Reconstitution of Arachin from Isolated Subunits

Six subunits of arachin were isolated in urea solution. They were then reassociated by removing urea by co-dialysis against 20 mM sodium phosphate buffer (pH 7.9), containing 30% sucrose, 0.1 M> sodium chloride and 7 mM β-mercaptoethanol, without agitation at 25°C. The reconstitution yield was greater than 90%. The reconstituted molecule was indistinguishable from intact arachin in disc electrophoretic mobility, subunit composition, sedimentation behavior depending upon ionic strength, circular dichroism, ultraviolet absorption and fluorescence emission spectra, and stabilities against heating, proteases and guanidine hydrochloride. The reconstituted arachin was, therefore, suggested to be in native state.

On the other hand, we found that co-dialysis of four or five subunits of arachin formed hexamer which contained the corresponding four or five subunits. These hexamers were more labile than intact arachin against heating. These facts suggest that the assembly of all six subunits to a hexamer will most advantage the quaternary structure of arachin.     

Protective Actions of l-Aspartate from Acid or Proteolytic Inactivation

1. l-Aspartate was found to replace l-asparagine in the protective action from acid inactivation of l-asparaginase (EC 3.5.1.1) produced by Escherichia coli A–1–3 and at the same time to inhibit the proteolytic inactivation by α-chymotrypsin.

2. l-Asparaginase changed in its chromatographic properties in the presence of l-aspartate and became to be absorbed on the CM Sephadex column.

3. The sedimentation patterns of l-asparaginase at pH 3.5 were identical either in the presence or absence of l-aspartate, showing partial dissociation. But the reversibility to the active state was observed only in the enzyme dissolved in the solution containing l-aspartate.

4. l-Aspartate did not prevent the enzyme either from the dissociation into subunits or from decrease in the activity by urea.

5. High concentration of l-aspartate was shown to inhibit the l-asparagine hydrolysis reaction.

6. l-Aspartate was suggested from ORD measurements to cause changes in the higher structure as well as the ionic properties or proteolytic inactivation.

     

Limited Tryptic Degradation of Urea Denatured Glycinin

Digestibilities of native, 5 m urea-denatured and 8 m urea-denatured glycinin were studied. Urea was removed by dialysis before digestion. The tryptic digestion of the proteins are influenced by ionic strength. Under low ionic strength condition (0 m NaCl), the proteins, even native glycinin, are well degraded. On the other hand, under high ionic strength condition (0.5 m NaCl), native glycinin resists the tryptic attack and 5 m urea-denatured glycinin is best degraded. The digestibility of 8 m urea-denatured glycinin is lower than that of 5 m urea-denatured one under the condition. The gel filtration and electrophoretic properties show that the digestion intermediate like glycinin-T (the intermediate from native glycinin) is contained in the digestion products. These suggest that the urea-denatured protein contains the almost renatured component after removal of urea. A larger amount of the glycinin-T-like protein was detected at 8 m urea denaturation than at 5 m urea. Therefore, glycinin renatures more readily from 8 m urea denaturation. Probably this is the cause of the decreased digestibility at 8 m urea denaturation.     

Dissociation into Subunits of a 7S Protein in Soybean Globulins with Urea and Sodium Dodecyl Sulfate

A 7S protein isolated from soybean globulins was dissociated into a similar slow-sedimenting material (subunit) by the treatment of urea and sodium dodecyl sulfate (SDS).

Sedimentation coefficients of the subunits obtained by treating with 8 m urea and 0.25% SDS were 1.35 S and 2.00 S, and molecular weights were 22,500 and 34,000, respectively. These subunits by the both treatments were apparently different in conformation from the results of optical rotatory dispersion, i.e., urea treatment caused the almost complete unfolding of the subunit structure. On the contrary, SDS treatment contributed new partial formation of a α-helical conformation for the subunits.

These dissociations were extremely disturbed by the presence of sodium chloride.     

Effect of Sodium Dodecyl Sulfate on D-Glucose-isomerizing Enzyme from Bacillus coagulans,Strain HN-68

Crystalline D-glucose-isomerizing enzyme from Bacillus coagulans, strain NH–68 has been shown to consist of subunits by the method of electrophoresis on sodium dodecyl sulfate (SDS) polyacrylamide gels.

