A sheet structure was prepared with the photoreaction unit (PRU), a complex of the reaction center and the light-harvesting chlorophyll proteins from Rhodopseudomonas viridis, by the dialysis method. The sheet had properties characteristic of a three-dimensional thin crystal. The absorption spectrum and the protein subunit composition of PRU were maintained during the sheet formation. Electron microscopy observation found that PRU molecules are regularly arrayed and stacked in the sheet. The sheet showed linear dichroism in the absorption spectrum, indicating also that chromophores in the sheet were arranged with orientation.

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Two pyridoxine compounds were found to be formed in a culture filtrate of Aspergillus niger and A. sydowi, when grown in a medium containing sucrose and pyridoxine. Each of the two compounds I and II was obtained as a white powdered preparation by preparative paper chromatography, gel filtration on Toyopearl HW-40S and Sephadex G-10 columns, DEAE-cellulose column chromatography, and lyophilization. Compounds I and II were identified as 5'-O-(β-D-fructofuranosyl)-pyridoxine and 5'-O-[β-D-fructofuranosyl-(2→1)-β-D-fructofuranosyl]-pyridoxine, on the basis of the various experimental results, viz., elementary analyses, UV, ¹H-, and ¹³C-NMR spectra, products by hydrolysis with acid and yeast β-D-fructofuranosidase, migration on paper electrophoresis, and Gibbs reaction in the presence and absence of boric acid. Levansucrase from Microbacterium laevaniformans and yeast β-D-fructofuranosidase did not catalyze the β-D-fructofuranosyl transfer from sucrose to pyridoxine to give rise to β-D-fructofuranosyl-pyridoxine.

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The reaction between dextran-dialdehyde, prepared by periodate oxidation, and glycine was examined in detail. In contrast to that of aldose with amino acids, the dialdehyde reaction proceeded very rapidly. Higher temperatures and pH were favorable for the reaction and the initial velocity was proportional to the reaction time and the concentration of dextran-dialdehyde. Although the dextran-glycine adduct was stable during the gel filtration step to remove excess glycine, the adduct was readily dissociated by 0.3M HCl. Among the amino acids tested, histidine and glycine gave higher reactivity than lysine and arginine. This basic information is useful for condensation of dextran-dialdehyde with various proteins and enzymes.

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Two haedoxan-analogs, (±)-(1S^*, 2R^*, 5R^*, 6S^*)-2-(2, 6-dimethoxyphenoxy)-1-hydroxy-6-[(±)-(2S^*, 3R^*)-7/6-methoxy-3/2-methoxymethyl-2/3-(3, 4-methylenedioxyphenyl)-3, 4-dihydro-2H-chromen-6/7-yl]-3, 7-dioxabicyclo[3.3.0]octanes, were synthesized. They both had chromanyl groups instead of the (2R, 3R)-6-methoxy-2-methoxymethyl-3-(3, 4-methylenedioxyphenyl)-1, 4-benzodioxan-7-yl group of the haedoxan molecule. In the synthesis, a (±)-(1S^*, 2R^*, 5R^*, 6R^*)-isomer was obtained. An assay of these chromans indicated that the 1-oxygen atom of the 1, 4-benzodioxane ring of haedoxans significantly contributed to their insecticidal activity.

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Cultured mouse fibroblasts synthesize and secrete nerve growth factor (NGF). To learn the mechanism of action of an iron-binding protein, lactoferrin (Lf), on cultured animal cells, we have examined the effects of bovine Lf (bLf) on NGF synthesis/secretion in mouse L-M cells, a line derived from L929 fibroblast cells. Both apo- and holo-bLf induced an increase in NGF content in the cell-conditioned medium (CM) with similar effectiveness. Neither apo- nor holo- bovine transferrin (bTf) was effective, indicating that the observed induction of NGF production cannot be attributed to stimulation of iron transport. A basic peptide fragment of bLf (residues 17-41) isolated from a pepsin hydrolyzate of bLf was found to be effective for stimulation of NGF synthesis/secretion in L-M cells. This observation demonstrates that the basic nature of Lf is important for its interaction with fibroblast cells.

