The enzymatically inactive but raw-starch-adsorbable peptide fragments designated as Gp-pan P and Gp-pan I were obtained from a tryptic digest of heat-inactivated hog pancreatic α-amylase. These two glycopeptide fragments were purified with Sephadex G-75, DEAE-Sephadex A-50, and HPLC and were found to be homogeneous on disc gel electrophoresis. Gp-pan P and I had molecular weights of 20, 000 and 30, 000 with SDS-PAGE, carbohydrate contents of 10% and 7%, N-terminal amino acids Gly-Trp and Ala-Val, and C-terminal amino acids Gly-Arg and Ile-Lys. Gp-pan P had promotive but Gp-pan I inhibitory effects on raw starch digestion by Aspergillus awamori var. kawachi glucoamylase I and Bacillus subtilis 65 α-amylase.

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The N-sparing action of methionine and threonine in rats fed with a non-protein diet seems to indicate that methionine and threonine are the endogenously most-limiting amino acids. The effect of a dietary supplementation of methionine and threonine to a non-protein diet on enzyme induction by polychlorinated biphenyls (PCB) was investigated. Liver microsomal drug-metabolizing enzymes were further induced by the methionine and threonine supplement to the non-protein diet in PCB-receiving rats. Although feeding with PCB did not increase the urinary ascorbic acid excretion in rats of the non-protein diet group, the supplementation of methionine and threonine to the non-protein diet markedly increased this excretion. From these observations, dietary methionine and threonine might promote the synthesis of drug-metabolizing enzymes and the enzymes related to ascorbic acid biosynthesis in PCB-receiving rats.

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Cuticle-bound phenoloxidase was found in the pupae of the housefly (Musca domestica L.), and its properties were studied. The time course of dopachrome formation from dopa, and the effects of the amount of cuticles and dopa concentration on the formation of dopachrome from dopa by cuticle-catalyzed oxidation were studied. Cuticle-catalyzed oxidation was found to be an enzyme-catalyzed oxidation dependent on the cuticle-bound phenoloxidase. Km and V_max values for the oxidation of dopa by cuticle-bound phenoloxidase were 4.70 mM and 1.68 × 10_-2U/mg cuticle, respectively. The optimal temperature of cuticle-bound phenoloxidase was approximately 45°C, and it was stable between 5 and 25°C. The optimal pH of cuticle-bound phenoloxidase was approximately 9.0, and it was stable from pH 7.0 to 9.5.

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An ethanol extract of Quercus acuta trunk showed antibacterial activity against both gram-positive and gram-negative bacteria. The extract was sequentially partitioned with n-hexane, chloroform, ethyl acetate and water, and the highest activity was observed in the ethyl acetate fraction. Two active compounds isolated from the ethyl acetate fraction were 4, 5-di-O-galloyl (+)-protoquercitol and 3, 5-di-O-galloyl protoquercitol, of which the former was the major active constituent. Gallic acid was also isolated from the same fraction, but it was not active.

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Bacillus macerans produced an extracellular endo-pectate lyase when cultivated with grated potato tuber. The enzyme activity was markedly activated by 1 mM calcium or manganese ion, but completely inactivated by ethylenediaminetetraacetate (EDTA). The activity was maximum at pH 9.0 and 60°C. The enzyme activity was stable up to 50°C and 55°C for 10min at pH 9.0 in the absence and presence of calcium ion, respectively, and between pH 6.5 and 9.5 for 2 hr at 37°C. The enzyme had a molecular weight of 35, 000 and a pI of 10.3. The Michaelis constant and maximum reaction velocity of the enzyme for polypectate were 0.11% and 74μmiol of unsaturated galacturonate formed per min per mg protein, respectively. The enzyme also attacked pectin that contained 4.3% methoxyl groups. The enzyme was stable in 100 mM ammonium chloride buffer (pH 9.0) for a month at 10°C, but 70% of its activity was lost in 20 mM ammonium chloride buffer (pH 9.0) after 24 hr at 10°C.

