Isolation and Characterization of 7S Protein-I of Phaseolus angularis (Adzuki bean)

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 (, 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.     

Purification and Some Properties of β-Xylosidase from Malbranchea pulchella var. sulfurea No. 48

A β-xylosidase of a thermophilic fungus, Malbranchea pulchella var. sulfurea No. 48, was purified 99-fold from the culture filtrate after ammonium sulfate fractionation, DEAE-cellulose column chromatography, column electrophoresis and gel filtration on Sephadex G–200. The purified enzyme was found to be homogeneous upon ultracentrifugal analysis, disc electrophoresis and gel filtration. The molecular weight of the enzyme was estimated to be 26,000 by gel filtration, and the sedimentation coefficient was calculated to be 2.78S. at 280 nm in phosphate buffer (pH 6.7) was 13.2. The optimum pH was found to be in the range of 6.2~6.8, and the optimum temperature was 50°C.     

Isolation and Physicochemical Properties of a Soluble Glycoprotein Fraction of Milk Fat Globule Membrane

A soluble apoprotein fraction was prepared from milk fat globule membrane lipoproteins by delipidation with a chloroform-methanol mixture and was fractionated into three fractions by gel filtration on Bio-Gel A–5m.

The major fraction, Fraction II, contained about 30% of carbohydrate, i.e. 13.9% of hexoses, 8.1% of hexosamines, 8.0% of sialic acid and 0.8% of fucose, and was therefore designated a soluble glycoprotein fraction. The fraction was apparently homogeneous on sedimentation velocity analysis and DEAE-Sephadex chromatography, and had 6.1, 3.79, 0.719, f/f⁰ 2.16 and molecular weight 139,000 daltons. However, the diffused pattern on disc electrophoresis and the occurrence of plural N-terminal amino acid residues suggest that the protein of this fraction is likely to be formed by intermolecular association of heterogeneous polypeptide chains.     

Substrate Specificity of α-Glucosidase II in Rice Seed

The substrate specificity of rice α-glucosidase II was studied. The enzyme was active especially on nigerose, phenyl-α-maltoside and maltooligosaccharides. The actions on isomaltose and phenyl-α-glucoside were weak, and on sucrose and methyl-α-glucoside, negligible. The α-glucans, such as soluble starch, amylopectin, β-limit dextrin, glycogen and amylose, were also hydrolyzed.

The ratio of the maximum velocities for hydrolyses of maltose (G₂), nigerose (N), kojibiose (K), isomaltose (I), phenyl-α-maltoside (?M) and soluble starch (SS) was estimated to be 100: 94.4: 14.2: 7.1: 89.5: 103.1 in this order, and that for hydrolyses of malto-triose (G₃), -tetraose (G₄), -pentaose (G₅), -hexaose (G₆), -heptaose (G₇), -octaose (G₈), and amyloses ( and ), 113: 113: 113: 106: 113: 100: 106: 106. The Km values for N, K, I, ?M and SS were 2.4 mm, 0.58 mm, 20 mm, 1.6 mm and 5.0 mg/ml, respectively; those for G₂, G₃, G₄, G₅, G₆, G₇, G₈, and , 2.4 mm, 2.2 mm, 2.1 mm, 1.5 mm, 1.0 mm, 1.1 mm, 0.95 mm, 1.5 mm and 1.1 mm.

Rice α-glucosidase II is considered an enzyme with a preferential activity on maltooligosaccharides.     

Isolation and Crystallization of Extracellular 3β-Hydroxysteroid Oxidase of Brevibacterium sterolicum nov. sp.

Among about 500 strains tested, a newly isolated soil bacterium, Brevibacterium sterolicum nov. sp. KY 3463 (ATCC 21387) showed the highest potency in production of 3β-hydroxysteroid oxidase in the culture fluid.

The 3β-hydroxysteroid oxidase was purified from the culture filtrate by a procedure involving ammonium sulfate fractionation, DEAE-cellulose and hydroxyapatite column chromatographies and Sephadex G–75 gel filtration. Crystals of the enzyme were obtained from solutions of the purified preparation by the addition of ammonium sulfate. The crystals appeared as fine rods, with a bright yellow color.

The enzyme is homogeneous by disc gel electrophoresis and ultracentrifugation. Sedimentation velocity yields a value of . It exhibits a typical flavoprotein spectrum of absorption maxima at 280, 390, and 470 mμ.     

