The land snail, Dyakia striata, bioluminesces to exhibit green light. Its luminescence spectrum was recently reported by using juvenile snails to show the maximum at ca. 515 nm. In the current studies, a green fluorescent substance was extracted from the separated anterior head-foot of the adult snails, which contained the photogenic organs. The fluorescent substance, which showed the maximum ca. 515nm in its spectrum, was purified by chromatography. The partially purified green fluorescent substance was soluble only in organic solvents and not in water. The chromophore of the green fluorescent substance was compared with isoalloxazine and riboflavin acetate in different organic solvents under reductive and re-oxidative conditions.

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A 5.3-kbp fragment of the KHS gene was cloned from a genomic bank of Saccharomyces cerevisiae No. 115 constructed with an E. coli as the host and YEpl3 as the vector. A non-killer yeast strain was transformed to a killer strain with the multi-copy vector containing the KHS gene, and the transformant could secrete 3-4 times more killer toxin into culture media than the donor, strain No. 115. The KHS toxin was purified 80-fold from the culture filtrate by gel filtration and column chromatography. The nucleotide sequence of a 2.8-kbp fragment of the KHS DNA that was enough for the expression of the killer activity was identified, and we found an open reading frame consisted of 2124 bp. Comparison of the open reading frame and N-terminal amino acid sequence of purified KHS toxin, suggested that the presumed peptide from the KHS gene might be processed between ³⁶Gin and ³⁷Ala before secretion.

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An enzyme component that hydrolyzes pNP-G₂ but not CMC has been isolated from a culture broth of Cellvibno gilvus by a multi-step procedure involving Butyl-Toyopearl, DEAE-Toyopearl, and CM-Toyopearl chromatographies. The purified enzyme gave a single protein band on native, SDS-, and IEF-PAGE. The enzyme had a molecular weight of 40, 000, an isoelectric point of 5.0, an optimum pH of 6.5, and an optimum temperature of 55°C. It was stable from pH 4.0 to 9.0 at 37°C for 1 hr and below 50°C for 30min. It hydrolyzed agluconic bonds not only of pNP-G₂ but also of pNP-G₃, pNP-G₄, and pNP-G₅. Cellooligosaccharides with D.P. of 3 to 5 were not hydrolyzed at all. Instead, the enzyme hydrolyzed xylan 4 times as fast as pNP-G₂. Both HgCl₂ and p-chloromercuribenzoic acid inhibited the two activities completely. Xylan inhibited the hydrolysis of pNP-G₂ competitively. From these results, the purified enzyme was considered to be a unique xylanase that hydrolyzed the agluconic bonds of pNP-G_n.

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A Cellulomonas sp. isolated from soil produced a novel lyase that degraded the acidic polysaccharide of Fusarium sp. M7-1 with the formation of mannose and O-β-D-mannopyranosyl-(1→2)-D-mannose. DEAE-Toyopearl 650M column chromatography showed three lyase activity peaks (fractions I, II, and III). The major fraction was purified to homogeneity by polyacrylamide gel electrophoresis analysis, and its molecular weight was 74, 000. The optimum pH was 6.5 to 8.0 and the stable pH range was 6.0 to 8.0. The purified enzyme did not degrade glucuronic or galacturonic acid-containing polysaccharides such as chondroitin, hyaluronic acid, pectin, or pectic acid. However, the purified enzyme specifically degraded various Fusarium and Gibberella acidic polysaccharides, and unsaturated sugars were produced with the release of mannose and O-β-D-mannopyranosyl-(1→2)-D-mannose. These results suggest that the acidic polysaccharides derived from Fusarium and Gibberella have similar structures.

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Four mating-type monokaryons, A₁B₁, A₁B₂, A₂B₁, and A₂B₂, were screened from basidiospore clones of one Pleurotus salmoneostramineus fruit body. Auxotrophic strains were induced by irradiating the monokaryotic protoplasts with ultraviolet light, and they were mated with each other to obtain active basidiospore clones with appropriate combinations of auxotrophy and mating-type. The auxotrophic protoplasts were fused in polyethyleneglycol solution between the four kinds of mating-types, A≠B≠, A≠B=, A=B≠, and A=B=. The frequencies of fusant colony formation by the four kinds of combinations were all similar, independent of mating-type combinations. Heterokaryotic conformation of all four kinds of fusant mycelia were confirmed by the heterogeneity of nutritional requirements of the re-protoplasted clones of fusants. Prototrophy of A≠B≠ and A≠B= fusants was stable in a complete medium, but those of A=B≠ and A=B= fusants could be preserved only in a minimal medium. Clamp structures were observed in A≠B≠ and A≠B= fusants, but not in A=B≠ and A=B= fusants.