The dissociation behavior of the enzyme has been characterized. The enzyme dissociates into inactive subunits by the preincubation with 0.05% SDS in the presence of 5 × 10^?3M MnCl₂ or CoCl₂, but not in the absence of these metal salts. In 8 м urea, however, the enzyme does not dissociate into subunits and the activity is completely recovered by dilution of the urea. Metal salts, such as MnCl₂ and CoCl₂, also do not affect activity in the presence of urea.     

Biochemical Studies on Rice Bran Lipase

Some chemical properties of the rice bran lipase were studied. The enzyme protein contained 14.98% nitrogen and consisted of 312 amino acid residues. It also contained a certain amount of lipid. The amino-terminal amino acids of the enzyme protein were shown to be glutamic acid and the carboxyl-terminal amino acids to be glycine and serine. The treatment of the enzyme protein with 8 m urea containing 1×10^?3m EDTA (ethyl-enediaminetetraacetic acid) seemed to cause dissociation of the subunits of the enzyme protein. From this observation and the results of the terminal amino acids analysis, it was presumed that the enzyme protein was composed of at least two types of subunits.     

Salt-induced Reconstitution of β-Conglycinin from Its Thermal Dissociates

The salt-free heat denaturated β-conglycinin, heated to 99°C for 5 min, exhibited dissociation of the protein into subunits (α, α′ and β). Heat-induced dissociates could be converted to β-conglycinin with an increase in ionic strength of the protein solution. The reassociation of these dissociates depended on the salt concentration and on the species of the constituent subunits. Adding salt above 0.1 m NaCl favored reassociation of the thermal dissociates. The β subunit has a tendency to form an aggregate of higher molecular size, while the α and α′ subunits have an ability to form the 7S aggregate. Reconstituted β-conglycinin possessed the characteristic of 7S ? 9S interconversion with a change of ionic strength, which has been considered as a feature of native conglycinin. The restoration of electrophoretical mobility, ultracentrifugal characteristics and the secondary structure of CD properties was distinct evidence supporting the reconstitution of β-conglycinin from its thermally denatured state. However, the reconstituted conformation differed from the native β-conglycinin in its quantitative precipitin curve and ultraviolet difference spectrum.     

Subunit Structure of Soybean 11S Globulin

The acidic and the basic subunits were shown to be present in equimolar amounts in the 11S globulin molecule by the densitometric scanning of the SDS gel and the molecular weight consideration. The four acidic subunits (A₁, A₂, A₃ and A₄) were found to be present in the approximate molar ratio of 1:1:2:2. Four basic subunits separated and designated as B₁, B₂, B₃ and B₄ based on the relative mobilities in the acidic gel in 7 m urea were found to be present in the approximate molar ratio of 1:1:2:2. The four basic subunits were fractionated in approximately same amounts into three different peaks, peak I (B₁ and B₂), peak II (B₃) and peak III (B₄) by CM-Sephadex C–50 column chromatography in the presence of 6 m urea. Three kinds of intermediary subunits of 11S globulin were fractionated with DEAE-Sephadex A–50 in the absence of reducing agents in 6 m urea, and disulfide bonds appeared to participate in the binding between the acidic and the basic subunits in the molar ratio of 1: 1 with the following combinations; A₁ and A₂ combined with B₃, A₃ with B₁ and B₂, and A₄ with B₄. In view of the above results and molecular weight consideration, a new model of subunit structure was proposed for 11S globulin.     

Mucopolysaccharides Isolated from Human and Cow Colostrums

Mucopolysaccharides were isolated from both human and cow colostrums. Each of the fractionated mucopolysaccharides was considered to be homogeneous from behaviors in chromatography, electrophoresis and sedimentation pattern. The fractions isolated from human colostrum were found to contain 51.0~78.3% carbohydrates consisting of d-galactose, 2-amino-2-deoxy-d-glucose, N-acetylneuraminic acid, l-fucose and d-glucose, and 31.6~11.0% peptides consisting of 16 kinds of amino acids. The sedimentation constants, s_{20, w}, of these fractions were in the range of 0.75 to 1.73 S. The fraction isolated from cow colostrum was found to contain 19.3% carbohydrates consisting of d-galactose, 2-amino-2-deoxy-d-glucose and N-acetylneuraminic acid, and 65.2% peptides or proteins consisting of 18 kinds of amino acids. The sedimentation constant, s_{20, w}, of the fraction was 3.68 S.     

