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Biomedical subjects

T Fukui

Publications and source records attributed to T Fukui.

At least 433 records · Page 24Linked to original sources
PubMed1982

Hydrogen exchange kinetics of nucleic acids. Double and triple helices with Hoogsteen-type basepairs.

The kinetics of hydrogen-tritium exchange reaction have been followed by a Sephadex technique of a double-helical poly(ribo-2-methylthio-adenylic acid) . poly(ribouridylic acid) complex with the Hoogsteen-type basepair. Only one hydrogen in every 2-methylthio-adenine . uracil basepair has been found to exchange at a measurably slow rate, 0.023 s-1 (at 0 degrees C), which is, however, much greater than that for a double-helix with the Watson-Crick type A . U pair. The kinetics of hydrogen-tritium exchange were also examined by triple-helical poly(rU) . poly(rA) . poly(rU) which involves both the Watson-Crick and Hoogsteen basepairings. Here, three hydrogens in every U . A. U base triplet have been found to exchange at a relatively slow rate, 0.0116 s-1 (at 0 degrees C). The kinetics of hydrogen-deuterium exchange reactions of these polynucleotide helices have also been followed by a stopped-flow ultraviolet absorption spectrophotometry at various temperatures. On the basis of these experimental results, the mechanism of the hydrogen exchange reactions in these helical polynucleotides was discussed. In the triple helix, the rate-determining process of the slow exchange of the three (one uracil-imide and two adenine-amino) hydrogens is considered to be the opening of the Watson-Crick part of the U. A. U triplet. This opening is considered to take place only after the opening of the Hoogsteen part of the triplet.

Base Composition↗
PubMed1982

Inhibition of murine leukaemia virus reverse transcriptase by 2-halogenated polyadenylic acids.

Several new analogues of polyadenylic acid [(A)n], i.e. poly(2-fluoroadenylic acid) [(fl2A)n], poly(2-chloroadenylic acid [(cl2A)n], poly(2-bromoadenylic acid) [(br2A)n] and poly(2-iodoadenylic acid) [(io2A)n] have been synthesized and evaluated for their effects on the RNA-directed DNA polymerase (reverse transcriptase) activity of Moloney murine leukaemia virus. All (A)n analogues were found to be potent inhibitors of reverse transcriptase, the order of (decreasing) potency being (fl2A)n greater than (io2A)n greater than (br2A)n greater than (cl2A)n. For all four (A)n analogues the inhibition of reverse transcriptase was competitive with respect to the template-primer. (A)n . oligo(dT). The K1 values were 0.02 microgram/ml for (fl2A)n, 0.1 microgram/ml for (io2A)n, 0.5 microgram/ml for (br2A)n and 8 microgram/ml for (cl2A)n. With a Ki of 0.02 microgram/ml (approx. 0.04 microM), (fl2A)n can be considered as one of the most, if not the most, potent polynucleotide inhibitor of reverse transcriptase that has been described so far.

Halogens↗
PubMed1982

Protein synthesis using poly(2'-halogeno-2'-deoxyadenylic acids) as messenger.

Poly(2'-fluoro-2'-deoxyadenylic acid), poly(2'-chloro-2'-deoxyadenylic acid) and poly(2'-bromo-2'-deoxyadenylic acid) are used as messenger RNAs in protein synthesizing systems in vitro. All polynucleotides were active as messengers and [14 C]lysine was incorporated into polypeptides. The initial velocity of polylysine formation was greater using poly(2'-fluoro-2'-deoxyadenylic acid) as messenger than in the case of poly(rA), and all synthetic messengers lived longer in the protein synthetic system.

Escherichia coli↗
PubMed1982

An extracellular polysaccharide produced by Zoogloea ramigera 115.

A weakly acidic polysaccharide was purified from the extracellular zoogloeal matrix produced by Zoogloeal ramigera 115. The purified polysaccharide was homogeneous as judged by sedimentation analysis, and the average molecular weight was estimated to be about 10(5) by gel permeation chromatography of the fully methylated preparation. The polysaccharide was composed of D-glucose, D-galactose and pyruvic acid in an approximate molar ratio 11:3:1.5. On the basis of methylation, periodate oxidation, Smith degradation and partial hydrolysis, the following highly branched structure was deduced for the polysaccharide: a long chain mainly consisting of beta 1 leads to 4-linked glucose residues branching at the C-3 or C-6 position of galactose residues which are present in beta 1 leads to 4 or beta 1 leads to 3 linkages as the minor component of the long chain; pyruvic acid residues, the sole acidic component, are linked to the nonreducing end and/or 1,3-linked glucose residues through 4,6-ketal linkages. The purified polysaccharide was not readily soluble in water and had a high affinity for several metallic ions (e.g, 0.25 mumol Fe3+/mg, and 0.17 mumol Fe2+ mg). Upon addition of metallic ions (1 mM) to a gelatinous aqueous solution of the polysaccharide (K+ form, 0.125%), more than 80% of it immediately coprecipitated out with them.