Asymmetric Reduction of Iminium Salt with Chiral Dihydropyridines

A gram positive bacterium (strain No. 109) isolated from soil as a producer of cyclodextrinase was identified as Bacillus coagulans. The cyclodextrinase from B. coagulans was purified to a homogeneous state by disc-electrophoresis after Streptomycin treatment, DEAE-Sephadex column chromatography, Ultrogel AcA44 gel filtration and hydroxyapatite column chromatography. The molecular weight of the enzyme was determined to be 6.2}10⁴ by sodium dodecyl-sulfate gel electrophoresis. The isoelectric point of the enzyme was pH 5.0. The enzyme was most active at pH 6.2 and 50°C, and stable up to 45°C at pH 7.0 and in the range of pH 6.0 ~ 7.3 at 40°C on 2 hr incubation. This enzyme hydrolyzed linear maltooligosaccharides (such as maltotetraose (G4), maltopentaose (G5) and maltohexaose (G6)) and α-, β- and α-cyclodextrins (CDs) faster than maltotriose (G3) and short chain amylose ( 18), but did not hydrolyze maltose. The rates of hydrolysis for polysaccharides (such as starch, amylose and amylopectin) were below 1 % as compared to that for β-CD. The Km values for G3, G4, G5, G6, short chain amylose ( 18) and α, β- and γ-CD were 4.5, 4.0,2.3,1.5,1.5,10,2.8 and 0.47 mM, respectively. The products with this enzyme had the α-configulation.     

Physical and Chemical Properties of Yeast Proteinase C

A simple purification method which enables us to obtain homogeneous proteinase C from S. cerevisiae was developed. Physical and chemical properties of the purified enzyme were determined. The extinction coefficient at 280 mμ, , of yeast proteinase C was 14.8, and its isoelectric point was pH 3.60. Partial specific volume, intrinsic viscosity and the sedimentation and diffusion coefficients of homogeneous protein were , 0.71 ml/g, [η], 4.83 × 10^?2ml/g, , 4.23 S and , w, 6.1 × 10^?7 cm²/sec. From these values, molecular weights, M_[·],D, M_S,D and M_[·],S, of 60,000, 59,000 and 58,000, respectively, were obtained. The sedimentation equilibrium experiment gave a molecular weight, M_equil, of 61,000. Yeast proteinase C contained 11.9% nitrogen and was a glycoprotein with 16.7% carbohydrate: The value of β-function, 2.163×l0⁶ or 2.20×l0⁶ indicates that the molecular shape of yeast proteinase C is a plorate with an axial ratio of 4.0, assuming 35% hydration. Furthermore, yeast proteinase C may be a compact, asymmetric ellipsoidal model having semi-axes 30Å × 30Å × 130Å.     

Degradation of Dextran by Free Radical Systems

Alcohol dehydrogenase (alcohol: NAD oxidoreductase, E.C. 1.1.1.1) from Thea sinensis seeds (variety: Zairai) was isolated and purified about 1,500-fold using preparative disc electrophoresis. The specific activity was about 3.4 units/mg protein against ethyl alcohol.

Its value was 6.96 S and its molecular weight was approximately 150,000 using gel filtration on Sephadex G–200. The physical, chemical and catalytic properties of the enzyme are described. The oxidoreduction products formed by the enzyme were identified by gas chromatography, and for the unsaturated compounds the conversion of double bond and geometrical isomerization was observed.

The substrate specificity of tea enzyme is discussed in comparison with the enzymes from Leuconostoc mesenteroides, and horse and human livers. Paticularly a tendency for reactivity in the oxidoreduction of unsaturated alcohols and aldehydes were discribed by comparing the effects of geometry, the position of the double bond and the length of chain in substrates.     

Analysis of Fluorescein-thiohydantoin Amino Acids by High Speed Liquid Chromatography

The distribution of choline oxidase activity was studied with cell-free extracts of yeasts, molds and actinomycetes. A fungus which was identified as Cylindrocarpon didymum M–1 showed the highest activity. The enzyme was purified from the cell-free extract of C. didymum M–1 by a procedure involving ammonium sulfate fractionation and DEAE-cellulose, hydroxyl-apatite and Sephadex G–150 column chromatographies. The enzyme preparation was homogeneous when subjected to disc gel electrophoresis and ultracentrifugation. Sedimentation velocity yielded a value of . The enzyme showed a typical flavoprotein spectrum of absorption maxima at 276,370 and 454 nm.     