Isopullulanase from Arthrobacter globiformis T6

The physico-chemical properties of the purified glucose isomerases [d-xylose ketol isomerase, EC 5.3.1.5] of Streptomyces olivochromogenes and Bacillus stearothennophilus were examined. The molecular size and shape of both enzymes were similar. The molecular weights, sedimentation coefficients, partial specific volumes, diffusion constants and Stokes’ radii of the Streptomyces and Bacillus enzymes were determined to be 120,000 and 130,000, 7.55 S and 9.35 S, 0.725 and 0.736 ml/g, 5.87 × 10^-7 and 6.82 × 10^-7 cm²/sec, and 51 and 53 Å, respectively. The Streptomyces glucose isomerase was found to consist of two subunits, each having a molecular weight of 56,000. Large differences were found in the amino acid compositions of these two enzymes, especially in their serine, proline, tyrosine, lysine and arginine contents. The enzymatic properties of both these purified glucose isomerases were also examined, and it was seen that they both displayed activity on d-xylose, d-xylulose, d-glucose, d-fructose, d-arabinose and d-ribose. The smaller Km values and the larger molecular activities for d-xylose and d-xyluIose indicated that both enzymes are essentially d-xylose isomerases. The optimum temperature was 80°C for both enzymes. The optimum pH was 8 to 10 for the Streptomyces enzymes and 7.5 to 8.0 for the Bacillus enzyme. The Bacillus enzyme was more thermostable than the Streptomyces enzyme, but required cobalt ions in addition to magnesium ions for the full expression of its activity.     

Free Amino Acid Contents of Plasma and Urine,and Liver Enzyme Activities in Rats Fed High Glycine Diets

The contents of plasma free amino acids, the amounts of urinary excreted amino acids and urea, and the activities of liver serine dehydratase, glutamic-oxalacetic transaminase and glutamic-pyruvic transaminase were determined in weanling rats fed ad libitum a 10% casein diet (control), a 10% casein diet containing 7% glycine and 10% casein diets containing 7% glycine supplemented with 1.4% L-arginine and/or 0.9% L-methionine for 14 days.

The remarkable increase of glycine and the moderate increase of serine in the plasma of animals fed excess glycine diets were observed. The amount of excreted glycine in the urine of animals fed the excess glycine diet supplemented with L-arginine and L-methionine was much greater than that of animals given the excess glycine diet. Urinary excreted urea of rats fed the excess glycine diet was a little greater and that of rats fed the excess glycine diet supplemented with L-arginine and L-methionine was much greater than the control. Liver serine dehydratase activity of animals given the excess glycine diets with or without L-arginine was higher than the control and the highest activity was observed in the liver of animals fed the excess glycine diet containing L-arginine and L-methionine. The activity of liver glutamic-oxalacetic transaminase of rats fed the excess glycine diet containing L-arginine and L-methionine was a little higher than that of rats given the other diets. Liver glutamic-pyruvic transaminase activity was a little higher in animals given the excess glycine diets with or without L-arginine and further higher in animals fed the excess glycine diet containing L-arginine and L-methionine than the control.     

Purification and Properties of d-Xylose Isomerase from Alkalophilic Bacillus No. KX-6

An alkalophilic Bacillus No. KX-6 isolated from soil produced a d-xylose isomerase in alkaline media. The striking characteristic of this bacterium was its especially good growth in alkaline media. The d-xylose isomerase of this bacterium was purified by ammonium sulfate fractionation, DEAE-Sepharose ion exchange column chromatography and G-200 gel Alteration. The molecular weight and sedimentation constant were approximately 120,000 and 9.35 S, respectively. The enzyme was most active at pH 7~10 and was stable at pH 6.0 to 11.0. Enzyme activity was stimulated by cobalt ion but inhibited by Hg^{2 +}, Ag^{2 +}, and Cu^{2 +}. Substrate specificity studies showed that this enzyme was active on d-xylose, d-glucose, d-ribose, and d-arabinose. The smaller Km value and larger V_max value for d-xylose indicated that this enzyme is essentially d-xylose isomerase.     

