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M Tatibana

Publications and source records attributed to M Tatibana.

At least 19 recordsLinked to original sources
PubMed1992

Promoter regions of the human X-linked housekeeping genes PRPS1 and PRPS2 encoding phosphoribosylpyrophosphate synthetase subunit I and II isoforms.

The 5' regions of the human phosphoribosylpyrophosphate synthetase subunit I and II genes (PRPS1 and PRPS2, respectively) were isolated and sequenced. A comparison of the nucleotide sequences between human and rat PRPS1 genes revealed that the sequences around the transcription initiation sites were conserved over 56 nucleotides, and that a TATA-like sequence, a CCAAT box and three putative Sp1 binding sites were present at almost the same positions in the GC-rich sequences. Two major transcription initiation sites were localized in the human PRPS1, one of the two was located 27 nucleotides downstream from the TATA-like sequence, while the upstream initiation site was in the TATA-like sequence. The promoter region of the human PRPS2 gene was also GC-rich and contained a TATA-like sequence, four Sp1 binding sites and a homopyrimidine stretch. The initiation sites were localized at 90 nucleotides upstream from the ATG initiation codon. Chloramphenicol acetyltransferase (CAT)/promoter fusion assays showed that a 2.0 kb region (human PRPS1) and a 1.1 kb region (human PRPS2) possessed the promoter activities in four cell lines. The CAT activities in the three human cell lines tended to correlate with the steady-state mRNA levels of the PRPS1 and PRPS2 genes. These results suggest that the 5' flanking regions cloned contribute to the cell-differential expression of these two genes.

Amino Acid Sequence↗
PubMed1991

Expression of rat phosphoribosylpyrophosphate synthetase subunits I and II in Escherichia coli. Isolation and characterization of the recombinant isoforms.

The 34-kDa subunit of rat liver phosphoribosylpyrophosphate synthetase is a mixture of the two highly homologous isoforms, PRS I and PRS II. Heretofore, it was not possible to separate the two. We now describe isolation and characterization of the recombinant isoforms, named rPRS I and rPRS II. The respective rat cDNAs were inserted into vectors constructed from pKK233-2 by replacing its replication origin with that of pGEM-1 and expressed in Escherichia coli. The rPRS I and rPRS II were purified to apparent homogeneity with specific activities of 33,400 and 46,200 milliunits/mg, respectively; these values were at least 2.5-fold higher than the highest value for the mammalian enzyme so far reported. Both isoforms showed a similar dependency on Pi as an absolute activator. Sulfate partially substituted for Pi. The maximal activities of rPRS I and rPRS II with sulfate were 43 and 7%, respectively, of those seen with Pi. The two isoforms differed in sensitivity to inhibition by ADP and GDP. Inhibition of rPRS I and rPRS II by 0.3 mM ADP was 87 and 54%, respectively, and inhibition by 1 mM GDP was 93 and 24%, respectively. rPRS II was 180-fold more sensitive than rPRS I to heat inactivation at 49 degrees C.

Animals↗
PubMed1991

Depression of liver-specific gene expression in regenerating rat liver: a putative cause for liver dysfunction after hepatectomy.

We carried out studies on the expression of liver-specific genes during regeneration of the liver and searched for changes in the expression of oncogenes and housekeeping genes. Albumin and ornithine transcarbamylase genes were the liver-specific genes examined by Northern blot analysis, using total RNAs isolated from residual livers of Sprague-Dawley rats subjected to a 68% partial hepatectomy. The mRNA levels of both genes began to decrease 8 hr after hepatectomy, both reaching the lowest levels at 24 hr, and then recovered to some extent at 48 hr. In contrast, these levels in the housekeeping and growth-related genes were augmented during this period. This would suggest that there is a selective expression of growth-related and housekeeping genes, in preference to liver-specific genes during liver regeneration. The expression of these genes in the regenerating liver was simulated in primary cultured hepatocytes during the dedifferentiation processes. It would appear that the first step in regeneration of the residual liver is dedifferentiation, in which the depression of liver-specific genes may be linked to liver dysfunction following hepatectomy.

Animals↗
PubMed1991

Mitogen-induced early increase in cytosolic free Mg2+ concentration in single Swiss 3T3 fibroblasts.

