Physiological Studies on Thiobacilli

The electrophoretically pure preparation of cytochrome c from Thiobacillus thiooxidans was obtained. The absorption spectrum exhibited maxima at 415, 521 and 550 mμ in reduced form. The various properties of the cytochrome were very close to these of mammalian cytochrome c, i. e., absorption spectrum, electrophoretic pattern, isoelectric point and E₀′.

Electrophoretically homogenous preparation of NADPH-cytochrome c oxidoreductase was isolated from the soluble fraction of Thiobacillus thiooxidans. The purification of the enzyme was carried out using the fractionation with ammonium sulfate, the treatment with Amberlite IRC-50 and the disk electrophoresis.     

Studies on Phyto-peroxidase

Both crystalline Japanese-radish peroxidase a and c could catalyze the aerobic destruction of indole-3-acetic acid in the presence of a small amount of hydrogen peroxide as an inducer. The pH dependences of the enzymic activities of peroxidase a and c were different from each other. The optimum pH of peroxidase a was found at 3.6, while that of peroxidase c lied in a broad range over 3 to 4.8. In lower concentrations of both peroxidases at pH 3.8 and 6.0, the major product of the reaction was identical with that presented by Ray with using an Omphalia enzyme, which would probably be methyldioxindole, while, in higher concentrations of both peroxidases at pH 3.8, the major product was found to be indole-aldehyde.     

Purification and Properties of Nitrite Reductase from Spinach Leaves

Nitrite reductase (EC 1.6.6.4) has been purified 730-fold from spinach leaves. The enzyme catalyzes the reduction of nitrite to ammonia, with the use of reduced form of methyl viologen and ferredoxin. A stoichiometry of one molecule of nitrite reduced per molecule of ammonia formed has been found. KCN at 2.5×10^-4 m inhibited nitrite reductase activity almost completely. Purified enzyme was almost homogeneous by disk electrophoresis with polyacrylamide gel. The molecular weight of the enzyme was estimated to be 61,000 from gel filtration. Nitrite reductase, in the oxidized form, has absorption maxima at 276, 388 and 573 mμ. Both methyl viologen and ferredoxin linked nitrite reductase activities of the enzyme were inactivated on exposure to low ionic strength.     

Micro Methods for Determination of 3-Ketosucrose and 3-Ketoglucose

Methods for differential determination of 3-ketosucrose and 3-ketoglucose were established. For determination of 3-ketosucrose, alkaline treatment with 0.1 N NaOH was found to be most effective. In this method, 3-ketosucrose gave a characteristic absorption spectrum with a molar extinction coefficient of 6.5 × 10³ m^?1cm^?1 at 340 mμ, while 3-ketoglucose did not show a significant absorption spectrum within a range from 300 to 400 mμ.

By mixing with 0.2 m phosphate buffer, pH 7.0, 3-ketoglucose gave a characteristic absorption spectrum with a molar extinction coefficient of 3.8 × 10³ m^?1cm^?1 at 310 mμ, while 3-ketosucrose showed little absorbance.

From the reduction rate of 2,6-dichloroindophenol with 3-ketoglucose, the ketosugar was determined. 3-Ketosucrose was not able to reduce the reagent at all.

The methods established here were not affected by fructose.     

Urocanic Acid,Produced by Sporogenous Bacteria

On the screening of microorganisms which accumulate ultra violet light absorbing substances, some sporogenous bacteria were selected as powerful strains for production of a crystalline substance having a maximum absorption at 267.5 m_μ (pH 6.0 in water). These strains were microbiologically examined and named B. subtilis var. thermophilus. Submerged fermentation was carried out for 48 hrs at 37°C in glucose bouillon medium and the isolation of substance was performed by absorption to active carbon and elution with ammonia water.

On the basis of chemical studies of this crystal, it was identified as urocanic acid.     

Studies on Phyto-peroxidase

Japanese-radish peroxidase c, a paraperoxidase exhibiting the optical absorption spectrum of low-spin nature, was found to transform to a high-spin state by removing a dissociable ligand of low molecular weight by the addition of the stoichiometric amount of p-chloro mercuribenzoate, as in the case of horseradish peroxidase I or wheat germ peroxidase 566. The reaction could be reversed by the addition of cysteine to remove p-chloromercuribenzoate. As this ligand would be possibly cyanide, the affinity of the high-spin form of the enzyme to sodium cyanide was determined, which was found to be much higher than that of Japanese-radish peroxidase a. The high-spin form of peroxidase c formed the usual Compound I by the addition of hydrogen peroxide, so that the peroxidatic reaction catalyzed by this enzyme should follow the common mechanism of plant peroxidases. However, Compound II was scarecely observed during the course of the stepwise reduction of Compound I by ascorbate, probably because of its more rapid conversion to the free enzyme.     