Gene cloning and biochemical characterization of eryngase,a serine aminopeptidase of Pleurotus eryngii belonging to the family S9 peptidases

Pleurotus eryngii serine aminopeptidase that has peptide bond formation activity, redesignated as eryngase, was cloned and expressed. Eryngase has a family S9 peptidase unit in the C-terminal region having a catalytic triad of Ser, Asp, and His. In the phylogenetic relations among the subfamilies of family S9 peptidase (S9A, prolyl oligopeptidase; S9B, dipeptidyl peptidase; S9C, acylaminoacyl peptidase; S9D, glutamyl endopeptidase), eryngase existed alone in the neighbor of S9C subfamily. Mutation of the active site Ser524 of the eryngase with Ala eliminated its catalytic activity. In contrast, S524C mutant maintained low catalytic activity. Investigation of aminolysis activity using l-Phe-NH₂ as a substrate showed that S524C mutant exhibited no hydrolysis reaction but synthesized a small amount of l-Phe-l-Phe-NH₂ by the catalysis of aminolysis. In contrast, wild-type eryngase hydrolyzed the product of aminolysis l-Phe-l-Phe-NH₂. Results show that the S524C mutant preferentially catalyzed aminolysis when on an l-Phe-NH₂ substrate.     

Sequential Oxidation and Reduction of Mono Methyl Ethers of Methyl 4,6-O-Benzylidene-α- and β-d-Glucopyranosides

(1) Both glutaminases A and B of Pseudomonas aeruginosa are inactivated by urea and guanidine hydrochloride, and the activities are partially restored by removal of the denaturants, while sodium lauryl sulfate denatured irreversibly the isozymes. (2) Glutaminase A consists of 4 identical subunits (mol. wt, 35,000) and B is composed of one polypeptide chain (mol. wt., 67,000). (3) Glutaminase A, which catalyzes the hydrolysis and also the hydroxylaminolysis of L and D isomers of glutamine and asparagine, does not act on γ-N-substituted glutamine e.g., γ-glutamylhydrazide. Some l- and d-γ-glutamyl derivatives, e.g., l- and d-γ-glutamyl-hydrazide, l- and d-γ-glutamylmethylester, and l-γ-glutamyl-l-alanine are substrates for glutaminase B, which does not catalyze the hydrolysis and hydroxylaminolysis of asparagine. α-Amino adipamic acid and α-amino substituted amino acids are inert for both the isozymes. (4) The acylation step is rate-limiting in the catalytic reactions by both the isozymes.     

Further Studies on the Effect of Amino Acid Supplementation on the Urinary Nitrogen Compounds in Rats Fed a Low-casein Diet

When 8% casein basal diet was supplemented with 0.3% dl-methionine or 0.3% dl-methionine plus 0.36% dl- or 0.18% l-threonine, the changes in urinary excretions of urea and allantoin were examined in weanling male rats of Wistar strain with the observations on the body weight gain and % nitrogen retention. Carbohydrate sources used were sucrose or an equimolar mixture of glucose and fructose (G-F) in place of pregelatinized starch used in the previous experiments.

In contrast to the previous results, differences in nitrogen utilization, expressed in term of growth rate or % nitrogen retention, became significant by the addition of 0.3% methionine to the basal diet and it was further increased by the simultaneous supplementation with 0.36% dl- or 0.18% l-threonine.

Urea excretion was the main variable in total urinary nitrogen output to cause the significant difference in % nitrogen retention between the groups. As postulated in the previous paper, thus, the use of sucrose or G-F mixture considerably exaggerated these group-differences in such various indices as body weight gain and % nitrogen retention, and this trend became more distinct in the urea and allantoin excretions.

Liver arginase activity inversely changed with urea excretion, but proportionately to the qualitative improvement of dietary protein by the addition of methionine or methionine plus threonine. Changes in liver glutamic dehydrogenase activity were also parallel with the improvement of dietary protein quality.     

Structure of the Hydrolyzed Product (F-2) Released from γ-Polyglutamic Acid by γ-Glutamyl Hydrolase YwtD of Bacillus subtilis

The structure of the hydrolyzed product (F-2) with a molecular mass of about 2 kDa released from γ-polyglutamic acid by the γ-glutamyl hydrolase YwtD of Bacillus subtilis was analyzed. The results showed that F-2 is an optically heterogeneous polymer consisting of D- and L-glutamic acid in an 80:20 ratio with D-glutamic acid on both the N- and C-terminal sides, suggesting that YwtD is an enzyme that cleaves the γ-glutamyl bond between D- and D-glutamic acid recognizing adjacent L-glutamic acid toward the N-terminal region.     

