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Biomedical subjects

K Soda

Publications and source records attributed to K Soda.

At least 235 records · Page 13Linked to original sources
PubMed1987

A spectrophotometric method for the determination of gamma-glutamyl cyclotransferase with alanine dehydrogenase in the presence of anthglutin.

gamma-Glutamyl cyclotransferase activity is assayed in tissues by a colorimetric method using gamma-glutamyl alanine as a substrate coupled with alanine dehydrogenase from B. sphericus, to measure the formation of NADH. In order to avoid interference by the reaction catalyzed by gamma-glutamyl transpeptidase, anthglutin, a specific inhibitor of the transpeptidase was included in the reaction mixture. The Km value of rat kidney gamma-glutamyl cyclotransferase with respect to gamma-glutamyl alanine appeared to be the same when determined by either the colorimetric or the radiometric method. This assay presents a reliable alternative to the use of radiolabeled substrate and is used for the assay of gamma-glutamyl cyclotransferase in a variety of physiological and experimental samples.

Acyltransferases↗
PubMed1987

Receptor-mediated O2- release by alveolar macrophages and peripheral blood monocytes from smokers and nonsmokers. Priming and triggering effects of monomeric IgG, concanavalin A, N-formyl-methionyl-leucyl-phenylalanine, phorbol myristate acetate, and cytochalasin D.

Receptor-mediated superoxide (O2-) release by alveolar macrophages and peripheral blood monocytes from smokers and nonsmokers was studied in vitro. When the cells were incubated with monomeric IgG or monomeric Fc(IgG) fragment, no cell O2- release was observed. However, when cytochalasin D (Cyto D) was subsequently added to the cell suspension, we observed a markedly enhanced O2- release. Neither Cyto D alone nor the double stimulation of following Cyto D with monomeric IgG induced O2- release. Concanavalin A (Con A) also had a priming effect on O2 release in combination with Cyto D, as did monomeric IgG or monomeric Fc(IgG) fragment. On the other hand, heat-aggregated IgG, N-formyl-methionyl-leucyl-phenylalanine (FMLP), or phorbol myristate acetate (PMA) induced O2- release without the addition of Cyto D. Thus, we observed 2 different mechanisms in the receptor-mediated O2- release by alveolar macrophages and peripheral blood monocytes. Alveolar macrophages from smokers, which had a higher affinity and a larger number of monomeric IgG binding sites per cell than those from nonsmokers, were more reactive to the double stimulation of following monomeric IgG with Cyto D than to that of Con A and Cyto D, FMLP, or PMA, but for peripheral blood monocytes it was the reverse. We conclude that the binding of monomeric IgG to the Fc(IgG) receptor of alveolar macrophages or peripheral blood monocytes results in a priming effect on the cells for O2- release, and that the regulation of receptor-mediated O2- release by alveolar macrophages differs at least in part from that of peripheral blood monocytes.

Adult↗
PubMed1987

Re-investigation of the protein structure of coenzyme B12-dependent diol dehydrase.

We have purified diol dehydrase, an adenosylcobalamin-dependent enzyme, from Klebsiella pneumoniae by two different procedures to re-investigate its protein structure; one including its extraction with detergent from the membrane fraction, and the other consisting of only chromatographic separations of the soluble fraction. The enzyme preparations obtained by these two methods were different in the subunit structure, but both are identical in molecular weight, and in-enzymological and immunochemical properties. In addition, the enzyme preparation obtained from the membrane fraction dissociated reversibly into two dissimilar protein components (F and S) in the absence of substrate, as did the preparation from the soluble fraction. Although the subunit multiplicity of component S might be partly due to proteolytic cleavage during the enzyme purification as revealed by limited digestion with trypsin, component F is not a product of proteolytic cleavage of component S, but a primordial and essential constituent of the enzyme.

Cell Membrane↗
PubMed1986

Thermostable alanine racemase from Bacillus stearothermophilus: molecular cloning of the gene, enzyme purification, and characterization.

The alanine racemase (EC 5.1.1.1) gene of a thermophilic bacterium, Bacillus stearothermophilus, was cloned and expressed in Escherichia coli C600 with vector plasmid pICR301, which was constructed from pBR322 and the L-alanine dehydrogenase gene derived from B. stearothermophilus. A coupled assay method with L-alanine dehydrogenase and tetrazolium salts was used to detect visually the alanine racemase activity in the clones. Alanine racemase overproduced in a clone carrying the plasmid pICR4, 12 kilobases of DNA, was purified from cell extracts about 340-fold to homogeneity by five steps including heat treatment. The overproduced enzyme was confirmed to originate from B. stearothermophilus by an immunochemical cross-reaction with the enzyme of B. stearothermophilus. The purified enzyme has a molecular weight of about 78 000 and consists of two identical subunits of Mr of 39 000. At the optimum temperature (50 degrees C), the enzyme has a specific activity of 1800 units/mg (Vmax, D- to L-alanine). Resolution and reconstitution experiments together with the absorption spectrum of the enzyme clearly indicate that alanine racemase of B. stearothermophilus is a pyridoxal 5'-phosphate enzyme.