Isolation and Characterization of Globulin from Seeds of Amaranthus hypochondriacus L.

Seeds of grain amaranths contain a high amount (about 60% of total nitrogen) of albumin and globulin and a trace amount of prolamin. From salt-soluble extracts of A. hypochondriacus seeds, a globulin (440,000 in apparent molecular weight and ) was purified by Sepharose 6B gel and DEAE-cellulose column chromatographies. The protein comprised at least four kinds of subunits whose molecular weights were 36,000, 32,000, 20,000 and 18,000, respectively. The amino acid composition of the globulin was almost similar to those of soybean and oat globulins.     

Determination of CGTase from Bacillus ohbensis and Its Optimum pH Using HPLC

Glucose is widely known to be required during superoxide generation in phagocytic cells. However, when an specific chemiluminescence probe with the Cypridina luciferin analog 2-methyl-6-(p-methoxyphenyl)-3, 7 -dihydroimidazo[ 1,2-a]pyrazin-3-one (MCLA) was used, about 60% of the chemiluminescence remained in stimulated macrophages in the presence of the glycolytic inhibitor 2-deoxyglucose. -nonspecific luminol-dependent chemiluminescence disappeared when the same drug was added. These results clearly demonstrate that the generation of by macrophages is not completely glucose-dependent, and strongly suggest that macrophages have both glucoseindependent NADPH-supplying pathway(s) and glucose dependent pathway(s) which generate reactive oxygen species other than .     

Structure of Possible Intermediate of Chlorogenic Acid Biosynthesis in Sweet Potato Root Tissue

Amino acid sequence and peptide-carbohydrate linkage of GP–I–a and GP–I–b, which are two glycopeptides out of three obtained from the saccharogenic amylase of Rhizopus javanicus sp. 3–46, were investigated. By the dinitrophenylation and the subtractive Edman degradation the sequence of GP–I–a and GP–I–b was determined to be respectively.

A glycosylamine type linkage between asparagine and N-acetylglucosamine residue was proved for GP–I–a by the use of the enzyme, β-aspartylglycosylamine amide hydrolase. It seems likely that the peptide-carbohydrate linkage of GP–I–b is also a glycosylamine type linkage.     

New 2-Acylcyclohexane-1,3-diones: Kairomone Components against a Parasitic Wasp,Venturia canescens,from Feces of the Almond Moth,Cadra cautella,and the Indian Meal Moth,Plodia interpunctella

As new kairomone components, 2-palmitoyl- (I) and 2-stearoylcyclohexane-1,3-dione (II) were identified for the parasitic wasp Venturia canescens from feces of the Indian meal moth larva, Plodia interpunctella. One more component, 2-arachidoylcyclohexane-1,3-dione (III), together with the afore mentioned two compounds were newly obtained from feces of the almond moth larva Cadra cautella.

Purified kairomone fractions from both species indicated similar activity to synthetic 2- oleoylcyclohexane-1,3-dione (active at 0.1 mg/ml). 2-Palmitoylcyclohexane-1,3-dione (I) was the most active (at 1 mg/ml) among the synthetic homologues with saturated 2-acyl groups.

No traces of 2-acylcyclohexane-1,3-dione were found in the rearing medium of the host; therefore, the kairomone was suggested to have been biosynthesized.     

Purification,Crystallization and Some Properties of β-Galactosidase from Saccharomyces fragilis

Crystalline β-galactosidase was prepared from the cell extract of Saccharomyces fragilis KY5463, by procedures including protamine sulfate treatment and DEAE-cellulose, hydroxylapatite and DEAE-Sephadex column chromatographies. Crystals were formed when solid ammonium sulfate was added to solutions of the purified enzyme. This procedure resulted in a 55-fold purification with an over-all yield of l5.4%. The crystalline enzyme appeared to be homogeneous on ultracentrifugation and electrophoresis.

The sedimentation coefficient, , was determined to be 10.0 S. The molecular weight was estimated to be approximately 203,000 by the sedimentation equilibrium method of Yphantis. Electrolysis with carrier ampholytes revealed that this enzyme has an isoelectric point at around pH 4.4.

The enzyme was activated by K⁺ in addition to bivalent cations, such as Mn²⁺, Mg^2? and Co²⁺. The Km values for o-NPG and lactose were 4.0×10^?3m and 21.0×10^?3m, respectively. The enzyme is sulfhydryl dependent and was completely inactivated by mercuric ions or p-chloromercuribenzoate.     

