β-1, 3-Glucanase was purified from the culture filtrate of a Streptomyces sp. by ammonium sulfate fractionation, hydroxylapatite and DE-52 column chromatographies, gel filtration on Biogel P-100 and isoelectric focusing. The purified β-1, 3-glucanase was homogeneous on polyacrylamide disc-gel elcctrophoresis. The isoelectric point was pH 3.7, and the molecular weight was 36, 000. The optimum pH and temperature for the activity of the enzyme were 5.5 and 55°C, respectively. The enzyme was stabilized by Ca²⁺, but inactivated by Hg²⁺, and was inhibited by N-bromosuccinimide. The glucanase did not hydrolyze laminaribiose or sophorose, cellobiose, or gentiobiose. The enzyme ultimately hydrolyzed laminari-oligosaccharides (laminaritriose to laminarihexaose) to glucose and laminaribiose, and their sugar alcohols (laminaritriitol to laminarihexaitol) mainly to laminaribiose and laminaribiitol via a disproportionation reaction.

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A glutamyl and two histidyl residues that are suspected to be part of the active site of a base non-specific, guanine-preferential ribonuclease from Aspergillus saitoi (RNase Ms) were located.
RNase Ms modified by radioactive iodoacetate was digested by α-chymotrypsin, then separated by paper chromatography and paper electrophoresis. The amino acid composition of the radioactive peptide indicated that the site of the modification was Glu₅₇.
A simple procedure to separate three histidyl-residue-containing peptides from RNase Ms was established. The procedure was used on the photooxidized RNase Ms, and the photooxidized histidyl residues were found to be His₃₉ and His₉₁.

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Rapid conformational changes of pepsin occurring within 5s due to pH-jump were studied by the stopped-flow method through the absorption change at 245nm which is caused by the ionization of tyrosine residues. Two distinct phases of conformational change involving the exposure of tyrosine residues, perhaps buried in native pepsin, were observed. The pH profile of absorption change caused by the alkaline denaturation, ΔA, suggests that the unfolding in the slower phase occurs in lower pH than that in faster phase and that tyrosine residues exposed in the faster phase are more perturbed in the folded state than those exposed in the slower phase.

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Earlier isolated plant polyphenol PI, which is carcinostatic on mice implanted with Ehrlich carcinoma [S. Takeuchi et al., Agric. Biol. Chem., 50, 569 (1986)], showed transient, nonspecific, non-cytotoxic growth inhibition in vitro on both normal and malignant cells. Examination of its effects on alteration of native and TPA-induced in vitro phenotypes revealed that PI suppress both the plasmin and ODC activities of the cells that increase with spontaneous malignancy, and also the TPA-induced ODC activity increase. TPA-induced decrease of ¹²⁵I-mEGF-binding ability was reversed by PI, to larger extent in the HeLa cells than in the untransformed RME-5-3-1 cells.

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An enzyme which catalyzes the hydrolysis of 5'-IMP to inosine and inorganic phosphate was purified from cod muscle by extraction of the acetone powder with 1% Triton X-100, and followed by two steps of affinity chromatography on Concanavalin A-Sepharose and 5'-AMP Sepharose 4B. The purified enzyme was homogeneous on disc polyacrylamide gel electrophoresis. The apparent molecular weight of the purified enzyme was estimated to be 2.7×10⁵ by gel filtration, and about 6.7×10⁴ by SDS-polyacrylamide gel electrophoresis. The enzyme was identified as 5'-nucleotidase (EC 3.1.3.5), since it hydrolyzed various 5'-nucleotides but not a number of phosphorylated compounds including 2'(3')-nucleotides. The optimum pH was 7.6, and there appeared a second peak at pH 9.1 in the presence of Mg²⁺. The enzyme was inhibited by Zn²⁺ and Cu²⁺, but was not inhibited by Ca²⁺, Ba²⁺, 2-mercaptoethanol, pCMB and inosine. The enzyme was competitively inhibited by ADP and ATP, and was also inhibited by EDTA, depending upon the concentration of EDTA and the incubation time. The EDTA-inactivated enzyme was partially reactivated by the addition of some divalent cations, Zn²⁺ being the most effective.