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We had investigated the enzymatic hydrolysis of soybean saponins and selected soybean saponin hydrolase from Aspergillus oryzae KO-2. We attempted purification of this enzyme for further characterization. This enzyme was purified 1500-fold using ammonium sulfate fractionation and Sephadex G-200 gel filtrations. The enzyme was electrophoretically homogeneous and a glycoprotein by PAS staining. By gel filtration, the molecular weight of enzyme was 158, 000 and SDS-PAGE showed the enzyme to have a tetrameric structure composed of heterogeneous subunits of 35, 000 and 45, 000. The enzyme activity was stable at temperatures below 40°C and stable from pH 5.0 to 8.0. The optimum pH was pH 4.5 to 5.0 and the optimum temperature was 50°C. The Km and V_max for soyasaponin I were 0.48 mM and 9.8 μmol/hr mg protein, respectively. After hydrolysis with the enzyme, soyasapogenol B and α-L-rhamnopyranosyl (1→2)-β-D-galactopyranosyl (l→2)-D-glucuronopyranoside were released from soyasaponin I.

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Structural requirements of N-blocked L-proline derivatives as specific inhibitors for prolyl endopeptidase were investigated using a series of substrate analogs. Replacement of L-proline by its D-isomer remarkably reduced the inhibition. Introduction of a sulfur atom in proline and/or in the penultimate pyrrolidine rings significantly increased the inhibition, but the introduction of oxygen rather diminished the activity. A peptide linkage (acid-amide bond) between the proline and the pyrrolidine ring was also required to keep the inhibitory activity. A benzyloxycarbonyl group was most effective as an N-blocked component of the inhibitors.

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Immobilization by covalent bonding of Cyclomaltodextrin glucanotransferase (CCTase) of Bacillus stearothermophilus was studied for the continuous production of glucosyl-cyclodextrins (G₁-CDs) from branched dextrins. A macroreticular hydrophilic resin containing a primary amine as the active group was synthesized as a candidate carrier of CGTase. The properties of CGTase immobilized on this carrier resin were examined and compared with those of the native enzyme.
The optimum pH of the native enzyme is 6.0, but that of the immobilized enzyme extends from 6.0 to 8.0. The optimum temperature for the immobilized enzyme shifts slightly to a lower range than that of the native enzyme. In a column test there was an optimum range of substrate flow rate that maximized the G₁-CDs content in the column effluent. The G₁-CD content in the column effluent was greatly influenced by the substrate concentration, but not by the activity of the immobilized enzyme. We produced G₁-CDs continuously using a pilot-scale CGTase bioreactor. We could do the continuous operation to obtain an average G₁-CDs content of 10% (w/w) in the reactor effluent for 70 days.

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We prepared several TNF mutants by protein engineering techniques and compared their biological properties with those of the wild-type TNF. The mutant that lacked 7 N-terminal amino acids had higher cytotoxicity and higher binding activity to receptors on tumor cells. In contrast, the mutagenesis of Arg³² or Ala⁸⁴, in combination with the deletion of 7 N-terminal amino acids, eliminated the cytotoxicity and the receptor binding. These mutants also lacked acute lethal toxicity in normal mice. Therefore, we concluded that the N-terminus, Arg³² and Ala⁸⁴ of TNF might be concerned with binding to the TNF receptor. It was also suggested that the receptor molecules on tumor cells bound to the same or neighboring sites on TNF molecules as normal cell receptors.

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The host-vector systems of an n-alkane-assimilating-yeast, Candida maltosa, that we previously constructed consisted of a vector replicating with an ARS region of this yeast, and C. maltosa strains J288 (leu2) or CH1 (his5) as hosts. Since each of these hosts has a single genetic marker, we have developed a new host-vector system using two genetic markers. By UV irradiation of strain CH1, an adenine auxotrophic mutant, CHA1, forming red colonies was isolated. A DNA fragment complementing this deficiency was isolated from the C. maltosa genome. Since the DNA fragment also complemented the ade1 mutation of S. cerevisiae, we termed a gene contained in this DNA fragment C-ADE1. The nucleotides of C-ADE1 were sequenced. The deduced amino acid sequence (291 residues) had 65.6% homology with that of ADE1 of S. cerevisiae (306 residues). Having the cloned C-ADE1 DNA, we improved the host-vector system of C. maltosa.

