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.     

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.     

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.     

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 .     

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.     

Hydrolysis Rate of Resistant Pentosan of White Birch Wood in Sulfuric Acid of Medium Strength

The purpose of this study is to find optimal conditions for pre-hydrolysis in the new wood saccharification process with strong sulfuric acid. In the experiment, the hydrolysis rate of resistant fraction of pentosan of white birch (Shirakamba, Betula platyphylla Sukatchev var. japonica Hara) wood and the decomposition rate of xylose are measured in acid concentrations ranging from 30 to 60% at temperatures ranging from 30 to 90°C. The hydrolysis of resistant pentosan of white birch and the decomposition of xylose are the first-order reactions. The first-order reaction constant of hydrolysis of resistant pentosan, k_B min^-1, is expressed by the following empirical equations as the function of percentage concentration of sulfuric acid, C, and reaction temperature described by absolute temperature, T°K, ranging from 40 to 80°C:

where sulfuric acid concentrations range from 30 to 50%;

where sulfuric acid concentration is 60%.

The first-order reaction constant of decomposition of xylose, k₂ min^-1, is expressed by the following empirical equation as the function of sulfuric acid strength described by acidity function, H₀, and reaction temperature described by absolute temperature, T°K, in sulfuric acid concentrations ranging from 30 to 60% at temperatures within the range of 40 to 100°C.

where C is sulfuric acid strength described by acidity function, H₀.     

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.     

New Strains of Botryodiplodin-producing Fungi

The light-emitting species of chemiluminescence produced in rat liver homogenate on adding autoxidized linseed oil (AOLO) were investigated. The chemiluminescent intensity of liver homogenate was strongly enhanced by the addition of AOLO and showed a proportional relationship to the amount of AOLO. The chemiluminescence was reduced with singlet oxygen (¹O₂) quenchers and free radical scavengers. Among them, β-carotene showed the most effective quenching. The emission spectrum had broad bands in the visible region with eminent chemiluminescent lines at 520, 575 and 640 nm due to the simultaneous transition, . An additional weak line was found at 480 nm corresponding to . In the presence of β-carotene, lines corresponding to the simultaneous transition of ¹O₂ disappeared. These results indicate that the liver homogenate with AOLO generated singlet molecular oxygen as one of the major light-emitters of the chemiluminescence. A possible mechanism for the generation of ¹O₂ is by decomposition of peroxy radicals derived from AOLO in the liver homogenate.     

Red Algae (Eucheuma muricatum) Mucilage- Complex Formation and Mechanism

Eucheuma muricatum mucilage which was extracted and purified after irradiation of the seaweed with γ-ray of ⁶⁰Co formed a complex with , and exhibited a new absorption band at 555 nm. The absorbancy observed at that time depended on the concentration of urea and on the temperature. The curves representing relations between absorbancy at 555 nm and the above factors have two inflection points. The fact that their inflection points shift toward the lower temperature side with the increase in urea concentration suggests that the coloring phenomenon may relate closely to the transition of the mucilage. It was also found that the absorbancy at 555 nm depended on the content of pyruvic acid residue in the same mucilages, the absorbancy decreased with the increase pyruvic acid residues, and that the steric hindrance caused by a sugar residue of large demension affected the stable from containing viscous polysaccharide.     

Microbial Transformation of Sterols

The ability of accumulating androsta-1, 4-diene-3, 17-dione (ADD) in the digestion of cholesterol by Arthrobacter simplex IAM 1660 was examined with 167 compounds and (i) chelating agents, (ii) Ni²⁺, Co²⁺, Hg²⁺, As³⁺, Sb³⁺, Bi³⁺, Cd²⁺, , and ions, and (iii) redox dyes were found effective for ADD accumulation. Ionic state of the chelating agents was unfavorable for ADD accumulation but inactive ethylaenediamine tetraacetic acid could be turned effective with aid of surface active agents and penicilline. Lipophilic structure of the chelating agents was required probably for its penetration through the cell membrane. The target process of the ADD accumulating agents was supposed as 9α-hydroxylation and their possible mechanism of inhibiting 9α-hydroxylation is discussed.     

Studies on Alginase

Chemical investigations were made on a new unsaturated crystalline diuronide isolated from alginase hydrolysate of alginic acid. This uronide has (in water), and m.p. 135.5~136.5°C (decomp.). The presence of an α/β-unsaturated carboxylic acid formulation is supported by the following evidences: (a) an ultraviolet absorption band at 232 m/μ, (b) infrared absorption bands at 1648 cm^-1 due to double bond and at 1720 cm^-1 due to conjugated carboxylic group, (c) the consumption of about 1 mole of bromine per mole of the compound, (d) the production of oxalic acid on oxidation with ozone, (e) the formation of a substance that shows absorption maximum at 550 mμ, caused by the addition of thiobarbituric test. After hydrolysis, crystalline mannuronic lactone was obtained from the unsaturated diuronide. Occurrence of mannuronic moiety in the reducing unit was observed by paper chromatography of the hydrolysate of borohydride-reduced unsaturated compound. From these results it can be seen that the possible structure of this unsaturated diuronide is 4-O- (β-d-Δ4,5 mannoseenpyranosyluronic acid) -d-mannuronic acid.     