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The P700-chlorophyll α protein complex purified from spinach consists of polypeptides of molecular mass 60-65, 23, 20, 17, 14, and 12kD. Proteolysis of the complex with trypsin for 24hr altered neither the photochemical activity of P700-chlorohyll α protein complex nor the characteristic effects of cations and pH on the electron transfer from plastocyanin to photooxidized P700. However, it was found that the trypsin-treated complex was photochemically inactive in the presence of 1% (v/v) Triton X-100, although 1% (v/v) Triton X-100 had no effect on the photochemical activity of the native complex. Upon lowering the concentration of Triton X-100 by dialysis, the digested complex regained the photochemical activity of P700. The results of SDS-polyacrylamide gel electrophoresis showed that proteolysis of the complex with trypsin for 24hr results in extensive degradation of polypeptides associated with this complex into small polypeptide fragments (M.W.≤14.000). Isolated reaction center protein (60-65kD polypeptide) was also treated with trypsin and we observed quite similar effects of trypsin treatment. All these data suggest that the structure of either trypsin digested P700-chlorophyll α protein complex or the reaction center protein required for its photochemical activity can be maintained through strong hydrophobic interactions among the nicked subunit polypeptides.

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The fluorometric reaction of sulfite with N-(9-acridinyl)maleimide (NAM) was applied to high performance liquid chromatography (HPLC). It improved the sensitivity and accuracy for the determination of sulfite to 0.01 nmol/ml or 0.1 pmol on the column. Three NAM-sulfite adducts were isolated, and their structures were identified as N-(9'-acridinyl)-2-sulfonyl succinimide (I) and its two isomeric hydrolysates, N-(9'-acridinyl)-2 or -3-sulfonyl succinamides (II and II'). Two-step reactions, involving the addition of sulfite to the maleimide ring of NAM and subsequent hydrolysis of the resulting succinimide ring to two amide-form adducts, were established.

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α-Hydroxyglutarate dependent NADH oxidizing activity was found in a cell-free extract of Alcaligenes No. 314. Succinate was identified as a product from L-α-hydroxyglutarate in the extract. Stoichiometric conversion of L-α-hydroxyglutarate to Succinate occurred under anaerobic conditions without the addition of any cofactors, while the reaction was stimulated by Fe²⁺. 4, 5-Dioxovaleric acid seems to be an intermediate of the reaction since it was converted to Succinate and L-α-hydroxyglutarate by the extract. Addition of formaldehyde, another possible product of the succinate forming reaction, to the extract caused oxidation of NADH, which might be responsible for the α-hydroxyglutarate dependent NADH oxidation. Considering the presence of L-α-hydroxyglutarate dehydrogenase in the Alcaligenes cells, an anaerobic by-path of the TCA cycle through which succinate is formed from α-ketoglutarate was proposed.

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Chemical modification of a polyether ionophore antibiotic, cationomycin, was attempted to clarify the structure-activity relationship and to obtain derivatives with higher activity. Deacylcationomycin showed the same ion selectivity as the parent compound, but it was less effective in sodium ion transport and anticoccidial activity. Thirteen acyl or alkyl derivatives were prepared by regioselective reactions. No derivatives were more effective in sodium ion transport. There are apparent relationships between sodium ion transport of acetyl derivatives and their biological activities.

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A chemically synthesized α-neo-endorphin (αNE) gene was fused to the PH05 gene which encodes repressible acid phosphatase (APase) of Saccharomyces cerevisiae. Three kinds of genes encoding APase-αNE chimeric proteins were cloned in the yeast. Nucleotide sequence analysis revealed that C-terminal amino acids comprising 6 to 17% of the intact enzyme were replaced by αNE. The APase-αNE chimeric proteins were expressed at the level of approximately 1 × 10⁶ molecules per cell under the derepressed conditions. The radioimmunoassay for αNE revealed that most of the APase-αNE chimeric proteins was not present in the periplasmic fraction but in the protoplast fraction.

