Pyridoxamine (pyridoxine) 5'-phosphate oxidase (EC. 1.4.3.5) has been purified from dry baker's yeast to an apparent homogeneity on a polyacrylamide disc gel electrophoresis in the presence of 10 μM of phenylmethylsulfonyl fluoride throughout purification.
1) The purified enzyme, obtained as holo-flavoprotein, has a specific activity of 27μmol/ mg/hr for pyridoxamine 5'-phosphate at 37°C, and a ratio of pyridoxine 5'-phosphate oxidase to pyridoxamine 5'-phosphate oxidase is approximately 0.25 at a substrate concentration of 285μm. Km values for both substrates are 18μM for pyridoxamine 5'-phosphate and 2.7μm for pyridoxine 5'-phosphate, respectively.
2) The enzyme can easily oxidize pyridoxamine 5'-phosphate, but when pyridoxamine and pyridoxine 5'-phosphate are coexisted in a reaction mixture the enzyme activity is markedly suppressed much beyond the values expected from its high affinity (low Km) and low V_max for the latter substrate.
3) Optimum temperature for both substrates is approximately 45°C, and optimum pH is near 9 for pyridoxamine 5'-phosphate and 8 for pyridoxine 5'-phosphate.
4) From the data obtained, the mechanism of regulation of this enzyme in production of pyridoxal 5'-phosphate and a reasonable substrate for the enzyme in vivo are discussed.

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The interaction of SDS with lysozyme was analyzed with enzyme activity and with NMR, fluorescence, and UV difference spectroscopies using various alkyl sulfates and variously modified lysozymes. SDS formed a stable complex with lysozyme without causing a gross conformational change in the enzyme molecule. Some SDS molecules bound to the active site cleft of lysozyme and therefore strongly inhibited the activity of lysozyme. Hydrophobic regions and positive charges for protein side, and a hydrophobic tail (possibly more than 8 carbons in alkyl chain) and a negative charge for detergent side were required for the formation of the complex.

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Digestibilities of native, 5M urea-denatured and 8M urea-denatured glycinin were studied. Urea was removed by dialysis before digestion. The tryptic digestion of the proteins are influenced by ionic strength. Under low ionic strength condition (0M NaCl), the proteins, even native glycinin, are well degraded. On the other hand, under high ionic strength con-dition (0.5M NaCl), native glycinin resists the tryptic attack and 5M urea-denatured glycinin is best degraded. The digestibility of 8M urea-denatured glycinin is lower than that of 5M urea-denatured one under the condition. The gel filtration and electrophoretic properties show that the digestion intermediate like glycinin-T (the intermediate from native glycinin) is contained in the digestion products. These suggest that the urea-denatured protein contains the almost renatured component after removal of urea. A larger amount of the glycinin-T-like protein was detected at 8M urea denaturation than at 5M urea. Therefore, glycinin renatures more readily from 8M urea denaturation. Probably this is the cause of the decreased digestibility at 8M urea denaturation.

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Moderate cell growth occurred after a long lag phase of about 100 hr when oxygen-sensitive hydrogen bacterium N34 was cultivated chemoautotrophically under 40% O₂. A decrease in cell growth or viable count was not observed during the lag phase. These cells grown under 40% O₂ were oxygen-resistant because when used as inocula for fresh 40% O₂-culture, the growth lag period was less than 10 hr. Nine oxygen-sensitive colonies developed from a single oxygen-sensitive cell respectively. When these colonies were inoculated into 40%. O₂-culture, they showed an almost equal lag period and growth rate. These results suggest that cell growth in 40% O₂-culture inoculated with oxygen-sensitive strain N34 occurred not by selection of oxygen-resistant variants which might preexist but by adaptation of very oxygen-sensitive cells to high oxygen tension. Oxygen-resistance thus developed was maintained after successive subcultures under 10% O₂ for more than one year.

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Hydrogenase activity (Tritium uptake activity) in cell-free extracts of bacteroids from soybean and lupin nodules was separated into two parts, whose activities were different from each other in responsiveness to ATP, by differential centrifugation, Sephadex gel filtration or DEAE-cellulose column chromatography. One was stimulated and the other was either inhibited or not affected by ATP.

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The amino acid residue(s) involved in the activity of buckwheat α-glucosidase was modified by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the presence of glycine ethyl ester. The modification resulted in the decrease in the hydrolytic activity of the enzyme following pseudo-first order kinetics. Competitive inhibitors, such as Tris and turanose, protected the enzyme against the inactivation. Protection was provided also by alkali metal, alkaline-earth metal and ammonium ions, though these cations are non-essential for the activity of the enzyme. Turanose or K⁺ protected one carboxyl group per enzyme from the modification with carbodiimide and glycine ethyl ester. Free sulfhydryl group of the enzyme was also partially modified with carbodiimide, but the inactivation was considered to be mainly attributed to the modification of essential carboxyl group rather than to that of free sulfhydryl group.