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A systematic method of extraction, fractionation, and purification of polysaccharides from Songshan Lingzhi (Ganoderma tsugae) with antitumor activity was established. Seven glycans with strong antitumor activities were obtained from 14 water-soluble, and 15 water-insoluble fractions: FI_o-a, FA-1, FII-1, FIII-2, and FIII-2-a, -b, and -c. FI_o-a and FA-1 were protein-containing glucogalactans associated with mannose and fucose. FII-1 was a (1→3)-β-D-glucan having a lower protein content. The water-insoluble fractions FIII-2-a, -b, and -c were extracted with alkali, and were found to be protein-containing (1→3)-β-D-glucans showing the strongest activity. Chemical properties and structure of each antitumor polysaccharide were compared with three fungi of the Ganoderma family, Kofukitake (G. applanatum), Mannentake (G. lucidum), and Songshan Lingzhi (G. tsugae).

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Protein-containing polysaccharides extracted from fruiting bodies of a Chinese fungus named Feng Wei Gu, were fractionated and purified, and their antitumor activities were tested, out of which the following active fractions were obtained. FI_o-a: A protein-containing xyloglucan, MW 280, 000, polysaccharide : protein=76 : 24 (w/w), polysaccharide consisting of Man : Gal : Xyl : Glc=2 : 12 : 42 : 42 (molar ratio). [α]²³_D +25.3°. FA-2: A protein-containing mannogalactan, MW 120, 000, polysaccharide : protein=76 : 16 (w/w), consisting of Xyl : Man : Gal=9 : 35 : 56 (molar ratio), [α]²³_D +98.5°. FII-1: A Protein-containing xylan (62 : 21 w/w). MW 200, 000, [α]²³_D +8.7°. FIII-1a: A protein-containing glucoxylan (15 : 71 w/w), [α]²³_D +30.7°, MW 90, 000, consisting of Glc : Xyl=40 : 44 (molar ratio). FIII-2a: A protein-containing xyloglucan, MW 70, 000, polysaccharide : protein=69 : 3 (w/w), polysaccharide consisting of Xyl : Glc=36 : 62 (molar ratio). [α]²³_D +38.6°.

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Stability of tea catechins during heat processing was examined. Effects of pH and temperature on reaction kinetics of degradation of tea catechins were investigated. Reaction of -EC was accelerated at pH higher than 6.0, inhibited at those lower than 5.0. A dominant reaction of -EC in slightly acidic media was isomerization. The apparent first order reaction proceeded in slightly acidic media under sterilization conditions. A similar reaction proceeded for -EC, -EGC, -ECg, and -EGCg in green tea infusion at temperatures below 95°C. A turning point temperature on an Arrhenius plot was observed at 82°C. A great difference of apparent activation energies was observed lower and higher than 82°C. The reaction rate constant of catechins in slightly acidified tea infusion was less than half as much as the constant in the original infusion. The stability of catechins in green tea infusion was subject to the turning point temperature.

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This study aims to improve the stickiness and brightness of cooked rice from aged rice grains by removing its albumins and globulins with proteases. In newly-harvested grains, the proteins were easily removed by extraction with water which contained minerals from rice. In aged grains, however, the proteins were already denatured and extracted with difficulty. Proteases hydrolyzed even the denatured proteins to make them extractable. As a result, starch granules on the surface of grains were liberated. Some proteases also hydrolyzed a starch granule-associated protein, and consequently, made the starch highly gelatinizable by heating. The protease treatment of aged grains was effective for two main reasons: liberation of starch granules and removal of the granule-associated protein. During cooking of rice the liberated starch without the associated proteins gelatinized to give rise to stickiness. The gelatinized starch is considered to form a more isotropic film on the surface of every cooked rice grain to give brightness as well.