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N-(9-Acrydinyl)maleimide (NAM) fluorometry was developed to selectively determine oxidized glutathione (GSSG) in a mixture of glutathione (GSH) and GSSG. It was applied to the enzymatic determination of fatty acid hydroperoxide (HPO), using glutathione peroxidase (GPX): reduction of HPO with GSH by GPX; masking excess GSH with N-ethylmaleimide; reduction of the resulting GSSG with sodium borohydride to GSH; and fluorometric determination of GSH with NAM. A linear relationship was obtained in the range from 0.1 to 10nmol of HPO with a sample size of 0.2 to 20 mg. The method is specific to fatty acid HPO.

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N-(9-Acrydinyl)maleimide (NAM) fluorometry was applied to the enzymatic determination of mono-, di- and triacylglycerol hydroperoxides (HPOs) by glutathione peroxidase (GPX) and lipase. Lipase hydrolysis made it possible to determine the acylglycerol HPOs. Two enzyme reactions were conducted simultaneously in one pot for 4hr at 25°C in 0.1 M phosphate buffer of pH 7.5 in a nitrogen atmosphere in the dark. The released glutathione-SH was measured by NAM fluorometry, the calibration curve giving a linear relationship in the range from 0.1 to 10nmol of HPO with a sample size of 0.2 to 20 mg. The sensitivity was about 250 times higher than that by conventional iodometry.

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Glycation of proteins was induced by incubation of high concentrations of D-glucose (1 M) and proteins (4 mg/ml) at 40°C for 7 days. The oxidative damage of glycated protein mediated by addition of traces of metal ion in phosphate buffer (1/15 M, pH 7.2) under aerated conditions was investigated. Free amino groups (mainly ε-NH₂ groups of lysine residues) of protein decreased during the glycation of protein but were regenerated in the oxidation of glycated protein induced with metal ion. Also in this process, an a-dicarbonyl compound was generated. Glycated protein was degraded to lower molecular weight fragments in the presence of Cu(II) but not in the absence of Cu(II). Moreover, the histidine residue of protein was selectively degradated in this oxidative process. The oxidative fragmentations of glycated protein were inhibited by EDTA or catalase, but not by urea, DMSO (scavengers of hydroxy radicals) or superoxide dismutase. A possible mechanism of oxidative fragmentation of glycated protein catalyzed by the metal ion is discussed.

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An examination of amines as catalysts for the first-step phosphorylation of nucleosides by a two-step phosphorylating agent, 2-methylthio-4H-1, 3, 2-benzodioxaphosphorin 2-oxide (MTBO, 2), revealed that, in the presence of 4-morpholine-N, N'-dicyclohexylcarboxamidine (MDC, 3) as the catalyst, MTBO phosphorylates and o-hydroxybenzylates adenosine borate complex (1) to give 1-(o-hydroxybenzyl)adenosine 5'-S-methyl phosphorothiolate (5), with subsequent Dimroth rearrangement to give N⁶-(o-hydroxybenzyl)adenosine 5'-S-methyl phosphorothiolate (6).
The guanosine borate complex (7) gave 1-(o-hydroxybenzyl)guanosine 5'-S-methyl phosphorothiolate (8) under the same conditions.
MTBO phosphorylated N⁶-benzyladenosine and N⁶-allyladenosine borate complexes (9a and b) in the presence of n-propylamine (11) to give their 5'-S-methyl phosphorothiolates (10a and b), respectively.
Thus, the 5'-S-methyl phosphorothiolates of cytokinin-like nucleosides were synthesized by the use of different amine catalysts from both intact and N⁶-substituted nucleosides.

