A new colorimetric method for the quantitative microanalysis of long-chain N-acylglutamic acids is proposed. This method consists of two steps in which a pinacyanol derivative derived from a long-chain N-acylglutamic acid and pinacyanol dye is extracted with an organic solvent, and the color intensity of the extract is spectrophotometrically measured. Long-chain N-acylglutamic acids were found to react speifically with pinacyanol chloride to form their pinacyanol derivatives under a weakly acidic condition. These derivatives were extracted with chloroform and determined by measuring the absorbancy of the solution at 610mμ within the range of 0.01_??_0.15mg. Several factors affecting the analysis and standard procedure of the determination were also investigated.

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As a new adsorbent of lysozyme-like enzymes, chitin coated (CC-)cellulose was prepared. CC-cellulose was stable and had good flow properties for use in column chromatography. Affinity chromatography with CC-cellulose showed that 3_??_5mg of lysozyme/ml resin was adsorbed specifically and desorbed quantitatively under mild conditions. The utilities of the method of affinity chromatography with CC-cellulose are discussed.

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Effect of 4-thiouridine, which was proved to inhibit selectively and “light-reversibly” the synthesis of chloroplast ribosomal RNAs in radish cotyledons, on the photo-induced development of photosystem I, II and a complete electron transport chain was investigated with plastids obtained from 4-thiouridine treated dark-grown radish cotyledons after various times of development in the light. It was demonstrated that the 4-thioridine treated chloroplasts showed a higher activity of photoreduction than the control untreated chloroplasts in every system on a chlorophyll basis during the development after 24 hr illumination. This specific activity decreased in both chloroplasts, as the chloroplasts matured with the time of illumination. The activity per g of fresh cotyledons treated with 4-thiouridine, especially in the early stage of development, was lower than that of ones untreated with the drug because total chlorophyll content was poor, but the activity of the former was enhanced with the increase of total chlorophyll content upon illumination while the activity of the latter decreased on 24hr illumination. Moreover, Hill reaction measurements showed that 4-thiouridine treated chloroplasts were saturated at lower light intensity than untreated ones inspite of the same content of chlorophyll in both the chloroplasts: photoreduction of NADP⁺ was saturated at 3000 lux for the former and at 5000 lux for the latter. Based upon these results, specific development of the chloroplast is discussed.

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Tryptic (LMM Fr 1) and CNBr-treated (LMM-C) rod portion of myosin molecule were prepared from rabbit skeletal muscle myosin. An attempt was made to clarify the difference between LMM Fr 1 and LMM-C as measured by solubility and some physico-chemical techniques. The rigid structure of LMM Fr 1 melted into a random coil as temperature of the solution increased and the value of b₀ and reduced viscosity did not show full recovery upon gradual cooling. The situation of LMM-C, on the other hand, showed the higher thermostability than LMM Fr 1. As stated in our previous paper, ¹ we have considered that the rod portion of myosin molecules is substantially thermostable.

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Culture conditions for the preparation of cells containing high tyrosine phenol lyase activity were studied with Erwinia herbicola ATCC 21434. Adding pyridoxine to the medium enhanced enzyme formation, suggesting that it was utilized as a precursor of the coenzyme, pyridoxal phosphate. Glycerol plus succinic acid; amino acids, such as, DL-methionine, DL-alanine and glycine; and metallic ion, ferrous ion promoted enzyme formation as well as cell growth. Adding L-tyrosine, as inducer, to the culture medium was essential for enzyme formation. However, when large amounts of L-tyrosine were added, the enzyme formation was repressed by the phenol liberated from L-tyrosine. In fact, formation of the enzyme was enhanced by removing phenol during cultivation. L(D)-Phenylalanine or phenylpyruvic acid had a synergistic effect on the induction of enzyme by L-tyrosine.
Cells with high enzyme activity were prepared by growing cells at 28°C for 28 hr in a medium containing 0.2% L-tyrosine, 0.2% KH₂PO₄, 0.1% MgSO₄•7H₂O, 0.001% FeSO₄•7H₂O, 0.01% pyridoxine•HCI, 0.6% glycerol, 0.5% succinic acid, 0.1% DL-methionine, 0.2% DL-alanine, 0.05% glycine, 0.1% L-phenylalanine and 120ml/liter hydrolyzed soybean protein in tap water with the pH controlled at 7.5 throughout cultivation.

