ATP and ADP increased in cut-injured sweet potato root tissue during the 3 to 6-hr incubation period, and showed the maximum for the 9 to 18-hr, and 6 to 9-hr incubation periods, respectively, then decreased. ATP was present in the highest amount among ATP, ADP and AMP throughout the 72-hr incubation period, while AMP was in the lowest. Total acid-soluble nucleotides increased gradually, and showed the peak content at the 12-hr incubation period, and decreased thereafter. Adenine mononucleotides such as ATP, ADP and AMP occupied about 40 to 65% of total acid-soluble nucleotides.

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Streptomyces peptidofaciens KY 2389, a new species isolated from a soil sample, exhibited the highest potencies in production of both aminopeptidase and carboxypeptidase among about 1, 300 strains tested.
Optimum pH values of both aminopeptidase and carboxypeptidase for Leu-β-naphthyl-amide and Cbz-Gly-Leu were 8.0. Aminopeptidase was thermostable and the activity was not lost by treatment at 70°C for 1hr, in the presence of Cal²⁺. Carboxypeptidase was heatlabile and over 50% of the activity was lost by treatment at 60°C for 1hr. Of the synthetic peptides tested, Leu-Gly-Gly and Cbz-Gly-Leu were the most suitable substrates for aminopeptidase and carboxypeptidase, respectively.

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Cells of a strain of Streptomyces sp. were incubated with an equivalent quantity of urate, xanthine, 6, 8-dihydroxypurine or hypoxanthine in a medium deprived of other nitrogen source. The amount of uricase produced by these cells was shown to differ significantly, increasing in the following order of purine bases added to the medium: urate, xanthine, 6, 8-dihydroxypurine and hypoxanthine. Of these was only urate indicated to be the inducer of uricase formation, and the difference in the quantity of uricase produced was found to be based on the duration of enzyme formation. The rate of uricase formation was essentially identical regardless of the purine bases supplied to cells.
Allantoin was accumulated in medium in remarkably different manners depending on the purine bases, which suggested the diversity in the mode of generation of urate in cells. Urate was generated at the slowest rate in the cells incubated with hypoxanthine, although the largest amount of uricase was produced. However, urate supplied to cells at the same rate but from medium failed to support the enzyme formation when the activity increased to a certain level. In order that the same amount of uricase was produced by the cells incubated with the different purine bases, the initial concentration of the purine bases should be raised so that they could remain in medium for the same incubation time.
Intracellular compartmentalization that might segregate endogenous and exogenous urate and might cause the difference in “effeciency” of these urate molecules as the inducer of uricase formation has been discussed.

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The state of water contained in emulsions, particularly in o/w emulsions, was studied as a model of water orientation at the peripheries of biomembranes. Dielectric measurements made at microwave frequency on emulsions containing water and liquid paraffin in various ratios with emulsifiers revealed that the o/w emulsions possessed considerably reduced dielectric loss as compared with theoretical values obtained in accordance with the MaxwellWagner model, while the dielectric properties of w/o emulsion were in good agreement with the theoretically expected values. The observations seem to be explained by assuming changes in the state of water in the oil-water interfacial layer in o/w emulsions. The preparation of stable emulsions for use in this study is also discussed.

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Polynucleotides could be synthesized from nucleoside diphosphates by microorganisms belonging to genera Pseudomonas, Serratia, Xanthomonas, Proteus, Aerobacter, Bacillus, and Brevibacterium. These strains were rich in polynucleotide phosphorylase easily extractable from cells and poor in both nuclease and nucleoside-diphosphate-degrading enzymes. Polynucleotide phosphorylase was effectively extracted from the bacterial cells, that had been once soaked in saturated saline solution, with hypotonic solution. Synthesis of polynucleotides was observed not only when the substrates were incubated with polynucleotide phosphorylase preparation isolated from the bacterial cells, but also when the substrates were added directly to the bacterial cultures.