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The effects of chemical modification of ε-amino groups (using the water soluble polyanionic copolymer ethylene/maleic anhydride) on the structure and function of various aspartyl proteinases (the proteinase from the fungus Mucor miehei (MMP), and the mammalian proteinases porcine pepsin and chymosin) were examined. Modification of 100% of the lysyl residues in the proteinases resulted in an increased milk-clotting to proteolytic activity ratio (MC/PA) for pepsin and chymosin, and a lower MC/PA for MMP. Amino-modified MMP was insensitive to the proteinase inhibitor pepstatin, but modified pepsin and chymosin were inactivated. Modification shifted the pH-activity optimum of the mammalian enzymes by 0.5 pH units to a more alkaline pH and to a more acidic optimum for MMP. Amino modification also decreased the thermostability of the fungal enzyme. Changes in tertiary structure and significant differences in the proportions of the secondary structure fractions α-helix and β-sheet were evident only for modified MMP. Results from this study suggested that MMP was more susceptible to destabilization by charge alteration than aspartyl proteinases of mammalian origin.

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The effects of chemical modification of amino groups on the conformational stability of Mucor miehei proteinase (MMP), porcine pepsin, and chymosin, the latter two being from mammalian sources, were examined using kinetic and thermodynamic analyses. Modification-induced stability reduction of only MMP was observed as demonstrated by a higher rate of guanidine hydrochloride-induced denaturation, lower activation energy, decreased enthalpy of denaturation, and a substantially reduced free energy of denaturation. Results from this study corroborate those of a companion investigation [J. L. Smith et al., Agric. Biol. Chem., 55, 2009 (1991)] indicating that MMP was more susceptible to destabilization by charge alteration than aspartyl proteinases of mammalian origin.

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Hairy root clones of tomato (Lycopersicon esculentum) were obtained by inoculation of the stems of sterile plants with Agrobacterium rhizogenes. The transformed root cultures produced a potato cyst nematode hatching stimulus, which was detected both in the extract of the cultured roots and in th culture medium. The hatching stimulus was also produced by non-transformed tomato root cultures. Large portions of the hatching stimuli synthesized in the hairy and non-transformed roots were found to be released into the medium. Five independent clones of the tomato hairy root cultures displayed considerable variation in the production of stimulus.

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The structures of the N-linked oligosaccharides of momordin-a, which is a ribosome-inactivating protein from the seeds of Momovdica chamntia, were analyzed. First, the N-linked oligosaccharides of this glycoprotein were liberated by hydrazinolysis. After N-acetylation, the reducing ends of the oligosaccharides were coupled with 2-aminopyridine and the pyridylamino (PA-) derivatives were purified by gel filtration and high performance liquid chromatography (HPLC) on an ODS-silica column. Three kinds of oligosaccharide fractions were separated by HPLC. The structure of each oligosaccharide isolated was analyzed by a combination of sugar component analysis, exoglycosidase digestion, another kind of HPLC using an amide-silica column, and 500-MHz ¹H NMR spectroscopy. The structures of two main oligosaccharides were established to be:
Xylβ2Manβ4GlcNAcβ4GlcNAc_Fucβ3
and Xylβ2Manβ4GlcNAcβ4GlcNAc
These two oligosaccharides were the first examples having xylose (or fucose) but no a-mannosyl linkage among the N-linked oligosaccharides of glycoproteins from both animal and plant origins.

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Fermentation of Nigrospora sphaerica on shredded wheat medium generated the novel metabolite, epoxyexserohilone, a congener of the known phytotoxin, exserohilone. Single crystal X-ray diffraction determined the structure and absolute stereochemistry of this metabolite which had the molecular formula C₂₀H₂₂N₂O₆S₂, melting point 190-192°C, and an EI-MS identical with its congener exserohilone. The metabolite did not exhibit biological activity in an etiolated wheat coleoptile bioassay, neither was it active against Gram-positive or Gram-negative bacteria.

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NADPH-Linked L-sorbose reductase from the cytosol fraction of Gluconobacter melanogenus N44-1, which is a 2-keto-L-gulonic acid producer from L-sorbose or D-sorbitol, was purified about 500-fold by DEAE-Cellulose, Blue Sepharose, and DEAE-Sepharose column chromatographies. The purified enzyme was entirely homogeneous on polyacrylamide gel and SDS gel electrophoresis. NADPH and NADP were specifically required for the reduction and oxidation of the substrate, respectively. The apparent molecular weight was 60, 000 by Sephadex G-200 column chromatography, and that of its subunit was 60, 000 by SDS-gel electrophoresis. The pH optimum for the reduction of L-sorbose and D-fructose was 7.0. The pH optimum for the oxidation of D-sorbitol to L-sorbose and D-mannitol to D-fructose was 10.0-10.5.