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Immature soybean seeds accumulate starch as a transient reserve material that declines later in seed development. Phosphorylase activity in cotyledons rapidly increased 40-50 days after flowering (DAF) corresponding to the starch breakdown. Decomposition of starch granules was intracellularly compartmented to arm loplasts which were associated with rough endoplasmic reticulum. Simultaneously with the phosphorylase activation, phytase activity appeared and increased till 45 DAF, and transitory decline of phytic acid was observed at the maximum of phytase activity. These results suggest that phosphorolysis may be involved in starch breakdown, and that phytic acid is mobilized in developing soybean seeds.

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Prothoracicotropic hormone (PTTH) of the silkworm, Bombyx mori, was purified and its primary structure determined for the most part. From sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified PTTH under non-reducing and reducing conditions, and the amino acid sequence and composition analyses of the carboxamidomethylated PTTH, we concluded that Bombyx PTTH has a dimeric structure consisting of two identical, or nearly identical subunits, held together by disulfide bond(s). There exist subunit variants that differ by deletion of only a few amino acid residues at the N-terminus, and possibly at the C-terminus also, giving rise to a high heterogeneity in the PTTH molecule. The amino acid sequence up to the 104th residue from the N-terminus of the longest subunit, except for the 41st residue, was determined by sequence analysis of fragment peptides produced by lysyl endopeptidase, chymotrypsin and V8 protease digestions, leaving only a short C-terminal sequence undetermined. Bombyx PTTH is expected to contain a carbohydrate chain bound to an asparagine at position 41, deduced from the cDNA sequence.

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A simple micromethod for measurement of CoASH was established. CoASH was quantitatively converted to acetyl-CoA with phosphate acetyltransferase (EC 2.3.1.8), then acetyl-CoA originated from CoASH was measuring by the acyl-CoA cycling method using malonate CoA-transferase (EC 2.8.3.3). This method is highly sensitive and can detect 10^-12 of CoASH without any loss of CoA-thioesters, and it is specific for CoASH and 3'-dephospho-CoA. The established method was useful to define the rapid changes in the size and composition of in vivo Co A pool (CoASH, acetyl-CoA, and malonyl-CoA) in Escherichia coli K12. It was found that the CoA pool changed rapidly and dramatically in a minute order in ranges of CoASH; 40-300μM (0.11-0.80 nmol/mg dry wt.), acetyl-CoA; 40-350μM (0.12-0.93 nmol/mg dry wt.), and malonyl-CoA; 0-290μM (0-0.79 nmol/mg dry wt.). When glucose in the medium was depleted, CoASH increased to be the major component (82%) of the CoA pool, while when sufficient amount of glucose (28 mM) was provided, CoASH was reduced to a minor component (15%). In contrast with this, acetyl-CoA increased to the maximal level of 350μM (0.93 nmol/mg dry wt.) and to be the major component (66%) lOmin after 28 mM glucose was added. A remarkable demonstration was achieved in confirmation of the reverse relationship between CoASH and short chain acyl-CoAs, particularly acetyl-CoA, of which the in vivo behavior has been mere conjecture until now.

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The accumulation of moniliphenone (3), a benzophenone biosy nthetic intermediate of an antibiotic chloromonilicin (1), by M. fructicola was enhanced 15-16 times with the presence of bromine ions in the medium. Two anthraquinone pigments, chrysophanol (4) and emodin (5), also isolated from the mycelia, were proved to be precursors of 3 by incorporation experiments of the deuterium derivatives of these pigments into 3.