Purification and Properties of Putrescine Oxidase of Micrococcus rubens

A procedure for obtaining a highly purified preparation of the putrescine oxidase of Micrococcus rubens has been developed. The method involved fractionation with ammonium sulfate and separation on successive columns of DEAE-cellulose, DEAE-sephadex and hydroxylapatite. The enzyme was purified about 200-fold from the cell extract and the final preparation showed a symmetric peak in the ultracentrifugal analysis. The purified enzyme was yellow in color and showed absorption maxima at about 380 and 460 mμ. The yellow color was lost by the substrate as well as by sodium dithionite, and was subsequently restored by oxygenation. The purified enzyme contained 12.2 mμ moles of flavin adenine dinucleotide (FAD) per mg of protein. The enzyme was inhibited by p-chloromercuric benzoate.

The putrescine oxidase could oxidatively degrade spermidine into 1,3-diaminopropane and γ-aminobutyraldehyde stoichiometrically, and no ammonia was liberated.     

Studies on the Sulfite Reducing System of Algae

Sulfite reductase using reduced methyl viologen as an electron donor was purified about 94-fold from a red alga, Porphyra yezoensis. The enzyme was ultracentrifugically homogenous and could reduce sulfite to sulfide quantitatively with an uptake of six electrons. The enzyme had a pH optimum in the vicinity of 7.5. The Km for sulfite was determined to be 6.5×l0^?4m. The purified preparation of the algal reductase showed its absorption maximum at 385 mμ and slight shoulders at 408, 456, 485, 600 and 664 mμ in addition to an intense peak at 278 mμ. Metal analysis of the purified enzyme suggested the presence of iron and copper in the molecule. NADPH, NADH or the reduced form of spinach ferredoxin could not be a direct electron donor for the purified algal sulfite reductase.     

Lipids of Algae

The carotenoid pigments prepared from acetone extracts of chlorella were separated into epiphasic and hypophasic fractions by partition between petroleum ether and 90% methanol. Each fraction was subjected to column chromatography, using aluminium oxide, magnesium oxide, calcium hydroxide or calcium carbonate as adsorbent. The absorption maxima of the separated pigments in hexane and in carbon disulfide were compared with those of the known pigments. Some of the separated pigments were identified as those previously known which follow: α-carotene, β-carotene, rhodoxanthin, sarcinaxanthin, lutein and neoxanthin. Two unknown pigments with absorption maxima not yet reported were separated. The first showed absorption maxima at 478 m_μ in hexane and 518 m_μ in carbon disulfide, and the second at 383, 402 and 425 m_μ in hexane and 428 and 450 m_μ in carbon disulfide.     

Properties of Crystalline Creatinine Deiminase from Corynebacterium lilium

The properties of creatinine deiminase (EC 3.5.4.21) were characterized with a crystalline preparation from Corynebacterium lilium ATCC 15990. The molecular weight was determined to be 195,000 by the sedimentation equilibrium method, and the isoelectric point was found to be 4.2 by isoelectric focusing. The enzyme was relatively thermostable and had a broad pH optimum of 7.5 to 10.0. It was specific for creatinine and showed a Km value of 1.27 mm. A compound from creatinine was isolated, with the release of ammonia, and identified as N-methylhydantoin. The enzyme activity was inhibited by heavy metal ions and p-chloromercuribenzoate. The enzyme may be useful in determinations of serum and urinary creatinine.     

Properties of the Crystalline Quinolinate Phosphoribosyltransferase from Hog Liver

Quinolinate phosphoribosyltransferase has an important role in the NAD de novo biosynthetic pathway. Crystalline quinolinate phosphoribosyltransferase could be obtained for the first time from mammalian tissue. The crystalline enzyme preparation was certified to be homogeneous by polyacrylamide gel disc electrophoresis. Catalytic properties of this enzyme preparation were investigated. Optimum pH for the reaction was 6.1. Divalent cations were absolutely required and Mg²⁺ was the most effective. Michaelis constants for quinolinic acid and PRPP were 1.2 × 10^?4 m and 1.8 × 10^?4 m, respectively. Quinolinic acid could not be replaced by nicotinic acid or 2-amino nicotinic acid in this reaction. Di- and tri-valent cations fairly inhibited the reaction, but mono-valent cations had no effects. The reaction product was identified as β-nicotinic acid mononucleotide by its ultraviolet absorption spectra, paper chromatography, paper electrophoresis and its ORD spectrum.     

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.     