Cleavage of Disulfide Bonds of Wheat Glutenin by Mercuric Chloride

The dissociation of wheat glutenin into subunits was observed by treatment with a small amount of mercuric chloride under moderate conditions, suggesting that the cleavage of inter-polypeptide chain disulfide bonds in the glutenin might occur. The dissociation into the subunits was examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The electrophoretic patterns of the glutenin treated with mercuric chloride were essentially similar to those of the glutenin treated with 2-mercaptoethanol. Silver nitrate also had the same effects as mercuric chloride, and p-chloromercuribenzoate and N-ethylmaleimide showed no effect on the dissociation of the glutenin. Complete dissociation was achieved when the glutenin solution containing 0.5% SDS and 0.01 m phosphate buffer (pH 7.0) was incubated with 10^?3 m mercuric chloride (about four moles per mole of disulfide groups) at 30°C for 20 hr. Partial dissociation was also observed after 30 min incubation. Increasing temperature and SDS concentration promoted the rate of the dissociation of the glutenin by mercuric chloride.     

Cysteine Formation from Methionine in Pseudomonas melanogenum and Mechanism of Methionine-inhibition of l-DOPA Production by the Bacterium

Using a Dowex 50–X2 column and eluting with a 1 mm sodium acetate buffer (pH 2.6), three l-β-aspartamido-carbohydrate (Asn-carbohydrate) fractions have been isolated in 19% yield from the pronase digest of a 7S soybean protein. The Asn-carbohydrates were composed of one asparagine, two glucosamine and seven, eight and nine mannose residues. Further, 1-l-β-aspartamido-2-acetamido-1,2-dideoxy-β-d-glucose (Asn-GlcNAc), which is derived from the protein-carbohydrate linkage, was produced by partial hydrolysis of the Asn-carbohydrates.     

Structure-Based Modification of D-Alanine-D-Alanine Ligase from Thermotoga maritima ATCC 43589 for Depsipeptide Synthesis

Depsipeptides are peptide-like polymers consisting of amino acids and hydroxy acids, and are expected to be new functional materials for drug-delivery systems and polymer science. In our previous study, D-alanyl-D-lactate, a type of depsipeptide, was enzymatically synthesized using D-alanine-D-alanine ligase from Thermotoga maritima ATCC 43589 (TmDdl) by Y207F substitution. Thereafter, in this study, further mutagenesis was introduced, based on structural comparison between TmDdl and a well-characterized D-alanine-D-alanine ligase from Escherichia coli. The S137A/Y207F mutant showed higher D-alanyl-D-lactate and lower D-alanyl-D-alanine synthesizing activity than the Y207F mutant. This suggests that substitution at the S137 residue contributes to product selectivity. Saturated mutagenesis on S137 revealed that the S137G/Y207F mutant showed the highest D-alanyl-D-lactate synthesizing activity. Moreover, the mutant showed broad substrate specificity toward D-amino acid and recognized D-lactate and D,L-isoserine as substrates. On the basis of these characteristics, various depsipeptides can be produced using S137G/Y207F-replaced TmDdl.     

Interaction Involving Disulfide Bridges between Subunits of Soybean Seed Globulin and between Subunits of Soybean and Sesame Seed Globulins

Native subunit proteins of glycinin, the acidic and the basic subunits designated as AS₁₊₂, AS₂₊₃, AS₄, AS₅, and AS₆ and BS, respectively, were isolated by DEAE-Sephadex A-50 column chromatography in the presence of 6 m urea and 0.2 m 2-mercaptoethanol.

Reconstitution of intermediary subunits involving a disulfide bridge from native acidic and basic subunits was investigated. Formation of the intermediary subunit was observed in combinations between BS and each acidic subunit except AS₆. The yields of the reconstituted intermediary subunits differed from one another.

Further, formation of the intermediary complexes was observed when native acidic and basic subunits of soybean glycinin and sesame 13 S globulin, respectively (or reverse combinations), were mixed under reductively denatured condition and subjected to the reconstitution procedure. Considerring the overall evidence, we may conclude that the complexes are probably a hybrid intermediary subunit.