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Superoxide dismutase (SOD) was purified from Aerobacter aerogenes IFO 3317 to an electrophoretically homogeneous state and partially characterized. SOD was purified by ammonium sulfate fractionation, column chromatography on DEAE-Sephadex A-50, gel filtration on Sephadex G-100, phenyl-Toyopearl 650 M hydrophobic chromatography, and hydroxyapatite adsorption chromatography. The molecular weight and subunit molecular weight of the purified enzyme were estimated to be 45, 000 and 22, 000, respectively. The isoelectric point of the enzyme was about pH 4.0. The purified enzyme remained stable at pH 7.0-11.0, 25°C for 36hrs, but was rapidly inactivated below pH 7.0. SOD was stable up to 35°C at pH 7.0, but was inactivated at temperatures above that. The absorption maximum in the visible range was found at 360 nm, and the enzyme was insensitive to cyanide and fluoride, and sensitive to hydrogen peroxide and azide. These results suggest that the enzyme is an iron-containing SOD. The amino acid composition and the N-terminal sequence of the first 15 amino acids of the enzyme exhibited close homology with the other iron-containing SODs.

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This paper describes the specificity of Aspergillus niger 5-16 α-galactosidase toward various oligosaccharides having terminal galactose or stub galactose or both on the oligosaccharide. The galactosidase rapidly hydrolyzed p-nitrophenyl-α-D-galactopyranoside, but hardly liberated galactose from melibiose, manninotriose, 6³-α-D-galactosylmannotriose, etc. On the other hand, the enzyme tore off the stub galactoses attached to the inner mannoses of the main-chain of galactomanno-oligosaccharides, but not the terminal galactoses attached to the non-reducing-end mannoses of the main-chain. Thus, the substrate specificity of A. niger 5-16 α-galactosidase is quite different from that of Mortierella vinacea α-galactosidase.

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Streptomyces nucleotide 3'-pyrophosphokinase, formerly known to synthesize a variety of 3'-pyrophospho nucleotides by 5'-β, γ-pyrophosphoryI transfer from ATP, ATP 3'-pyrophosphate, and dATP, can also synthesize 2', 3'-cyclic monophospho nucleotides using diadenosine 5', 5'-poly (tri, tetra, and penta) phosphates as the phosphate donor. The examples are pA > p, ppA > p, pG > p, and CpG > p. Transient formation of an -N(3')pp(5')A structure via ADP transfer from the donor followed by AMP elimination is proposed as their synthetic mechanism.

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2-Oxobutyric acid (OBA) was produced from crotonic acid by two steps. OBA was produced from threo-(±)-dihydroxybutyric acid ((2RS, 3SR)-2, 3-dihydroxybutyric acid, DHB) by the action of cells of Pseudomonas putida. DHB is easily prepared by oxidation of crotonic acid with N-methylmorpholine-N-oxide. The microorganism was screened from a soil sample as the best producer of OBA, although the strain could not assimilate DHB as a sole carbon source. The culture conditions of the microorganism and the reaction conditions were optimized. In the presence of 80 g/1 of the cells of P. putida, the amount of OBA produced from DHB was 4.8 g/1 under the best conditions examined, with a molar yield of 47%.

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An α-amylase hyper producer A. oryzae and a protease hyper producer A. sojae were electrofused to breed more excellent koji-molds. Fusion between the yellow-spored, pant mutant or the yellow-spored, met mutant of A. oryzae and the white-spored, bio mutant or the white-spored, ade mutant of A. sojae produced heterokaryons with a frequency of 2 × 10^-5 per protoplast pair. The optimal parameters for the fusion of Aspergillus protoplasts were 1.1 MHz AC frequency, 400 V/cm AC voltage, 4kV/cm DC pulse voltage, 60 μsec pulse duration, and 2 or 3 pulses. In this combination of the fusion-parents, all the fusants with stable green conidia formed an alkaline proteinase with the mobility of A. oryzae-type, and none of them formed any intermediate type. The productivity and molecular specificity of proteinases behaved independently from each other in the processes of fusion and haploidization.

