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K Soda

Publications and source records attributed to K Soda.

At least 217 records · Page 12Linked to original sources
PubMed1989

Overproduction of thermostable leucine dehydrogenase of Bacillus stearothermophilus and its one-step purification from recombinant cells of Escherichia coli.

We have cloned the leucine dehydrogenase (EC 1.4.1.9) gene from a thermophile, Bacillus stearothermophilus, into Escherichia coli MV1184 with a vector plasmid, pUC119. The cloned cells produced a large amount of the thermostable enzyme, which corresponds to about 60% of the total soluble protein. The enzyme was purified to more than 95% homogeneity by only one step, heat treatment of the cell-extracts, with an average yield of 75 mg/g of wet cells (obtained from 100 ml of the culture).

Amino Acid Oxidoreductases↗
PubMed1988

Gene cloning and sequence determination of leucine dehydrogenase from Bacillus stearothermophilus and structural comparison with other NAD(P)+-dependent dehydrogenases.

The gene for leucine dehydrogenase (EC 1.4.1.9) from Bacillus stearothermophilus was cloned and expressed in Escherichia coli. The selection for the cloned gene was based upon activity staining of the replica printed E. coli cells. A transformant showing high leucine dehydrogenase activity was found to carry an about 9 kilobase pair plasmid, which contained 4.6 kilobase pairs of B. stearothermophilus DNA. The nucleotide sequence including the 1287 base pair coding region of the leucine dehydrogenase gene was determined by the dideoxy chain termination method. The translated amino acid sequence was confirmed by automated Edman degradation of several peptide fragments produced from the purified enzyme by trypsin digestion. The polypeptide contained 429 amino acid residues corresponding to the subunit (Mr 49,000) of the hexameric enzyme. Comparison of the amino acid sequence of leucine dehydrogenase with those of other pyridine nucleotide dependent oxidoreductases registered in a protein data bank revealed significant sequence similarity, particularly between leucine and glutamate dehydrogenases, in the regions containing the coenzyme binding domain and certain specific residues with catalytic importance.

Amino Acid Oxidoreductases↗
PubMed1988

Specific labeling of the essential cysteine residue of L-methionine gamma-lyase with a cofactor analogue, N-(bromoacetyl)pyridoxamine phosphate.

L-Methionine gamma-lyase from Pseudomonas putida is composed of four identical polypeptide chains and contains four cysteinyl residues per subunit. We have found one of them catalytically essential by its specific cyanylation with 2-nitro-5-thiocyanobenzoic acid. We have shown its essentiality also with N-(bromoacetyl)pyridoxamine 5'-phosphate (BAPMP), which is a cofactor analogue and also an affinity-labeling agent. The kinetic data show that the apoenzyme forms a binary complex with BAPMP prior to covalent binding. The stoichiometry of inactivation was 1 mol of BAPMP per subunit. We have shown that the cysteine residue modified with BAPMP is identical with that labeled specifically with [14C]iodoacetic acid. The amino acid sequences of the peptides containing the essential cysteine residue and the lysine residue to which pyridoxal 5'-phosphate is bound were determined by automated Edman degradation.

Affinity Labels↗
PubMed1988

Thermostable alanine racemase from Bacillus stearothermophilus: DNA and protein sequence determination and secondary structure prediction.

The nucleotide sequence of the alanine racemase (EC 5.1.1.1) gene from a thermophile, Bacillus stearothermophilus, was determined by the dideoxy chain termination method with universal and synthetic site-specific primers. The amino acid sequence of the enzyme predicted from the nucleotide sequence was confirmed by peptide sequence information derived from the N-terminal amino acid residues and several tryptic fragments. The alanine racemase gene consists of 1158 base pairs encoding a protein of 386 amino acid residues; the molecular weight of the apoenzyme is estimated as 43,341. The racemase gene of B. stearothermophilus has a closely similar size (1158 vs 1167 base pairs) to that of the gene of a mesophile, B. subtilis, but shows a higher preference for codons ending in G or C. A comparison of the amino acid sequence with those of Bacillus subtilis and Salmonella typhimurium dadB and alr enzymes revealed overall sequence homologies of 31-54%, including an identical octapeptide bearing the pyridoxal 5'-phosphate binding site. Although the residues common in the four racemases are not continuously arrayed, these constitute distinct domains and their hydropathy profiles are very similar. The secondary structure of B. stearothermophilus alanine racemase was predicted from the results obtained by theoretical analysis and circular dichroism measurement.