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We expressed human macrophage colony-stimulating factor (M-CSF) in Chinese hamster ovary (CHO) cells by introducing an expression plasmid coding for a 554-amino-acid M-CSF precursor and dihydrofolate reductase (DHFR) gene, and by amplifying the sequence. A cell line was obtained that secreted approximately 200, 000 units/ml after 6 days in culture. The expressed recombinant human M-CSF (rhM-CSF) primarily consisted of two molecular species, a main 80-90kD M-CSF as a homodimer and a molecular form higher than 150kD. Purification of a main rhM-CSF gave an apparently homogeneous protein disulfide-bonded from 42-kD subunits, but one of the purified rhM-CSFs was composed of two subunit species with molecular masses of 44 and 42kD. These purified rhM-CSFs had substantially the same specific activity (1 to 4×10⁷ units/mg protein). Deglycosylation experiments with the latter rhM-CSF using chemical (trifluoromethanesulfonic acid) and enzymatic methods found a terminal neuraminic acid in addition to N- and O-glycosylation, but the two subunit species did not coalesce into a single molecule.

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Hydrolyzates which inhibit the angiotensin I-converting enzyme (ACE) were prepared from sardine muscle by Bacillus licheniformis alkaline protease. Considering the practical application of preparations as a functional food material, the best proteolytic conditions with respect to taste, solubility and ACE inhibitory activity were a 0.3 wt% addition of the enzyme and 17-h proteolysis at 50°C and pH 9.0. The preparations under these conditions had potent activity (IC₅₀=0.26 mg protein/ml). Fractionation of the preparations on an ODS column with ethanol resulted in the production of more potent inhibitors. The most potent activity was obtained when eluting with 10% ethanol (IC₅₀=0.015 mg protein/ml). This fraction was apparently rich in acidic amino acids, poor in hydrophobic ones, and effective for use as a physiologically functional food material by virtue of little bitterness, a fish odor and powerful ACE inhibitory activity.

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From the genomic library of Pseudomonas paucimobilis TMY1009 constructed with the cosmid pHC79, an 8-kb BamHI-KphI fragment encoding lignostilbene-α, β-dioxygenase (LSD) was cloned into pUC19 (designated pKSN2510). E. coli JM109 having pKSN2510 produced a small amount of LSD only when the lac promoter was induced by isopropyl-β-D-thio-galactopyranoside (IPTG). The 1.9-kb SalI fragment containing the LSD gene on pKSN2510 was subcloned into pUC119 in opposite directions (designated pKHN2560 and pKHN2590). The LSD gene on pKHN2590 was expressed in E. coli MV1184 using the lac promoter. LSD produced by E. coli MV1184 transformed with pKHN2590 (cloned LSD) was purified and found to be identical with LSD-I, which was the major isozyme produced by P. paucimobilis TMY1009. Cloned 1.9-kb nucleotide was sequenced and one open reading frame composed to 486 codons was found.

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Three isozymes of lignostilbene-α, β-dioxygenase (LSD) from Pseudomonas paucimobilis TMY1009 were separated on QAE-Toyopearl chromatography. All active fractions were further chromatographed on DEAE-Toyopearl, Butyl-Toyopearl, and Sephacryl S-300 columns. Then the isozymes I, II, and III were purified homogeneously. All three isozymes consisted of two subunits with the same mol. mass. According to the N-terminal amino acid sequences up to 25 residues of these three isozymes and the reversed-phase HPLC patterns of peptidase-digested them, it was found that LSD-I, II, and III consisted of αα, αβ, and ββ subunits, respectively. They showed different specificities for several substrates that are stilbene and styrene derivatives.

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An antioxidative substance produced by Aspergillus sojae K is absolutely soluble in water and strongly inhibits autoxidation of Na-ascorbate. The substance, produced extracellularly in the culture fluid by the mold, was purified by ion exchange chromatography, gel filtration, and HPLC. The substance was purified 34-fold with an activity recovery of 38% from culture fluid. Purity of the substance was confirmed with a single peak through HPLC using an analytical column as well as a single spot on TLC. The purified substance consists of equimolar aspartic acid and glycine, indicating that the substance is a peptide. From mass spectral analysis the molecular weight was 710, but the precise sequence of the amino acids is not clear. The substance is stable at 70°C, but about 80% of the activity was lost at 80°C after 60 min. Besides, the substance is completely stable at pH 3-14. This substance efficiently suppressed the oxidation of fish oil.

