Formation of Pseudo- and Hybrid-11S Globulins from Subunits of Soybean and Broad Bean 11S Globulins

Pseudo- and hybrid-11S globulins were reconstituted from native acidic and basic subunits of soybean and broad bean 11S globulins. The subunit structures of these two globulins are known to be similar to each other. Pseudo-11S globulins were formed in combinations between glycinin acidic subunit (G-AS_{1 + 2}) and glycinin basic subunit (G-BS) and between legumin acidic subunit (L-ASII) and legumin basic subunit (L-BS). Hybrid-11S globulins were formed in combinations between G-AS_{1 + 2} and L-BS and between L-ASII and G-BS. The yields of the reconstituted 11S components of G-AS_{1 +2} + G-BS and G-AS_{1 + 2} + L-BS were lower than those of L-ASII + G-BS and L-ASII + L-BS. These pseudo- and hybrid-11S globulins were similar to native 11S globulins; they all consisted of reconstituted intermediary subunits which were composed of acidic and basic subunits linked by disufide bridges and had molecular weights similar to those of native 11S globulins. However, the dissociation-association behaviors of pseudo-glycinin and hybrid-11S globulins were different from those of native 11S globulins.     

Factors Influencing Conformation Changes in a 7S Protein of Soybean Globulins by Ultracentrifugal Investigations

Effects of pH, ionic strength, kind of salts and disulfide bond cleaving agent (2-mercaptoethanol) on conformation changes revealed on ultracentrifugal patterns of a 7S protein in soybean globulins were investigated. In the solution with lower pH than isoelectric point, this protein dissociated into two components in low ionic strength, but showed a 7S sedimentation pattern in higher ionic strength than 0.1. On the other hand, in the solution with higher pH than isoelectric point, this protoin showed aggregation to a 9S isomer in lower ionic strength than 0.1. Between ionic strength of 0.1 and 0.5, the mixture of 7S and 9S forms existed and in higher ionic strength than 0.5, the protein kept a 7S form stablely. These reactions were reversible and effect of 2-mercaptoethanol was scarcely observed but those of salts were observed.

The molecular weight of the 9S isomer was approximately 370,000 and the s_20,w value was 12.30S. Therefore, the 9S isomer was considered to be a dimer of the 7S protein.     

The Dissociation of Wax Bean Hemagglutinin by Polyacrylamide Gel Electrophoresis in Sodium Dodecyl Sulfate

(+)-Biotin was synthesized from D-glucosamine in a sequence of 13 reactions.     

Relationship between Gelation and Aggregation of Alkali-treated Soybean 7S and 11S Globulins

Gel was obtained when alkaline dope solutions of the 7S and 11S globulins (8% protein concentration) prepared at pH above 11 were dialyzed against phosphate buffer, pH 7.6, µ= 0.3. To make clear the mechanism of gelation, the relationship between changes in viscosity and aggregation phenomena of the neutralized dope solutions was investigated by means of viscosity measurement, disc electrophoresis and gel filtration, comparing the 7S and 11S fractions. In conclusion, it is revealed that the gel is constituted with macromolecule aggregates, and to form the aggregates which are suitable for gelation, all of the following conditions must be satisfied at least : 1); Unfolding and dissociation into subunits once (above pH 11), 2); High ionic strength in the media (µ=0.3), 3); Formation of hydrogen, hydrophobic and disulfide bonds, 4); High protein concentration (above 8%).     

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.     

Isolation and Characterization of the Multiple 7S Globulins of Soybean Proteins

Two major proteins (the 7S and 11S globulins) of soybean (Glycine max) were simultaneously isolated by a simple method based on their different solubilities in dilute tris (hydroxymethyl) aminomethane buffers. The purified 7S globulins, which represented essentially the entire 7S soybean protein fraction capable of dimerization at 0.1 ionic strength, were fractionated into five components by diethylaminoethyl Sephadex A-50 column chromatography. The five 7S components were characterized by disc-electrophoresis.     