Events related to the early mobilization of Mg2+ in mammalian cells in response to external stimuli are not well characterized. We examined changes in cytoplasmic free Mg2+ concentrations ([Mg2+]i) after mitogenic stimulation in single mouse Swiss 3T3 fibroblasts, using digital ratio imaging microscopy of the fluorescent probe mag-fura-2. Stimulation with bombesin or epidermal growth factor (EGF) in combination with insulin led to a significant increase in mean [Mg2+]i levels from basal 0.22 mM to 0.29-0.35 mM after 30-60 min. The response showed some heterogeneity among individual cells with respect to the extent of the increase; approximately 10% of the cells showed no [Mg2+]i response. Bombesin or EGF alone induced a significant increase in [Mg2+]i but was less effective than when combined with insulin. In medium without added Mg2+, the increase in [Mg2+]i was considerably decreased, either with bombesin plus insulin or EGF plus insulin. These results provide direct evidence for the mobilization of Mg2+ as an early cellular response to growth factors.

3T3 Cells↗
PubMed1991

Complete nucleotide sequence of human phosphoribosyl pyrophosphate synthetase subunit I (PRS I) cDNA and a comparison with human and rat PRPS gene families.

cDNA clones for human phosphoribosyl pyrophosphate synthetase subunit I (PRS I) were isolated from a glioblastoma cell line MGC 1 cDNA library. The longest clone contained 2,075 base pairs (bp) almost covering the 2.3-kb mRNA and the base sequence of the coding region (954 bp) had a 92.0% sequence homology with that of rat PRS I cDNA. The deduced amino acid sequences were identical between human and rat PRS I. This perfect conservation has heretofore not been reported for other enzymes involved in nucleotide metabolism and glycolysis. A comparison with other isoforms of this enzyme, PRS II and PRS III, showed that the human PRS I was 79.9 and 92.2% homologous in the coding sequence and 95.3 and 94.0% in the deduced amino acid sequence to human PRS II and PRS III, respectively. The high value of the synonymous difference between PRS I and PRS II cDNAs places their time of divergence long before that of the radiation of mammals. Based on the evolutionary rate of amino acid substitution, the PRS I and II genes probably diverged about 760 million years ago.

Amino Acid Sequence↗
PubMed1990

A human testis-specific mRNA for phosphoribosylpyrophosphate synthetase that initiates from a non-AUG codon.

Two highly homologous subunits for phosphoribosylpyrophosphate synthetase are encoded by human X-linked genes, PRPS1 and PRPS2 (Taira, M., Kudoh, J., Minoshima, S., Iizasa, T., Shimada, H., Shimizu, Y., Tatibana, M., and Shimizu, N. (1989b) Somat. Cell Mol. Genet. 15, 29-37). These genes are expressed in most tissues, whereas an additional unique mRNA (1.4 kilobases) is present in the testes of rats as well as mice and humans (Taira, M., Iizasa, T., Yamada, K., Shimada, H., and Tatibana, M. (1989a) Biochim. Biophys. Acta 1007, 203-208). In this paper, cDNA cloning revealed that the human testis-specific mRNA was encoded by an autosomal gene, termed PRPS3. RNA blot analysis showed that the expression of this gene began at 4 weeks of age in rats, coinciding with the reported appearance of primary spermatocytes. A cDNA clone of PRPS3 was sequenced and found to encode a predicted product of 317 amino acids which was highly homologous to those of PRPS1 and PRPS2 (94.3% and 91.2% identities, respectively). However, the PRPS3 cDNAs lacked an ATG initiator for translation at the expected position, and instead contained an ACG triplet. In vitro transcription/translation studies, combined with in vitro site-directed mutagenesis experiments, suggested that the ACG codon at this position did serve as a start codon. Analysis of amino-terminal sequence of the radiolabeled PRPS3 product, prepared by in vitro translation, supported the predicted sequence starting with Pro-1, and, in addition, this product was labeled with N-formyl[35S]methionyl-tRNAi. These results suggested that the synthesis of the nascent polypeptide could initiate with methionine at the position corresponding to the ACG codon.

Amino Acid Sequence↗
PubMed1990

Selective activation of testis-specific genes in cultured rat spermatogenic cells.