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Our previous study [Takahashi et al., J. Biochem., 109, 846-851 (1991)] has shown that the disulfide-reduced form of ovalbumin was proteolyzed by subtilisin into three major fragments. It was investigated whether or not these three fragments would be folded into one molecule. Gel permeation and ion-exchange chromatography indicated that the three fragments were eluted in a single peak. The proteolyzed protein had a CD spectrum that was almost indistinguishable from the disulfide-reduced, non-proteolyzed form of ovalbumin. Differential scanning calorimetry, however, revealed that the proteolyzed ovalbumin was denatured at a lower temperature than that of the disulfide-reduced, non-proteolyzed protein. Thus, it is concluded that the three fragments were folded into a native-like conformation with decreased stability. Chemical analyses of the fragments purified by reverse-phase HPLC revealed that there was a cleavage site in the disulfide-reduced form of ovalbumin, at least at the amino-terminal side of Cys⁷³, in addition to the well-known cleavage sites in plakalbumin.

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Glutamate dehydrogenase (L-glutamate: NADP oxidoreductase, deaminating, EC 1.4.1.4) from the hyperthermophilic archaebacteria Pyrococcus woesei and P. furiosus were purified to homogeneity from crude extracts. The enzymes had similar enzymological properties: molecular mass, subunit structure, optimum pHs for the oxidative deamination and reductive amination, substrate specificity and coenzyme specificity as well as thermostability; the activity was not lost after incubation at 105°C for 30 min. However, some differences were detected in resistance to urea denaturation and effects of salts on their activity and stability. The N-terminal 20 amino acid sequences of the two enzymes were identical.

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Bovine serum albumin (BSA) was hydrolyzed with proteases, and the emulsifying and oil-binding properties of the hydrolyzates were then studied. By limiting hydrolysis with trypsin, the emulsifying activity index (EAI) increased about 40% above its original level. In order to find out which peptides contributed to the good emulsifying properties of the hydrolyzate, the peptides adsorbed onto the emulsified oil globule surface were extracted and analyzed. SDS-polyacrylamide gel electrophoresis showed that a peptide with a molecular weight of about 24 kDa was preferentially adsorbed onto the oil surface. This 24 kDa peptide was found to be fairly hydrophobic and corresponds to the third domain of BSA, residues 377-582. In spite of the preferential adsorption characteristics of the 24 kDa peptide, this peptide itself had a lower EAI value than that of the whole hydrolyzate. The contribution of some small hydrophilic peptides, as well as the 24 kDa peptide, to the good emulsifying properties of the BSA hydrolyzate was suggested.

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Pectate lyase was purified approximately 29-fold to electrophoretic homogeneity from Pseudomonas marginalis N6301. A pectate lyase (PL; EC4.2.2.2) gene of the strain was cloned and expressed in Escherichia coli. The nucleotides of the PL gene (pel) were sequenced. An open reading frame that encodes a polypeptide (molecular weight: 40, 812) composed of 380 amino acids including a 29 amino acid signal peptide was assigned. The structural gene of pel consisted of 1140 base pairs. The nucleotide sequence of the 5'-flanking region of pel showed a consensus sequence of the promoter region of the pectin lyase gene (pnl) in P. marginalis N6301, a Pribnow box, and a ribosome binding site as found in E. coli.

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Two inhibitors of chitin synthase were isolated from submerged cultures of an osmophilic Aspergillus fumigatus strain and identified as pseurotin A (1) and its 8-O-demethyl derivative (2). The two compounds inhibited both the solubilized and the membrane-bound forms of the enzyme, but do not have antifungal or antibacterial activities. The IC₅₀ of pseurotin A was 81 μM for the solubilized enzyme. 8-O-Demethylpseurotin was less active with an IC₅₀ of 192 μM. The structures of the compounds were elucidated by spectroscopical methods.

