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.     

The characterization of myosin–product complexes and of product-release steps during the magnesium ion-dependent adenosine triphosphatase reaction

Evidence is presented that the myosin subfragment-1–ADP complex, generated by the addition of Mg²⁺ and ADP to subfragment 1, is an intermediate within the myosin Mg²⁺-dependent adenosine triphosphatase (ATPase) turnover cycle. The existence of this species as a steady-state intermediate at pH8 and 5°C is demonstrated by fluorescence measurements, but its concentration becomes too low to measure at 21°C. This arises because there is a marked temperature-dependence on the rate of the process controlling ADP dissociation from subfragment 1 (rate=1.4s⁻¹ at 21°C, 0.07s⁻¹ at 5°C). In the ATPase pathway this reaction is in series with a relatively temperature-insensitive process, namely an isomerization of the subfragment-1–product complex (rate=0.055s⁻¹ at 21°C, 0.036s⁻¹ at 5°C). By means of studies on the P_i inhibition of nucleotide-association rates, a myosin subfragment-1–P_i complex was characterized with a dissociation equilibrium constant of 1.5mm. P_i appears to bind more weakly to the myosin subfragment-1–ADP complex. The studies indicate that P_i dissociates from subfragment 1 at a rate greater than 40s⁻¹, and substantiates the existence of a myosin-product isomerization before product release in the elementary processes of the Mg²⁺-dependent ATPase. In this ATPase mechanism Mg²⁺ associates as a complex with ATP and is released as a complex with ADP. In 0.1m-KCl at pH8 1.0mol of H⁺ is released/mol of subfragment 1 concomitant with the myosin-product isomerization or P_i dissociation, and 0.23 mol of H⁺ is released/mol of subfragment when ATP binds to the protein, but 0.23 mol of H⁺ is taken up again from the medium when ADP dissociates. Within experimental sensitivity no H⁺ is released into the medium in the step involving ATP cleavage.     

Ethylene Formation from Acrylic Acid by a Banana Pulp Extract

An enzyme extract from ripe banana pulp catalyzed the formation of ethylene in the presence of Mg²⁺, TPP and acrylic acid under the gas phase of argon. Reaction conditions, cofactor requirement and optimum pH are presented.

Ethylene formation from acrylic acid catalyzed by TPP at pH 9.0 or by yeast decarboxylase was also demonstrated.

The following route was proposed as a biosynthetic pathway of ethylene in banana fruit;      

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.     

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.     

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.     

Comparison of ATPase activities and heavy chains of rabbit atrial and thyrotoxic ventricular myosin subfragment-1

Summary Subfragment-1 of rabbit atrial and thyrotoxic ventricular myosin (V₁ isomyosin) has been prepared and purified by DEAF-cellulose column chromatography. Pyrophosphate-polyacrylamide gel electrophoretic patterns and column chromatographic profile of the atrial subfragment differ from those of thyrotoxic ventricular myosin subfragment-1. On the other hand, Ca²⁺, Mg²⁺ and actin-activated ATPase activities of these subfragments are identical. Comparison of the peptide mapping by limited proteolysis in the presence of sodium dodecyl sulfate of the heavy and the light subunits of these subfragments reveals that the patterns for the heavy chain peptides of these subfragments are substantially similar but their light chain peptide patterns differ. The results suggest that the enzymatic and structural similarities that have been recognized between these isoenzymes using intact myosin hold true for the myosin subfragment-1.The differences between these subfragments are due to the differences in the light chains associated with them.Abbreviations EDTA Ethylene Diamine Tetra-acetic Acid - SDS Sodium Dodecyl Sulfate - S₁ myosin subfragment-1 - HC Heavy Chain - LC Light Chain     

Fukiic Acid Isolated from the Hydrolysate of a Polyphenol in Petasites japonicus

d-Glucose-isomerizing enzyme was purified in a crystalline form with a good yield from the cells of Bacillus coagulans, strain HN-68, and some phsicochemical properties were investigated.

The purified enzyme was homogeneous on both ultracentrifugal and disc-electrophoretical analyses. The molecular weight of the enzyme was determined to be 175,000 and 160,000 from the sedimentation-viscosity method and the gel filtration method, respectively.

The sedimentation coefficient , partial specific volume, at 280 mμ, and the nitrogen content of the enzyme were determined to be 10.2×10^?13 sec, 0.705 cm³g^?1, 10.6 and 16.2%, respectively. The integral numbers of amino acid residues per molecule calculated on the basis of 160,000 were as follows; Lys₁₂₀, His₄₉, Arg₆₁, Asp₁₈₂, Thr₈₇, Ser₇₀, Glu₁₃₆, Pro₄₄, Gly₁₀₆, Ala₁₄₀, Half-Cys₀, Val₅₃, Met₂₇, Ileu₅₁, Leu₁₃₄, Tyr₅₈, Phe₉₆, Try₁₃, and amide-ammonia₈₀.

Purified enzyme preparation obtained from Bacillus coagulans, strain HN-68 requires Co²⁺ for d-glucose- and d-ribose-isomerizing activities and Mn²⁺ for d-xylose-isomerizing activity. The values of Km for d-glucose, d-xylose and d-ribose were 9×10^?2, 1.1×10^?3, 7.7×1O^?m and of the relative V_max were 0.52, 1.1 and 0.25 mg/min at 40°C, respectively. d-Glucose-isomerizing activity was inhibited by d-xylose and d-ribose. However, there was not a difference among three activities of the enzyme with respect to following properties: Activation energy was 14,600 cal per mol. The enzyme was inhibited in a competitive manner by tris(hydroxymethyl)aminomethane, d-xylitol, d-sorbitol and d-mannitol, and the Ki values for these inhibitor were 3×10^?4, 2.5×10^?3, 2.9×10^?2 and 7×10^?2m, respectively. The ratio of three activities did not change by heat- and pH-treatments. Mn²⁺, Co²⁺ and Ni²⁺ protected strongly the enzyme from heat denaturation. The enzyme can isomerize d-glucose, d-xylose and d-ribose to their corresponding ketose, but the kinetic constants and induction studies indicated that ^d-xylose is the natural substrate for the enzyme.     