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Metabolism and residual fate of O-ethyl S, S-diphenyl phosphorodithiolate (Hinosan^®) applied on rice plant was examined by using ³⁵S-labeled or ³²P-labeled compound. Ion exchange chromatography, thin-layer chromatography and gas-liquid chromatography with flame thermionic detector or flame photometric detector were applied for identification of water soluble and toluene soluble metabolites of Hinosan. Degradation of Hinosan at the initial stage of metabolism was mainly the cleavage of P-S linkage, and a large portion of phenyl dihydrogen phosphorothiolate and a minor portion of O-ethyl S-phenyl hydrogen phosphorothiolate were found as water soluble metabolites. Phenylthio radical released on the production of the above mentioned metabolites was recovered as diphenyl disulfide, which was finally converted to sulfuric acid through benzenesulfonic acid. Triphenyl phosphorotrithiolate and O, O-diethyl S-phenyl phosphorothiolate were produced by transesterification between molecules of Hinosan at the initial stage of metabolism. Examination of metabolites in rice grains showed that sulfur and phosphorus atoms in Hinosan were incorporated into neutral or cationic substances probably after several steps of chemical transformation.

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Behavior and metabolism of O, O-diisopropyl S-benzyl phosphorothiolate (Kitazin P^®) in rice plant were examined using ³²p, ³⁵S-double labeled compound. Uptake of Kitazin P by the plant was different with the growth stages of the plant, and the rate of uptake was rapid in early growth stage. Kitazin P penetrated into plant tissues was gradually hydrolyzed to produce O, O-diisopropyl hydrogen phosphorothioate which was converted to diisopropyl hydrogen phosphate, isopropyl dihydrogen phosphate and phosphoric acid. As toluene soluble metabolites, eight spots were detected by thin-layer chromatography, but their percentages in toluene soluble fraction were extremely low as compared with that of Kitazin P. Only two metabolites, dibenzyl disulfide and O, O-diisopropyl O-benzyl phosphorothionate were identified by a gas-liquid chromatography with a flame thermionic detector or a flame photometric detector. Diisopropyl hydrogen phosphorothioate was detected as a persistent metabolite even in rice grains.

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³²P-labeled organophosphorus fungicide Hinosan was sprayed on rice plants at various growth stages, and metabolic fate of the pesticide in the rice plants was studied. Identification of Hinosan and its metabolites in the n-hexane and water extracts was conducted by TLC and GLC (FPD).
The rate of hydrolysis of Hinosan in rice plants seemed to be slower than that of other organophosphorus pesticides. Hexane-soluble components, which were detected throughout the experimental period, consisted mainly of Hinosan. Among the water-soluble metabolites identified were O-ethyl S-phenyl phosphorothioic acid and S, S-diphenyl phosphorodithioic acid, which were detected one to four days after the application, and ethyl phosphate and phosphoric acid which increased with the lapse of time.
Upon examination of the radioactivity of Hinosan and its metabolites in rice grains, a certain level was detected in husk, but very little in hulled rice and polished rice.

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It was, using the particulate enzyme from Micrococcus lysodeikticus, revealed that most of 2-¹⁴C-mevalonic acid incorporated into the cell of Lactobacillus heterohiochii H-1 was incorporated into the lipid intermediate of cell wall biosynthesis. About 10% of the radioactivities incorporated into the cells was, however, found in nucleic acid fraction which was extracted from lysozyme treated cells with phenol. Most of the radioactivities in the nucleic acid fraction was eluted at the beginning of the elution pattern from Sephadex G-200 or MAK-column. The material is different from tRNA and rRNA.

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In the course of the study on uptake of mercury by plants, tobacco was grown in the presence of mercury vapours and the amino acidic composition of the leaves was examined. The results showed that tobacco can take up more than 5, 000 ppm of mercury through the leaves and store it, showing no symptom of poisoning. In relation to the absorption of mercury, an abnormal increase of cystine, glutamic acid and glycine was observed in the proteins of the leaves. A possible role of cystine in the metabolism of mercury is also discussed.

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Extracellular polysaccharides produced by 3 strains of Pullularia pullulans were fractionated by treating with cetyl trimethylammonium hydroxide into soluble and insoluble fractions, and the structure of the former fraction, i.e., pullulan, was studied. The yield and the ratio of 2 fractions varied widely according to the strains. But the structure of pullulan was found to be uniform irrespective of the strains used. All 3 samples of pullulan gave only glucose on complete acid hydrolysis, and 93_??_95% maltotriose and 5_??_7% maltotetraose after isoamylase (pullulanase) action. The ratio of α-1, 4- to α-1, 6-glucosidic linkages calculated from periodate oxidation data coincided very well with the value expected from the ratio of maltotriose to maltotetraose units. An evidence for the complete absence of branch structure in pullulan was presented from the results of hydrolysis by pullulan 4-glucanohydrolase.

