Bacterial strain No. 655 was isolated from soil by screening of microorganisms producing xanthine oxidase inhibitor. The taxonomical properties indicated that it belonged to a variety of Alcaligenes aquamarinus. The xanthine oxidase inhibitor produced by Alcaligenes aquamarinus No. 655 was isolated from the culture broth and purified and the properties of the purified inhibitor were investigated. The inhibitor was identified as 2, 8-dihydroxyadenine. The inhibitor showed 50% inhibition at a concentration of 3×10^-6M (0.5 μg/ml) on xanthine oxidase from rat liver and 2×10^-6M (0.33 μg/ml) on that from milk, respectively. The strain had the following Ki values; 7.0×10^-7M and 5.4×10^-7M against rat liver and milk xanthine oxidase respectively. The mode of action of the inhibitor was of the competitive type.

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Nuclease P₁ from Penicillium citrinum was found to be produced in a form of complex with malonogalactan (a galactan, 1, 5-β-galactofuranoside polymer esterfied with malonic acid at position 3) in the culture on wheat bran. Neither nuclease P₁-malonogalactan complex nor malonogalactan was produced in a liquid medium. Nuclease P₁-malonogalactan complexes, P₁-MG I, II, and III were purified from an aqueous extract of the culture on wheat bran. The most anionic complex, P₁-MG III, was composed of the protein, carbohydrate and malonic acid in the ratio of 1:2.6:0.5 (w/w). The complex was not dissociated by purification procedures including fractionations with acetone and ammonium sulfate, gel filtration and DEAE-cellulose chromatography. A malonogalactan-specific carboxylesterase was found in culture of the same mold on wheat bran. Nuclease P₁-malonogalactan was demalonylated by the esterase to yield nuclease P₁-galactan. The binding of nuclease P₁ to galactan was rather loose so that nuclease P₁-galactan complex was partially dissociated by DEAE-cellulose chromatography. Attempt to reconstitute the complex from nuclease P₁ and malonogalactan upon mixing was unsuccessful. Exogenously supplemented nuclease P₁ did not associate with malonogalactan in the growing culture on wheat bran, either.
Several extracellular enzymes such as RNase, β-galactosidase and protease were also found in a form of complex with malonogalactan in the culture on wheat bran.

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Properties of nuclease P₁-malonogalactan complex (P₁-MG) were compared with those of the polysaccharide-free nuclease P₁. Significant difference was not observed between them in phosphomonoester splitting activity, but marked differences were observed in nucleolytic activity as follows: (1) The pH optima of P₁-MG for RNA and heat-denatured DNA were lower than those of nuclease P₁. (2) At lower than 0.001 of ionic strength, RNA and heatdenatured DNA were attacked hardly by P₁-MG, but attacked well by nuclease P₁. (3) The increase in hydrolysis rate of RNA or heat-denatured DNA with an elevation of temperature from 37°C to 70°C was not so marked in P₁-MG as compared with nuclease P₁. (4) P₁-MG hydrolyzed polynucleotides, especially native DNA, much slower than nuclease P₁.
Influence of ionic strength, pH and temperature on the nucleolytic activity of nuclease P₁-galactan (P₁-G), which was prepared by demalonylating P₁-MG enzymatically, was similar to that of nuclease P₁, except that the activity of P₁-G for native DNA was much lower than nuclease P₁.

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P₁ type nuclease, which hydrolyzes RNA and heat-denatured DNA completely into 5'-mononucleotides and also shows 3'-nucleotidase activity, was widely distributed among various species belonging to the genus Penicillium such as P. expansum, P. notatum, P. steckii and P. meleagrinum. P₁ type nucleases isolated from these strains were produced in a form of complex with malonogalactan when molds were grown on wheat bran. These enzymes showed similar characters in heat-stability (stable at 60°C), temperature optimum (60 to 70°C for RNA and heat denatured DNA, and 70°C for 3'-AMP) and sensitivity to EDTA. The enzymes from P. steckii and P. expansum were immunologically co-related to nuclease P₁.
In addition, many strains of Penicillium produced base-nonspecific RNases forming 3'-mononucleotides via 2':3'-cyclic nucleotides. These RNases showed similarity in heat-lability (completely inactivated at 60°C), temperature optimum (45 to 50°C), sensitivity to Zn²⁺ and Cu²⁺, and relative hydrolysis rate toward 2':3'-cyclic nucleotides (A_??_;C>U_??_;G).