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The amounts of limonoid glucosides were measured in the seeds of grapefruit (Citrus paradisi), lemon (C. limori), Valencia orange (C. sinensis) tangerine (C. reticulata), Fukuhara (C. sinensis Osbeck Hort.), Hyuganatsu (C. tamurana Hort. ex Tanaka), Shimamikan (C. kinokuni Hort. ex Tanaka) and Sanbokan (C. sulcata Hort. ex Takahashi). All the seeds contained 17-β-Dglucopyranosides of limonin, nomilin, obacunone, deacetylnomilin, nomilinic acid and deacetylnomilinic acid. The total limonoid glucoside content ranged from 0.31 to 0.87% of the dry weight. The concentration of nomilin glucoside was highest among the glucosides found in the seeds. All the seeds also contained the major neutral limonoid aglycones commonly found in citrus, namely limonin, nomilin, deacetylnomilin, obacunone and ichangin. As previously reported, limonin was the predominant aglycone.

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Measurements of 3-hydroxyanthranilic acid and anthranilic acid in urine are described. The urine adjusted to pH 3.0 was saturated with NaCl, and 3-hydroxyanthranilic acid and anthranilic acid were extracted with diethyl ether. The method for 3-hydroxyanthranilic acid measurement employed a Shim-pack CLC-ODS (150 mm × 6.0 mm i.d.) column eluted with 100 mM potassium dihydrogenphosphate (pH 4.5)-acetonitrile (9:1, v/v) at a flow rate of 1.5 ml/min. The applied volage was set at +300mV vs. Ag/AgCl, the detection limit being 0.2pmol (30.62 pg) at a signal-to-noise ratio of 5:1. The method for anthranilic acid measurement employed a Chemcosorb 5-ODS-H (150 mm × 4.6 mm i.d.) column eluted with 80 mM potassium dihydrogenphosphate (pH adjusted to 3.0 by phosphoric acid)-acetonitrile (65:35, v/v) at a flow rate of 1.0 ml/min, with estimation at an excitation wavelength of 340 nm and an emission wavelength of 410 nm. The detection limit was 0.3pmol (41.13pg) at a signal-to-noise ratio of 5:1. These techniques were applied to the analyses of riboflavin-deficient rat urine. The total analysis times for 3-hydroxyanthranilic acid and anthranilic acid were ca. 12 min and 7 min, respectively.

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The relationship between the limited proteolysis and conformation of native glycinin was studied by the following methods: analyses of the proteolytic digestion of chemically modified glycinin, circular dichroism (CD) measurements, a secondary structure prediction from the amino acid sequence and a hydropathy index analysis. The locations of tryptic fragments of glycinin were confirmed from the N-terminal amino acid sequences of the fragments. T fragments and P fragments were located at the N-terminal and central area of the acidic polypeptide chains, respectively. One of the cleavage sites was the Arg residue of around the 100th from the N-terminal side. The regions digested by limited trypsinolysis were presumed to be flexible, hydrophilic and near the surface of the molecule by prediction methods from the amino acid sequence. The other regions were predicted to be compact and β-sheet in nature, almost 40-50% of the amino acid residues being predicted as β-sheet from the amino acid sequence and from CD data.

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Reaction rate constants for Superoxide (O₂^-) with Cypridina luciferin analogues (CLAs), 2-methyl-6-phenyl- and 2-methyl-6-(p-methoxyphenyl)-3, 7-dihydroimidazo[1, 2-a]pyrazin-3-ones (CLA and MCLA), and 2-methyl-6-[p-[2-[sodium 3-carboxylato-4-(6-hydroxy-3-xanthenon-9-yl)-phenylthioureylene]ethyleneoxy]phenyl]-3, 7-dihydroimidazo[1, 2-a]pyrazin-3-one(FCLA), were determined for the first time by using superoxide dismutase (SOD) for a quencher as 1.08 × 10⁸, 2.54 × 10⁸ and 8.5 × 10⁷ M^-1 sec^-1 in pH 7.1 buffer solutions at 25°C.

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Linecin A is produced by Brevibacterium linens ATCC 9175 and kills some strains of B. linens. Maximum extracellular linecin A activity was 8 units/ml at 48 hr of cultivation, but intracellular linecin A activity rapidly increased after 18 hr of cultivation, reaching 160 units/ml at 36 hr. To release intracellular linecin A from cells, mitomycin C was added to a culture of B. linens ATCC 9175 at a final concentration of 0.3 μg/ml. The extracellular linecin A activity increased to nearly 15-fold that of normal cultivation.
The extracellular linecin A was purified to a homogeneous state on polyacrylamide gel electrophoresis by DEAE-cellulofine, Sephacryl S-500, and Sephacryl S-300 column chromatography. Linecin A consisted of mostly protein and the molecular weight was estimated to be about 95, 000 by gel filtration. Linecin A was a thermolabile protein as the activity was completely lost by incubating at 45°C for 60 min.