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Effect of daily oral feeding of 33mg retinol for nine days on the liver phospholipids of rats has been studied. As early as two days after feeding retinol an increase in the amounts of liver triglycerides, proteins, phospholipids, and cholesterol was noted which kept increasing and reached the peak concentration 6 days after daily retinol feeding and thereafter a decrease in their amounts was noted. Hepatic phospholipid fractions viz. phosphatidyl choline, phosphatidyl ethanolamine, phosphatidic acid and polyglycerol phosphatide, phosphatidyl inositol, phosphatidyl serine, sphingomyelin, lysophosphatidyl ethanolamine and lysophosphatidyl choline showed the same pattern. Labelling of these phospholipids with NaH₂³²PO₄ in rats fed daily 33mg of retinol for a period of two days also exhibited the pattern which was observed in their amounts two days after daily feeding of retinol. The results suggest a close relationship between the metabolism of hepatic triglycerides and phospholipids of rats fed excessive amounts of retinol.

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Among various nutritional mutants with weak 5'-nucleotidase derived from Bacillus subtilis IAM 1145, the adenine-requiring mutants could convert exogenously added hypoxanthine, guanine, xanthine and their ribosides to 5' inosinic acid (IMP) and accumulate it in the medium. Synthesis of IMP from purine derivatives was observed predominantly in an early stage of the cultivation. The conversion was stimulated by Fe²⁺ or Mn²⁺, and markedly depressed by an excess amount of adenine in the production-medium.

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Two hundreds and fifty eight strains of microorganisms have been isolated from 526 samples (soil, leaf and river water gathered from 17 prefectures) by repeating liquid enrichment culture techniques in the medium containing biphenyl, diphenylmethane, diphenylethane or terphenyl, as the sole source of carbon.
In the course of investigation, several strains were found to produce a large amount of γ-benzoylbutyric acid from biphenyl. Furthermore these strains utilized p-Cl-biphenyl and produced p-Cl-benzoic acid in good yield.
Microorganisms obtained were almost short rod, motile bacteria, and fungi were also found from the screening medium of diphenylethane.

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The respiratory chain system of Brev. thiogenitalis grown in the presence of copper ions contained cytochromes a, b and c. The cytochrome a was solubilized and purified from the cell-free extracts by means of Triton X-100 and cholate extraction, and DEAE-cellulose chromatography. It was purified about 130-fold from the cell-free extracts and was free from other cytochromes. The purified preparation contained 1.4mμatom copper and 1.9mμatom iron per mμmole heme a, respectively, and approximately 5mμmoles heme a per mg protein.

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The reaction of tri- and tetrapeptides with glyoxal at 100°C and pH 5.0 was studied. A series of new pyrazinone compounds was isolated from the reaction solutions of tri- and tetrapeptides with glyoxal: N-[2 (3-alkylpyrazin-2-on-1-yl) acyl] amino acids (I) and 2-(3-alkylpyrazin-2-on-1-yl) alkyl acids (II) from tripeptides, (I), (II) and N-[2 (3-alkylpyrazin-2-on-1-yl) acyl]-dipeptides (III) from tetrapeptides. Their chemical structures were determined by UV, IR, MS and NMR spectroscopy.
Aldehydes and free amino acids were also detected as reaction products. The amino acids were proved to be derived from the C-terminals of the peptides. Reaction mechanisms for the reaction of tri- and tetrapeptides with glyoxal were also proposed.

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A glucanase was isolated from a culture fluid of an Arthrobacter bacterium. The purified enzyme preparations consisted of the glucanase components having the same enzymatic activity. The enzyme was stable in a broad pH range, but lost its activity rapidly at above 60°C. Optimum pH values were found to be 5.5_??_6.5.
The glucanase attacked the following glucan preparations and liberated a relatively small amount of reducing power: Saccharomyces cerevisiae glucan, Candida albicans glucan, Saccharomyces fragilis glucan, pachyman, curdlan and laminaran. The most prominent sugar spot on the chromatogram of the digest from yeast glucan was identified with laminaripentaose, and the other faint spots with a series of laminaridextrins. The β-1, 6 glucosidic bonds in yeast glucan were not hydrolyzed and concentrated in a soluble fraction which was found near the origin of the chromatogram.

