sequence and the initiation codon, facilitating an interaction with 30 S ribosomal subunits.

[1999-16] Nishimura, K. et al., Gene 238, 343-350 (1999)

The structure of the mouse S-adenosylmethionine decarboxylase (AdoMetDC) gene has been determined. The mouse gene (AMD1) consisted of eight exons and seven introns, similar to the rat AdoMetDC gene, and was mapped to chromosome 10. The characteristics of AMD1 gene were as follows: (1) The region of the promoter necessary for maximal transcriptional activity was located about 400 nucleotides upstream of the transcriptional initiation point, and contained a TATA box and two GC boxes. The transcriptional activity of the promoter was nearly equal to that of the SV40 promoter. (2) Two polyadenylation signals for transcription were observed, and the larger AdoMetDC mRNA, which is the dominant form of mRNA, corresponded to mRNA that is generated using the second polyadenylation signal. (3) Using stable trasfectants, we confirmed that the upstream open reading frame (uORF) in the 5'-untranslated region (5'-UTR) of AdoMetDC mRNA functioned as a negative regulatory element. Lower concentrations of polyamines affect both stimulation and inhibition of AdoMetDC synthesis, through the uORF in the mRNA, that affect general protein synthesis.

[1999-17] Apirakaramwong, A. et al., Biochem. Biophys. Res. Commun. 264, 643-647 (1999)

Accumulation of spermidine in Escherichia coli causes a decrease in cell viability at the late stationary phase of cell growth. The mechanism underlying this effect has been studied. Spermidine accumulation caused an increase in the level of ppGpp and a decrease in ribosome modulation factor (RMF) and stationary phase-specific sigma factor sigmaS, both of which are believed to be involved in cell viability. Transformation of E. coli with the gene for stringent factor, which synthesizes ppGpp, also caused a significant decrease in the levels of RMF and sigmaS factor and a decrease in cell viability. The results strongly suggest that the accumulation of ppGpp is also involved in the decrease in cell viability and that the sigmaS factor assists the function of RMF in cell viability.

[1999-18] Igarashi, K., and Kashiwagi, K., Biochem. J. 344, 633-642 (1999)

The polyamine content of cells is regulated by biosynthesis, degradation and transport. In Escherichia coli, the genes for three different polyamine transport systems have been cloned and characterized. Two uptake systems (putrescine-specific and spermidine-preferential) were ABC transporters, each consisting of a periplasmic substrate-binding protein, two transmembrane proteins and a membrane-associated ATPase. The crystal structures of the substrate-binding proteins (PotD and PotF) have been solved. They consist of two domains with an alternating beta-alpha-beta topology, similar to other periplasmic binding proteins. The polyamine-binding site is in a cleft between the two domains, as determined by crystallography and site-directed mutagenesis. Polyamines are mainly recognized by aspartic acid and glutamic acid residues, which interact with the NH2- (or NH-) groups, and by tryptophan and tyrosine residues that have hydrophobic interactions with the methylene groups of polyamines. The precursor of one of the substrate binding proteins, PotD, negatively regulates transcription of the operon for the spermidine-preferential uptake system, thus providing another level of regulation of cellular polyamines. The third transport system, catalysed by PotE, mediates both uptake and excretion of putrescine. Uptake of putrescine is dependent on membrane potential, whereas excretion involves an exchange reaction between putrescine and ornithine. In Saccharomyces cerevisiae, the gene for a polyamine transport protein (TPO1) was identified. The properties of this protein are similar to those of PotE, and TPO1 is located on the vacuolar membrane.

[1999-19] Nishimura, K. et al., Yeast 15, 1711-1717 (1999)

Spt7p is a new global transcription factor in Saccharomyces cerevisiae (Gansheroff et al., 1995). We report here that the activities of high affinity phosphate transport and acid phosphatase in particular were decreased in a spt7 null mutant. Northern blot experiments recealed that transcription of the PHO84 and PHO5 genes was impaired in this mutant; expression of the PHO regulatory genes, PHO4 and PHO2, was normal. Spt7p is thus linked with expression of several structural genes of the PHO regulon in yeast.