Texture of Cooked Rice Prepared from Aged Rice and Its Improvement by Reducing Agents

The textures of cooked rice prepared from aged rice grains and their improvement by reducing agents were investigated. For aged rice that was stored for 5 months without air by the operation of a vacuum packing machine, the stickiness/hardness ratio of cooked rice was as low as that of aged rice stored in air. The results of electrophoresis showed that oxidation of proteins in the former was advanced to the same degree as in the latter. The stickiness/hardness ratios of the aged rice were increased by the addition of sodium sulfite, cysteine, and dithiothreitol to the cooking water. Sodium sulfite, cysteine, and dithiothreitol cleave disulfide bonds to sulfhydryl groups. Therefore, cleaving disulfide bonds to sulfhydryl groups improved the texture. The addition of them to the cooking water also increased the extractable solids at the time of heating. Hence cleaving disulfide bonds to sulfhydryl groups must increase extractable solids. Consequently, the gelatinized paste layer thickened and the thick paste layer softened the cooked rice.     

Enzymatic Improvement of the Cooking Quality of Aged Rice: A Main Mode of Protease Action

This study aims to improve the stickiness and brightness of cooked rice from aged rice grains by removing its albumins and globulins with proteases. In newly–harvested grains, the proteins were easily removed by extraction with water which contained minerals from rice. In aged grains, however, the proteins were already denatured and extracted with difficulty. Proteases hydrolyzed even the denatured proteins to make them extractable. As a result, starch granules on the surface of grains were liberated. Some proteases also hydrolyzed a starch granule-associated protein, and consequently, made the starch highly gelatinizable by heating. The protease treatment of aged grains was effective for two main reasons: liberation of starch granules and removal of the granule-associated protein. During cooking of rice the liberated starch without the associated proteins gelatinized to give rise to stickiness. The gelatinized starch is considered to form a more isotropic film on the surface of every cooked rice grain to give brightness as well.     

Comparison of eating quality and physicochemical properties between Japanese and Chinese rice cultivars

In this study, we evaluated 16 Japanese and Chinese rice cultivars in terms of their main chemical components, iodine absorption curve, apparent amylose content (AAC), pasting property, resistant starch content, physical properties, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, and enzyme activity. Based on these quality evaluations, we concluded that Chinese rice varieties are characterized by a high protein and the grain texture after cooking has high hardness and low stickiness. In a previous study, we developed a novel formula for estimating AAC based on the iodine absorption curve. The validation test showed a determination coefficient of 0.996 for estimating AAC of Chinese rice cultivars as unknown samples. In the present study, we developed a novel formulae for estimating the balance degree of the surface layer of cooked rice (A3/A1: a ratio of workload of stickiness and hardness) based on the iodine absorption curve obtained using milled rice.     

A method for the controlled cleavage of disulfide bonds in proteins in the absence of denaturants

A simple method was developed for the controlled cleavage of protein disulfide bonds and the simultaneous blockage of the free sulfhydryl groups in the absence of a denaturant. The disulfide bonds of bovine serum albumin were cleaved unsymmetrically at pH 7.0 using 0.1 M sulfite in 0.1 M phosphate buffer and the free sulfhydryl groups formed were sulfonated in an oxidation-reduction cycle using molecular oxygen and 400 microM cupric sulfate as a catalyst. The reaction was affected by cupric ion concentration, sulfite concentration, reaction pH and temperature. The standardized method was successfully used to cleave the disulfide bonds of other proteins pepsin, trypsin, and chymotrypsin. The method is reliable and can be used for achieving progressive cleavage of disulfide bonds in proteins without employing a denaturant.     

Mitochondrial adenosine triphosphatase. Location of sulfhydryl groups and disulfide bonds in soluble enzyme from beef heart.

The soluble beef heart mitochondrial ATPase (F1) contains eight sulfhydryl groups and two disulfide bonds. N-Ethylmaleimide has been used to radioactively label the sulfhydryl groups before and after cleavage of the disulfide bonds by dithiothreitol. After subjecting the labeled protein to polyacrylamide gel electrophoresis in sodium dodecyl sulfate and measuring radioactivity in each of the separated subunits the location of all the sulfhydryl groups and the disulfide bonds may be specified. The conclusions are supported by direct examination of depolymerized, unreduced, enzyme by polyacrylamide gel electrophoresis. The results also indicate that current ideas regarding the overall subunit structure of this enzyme may be incorrect, and this is discussed in light of new data presented here.     