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Bacillus subtilis FHC 402-derived antibacterial factor (abbreviated to BAF), which showed antibacterial activity by its combined use with hexametaphosphate, was purified from a culture broth by means of column chromatography on octyl-Sepharose CL-4B, DEAE-Cellulofine AL and Bio-Gel P-6. BAF was purified 63-fold with an activity yield of 17%. The purified preparation gave a single band on the electrophoregram. BAF may be a glycopeptide containing 22 molecules of 8 different amino acids, 3 hexoses, 1 hexosamine, 1 deoxyribose, and unknown substances. The N-terminal amino acid of the BAF preparation was determined to be tyrosine and the C-terminal amino acid to be proline. Gel filtration estimated the molecular weight of the BAF preparation at 3, 200. The purified BAF preparation was stable in a pH range from 5.0 to 11.0, was also stable on heating, but was unstable at pH 3.0.

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Transformation of the yeast, Saccharomyces carlsbergensis, with plasmids carrying the kanamycin (G418) resistance gene of Tn903 was attempted. Transformants resistant to the antibiotic, G418, were obtained from two strains of this species as well as a strain of S. cerevisiae after treatment of intact cells with lithium acetate and polyethylene glycol. The optimum period of post-incubation for expression of the kanamycin resistance gene was found to be four hours at 30°C for both S. carlsbergensis and S. cerevisiae. The transformation efficiency of S. carlsbergensis was lower than that of S. cerevisiae. The mitotic stability of the four plasmids used was determined; that of a plasmid containing the replication origin of 2 μm-DNA was lower in S. carlsbergensis than in S. cerevisiae. The mitotic stabilities of the three other plasmids containing ars 1, a centromere and ars 1, and no replication origin, respectively, in yeast cells were almost the same in S. carlsbergensis and S. cerevisiae.

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A β-1, 3-xylan was prepared by sodium hydroxide extraction from seaweeds of Bryopsis maxima and Caulerpa sp. The seaweed xylan obtained contains xylose residues only and β-1, 3-linkage was characterized by the method of ¹³C-NMR spectroscopy and methylation analysis. A strain of Aspergillus terreus A-07 isolated from soil produced an endo-β-1, 3-xylanase, decomposing β-1, 3-xylan to D-xylose and D-xylooligosaccharides. Six different xylanases in the culture filtrate of A. terreus A-07 have been found and purified to homogeneity from 40- to 95-fold by ammonium sulfate fractionation, SP-Sephadex C-25 ion exchange chromatography, Biogel P-100 gel filtration, and isoelectric focusing. Each of the purified enzymes gave a single band on polyacrylamide disc gel electrophoresis, indicating that all of the purified β-1, 3-xylanases were electrophoretically homogeneous.
The six kinds of purified enzymes on SDS polyacrylamide gel electrophoresis indicated that two of the β-1, 3-xylanases have molecular weights of 11, 000, another two of the β-1, 3-xylanases have molecular weights of 14, 500, and the last two β-1, 3-xylanases have molecular weights of 13, 500 and 20, 000. The pH optima of the β-1, 3-xylanases were from 4.0 to 5.5. The optimum temperature for activity was from 40 to 55°C. All of the enzyme activities of the six β-1, 3-xylanases were inhibited by Hg²⁺, Mn²⁺, and N-bromosuccinimide. With respect to the hydrolysis patterns, the purified β-1, 3-xylanases hydrolyzed β-1, 3-xylan to xylose and xylooligosaccharides. The β-1, 3-xylanases hydrolyzed β-1, 3-xylan and rhodymenan but did not hydrolyze β-1, 4-xylan, cellulose powder, laminaran, β-1, 3-xylobiose or β-1, 3-xylotriose. By their action patterns and substrate specificities, all of the six purified enzymes were endo-type β-1, 3-xylanases.