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The key enzymes, 3-hexulose phosphate synthase and phospho-3-hexuloisomerase, of the pentose monophosphate pathway for formaldehyde fixation were purified from a methanolgrown bacterium 77 a, and their enzymatic properties were investigated. The condensation product between formaldehyde and D-ribulose 5-phosphate by 3-hexulose phosphate synthase was identified as D-arabino-3-hexulose 6-phosphate, on the bases of UV-absorption spectrum, pH stability and color reactions with several reagents on thin-layer chromatography. The apparent Km value of 3-hexulose phosphate synthase for each substrate was determined: formaldehyde, 0.74; D-ribulose 5-phosphate, 0.081; D-arabino-3-hexulose 6-phosphate, 0.036mM. The apparent Km values of phospho-3-hexuloisomerase for D-arabino-3-hexulose 6-phosphate and D-fructose 6-phosphate were found to be 0.029 and 0.67mM, respectively.
No activity of phospho-3-hexuloisomerase was able to detect in the cell-free extract of methanol-utilizing yeasts, Kloeckera sp. No. 2201 and Hansenula polymorpha DL-1, under the condition tested.

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Acetate: CoA ligase (AMP) (EC 6. 2. 1. 1, acetyl CoA synthetase, ACS) was found in sweet potato root tissue. When the tissue was infected by Ceratocystis fimbriata, activity of the enzyme was increased several-fold compared to that in fresh tissue and decreased rapidly after reaching a maximum. When cut tissue was incubated without inoculation, the activity was rather decreased.
The fact that terpenes such as ipomeamarone are synthesized in diseased tissue but not in cut tissue, strongly suggests that increased activity of ACS in diseased tissue mainly participates in terpene biosynthesis supplying acetyl CoA.

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Biotin requirements for the growth of a mutant (KY-21-1-25-43-101) of a biotin-producing bacterium (Bacillus sp. KY-21-1-25) was examined and the characteristics of the biotinvitamers produced by the mutant was also examined. Either biotin or dethiobiotin supported the growth of the mutant, but pimelic acid, 7-keto-8-aminopelargonic acid and 7, 8-diamino-pelargonic acid did not support the growth of the mutant. Biotin-vitamers produced from pimelic acid by the mutant were identified as 7-keto-8-aminopelargonic acid and 7, 8-diamino-pelargonic acid. Dethiobiotin was not produced from pimelic acid by the mutant. Intact cells of the mutant had no capacity to synthesize dethiobiotin from 7, 8-diaminopelargonic acid, while the parent strain had a high potency for dethiobiotin synthesis from 7, 8-diamino-pelargonic acid. The mutant had high potency for biotin production from dethiobiotin that was the same as that of the parent strain.

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Biotin-vitamers produced from pimelic acid by a biotin-producing bacterium, Bacillus sp. KY-21-1-25, were purified from the culture filtrate and some properties of the isolated vitamers were examined. The dominant component of biotin-vitamers was purified by the column chromatography, crystallized and identified as dethiobiotin. Three components among several minor components observed were partially purified. One of them was assumed to be a combined form of biotin-d-sulfoxide. Two other components which were assumed to be combined form of dethiobiotin were mainly converted to biotin and partly converted to dethiobiotin by intact cells of the bacterium.

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The regulation of xanthine dehydrogenase formation in a strain of Streptomyces cyanogenus was studied in the presence and absence of various carbon and nitrogen sources. If glucose and ammonium were added together to medium, almost no increase was observed in the enzyme activity. The enzyme formation appeared to be influenced at the level of transcription. If glucose but no ammonium was added, the increase in the activity was also reduced although moderately, and the effect seemed to be at the level of translation. Glucose was not only inhibitory to the enzyme formation but also transitorily promotive upon the addition to medium. If glucose or glucose and ammonium were removed from medium, the formation of xanthine dehydrogenase was relieved of the repression and induced by hypoxanthine, xanthine and 6, 8-dihydroxypurine. Intracellular free amino acids were found to decrease considerably under the conditions where the enzyme could be synthesized. Hypoxanthine appeared to be utilized as nitrogen source by the organism, and, therefore, the role of the regulation of xanthine dehydrogenase formation has been discussed in terms of nitrogen metabolism of this organism.