Gelation Phenomena Induced by Alkali-alcohol Treatment of 7S and 11S Components in Soybean Globulins

Transparent gels containing about 2% protein were obtained by mixing alkaline dope solution of 7S or 11S soybean proteins with alcohol. The 7S component showed the ability to form a stronger gel than the 11S. This phenomenon depended on pH and alcohol concentration. In 66 % ethanol, the viscosity of the 7S and 11S reached maxima at pH 11.4 and 11.2, respectively. Above these pH levels where further unfolding and dissociation into subunits of the protein molecules occur, the viscosity decreased rather. The effectiveness of alcohol to increase viscosity increased in the order; n-butanol < tert-butanol < n-propanol < iso-propanol < ethanol < methanol. Alcohols having minor hydrophobicity were more effective for increasing viscosity, but ethylene glycol was ineffective. The addition of NaCl or 2-mercaptoethanol to ethanol-mixed alkaline dope solutions resulted in the remarkable increment of the viscosity, especially for the 7S.     

Enzymes Induced in a Bacterium by Growth on Sodium Dodecyl Sulfate

Alkyl sulfatase was induced by growth on nutrient broth plus sodium dodecyl sulfate (SDS) in a bacterium we have designated Pseudomonas C12B. Measurement of the radioactivity of S³⁵O₄⁼ released from SDS³⁵ by the enzyme in cell-free extracts provided an effective assay technique. The barium chloranilate assay for release of SO₄⁼ from SDS was somewhat less sensitive but effective if analyzed at 332 mμ. This test was only approximately 55% as sensitive if analyzed at 530 mμ. The activity of the glyoxylate bypass enzymes, isocitrate lyase and malate synthetase, was significantly stimulated by growth of the bacteria on SDS as the sole carbon source, but not by growth on nutrient broth or nutrient broth plus SDS.     

Distribution of the 7S Proteins in Soybean Globulins by Gel Filtration with Sephadex G-200

The level of isocitrate lyase, an enzyme of glyoxylate cycle, in Candida tropicalis was enhanced at the later period of growth when the yeast was cultivated in a semisynthetic glucose medium. On the other hand, such increase in the enzyme activity was not observed in C. lipolytica grown under the same conditions. In the case of C. tropicalis, high concentrations of glucose remaining in the medium permitted the increase in the enzyme activity and the addition of ethanol, one of the major products from glucose, to the glucose medium did not stimulate the enzyme formation, indicating that the enhanced enzyme level in the yeast was not merely attributable to the release from the repression by glucose or to the induction by ethanol. Biotin, one of the growth-stimulating factors for C. tropicalis, affected markedly the level of isocitrate lyase. That is, the supplementation of biotin to the synthetic glucose medium inhibited completely the increase in the enzyme activity, and reversely the absence of biotin stimulated the enzyme formation in the glucose-assimilating cells. Thiamine, another growth-stimulating factor for C. tropicalis, did not show any effect on the level of isocitrate lyase in the yeast. The level of isocitrate lyase in C. lipolytica growing on glucose was not affected by biotin added exogenously.     

On Basic Subunits Dissociated from C (11S) Component of Soybean Proteins with Urea

Galactolipase (galactolipid acyl hydrolase, EC 3.1.1.26) was purified 147-fold in good yield (91 %) from rice bran by affinity chromatography, in which the enzyme was adsorbed on a palmitoylated gauze column at pH 5.5 and then was eluted with a buffer solution containing a detergent such as sodium deoxycholate or Triton X–100 at pH 8.0. The preparation obtained was further purified by gel filtration on a Sephadex G–100 column and isoelectric focusing. After electrophoresis, the enzyme separated into four components with different isoelectric points. It seems that galactolipase in rice bran exists in multiple forms. The major component (G–2) with isoelectric point of 7.3, one of them, was purified 268-fold and electrophoretically homogeneous. The enzyme (G–2) hydrolyzed rapidly galactolipid and also slowly phospholipid, but hardly triglyceride.     