Studies on Oxidation-Reduction Potentials (ORP) of Microbial Cultures

At maximum production of l-glutamic acid, the oxidation-reduction potential of the culture broth in l-glutamic acid fermentation showed a stable value of 9.0 to 9.6 as rH value. When biotin concentration in the medium was high (40γ/liter), the production of l-glutamic acid decreased, and the rH was 8.0 and it was out of accordance with that of the control (biotin-poor; 2γ/liter). Under “less-aerobic” conditions, its rH rose to 10.4.

From these results, it was concluded that the rH during maximum production of l-glutamic acid showed a stable value affected actively by the redox system, l-glutamic acid/α-ketoglutaric acid and      

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.     

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.     

The Initial Attack Sites of a Streptomyces Alkalophilic Proteinase on Oxidized Insulin B-Chain

The sites on oxidized insulin B-chain substrate initially attacked by an alkalophilic proteinase from a Streptomyces sp., were investigated under incubation conditions employing one part enzyme to one thousand parts of substrate at 0°C.

Analysis of the peptides produced after 10 to 40 seconds of incubation revealed that the enzyme, which has an optimum pH of around 13, first attacks two peptide linkages “-Leu (15)Tyr (16)-Leu (17)-” of the oxidized insulin B-chain with equal efficiency.     

Changes in Amino Acid Composition during Germination of Soybean

Malonogalactan, a malonylated polysaccharide (—74° (c=1.6, H₂O)) produced by Penicillium citrinum, consisted of d-galactose and malonic acid in the approximate molar ratio of 3:1. Molecular weight of the demalonylated galactan (-99° (c=4.6, H₂O)) was about 40,000. From the data regarding optical rotation, nuclear magnetic resonance spectrum, infrared spectrum, glycosidase susceptibility, periodate oxidation, Smith degradation, methylation and acid hydrolysis, the possible structure of the Penicillium malonogalactan is deduced as follows: A galactan, 1,5-β-galactofuranoside polymer esterified with malonic acid at the position of 2 or 3.     

Lipolysis by Intracellular Lipase of Streptococcus lactis against Its Neutral Lipids Obtained by Growth at Low Temperature

Lipolytic activities of intracellular lipase obtained from Streptococcus lactis 527 cells grown at 30°C were determined using bacterial neutral lipids extracted from cells grown at 10 and 30°C. The amounts of free fatty acids liberated from lipids by lipase were in the order: 30°C neutral lipid > 10°C neutral lipid > triolein > intracellular membrane fraction. Glycerides hydrolyzed partially by lipase were detected on thin-layer plates and were composed of 1,3- and 1,2-diglycerides, fatty acids and unhydrolyzed triglycerides. Fatty acids liberated from neutral lipids by lipase were determined by gas chromatography. It was found that the major acid was cy-C₁₀ and the minor among the acids liberated from 10°C neutral lipid, whereas the major acid was and the minors and cy-C₁₀ from 30°C lipid.     

Guanosine Diphosphate Mannose in Conformational Relation to Other Suger Nucleotides and Phosphates in Solution

Molecular conformational transition of GDPMan and solution conformation of α-d- mannopyranose moiety in Man-l-P and GDPMan were examined in relation to other sugar nucleotides and phosphates. GDPMan and other sugar nucleotides examined revealed changes in the optical rotation in sigmoidal curve in water by addition of urea. The change was reversible without significant decomposition and is attributable to dissociation of an ordered form into a random form. Optical conformational values in 8m urea solution were+116° for GDPMan, +58°~+79° for UDPGlc, +79° for UDPGal, +135°~+143° for UDPGlcNAc, and +138°~ +155° for UDPGIcA.

NMR analysis and periodate oxidation study revealed the ⁴C₁ conformation of α-d-hexopyranose moieties in Man-1-P, Glc-l-P, GDPMan, UDPGlcNAc and UDPGalNAc.     

Purification and Substrate Specificity of Yeast Isoamylase

Yeast isoamylase was highly purified by means of salting-out with ammonium sulfate, chromatography on DEAE-cellulose, and gel filtration on Sephadex G-100. More than 200-fold purification was achieved through these procedures from crude yeast extract. While the purified enzyme did not attack α-1, 6-glucosidic linkages in panose, isopanose (6-malto-sylglucose), branched triose (4,6-diglucosylglucose), and isomaltosylmaltose (6³-α-glucosylmaltotriose), it acted on α,β-limit dextrin to liberate glucose as well as maltose and higher oligosaccharides. Substrate specificity of the yeast isoamylase was discussed in comparison with that of plant and bacterial isoamylases (R-enzvme and pullulanase), and the mechanism of debranching of glycogen by yeast enzymes was also discussed.