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Lactobacillus acidophilm strain TK8912 was found to carry six pi asm ids having molecular masses of 40, 17, 10.5, 5, 2, and 1.8 megadaltons (Mdal). An acriflavin-induced, Lac^- mutant had lost a 17-Mdal pi asm id (pLA102) and phospho-β-galactosidase (P-β-gal) activity. Since strain TK1-2TL (Lac⁺ transformant) restored P-β-gal activity, pLA102 is likely to encode a P-β-gal gene required for lactose metabolism in strain TK8912. It is also suggested that the gene for the tagatose 6-phosphate pathway, which is responsible for galactose metabolism, is encoded by the 40-Mdal plasmid pLA101.

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A previous report [M. Hirose, Y. Nishizawa and J. Y. Lee, J. Food Sci., 55, 915 (1990)] has shown that the thio-dependent gelation of bovine serum albumin was strongly inhibited by high concentrations of salts. The inhibition mechanism was investigated in relation to the conformation and disulfide cleavage of the protein. The effects on the thiol-dependent disulfide cleavage of the protein varied with the salt species, especially with the anion species. The circular dichroism spectra and difference UV-spectra showed that anions induced some conformational changes in the tertiary structure, but not in the secondary structure. With regard to a chaotropic anion, perchlorate, this conformational change was closely correlated with the inhibition of gelation and disulfide reduction. In contrast, a lyotropic anion such as sulfate inhibited the gelation at a higher concentration than the concentrations that induced the conformational change. In addition, this anion showed no inhibition effect on disulfide reduction. Thus, we conclude that there are two inhibition sites for the thiol-dependent gelation of BSA. One involves the reductive cleavage of disulfide bonds, and the other probably concerns intermolecular interaction of the disulfide-reduced protein.

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The sugar-yeast (SY) addition method, which was developed for the production of fermented protein foods, was applied to the preservation of raw fish and meat and compared with the NaCl and ethanol addition methods. The preservation mixture, consisting of minced raw meat of fish or livestock, 10% glucose, and 10⁶ yeast cells/g, was incubated at 12°C. Glucose was consumed quickly and about 2-4% ethanol was produced after 3-5 days of preservation. About 10⁵ cells/g of contaminating bacteria present in the preservation mixture increased to about 10⁶ cells/g after 3 day of preservation, and then the cell number of bacteria showed a tendency to decrease. The preservation mixture did not putrefy. The amount of eicosapentaenoic acid contained in sardines was preserved effectively by the SY addition method and by the ethanol addition method, but not well by the NaCl addition method. The freshness (K value) of sardines preserved by the SY addition method was maintained at the same level as that using the NaCl method. However, deterioration of freshness was observed in the ethanol addition method. The water holding capacity (WHC) of the NaCl addition method decreased, but WHC was well maintained by the SY and ethanol addition methods.

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Reaction velocities of two reactions of the Maillard reaction, the condensation reaction of amino compounds with carbonyl compounds and the successive browning reaction along with melanoidin formation, were measured under 50-500 MPa at 50°C. The condensation reactions of glyceraldehyde with di- or tri-peptides were little affected by the applied hydrostatic pressure, and the reactions of glyceraldehyde, glycolaldehyde, or xylose with amino acids were suppressed very weakly by the pressure. On the contrary, the browning reactions were significantly suppressed by the pressure at 50-200 MPa with the activation volume of 13-27 ml/mol. These results indicate that the high pressure suppresses the browning process rather than the initial condensation process in the total course of the Maillard reaction.