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β-N-Acetyl-D-hexosaminidase was isolated from the mid-gut gland of Patinopecten yessoensis. The enzyme was purified by making an acetone-dried preparation of the mid-gut gland, extracting with 50 mM citrate-phosphate buffer (pH 4.0) (about 13% of the extracted proteins was β-N-acetyl-D-hexosaminidase), ammonium sulfate fractionation, and column chromatographies on CM-Sepharose and DEAE-Sepharose. The purified β-N-acetyl-D-hexosaminidase was homogeneous on SDS-PAGE, and sufficiently free from other exo-type glycosidases. The molecular weight was 56, 000 by SDS-PAGE. The enzyme hydrolyzed both p-nitrophenyl β-N-acetyl-D-glucosaminide and p-nitrophenyl β-N-acetyl-D-galactosaminide. For p-nitrophenyl β-N-acetyl-D-glucosaminide, the pH optimum was 3.7, the optimum temperature was 45°C, and the K_m was 0.24 mM. For p-nitrophenyl β-N-acetyl-D-galactosaminide, these were pH 3.4, 45°C, and 0.15 mM, respectively. The enzyme liberated non-reducing terminal β-linked N-acetyl-D-glucosamine or N-acetyl-D-galactosamine from various 2-aminopyridyl derivatives of oligosaccharides of N-glycan or glycolipid type except of G_M2-tetrasaccharide. As the enzyme was stable around pH 3.5-5.5, it may be useful for long time reactions around the optimum pH.

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In the course of our screening for new immunomodulators, a novel compound, melastin, was purified from the culture broth of Streptomyces. Melastin was purified through adsorption to Diaion HP-20, ethanol precipitation, and anion exchange column as a brown powder. The molecular weight was estimated as 5000±3000 by gel filtration HPLC. Melastin suppressed lipopolysaccharide (LPS)-induced blastogenesis of B cells more profoundly than concanavalin A (con A)- or phytohemagglutinin (PHA)-induced blastogenesis of T cells. Moreover, it selectively inhibited the growth of several leukemia cells as compared with interleukin-dependent nontransformed leukocytes. No selectivity was observed between nontransformed fibroblasts and their oncogene-transformed variants. Melastin did not selectively inhibit macromolecule synthesis of leukemia cells.

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Several properties of ATPase bound to the inner membrane of a psychrophilic marine bacterium Vibrio sp. strain ABE-1 were examined. The membrane-bound ATPase had two optimal peaks of the activity at pH 5.8 and 7.3. The ATPase activity was strongly inhibited by N, N'-dicyclohexylcarbodiimide (DCCD) and NaN₃ at pH 5.8 and 8.0, and stimulated by MgCl₂ and CaCl₂ at pH 8.0. At pH 8.0, the enzyme hydrolyzed GTP and ITP as well as ATP but not AMP or p-nitrophenylphosphate. CTP, UTP, and ADP were poor substrates. These characteristics indicate that there is a F₀F₁-type ATPase in the inner membrane of this bacterium. In addition, the ATPase activity was also significantly inhibited by Na₃VO₄, suggesting the coexistence of a P-type ATPase as a minor constituent. The membrane-bound ATPase activity was maximum at 50°C, but the strong DCCD-sensitivity observed at 20°C was greatly reduced at this temperature.

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Pharmacological studies on the antihypertensive effect of a polysaccharide-glycopeptide complex (SG-1) isolated from Lactobacillus casei were carried out by using spontaneously hypertensive rats (SHR). An antihypertensive effect of SG-1 was observed by oral, but not by intravenous or intraperitoneal administration, and the effect was attenuated by orally pre-treating with indomethacin. A single oral administration of SG-1 (20 mg/kg) decreased the peripheral vascular resistance (PR). The daily oral administration of SG-1 (10 mg/kg) for 14 days had no effect on either the urine volume or urinary electrolytes (Na⁺, K⁺, and Cl^-), but it did increase the excretion of 6-keto-PGF₁α, a metabolite of PGI₂, in the urine. Moreover, a single oral administration of SG-1 (20 mg/kg) also increased the biliary 6-keto-PGF_1α excretion. These results suggest that the antihypertensive effect of orally administered SG-1 resulted from an enhancement of PGI₂ biosynthesis and the subsequent decrease in PR.

