Arylsulfatases reacting in alkaline condition have been detected in many Aspergillus fungi grown on defatted soybean meal or wheat bran. The activity was produced being associated with fungal growth but the activity seemed to be unstable under the optimum incubation temperature for growth, The arylsulfatase (arylsulfatase III) active in the pH range from 7 to 10 with an optimum pH of 8.5 has been partially purified from the solid culture of A. sojae SH 10-6 grown on defatted soybean meal. Arylsulfatase III was unstable above 20°C, stable in the pH range from 8 to 10, and inhibited by Ag⁺, Zn²⁺, Hg²⁺, fluoride, borate, and PCMB, but not by cyanide, molybdate, and tungstate. Another fraction obtained from A. awamori R-0827, which was purified with the same manner as the case of A. sojae SH 10-6, showed closely similar enzymatic properties to arylsulfatase III of A. sojae SH 10-6. These enzymatic properties are clearly different from those of arylsulfatase I and II previously noted.

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The metabolic responses to dietary fat to carbohydrate ratios have been investigated in growing rats fed with the diets at various fat calorie percents (FC%), containing a purified whole egg protein at 12.5 protein calorie percent.
The growth rate and the efficiencies of protein and energy utilization were not considerably affected by the fat content of the diets in a wide range from 0 to 87.5 FC %. However, the concentration of liver lipid was significantly increased in the 87.5 FC % group, whereas that of liver glycogen was markedly decreased.
At 12hr after the intraperitoneal injection of L-(U-¹⁴C) serine, the percentage recoveries of the radioactivity in body protein, lipid and soluble fractions were approximately 50, 6 and 14 %, respectively, of the injected dose. The radioactivity incorporated into body protein was distributed both in serine and glycine, and the radioactivity in body lipid was recovered mainly in phospholipids. The overall oxidative degradation of ¹⁴C-serine to expired ¹⁴CO₂ was amounted to about 20% of the dose in the 0 and 30 FC% groups, while it was lowered to 14%. in the 87.5 FC % group.
The activities of hepatic serine dehydratase and alanine aminotransferase were significantly increased in the 87.5 FC% group.

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Changes in glycine uptake activity were examined throughout sporulation stages of Bacillus subtilis. The maximal activity was attained at the late stages in sporulation. On the other hand, the uptake activity for L-leucine decreased rapidly when sporulation commenced. A similar increase was observed in glycine uptake activity in asporogenous mutants which were blocked at stage III or IV, but not in other mutant strains blocked at stage 0 or II. It was found that most glycine taken up was incorporated into the protein fraction. This suggests that the use of radioactive glycine as a marker is useful in protein synthesis studies of sporulation. Proteins labeled with radioactive glycine were fractionated, and specific proteins insoluble in aqueous solution but soluble in alkail solution with sodium dodecyl sulfate increased in concentration during sporulation. In kinetic studies of spore coat synthesis, the increased glycine uptake activity was related to spore coat synthesis.

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Forty nine N-substituted-α-aminonitriles were prepared and their preventive activities against Fusarium diseases were determined. Relationships between chemical structure and preventive activity were examined and the systemic movement in plants was investigated with ¹⁴C-N-allylaminoacetonitrile. Of the compounds tested, N-sec-butyl-α-aminoisobutyronitrile and N-allylaminoacetonitrile were most effective in controlling yellows of the Japanese radish which were caused by Fusarium oxvsporum f. sp. raphani. N-Allylaminoacetonitrile showed remarkable systemic movement and a potent preventive activity against Fusarium wilts of cucumber and tomato not only by the soil drench test but also by the foliar spray assay.

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The structure-activity relationships of N-alkylaminoacetonitriles and analogs possessing preventive activity against Fusarium diseases were investigated by the Hansch method. The variation in the preventive activity against yellows of the Japanese radish caused by Fusarium oxysporum f. sp. raphani was shown to be related to the variation in the steric dimension of the N-alkyl group. The amide, carboxylic acid and alcohol derivatives of N-sec-butylamino-acetonitrile were inactive while those of N-allylaminoacetonitrile showed the potent activity.