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Among three alkaline protease inhibitors (API-2a, -2b, -2c) produced by Streptomyces griseoincarnatus strain No. KTo-250, API-2b was converted to API-2c in the growing system.
The cultural conditions were examined exclusively for the production of API-2b→API-2c converting protease in the culture filtrate. The protease was purified about 1080-folds by salting-out with ammonium sulfate, column chromatography on DEAE-cellulose and gel filtration on Sephadex G-100.
The optimal and maximal caseinolytic activities of the protease were around pH 9.0 and at 28°C, respectively. The protease activity was inhibited by EDTA and DFP, but not by PCMB, o-phenanthroline, TPCK, TLCK, AP-I and S-SI. The protease was a DFP and EDTA-sensitive alkaline protease, and it required Ca²⁺ ion for its activity and stability.

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It was found that there was a fairly well correlation between the soybean disintegrating activity (SD activity) of Driselase, a cellulase preparation from Irpex lacteus, and the improvement of feed efficiency caused from its supplementation to a ration.
When the seed coat of soybean was hydrolysed by Driselase, arabinose, galactose, and other aldoses were liberated; on the other hand, some ketoses such as fructose, sucrose, raffinose and such were detected as a result of the hydrolysis of the cotyledon. On the fractionation of Driselase with column chromatography, acid proteinase was appeared to be parallel, in a certain extent, with SD activity. These suggested that Driselase partially attacked the cell walls of the cotyledon and led to the leakage of intracellular substances such as ketoses and protein.
Since it was revealed, however, that only a kind of pectin hydrolase was detected in the fraction with high SD activity, the maceration of soybean by a pectin hydrolase was thought to be chiefly concerned with SD activity.

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Some analogs of stevoiside (I), a sweet diterpene glycoside, were synthesized. Coupling of hepta-O-acetylsteviolbioside with hexa-O-acetyl-2-O-a-L-rhamnopyranosyl-a-D-glucopyranosyl bromide, bexa-O-acetyl-2-O-a-L-rhamnopyranosyl-a-D-galactopyranosyl bromide and hexa-O-acetyl-2-O-a-L-quinovopyranosyl-a-D-galactopyranosyl bromide, in the presence of silver carbonate-celite in dichloroethane followed by deacetylation yielded steviol-13-O-β-sophoroside-19-O-(2-O-a-L-rhamnopyranosyl-β-D-glucopyranosylester) (VIII), steviol-13-O-β-sophoroside-19-O-(2-O-a-L-rhamnopyranosyl-β-D-galactopyranosylester) (IX) and steviol-13-O-β-sophoro-side-19-O-(2-O-a-L-quinovopyranosyl-β-D-glueopyranosylester) (X), respectively. Methylation of steviolbioside heptaacetate followed by deacetylation yielded steviolbioside-19-O-methylester (XII). Reduction of stevioside and XII with hydrogen gave dihydrostevioside (XIV) and dihydrosteviolbioside-19-O-methylester. The relative sweetness of I, VIII, IX, X, steviolbioside, XII, dihydrosteviolbioside, XIV and XV as expected by the percentage of that of sucrose were 210, 300, 230, 90, 6, 30 and 3, respectively.

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Direct experimental evidence is given for the presence of methionine sulfoxide reducing enzymatic activity in rat liver and kidney. Experiments were performed with L(U.¹⁴C) methionine RS sulfoxide, incubated with liver or kidney homogenates or subcellular fractions, the formation of methionine being determined by ion-exchange chromatography and radioactivity measurement. The methionine sulfoxide reducing activity is thermolabile and enhanced by the addition of NADH to the incubation medium. The subcellular distribution patterns of the enzymatic activities from the liver and from the kidney are quite different.

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Bovine liver mitochondrial aldehyde dehydrogenase (aldehyde: NAD⁺ oxidoreductase, EC 1.2.1.3) has been purified to homogeneity by conventional purification procedures. The enzyme was found to have a molecular weight of 215, 000 based on gel filtration. The protein is composed of polypeptides having the same molecular weight, 54, 000 and thus it appears to consist of four subunits of equal size. The enzyme exhibited a broad aldehyde specificity, oxidizing irreversibly a wide variety of aliphatic and aromatic aldehydes to corresponding carboxylic acids. Km values for straight-chain saturated aldehydes were below 0.1μM, and relatively constant independent of the carbon chain lengths of the aldehydes. The maximum velocities for saturated aldehydes also did not vary appreciably with their carbon chain lengths. Maximum activity was observed at pH 9.3 and 50°C. The enzyme activity was affected by some divalent cations. Ca²⁺ enhanced the activity, while Mg²⁺ inhibited it. The enzyme was quite stable at neutral pH, but was unstable above pH 9 or below pH 6. Bovine liver has three isozymes of aldehyde dehydrogenase which are located in the mitochondrial, cytosolic, and microsomal fractions. Comparison of enzymic properties among these isozymes and yeast enzyme indicates that the mitochondrial enzyme is very suitable for improving the objectionable flavor due to aldehydes in foods.