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Reaction conditions for the synthesis of L-tyrosine or L-dopa from DL-serine and phenol or pyrocatechol were studied with intact cells of Erwinia herbicola (ATCC 21434) containing high tyrosine phenol lyase activity. The optimum pH for this reaction was around 8.0, and the optimum temperature range was between 37_??_40°C for the synthesis of L-tyrosine and between 15_??_25°C for that of L-dopa. Sodium sulfite and EDTA were added to protect the synthesized L-dopa from decomposition. As high concentrations of phenol or pyrocatechol denatured the enzyme, each substrate was fed to maintain the optimum concentration during incubation.
The reaction mixture (100ml) containing 4.0g of DL-serine, 1.0g of phenol or 0.7g of pyrocatechol, 0.5g of ammonium acetate and the cells, was incubated. During incubation, phenol or pyrocatechol was fed at intervals to maintain the substrate at the initial concentration. 5.35g of L-tyrosine or 5.10g of L-dopa was synthesized in 100ml of the reaction mixture.

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Susceptibility to hydrogen peroxide of the erythrocytes from chicks and rats fed dilauryl succinate and related compounds with and without supplementation of dl-α-tocopheryl acetate was determined.
Dilauryl succinate, lauryl alcohol, n-decyl alcohol, myristyl alcohol, and lauraldehyde were confirmed to make the erythrocytes from the chicks fragile. Supplemented dl-α-tocopheryl acetate of 200mg per kg of diet completely prevented the hemolysis induced by these compounds. Dilauryl succinate also makes the rat's erythrocytes fragile and supplemented dl-α-tocopheryl acetate prevented the hemolysis of the rats, but ethoxyquin was not. The symptoms of encephalomalacia in the chick is preceded by increased hemolysis value of the erythrocytes, and this hemolysis value dropped after the appearance of encephalomalacia.

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Relation between fatty acid composition of cellular phospholipids and the excretion of L-glutamic acid was investigated using Corynebacterium alkanolyticum GL-21 (a glycerol auxotroph).
When grown on n-hexadecane, the proportion of unsaturated fatty acids was higher in L-glutamic acid-accumulating cells than in L-glutamic acid-nonaccumulating cells. When grown on fructose or acetic acid, the reverse relation was observed. Moreover, cells containing no oleic acid produced L-glutamic acid from n-pentadecane.
These results suggest that the membrane permeability to L-glutamic acid is not always controlled by the cellular content of unsaturated fatty acids.

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Relation between cellular phospholipids and L-glutamic acid excretion was investigated using Corynebacterium alkanolyticum GL-21 (a glycerol auxotroph).
When strain GL-21 was cultured in glycerol-limited medium which contained n-hexadecane, acetic acid or fructose as carbon source, there occurred the limitation of cellular phospholipid content and the over-accumulation of L-glutamic acid in the broth. Two-dimensional thin-layer chromatograms provided evidence that both the parent and the mutant strains contained the same phospholipids such as cardiolipin, hosphatidylethanolamine, phosphaditylglycerol, phosphatidylinositol and phosphatidic acid. Limited supply of glycerol to the mutant did not greatly alter the proportions of the individual phospholipids.

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L-Arabinose isomerase (L-arabinose ketol-isomerase, EC 5. 3. 1. 4) was demonstrated from the L-arabinose-grown cells of Streptomyces sp. which was isolated from sea water. The enzyme was purified by MnCl2 treatment, fractionation by polyethylene glycol and by column chromatographies on Sephadex G-150 and DEAE-cellulose. The purified enzyme was specific only for L-arabinose and the Michaelis constant for L-arabinose was 40mm at pH 7.5. Manganese or cobalt ions were effective for the enzyme activity after dialysis against EDTA. The enzyme activity was inhibited competitively by L-arabitol, ribitol and xylitol, of which inhibition constants were 1.1, 1.0, and 15mm, respectively.

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We examined the effect of various agents on the cell division of E. coli B(Sm^r) irradiated with ultraviolet (UV) light. It was found that the impaired cell division was reversed by one of various agents such as higher fatty acids, lower alcohols, terpineol, phenethyl alcohol, streptomycin, ribonuclease and EDTA. These agents, except RNase, showed the maximum activity of recovery just below a concentration causing a complete suppression of cell growth.

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UDP-glucose pyrophosphorylase of Jerusalem artichoke tubers was purified 90-fold over the crude extract. The purified enzyme preparation absolutely required magnesium ions for activity. Cobalt ions were 60% as effective as magnesium ions; other divalent cations including manganese showed little or no effect. This enzyme had a pH optimum of 8.5 and a temperature optimum of 40°C. ATP and UDP inhibited the activity of this enzyme in both forward and backward directions. Km values for UDP-glucose, inorganic pyrophosphate, glucose-l-phosphate and UTP were determined to be 4.45×10^-4M, 2.33×10^-4M, 9.38×10^-4M and 2.98×10^-4M, respectively. These results are discussed in comparison with those of UDP-glucose pyrophosphorylases isolated from other plants.

