Biomedical subjects

J Fukuchi

Publications and source records attributed to J Fukuchi.

7 recordsLinked to original sources

PubMed1996

An aspartate residue in the extracellular loop of the N-methyl-D-aspartate receptor controls sensitivity to spermine and protons.

To study the role of acidic residues in modulation of NMDA receptors by spermine, we used site-directed mutagenesis of receptor subunits and voltage-clamp recording in Xenopus oocytes. Sixteen glutamate and aspartate residues, located in the first two thirds of the putative extracellular loop of the NR1A subunit, were individually mutated. This region of NR1A shows homology with bacterial amino acid binding proteins, a bacterial polyamine binding protein, and a bacterial spermidine acetyltransferase. Mutation of D669 to asparagine (D669N), alanine (D669A), or glutamate (D669E) abolished the "glycine-independent" form of spermine stimulation in heteromeric NR1A/NR2B receptors. These mutations also markedly reduced inhibition by ifenprodil and by protons at NR1A/NR2B receptors. Mutations at the equivalent position (D690) in NR1B, which contains the insert encoded by exon 5, reduced the pH sensitivity of NR1B/NR2B receptors. Thus, the effects of mutations at D669 are not prevented by the presence of exon 5, and the influence of exon 5 is not prevented by mutations at D669 (D690 in NR1B). Mutations at NR1A (D669) had little or no effect on the potencies of glutamate and glycine and did not alter voltage-dependent block by Mg2+ or the "glycine-dependent" form of spermine stimulation. Surprisingly, the D669N and D669A mutations, but not the D669E mutation, reduced voltage-dependent block by spermine at NR1A/NR2 receptors. Mutations in NR2B at a position (D668) equivalent to D669 did not alter spermine stimulation or sensitivity to pH and ifenprodil. However, mutations D668N and D668A but not D668E in NR2B reduced voltage-dependent block by spermine. Screening of the negative charges at NR1A(D669) and NR2B(D668) may be involved in voltage-dependent block by spermine. D669 in NR1A could form part of a binding site for polyamines and ifenprodil and/or part of the proton sensor of the NMDA receptor. Alternatively, this residue may be critical for coupling of modulators such as spermine, protons, and ifenprodil to channel gating.

Amino Acid Sequence↗

PubMed1995

Decrease in cell viability due to the accumulation of spermidine in spermidine acetyltransferase-deficient mutant of Escherichia coli.

Physiological functions of spermidine acetyltransferase in Escherichia coli have been studied using the spermidine acetyltransferase (speG) gene-deficient mutant CAG2242 and the cloned speG gene. The growth of E. coli CAG2242 in the defined M9 medium was normal in the presence and absence of 0.5mM spermidine. However, cell viability of E. coli CAG2242 at 48 h after the onset of growth decreased greatly by the addition of 0.5 mM spermidine. The amount of spermidine accumulated in the cells was approximately 3-fold that in the cells grown in the absence of spermidine. Transformation of the cloned speG gene to E. coli CAG2242 recovered the cell viability. Decreased in cell viability of E. coli CAG2242 was observed even when 0.5mM spermidine was added at 24 h after the onset of growth. The results indicate that accumulated spermidine functions at the late stationary phase of growth. The accumulation of spermidine caused a decrease in protein synthesis but not in DNA and RNA synthesis at 28 h after the onset of growth. The synthesis of several kinds of proteins was particularly inhibited. They included ribosome modulation factor and OmpC protein. Since the ribosome modulation factor is essential for cell viability at the stationary phase of growth (Yamagishi, M., Matsushima, H., Wada, A., Sakagami, M., Fujita, N., and Ishihama, A. (1993) EMBO J. 12, 625-630), the decrease in the protein was thought to be one of the reasons for the decrease in cell viability. The decrease in the ribosome modulation factor mainly occurred at the translational level.

Acetyltransferases↗

PubMed1995

An acidic amino acid in the N-methyl-D-aspartate receptor that is important for spermine stimulation.

The polyamine spermine has multiple effects on N-methyl-D-aspartate (NMDA) receptors, including "glycine-independent" stimulation, which is seen in the presence of saturating concentrations of glycine; "glycine-dependent" stimulation, which is due to an increase in the affinity of the receptor for glycine; and voltage-dependent block. These effects may involve three separate polyamine binding sites on the receptor. To identify amino acid residues that are important for spermine binding, we used site-directed mutagenesis to alter amino acids in and around a region of the NR1 subunit of the NMDA receptor that shows homology with PotD, a polyamine binding protein from Escherichia coli. Mutated subunits, expressed in heteromeric and homomeric NMDA receptors, were studied by voltage-clamp recording in Xenopus oocytes. Mutation of two acidic residues (E339-E342) to neutral amino acids reduced or abolished glycine-independent stimulation by spermine without affecting glycine-dependent stimulation or voltage-dependent block by spermine. Mutation of these residues also had modest effects on sensitivity to protons and to ifenprodil but did not alter sensitivity to glutamate and glycine or to voltage-dependent block by Mg2+. Residue E342 in NR1 appears to be critical for glycine-independent spermine stimulation. Mutations at equivalent positions in NR2A(E352Q) or NR2B(E353Q) had no effect on sensitivity to spermine, pH, or ifenprodil. Residue E342 in NR1 may form part of a discrete spermine binding site on the NMDA receptor or be involved in the mechanism of modulation by polyamines. This residue may also be involved in modulation by protons and ifenprodil.

Amino Acid Sequence↗

PubMed1994

Properties and structure of spermidine acetyltransferase in Escherichia coli.