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Aldehyde dehydrogenase catalyzes the irreversible conversion of aldehydes into their corresponding acids. NAD-dependent aldehyde dehydrogenase purified from bovine liver mitochondria was used to remove the green beany flavor of soybean products. Incubation of the enzyme, in the presence of NAD⁺, with defatted soybean extracts or with soybean milk, resulted in the almost complete disappearance or in a great reduction of the flavor. It was found from experiments with pyrazole, an inhibitor of alcohol dehydrogenase, was used, that alcohols contributing to the beany flavor were converted into acids by the cooperative action of alcohol dehydrogenase and aldehyde dehydrogenase. The protein isolate prepared from the soybean extract after treatment with these enzymes produced no substantial beany flavor after storage in powdered form. Aldehyde dehydrogenase improved flavor in extract of mutton.

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Defatted soybean extract was fractionated into protein fractions and low molecular weight fractions with gel filtration. NAD-dependent aldehyde dehydrogenase from bovine liver mitochondria and from yeast was found to oxidize aldehyde in both fractions. These enzymes, therefore, were used to determine the quantity of aldehyde. When the protein fraction obtained by gel filtration was subjected to gel filtration again, aldehyde was recovered in the protein fractions. The level of aldehyde in the protein fractions was unchanged before and after digestion of the protein with pepsin. When the soybean extract was incubated beforehand with aldehyde debydrogenase and NAD⁺ and the subjected to gel filtration, no aldehyde was detected in the protein fractions. These results indicate that aldehyde dehydrogenase acts on the soybean protein-bound aldehyde. Alcohol dehydrogenase from horse liver in the presence of NADH did not convert the bound aldehyde to alcohol.
A large portion of the aldehyde in the extract was separated from the protein by acid precipitation of the protein. Aldehyde dehydrogenase acts on the aldehyde remaining in the protein after acid precipitation. Thus acid precipitation helps to save NAD⁺ required for complete removal of aldehyde from the soybean protein by aldehyde dehydrogenase.

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An N-acetylglutamate-acetylornithine acetyltransferase-deficient arginine-requiring mutant AA-1, was derived from an L-arginine producer of Corynebacterium glutamicum. It accumulated a large amount (30mg per ml) of L-glutamic acid and a small amount (1.2mg per ml) of N^α-acetylornithine, an intermediate of arginine biosynthesis, in the culture medium.
The production of N^α-acetylornithine by AA-1 was not affected by the concentration of L-arginine in the medium, whereas that of L-glutamic acid was inhibited by a high concentration of L-arginine in the medium containing excess biotin.

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Discrimination of soy sauce samples consist of different 8 brands on the basis of principal components extracted from GC profiles of aroma concentrates was performed by stepwise discriminant analysis. Considering the results from sensory evaluation, classification of samples into 8, 3, and 2 groups was examined. Statistically significant difference was found among the 8 brands on the basis of principal components. The three groups consist of good, common, and inferior samples also classified correctly. Completely correct two way discrimination, good and bad, was accomplished in the same manner. These clear classifications suggested that evaluation and discrimination in sensory tests were performed by comparing the profiles of aroma substances.

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A mixture of heavy and light membranes could be fragmented in the presence of 20% sucrose with only limited hybrid formation. The limiting size to which membranes can be fragmented without hybridization was approximately 500 Å. The membranes from the density shift or from both the density and temperature shifts gave no indication for distinctly light or heavy membrane patches within the limit of detection.

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(Z)-3-Hexene-1, 6-diol, a new synthon for the synthesis of (Z)-olefinic pheromones, was prepared from 1, 4-cyclohexadiene. This synthon was used in the synthesis of (Z, Z)-3, 13-octadecadienyl acetate, whose high stereochemical purity at C-3 was essential in attracting male Synanthedon tenuis Butler.

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Blue seed-coats of Ophiopogon jaburan have been found to contain kaempferol, kaempferol 3-glucoside (astragalin), two new glucosides of kaempferol, and a trace amount of an unknown flavonol-like compound. One of the new glucosides was determined to be kaempferol 4'-glucoside and the other to be kaempferol 3, 4'-diglucoside by means of paper-chromatographic and spectral analyses.