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.     

Certain Properties of Crystalline α-Glucosidase from Mucor javanicus

The crystalline α-glucosidase from Mucor javanicus has a sedimentation constant () of 6.1 S, a diffusion constant (D_{20, w}) of 4.8 × 10^?7 cm² · sec^?1, and an average molecular weight, as determined by two different methods, of 124,600. The α-glucosidase is a glycoprotein containing the following constituents; tryptophan₂₃, lysine₈₁, histidine₃₉, arginine₃₄, aspartic acid₁₀₂, threonine₆₉, serine₄₆, glutamic acid₇₈, proline₅₅, glycine₇₈, alanine₅₅, half cystine₈, valine₅₃, methionine₁₇, isoleucine₅₈, leucine₈₁, tyrosine₅₁, phenylalanine₄₁, glucosamine₁₂, and mannose₃₈.

The low content of half cystine, the high contents of aspartic acid, lysine, and histidine, and the presence of mannose as the sole constituent of neutral sugar are the characteristics of this enzyme.     

Some Properties of GTP Cyclohydrolase from Serratia indica and the Pterin Product by Its Enzymatic Reaction

GTP cyclohydrolase which catalyzes the formation of formic acid and a pterin compound from guanosine-5′-triphosphate (GTP) has been partially purified from extracts of Serratia indica IFO 3759. ¹⁴C-Formic acid eliminated from (8-¹⁴C)GTP is oxidized with mercury acetate to ¹⁴CO₂, which is trapped by β-phenylethylamine. The molecular weight of the enzyme is approximately 170,000 and the enzyme is relatively heat-stable. The enzyme activity is strongly inhibited by GDP and ATP, but not by other nucleotides. Inhibition by GDP is competitive with GTP. Metals, such as Fe²⁺, Co²⁺, Ni²⁺, Zn²⁺, Cd²⁺, Al³⁺, Hg²⁺ and p-chloromercuribenzoate strongly inhibit the enzyme activity. The activity is also inhibited by . The pterin product has been characterized as a derivative of neopterin triphosphate by enzymatic degradations, ultraviolet spectra, fluorescence and excitation spectra, thin-layer chromatography and thin-layer electrophoresis. The product is estimated to differ from d-erythro-neopterin triphosphate prepared from the enzyme system of Escherichia coli B, since (1) only one mole of phosphate can be liberated by alkaline phosphatase and two moles of phosphates by phosphodiesterase and alkaline phosphatase from the product, and (2) the retention time of the product on high-performance liquid chromatography is different from that of d-erythro-neopterin triphosphate.     

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.     

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.     

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.     

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.     

The tryptic digestion of spin-labelled heavy meromyosin

The S₁ thiol groups of heavy meromyosin (HMM) have been selectively spin labeled with a paramagnetic analog of iodoacetamide (10) and the effects of tryptic digestion on the ESR spectrum and ATPase activity have been studied. The loss of ATPase activity on tryptic digestion occurs at the same rate with spin-labeled or unlabeled HMM suggesting that spin labeling produces no major change in the conformation of HMM. ESR spectra indicate that spin labels bound to S₁ groups of HMM are strongly immobilized; spectra of subfragment-1 isolated from tryptic digests of spin-labeled HMM are the same as those of labeled HMM. ESR spectra of S₁-spin-labeled peptides produced by tryptic digestion of HMM indicate essentially no immobilization of labels, the spectra being similar to that of a solution of free labels. The ESR spectrum of an unfractionated digest of HMM exhibits a peak attributable to strongly immobilized labels on HMM and subfragment-1 and a peak attributable to weakly immobilized labels bound to peptides. The rate at which spin-labeled peptides are released on tryptic digestion can be measured on the unfractionated digest by the decrease in the ESR peak corresponding to HMM and subfragment-1. The appearance of peptides containing spin-labeled S₁ groups parallels the loss of ATPase activity. No evidence has been found for the existence of an enzymatically active subfragment-1 lacking S₁ thiol groups.     

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.     

Structural Studies on “Isosclerotan”, a New Glucan Isolated from Sclerotinia Fungus,by Physical,Chemical and Enzymatic Methods

“Isosclerotan”, a polysaccharide constituent extracted with a sodium hydroxide solution from sclerotia of Sclerotinia libertiana, could be purified by the successive precipitation with the followings; a mixture of copper sulfate and sodium hydroxide, ammonium sulfate, and ethyl alcohol. The preparation proved homogeneous by ultracentrifugal analysis. From sedimentation and viscosity measurements, the molecular weight of isosclerotan was calculated as 6.13 × 10⁶, andas 1.60 × 10⁵ after treatment with a dilute oxalic acid solution. Isosclerotan was scarecely soluble in cold water but soluble in hot water, yielding a highly viscous solution. It exhibited a low positive optical rotation, + 23.0° (in water), and infrared spectrum had a sharp absorption at 890~898 cm^?1, which indicated the prevalence of the β-glycosidic linkage in isosclerotan. Through degradation by acids and enzymes of isosclerotan, there were obtained various oligosaccharides containing β-1.3, β-1.4, and β-1.6 linkages. From results obtained by periodate oxidation and methylation, it is assumed that the polysaccharide involves the 1.3, 1.4, and 1.6 linkages in 47.7%, 16.6% and 35.7%, respectively, and a branching structure about 12.5%.