Spermidine acetyltransferase (SAT) from Escherichia coli was purified about 40,000-fold. The molecular mass of native SAT was 95 kDa, and it consisted of four identical subunits. The products formed from the reaction of acetyl-CoA with spermidine by SAT were N1- and N8-acetylspermidine. The Km values for acetyl-CoA, spermidine, and spermine were 2 microM, 1.29 mM, and 220 microM, respectively. The enzymatic activity increased by 2.5-3.5-fold under the condition of poor nutrition but not in response to cold shock or high pH. By using synthetic oliogonucleotides deduced from amino acid sequences of the peptides in SAT, a polymerase chain reaction product with a length of 250 nucleotides was obtained. Using this polymerase chain reaction product, the gene encoding SAT (speG) was cloned and mapped at 35.6 min in the E. coli chromosome. E. coli cells transformed with the cloned speG gene increased SAT activity by 8-40-fold. The gene encoded a 186-amino acid protein, but SAT consisted of 185 amino acids because the initiator methionine was liberated from the protein. Thus, the predicted molecular mass was 21,756 Da. Significant similarity to aminoglycoside acetyltransferase and peptide N-acetyltransferase was observed in the amino acid sequence 87-141, and some similarity with spermidine-preferential binding protein (potD protein) in the spermidine-preferential uptake system was observed in the amino acid sequence 122-141. The results suggest that the active center of SAT may be located in the COOH-terminal portion.

Acetyl Coenzyme A↗

PubMed1993

Correlation between the inhibition of cell growth by accumulated polyamines and the decrease of magnesium and ATP.

The mechanism of the antiproliferation effect of spermidine and spermine was studied using a cell culture system of mouse FM3A cells. The addition of either 10 mM spermidine or 2 mM spermine to the growth medium containing 0.9 mM Mg2+ greatly inhibited cell growth (more than 90%). A decrease in the Mg2+ concentration to 50 microM in the growth medium, but without the polyamine addition, did not influence cell growth. However, the concentrations of spermidine and spermine necessary for the inhibition of cell growth when cells were cultured in the presence of 50 microM Mg2+ were much smaller (2 mM spermidine and 0.15 mM spermine). Nevertheless, the amount of polyamines accumulating in cells which could cause the inhibition of cell growth was almost the same, regardless of the large difference in the added polyamine concentrations. At the early stage of polyamine accumulation, the inhibition of cell growth correlated with the decrease of Mg2+ content, but not with a decrease of the ATP content. The decrease in Mg2+ content correlated well with the inhibition of macromolecular synthesis, especially protein synthesis. Thus, the inhibition of cell growth at the early stage of polyamine accumulation was thought to be due to the inactivation of ribosomes through the replacement of Mg2+ on magnesium-binding sites by polyamines. The decrease in Mg2+ content was mainly caused by the inhibition of Mg2+ transport by polyamines. At the later stage of polyamine accumulation, a decrease in ATP content was also observed. This was followed by swelling of the mitochondria, which may be a symptom of the subsequent cell death.

Adenosine Triphosphate↗

PubMed1992

Mechanism of the inhibition of cell growth by N1,N12-bis(ethyl)spermine.

The mechanism of the antiproliferation effect of N1,N12-bis(ethyl)spermine (BESPM) was studied in detail using mouse FM3A cells, since this polyamine analogue mimics the functions of spermine in several aspects [Igarashi, K., Kashiwagi, K., Fukuchi, J., Isobe, Y., Otomo, S. & Shirahata, A. (1990) Biochem. Biophys. Res. Commun. 172, 715-720]. Our results indicate that not only the decrease in sperimine and spermine caused by BESPM but also its accumulation play important roles on the inhibition of cell growth by BESPM, since BESPM accumulated in cells at a concentration fivefold that of spermidine in control cells. In comparison with the polaymine-deficient cells caused by alpha-difluoromethylornithine, an inhibitor of ornithine decarboxylase, and ethylglyoxal bis(guanylhydrazone), an inhibitor of S-adenosylmethionine decarboxylase, the behavior of polyamine-deficient cells caused by BESPM was different as follows: the inhibition of cell growth by BESPM was not abrogated by spermine or spermidine; polyamine uptake, which is stimulated during polyamine deficiency, was greatly inhibited, while spermidine/spermine N1-acetyltransferase activity, which is inhibited during polyamine deficiency, was enhanced in BESPM-treated cells; thymidine kinase activity did not decrease in BESPM-treated cells; inhibition of cell growth and macromolecule synthesis by BESPM correlated with the swelling of mitochondria and the decrease in ATP content; BESPM caused cell death when incubated together for several days. The role of BESPM accumulation on inhibition of cell growth is discussed.

Adenosine Triphosphate↗

PubMed1990

Spermine-like functions of N1, N12-bis(ethyl)spermine: stimulation of protein synthesis and cell growth and inhibition of gastric ulceration.

The spermine analogue N1, N12-bis(ethyl)spermine (BESPM) could mimic the functions of spermine in the following aspects: 1) BESPM could stimulate globin and ornithine decarboxylase synthesis in a rabbit reticulocyte cell-free system; 2) the addition of BESPM to the culture medium could recover cell growth of polyamine-deficient bovine lymphocytes; 3) spermidine uptake by bovine lymphocytes was inhibited by BESPM and spermine to a comparable degree; and 4) stress-induced gastric ulceration was inhibited by subcutaneous administration of BESPM. Since BESPM was less toxic than spermine for mice, BESPM or its derivatives may be useful for diseases which can be cured by polyamines.

Animals↗