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We established fourteen monoclonal antibodies (MAbs) reactive to bovine ephemeral fever virus YHL strain, and characterized six representatives including three IgGls (YG3/4, YG5/8, and YG6/7) and three IgMs (YM4/9, YM2/6, and YM6/8). Among them, YG3/4 and YM4/9 gave especially strong reactivities to the virus. YM4/9 reacted specifically with a 43K antigen of the virus, corresponding to the matrix protein 1. The other MAbs reacted most strongly with the 43K antigen, but also reacted with unknown 23K and 21K antigens. By a simultaneous two-site method using YM4/9 and YG3/4, it was possible to detect 10^4.10TCID₅₀/ml of the virus, in the presence of serum.

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Until recently, it was assumed that any short DNA segment could be regarded as a straight rod. Many instances, however, have been reported in which the helical axis was curved. In this study, to gain a general insight into the structural features of sequence-directed DNA curvatures, we constructed a set of plasmids carrying curved DNA segments, which were randomly cloned from Streptomyces total DNA with extremely high G/C-contents. The results of primary characterization of these curved DNA segments are presented. Although the cloned DNA segments had high G/C-contents, in all cases, several homopolymeric [dA] and [dT] stretches were found to be clustered around the centres of the determined nucleotide sequences. Furthermore, the clustered [dA] and [dT] stretches were located nearly in phase with the DNA helical screw. One of the DNA segments characterized in this study appeared to be derived from the giant linear plasmid, SCP1, and another from the circular fertility plasmid, SCP2.

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A water-soluble derivative of rutin was found to be formed in a high yield from dextrin and rutin in 50% methanol by the successive actions of Bacillus stearothermophilus cyclomaltodextrin glucanotransferase and Rhizopus sp. glucoamylase. The derivative was isolated from the reaction mixture by paper chromatography, Toyopearl HW-40S column chromatography, lyophilization, and successive treatment with methanol and ethanol, and then obtained as yellowish needle crystals [232-235°C, decomp. ¹⁶_D +65.3° (c = 3.77, H₂O)]. The compound was identified as 4^G-α-Dglucopyranosyl-rutin on the basis of the various experimental results, viz., UV, IR, ¹H-NMR, and ¹³C-NMR spectra, products by hydrolysis with acid and with α- and β-glucosidases. The solubility of the compound in water was about 30×10³ times higher than that of rutin.

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The cell wall protein gene operon of Bacillus brevis 47 has multiple and tandem promoters. The precise locations of the two major promoters (P2 and P3) of the operon were determined by deletion analysis. This together with results of oligonucleotide-directed mutagenesis of the promoter regions confirmed that the -35 and -10 sequences of the two promoters proposed previously are essential for their promoter activity. A G + C-rich sequence upstream of the - 35 region of P2 and an A-rich sequence upstream of the - 35 region of P3 facilitate the transcription from P2 and P3, respectively.

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A rapid, selective and sensitive method was developed for determining glyphosate and (aminomethyl)phosphonic acid (AMPA) by gas chromatography (GC). These compounds were converted into their N-isobutoxycarbonyl methyl ester derivatives and measured by GC with flame photometric detection, using a fused-silica capillary column with a cross-linked DB-1701. The calibration curves for glyphosate and AMPA in the range of 5-200 ng were linear, and the detection limits were about 10 and 15 pg as injection amounts, respectively. This method was applied to water and soil samples without a preliminary clean-up procedure, and glyphosate and AMPA were measured without any influence from coexisting substances. The recoveries of these compounds in potable water and river water samples were 96.2-100.3%, and those in soil samples were 81.7-99.1%.