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Branched chain amino acid aminotransferase was partially purified from Pseudomonas sp. by ammonium sulfate fractionation, aminohexyl-agarose and Bio-Gel A-0.5m column chromatography.
This enzyme showed different substrate specificity from those of other origins, namely lower reactivity for L-isoleucine and higher reactivity for L-methionine.
Km values at pH 8.0 were calculated to be 0.3mM for L-leucine, 0.3mM for α-ketoglutarate, 1.1mM for α-ketoisocaproate and 3.2mM for L-glutamate.
This enzyme was activated with β-mercaptoethanol, and this activated enzyme had different kinetic properties from unactivated enzyme, namely, Km values at pH 8.0 were calculated to be 1.2mM for L-leucine, 0.3mM for α-ketoglutarate.
Isocaproic acid which is the substrate analog of L-leucine was competitive inhibitor for pyridoxal form of unactivated and activated enzymes, and inhibitor constants were estimated to be 6mM and 14mM, respectively.

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Besides reticulol, the strain MD611-C6 produced two compounds which inhibited cyclic nucleotide phosphodiesterases [EC 3. 1. 4. C.] These substances were isolated and their structures were elucidated to be 8-hydroxy-6, 7-dimethoxy-3-hydroxymethylisocoumarin (II) and 6, 8-dihyroxy-7-methoxy-3-hydroxymethylisocoumarin (III). Concentrations of II and III for 50% inhibition of cAMP phosphodiesterase were 3.97×10^-4M and 1.26×10^-3M, respectively.

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The identity of α-ovomucins obtained from the sonicated insoluble ovomucin (Sα-OM (I)) and soluble ovomucin (Sα-OM (S)) was demonstrated by carbohydrate and amino acid analyses, circular dichroism, ultracentrifugation and electrophoresis, and it was concluded that Sα-OM (I) was the same component as Sα-OM (S).
Physicochemical properties of β-ovomucins obtained from the two kinds of ovomucin (Sβ-OM (I), Sβ-OM (S)) were compared with each other by the same methods as described above. Sβ-OM (S) consisted of β-ovomucin and a small amount of α-ovomucin bound with it, while Sβ-OM (I) consisted of only β-ovomucin.
Since the insoluble ovomucin contained β-ovomucin two and a half times as much as the soluble ovomucin did, it was suggested that the difference in the content of β-ovomucin was mainly responsible for the differences in viscous property, molecular weight and solubility between the insoluble and the soluble ovomucins.

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Dibenzothiophene (DBT) was oxidized with supplement of energy source such as lactate or glycerol by resting cells of Pseudomonas jianii. Necessity of the supplement was in agreement with the phenomenon of co-metabolism. DBT oxidizing enzymes were induced by benzene ring having no side chain such as DBT, naphthalene, and anthracene. Supplemental substance, co-substrate, was used as energy source for synthesis of the enzymes. Induction of the enzymes was significantly repressed by presence of glucose, and the repression could be overcome by addition of cyclic-3', 5'-adenosine monophosphate (cAMP).

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In order to obtain basic information on ubiquinone (UQ) formation by BY-2 cells in suspension culture, effects of inorganic phosphate and nitrogen sources and such physical factors as initial pH, light irradiation, shaking condition and temperature were investigated. The concentration of phosphate and nitrogen sources had no marked effect, but the ratio of ammonium nitrogen to nitrate nitrogen was significantly effective on UQ formation. The UQ content in BY-2 cells tends to increase at higher ratios of ammonium nitrogen. The increase in the UQ content was recognized at higher concentrations of 2, 4-D, especially with lower concentrations of sucrose. Physical factors had no marked effect on UQ formation except temperature. The UQ content was considerably raised at higher temperatures.

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IgG type of immunoglobulins was isolated from normal bovine colostrum and further fractionated by anion-exchange chromatography to yield IgG2 and IgG1 fractions. Immunoelectrophoresis showed that the IgG1 fraction contained a heterogeneous population with different charges, but no further structural differences were found on both digestion and reduction experiment within this IgG1 population.
The IgG2 fraction showed strong resistance to digestion with pepsin and papain, while the IgG1 fraction was easily digested. Treatment with cysteine and DTT showed that the IgG2 fraction was easily reduced, producing an appreciable amount of H-L half molecules, H chains, and L chains. Under identical conditions, however, only a negligible amount of H-L and H was produced from the IgG1 fraction. Thus it was suggested that the differential susceptibility to proteolytic enzymes found between IgG2 and IgG1 should not be correlated with the difference in the number of interheavy chain disulfide binding.