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Thirty-two N-allylamino acids were prepared, and their preventive activity against Fusarium diseases was determined. Esters of N-allyl-glycine and -sarcosine showed a strong effect in preventing yellows of the Japanese radish caused by Fusarium oxysporuwn f. sp. raphani. These compounds were also effective against Fusarium wilt of tomato by foliage treatment. The preventive activity of N-allylsarcosinates varied insignificantly with the variation of the alcohol moiety of the esters. Although n-dodecyl N-allylsarcosinate was shown to effectively control Fusarium diseases, it did not affect the growth of Fusarium on an agar medium. The preventive effect was dependent on the application time. Enhancement of the peroxidase activity and the accumulation of total phenols were seen in the plants treated with these chemicals.

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An alkalophilic bacterium, strain No. 93-1, was isolated from soil. On the basis of the morphological and biochemical characteristics, this strain was identified to belong to the genus Corynebacterium. A crude extract of this strain showed NAD-dependent dehydro-genase activity on maltose (NAD-MalDH) and glucose (GlcDH). The ratio between these two dehydrogenase activities varied by cultural conditions. And NADP was also a suitable co-factor for dehydrogenase activity on maltose (NADP-MalDH). There were remarkable differences in an optimal temperature and pH stability among these three activities. According to these experimental results, we expected that, at least, three kinds of NAD (P)-dependent dehydrogenase acted on maltose and glucose in the crude extract of this microorganism.

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Antigenicity of mannan preparation obtained by the usual method so far published and that prepared by our method was investigated with the rabbit antiserum against the intact yeast cell. The antigenicity of the former was a little less than the latter, but both mannan preparations were much less in the antigenicity as compared with the intact yeast cell. Thus the preparation of an intact surface antigen was attempted through treating the yeast whole cells successively with acetic acid, pepsin, acetone and ether. The cell envelope obtained by the procedure was almost intact microscopically and maintained the antigenic activity similar to that of the intact cells. The dry weight of the cell envelope was a half of the whole cell and its composition was sugar 60%, crude protein 32% and lipid 5%.

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Phenoloxidase was purified from the larvae of housefly, Musca domestica vicina Maquart, by a procedure involving ammonium sulfate precipitation, extensive dialysis against deionized water, hydroxylapatite column chromatography and Sephadex G-200 gel filtration. Purified phenoloxidase was homogeneous on polyacrylamide gel disc electrophoresis and ultracentrifugation. The sedimentation coefficient of phenoloxidase was 13.5 S. The molecular weight of phenoloxidase was 3.4×10⁵ by Archibald's method and 3.1×10⁵ by polyacrylamide gel disc electrophoresis. The isoelectric point of phenoloxidase was 4.7 by ampholine electrophoresis. The maximum and minimum absorptions were found at 280nm and 251nm, respectively, with purified phenoloxidase.

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A new nicotinamid adenine dinucleotide (NAD) kinase which synthesizes nicotinamid adenine dinucleotide phosphate (NADP) from NAD and metaphosphate was found in some microorganisms. The activity of this enzyme, designated tentatively as metaphosphate-dependent NAD kinase, was detected in Acetobacter, Achromobacter, Brevibacterium, Corynebacterium and Micrococcus species, but was not detected in Escherichia, Proteus and Aerobacter species. The metaphosphate-dependent NAD kinase activity of Brevibacterium ammoniagenes IAM 1645 was not affected by culture conditions, though the adenosine-5'-triphosphate (ATP)-dependent NAD kinase activity was did. The metaphosphate-dependent NAD kinase activity from B. ammoniagenes also differed from the ATP-dependent NAD kinase activity in optimal pH of reaction and stability in heating and in freezing and thawing. Of phosphate polymers tested, the potent phosphoryl donor was metaphosphate alone, and other chain and ring phosphate polymers of different degrees of condensation were not utilized by this enzyme.

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It was found that certain substances which made the bound saccharase insoluble were released together with the enzyme by treating cell wall of sugar beet seedlings with salt.
Insolubilization of the enzyme was extremely reduced by pretreatment of the substance with either deoxyribonuclease or hot perchloric acid, but not affected by the substance pretreated with ribonuclease, alkali or Pronase. UV spectrum of the purified substance showed a maximal absorption at 260nm and a minimal at 238nm. The diphenylamine test for deoxyribose and the Fiske-Subbarow test for phosphorus were both positive. These results indicate that the substance causing insolubilization of the enzyme is DNA.
Calf thymus DNA and Yeast RNA as well as the purified DNA from sugar beet seedlings were all effective in insolubilization of the enzyme. Association of the enzyme with these nucleic acids depended on the concentration of NaCl. The DNA's from sugar beet seedlings and calf thymus insolebilized the enzyme at NaCl concentrations below 0.16M, while yeast RNA below 0.04M.