Optical rotatory dispersion studies of poly-O-acetyl-gamma-hydroxy-L-proline forms I and II

Poly-O-acetyl-hydroxy-L -proline, forms I and II have been studied by optical rotatory dispersion (ORD) and ultraviolet spectrophotometry in solution and in the solid state. Cotton effects of opposite sign, but not mirror images, were observed in the 250 mμ region for the two forms (Form I, peak 278 mμ; crossover, 254 mμ; trough, 244 mμ: Form II, trough 270 mμ; crossover, 248 mμ; peak, 238 mμ). Thus, the Cotton effects for a right-handed and left-handed helix have been shown to be opposite for the proline type helices I and II. The ORD of films of form I was found to have a positive Cotton effect further into the ultraviolet region with peak at 218 mμ. Absorption spectra showed a shift of 8 mμ in the absorption peak in the 200 mμ region for the two forms (form. I, 211 mμ; form II, 203 mμ). A shoulder was demonstrated in the film absorption spectra in the 250 mμ region where the Cotton effects are found. The mixing of the n, π* and π, π* states of the amide chromophore and n, π* state of the ester chromophore was suggested as being responsible for the Cotton effects in the 250 mμ region.     

Purification of Cytochrome a of an L-Glutamate-producing Microorganism,Brevibacterium thiogenitalis

The respiratory chain system of Brev. thiogenitalis grown in the presence of copper ions contained cytochromes a, b and c. The cytochrome a was solubilized and purified from the cell-free extracts by means of Triton X-100 and cholate extraction, and DEAE-cellulose chromatography. It was purified about 130-fold from the cell-free extracts and was free from other cytochromes, The purified preparation contained 1.4 mμatom copper and 1.9 mμatom iron per mμmole heme a, respectively, and approximately 5 mμmoles heme a per mg protein.     

Adsorption of peroxidase on Celite 545 directly from ammonium sulfate fractionated white radish (Raphanus sativus) proteins

This paper demonstrates the direct immobilization of peroxidase from ammonium sulfate fractionated white radish proteins on an inorganic support, Celite 545. The adsorbed peroxidase was crosslinked by using glutaraldehyde. The activity yield for white radish peroxidase was adsorbed on Celite 545 was 70% and this activity was decreased and remained 60% of the initial activity after crosslinking by glutaraldehyde. The pH and temperature-optima for both soluble and immobilized peroxidase was at pH 5.5 and 40°C. Immobilized peroxidase retained higher stability against heat and water-miscible organic solvents. In the presence of 5.0 mM mercuric chloride, immobilized white radish peroxidase retained 41% of its initial activity while the free enzyme lost 93% activity. Soluble enzyme lost 61% of its initial activity while immobilized peroxidase retained 86% of the original activity when exposed to 0.02 mM sodium azide for 1 h. The K_m values were 0.056 and 0.07 mM for free and immobilized enzyme, respectively. Immobilized white radish peroxidase exhibited lower V_max as compared to the soluble enzyme. Immobilized peroxidase preparation showed better storage stability as compared to its soluble counterpart.     

Preparation and General Properties of Glucoamylase Bound to Halogenacetyl Cellulose

The coupling reaction of glucoamylase and halogenacetyl cellulose (HAC) without pretreating with organic solvent led to form large particles of immobilized glucoamylase and the activity and the specific activity of the preparation were very low. However, the coupling reaction with HAC pretreated with organic solvents allowed to form very fine particles and the activity was increased by five times. The latter contained 3~6% of enzyme protein and the specific activity to maltose reached to 80~90% of native glucoamylase. Since the specific activity of the preparation was presumed to be much influenced by the particle size, the specific activity and general properties of different particle sizes were compared with those of native enzyme. The specific activities of particles of 0~15μ, 15~55μ, 70~190μ and 130~270μ showed 82%, 33%, 27% and only 7% of native enzyme, respectively. Km values of native form, 0~15μ, 15~55μ and 70~190μ particles were 0.90×10^?3m, 1.35×10^?3m, 1.60×10^?3m and 2.1×10^?3m in the case of maltose as substrate, respectively. The other properties of particles of 0~15μ were almost identical to those of native enzyme except for the effect of temperature on the reaction rate. However, pH activity, pH stability and urea stability of particles of 70~190μ were much inferior to those of native enzyme and particles of 0~15μ.     

Differential effects of nitric oxide on peroxidase and H2O2 production by the xylem of Zinnia elegans

IWF, intercellular washing fluid
pCMB, p-chloromercuribenzoic acid
SNAP, S-nitroso-N-acetyl-penicillamine SNP, sodium nitroprusside
TMB, 3,3’,5,5’- tetramethylbenzidine