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Two constituent polypeptide chains were isolated from performic acid-oxidized ricin D by DEAE-cellulose column chromatography in phosphate buffer, pH 7.0, containing 8M urea or from reduced-carboxymethylated ricin D by gel filtration on Sephadex G-75 followed by DEAE-cellulose column chromatography in Tris-HCl buffer, pH 8.5. Amino acid analyses of the isolated two chains revealed that they were distinct molecules possessing similar molecular weights of near 30, 000 and linked by one disulfide bond in ricin D.

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Two constituent polypeptide chains of native or iodinated ricin D were separated by affinity chromatography on Sepharose 4 B after reduction with β-mercaptoethanol, and purified by DEAE-cellulose column chromatography.
The hybrid molecule between the native Ile chain and the iodinated Ala chain was easily formed by reduction-reoxidation of a mixture of both isolated chains. Its toxicity to mice was about 20% of that of ricin D and about ten fold that of the iodinated ricin D.
On the other hand, the hybrid molecule between native Ala chain and maleyl Ile chain was obtained by reduction-reoxidation of a mixture of native and maleyl ricin D, and its toxicity to mice was about 15% of that of ricin D and about four fold that of maleyl ricin D.
These results suggest that tyrosine and lysine residues in each chain of ricin D are involved in the toxic action of ricin D.

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Twenty tryptic peptides were isolated from the performic acid-oxidized Ile chain of ricin D by Dowex 1×2 column chromatography followed by paper chromatography. The amino acids contained in these peptides accounted for 218 out of 266 residues in the whole protein. The amino acid sequences of nine peptides were determined by manual liquid phase or automatic solid phase Edman degradation, and N- and C-terminal sequences of the Ile chain of ricin D were established to be NH₂-Ile-Phe-Pro-Lys-Gln-Tyr-Pro-Ile-Ile- and Cys-Ala-Pro-Pro-Pro-Ser-Ser-Gln-Phe, respectively.

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The effects of temperatures ranging from 10°C to 35°C on sterol and fatty acid production and hydroxymethylglutaryl CoA reductase (EC 1. 1. 1. 34, HMGCoA reductase) activity have been examined. Growth, based on dry weight, was maximal at 25°C to 30°C. Sterol production and the reductase activity were highest at 15°C after 28_??_32hr incubation when the total fatty acids were minimal. Fatty acid unsaturation generally increased with decrease in temperature.

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To elucidate the mechanism of light-activation of pyruvate P₁ dikinase in maize leaf, the nactive form was purified to homogeneity from dark-treated leaves using an activation system to locate it. The purification procedure included ammonium sulfate-fractionation followed by conventional chromatography.
The homogeneous enzyme after maximal activation had a specific activity comparable to that of the active enzyme obtained from non-dark-treated plants. The enzyme was indistinguishable from the active one in its molecular size and charge and in the amino acid composition of its acid-hydrolysate.

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Kinetic studies on the active site of pig serum α-glucosidase and buckwheat α-glucosidase catalyzing the hydrolyses of maltose and soluble starch were made. The kinetic features obtained by the experiments with the mixed substrates, the linearity of Lineweaver-Burk plots, and the dependence of the apparent Michaelis constants and the apparent maximal velocities on the fraction ƒ of maltose in the mixed substrate solutions, that is, ƒ=maltose/(maltose+soluble starch), were in good agreement with those expected for the single active site catalyzing the hydrolyses of both substrates. From the results it was concluded that these enzymes attacked maltose and soluble starch by the single active site mechanism.

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Gallic acid, methyl gallate, dehydrodigallic acid, three tannic constituents named MP-2, MP-3, MP-4 and a related substance MP-10 were isolated from chestnut galls by solvent fractionation and column chromatography. Hydrolysis with tannase revealed the components of these tannic substances as follows, MP-2: D-glucose, gallic acid and compound I (3, 4, 5-trihydroxybenzyl alcohol); MP-3 and MP-4: D-glucose, compound I and compound II (dehydrodigallic acid); MP-10: D-glucose and compound I.

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The chemical structures of the tannic substance MP-2 and the related compound MP-10 isolated from chestnut galls were established to be 2, 6-dihydroxy-4-galloyloxymethylphenyl-β-D-glucoside (4) and 2, 6-dihydroxy-4-hydroxymethylphenyl-β-D-glucoside (1), respectively, on the basis of spectral data and enzymatic degradation products.