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A transfer reaction catalyzed by an exo-β-1, 4-galactanase from Bacillus subtilis was studied. The enzyme had a broad acceptor specificity and transferred galactobiosyl residues to acceptors such as various alcohols, including hydroxy benzenes and saccharides. Transfer products of glycerol formed by the enzyme were compared with those formed by Escherichia coli β-galactosidase and by Penicillium citnnum endo-galactanase. E. coli enzyme transferred 90% of galactose residues to the primary hydroxyl groups of glycerol and P. citnnum endo-enzyme transferred 80% of saccharide residues to the secondary hydroxyl group. The B. subtilis exo-galactanase was less specific than the other two enzymes and formed two products (1-DG and 2-DG) with a 2-DG/l-DG ratio of about 2. The structures of the saccharides were examined by ¹³C-nuclear magnetic resonance analysis and by enzymatic hydrolysis. 1-DG and 2-DG were elucidated to be O-β-D-galactosyl-(1→4)-O-β-D-galactosyl-(1→1)-glycerol and O-β-D-galactosyl-(1→4)-O-β-D-galactosyl-(1→2)-glycerol, respectively. The efficiency of the transfer reaction was measured at various concentrations of glycerol using galactotriose as a donor. About 40-75% of galactobiosyl residues were transferred at an acceptor concentration range of 20-100 mg/ml.

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Glycosides, the reducing ends of which were glycerol and 2-ethyl-2-(hydroxymethyl)-1, 3-propanediol (trimethylolpropane, TMP), were esterified with oleic acid (OA) by a lipase from Candida cylindmcea. Two glycosides, 1-Oβ-D-galactosyl glycerol (Gal-1-Gl) and O-α-D-glucosyl TMP (Glc-TMP), were enzymatically synthesized by transglycosylation. Glc-TMP was esterified at a higher water content than Gal-1-GI. When an equal amount of the glycosides and OA were used as substrates, almost 20% and 50% of OA were consumed for the esterification of Gal-1-GI and Glc-TMP, respectively. Laurie, capric, and caprylic acids also gave esters. Oleic ester synthesis was compared among several related glycosides. The enzyme esterified the glycosides with a TMP moiety better than those with a glycerol moiety. The reaction was scarcely affected not only by the kind of monosaccharide moieties but also by anomeric linkage types between the moieties.

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A hydrogen sulfide: ferric ion oxidoreductase (SFORase), which is crucial in sulfur oxidation of T. ferrooxidans AP19-3, oxidizes hydrogen sulfide with Fe³⁺ as an electron acceptor to give sulfite ion and Fe²⁺. When washed intact cells of T. ferrooxidans AP19-3 were treated with hydrogen sulfide solution (100 mM) at pH 6.5 for 1 hr, a heat stable reduced sulfur containing protein, which serves as a reduced sulfur compound for purified SFORase to give sulfite, was formed in the plasma membrane of this strain. The protein, which can bind hydrogen sulfide reversibly, was solubilized from plasma membrane with 1% SDS and purified to an electrophoretically homogeneous state. We tentatively named the protein hydrogen sulfide-binding protein (SBP). SBP had an apparent molecular weight of 16, 000 in the presence of 1% SDS. One molecule of SBP had 4.5 molecules of iron and could bind 2.3 molecules of hydrogen sulfide. The hydrogen sulfide bound to SBP was oxidized by a purified SFORase to give sulfite ion, suggesting that SBP is involved in sulfur oxidation of this bacterium.

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The neutral protease of Bacillus subtilis var. amylosacchariticus was photooxidized in the presence of methylene blue, by which treatment the enzyme was rapidly inactivated. The inactive enzyme was digested with endoproteinase Asp-N, the resultant peptides were separated by HPLC, and their amino acid sequences were compared with those obtained from the unmodified enzyme. Of four peptides that contained histidine residues, only the recovery of one peptide was found to be decreased by the photooxidation with the appearance of a new peptide. Comparisons of amino acid compositions and sequences between these two peptides showed that the latter peptide lacked His²²⁸ of the former one, indicating that His²²⁸ was photooxidized. This result suggests that His²²⁸ is involved in the catalytic reaction of the neutral protease or interaction with substrates.