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Sulfur-containing amino acids (L-cysteine or L-cystine) were reacted with D-glucose or pyruvaldehyde at various temperatures and submitted to flavor evaluation. The nuance of the aroma was changed with temperature, and the most acceptable aroma (Japanese rice cracker with sesame-like) was produced at 160°C in all the samples. Volatile compounds produced at 160°C were investigated by gas chromatography and GC-MS coupling. Many compounds such as thiazoles and thiophenes found in the volatiles of some foodstuffs were identified.

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A growth factor (TJF) for a malo-lactic fermentation bacterium (Lenconostoc sp.) has been found to be 4'-O-(β-D-glucopyranosyl)-D-pantothenic acid with structural and synthetical studies. Now other 4'-O-glycosides (β-D-ribofuranosyl, α-D-glucopyranosyl, β-D-galactopyranosyl, β-maltosyl and β-cellobiosyl) and 2', 4'-O-di-β-D-glucopyranoside of DL-pantothenic acid, and 4'-O-β-D-glucopyranoside of DL-pantethine were synthesized to examine their biological activities. The improved syntheses of TJF were also examined.

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The accumulation of vitamin B₆ by Pichia guilliermondii Wickerham NK-2 strain grown on hydrocarbon was investigated. Ammonium acetate was more effective than other nitrogen sources tested. Satisfactory utilization by the yeast strain was observed in n-alkanes of C₁₀-C₁₈, and n-pentadecane was the best for vitamin B₆ production. Vitamin B₆ was excreted in the cultural broth mainly in the form of pyridoxal. The maximal vitamin B₆ production was approximately 25mg per liter of the culture broth.

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In order to elucidate the mechanism of anthocyanin formation by Populus cells in suspension culture, the favourable conditions for anthocyanin formation were investigated. The influence of some factors affecting the anthocyanin formation, i.e. light, sucrose and riboflavin etc. were also examined. Light irradiation and high sucrose concentrations brought about a marked increase of PAL activity, which increased rapidly at the lag phase preceding the anthocyanin formation. The effect of blue light on anthocyanin formation was markedly superior to other kinds of monochromatic light (green and red) or white light. Riboflavin was effective only under light exposure. It was inferred that light, sucrose, riboflavin and PAL activity etc. were closely related with the anthocyanin formation. Especially, light and sucrose cooperated in the increase of PAL activity which was a key enzyme in the iosynthesis of anthocyanin.

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1) L-Asparaginase (EC 3. 5. 1. 1) from Escherichia coli A-1-3 was acetylated using acetic anhydride as a modifying chemical. The fully acetylated L-asparaginase retained 60% of the activity of the unmodified L-asparaginase.
2) The acetylated L-asparaginase hydrolyzed D-asparagine and L-glutamine as well as L-asparagine in the same ratio as the unmodified L-asparaginase did.
3) However, the effects of pH on the activity of the acetylated L-asparaginase showed very interesting differences from that of L-asparaginase. On the other hand, both L-aspara-ginase and the acetylated L-asparaginase exhibited similar pH activity curves on L-glutamine hydrolysis.
4) The acetylated L-asparaginase was found to become more stable against acid or heat in the presence of L-aspartate than in its absence in the same manner as L-asparaginase was.

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3, 4-Diaryl-5-mercapto-1, 2, 4-triazoles have been prepared by the cyclization of the corresponding 1-aroyl-4-Substituted thiosemicarbazide with alkali. The resulting mercapto triazoles were converted into 3, 4-disubstituted-5-alkylthio-1, 2, 4-triazoles with the action of different alkyl halides and some of the sulphides were converted into sulphones by oxidation with aqueous potassium permanganate.

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In order to clarify the magnitude of different labile body proteins and the over-all catabolizable body protein, the catabolic rate of total body nitrogen in adult rat was measured by nitrogen balance method up to the time of death due to protein depletion.
The more labile body protein having 83.3% of fractional catabolic rate per day and occupying 2.8% of the whole body protein mainly represented the so-called protein reserves at the beginning of protein depletion. The less labile one, the remainder, namely 97.2%, having 0.79% of fractional catabolic rate almost wholly represented the exponential decrease of body protein during the first 40 days of protein depletion. Urinary and fecal nitrogen in this period showed a similar exponential decrease. In the next 40 days of the depletion, body protein decreased almost linearly giving the constant excretions of urinary and fecal nitrogen. In the last 40 days, it decreased drastically accompanied by a remarkable increase in urinary nitrogen.
After 118 days, on the average, the animals died of protein depletion at 35.7% level of the initial body nitrogen. Contributions of various organs to the total nitrogen deficit up to the time of death, were considerablly different in different organs, where muscle was the greatest in total amount but with less catabolizability than the viscera, such as liver, pancreas and spleen.

