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Biomedical subjects

T Fukui

Publications and source records attributed to T Fukui.

At least 19 recordsLinked to original sources
PubMed1979

Inhibitory effect of methylated derivatives of guanylic acid for protein synthesis with reference to the functional structure of the 5'-'cap' in viral messenger RNA.

Guanylic acid modified variously with methyl groups on base or sugar moieties were synthesized chemically and their inhibitory effects on protein synthesis were tesetd in a wheat germ cell-free system using mRNAs from cytoplasmic polyhedrosis virus and tobacco mosaic virus. The confronting dinucleotide m7G5' pppA that corresponds to the most simple 'cap' structure of an eukaryotic mRNA is a strong inhibitor of protein synthesis, but non-methylated G5' pppA or G5' ppA is not inhibitory. The strong inhibitory effect is observed only by 7-methylguanylic acid (pm7G). Among 11 derivatives of pG, the most effective inhibitors are methylated at the 7-position. Further methylation at the other position sometimes cancels the inhibitory effect. Although pm7G carries a positively charged base, other nucleotides which carry a plus charged base (1-methyladenylic acid and 2-methylthio-7-methylinosinic acid) were not inhibitory. Thus, methylation at the 7-position on guanylic acid is specifically required for the inhibitory effect. Addition of pm7G was inhibitory for the formation of the initiation complex for eukaryotic protein synthesis. These results suggest that the 'cap' component containing 7-methylguanylic acid in viral mRNA participates during protein synthesis, especially in its initial steps. Protein synthesis in a bacterial cell-free system was not inhibited by addition of m7GpppA or pm7G when either TMV RNA or phage MS2 RNA was used as an mRNA.

Guanine Nucleotides↗
PubMed1979

Polynucleotides. LVII. Synthesis and properties of poly (2'-chloro-2'-deoxyinosinic acid).

Poly (2'-chloro-2'-deoxyinosinic acid) [poly(Icl)] was synthesized from Icl 5'-DP by polymerization with polynucleotide phosphorylase. UV absorption properties of poly(Icl) are very similar to those of poly(I). Poly(Icl) adopted a multi-stranded ordered form in the presence of 0.95M Na ion. The Tm value of this form was 36 degrees, which resembles that of poly(I) quadruple-stranded form at high salt. CD spectra also suggested presence of these two forms. Upon mixing with poly(C), poly-(Icl) forms a double-stranded 1 : 1 complex, which had very similar Tm-log[Na+] relationship to that of poly(I) . poly(C). Thus it was concluded that the chlorine substitution at 2'-position of the polynucleotide had the similar effect to OH on physical properties of polynucleotides.

Circular Dichroism↗
PubMed1979

Polynucleotides XLVII. Synthesis and properties of poly(2-methylthio- and 2-ethylthioadenylic acid). Formation of non-Watson-Crick type complexes.

Poly(2-methyl- and 2-ethylthioadenylic acid) were prepared by polymerization of corresponding diphosphates with Escherichia coli polynucleotide phosphorylase. These polynucleotides have relatively large hypochromicity of 30-35%. Acid titration of these polymers showed abrupt transition at pH 5.34-5.4, which may indicate that the introduction of alkylthio group at 2-position of adenine bases reduced their basicity. Thermal melting of these polymers showed no clear transition points at neutral pH, but in acidic media they have Tm values of 57 and 56 degrees C, somewhat lower than that of poly(A). Upon complex formation with poly(U), these poly(A) analogs showed only one poly(rs2A) . poly(U) type double-strand complexes, similar to that found in the case of poly(m2A) . poly(U).

Circular Dichroism↗
PubMed1979

Influence of various 2- and 2'-substituted polyadenylic acids on murine leukemia virus reverse transcriptase.

Several newly synthesized polyadenylic acid [(A)n] analogues, including poly(2-methyladenylic acid) [(m2A)n], poly(2-ethyladenylic acid) [(e2A)n], poly(2-isopropyladenylic acid) [(i-pro2A)n], poly(2-methylthioadenylic acid) [(ms2A)n], poly(2-ethylthioadenylic acid) [(e2A)n], poly(2'-fluoro-2'-deoxyadenylic acid) [(dAfl)n] and poly(2'-azido-2'-deoxyadenylic acid) [(dAz)n] have been evaluated for their effects on the RNA-directed DNA polymerase (reverse transcriptase) activity of Moloney murine leukemia virus; (m2A)n and (e2A)n did not markedly affect reverse transcriptase activity, (dAfl)n served as an efficient template for the reverse transcriptase reaction, and (i-pro2A)n, (ms2A)n, (es2A)n and (dAz)n strongly inhibited reverse transcriptase activity. (dAfl)n also served as an efficient template (Km : 0.025 micron) for the reverse transcriptase of avian myeloblastosis virus.

Avian Myeloblastosis Virus↗
PubMed1978

Polynucleotides. XLIV. Synthesis and properties of poly (2-azaadenylic acid) and poly(2-azainosinic acid).