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The action pattern of lytic β-1, 3 glucanase (glucanase I) from Arthrobacter which liberates predominantly laminaripentaose from various β-glucans has been studied. The enzyme was not active on short linear laminaridextrins, but was active on an enzymatically synthesized, linear β-1, 3 oligoglucan preparation. Any intactness of the glucose residues of the chain ends of a substrate did not seem to be necessary for the action of the enzyme. The results of determination of laminaripentaose during a relatively early phase of the reaction suggested that about half of the reducing power liberated in the medium might be explained by the formation of the sugar. It seems that the formation of laminaripentaose relates to the initial attack of glucanase I on β-1, 3 glucan chains.

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It was demonstrated that the polysaccharide, pullulan, was synthesized from sucrose by acetone-dried cells of Pullularia pullulans or from UDPG by cell-free enzyme preparations prepared from the organism. The pullulan formed was estimated by precipitation with ethanol, and determining maltotriose produced after treating the precipitate with Aerobacter isoamylase (pullulanase). Acetone cells (5g) shaken with 200ml of 10% sucrose produced over 250mg of pullulan per 100ml after 90 hr at 30°C and pH 6.0. Cell-free enzyme produced pullulan from UDPG in the presence of ATP. ATP was essential for the biosynthesis, and ADPG could not replace for UDPG.
In addition, it was observed that a lipid containing glucose residue was formed during. the reaction. The nature of this glucolipid was examined, and possible participation of a lipid intermediate was assumed in the pullulan biosynthesis.

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An isoleucine leaky auxotroph of Arthrobacter paraffineus, which was isolated by Takayama et al.³⁾ as a mutant producing L-threonine and L-valine from n-paraffin, was subjected to further mutagenesis in an attempt to obtain better L-threonine producers. Some of the double auxotrophs derived from the isoleucine auxotroph and some of their revertants with respect to isoleucine requirement produced more L-threonine than the original isoleucine auxotroph. In contrast to the original isoleucine auxotroph, a revertant derived from a methionine plus isoleucine double auxotroph, KY7135, produced an increased amount of L-threonine and a decreased amount of L-valine. The optimum level of L-methionine for L-threonine production in KY7135 was much higher (1000_??_2000μg/ml) with n-paraffin medium than with sorbitol or mannitol medium (10_??_50μg/ml). L-Threonine production reached a maximum level (11.5mg/ml) in 7 days incubation with the medium containing 10% n-paraffin (C₁₂_??_C₁₄ rich). Several mutants which produce L-threonine more than 12mg/ml were obtained from KY 7135 by monocolony isolation procedure.

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Occurrence of cellulase activity was demonstrated in the filtrates of germinating conidiospores and growing mycelia of P. oryzae. Activity and some properties of cellulase in the filtrate of mycelia grown on rice plant powder as carbon source were compared among various strains.
Cellulase activity (C₁ and C_x enzymes; cellulose and carboxymethylcellulose as substrates, respectively) in the filtrate of germinating conidiospores was detected in the pathogenic T-1 (Ken 53-33) strain as well as nonpathogenic 0 (THU 3×1) strain of P. oryzae. The activity was higher in the former than the latter strains. Cellulase activity (C_x. enzyme) in the filtrate of growing mycelia was detected in the four strains used, T-1 (Ken 53-33), C-3 (N 87), N-1 (H373), and 0 (THU 3×1). Cellulase activity (C_x. enzyme) in the filtrate of mycelia was optimal at pH 5.0 and 40°C, and stable up to 40°C. Their properties did not differ significantly except for the pH-activity curve at alkaline side among various strains; but cellulase activity (C₁ enzyme) was found to be correlated with their pathogenicity except for the case of C-3 strain.