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A bacterial strain, NS671, which converts DL-5-(2-methylthioethyl)hydantoin stereospecifically to L-methionine, was isolated from soil and was classified into the genus Pseudomonas. With growing cells of Pseudomonas sp. strain NS671, DL-5-(2-methylthioethyl)hydantoin was effectively converted to L-methionine. Under adequate conditions, 34 g of L-methionine per liter was produced with a molar yield of 93% from _DL-5-(2-methylthioethyl)hydantoin added successively. In addition to L-methionine, other amino acids such as L-valine, L-leucine, L-isoleucine, and L-phenylalanine were also produced from the corresponding 5-substituted hydantoins, but these L-amino acids produced were partially consumed by strain NS671. The hydantoinase, by which 5-substituted hydantoin rings are opened, was ATP-dependent. The N-carbamyl-amino acid amidohydrolase was found to be strictly L-specific, and its activity was inhibited by high concentration of ATP.

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2-Chloromethyl-5H/methyl benzimidazoles were condensed with different 2-substituted 5-mercapto-1, 3, 4-oxadiazoles and thiadiazoles, and 4-amino 2-substituted 5-mercapto-1, 2, 4-triazoles. The synthesized compounds were characterized and evaluated for their fungicidal activities, and few were found to be better fungicides than those commercially used.

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The synthesis of (-)-muricatacin starting from 1-bromododecane and 2-pentyn-1-ol is described. 2-Pentadecyn-1-ol (4), which was prepared from 1-bromododecane (2) and 2-pentyn-1-ol (3), was converted to epoxy alcohol 6 through a two-step reaction sequence, 6 being successively submitted to tosylation, iodination, chain extension with tert-butyl lithioacetate, and acid-catalyzed cyclization to give (-)-muricatacin (1a). Recrystallization afforded optically pure 1a.

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The soybean allergenic protein, Gly m Bd 30K [Ogawa et al., J. Nutr. Sci. Vitamihol., 37, 555-565(1991)] which is most strongly and frequently recognized by the IgE antibodies in sera of soybean-sensitive patients with atopic dermatitis, has been characterized. The allergen was isolated from the crude 7S-globulin fraction as an oligomeric form with a molecular weight of more than 3000, 000 by gel-filtration chromatography. On two-dimensional gel electrophoresis, the native oligomeric allergen had an isoelectric point of about pH 4.5 and was dissociated into a monomeric form with a molecular weight of about 32, 000 by the treatment with sodium dodecyl sulfate and 2-mercaptoethanol. The monomeric allergen had an N-terminal amino acid sequence and amino acid composition identical with those of the soybean seed 34-kDa oil-body-associated protein or the soybean vacuolar protein P34 with close homology to papain-like thiol proteinases [Kalinski et al., J. Biol. Chem., 267, 12068 (1992)]. The identity was further confirmed by the immunological cross-reactivity to the antibodies produced against each of the purified allargen and the 34-kDa oil-body-associated protein. By this observation, Gly m Bd 30K was shown to have about 30% sequence homology with Der pI, a house dust mite allergen that is a thiol proteinase from Dermatophagoides pteronyssius.

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Peroxidase (POD) plays an important role in the biosynthesis of disease-lignin, which protects plants from fungal invasion. A novel POD inhibitor, ST-1 (1), was isolated from a fungus, and its chemical structure was elucidated to be 1, 3-dihydro-4, 5, 6-trihydroxy-1, 3-dimethoxy-7-methylisobenzofuran by a combination of chemical modification and spectroscopic techniques. ST-1 effectively scavenged oxygen radicals and also suppressed the phytoalexin accumulation in kidney bean.

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