Sodium nitroprusside (SNP) and S-nitroso-N-acetyl-penicillamine (SNAP) are two nitric oxide (NO)-releasing compounds that, when used at 5·0 mol m^–3 concentrations, are capable of releasing NO in the aqueous phase at a rate of 35 ± 4 and 47 ± 5 μmol m^–3 s^–1, respectively. For this reason, the effect of SNP and SNAP on coniferyl alcohol peroxidase and on H₂O₂ production by the lignifying xylem of Zinnia elegans (L.) has been studied in order to ascertain whether NO, which is a synchronizing chemical messenger in animals and an air pollutant, has any effect on these plant-specific metabolic aspects. The results showed that both SNP and SNAP provoke an inhibition in the mol m^–3 concentration range of the coniferyl alcohol peroxidase activity of a basic peroxidase isoenzyme present in the intercellular washing fluid of Z. elegans. The effect of these NO-releasing compounds on peroxidase was confirmed through histochemical studies, which showed that xylem peroxidase was totally inhibited by treatment with these NO donors at 5·0 mol m^–3, and by NO at a concentration change rate of 55 ± 5 and 110 ± 9 μmol m^–3 s^–1. However, SNP, at 5·0 mol m^–3, does not have any effect on H₂O₂ production by the xylem of Z. elegans. The fact that SNP and SNAP are two structurally dissimilar compounds which only share the common ability to release NO in aqueous buffer, and that similar results were obtained when using NO itself, suggest that NO could be considered as an inhibitor of coniferyl alcohol peroxidase which does not affect H₂O₂ production in the xylem of Z. elegans.     

Distribution of Selenium in Bovine Milk and Selenium Deficiency in Rats Fed Casein-based Diets,Monitored by Lipid Peroxide Level and Glutathione Peroxidase Activity

A major proportion of selenium in bovine milk was found in fluorometric analysis to be associated with the casein fraction, largely in alkali-labile form, and the rest with the whey fraction mostly in free selenite form. This uneven distribution of milk selenium seems to provide an explanation for selenium deficiency in purified caseins. The activity of glutathione peroxidase, a selenoprotein, in the liver of growing male rats fed ad libitum low-selenium diets containing either vitamin-free casein or Torula yeast 0.065 ± 0.012 or 0.015 ± 0.004 μ g Se/g diet, respectively) for 3 weeks decreased to 4 to 6% of that of the control rats fed a commercial stock diet (0.185 ± 0.092 μ g Se/g diet). Selenium status was evaluated by three different parameters for the rats assigned under pair-feeding regimen to those vitamin-free casein-based diets which were supplemented with graded levels of selenium as sodium selenite. The hepatic levels of the thiobarbituric acid-reactive substance, an indication of lipid peroxidation, decreased to control level with selenium supplementation per g diet of 0.1 μ g and over. The hepatic glutathione peroxidase activity reached a plateau above a 0.1 μ g/g diet of selenium supplementation, whereas the erythrocyte enzyme activity increased with increasing levels of supplementary selenium. These results support the notion that semi-purified diets containing vitamin-free casein as a prime protein source would not satisfy the selenium requirement of growing animals unless deliberately supplemented with additional selenium.     

Manganese Absorption and Transport in Rice

The absorptive patterns of Mn²⁺ in excised rice roots, leaf tissues and intact plants, were studied. The rates of absorption of Mn²⁺ followed different patterns in the roots and the leaf tissues. The uptake from 0.1 and 5 mM MnSO₄ was found to be sensitive to metabolic inhibitors. The time course of uptake from 0.1 mM and 5 mM MnSO₄ followed a biphasic pattern which represented only the metabolic component of absorption. A secondary biphasic pattern of uptake at 5 mM MnSO₄ (one at 20 min and another at 80 min) appears quite anomalous and is probably related to structural virations or cellular compartments. When absorption and transport of Mn²⁺ were measured in intact rice and wheat plants, it was found that Mn²⁺ was easily translocated to shoot from roots and the transport of Mn²⁺ was comparable to that of K⁺.     

Isolation of 2′-Hydroxy-4′-methoxy propiophenone from Tribolium castaneum and T confusum

The copper binding properties were influenced by growth phase of cells, pH and concentration of copper in reaction mixtures. The efficiency of copper absorption increased with growth time and was largest at the mid-logarithmic growth phase. The time course of copper absorption was biphasic, that copper rapidly bound to cell surface for initial few minutes after addition of copper and then the copper was slowly transported into cells. The copper binding to the cell surface depended on the molecular form of copper complex in the reaction mixture and the ligand residue to copper on the cell surface. Double reciprocal plots of absorption velocity of copper vs. copper concentration gave straight lines at low concentration between 0.01 to 0.1 mm. The apparent affinity of copper to the cells of stationary growth phase was the same as that of logarithmic growth phase, that is, the Km values were about 0.01 mm. On the other hand, at high concentration of copper between 0.1 to 5.0 mm the apparent affinity decreased but the absorption velocity of copper remarkably increased. Zinc sulfate most strongly inhibited the copper absorption in this test. It was assumed that zinc competitively bound to the copper binding sites of cell surface.