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Isopimpinellin, bergapten, xanthotoxin, kokusagine, evoxine and japonin were isolated and identified in the leaves of Orixa japonica (Rutaceae) as feeding inhibitors against Spodoptera litura F. (Noctuidae). Isopimpinellin exhibited the most potent activity among above substances at the feeding inhibitory test. The isolated evoxine was the optical antipode of the previously reported one.

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The aim of this research was to examine the inhibitory effect of aflatoxin B₁, one of the most potent hepatocarcinogen, on the translational step in mouse liver. It has been shown that polysomes were released in vitro from microsomal membrane prepared from rat liver by incubation with aflatoxin B₁ and that this release of ribosomes was prevented by addition of corticosterone in the incubation medium.
In this paper, the same phenomenon was proved to occur in vivo by an improved fractionation methods, in which ribosome-distributions can be analyzed quantitatively, not only between free and membrane-bound states but also between monosomes and polysomes. Administration of aflatoxin B₁ to mice induced reductions of membrane-bound ribosomes and polysomes, with concomitant increases of free ribosomes and monosomes in liver. Simultaneous administration of corticosterone prevented this alteration of ribosome-distributions.
From these results, it was deduced that a release of polysomes from membrane occurred primarily by administrating aflatoxin, which then caused a shortening of half-life of mRNA on polysomes, resulting in an increase of the amount of monosomes.

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Several N¹-aryl-N⁴-(O, O-diethylthiophosphoryl) sulphanilamides and N¹-aryl-N⁴-(N, N-diarylthiophosphoramidic) sulphanilamides have been prepared with a view to study their pesticidal properties. Six such compounds have been tested against two species of fungi.

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While the isolated soil bacterium, Arthrobacter aurescens, had been found to secrete a chondroitinase AC into the culture medium, it was recognized that the chondroitinase preparation obtained from the broth of the strain cultured in a jar fermentor contained at least three electrophoretically separable components of the enzyme. Each component (named chondroitinase I, II, and III, respectively) was separated from the others by use of isoelectric focusing and then the enzymic properties of each component were examined.
The value of isoelectric point of each component differed from one another (I, 5.5; II, 5.9; III, 6.4). However, no difference could be detected in pH-stability (pH 5.0 to 7.5), optimum pH (pH 6.0), thermal stability (below 45°C), optimum temperature (50°C), substrate specificity (chondroitin A and C lyase), mode of action (endo type, the degree of multiple attack was 3.0 to 3.1), and the dissociation constant of the enzyme-substrate complex (Km for chondroitin sulfate C was 3.3_??_3.6×10^-4M). Thus the electrophoretically separable components with the chondroitinase activity were thought to be the multiple forms of the enzyme, chondroitinase AC. There was also no appreciable difference in the molecular weight values of those chondroitinase components, however, considerable difference was detected in the carbohydrate content of those components.

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The pD dependence of chemical shifts of the methylene protons in glycine and glycinepeptides (up to tetraglycine) in D₂O solution has been investigated by proton magnetic resonance spectroscopy. The different kinds of the methylene protons in glycinepeptides show different chemical shifts. Furthermore, their chemical shifts significantly depend on the pD of the solutions, and reflect the dissociations of the carboxylic and amino groups. The chemical shifts of the methylene protons in peptides in D₂O solution have been determined in the pD region from ca. 1 to 14. In addition, the dissociation constants, pK₁ and pK₂, in D₂O solution are estimated from the present results as follows; pK₁=2.84 and pK₂=10.84 for glycine, pK₁=3.74 and pK₂=9.34 for diglycine, pK₁=4.04 and pK₂=9.04 for triglycine, and pK₁=3.94 and pK₂=8.94 for tetraglycine.

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Mutants of Saccharomyces cerevisiae defective in acyl-CoA synthetase (EC 6. 2. 1. 3) were isolated. The mutants were concentrated by the radiation-suicide technique with the use of tritiated palmitic acid. Selection of the mutants was based on the premise that acyl-CoA synthetase activity would become indispensable when yeast cells in which fatty acid synthesis de novo is blocked are grown in a medium supplemented with fatty acid. The mutant strains isolated exhibited low acyl-CoA synthetase activity in vitro. Furthermore, they accumulated markedly more of the incorporated palmitic acid in the nonesterified form than did the wildtype strain. Some of the mutants showed thermosensitive acyl-CoA synthetase activity, indicating a mutation of the structural gene of the enzyme. Genetic studies on these mutants indicated that their phenotype resulted from a single, recessive mutation of a nuclear gene, designated faa 1 (fatty acid activation).

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