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Effects of dietary β-sitosterol (S) and β-sitostanol (HS) on the metabolism and fate of labeled cholesterol intravenously injected were compared in rats fed diets high in cholesterol. Kinetic behavior of the decay curve for serum cholesterol in the HS supplemented (C+HS) group approximated to that in the cholesterol-free (control) group. The largest dilution of the label was observed in rats of the cholesterol (C) group and the least in the C+HS group, the C+S group being intermediate. The specific activity of hepatic cholesterol was in the decre-asing order of the C+HS, C+S and C groups, while the situation was reversed when expressed in terms of net incorporation. Thus, cholesterol pool seemed to be much smaller in the C+HS group than in the C+S group.
In a long term feeding experiment with diets free of cholesterol, HS exhibited significantly greater hypocholesterolemic activity than S did.
These data, together with those reported previously, indicated that inhibitory effect on the absorption of both endogenous and exogenous cholesterol was much more greater in HS than in S.

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The relationships between GC data of volatiles and sensory score of the heighest grade of soy sauce (Tokusen shoyu) were analyzed by multivariate analyses. Samples belonging to four brands could be unambiguously classified into the correct brands and the statistical distance among them suggested a close relation with sensory evaluation. The precise predictive equations for the aroma quality were calculated from GC data and sensory evaluation by multiple regression analysis. Eight principal components were extracted from 39 GC peaks as significant factors constituting soy sauce aroma. The second PC can explain 59% of the variation among total variation contained in the sensory score. Discriminant functions on the basis of the 8 PCs can clearly classify all samples.

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Five new derivatives of adenosine, N⁶-[(1-methylethyl)thiomethyl]-(1), N⁶-methylthio-methyl-(2), N⁶-phenylthiomethyl-(3), N⁶-[(3-amino-3-carboxypropyl)thiomethyl]-(4), and N⁶-[(2-amino-2-carboxyethyl)thiomethyl]adenosine (5), were synthesized and their cytokinin activity was tested in the Amaranthus betacyanin assay and the soybean callus growth.
1, 2, and 3 were active in the former assay and all five compounds were active in the latter assay. The activities of the compounds were, however, weaker than those of the reference derivatives, in which sulfides were replaced by methylenes, N⁶-isopentyl-, N⁶-n-propyl-, N⁶-benzyl-, and N⁶-(5-amino-5-carboxypentyl)adenosine. This fact indicates that the sulfide structure introduced into the N⁶-side chains had the effect of reducing cytokinin activity.

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The major storage protein of Phaseolus angularis, 7S protein-I, was purified by gel filtration on Sephadex G-200 and ion exchange chromatography on DEAE-cellulose. The purified 7S protein-I was homogeneous on disc electrophoresis, isoelectric focusing and ultracentrifugation. The sedimentation coefficient (S⁰₂₀, w) and Stokes radius of 7S protein-I were estimated to be 7.5 S and 48 Å, respectively. The molecular weight of 7S protein-I was calculated to be 150, 000±15, 000 from these values. Polyacrylamide gel electrophoresis of 7S protein-I in the presence of sodium dodecyl sulfate showed one main (55, 000±3000 daltons) and two minor protein bands (28, 000±1400 and 25, 000±1300 daltons). 7S protein-I contained large amounts of glutamic acid and aspartic acid but no cysteine and low amounts of methionine. Carbohydrate analysis of 7S protein-I revealed the presence of 5.0% of neutral sugars and 0.5% of amino sugars. Circular dichroism measurements indicated that this protein is a β-form rich protein.

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The metabolic pathway of O-alkylhomoserine in Corynebacterium acetophilmn was determined using mutants with defects in methionine biosynthesis and purified O-acetylhomoserine sulfhydrylase. The mutant strain M-74, defective in homoserine transacetylase, utilized O-alkyihomoserines and O-acetylhomoserine instead of methionine, but strain M-933, lacking cystathionine γ-synthase, did not. The incorporation of radioactive O-ethylhomoserine into cells was inhibited competitively by O-acetylhomoserine. Analysis of autoradiograms of two-dimensional thin-layer chromatograms showed that labeled O-ethylhomoserine was converted to O-acetylhomoserine by strain M-933 and finally metabolized to methionine by strain M-74. Furthermore, results showed that O-acetylhomoserine was synthesized from O-ethylhomoserine and acetic acid by a reversible side reaction of purified O-acetylhomoserine sulfhydrylase of C. acetophilum. These findings show that O-alkylhomoserine is converted to O-acetylhomoserine and then metabolized to methionine via cystathionine and homocysteine in cells of C. acetophilum starved of methionine.

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In connection with our investigation on the structure-activity relationships of the American cockroach sex pheromone mimic. (+)-trans-verbenyl acetate, a number of alcohols having 6, 6-dimethylbicyclo[3. 1. 1]heptane skeleton were synthesized, and measured their PMR spectra in the presence of the shift reagent, Eu(dpm)₃. The induced shifts of the 6, 6-gem-dimethyl protons and a-proton at the C-7 guided us to distinction between cis- and trans-alcohol. The configuration of secondary methyl groups was discussed from the induced shift values of the methyl protons.

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