During mammalian spermatogenesis the isozyme pattern of a glycolytic enzyme, phosphoglycerate kinase (PGK; ATP: 3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3), changes from the somatic-type PGK-1 to the testis-specific PGK-2, and this change has been suggested to involve transcription switch. We have isolated genomic DNA fragments which code for the mouse PGK isozymes and determined the transcription start site of each gene. The results demonstrate that transcriptions of the two PGK genes are initiated at multiple sites under the control of TATA box-lacking promoters. The putative promoter regions of the two genes contain several distinct sequences known as the CCAAT box and the GC box which possibly bind CCAAT-binding proteins and Sp1, respectively. We next developed a culture system in which spermatogenic gene expression is partly reproduced. When spermatogenic cells of 20-day-old rats were cultured, transcripts from PGK-2 and another spermatogenic gene PRPS3 became detectable, while expression of other non-spermatogenic genes did not significantly change during culture. These results suggest that two spermatogenic genes PGK-2 and PRPS3 were activated in culture according to a developmental program of spermatogenesis. Thus, this culture system may be useful for studying the molecular mechanism underlying mammalian spermatogenic gene expression.

Animals↗
PubMed1990

Insulin-resistant diabetes associated with partial deletion of insulin-receptor gene.

The insulin-receptor genes from a 16-year-old girl with type A insulin resistance, who presented with fasting hyperinsulinaemia, acanthosis nigricans, and reduced insulin binding, and from her family were examined. One allele of her insulin-receptor gene inherited from her mother contained a 1.2 kb deletion arising from a recombination between two Alu elements. The deletion removed the 14th exon in the beta subunit and altered the reading frame, to produce a stop codon after aminoacid 867. Pedigree analysis indicated that this mutation alone will not cause diabetes, and the proband is possibly a compound heterozygote. 4 other members of her family were heterozygous for the same mutation; all 4 had a decrease in insulin binding and slight impairment of glucose tolerance. Perhaps the same mutation is an underlying feature of some cases of non-insulin-dependent diabetes mellitus.

Adolescent↗
PubMed1990

Structure of the rat PRPS1 gene encoding phosphoribosylpyrophosphate synthetase subunit I.

Phosphoribosylpyrophosphate (PP-Rib-P) synthetase (EC 2.7.6.1) subunit I gene (PRPS1) is constitutively expressed in various tissues (Taira, M., Iizasa, T., Yamada, K., Shimada, H., and Tatibana, M. (1989) Biochim. Biophys. Acta 1007, 203-208). We report here the exon-intron organization and the transcription promoter sequence of rat PRPS1 gene. This gene has 22 kilobases and is split into 7 exons ranging in size from 99 to 251 base pairs (bp), except for exon 7 (1008 bp). A putative PP-Rib-P binding site is encoded in exon 5. The exon-intron boundaries are similar to the consensus sequences for mammalian introns. S1 nuclease and primer extension assays with the use of RNA from rat Yoshida ascites sarcoma cells led to the identification of four possible transcription start points closely spaced between 126 and 129 bp from the ATG initiation codon. In the upstream region from the transcriptional start sites, we observed a TATA-like sequence (TAATTTAAT) at nucleotides -28, a CCAAT element (AGCCAATC) at nucleotides -80, and three GC boxes (putative Sp1-binding sites) at nucleotides -103, -43, and -10. A comparison of the promoter region for PRPS1 with those of other housekeeping genes revealed a homology resembling that of the beta-actin gene.

Actins↗
PubMed1990

An improved method for determination of N-acetyl-L-glutamate by its function as an activator of carbamoyl phosphate synthetase I.

N-Acetyl-L-glutamate has been examined with regard to its ability to activate carbamoyl phosphate synthetase I (EC 6.3.4.16). Substance(s) inhibitory to carbamoyl phosphate synthetase, present even in the partially purified preparation of rat liver extracts, interfered with the measurement of acetylglutamate. In the experiments using chelating agents, metals were apparently involved in this inhibition. When the partially purified preparation of liver extract was placed on a Chelex 100 column, the inhibitor was eliminated and accurate measurements of acetylglutamate content could be made. Evidence supporting the validity of this improved method is given. A significant difference was observed between acetylglutamate levels determined by the present method and by the one using aminoacylase I (N-acylamino acid amidohydrolase, EC 3.5.1.14) to hydrolyze acetylglutamate followed by assay of the glutamate generated. We searched for the presence of glutamate derivatives other than acetylglutamate. When impure tissue preparations containing acetylglutamate were treated with a commercial preparation of aminoacylase, there was an excess amount of glutamate apparently derived from compounds other than acetylglutamate. This can lead to an overestimation of the tissue levels of acetylglutamate.

Animals↗
PubMed1989

Early mitogenic stimulation of metabolic flux through phosphoribosyl pyrophosphate into nucleotides in Swiss 3T3 cells and requirement of external magnesium for the response.