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The effect of supplementing xylooligosaccharides (XO) to purified diets on growth performance and several metabolic parameters was examined in streptozotocin-induced diabetic rats. The dietary XO improved growth retardation, hyperphagia, polydipsia, elevation of serum glucose, triglyceride and cholesterol, reduction of liver triglyceride, and fatty acid composition (reduction of the desaturation index) of liver phosphatidylcholine. In addition, the XO diet yielded an acidic environment in the cecum by increasing the pool size of acetic acid. Thus, XO, which is half as sweet as sucrose, can be applicable to foods as a sweetener that is capable of improving diabetic symptoms.

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The primary structure of the ribosomal protein S7 from Bacillus stearothermophilus (BstS7) was analyzed by a combination of amino acid and DNA sequence analysis. Peptide sequence information was derived from tryptic peptides of BstS7 by manual Edman degradation. The nucleotide sequence of the 1.5-kb Sail fragment from B. stearothermophilm genome, cloned by the Escherichia coli S12 gene (rpsL) as a hybridization probe, was determined. Comparison of deduced amino acid sequence with the corresponding sequences of E. coli ribosomal proteins showed that this fragment contains the genes encoding S12, S7, and the N-terminus of the elongation factor G. Thus, the organization of this gene cluster is same as that in the str operon of E. coli. The amino acid sequence of B. stearothermophilus S12 (BstS12) deduced from the nucleotide sequence information agrees with the published amino acid sequence of BstS12 [M. Kimura and J. Kimura, FEES Lett., 210, 91 (1987)]. The deduced sequence of B. stearothermophilus S7 (BstS7), together with sequence information of tryptic peptides, showed that protein S7 consists of 155 amino acid residues with a calculated molecular weight of 17933 and has 56% amino acid identity with the E. coli S7 (EcoS7).

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Succinate-semialdehyde dehydrogenase (EC 1.2.1.16) was purified to apparent homogeneity from L-arginine-grown cells of Brevibacterium helvolum IFO 12073 (ATCC 11822). The molecular weight and the subunit molecular weight of the enzyme were approximately 250, 000 and 59, 000, respectively, suggesting that the enzyme is a tetramer of identical subunits. The apparent Michaelis constant (Km) for succinate-semialdehyde was approximately 30μM. The enzyme used both NAD⁺ and NADP⁺ as the coenzyme almost equally. The apparent Km values for NAD⁺ and NADP⁺ were 0.5mM and 0.15 HIM, respectively. The maximum reaction rate (V_max) of about 110μmol/min/nig was shown with NAD⁺ and a very close value was obtained with NADP⁺. Malonate-semialdehyde and other aldehydes tested were inert as substrates. The optimum pH was 9.0-9.5. The enzyme was sensitive to sulfhydryl reagents.

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The xylose isomerase gene from the thermophile Clostridium thermohydrosulfuricum has been cloned, using a fragment of the Bacillus subtilis gene as a probe. The complete nucleotide sequence of the gene was analyzed. C. thermohydrosulfuricum is the most thermophilic organism from which a xylose isomerase gene has been cloned and characterized. Comparison with amino acid sequences from other xylose isomerases showed that amino acitfs involved in substrate binding and isomerization are well conserved. Purification of the enzyme produced in E. coli was done by heating a cell-free extract at 85°C for 10 min, giving a 20-fold purified enzyme. The native enzyme is a homomeric tetramer with a molecular weight of 200, 000.

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The complete amino acid sequence of luffin-b has been determined. All the twenty-seven tryptic peptides were isolated by reverse-phase HPLC from the tryptic digests of intact luffin-b and one of its CNBr fragments (CB4), and sequenced using the DABITC/PITC double coupling method. The overlap of these peptides was achieved by analyzing the CNBr fragments and their chymotryptic peptides. Luffin-b consists of 250 amino acid residues with a relative molecular mass of 27, 275 Da. Investigation for glycosylation sites indicated that Asn at positions 2, 78, and 85 might carry sugars. Sequence comparison with luffin-a showed that amino acid substitution occured in 55 positions. Luffin-b contains three glycosylation sites instead of the six sites in luffin-a, of which two were found to be conserved.

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