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A purified extracellular endo β-1, 3-xylanase (EC 3.2.1.32) from an isolated strain, Aspergillus reus A-07, was found to hydrolyze 1, 3-xylosyl linkages only. When rhodymenan (β-1, 4 and β-1, 3-linked xylan) was hydrolyzed by β-1, 3-xylanase (EF-6), four β-1, 4-linked xylooligosaccharide ictions were produced. The main product was β-1, 4-xylotriose, with trace amounts of other β-1, 4-ked xylooligosaccharides. Successive degradation by β-1, 4-xylosidase of the β-1, 4-looligosaccharides that were produced from hydrolysis of β-1, 3-xylanase on rhodymenan yielded ly xylose as the final product.
We compared the action pattern of this enzyme with that of an extracellular endo β-1, 4-lanase (EC 3.2.1.8) of Streptomyces. From a mixture of products of β-1, 4-xylanase hydrolysis on adymenan, an isomeric xylotriose was isolated by charcoal chromatography after treating with β-1, 4-xylosidase. The structure of this isomeric xylotriose was elucidated by methylation analysis and susceptibility to β-1, 4-xylanase, β-1, 3-xylanase, and β-1, 4-xylosidase. The obtained isomeric lotriose was identified as 3-O-β-xylopyranosyl-4-O-β-D-xylopyranosyl-D-xylose (XI→3X1→4X). It has a melting point of 224-225°C and [α]²⁰_D(c=1, H₂0)= -46°.

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Comparative study on the aromas of three kinds of fish sauces, Shottsuru, Nampla and Noucmam was conducted. Volatile compounds of all samples were separately collected by steam distillation, and separated and identified by gas chromatography and gas chromatography massspectrometry, respectively.
Subjectively, these three fish sauces revealed aromas different from each other. A total of 50, 44 and 49 volatile compounds were identified in Shottsuru, Nampla and Noucmam respectively. Acids, alcohols, nitrogen-containing compounds, sulfur-containing compounds, lactones, esters, phenols, carbonyls and hydrocarbons were among the main groups of volatile compounds identified. The differences in the aromas of the samples were thought to be due to the differences in the level of concentrations of the major acids. Moreover, some differences in the kinds of the minor volatile compounds were possible contributing factors in the differences of the total aromas.

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Modification of butter oil was attempted in order to lower the cholesterol level and to improve the spreadability using extraction with supercritical carbon dioxide (SC-CO²). Although SC-CO² extraction successfully fractionated the triglycerides according to their carbon numbers, cholesterol could not be removed from the triglycerides by this simple extraction. The cholesterol content of SC-CO²-extracted butter oil could be decreased by passing the extract through a silicic acid column. This process succeeded in lowering both the melting point and cholesterol level of the extracted oil. The flavor of the extracted oil was similar to the original butter.

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Peptides remaining or appearing during protein digestion were examined for a possibility of their involvement in the regulation of cholesterol levels in plasma. Plant proteins were somewhat inferior in digestibility to casein and their digestive products were abundant in 'hydrophobic' peptides relative to casein. The 'hydrophobic' peptides bound well to bile acid. There was a correlation between the plasma cholesterol level in rats given various food proteins and the hydrophobicity of their digestive products (peptic-pancreatic digests). The preponderance of hydrophobicity seems to favor an explanation for the cholesterol-lowering effects of plant proteins such as soy protein isolate and wheat gluten.

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We examined the structure of lysine residues which had been impaired due to exposure of proteins to hexanal, using a free lysine/hexanal system (I), a Gly-His-Lys/hexanal system (II) and a lysozyme/hexanal system (III). The most abundant ninhydrin positive compound (N-50) was isolated from the products with system I and identified as l-(5-carboxy-5-aminopentyl)-2-pentyl3, 5-dibutylpyridinium betaine, which was formed through condensation of e-amino groups of lysine with three molecules of hexanal.
A new ninhydrin positive compound (N-51) was isolated from hydrolysates of the products with systems II and III, and identified as the same compound as N-50 from the UV and mass spectra and thin layer chromatogram.
These results indicated that the E-amino groups of lysine residues of proteins are modified to alkyl-substituted pyridinium rings on exposure to hexanal.

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Two β-glucosidases (EC 3.2.1.21) from Euphausia superba designated as I and II were purified by ammonium sulfate fractionation and chromatographies on DEAE-cellulose and Con A-Sepharose 4B. They had the following similar properties: pH optima at 6.0; temperature optima at 55°C; molecular weight of about 155, 000; stability at low temperature in pH 5.6-9; and kinetic parameters. Both enzymes had β-glucosidase, β-fucosidase, β-galactosidase, and β-xylosidase activities. Inhibition studies indicated that these four activities shared a common active site on β-glucosidase I. These two enzymes also had very low α-L-arabinosidase activities.

