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.     

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.     

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 .     

Kinetics of Carbon Dioxide Evolution in Agitation System

Kinetics of carbon dioxide evolution was investigated in agitation system. Reaction steps of carbon dioxide evolution in submerged fermentations may consist of three steps; the first, hydration of carbon dioxide liberated from living cells, the second, dehydration of bicarbonate ions and the third, formation of carbon dioxide bubbles. Taking into account the equilibrium between hydration of carbon dioxide and dehydration of bicarbonate ions at physiological pH value, the fallowings may be rate-limiting steps in mass transfer of carbon dioxide in submerged fermentations, dehydration of bicarbonate ions and the bubble formation. The overall velocity constant of these two reaction steps was determined in the agitation vessel This reaction obeyed good first-order kinetics and the term of was introduced as a velocity constant. This value was influenced by agitation speed, temperature, viscosity of the fluid and carbonic anhydrase. The value of carbon dioxide coefficient (Kd)_CO2 was higher than the oxygen absorption coefficient Kd. The driving force of mass transfer for carbon dioxide, DCO₂—pCO₂, therefore, was lower than that for oxygen, P_B—P_L. The relationship between the overall coefficient of oxygen transfer across gas-liquid interface K_La and the overall velocity constant of carbon dioxide evolution was expressed in the formula      

Two Forms of Glucoamylase from Mucor rouxianus 1. Purification and Crystallization

Mucor rouxianus produced two forms (isoenzymes) of glucoamylase which could be separated from each other by polyacrylamide gel electrophoresis or by chromatography on SP-Sephadex C-50, and they were designated glucoamylase I and glucoamylase II. Glucoamylases I and II were isolated in crystalline form, and were homogeneous in poly acrylamide gel electrophoresis and in ultracentrifugation, respectively. The sedimentation coefficient () molecular weight of glucoamylase I were 4.39 S and 59,000, and those of glucoamylase II were 4.29 S and 49,000, respectively.     

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.     

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.     

Aromatic Amino Acid Residues in Japanese-radish Peroxidase a and Apoenzyme†

The nature of aromatic amino acid residues in Japanese-radish peroxidase a and the apoprotein was investigated by means of spectrophotometry and fluorospectrophotometry. The tyrosine residues in the holoenzyme were masked in the alkali-titration, giving an abnormally high value of 12.6, while they were exposed in the apoenzyme, exhibiting a value of 10.8. The difference spectra in the ultraviolet region between the holo-and apo-enzyme showed characteristic bands of tryptophan and phenylalanine as well as tyrosine. The perturbation of the aromatic amino acid residues by 50% ethyleneglycol was observed in the apoenzyme but not in the holoenzyme. The fluorophotometric experiments also revealed that the aromatic amino acid residues were in different environments in the holo- and apoenzyme. The difference between the conformation of peroxidase and that of the apoprotein was discussed.     

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.     

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.     

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.     

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.     

Cell-wall Polysaccharides of Immature Barley Plants. II. Characterization of a Xyloglucan

A xyloglucan occurring in the hemicellulose II fraction of cell walls of immature barley plants was characterized by methylation and fragmentation analyses. The results indicated that the xyloglucan was mainly composed of the following repeating units:      

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.     

Isosclerone,a New Metabolite of Sclerotinia sclerotiorum (Lib.) De Bary

Isosclerone, mp 74~76°C, , a new metabolite of Sclerotinia sclerotiorum, was isolated and its absolute structure was determined as (4S)-3,4-dihydro-4,8-dihydroxy-1(2H)-naphthalenone. It stimulated the root elongation of rice seedlings by ca. 30% at concentrations of 1~10 ppm, and inhibited the growth of shoots and roots at high concentrations above 50 ppm.     

Production and Isolation of Dopastin,an Inhibitor of Dopamine β-Hydroxylase

A new inhibitor of dopamine β-hydroxylase, dopastin, has been isolated. The dopastin-producing strain was found in a mushroom culture, and after being separated, it was confirmed to be a member of Pseudomonas. Dopastin was obtained as colorless needles, mp 116~119°C, (c=0.5, С₂Н₅ОН), C₉H₁₇N₃O₃. The catalytic hydrogenation afforded dihydro-dopastin which also inhibits dopamine β-hydroxylase.     

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.     

The Chemistry of Ilexol. IV.

It has now been shown that Acetate A previously obtained by isomerizing ilexol acetate is nothing other than urs-13 (18)-en-3β-yl acetate prepared from α-amyrin, furnishing conclusive evidence for its conversion into one of the compounds of the ursane series.

Isoilexol, oxidized with chromium trioxide at room temperature, afforded a ketone named isoilexone, C₃₀H₄₈O, m.p. 194–195°, +75.90° (c, 0.527), and oxidized with the same oxidant at 70–80°, another ketone named isoilexenedione, C₃₀H₄₆O₂, m.p. 221-223°, +16.28° (c, 0.307).

Huang-Milon reduction of these ketones afforded one and the same deoxy compound named isoilexene, C₃₀H₅₀, m.p. 183–185°, +48.34° (c, 0.290), which might well be assumed to constitute a pair of optical antipodes with olean-13 (18)-ene, C₃₀H₅₀, m.p. 184-185°, ?48.50° (c, 0.545).     

Isolation and Structure of Maingayic Acid,a Piscicidal Constituent of Callicarpa maingayi

A piscicidal constituent (1), C₂₀H₂₈O₃, (chloroform), which was named maingayic acid, was isolated from the leaf of Callicarpa maingayi. On the basis of the chemical spectral studies, the pK_MCS evaluation and the octant rule on the ORD curves, we have and deduced that maingayic acid is a furanoid diterpene acid possessing a rearranged labdane skeleton shown as 1’a.     

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.