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A simplified procedure for the purification of pea phytohemagglutinin, which involves fractionation with ammonium sulfate and column chromatography to give two active components, has been developed. The main component of the purified hemagglutinin gave one single band with disc electrophoresis. Molecular weight of the hemagglutinin was calculated as 61, 000 (Vρ=0.75). Using this hemagglutinin, the experiment was carried out to aim from the viewpoint of conformational feature of sugars at an explanation of the role of a hydroxyl group on gluco- or mannopyranoid moiety of the sugars capable of interaction with the hemagglutinin. A possible concern of orientation of hydroxyl group at the C-2 position on pyranoid moiety of sugars is proposed to interpret the hemagglutination with the phytohemagglutinin from the results of inhibition tests and from the consideration on conformational feature of sugars reported previously.¹⁾

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About 2, 000 strains of streptomycetes and 200 strains of molds were tested for egg yolkdegrading activity. A strain isolated from soil was found to produce the strong activity in the culture medium.
The egg yolk-degrading substance was found to be phospholipase D and the producing strain was identified as Streptomyces hachijoensis. It was found out that the strains producing egg yolk-degrading substance appeared with high frequency in whirl-forming species in genus Streptomyces.

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An automated method for rapid and convenient measurement of L-glutamate has been developed by using a discrete analyzer, EEL Auto Chemist. It is based on the colorimetric measurement of NADH produced on a mole-mole basis by enzymatic dehydrogenation of L-glutamate using L-glutamate dehydrogenase from bovine liver. The values of L-glutamate obtained by this method were well agreed with those obtained by the routine Waruburg manometric method using L-glutamate decarboxylase from Escherichia coli.

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The color tone of melanoidins prepared from model systems by heating mixtures of 1 mmole amino acid or peptide and 1 mmole sugar in aqueous solution was investigated using ΔA (change of log absorbance per 100mμ) as a parameter of color tone.
Amino acids and peptides were responsible for the color tone of melanoidins while sugar had no effect on the color tone. In addition, the color tone of melanoidin varied as the concentration ratio of amino acid to sugar changed. Melanoidins from tryptophanand proline-xylose systems exhibited the lightest color tone. Melanoidins from peptides generally exhibited dark tones compared with those from amino acids.
Using the distribution pattern of color components fractionated by DEAE-cellulose chromatography, melanoidins have been classed into 4 groups based on the percentage of PI (non-adsorbed fraction on DEAE-cellulose): group 1, melanoidins containing 90_??_100% P1; group 2, 70_??_80% Pl; group 3, 30_??_50% Pl; group 4, less than 20% P1. Melanoidins derived from most amino acids examined, those from basic amino acids and proline, those from glutamic acid and glycine, and those from peptides and amino terminal monocarboxylic acids belonged to groups 2, 1, 3, and 4, respectively.
The color tone of each color component except for P1 was generally very similar in most melanoidins. However, the color tone of P1 was variable. Therefore, melanoidin is composed of various color components having intrinsic color tones from yellowish-brown to dark brown and their color tone depends upon the variation of the amount of color components and upon the color tone of P1.

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By microorganisms dl-isopulegyl acetate was hydrolyzed asymmetrically to give a mixture of l-isopulegol and d-isopulegyl acetate, but dl-neo isopulegyl acetate was not hydrolyzed. Since d-citronellal can be obtained by the thermal decomposition of l-isopulegol, the asymmetrical hydrolysis of dl-isopulegyl acetate means the resolution of dl-citronellal.

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By selected microorganisms dl-carvomenthyl acetate, dl-isocarvomenthyl acetate and dl-neo isocarvomenthyl acetate were asymmetrically hydrolyzed to l-carvomenthol with dcarvomenthyl acetate, l-isocarvomenthol with d-isocarvomenthyl acetate and d-neo isocarvomenthol with l-neo isocarvomenthyl acetate respectively; dl-neo carvomenthyl acetate was not hydrolyzed.

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By using microorganisms (their esterase), (±)-trans and cis-2-methylcyclohexyl acetates were asymmetrically hydrolyzed to (-)-traps-2-methylcyclohexanol with (+)-trans-2-methylcyclohexyl acetate and (-)-cis-2-methylcyclohexanol with (+)-cis-2-methylcyclohexyl acetate. Similarly (±)-traps and cis-3-methylcyclohexyl acetates were hydrolyzed by the same microorganisms to give (+)-trans-3-methylcyclohexanol with (-)-tians-3-methylcyclohexyl acetate and (-)-cis-3-methylcyclohexanol with (+)-cis-3-methylcyclohexyl acetate.