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The crystal structure of SF-1836, C₅H₇NO₂, has been determined by direct methods. The crystals are orthorhombic with the space group P2₁2₁2₁ and the unit-cell dimensions are a=11.149 Å, b=9.059 Å, c=5.265 Å, z=4. SF-1836 was determined to be trans-2-azabicyclo[2.1.0]pentane-3-(S)-carboxylic acid.

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Germinating rice contained three carboxypeptidases or carboxypeptidase-like enzymes which hydrolyzed CPA (carbobenzoxy-L-phenylalanyl-L-alanine) at different pH ranges. The three CPA hydrolases which acted optimally at pH 4, 5_??_5.5, and 7. were tentatively termed CPAase-4, CPAase-5, and CPAase-7 respectively. CPAase-4 was contained in resting seeds and endosperms of germinating seeds. Its activity was maximum in resting seeds and gradually decreased during germination. CPAase-5 was absent in resting seeds, and appeared in endosperms and seedlings in the later stages after germinaion. This enzyme was predominant in mature plant leaves. CPAase-7 was detected in the young shoots and young roots during a limited period of germination. Enzymatic properties of CPAase-5 partially purified from rice leaves were very similar to those of carboxypeptidases in various plants.

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Resting rice seeds contained a carboxypeptidase which hydrolyzed CPA(carbobenzoxy-L-phenylalanyl-L-alanine) at pH 4 and this enzyme was tentatively termed CPAase-4. This enzyme is distributed in endosperm of rice grain, and its distrubution pattern was similar to that of glutelin. CPAase-4 was purified from rice bran by chromatography on CM-Sephadex and gel filtration. The molecular weight of the enzyme was estimated to be 110000. By SDS gel electrophoresis, the presence of two subunits (MW=35000 and 21000) was detected. The activity was inhibited when the enzyme had been preincubated with DFP at pH 7. The enzyme was optimally active for CPA at pH 4 and for acetyl-L-phenylalanine ethyl ester at pH 8, and was stable in a wide pH range (2.5_??_8.5).

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In order to confirm the primary structure of a delicious peptide which was isolated from extracts of the beef meat, a peptide, H-Lys-Gly-Asp-Glu-Glu-Ser-Leu-Ala-OH, was synthetized. Both of the synthetized peptide and isolated one were identical as shown in many respects.

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Influence of the components of fermentation medium upon protein production by Bacillus brevis No. 47 was investigated. Bacterial growth was highly stimulated with yeast extract or meat extract without any appreciable increase in protein accumulation. But, both growth and protein production was enhanced with increasing amount of polypepton, a commercial product of peptone. Mg ion and potassium phosphate appeared to have important roles in protein production. Higher concentrations of Mg ion stimulated growth, but prevented protein excretion. An optimal pH for protein production was about 8.
Factor(s) stimulating protein production in polypepton were fractionated by Sephadex G-25. Such an activity was detected in a fraction consisting of peptide(s) with molecular weight of 2_??_3000. Based on the time course of protein production and growth curve, it was suggested that protein accumulation is not the result of cellular autolysis.

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Among factors affecting the protein production by Bacillus brevis No. 47, glycine and L-isoleucine were found to be prominent in stimulating protein production. The simultaneous addition of appropriate amount of these amino acids resulted in the largest accumulation of proteins; namely, 12g/liter.
The mode of action of glycine and isoleucine appeared different. Isoleucine stimulated the synthesis of both extracellular and intracellular proteins, while glycine caused a considerable increase of extracellular protein accumulation with a concomitant decrease in the amount of intracellular protein. Therefore, glycine may have a function in stimulating protein excretion. Glycine made cells more sensitive to lysozyme and caused a large decrease in alanine content of the cell wall fraction. These findings supported the possibility that glycine alters cell wall structure in such a way so as to facilitate protein excretion.
The proteins produced in the presence of glycine as a whole were smaller in molecular weight than those without glycine. Amino acid composition of the proteins produced was same regardless of the presence or absence of glycine.