A New Growth Effecting Peptide of Bacillus Species from Soybean Cake

A new peptide, which has a marked effect on the growth of Bacillus species, was purified from soybean cake by a procedure employing ammonium sulfate fractionation, and active charcoal-, DEAE-Sephadex A–25- and Sephadex gel-column chromatographies. The purified peptide gave a single band on paper electrophoresis. The peptide possessed the following properties: specific extinction (E at 280nm), 11.9; N-terminal amino acid, glutamate; molecular weight, 8058; total number of amino acid, 70; chemical composition (%), C, 49.12; H, 6.26; N, 16.44; and S, 2.34; isoelectric point, pH4.3. The hydrolyzed peptide had no stimulative effect on the growth of Bacillus species. Especially, those microorganisms belonging to the genera of Bacillus, Arthrobacter and Xanthomonas were remarkably sensitive to the peptide.     

Acid Catalyzed Isomerization of Monocyclic Monoterpenes

The purpose of the present investigation was to study the acid-isomerization of monoterpene aldehydes and alcohols, such as dihydroperillaldehyde (I), phellandral (II), perillyl alcohol (III), dihydroperillyl alcohol (IV) and phellandrol (V), in connection with a previous report on the isomerization of perillaldehyde (VI).

Isomerization of each compound was conducted in 10% aqueous sulfuric acid in the same manner as previously described. Phellandral (II), however, was never isomerized within the limits of our experimental conditions.     

DNA Strand Breakage In Vitro by Autoxidized Unsaturated Fatty Acids

Nitrate and nitrite were successfully extracted from deproteinized chicken egg with aqueous solution, and analyzed by gasliquid chromatography with an electron capture detector without further cleaning. The distribution of these anions in 50 egg samples was the logarithmic normal distribution in each case, that is, N and p{0.052 ppm ≤ ¼ ≤ 0.076ppm} = 0.95 for nitrate-N, and N and p{0.026ppm ≤ ¼ ≤ 0.034 ppm} = 0.95 for nitrite-N. When the chickens were fed with a commercial diet containing elevated levels (1,000 or 5,000 ppm) of nitrate- or nitrite-N, the concentration of these anions in their eggs markedly increased and proceeded to the steady state within 2 or 3 days, where the level was proportional to that of anions added to the diet. After withdrawing the excess of anions from the diet, the concentrations of anions in the eggs decreased exponentially, where the rate constants for nitrate and nitrite were about 0.6 day^?1 and 1.0 day^?1, respectively. In the series of experiments, it was assumed that the reactions proceed simultaneously in the body of chickens.     

Radiation Chemistry of Foods

When 10^?3m cysteine solution was irradiated in the presence of glucose at the concentration of ten-fold of cysteine, the G-values of products produced from cysteine were similar to those from 10^?3m cysteine solution. On the other hand, the yield of carbonyl compound from glucose was suppressed completely by interaction between cysteine and radicals which are secondarily produced from glucose.

Methionine could not suppress the yield of carbonyl compound from glucose, and, G-values of products from methionine varied in comparison with those from solution containing methionine only.

From the results using scavenger, it was concluded that oxidation to methionine sulfoxide and cleavage to α-aminobutyric acid was caused by OH and attack, respectively.     

Studies on Bacterial Protease

A crystalline alkaline protease was prepared from B. amylosacchariticus, which was isolated as a strain of saccharogenic α-amylase-producing Bacillus subtilis. The enzyme was most active at pH values between 10.3 and 10.7 towards casein and was stable at pH values from 6 to 11 on twenty hour incubation at 30°C. Calcium ions were effective to stabilize the enzyme especially at higher temperatures. The enzyme was markedly inactivated by DFP as well as protease inhibitor from potato and slightly by surface active agents, but not affected by sulfhydryl reagents and divalent metal ions except Hg⁺⁺ .Hemoglobin was the best substrate for the enzyme and more than 20% of the peptide bonds were hydrolyzed. Of numerous synthetic peptides tested, only the two compounds, and , were found to be hydrolyzed. A cyclic peptide, gramicidin S, was split by the enzyme only at the peptide bond of -l-valyl-l-ornithyl-. Methyl n-butyrate and tributyrin were also good substrates for the alkaline protease obtained here.