An Analysis of the Interaction of Sodium Dodecyl Sulfate with Lysozyme

The interaction of SDS with lysozyme was analyzed with enzyme activity and with NMR, fluorescence, and UV difference spectroscopies using various alkyl sulfates and variously modified lysozymes. SDS formed a stable complex with lysozyme without causing a gross conformational change in the enzyme molecule. Some SDS molecules bound to the active site cleft of lysozyme and therefore strongly inhibited the activity of lysozyme. Hydrophobic regions and positive charges for protein side, and a hydrophobic tail (possibly more than 8 carbons in alkyl chain) and a negative charge for detergent side were required for the formation of the complex.     

A Comparison of Soybean Globulins and the Protein Bodies in the Protein Composition

The protein compositions between soybean globulins and the protein bodies were compared by gel filtration with Sephadex G-200, sedimentation analyses and disc isoelectric focusing.

From the results of the three comparisons, it was difficult to find an essential difference in the protein compositions of the both. And, the 7S and the 11S globulins were the main components in the both. This fact was more strongly suggested that these globulins were the typical reserve proteins.     

海藻酸钠与十二烷基聚氧乙烯醚硫酸钠复配体系泡沫及流变性能的研究

采用罗氏泡沫仪和流变仪对海藻酸钠(NaAlg)/十二烷基聚氧乙烯醚硫酸钠(AES)复配体系的起泡稳泡性能及流变行为进行研究,以探索NaAlg对AES起泡稳定性的影响及作用机理。泡沫性能试验显示,NaAlg的加入,提高了AES溶液的起泡能力及泡沫稳定性,溶液的pH值由7.0降到5.0,混合体系的起泡稳泡性明显增加。流变性研究展示,随着NaAlg浓度的增加,溶液的粘度呈增大的趋势,溶液总体呈现牛顿流体性质,当NaAlg浓度大于0.5%时,低剪切速率区,溶液仍呈现牛顿流体性质,而在高剪切速率区,有剪切变稀的现象出现。总体来看,各试液的G’相似文献   

Non-Immunological Precipitation of Serum by Sodium Dodecyl Sulfate in Agar Diffusion

Marmoset serum or serum of other species of animal may react with sodium dodecyl sulfate and forms nonspecific precipitin lines in agar diffusion. The protein detergent complexes are not readily dialyzable. Therefore precipitin lines derived from studies that use sodium dodecyl sulfate-treated antigens in agar diffusion must be interpreted with caution.     

Limited Hydrolysis of Ricin D with Trypsin in the Presence of Sodium Dodecyl Sulfate

An α-amylase which produces both maltotetraose and maltopentaose from starch as the main products was found in the culture filtrate of a strain of Bacillus circulans which was newly isolated from soil. The enzyme was purified to be almost homogeneous on disc electrophoresis in polyacrylamide gel by means of ammonium sulfate fractionation, DEAE-Sepharose column chromatography and Sephadex G-200 gel filtration.

The optimum pH and temperature of the enzyme were around pH 7.0 and around 50°C, respectively. Metal ions such as Hg²⁺, Cu²⁺, Ni²⁺, Zn²⁺, Fe²⁺ and Co²⁺ strongly inhibited the enzyme activity. The molecular weight was about 45,000. The yields of maltotetraose and maltopentaose from potato starch were 30 ~ 40% and 20 ~ 30%, respectively.     

Effects of Treatment with Sodium Dodecyl Sulfate on the Ultrastructure of Escherichia coli