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API-2 degrading protease produced by Streptomyces griseoincarnatus strain No. KTo-250 was purified. Optimal pH of the protease was 9.0 which was the same as that of API-2b→API-2c converting protease. But the protease was clearly distinguishable from API-2b→API-2c converting protease: The molecular weight of the degrading protease and the converting protease were 28000 and 48000, respectively, and the former was inhibited by AP-1 and S-SI, whereas the latter was not inhibited by either. Moreover, API-2 degrading protease degraded or digested API-2c, although API-2b→API-2c converting protease did not degrade it.

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Effects of various substances on crystalline quinolinate phosphoribosyl transferase (EC 2.4.2.19) activity from Alcaligenes eurrophus subsp. quinolinicus were investigated. The enzyme activity was inhibited by divalent cations but not by mono- and trivalent cations. Phosphorous compounds like ATP inhibited the enzyme activity but this inhibition was completely recovered by raising the Mg²⁺ ion concentration. With regard to the effect of reaction products, nicotinic acid mononucleotide and inorganic pyrophosphate inhibited but CO₂ did not. Nicotinic acid mononucleotide inhibited competitively for quinolinic acid and 5-phosphoribosyl-1-pyrophosphate. The effect of glycerol on the enzyme activity was different by pH. Glycerol caused a decrease in Km values for quinolinic acid and 5-phosphoribosyl-1-pyrophosphate and the turnover rate. The enzyme activity was not affected by 2-mer-captoethanol even at a final concentration of 0.3_M.

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A method is presented for the isolation of a lectin from a Japanese cultivar of Phaseolus vulgaris seed by an affinity chromatography on the heat-denaturated porcine thyroglobulin-Sepharose. The lectin is a glycoprotein whose molecular weight is 120000; it consists of four apparently identical subunits, held together by non-covalent forces. The isoelectric point is 5.5 and the sedimentation constant is 6.66 S at pH 5.3. The lectin is nonspecific in agglutination for any types of human erythrocytes. The lectin induces mitosis in human lymphocytes at concentration between 10 and 100μg per 3×10⁸ lymphocytes. The hemagglutinating and mitogenic activities are inhibited by N-acetyl-D-galactosamine and some simple sugars.

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The intracellular distribution of UQ in BY-2 cells was examined. The distribution of UQ is well correlated with the distribution of Cyt c oxidase activity in mitochondria. Mitochondrial preparations from BY-2 cells examined showed a high degree of both coupling and respiratory control. They were further purified by sucrose density gradient centrifugation and UQ and Cyts contents in the purified mitochondria were determined. The UQ and Cyts (b and c) contents and their molar ratios in BY-2 cell mitochondria are similar to those of mung bean seedling mitochondria.

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The chemical modification of validamycin A was examined. One of the synthetic validamy-cin analogs, N[(1S)-(1, 4, 6/5)-4, 5, 6-trihydroxy-3-hydroxymethyl-2-cyclohexenyl] [O-β-2-amino-2-deoxy-D-glucopyranosyl-(1→3)-(1S)-(1, 2, 4/3, 5) -2, 3, 4- trihydroxy-5-hydroxymethyl-cyclohexy] amine monohydrochloride had almost the same activity against the sheath blight of rice plants as that of validamycin A. Partial synthesis of validamycin A was also examined.

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To study the mechanism of the formation of the heat-stable form of ovalbumin (s-ovalbumin), comparisons were made about the properties of ovalbumin and s-ovalbumin in native state. Although gross structural difference could not be found, some minor differences were clearly noted in some cases such as the DEAE-cellulose chromatographic elution pattern, the isoelectric focusing and the titration curve of both proteins. All these results seem to show that changes in the surface charge occur during ovalbumin-s-ovalbumin transformation.

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NADP-dependent aldehyde dehydrogenase (EC 1.2.1.4) has been crystallized for the first time from cytosol fraction of Gluconobacter melanogenus IFO 3293. Purification of the enzyme was performed by column chromatographies on DEAE-Sephadex A-50 and hydroxylapatite. The enzyme was purified about 60-fold with an overall yield of 33%. Crystalline enzyme was found homogeneous in disc gel electrophoresis and analytical ultracentrifugation. The enzyme was highly specific for NADP and completely inactive with NAD. Abundance of the enzyme in cytosol fraction and its predominant occurrence through acetic acid bacteria indicated the importance of the enzyme in ethanol assimilation. NADPH formed in aldehyde oxidation was reoxidized into NADP by the old yellow enzyme system which existed in the same cytosol fraction of the organism. Cyclic regeneration of NADP smoothly occurred in the presence of acetaldehyde dehydrogenase, old yellow enzyme and catalase, even when a limited amount of NADP or NADPH was present in the reaction mixture.