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18β-Glycyrrhetinic acid (3β-hydroxy-11-oxo-18β-olean-12-en-30-oic acid) was converted into two 3, 4-seco-oleanane-type compounds, 4-hydroxy-3, 4-seco-11-oxo-18β-olean-12-ene-3, 30-dioic acid and 4, 7β-dihydroxy-3, 4-seco-11-oxo-18β-olean-12-ene-3, 30-dioic acid, by Chainia antibiotica, and 22α-hydroxy-18β-glycyrrhetinic acid (3β, 22α-dihydroxy-11-oxo-18β-olean-12-en-30-oic acid) was converted into 4, 22α-dihydroxy-3, 4-seco-11-oxo-18β-olean-12-ene-3, 30-dioic acid. The structures of these conversion products were determined by a combination of chemical and spectroscopic methods.

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The bitter components of cheese are hydrophobia peptides which are produced during the process of enzymatic digestion, and some of the isolated bitter peptides are derived from the middle portion of β-casein. However, quantitative examination of the bitter taste is seldom performed. We synthesized two hydrophobic peptides, H-Pro⁶¹-Phe-Pro-Gly-Pro-Ile-Pro⁶⁷-OH and H-Tyr⁶⁰-Pro-Phe-Pro-Gly-Pro-Ile⁶⁶-OH, which correspond to common portions among the isolated bitter peptides, in order to determine how bitter they were. From the results of sensory analysis, it was found that the synthetic peptides exhibited a bitter taste with threshold values 0.25 and 0.16mM, respectively. We also synthesized their fragments and analogs, and discussed the structurebitterness relationship.

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A heptapeptide, H-Val-Val-Val-Pro-Pvo-Phe-Leu-OH, corresponding to the partial sequence of bovine β-casein, positions 82-88, was synthesized and its bitter taste was measured. Its threshold value of bitterness was 0.14mM. We previously reported that H-Arg-Gly-Pro-Pro-Phe-Ile-OH showed strong bitterness with a threshold value of 0.025mM. The difference in the characteristic natures of these bitter peptides was found in their N-terminal moiety. We could confirm that, for an increase in the bitterness of peptides, the presence of a basic amino acid in the N-terminal was more effective than an increase in the bulkiness and hydrophobicity with a valine residue.

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Reduction of benzaldehyde and substituted benzaldehydes with actively fermenting baker's yeast afforded optically active 1-arylpropane-1, 2-diols in fairly good yields, as well as benzylic alcohols. It was revealed that benzaldehydes substituted with electron-withdrawing groups, which were previously reported to result only corresponding benzylic alcohols, were also reduced to give propanediol compounds in moderate yields. The reaction was shown to be highly diastereo-selective for erythro diols (>97% d.e.). The optical purities of the erythro isomers (1R, 2S) were also extremely high (>97% e.e.), and single recrystallization of dibenzoates gave optically pure diol derivatives within the limits of HPLC analysis.

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The mode of action of a novel macromolecular antibiotic, SN-07, was studied using Salmonella typhimur.ium TA1535, highly susceptible strain to the antibiotic. Thirty min of treatment with SN-07 at a concentration of 25μg/ml caused irreversible growth inhibition of TA1535 cells. This concentration of the antibiotic severely inhibited DNA synthesis but no effect on either RNA or protein synthesis was observed. Further, SN-07 broke down chromosomal DNA into the acidsoluble fraction.

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An extracellular acidic polysaccharide elaborated by Acetobacter sp. NBI 1005 was composed of D-glucose, D-galactose, D-mannose, and D-glucuronic acid (approximate molar ratio, 6:2:1:1). Methylation and fragmentation analysis by partial acid hydrolysis indicated that the polysaccharide has a branched structure containing a backbone chain of β-(1→4)-linked D-glucose residues, two out of every four glucose residues being substituted at the O-3 positions to form two kinds of branches, one consisting of D-mannose and D-glucuronic acid residues and the other of (1→6)-linked D-galactose and D-glucose residues.