Alanine Racemase↗
PubMed1988

Thermostable dipeptidase from Bacillus stearothermophilus: its purification, characterization, and comparison with aminoacylase.

Dipeptidase (dipeptide hydrolase [EC 3.4.13.11]) has been purified to homogeneity and crystallized from the cell extract of Bacillus stearothermophilus IFO 12983. The enzyme has a molecular weight of about 86,000, and is composed of two subunits identical in molecular weight (43,000). The enzyme contains 2 g atoms of zinc per mol of protein. A variety of dipeptides consisting of glycine or only L-amino acids serve as substrates of the enzyme; Km and Vmax values for L-valyl-L-alanine are 0.5 mM and 68.0 units/mg protein, respectively. The enzyme is significantly stable not only at high temperatures but also on treatment with protein denaturants such as urea and guanidine hydrochloride. The enzyme also catalyzes hydrolysis of several N-acylamino acids with Vmax values 3-30% of those for the hydrolysis of dipeptides. The thermostable dipeptidase shares various properties with bacterial aminoacylase [EC 3.5.1.14]: their subunit molecular weight, metal content and requirement, amino acid composition, and amino acid sequence in the N-terminal region are very similar.

Amidohydrolases↗
PubMed1988

Glutamine synthetase from a cyanobacterium, Phormidium lapideum: purification, characterization, and comparison with other cyanobacterial enzymes.

Glutamine synthetase has been purified to homogeneity from cell extracts of a non-N2-fixing filamentous cyanobacterium, Phormidium lapideum. The subunit molecular weight of the enzyme was determined as about 59,000 by sodium dodecyl sulfate gel electrophoresis. Electron micrographs of the Phormidium enzyme revealed a two-layered structure of regular hexagons (12 subunits per molecule), which markedly resembles the three-dimensional polypeptide backbone structure of the Salmonella typhimurium glutamine synthetase established by X-ray crystallography (Almassy, Janson, Hamlin, Xuong, & Eisenberg (1986) Nature 323, 304-309). The N-terminal amino acid sequence of the Phormidium enzyme shows very high similarity with that of the enzyme from an N2-fixing cyanobacterium, Anabaena 7120; 18 residues are common in 23 residues compared. Strong immunocross-reactions between the antibody against the purified Phormidium glutamine synthetase and other cyanobacterial enzymes except the Anacystis enzyme were observed. The apparent Michaelis constants for NH3, L-glutamate, and ATP were determined to be 0.29, 7.4, and 1.7 mM, respectively. Divalent metal ions such as Mg2+ and Mn2+ activated the enzyme in the biosynthetic reaction, whereas various amino acids and glutamate analogs strongly inhibited the enzyme.

Amino Acid Sequence↗
PubMed1988

Chemical synthesis and expression of copper metallothionein gene of Neurospora crassa.

The gene coding for the Neurospora crassa copper metallothionein (MT) was synthesized and inserted in the lacZ' gene of pUC18 plasmid to give the same translational reading frame as the latter gene. The MT-beta-galactosidase fused gene was expressed in Escherichia coli to produce a fused protein in which the amino and carboxy termini of MT are linked to the beta-galactosidase through methionine residues. An MT derivative containing an extra homoserine residue at the carboxy terminus was prepared by cyanogen bromide cleavage of the fused protein followed by a reverse-phase HPLC separation. The spectral features of the MT derivative and its copper complex were similar to those of the corresponding native MTs.

Amino Acid Sequence↗
PubMed1988

Purification and characterization of Clostridium sticklandii D-selenocystine alpha, beta-lyase.