5-Phosphoribosyl 1-pyrophosphate (PRPP) is a common precursor for the synthesis of all nucleotides and also serves as a critical regulator for the synthesis. In spite of a number of studies in vitro on mammalian PRPP synthetase, our understanding of the regulation of PRPP synthesis in situ is very limited. Various mitogens are known to activate purine and pyrimidine de novo biosynthesis and purine base phosphoribosylation as an early response in quiescent mouse fibroblasts. We aimed at elucidation of the underlying mechanism for the possible increase in PRPP synthesis in mitogen-stimulated mouse fibroblasts in culture. In order to quantitatively follow metabolic flux through PRPP into nucleotides, [ribosyl-14C]inosine was enzymatically prepared and used as a tracer to preferentially label intracellular ribose phosphate. The radioactivity incorporation into cellular nucleotides was measured. Evidence supported the validity of the method. Prior exposure of quiescent Swiss 3T3 cells in culture to epidermal growth factor (EGF) plus insulin for 45-60 min enhanced approximately 2-fold the radioactivity incorporation from [ribosyl-14C]inosine into nucleotides, without increasing the specific radioactivity of intracellular free ribose 5-phosphate. [14C]Uracil incorporation into nucleotides, a measure for PRPP-independent ribose phosphate utilization for nucleotide synthesis, was not increased. These and other results indicate that EGF plus insulin stimulates the metabolic flux through PRPP. A similar stimulation was induced by bombesin and melittin in combination with insulin and by fibroblast growth factor alone. Quiescent Swiss 3T6 cells and human fetal fibroblasts showed a similar stimulation of nucleotide synthesis in response to exposure to serum. For characterization of intracellular signaling pathways, we examined effects of several inhibitors and agents on the stimulation. The divalent cation ionophore A23187 mimicked the response to EGF and insulin in Swiss 3T3 cells, thereby suggesting involvement of divalent cation mobilization in this increase. The effect of the ionophore was not additive to that of the growth factors. Omission of Ca2+ from the incubation medium did not affect the response to EGF and insulin, whereas the omission of Mg2+ did abolish the response. Furosemide, an inhibitor of Mg2+ influx, partially inhibited the stimulated synthesis of nucleotides. Thus, the entry of external Mg2+ into the cells may play a critical role in this signal transduction. These results provided an important access to elucidation of the intracellular mechanisms for the mitogen-induced increase in PRPP and nucleotide syntheses.

Animals↗
PubMed1989

External Mg2+-dependent early stimulation of nucleotide synthesis in Swiss 3T3 cells.

In quiescent Swiss 3T3 cells, metabolic flux through phosphoribosyl pyrophosphate into nucleotides increased within 1 h when various growth factors were added (S. Ishijima, K. Kita, N. Kinoshita, T. Ishizuka, N. Suzuki, and M. Tatibana. J. Biochem. 104: 570-575, 1988). The divalent cation ionophore A23187 mimicked the stimulatory effect of epidermal growth factor plus insulin, thereby suggesting involvement of divalent cation mobilization in signaling the stimulation by the growth factors. The stimulation induced by the growth factors was nil in medium devoid of added Mg2+ but was not affected by the omission of Ca2+. The dependency on external Mg2+ was also observed with the stimulations by bombesin plus insulin, fibroblast growth factor, and A23187. In contrast, the mitogenic stimulation of glycolysis was observed irrespective of the presence or absence of Mg2+, indicating that initial events in the signaling process, including mitogen binding to receptors, took place in the absence of exogenous Mg2+. These results suggest that a Mg2(+)-dependent process, subsequent to growth factor binding to receptors, plays an essential role in signaling the early mitogenic stimulation of nucleotide synthesis.

Amiloride↗
PubMed1988

Stimulation of de novo biosynthesis of purine and pyrimidine nucleotides in the liver of rats following burn injury.

Third-degree burn injury covering 25% of the body surface was imposed on rats. The de novo biosynthesis of purine and pyrimidine nucleotides in the liver of these rats was measured by the incorporation of labeled glycine and bicarbonate into the respective bases. They were increased one day after injury (day 2) and returned to the control values three days after injury (day 4). As expected, the metabolic flow through 5-phosphoribosyl l-pyrophosphate (PPRibP), estimated using [14C]ribose as a tracer, varied in a similar manner. The activities of glutamine phosphoribosylpyrophosphate amidotransferase and carbamoyl-phosphate synthetase II on day 2 did not change significantly. The nucleotide concentrations, effectors of the enzymes, also did not change significantly. The concentrations of PPRibP on days 2 and 4 were 85% higher (P less than .02) and similar to that of controls, respectively, and the elevated concentration was a major factor responsible for the increased nucleotide biosynthesis. Increased synthesis caused the elevation of PPRibP concentrations. There were, however, no significant changes in the factors so far known to regulate PPRibP synthesis.