Chemically synthesized 2-azaadenosine 5'-diphosphate (n2ADP) and 2-azainosine 5'-diphosphate (n2IDP) were polymerized to yield poly(2-azaadenylic acid), poly(n2A), and poly(2-azainosinic acid), poly(n2I), using Escherichia coli polynucleotide phosphorylase. In neutral solution, poly(n2A) and poly(n2I) had hypochromicities of 32 and 5.5%, respectively. Poly(n2A) formed an ordered structure, which had a melting temperature (Rm) of 20 degrees C at 0.15 M salt concentration. Upon mixing with poly(U), poly(n2A) formed a 1 : 2 complex with Tm of 41 degrees C at 0.15 M salt concentration. Poly(n2A) and poly(n2I) formed three-stranded complexes with poly(I), and poly(A), respectively. Poly(n2A) . 2poly(I), poly(A) . 2poly(n2I), and poly(n2A) . 2poly(n2I) complexes had Tm values of 23, 48, and 31 degrees C at 0.15 M salt concentration, respectively. Poly(n2I) formed a double-stranded complex with poly(C), but its Tm was very low.

Circular Dichroism↗
PubMed1978

Polynucleotides. LII. Synthesis and properties of poly(2'-deoxy-2'-fluoroadenylic acid).

2'-Deoxy-2'-fluoroadenosine was chemically transformed to its 5'-diphosphate and polymerized with polynucleotide phosphorylase to give poly(2'-deoxy-2'-fluoroadenylic acid) [poly(Af)]. Polymerization proceeded smoothly as in the case of poly(A) and the yield of the polymerization was 55%. The UV absorption spectra of poly(Af) closely resembled those of poly(A) and the hypochromicity was 32% at pH 7.0. The CD profile at 25 degrees and neutrality showed similar pattern to that of other poly(2'-deoxy-2'-halogenoadenylic acids) with somewhat larger [theta] values both in the positive and negative maxima. Acid titration of poly(Af) showed a transition point at pH 5.2 and the Tm of the acid form was 37 degrees which was significantly lower than that of poly(A), but similar to that of poly(2'-azido-2'-deoxyadenylic acid). Poly(Af) formed 1:1 and 1:2 complexes with poly-(U) having Tm of 49 degrees and 62 degrees at 0.04M and 0.15M Na(+) concentration, respectively. Poly(Af) also formed a 1:2 complex with poly(I) and its Tm was 36 degrees at 0.05M Na(+) concentration. These data showed that poly(Af) has rather similar properties to those of poly(A), but not to poly(dA).

Circular Dichroism↗
PubMed1978

Polynucleotides. LVI. Synthesis and properties of poly(2-deoxy-2'-fluoroinosinic acid).

Poly (2'-deoxy-2'-fluoroinosinic acid) [ poly(If)] was synthesized by polymerization of 2'-deoxy-2'-fluoroinosine 5'-diphosphate catalyzed by Escherichia coli polynucleotide phosphorylase. Although the UV absorption properties of poly(If) closely resembled those of poly(I), thermal melting curves at Na+ concentrations of 0.15M and 0.75M suggested two ordered structures for poly(If) neutral form. CD psectra taken at 0.15M Na+ concentration showed rather larger amplitudes in both a peak at 273 nm and a trough at 246 nm, suggesting rather strong vertical stacking of bases. When complexed with poly(C), poly(If) forms a double-stranded complex, poly(If).poly(C) which has Tm's higher by 10-20 degrees than those of poly(If).poly(C) measured under the same conditions. The CD spectrum of this complex resembled that of poly(I).poly(C). The effect of the fluorine atom at the 2'-position on thermal stability of polynucleotides is discussed.

Circular Dichroism↗
PubMed1978

Modification of rabbit muscle phosphorylase b by a water-soluble carbodiimide.

Glycogen phosphorylase b from rabbit muscle was rapidly inactivated by incubation with 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide metho-p-toluenesulfonate (CMC) at pH 5.1. The inactivation was pH-dependent and was not restored by treatment with hydroxylamine. The addition of glycine ethyl ester or N-(2,4-dinitrophenyl)-ethylenediamine (DNP-EDA) markedly increased the rate of inactivation. Of the various amino analogs of glucose tested, only glucosyl amine accelerated the inactivation, although they are all bound to the glucose 1-phosphate site of the enzyme. In the absence of amines, incorporation of about 3 mol of [metho-14C]CMC per protein monomer was observed on complete inactivation. In the presence of DNP-EDA, however, only 2 mol of [metho-14C]CMC and 1 mol of DNP-EDA were incorporated before the activity was completely lost. The treatment of phosphorylase b with CMC did not change the Km values of the enzyme for glucose 1-phosphate and AMP, in spite of the 56% inactivation. It is suggested that, in the phosphorylase-catalyzed reaction, an essential carboxyl group of the enzyme plays a role in the protonation of the glucosidic oxygen of glucose 1-phosphate.