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Microbial transaminase which catalyzes the reaction between 3, 4-disubstituted phenyl pyruvate and certain amino acids to produce 3, 4-disubstituted phenyl-L-alanine was investigated. Wide distribution of this enzyme among the various kinds of microorganisms was confirmed.
3, 4-Dihydroxyphenyl-L-alanine (L-Dopa) or 3, 4-dimethoxyphenyl-L-alanine was isolated from corresponding keto acids using three strains of selected microorganisms.

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Contrary to the exopectic acid transeliminase of Clostridium multifermentans, that of Erwinia sp. was activated strongly by Na⁺ and to a much less extent by Ca²⁺. K⁺ had a small stimulating effect on the enzyme activity. Mn²⁺ and Co²⁺, like Ca²⁺, activated the enzyme weakly. Ba²⁺ and Mg²⁺ showed no and a slight inhibitory effect, respectively, on the activity.
An almost total loss of activity was caused by the addition of EDTA to the reaction mixture. In the presence of Na⁺ the enzyme activity was restored by addition of divalent cations. Individual monovalent cations or each of the divalent cations was ineffective in restoring the activity.

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Though sclerin (SCL) only slightly inhibited the activity of pyruvate kinase (PK) in crude extract of Candida utilis, markedly repressed the level of that in the growing cells in a glucose medium. The repression of PK was largely recovered by 2, 4-dinitrophenol (DNP), and SCL rather raised the level in the cells growing on gluconate.
SCL also slightly inhibited the activity of a partially purified PK from rat liver, and, when orally administered, or incubated with the liver slices, obviously lowered the level of PK in the liver and liver slices. The effect of SCL in the liver slices was reversed by DNP. SCL stimulated the oxidative phosphorylation in mitochondria prepared from the fresh liver, and served to maintain the activity in the liver slices during incubation.
Both activity of PK from Candida utilis and rat liver was remarkably inhibited by adenylate energy charge in vitro.
It is concluded that SCL represses the level of PK in these cells and tissues through a high energy charge by stimulating the oxidative phosphorylation.

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The cultivation of Brevibacterimn ammoniagenes IFO 12071 with pantothenic acid, cysteine, and 5'-adenylic acid gave coenzyme A in a high yield. The organism was stabilized by repeated single colony isolations. The culture conditions optimal for the production of coenzyme A were investigated, and the yield of coenzyme A in the culture broth reached more than 3mg/ml.
The advantages and disadvantages of the present method were discussed by comparing them with our original dried cell method.

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A new process has been described for the preparation of coenzyme A of high purity from the cultured broth of Brevibacterium mnmoniagenes IFO 12071. The product was obtained in a high yield by the use of Duolite S-30, charcoal, and Dowex 1×2, and identified chemically and enzymatically. This method is simple, rapid, and compact, requires no special equipment, and has been shown to be adaptable for preparing large amounts of highly pure coenzyme A.

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From the intact cells of “Rhodotorula lactosa” R1 (IFO 1058), a new coenzyme Q, which has a different mobility on paper chromatograms from other five naturally occurring homologs of the coenzyme Q series, was isolated and purified as a crystalline state. The chemical analyses such as UV and IR absorption spectrophotometries, and NMR and mass spectrometries revealed that the material, mp 28.7_??_28.9°C, was identified as a Co Q₁₀ derivative with the reduced C₅ unit in the isoprenoid side chain terminal remote from the quinone nucleus, Co Q₁₀ (H-10). The strain R 1 with such a unique coenzyme Q system is, concerning its taxonomic position, discussed in connection with other criteria.

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A fungal rhodanese from the spray-dried powder of a culture filtrate of Trametes sanguinea was purified to 142-fold by ammonium sulfate precipitation and DEAE-cellulose and Sephadex G-100 column chromatography. The purified rhodanese (pl 5.10) showed a single band on disc electrophoresis, and its molecular weight was estimated to be 51, 700 by gel filtration technique. The enzyme had a broad pH optimum between 7.5 and 8.5 and was stable at pH values from 4 to 8 at 30°C for 44 hr. Its activity was inhibited by p-chloromercuribenzoate at pH 9.5, but not at pH 8.0, and was inhibited by cysteine, β-mercaptoethanol and sodium borohydride at pH 8.0. Both thiosulfate and cyanide showed substrate inhibition at high concentrations. Dihydrolipoate and benzenethiosulfonate were also good substrates.