Chemical Phenomena↗
PubMed1982

Potato and rabbit muscle phosphorylases: comparative studies on the structure, function and regulation of regulatory and nonregulatory enzymes.

Phosphorylases (EC 2.4.1.1) from potato and rabbit muscle are similar in many of their structural and kinetic properties, despite differences in regulation of their enzyme activity. Rabbit muscle phosphorylase is subject to both allosteric and covalent controls, while potato phosphorylase is an active species without any regulatory mechanism. Both phosphorylases are composed of subunits of approximately 100 000 molecular weight, and contain a firmly bound pyridoxal 5'-phosphate. Their actions follow a rapid equilibrium random Bi Bi mechanism. From the sequence comparison between the two phosphorylases, high homologies of widely distributed regions have been found, suggesting that they may have evolved from the same ancestral protein. By contrast, the sequences of the N-terminal region are remarkably different from each other. Since this region of the muscle enzyme forms the phosphorylatable and AMP-binding sites as well as the subunit-subunit contact region, these results provide the structural basis for the difference in the regulatory properties between potato and rabbit muscle phosphorylases. Judged from CD spectra, the surface structures of the potato enzyme might be significantly different from that of the muscle enzyme. Indeed, the subunit-subunit interaction in the potato enzyme is tighter than that in the muscle enzyme, and the susceptibility of the two enzymes toward modification reagents and proteolytic enzymes are different. Despite these differences, the structural and functional features of the cofactor, pyridoxal phosphate, site are surprisingly well conserved in these phosphorylases. X-ray crystallographic studies on rabbit muscle phosphorylase have shown that glucose-1-phosphate and orthophosphate bind to a common region close to the 5'-phosphate of the cofactor. The muscle enzyme has a glycogen storage site for binding of the enzyme to saccharide substrate, which is located away from the cofactor site. We have obtained, in our reconstitution studies, evidence for binding of saccharide directly to the cofactor site of potato phosphorylase. This difference in the topography of the functional sites explains the previously known different specificities for saccharide substrates in the two phosphorylases. Based on a combination of these and other studies, it is now clear that the 5'-phosphate group of pyridoxal phosphate plays a direct role in the catalysis of this enzyme. Information now available on the reaction mechanism of phosphorylase is briefly described.

Adenosine Monophosphate↗
PubMed1982

Independent roles of prostaglandins and the renin-angiotensin system in abnormal vascular reactivity in Bartter's syndrome.

To clarify the independent roles of prostaglandins and the renin-angiotensin system in the pressor resistance to angiotensin II in Bartter's syndrome, the pressor responsiveness to exogenous angiotensin II was investigated in three patients with the syndrome during the administration of indomethacin synthesis, and captopril is an angiotensin-converting enzyme inhibitor. All the patients showed high plasma renin activity, increased urinary excretion of prostaglandin E, and pressor resistance of angiotensin II. An analogue of angiotensin II that had weak agonistic properties induced a marked fall in blood pressure. Pretreatment with indomethacin (150 mg/day) decreased baseline plasma renin activity and reversed the hypotensive effect of the analogue of angiotensin II. Apparently, our data support the concept that pressor resistance ultimately results from the increase in the concentration of endogenous angiotensin II. However, the augmentation of indomethacin was significantly (p less than 0.01) greater in magnitude than the response obtained with captopril, although the concentration of plasma angiotensin II prior to each infusion of angiotensin II was the same. This observation could be explained by the finding that indomethacin suppressed both systems, but captopril inhibited only the renin-angiotensin system. Evidence presented herein suggests that the abnormalities in the vascular reactivity to angiotensin II may result from, not only the decreased number of receptor sites as a results of the increased concentration of endogenous angiotensin II, but also from the alteration of the end-organ sensitivity to angiotensin II via overproduction of prostaglandins.

Adult↗
PubMed1982

Catalytic mechanism of glycogen phosphorylase: pyridoxal(5')diphospho(1)-alpha-D-glucose as a transition-state analogue.