Adenosine Monophosphate↗
PubMed1978

Amino acid sequence around the pyridoxal 5'-phosphate binding site in potato phosphorylase.

The amino acid sequence around the pyridoxal 5'-phosphate binding site in potato phosphorylase was determined in order to compare it with those in phosphorylases from other sources having different regulatory properties. The potato enzyme was reduced by NaBH4 in the presence of urea, carboxymethylated, and digested with chymotrypsin and trypsin. Pyridoxyl peptides were isolated by the differential procedure using paper electrophoresis or DEAE-cellulose column chromatography. In Edman degradation of these peptides, pyridoxyllysine was identified as the phenylthiohydantoin derivative of pyridoxyllysine using a combination of thin-layer chromatography and the Pauli reaction. The sequence around pyridoxyllysine, comprising 57 amino acid residues, was determined except for a region with 6 amino acid residues. The pyridoxal 5'-phosphate binding site in potato phosphorylase showed a high homology with those of the rabbit muscle and yeast enzymes. This finding suggests that the cofactor should be directly related to the essential process of phosphorylase action.

Amino Acid Sequence↗
PubMed1978

Phosphoglucomutase from potato tubers. Chemical and catalytic properties.

The chemical and catalytic properties of potato phosphoglucomutase [EC 2.7.5.1] were studied using various enzyme species (Peaks Ia, Ib, Ic, and II; Takamiya, S. & Fukui, T. (1978) Plant Cell Physiol. 19, 319--328). The molecular weights of the species are all approximately 60,000. No indication of the presence of subunit structure was obtained under various conditions. The amino acid composition of Peak Ia is generally similar to those of the enzymes from other sources, though it has some peculiarities. The Peak Ia and Peak II enzymes both absolutely require alpha-D-glucose 1,6-bisphosphate and Mg2+ for activity, and appear to have a "ping-pong" mechanism. A low concentration of Be2+ inhibits their action, the inhibition being retarded either by Mg2 or EDTA. Although the inhibition patterns by various metabolites, are similar for Peaks Ia and II, they differ in their kinetic parameters and optimal pH values.

Amino Acids↗
PubMed1977

An NADP-linked acetoacetyl CoA reductase from Zoogloea ramigera.

Zoogloea ramigera I-16 M was found to contain two stereospecific acetoacetyl CoA reductases; one was NADP+-linked and D(-)-beta-hydroxybutyryl CoA specific and the other was NAD+-linked and L(+)-isomer specific. The NADP+-linked enzyme, purified approximately 150-fold, had a pH optimum for the reduction of acetoacetyl CoA at 8.1, but no definite pH optimum for the oxidation for beta-hydroxybutyryl CoA. The apparent Michaelis constants for acetoacetyl CoA and NADPH were 8.3 and 21 micrometer, respectively. The enzyme was markedly inhibited by acetoacetyl CoA at concentrations higher than 10 micrometer. The incorporation of [1-14C]acetyl CoA into poly-beta-hydroxybutyrate (PHB) by bacterial crude extract (containing beta-ketothiolase, acetoacetyl CoA reductases, enoyl CoA hydratases and PHB synthases) or by a system reconstituted from purified preparations of beta-ketothiolase, acetoacetyl CoA reductase and PHB synthase, was observed only in the presence of NADPH, but not NADH. Among various enzymes involved in PHB metabolism, only the specific activity of glucose 6-phosphate dehydrogenase was elevated 5-fold within 2 h after the addition of glucose to the cells grown in the basal medium. These findings suggest that, in Z. ramigera I-16M, acetoacetyl CoA is directly reduced to D(-)-beta-hydroxybutyryl CoA by the NADP+-dependent reductase, and PHB synthesis is at least partially controled by NADPH availability through glucose 6-phosphate dehydrogenase.

Acetoacetates↗
PubMed1977

Biologic activities of poly (2-azaadenylic acid) and poly (2-azainosinic acid).

Poly (2-azaadenylic acid) [(aza2A)n] and poly(2-azainosinic acid [(aza2I)n], two newly synthesized analogues of (A)n and (I)n, in which CH-2 of the purine ring is replaced by a nitrogen atom, have been evaluated in various biological assay systems. (Aza2A) n formed a complex with (U)n and (br5U)n, and (aza2I)n formed a complex with (C)n and (br5C)n, but these complexes were markedly destabilized relative to the corresponding (A)n or (I)n complexes. The (aza2A)n-and (aza2I)n-derived complexes failed to stimulate the production of interferon in primary rabbit kidney cells and human diploid fibroblasts, under conditions (A)n. (U)n, (I)n. (C)n and (I)n. (br5C)n induced high amounts of interferon. both (aza2A)n and (aza2I)n exerted a marked inhibitory effect on the endogenous RNA directed DNA polymerase (reverse transcriptase) activity associated with murine leukemia virus. They caused a relatively mild inhibition of complement activity in an hemolytic assay system.

Aza Compounds↗