We have found a novel enzyme that decomposes D-selenocystine into pyruvate, ammonia, and elemental selenium in extracts of Clostridium sticklandii and C. sporogenes. The enzyme of C. sticklandii has been purified to homogeneity. It has a molecular weight of 74,000 and consists of two subunits identical in molecular weight (35,000). Pyridoxal 5'-phosphate is required as a cofactor. In addition to D-selenocystine, D-cystine, D-lanthionine, meso-lanthionine, and D-cysteine serve as substrates. However, D-selenocysteine, D-serine, DL-selenohomocystine, and L-amino acids are inert. The enzyme also catalyzes the beta-replacement reaction between D-selenocystine and a thiol to produce S-substituted D-cysteine. L-Selenohomocysteine also can serve as a substituent donor in the beta-replacement reaction to yield selenocystathionine.

Chemical Phenomena↗
PubMed1988

C1q and C3 in bronchoalveolar lavage fluid from patients with summer-type hypersensitivity pneumonitis.

Immune complexes have been thought to participate in the pathogenesis of hypersensitivity pneumonitis, but the role of complement components is not defined. In our study of nine patients with summer-type hypersensitivity pneumonitis (summer-type HP), C1q in bronchoalveolar lavage fluid (BALF) was strikingly increased (mean 3.7, range 0.4 to 10 micrograms/ml). The value of C1q/albumin was several to 20 times greater in BALF than in serum samples from individual patients. In contrast, BALF samples from control subjects (ten patients with sarcoidosis and nine normal subjects) contained an undetectable amount (less than 0.02 micrograms/ml) of C1q. C3 in BALF also increased in the summer-type HP patients. Furthermore, C1q (as well as specific IgG and IgA antibody activities to Trichosporon cutaneum antigen) in BALF correlated with clinical symptoms and diffusing capacity (DCO), while the BAL lymphocytosis or the change of OKT4/OKT8 ratio did not. These findings are indicative of local secretion or concentration mechanism of C1q and C3, supporting the involvement of immune complexes in the respiratory tract of the patients.

Alveolitis, Extrinsic Allergic↗
PubMed1988

Changes in the molecular sieve of the glomerular basement membrane of rats with chronic serum sickness.

In order to explore the pathogenic mechanism of proteinuria in glomerulonephritis, ultrastructural changes of the glomerular basement membrane were investigated in rats with chronic serum sickness induced by repeated intravenous injections of bovine serum albumin (experimental rats). Rats injected with saline served as controls. The animals were sacrificed and examined 13 weeks after treatment, when the mean urinary protein of experimental animals reached 206 mg/24h/100g body weight. Enhanced transcapillary passage of anionic ferritin was observed in experimental rats. Purified glomerular basement membranes of control and experimental rats were examined by electron microscopy after negative staining. The glomerular basement membrane of experimental rats had enlarged pores. The results suggest that an increase in the radius of glomerular pores may be responsible for proteinuria in glomerulonephritis.

Animals↗
PubMed1988

Experimental hypersensitivity pneumonitis in rabbits induced by Trichosporon cutaneum: role of local cellular and humoral immune responses.

We evaluated the immunopathologic properties of Trichosporon cutaneum, an important etiologic agent of summer-type hypersensitivity pneumonitis in Japan. Rabbits were sensitized and challenged by either a culture-filtrate antigen or a particulate antigen prepared from a TIMM 1318 strain of the fungus. Group 1 animals received repetitive intratracheal injections of a particulate T. cutaneum antigen without use of any adjuvant. Group 2 animals were sensitized by foot-pad injections of particulate antigen followed by an intratracheal challenge with the same antigen. Group 3 animals were sensitized by a culture-filtrate antigen emulsified in Freund's incomplete adjuvant followed by a challenge with the intratracheal injection of the same antigen. In group 1, we observed interstitial pneumonitis with prominent granulomatous reactions which resembled those in human hypersensitivity pneumonitis. Group 2 animals also showed granulomatous inflammation in a some minor extent, but they were accompanied with excessive infiltration of neutrophils. In contrast, group 3 showed mild alveolitis in a cell population consisting predominantly of macrophages. Lymphocytes from mediastinal nodes responded to the antigen in groups 1 and 2 but not in group 3. Interestingly, the specific IgA antibody activity in the bronchoalveolar lavage fluids (BALF) of group 1 was significantly higher than those of group 2, despite the IgG antibody activity was the same. The ratio of IgA/IgG antibody activity in individual BALF sample in these two groups correlated inversely with the grade of neutrophil infiltration. Thus, the presence of IgA antibody in the respiratory tract affects the pathological outcome.(ABSTRACT TRUNCATED AT 250 WORDS)