Pyridoxal(5')diphospho(1)-alpha-D-glucose was used to reconstitute glycogen phosphorylase beta (1,4-alpha-D-glucan:orthophosphate alpha-D-glucosyltransferase, EC 2.4.1.1) from rabbit muscle, replacing the natural pyridoxal 5'-phosphate coenzyme. Incubation of the reconstituted enzyme alone resulted in the gradual cleavage of the synthetic cofactor to pyridoxal 5'-phosphate, which caused slow reactivation of the enzyme. The addition of maltopentaose or glycogen altered the mode of cleavage; the cofactor was rapidly decomposed to pyridoxal 5'-diphosphate. The radioactive glucose moiety released from pyridoxal(5')diphospho(1)-alpha-D-[14C]glucose was incorporated into the outer chain of glycogen, forming an alpha-1,4-glucosidic linkage. These results show that the glucosyl transfer reaction discovered mimics the normal catalysis of this enzyme, and they strongly support the catalytic mechanism in which the coenzyme phosphate acts as a catalyst by direct interaction with the phosphate of the substrate, forming the pyrophosphate-like transition intermediate.

Catalysis↗
PubMed1982

A monomeric intermediate in the reconstitution of potato apophosphorylase with pyridoxal 5'-phosphate.

The process of reconstitution of potato apophosphorylase with pyridoxal 5'-phosphate (PLP) has been investigated to elucidate the structure-function relationship in phosphorylase [EC 2.4.1.1]. In time-course studies, the recovery of enzyme activity was found to be delayed, especially at low temperatures, compared with the binding of PLP to protein. On polyacrylamide gel electrophoresis, an intermediary enzyme species was detected which bound PLP in the same binding mode as the native holoenzyme does, but which had neither enzyme activity nor affinity for the substrate amylopectin. The intermediate is monomeric and is converted to the active holoenzyme with concomitant dimerization. NaBH4-treatment of the monomeric intermediate produced the reduced monomeric enzyme, which could be converted into the reduced dimeric enzyme with considerable enzyme activity. The findings support the view of the catalytic activity of phosphorylase requires the dimeric structure of protein. As in the animal enzyme, the aldimine bond between the PLP and the epsilon-amino group of the lysyl residue is also not essential for enzyme activity in plant phosphorylase.

Apoenzymes↗
PubMed1982

Structural similarities in the active-site region between potato and rabbit muscle phosphorylases: a lysyl residue located close to the pyridoxal 5'-phosphate.

P1,P2-bis(5'-pyridoxal)diphosphate crosslinks between the original cofactor (pyridoxal 5'-phosphate) linking residue and Lys-573 in rabbit muscle phosphorylase (Shimomura, S., Nakano, K., & Fukui, T. (1978) Biochem. Biophys. Res. Commun. 82, 462-468). We have applied the same technique to potato phosphorylase to compare the structures of the active-site regions of the two enzymes, which have different regulatory properties. The reagent was bound to the potato enzyme in the same binding mode as the rabbit muscle enzyme. A sequence study on the potato enzyme labeled with this reagent revealed that it crosslinks between the original cofactor-linking lysyl residue and another lysyl residue, respectively corresponding to Lys-679 and Lys-573 in the rabbit muscle enzyme, and that the sequence Lys-573 to Leu-577 in the rabbit muscle enzyme is conserved in the potato enzyme. These findings indicate structural similarities in the active-site region between the phosphorylases, and suggest the importance of a lysyl residue in the catalytic mechanism of the phosphorylase reaction.

Amino Acid Sequence↗
PubMed1982

Comparative glucan specificities of two types of spinach leaf phosphorylase.

Two types of alpha-glucan phosphorylase [EC 2.4.1.1] from spinach leaves have been separately purified to near homogeneity. Type I enzyme shows a subunit molecular weight of 92,000 and Km values for amylopectin, glycogen and amylose much smaller than that for maltopentose. Cyclodextrin is a normal competitive inhibitor with respect to maltopentose, while it is a multi-site competitive type inhibitor with respect to amylopectin, glycogen or amylose. Type II enzyme shows a subunit molecular weight of 108,000, and utilizes amylopectin, amylose and maltopentose well, but glycogen very poorly. On affinity electrophoresis, Type II enzyme shows no affinity for glycogen and the dissociation constant for amylopectin is more than a thousand-fold greater than that of Type I enzyme. Type II enzyme has similar characteristics (subunit size, glucan specificity, and mode of cyclodextrin inhibition) to potato phosphorylase, for which cyclodextrin is a normal competitive inhibitor with respect to either maltopentose or amylopectin. Enzyme-glucan binding models have been proposed to explain these different kinetic properties. In spinach Type I phosphorylase, multiple glucan binding sites are located on the same face of the enzyme molecule to enable a single large substrate to be bound by riding on all the site; in spinach Type II and potato phosphorylases, two glucan binding sites are three-dimensionally arranged in a manner that excludes the possibility of the binding of amylopectin by riding on the two sites.