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Some enzymatic properties were examined on the transaminase (DOPA transaminase) which catalyzes the reaction between 3, 4-dihydroxyphenyl pyruvate (DOPP) and certain amino acids to form 3, 4-dihydroxyphenyl-L-alanine (DOPA). The cell-free extract from Alcaligenes faecalis IAM 1015 was used as the DOPA transaminase. L-Aspartate, L-gluta-mate, and L-phenylalanine were utilized efficiently as amino donor. The occurrence of three kinds of transaminase-apartate-DOPP transaminase (ADT), glutamate-DOPP transaminase (GDT), and phenylalanine-DOPP transaminase (PDT)-was postulated.
The pH optima of these enzymes were observed in the alkaline pH range. The enzymes were unstable in the acidic range and inactivated above 60°C. Ca²⁺, Mg²⁺, and Mn²⁺ protected PDT from heat denaturation. Fe²⁺, Cu²⁺, and Al³⁺ remarkably inhibited the enzyme reaction.

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Mechanism of inactivation of a double-stranded DNA phage, phage J1 of Lactobacillus casei, by reduced form of glutathione (GSH) was studied.
Air (oxygen) bubbling, oxidizing agents and transition metal ions enhanced the rate of inactivation of the phage by GSH. Partial oxidation of GSH resulted in a more rapid rate of inactivation. In contrast, nitrogen bubbling, reducing agents, chelating agents and radical scavengers prevented the inactivation. Fully oxidized GSH had no phagocidal effect. These results indicate that the inactivating effect of GSH requires the presence of molecular oxygen and is caused by free radical involved in the mechanism of GSH oxidation.
The target of GSH in the phage particle was not the tail protein but DNA. GSH reacted with phage DNA and caused single-strand scissions in the DNA, as exhibited by alkaline sucrose gradient centrifugation; thus inactivating phage.

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Seven optical active 2-benzylamino alcohols were synthesized by reduction of N-benzoyl derivatives of L-alanine, L-valine, L-Ieucine, L-phenylalanine, L-aspartic acid, L-glutamic acid and L-lysine and applied for the resolution of (±)-trans-chrysanthemic acid. d-trans-Chrys-anthemic acid was obtained by resolution via the salts of 2-benzylamino alcohols derived from L-valine and L-leucine, while (-)-trans-chrysanthemic acid was prepared through the salts of the amino alcohols derived from L-alanine and L-phenylalanine.

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A new synthetic route of rotenoids was developed and (±)-munduserone (15) was synthesized from acetylenic ketone (11).

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Some physicochemical properties and substrate specificity of crystalline acid protease obtained from Cladosporium sp. No. 45-2 were investigated.
The molecular weight determined by the sedimentation equilibrium method and Sephadex G-75 gel filtration was 32, 000 and 30, 000, respectively. The isoelectric point was determined as 4.6 by using the Tiselius electrophoresis apparatus and the amino terminal amino acid was found to be alanine. The enzyme did not contain histidine and was composed of 294 amino acid residues. Substrate specificity against synthetic peptides was similar to that of pepsin. The enzyme was remarkably inactivated by a synthetic pepsin inhibitor such as a-Diazo p-bromo acetophenone, Diazo acetyl glycine ethylester and N-Diazo acetyl norleucine methylester.

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During the investigation for dephosphorylation of 4-hydroxy-1-β-D-ribofuranosylpyrazolo[3, 4-d] pyrimidine 5'-phosphate, it was found that the compound was converted to an unknown substance by alkaline hydrolysis for 3hr at 140°C. The structure of the substance was assigned to be 5-amino-1-β-D-ribofuranosylpyrazole-4-carboxamide 5'-phosphate. 5 (or3)-Amino-pyrazole-4-carboxamide and its riboside were also obtained from 4-hydroxypyrazolo [3, 4-d] pyrimidine and its riboside, respectively, under the similar conditions.
5-Amino-1-β-D-ribofuranosylpyrazole-4-carboxamide and 5-amino-1-β-D-ribofuranosyl-pyrazole-4-carboxamide 5'-phosphate are new compounds.

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(±)-Millettone (14) was synthesized.

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