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Three peptides, α^sk1, α^sk2 and α^sk3 pherornones, have been isolated as α-mating pheromones of Saccharomyces kluyveri, the primary structure of the main active component, α^sk2 pheromone, having already been determined. The unknown N-terminus of α^sk1 pheromone was elucidated to be l, 2, 3, 4-tetrahydro-β-carbolme-3-carboxylic acid (β-CAR) by mass and NMR spectrometric analyses. Synthetic β-CAR-His-Trp-OH was identical with N-terminal tripeptide fragment obtained from α^sk1 pheromone, and the primary structure of α^sk1 pheromone was determined as β-CAR-His-Trp-Leu-Ser-Phe-Ser-Lys-Gly-Glu-Pro-Met(O)-Tyr-OH. The amino acid sequence of α^sk3 pheromone was determined as H-Trp-His-Trp-Leu-Ser-Phe-Ser-Lys-Gly-Glu-Pro-Met-OH by comparing the enzymatic fragments with those of α^sk2 pheromone.

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The gelation of 7S globulin when heated at 100°C and 0.5 ionic strength was investigated. The minimum protein concentration required for gel formation was 7.5%. The minimum heating times with protein concentrations of 7.5% and above 10% were 20min and 15sec, respectively. The hardness of gel formed above 12% protein concentration became maximum after 15 sec heating and then remained constant on subsequent heating. The gels were transparent. The gelation mechanism was presumed to be as follows. Soluble aggregates with molecular weights of around 1 million, the shapes of which are not uniform, are formed at first which then associate with each other randomly to form a cluster. Finally a gel, the structure of which seems to comprise aggregates of clusters, is formed. No SH/S-S exchange reaction participates in this process. These results show that the gel properties and gelation mechanism for 7S globulin are different from those for IIS globulin.

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We constructed a hybrid plasmid to allow controlled expression of a gene and the subsequent secretion into the culture medium of the gene product in Escherichia coli. This was achieved by the use of five trp promoter-operator regions in tandem followed by the DNA fragment coding for the signal peptide and the N-terminus of the OmpF protein, and the trpR gene coding for the Trp represser. Multiplication of the trp promoter-operator appreciably enhanced expression of the gene that followed. A single copy of the trpR gene on the chromosome was insufficient for controlling the enhanced expression. The expression was, however, completely controlled when the trpR gene was cloned onto the same plasmid. When the multiple trp promoter-operator was followed by the DNA fragment coding for the signal peptide and the N-terminus of the OmpF protein that was further followed by the gene for human β-endorphin, a β-endorphin-containing polypeptide was synthesized under the complete control of the trp promoter-operator, and secreted to the culture medium across both the cytoplasmic membrane and the outer membrane. Controlled expression of a foreign gene and subsequent secretion into the medium of the product were thus achieved.

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Extracts from brains of mouse, rat, rabbit, pig, chicken, and duck were assayed for peptidylarginine deiminase, which catalyzes the deimination of arginyl residues. There was peptidylarginine deiminase in the extracts from all the brains that we tested, suggesting the widespread occurrence of this enzyme in vertebrate brains. The levels of the activity in the brains of 8 different inbred strains of mice could be divided into two groups. There is a difference of over 300% between the high and low groups. Furthermore peptidylarginine deiminase purified from the brains of mouse, pig, and chicken showed that there is no great difference in their MW and the optimal conditions for the activity. However, the substrate specificity of the enzyme from chicken brain was somewhat different from those of the enzymes from mammalian brains.

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Insecticidal synthetic amides related to a novel lead compound, (2E, 4E)-N-isobutyl-6-phenyl-hexa-2, 4-dienamide, and to some natural isobutylamides are up to four times as effective for both knockdown and kill against houseflies with the super-kdr mechanism of resistance (which operates against DDT and pyrethroids) than against the S-strain. This selectivity in the response to N-alkylamides demonstrates that the non-metabolic resistance mechanism, super-kdr, is vulnerable to and might be combated effectively with N-alkylamides.

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