An electron microscopy study has been made of the effects of dissolution of the plasma membrane of Escherichia coli with sodium dodecyl sulfate (SDS) on the organization of the nucleoplasm and the cytoplasm. The alterations observed in time course experiments were related to absorbance changes and to release of macromolecules from the cells. As the cells became plasmolyzed, under the conditions used, the first visible effect of SDS was a collapse of the plasmolysis spaces. This was accompanied by a displacement of the nuclear material which then appeared in broad contact with the redeployed plasma membrane. This initial displacement of nuclear material to the cell border may indicate an association between the nucleoplasm and the plasma membrane. Upon further dissolution of the plasma membrane, the nuclear material receded from the cell margin and contracted into an axial filament. Meanwhile, the cytoplasm dissociated into an amorphous, Pronase-sensitive component and an electron-opaque, granular one sensitive to ribonuclease. The latter represented one continuous area of ribosomal structures surrounding the nucleoplasm, an organization which did not occur when the cells were inhibited with rifamycin before SDS treatment. During prolonged SDS interaction, approximately 65% of the cellular protein, 25% of the ribonucleic acid and 40% of the deoxyribonucleic acid were released from the cells concomitant with the disappearance of the amorphous cytoplasmic part, expansion of the ribosomal aggregate, and rearrangement of the nuclear material at the cell periphery. The observations support the contention that all ribosomal structures bear a direct relationship with the nucleoplasm.     

Curing Action of Sodium Dodecyl Sulfate on a Proteus mirabilis R+ Strain

Growth of Proteus mirabilis harboring R100-1 (fi(+)drd str(r)cml(r)tet(r)sul(r)) factors in Penassay broth containing sodium dodecyl sulfate (SDS) leads to the loss of all or part of the genetic elements in high frequencies. In media containing SDS at concentrations as low as 0.03%, both lysis of R(+) cells and elimination of the R factors occur at high frequencies. Appearance of drug-susceptible cells in R(+) cultures occurs during the exponential phase of growth; however, the frequencies of susceptible cells increase substantially after the culture reaches the stationary phase. Reconstruction experiments, coupled with other observations, suggest that the major factor in altering the frequency of drug-susceptible variants is the greater resistance of the variants to the lytic action of SDS. This resistance correlates in most cases with the loss of the transfer functions in the resistance transfer factor.     

Extraction of Wheat Flour Proteins with Sodium Dodecyl Sulfate and Their Molecular Weight Distribution

By extraction of wheat flour with sodium dodecyl sulfate (SDS) solution at pH 6.8, about 76% of the total flour nitrogen solubilized into clear supernatant. This solvent was more effective for extraction of wheat protein than 0.01 m acetic acid, aluminium lactate-lactic acid buffer (pH 3.1), AUC-solvent (0.1 m acetic acid, 3 m urea and 0.01 m cetyltrimethyl-ammomum bromide) and 3,5-diiodosalicylic acid lithium salt etc. The molecular weight distribution of the SDS-soluble proteins was studied by SDS-polyacrylamide gel electrophoresis and by molecular sieve chromatography on controlled pore glass (CPG–10–500) without prior reduction of disulfide linkages of the proteins. Most of the SDS-soluble proteins had molecular weight of less than 75,000, suggesting single-chained proteins. A small amount of relatively high molecular weight proteins which contained intermolecular disulfide linkages was also detected in the gel of electrophoresis, while high molecular weight protein which did not migrate into gel matrix during electrophoresis without prior reduction of disulfide linkages existed in trace amount in the SDS-soluble fraction.

The SDS-insoluble proteins were almost completely extracted by further extraction with SDS in combination with 2-mercaptoethanol or with mercuric chloride.     

Sodium Dodecyl Sulfate Hypersensitivity of clpP and clpB Mutants of Escherichia coli

We studied the hypersensitivity of clpP and clpB mutants of Escherichia coli to sodium dodecyl sulfate (SDS). Both wild-type E. coli MC4100 and lon mutants grew in the presence of 10% SDS, whereas isogenic clpP and clpB single mutants could not grow above 0.5% SDS and clpA and clpX single mutants could not grow above 5.0% SDS. For wild-type E. coli, cellular ClpP levels as determined by Western immunoblot analysis increased ca. sixfold as the levels of added SDS increased from 0 to 2%. Capsular colanic acid, measured as uronic acid, increased ca. sixfold as the levels of added SDS increased from 2 to 10%. Based on these findings, 3 of the 19 previously identified SDS shock proteins (M. Adamowicz, P. M. Kelley, and K. W. Nickerson, J. Bacteriol. 173:229-233, 1991) are tentatively identified as ClpP, ClpX, and ClpB.