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Crystalline polymorphs of four glucomannans with a different mannose/glucose ratio from 1.5 to 4.0 and their acetyl derivatives was studied by X-ray diffraction measurements. The glucomannans showed an amorphous or similar crystal structure (Mannan I or Mannan II polymorph) of β-1, 4-D-mannan depending on their mannose content, indicating the occurrence of isomorphous replacement of mannose by glucose during glucomannan crystallization. When these glucomannans were annealed, however, Konjac glucomannan (mannose/glucose ratio = 1.6) showed a different powder pattern from both Mannans I and II. The fiber pattern of the annealed Konjac glucomannan indicated that it took an extended two-fold helical structure. Among the acetylated glucomannans, Higanbana glucomannan with a high mannose content (mannose/glucose = 4.0) showed a similar diffraction pattern to that of β-1, 4-D-mannan triacetate, indicating that the isomorphous replacement also occurred in the acetyl derivative. However, glucomannan triacetate with a low mannose content (1.5 or 1.6) showed a different crystalline form from that of mannan triacetate. The fiber diffraction pattern of Konjac glucomannan acetate suggested that it took a three-fold helix.

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Mutants resistant to L-ethionine, an analogue of methionine, were derived from Zygosaccharomyces rouxii MAr1. Some of them produced large amounts of 3-(methylthio)-1-propanol (methionol); an important flavor component of soy sauce. One of these mutants, M33, produced 60-fold as much methionol as the parental strain MAr1 in a minimum medium without methionine. The intracellular methionine pool of this mutant was increased 5-fold. S-Adenosylmethionine (SAM) synthase (EC 2.5, 1.6) in the mutant was reduced to 45% of that in the parent and a decrease in intracellular SAM level was also confirmed. The reduction of the SAM level due to the deficiency of SAM synthase, accompanied by derepression of enzymes of the methionine synthetic pathway, was considered to be responsible for the hyper-production of methionol in the mutant.

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2-Acetylpyrrole, which was isolated from a culture broth of Streptomyces sp. A-5071, was found to protect primary cultured rat hepatocytes against D-galactosamine (GalN) cytotoxicity. This compound was also effective against GalN-induced liver injury in rats and against carbon tetrachloride (CCl₄)-induced liver injury in mice.

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The trisaccharide segment of the phenolic glycolipid (PGL) of Mycobacterium tuberculosis, 2-O-methyl-3-O-[3-O-(2, 3, 4-tri-O-methyl-α-L-fucopyranosyl)-α-L-rhamnopyranosyl]-α-L-rhamnopyranose, was synthesized in the form of the p-(2-methoxycarbonylethyl)phenyI glycoside by a stepwise condensation. 2, 4-Di-O-benzyl-3-O-acetyl-α-L-rhamnopyranosyl chloride was coupled to p-(2-methoxycarbonylethyl)phenyl 4-O-benzyl-2-O-methyl-α-L-rhamnopyranoside in the presence of silver triflate, and 2, 3, 4-tri-O-methyl-α-L-rhamnopyranosyl chloride was then coupled to the deacetylated disaccharide by the same procedure. The trisaccharide was deblocked and coupled to BSA, giving the neoglycoconjugate TB-NT-P-BSA. TB-NT-P-BSA showed its possibility as a useful tool for the serodiagnosis of tuberculosis.

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The chromatographic behaviors of proteins and amino acids on a gel filtration matrix, TSK-GEL Toyopearl have been examined, and the effects interfering with their elution from Toyopearl gel were analyzed individually. Many proteins are retarded in elution to various extents in 25 mM Tris-HCl buffer at pH 7.5. All the proteins examined of isoelectric points (pI) higher than the pH value of elution buffer (pH 7.5) are retarded in comparison with the elution of the proteins of pI less than 7.5. The retardation can be diminished almost to nothing by the addition of 0.3-0.5 M NaCl, suggesting that the electrostatic interaction works between the proteins and gel matrix. On the other hand, the adsorption of some proteins can be reduced by the addition of 30% ethanol (v/v) to the eluent. These proteins were supposed to be adsorbed to the gel matrix by hydrophobic interaction. Aromatic amino acids such as tryptophan and tyrosine are adsorbed to the gel matrix strongly, but no aliphatic hydrophobic amino acids (e. g.., leucine and isoleucine) and no charged amino acids (e. g., lysine, arginine, aspartic acid and glutamic acid) are adsorbed to the matrix at all. By considering the effects interfering with the elution of proteins and amino acids from Toyopearl gel, it is possible to separate them more effectively by the subtle differences in their properties by changing pH, ionic strength, and dielectric constant of the elution buffer.

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