Alanine Racemase↗
PubMed1986

Time-dependent inhibition of Bacillus stearothermophilus alanine racemase by (1-aminoethyl)phosphonate isomers by isomerization to noncovalent slowly dissociating enzyme-(1-aminoethyl)phosphonate complexes.

An alanine racemase encoded by a gene from the thermophilic Gram-positive bacterium Bacillus stearothermophilus is overproduced to 0.3% of the soluble protein when carried on plasmid pICR4 in Escherichia coli [Inagaki, K., Tanizawa, K., Badet, B., Walsh, C. T., Tanaka, H., & Soda, K. (1986) Biochemistry (third paper of four in this issue)]. Purification of large quantities (50 mg) of racemase permits study of time-dependent inactivation by D and L isomers of the antibacterial (1-aminoethyl)phosphonate (Ala-P), the phosphonate analogue of alanine. The time-dependent activity loss by this compound now appears general to Gram-positive but not to Gram-negative racemases [Badet, B., & Walsh, C. (1985) Biochemistry 24, 1333] and is shown to occur by extremely slow dissociation of a noncovalent E X Ala-P complex. Ala-P binds initially in a weak, reversible (KI = 1 mM) competitive manner but is slowly isomerized (kinact = 6-9 min-1) to a stoichiometric enzyme complex, which in turn dissociates extremely slowly, with a half-time about 25 days. Thus, Ala-P is a slow but not a tight-binding inhibitor. The E X Ala-P complex is not reducible by borohydride but does perturb the fluorescence of bound pyridoxal 5'-phosphate coenzyme. Determination of the sequence of an active site octapeptide of the B. stearothermophilus alanine racemase shows homology with the sequence of a Gram-negative Salmonella typhimurium alanine racemase that is not susceptible to time-dependent inhibition by Ala-P. Studies with Ala-P analogues suggest the phosphonate dianion is crucial for stable formation of an isomerized long-lived E X Ala-P-inhibited complex.

Alanine Racemase↗
PubMed1986

Production of a novel tryptophan analog, beta-1-indazole-L-alanine with tryptophan synthase of Escherichia coli.

The tryptophan synthase alpha 2 beta 2 complex from Escherichia coli has been found to catalyze the beta-replacement reaction of L-serine with indazole, an indole analog which has a nitrogen atom at the 2-position (pyrazole ring). The reaction product was isolated and identified as beta-indazolealanine by mass spectrometric, elemental and NMR analyses. Careful assignment of 1H- and 13C-signals with several NMR techniques revealed that the beta-carbon of the product alanine moiety was bound to the 1-N-position of the indazole ring. This is the first example of the beta-replacement reaction catalyzed by tryptophan synthase occurring at any other position than the 3-position of indole analogs.

Chemical Phenomena↗
PubMed1986

Mechanism of alpha-amino-epsilon-caprolactam racemase reaction.

alpha-Amino-epsilon-caprolactam racemase catalyzes the exchange of the alpha-hydrogen of the substrate with deuterium during racemization in deuterium oxide. The rate of the hydrogen exchange measured by 1H NMR is lower than that of racemization in deuterium oxide for both the enantiomers. Both the enantiomers of alpha-amino-epsilon-caprolactam show an overshoot of the optical rotation during the enzymatic racemization in deuterium oxide (but not in water). This phenomenon may be attributable to a primary deuterium isotope effect at the alpha-position: alpha-deuterium isotope effects of 3.6 and 2.0 were observed for the racemization of the D and L enantiomers of alpha-amino-epsilon-caprolactam, respectively. Results of tritium-labeling experiments showed that the enzyme catalyzes both retention and inversion of configuration of the substrate with a similar probability in each turnover. Conversion of [alpha-2H]-D-alpha-amino-epsilon-caprolactam in water and unlabeled D-alpha-amino-epsilon-caprolactam in deuterium oxide into the L isomer under nearly single turnover conditions with the enzyme showed significant internal return of the alpha-hydrogen. These results support a single base mechanism for the racemization reaction catalyzed by the enzyme.

Alcaligenes↗