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A facile procedure to synthesize cyclitols from cyclohexene was examined. 1-O-Benzyl-cyclohex-2-enol (1-benzyloxycyclohexene-2) (I) was prepared from cyclohexene by bromination and successive treatment with sodium benzyloxide. Compound I was converted, by using the same procedure, into DL-trans-1, 2-di-O-benzylcyclohex-3-enediol (IIIa), DL-trans- and meso-cis-1, 4-di-O-benzylcyclohex-2-enediols (IIIb and IIIc). The main product (IIIa) was converted into dihydroconduritols (IXc and Xc) via the corresponding di-O-benzylcyclohexanetetrols.

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A facile method to synthesize a variety of aminocyclitols from cyclohexene was examined. DL-traps-2-acetamido-1-O-benzylcyclohex-3-enol (IIIa) and DL-trans- and cis-4-acetamido-l-O-benzylcyclohex-2-enols (IIIb and IIIc) were synthesized from cyclohexene via the corresponding azide derivatives (IIIa-c). The major products IIIa and IIIb were converted into dyhydro-conduramine derivatives via epoxides (VIII and IX) or by cis-hydroxylation with osmium tetroxide.

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A broad range of conditions was used for lysinoalanine formation in ribonuclease A and lysozyme. High alkali concentrations and high temperatures accelerated the production of lysinoalanine, but only limited amounts of lysinoalanine, less than that corresponding to the lysine and cystine residues in the original protein, were generated in the proteins tested. Neither aggregation nor extensive hydrolysis of protein was observed when the maximum amounts of lysinoalanine were generated. Tryptic hydrolysis of proteins decreased lysinoalanine formation. Addition of derivatives of lysine or cystine to the protein solution prior to alkaline treatment did not increase the amount of lysinoalanine formed. These data support the suggestion of Bohak (Z. Bohak, J. Biol. Chem., 239, 2878_??_2887 (1964)) that lysinoalanine formation is an intramolecular reaction in the protein molecule and that some intrinsic features in the amino acid sequence would facilitate lysinoalanine formation. These suggestions are consistent with the results found for soybean protein and casein.

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Soybean seedlings were grown at 28°C in the dark or the light for 12 days, and four classes of sterol lipids, sterol esters (SE), free sterols (St), acylated steryl glycosides (ASG) and steryl glycosides (SG), were isolated from the axis and root, respectively. Each sterol lipid (SE, ASG and SG) obtained was hydrolyzed and then divided into sterol, fatty acid and/or sugar fractions. The hydrolysates and St were analyzed mainly by gas-liquid chromatography (GLC).
During germination, the amounts of ASG markedly increased, especially in the light-grown seedlings, whereas SE maintained almost constant values. The compositions and changing patterns of the sterol in sterol lipids were similar between the dark-grown and light-grown seedlings, and among epicotyl, hypocotyl and root. The changing patterns of fatty acid composition differed between SE and ASG. SE was found to have high proportions of unsaturated fatty acids, whereas ASG had high proportions of saturated fatty acids. In general, the changes in constituents of sterol lipids were more marked in the light-grown seedlings than in the dark-grown ones.

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Three 7-hydroxylkanoic acids not previously reported in microorganisms were found in extracts from saponified whole cells of Mucor spp. grown under oxygen limiting conditions. On the basis of evidence from mass spectrometry, infrared spectroscopy, gas chromatographic procedures and chemical conversions, they were confirmed to be 7-hydroxydecanoic, 7-hydroxydodecanoic and 7-hydroxytetradecanoic acids. The racemates of the former two acids were synthesized and compared with the isolates. These acids were found in the hydrolyzates of extractable and residual parts of lyophilized cells with a chloroformmethanol (2:1) mixture. The hydroxy acid contents of eight species of the genus Mucor grown in stationary liquid medium were also determined.

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The synthesis of (S)-(+)-methyl β, γ-dihydroxy-α-methylenebutyrate and (S)-(-)-tulipalin B, antibacterial substances isolated from Spiraea thunliergii Sieb. has been accomplished starting from isopropylidene-D-glyceraldehyde.

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