Alveolitis, Extrinsic Allergic↗
PubMed1987

X-ray crystallographic studies of the alanine-specific racemase from Bacillus stearothermophilus. Overproduction, crystallization, and preliminary characterization.

To facilitate large-scale purification and crystallographic study, we have subcloned the gene for the alanine racemase of Bacillus stearothermophilus from pICR401 (Inagaki, K., Tanizawa, K., Badet, B., Walsh, C. T., Tanaka, H., and Soda, K. (1986) Biochemistry 25, 3268-3274) and overproduced the enzyme in Escherichia coli W3110 lacIq using the tac promoter of PKK223-3. This system yields alanine racemase as 6% of the bacterial cytosolic protein. Purification by a modification of the procedure of Inagake et al. yielded 75 mg of homogeneous alanine racemase from 30 g of cells (wet weight). Large, well-formed crystals of alanine racemase have been grown from polyethylene glycol 8000 using vapor diffusion. These crystals have unit cell dimensions a = 85.3 A, b = 110.0 A, and c = 89.9 A. The crystals belong to space group P2(1), with beta fortuitously equal to 90 degrees within experimental error; however, they are frequently twinned by second order pseudomerohedry with twin fraction (the ratio of the volume of the smaller twin domain to the total volume of the crystal) ranging from about 0 to 0.5. Fortunately, for crystals with low twin fraction, computational methods have been developed for the analysis and correction of simple twinning (Fisher, R. G., and Sweet, R. M. (1980) Acta Crystallogr. A36, 755-760). The crystals contain two alpha 2 dimers of alanine racemase in the asymmetric unit. We have identified several potentially useful heavy atom derivatives in low resolution screening experiments and are proceeding with high resolution data collection.

Alanine Racemase↗
PubMed1987

A spectrophotometric rate assay of aminoacylase.

Acetamidoacrylate, a synthetic N-acetyl unsaturated amino acid, was hydrolyzed to acetate, ammonia, and pyruvate by hog kidney, fungal, and bacterial aminoacylases. A spectrophotometric procedure for rate assay of aminoacylase has been established with this substrate on the basis of the simultaneous reduction of pyruvate with NADH and alanine dehydrogenase. This assay is linear with time and enzyme concentration and is useful for kinetic studies of aminoacylase. This procedure is not influenced significantly by amino and thiol compounds and metal ions, which interfere with the ninhydrin methods traditionally used. Alanine dehydrogenase can be replaced by lactate dehydrogenase in the reaction system.

Alanine Dehydrogenase↗
PubMed1987

Preliminary X-ray data for a D-amino acid amino-transferase from a novel thermophilic Bacillus.

Crystals of the D-amino acid aminotransferase (D-ATA) from a novel thermophilic Bacillus species (Escherichia coli pICT113 cloned gene product) have been examined by X-ray analysis. The crystals grow as hexagonal prisms, with the symmetry of space group P61 or P65 (indistinguishable crystallographically). The cell dimensions are a = b = 135 A, c = 53 A, alpha = beta = 90 degrees, and gamma = 120 degrees. The unit cell has a volume of 850,000 A3 with six asymmetric units per unit cell. There is one dimer of molecular weight 62,000 per asymmetric unit, and the crystals diffract to 2.7 A.

Bacillus↗
PubMed1987

Selenocysteine.

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Chromatography↗
PubMed1987

Selenocysteine.

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Cysteine↗