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Dehydrodiferulic acid (II), a dimeric lactone obtainable from ferulic acid (I) by oxidative coupling, was converted into two types of lignans. Hydrogenolysis of II coupled with three subsequent operations gave dl-matairesinol dimethyl ether (IIIb). Acid-catalyzed cyclization of II followed by seven steps afforded dehydrodimethylconidendrin (X) and dehydrodimethylretrodendrin (XI).

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Three metabolites designated Cyl-1, -2 and -3 were isolated as plant growth regulators from culture filtrate of Cylindrocladiunr scoparium Morgan, a phytopathogenic fungus. Cyl-2 was obtained as pure crystals, and it revealed marked inhibitory activity on the root growth of lettuce seedlings. Cyl-1 showed inhibition on the same organ, while Cyl-3 showed promotion.

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(±)-Azetidine-2-carboxylic acid and 3-substituted-2-pyrrolidinones were synthesized from 2-pyrrolidinone via 3-bromo-2-methoxy-1-pyrroline (IIIa). The bromide (IIIa) was obtained by the bromination of 2-methoxy-1-pyrroline using NBS.

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Growth of Brevibacterium flavum FA-1-30 and FA-3-115, L-lysine producers derived from Br. flavum No. 2247 as S-(2-aminoethyl)-L-cysteine (AEC) resistant mutants, was inhibited by α-amino-β-hydroxyvaleric acid (AHV), and this inhibition was reversed by L-threonine. All the tested AHV resistant mutants derived from FA-1-30 accumulated more than 4g/liter of L-threonine in media containing 10% glucose, and the best producer, FAB-44, selected on a medium containing 5mg/ml of AHV produced about 15g/liter of L-threonine. Many of AHV resistant mutants selected on a medium containing 2mg/ml of AHV accumulated L-lysine as well as L-threonine. AHV resistant mutants derived from FA-3-115 produced 10.7g/liter of L-threonine maximally. AEC resistant mutants derived from strains BB-82 and BB-69, which were L-threonine producers derived from Br. fiavum No. 2247 as AHV resistant mutants, did not produce L-threonine more than the parental strains, and moreover, many of them did not accumulate L-threonine but L-lysine. Homoserine dehydrogenases of crude extracts from L-threonine producing ARV resistant mutants derived from FA-1-30 and FA-3-115 were insensitive to the inhibition by L-threonine, and those of L-threonine and L-lysine producing AHV resistant mutants from FA-1-30 were partially sensitive.
Correlation between L-threonine or L-lysine production and regulations of enzymatic activities of the mutants was discussed.

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In the course of a screening study for antibiotics using psychrophilic microorganisms a water-insoluble antibiotic complex, SP-351, was found in the culture filtrate of a psychrophilic actinomycete, strain No. 351. This active principle was isolated and characterized as a cyclicpolylactone antibiotic. The SP-351-producing strain was classified as a facultative psychrophile and identified as Streptomyces phaeochromogenes.
The main components of the antibiotic complex SP-351 changed with the composition of the culture medium but not with culture temperatures. Component A was exclusively produced in a medium composed of roasted soybean powder and glycerol; components B and C in a medium composed of soybean, glycerol and potassium nitrate; and components A and D in a synthetic medium containing a hydrocarbon, alcohol or ester as the sole carbon source. Maximum production of SP-351 from n-paraffin and methyl acetate was 10 and 15mcg/ml, respectively.
SP-351 showed strong antibacterial activity against Gram-positive bacteria and acidfast bacteria at 0.1_??_0.3mcg/ml concentrations.

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Sclerotinin A (3, 6, 8-trihydroxy-3, 4, 5, 7-tetramethyl-3, 4-dihyroisocoumarin: 1), a metabolite of Scierotinia sclerotiorum, was first totally synthesized. The pentenone ring of 2, 3, 4, 6-tetramethyl-5, 7-dimethoxyindanone (VII_a) which was obtained from 2, 4-dimethylresorcinol dimethyl ether (VI) through a new synthetic process, was cleavaged oxidatively, and demethylated to I. Sclerotinin A was also yielded from indanone dibenzyl ether (VII_b) by the same method and reductive debenzylation.

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