Animals↗
PubMed1988

Evidence for early mitogenic stimulation of metabolic flux through phosphoribosyl pyrophosphate into nucleotides in Swiss 3T3 cells.

Various mitogens activate purine and pyrimidine de novo biosynthesis and purine base phosphoribosylation as an early response in quiescent fibroblasts. Increased synthesis of 5-phosphoribosyl 1-pyrophosphate (PRPP) may precede or underlie these activations, but little direct evidence has been presented for this notion, due to lack of suitable analytical methods. To preferentially label intracellular ribose phosphate and quantitatively follow metabolic flux through PRPP into nucleotides, we prepared [ribosyl-14C]inosine and used it as a tracer. Evidence showed the validity of this method. Prior exposure of quiescent Swiss 3T3 cells in culture to epidermal growth factor plus insulin for 45-60 min enhanced approximately 2-fold the radioactivity incorporation from [ribosyl-14C]inosine into nucleotides, without increasing the specific radioactivity of intracellular free ribose 5-phosphate. [14C]Uracil incorporation into nucleotides, a measure of PRPP-independent ribose phosphate utilization for nucleotide synthesis, was not increased. These and other results indicate that epidermal growth factor plus insulin stimulates the metabolic flux through PRPP. Similar extents of stimulation were induced by bombesin and melittin in combination with insulin and by fibroblast growth factor alone, suggesting the presence of an unknown signaling pathway common to these mitogens. This system is highly useful for studies of the mechanisms that stimulate in situ activity of PRPP synthetase.

Animals↗
PubMed1979

Inhibition of urea synthesis by pent-4-enoate associated with decrease in N-acetyl-L-glutamate concentration in isolated rat hepatocytes.

Pent-4-enoate at 0.1 to 1.0 mM strongly inhibited urea synthesis in isolated rat hepatocytes. Pent-4-enoate at the same concentrations markedly decreased concentrations of N-acetyl-L-glutamate, an essential activator of carbamoyl-phosphate synthase-I (EC 2.7.2.5), and the decrease was well in parallel with the inhibition of urea synthesis by pent-4-enoate. This compound also lowered cellular concentrations of acetyl-CoA, a substrate of acetylglutamate synthase (EC 2.3.1.1). Pent-4-enoate in a dose of 1 mM did not significantly affect cellular concentrations of ATP, and had no direct effect on acetylglutamate synthase activity. These results suggest that the inhibition of urea synthesis by pent-4-enoate is due to decrease in N-acetyl-L-glutamate concentration and that the decrease is probably brought about by decreased rate of its synthesis due to the lowered concentration of cellular acetyl-CoA.

Acetyl Coenzyme A↗
PubMed1979

Cell-free synthesis and processing of a putative precursor for mitochondrial carbamyl phosphate synthetase I of rat liver.

Total RNA or poly(A)(+) RNA of rat liver was translated in a rabbit reticulocyte or wheat germ protein-synthesizing system and the carbamyl phosphate synthetase I [carbamoyl-phosphate synthetase (ammonia); carbon dioxide: ammonia ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.4.16] synthesized was isolated by indirect immunoprecipitation by using antibody purified on enzyme-bound Sepharose and Staphylococcus aureus cells. The in vitro product moved on sodium dodecyl sulfate/polyacrylamide gels as a polypeptide that was about 5000 daltons larger than the subunit of the mature enzyme (160,000 daltons). The same polypeptide was also obtained by direct immunoprecipitation or by a double-antibody precipitation method. The mature enzyme competed effectively with the in vitro product for interaction with anti-carbamyl phosphate synthetase I antibody. Digestion of the in vitro product by S. aureus protease gave a pattern of peptide fragments similar to that of the mature enzyme. A mitochondrial membrane preparation from rat liver converted the in vitro product into a polypeptide that comigrated with the mature subunit on sodium dodecyl sulfate gel electrophoresis. Similar proteolytic activity was not detected in either a cytosol or a microsomal fraction of rat liver. These results indicate that the enzyme is synthesized as a larger precursor which is converted to the mature form of enzyme by posttranslational processing.

Animals↗