Animals↗
PubMed1982

An extracellular poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis.

A strain of Alcaligenes faecalis T1, which was isolated from activated sludge, excreted an extracellular poly(3-hydroxybutyrate) depolymerase as it grew in a medium containing poly(3-hydroxybutyrate) as the sole carbon source. The molecular weight of the enzyme, purified from the culture medium to electrophoretic homogeneity, was 48 000 as determined by Sephadex G-100 filtration, and 50 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. The pH optimum for the enzyme reaction was 7.5. The purified enzyme depolymerized poly(3-hydroxybutyrate) purified from Zoogloea ramigera 1-16-M, but did not attack the bacterial native poly(3-hydroxybutyrate)-containing granules. Km values were 13.3 micrograms/ml (= 0.78 microM, based on an estimated average molecular weight of 17 000) for poly(3-hydroxybutyrate) and 5.4 mM for the trimeric ester of D(--)-3-hydroxybutyric acid. Analysis of hydrolytic products of poly(3-hydroxybutyrate), several oligomeric esters of D(--)-3-hydroxybutyric acid, and the methyl ester of the trimeric ester indicated that the enzyme hydrolyzed these substrates from the free hydroxyl terminus, releasing D(--)-3-hydroxybutyrate dimer units one at a time.

Alcaligenes↗
PubMed1982

Purification and characterization of acetoacetyl-CoA synthetase from Zoogloea ramigera I-16-M.

Acetoacetyl-CoA synthetase was purified to electrophoretic homogeneity from Zoogloea ramigera I-16-M, a poly(3-hydroxybutyrate)-accumulating bacterium, which lacks 3-ketoacid CoA-transferase. The purified enzyme had a specific activity of 52.2 mumol acetoacetyl-CoA formed min-1 mg protein-1, which constituted a 680-fold purification compared to the crude extract, with a 5.1% yield. The enzyme absolutely required ATP, CoA, a monovalent cation (K+, Rb+, Cs+ or NH+4) and a divalent cation (Mg2+, Mn2+, Ca2+ or Ni2+) for the activation of acetoacetate, yielding acetoacetyl-CoA, AMP and pyrophosphate in equimolar amounts. The pH optimum of the enzyme reaction was 8.4. The molecular weight of the enzyme was approximately 70 000 as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and 72 000 by Sephadex G-200 gel filtration. The enzyme was active only on acetoacetate and to a lesser extent on L(+)-3-hydroxybutyrate, and the Km values for acetoacetate, L(+)-3-hydroxybutyrate, ATP and CoA were 7.6 X 10(-5) M, 1.4 X 10(-3) M, 3.3 X 10(-5) M and 9.1 X 10(-5) M respectively.

Chemical Phenomena↗
PubMed1982

X-ray diffraction studies of poly(I) . poly(C) analogues with substitution of the 2'-hydroxyl group by a fluorine atom.

From X-ray results of 2'-deoxy-2'-fluoronucleosides, characterization of the sugar conformation was summerized in comparison with the related ribo- and deoxyribonucleosides. It is emphasized that twisted forms of the sugar involving C(4')-exo puckering may be one of the characteristic feature of 2'-deoxy-2'-fluoronucleosides. In context of the characterization, molecular structure of duplex of poly(2'-deoxy-2'-fluoroinosinic acid) and poly(C), poly(dIf1) . poly(C), was analyzed by x-ray fiber diffraction methods. It is shown that the duplex has a strong preference of A'-RNA form than poly (I) . poly (C) and the preference could be interpreted by the sugar conformation of C(3') -endo-C(4')-exo pucker. Comparative studies of X-ray fiber diffraction by poly(I) . poly(C), poly(dIf1) . poly(C), poly(I) . poly(dCf1) and poly (dIf1) . poly (dCf1) are also given.

Deoxyribonucleosides↗