Beta subunit of (Na+ + K+)-ATPase contains three disulfide bonds

Previous studies of titratable (Na+ + K+)-ATPase sulfhydryl groups have indicated the presence of one disulfide bond per mole of holoenzyme. This single disulfide cross-link was assigned to the beta subunit on the basis of the difference between the number of titrated "free" sulfhydryl groups and the total number of titrated sulfhydryl groups for each subunit [Esmann, M. (1982) Biochim. Biophys. Acta 688, 251; Kawamura, M., & Nagano, K. (1984) Biochim. Biophys. Acta 694, 27]. In the present study, beta-subunit tryptic peptides containing disulfide cross-links were identified and purified by HPLC. Two new peptides were generated from each disulfide-bonded peptide by reduction with dithiothreitol, and the amino acid compositions of these reduced peptides were determined. The data demonstrate that there are three disulfide bonds in the native beta subunit: 125Cys-148Cys, 158Cys-174Cys, and 212Cys-275Cys. The number of disulfide bonds in the beta subunit was also estimated by titration of sulfhydryl groups with [14C]iodoacetamide. Six sulfhydryl groups were identified: two sulfhydryl groups were titrated without prior reduction, and four were identified only after reduction of the protein with dithiothreitol. These data, suggesting that the beta subunit contains two disulfide bonds, are inconsistent with the peptide isolation experiments, which directly identified three disulfide bonds in the beta subunit. This inconsistency was resolved by demonstrating that approximately 20% of each disulfide bond in the beta subunit was reduced prior to the start of the experiment, resulting in an underestimation of the number of disulfide-bonded sulfhydryl groups in the beta subunit from the titration experiments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sulfhydryl group content of chicken progesterone receptor: effect of oxidation on DNA binding activity

DNA binding activity of chicken progesterone receptor B form (PRB) and A form (PRA) has been examined. This activity is strongly dependent upon the presence of thiols in the buffer. Stability studies showed that PRB was more sensitive to oxidation than was PRA. Receptor preparations were fractionated by DNA-cellulose chromatography to DNA-positive and DNA-negative subpopulations, and sulfhydryl groups were quantified on immunopurified receptor by labeling with [3H]-N-ethylmaleimide. Labeling of DNA-negative receptors with [3H]-N-ethylmaleimide showed 21-23 sulfhydryl groups on either PRA or PRB form when the proteins were reduced and denatured. A similar number was seen without reduction if denatured DNA-positive receptor species were tested. In contrast, the DNA-negative PRB had only 10-12 sulfhydryl groups detectable without reduction. A similar number (12-13 sulfhydryl groups) was found for PRA species that lost DNA binding activity after exposure to a nonreducing environment in vitro. We concluded that the naturally occurring receptor forms unable to bind to DNA, as well as receptor forms that have lost DNA binding activity due to exposure to a nonreducing environment in vitro, contain 10-12 oxidized cysteine residues, likely present as disulfide bonds. Since we were unable to reduce the disulfide bonds when the native DNA-negative receptor proteins were treated with dithiothreitol (DTT), we speculate that irreversible loss of DNA binding activity of receptor in vitro is due to oxidation of cysteine residues that are not accessible to DTT in the native state.     

Contrasting effects of various sulfhydryl reagents on the activity of lecithin: Cholesterol acyltransferase

Lecithin:cholesterol acyltransferase, E.C. 2.3.1.43, can be either stimulated or inhibited by reducing reagents containing free sulfhydryl groups. Mercaptoethanol and dithiothreitol stimulate enzyme activity while cysteine and reduced glutathione inhibit activity. The oxidizing disulfide reagent 2-pyridine disulfide has minimal effects on enzyme activity by itself but suppresses the stimulatory effect of mercaptoethanol.     

Sensitive quantitative analysis of disulfide bonds in polypeptides and proteins

A sensitive quantitative method has been developed to determine the number of disulfide bonds in peptides and proteins. The disulfide bonds of several peptides and proteins were cleaved quantitatively by excess sodium sulfite at pH 9.5 and room temperature. Guanidine thiocyanate (2 M) was added to the protein solutions in order to denature them and thereby make the disulfide bonds accessible. The reaction with sulfite leads to a thiosulfonate and a free sulfhydryl group; the concentration of the latter was determined by reaction with disodium 2-nitro-5-thiosulfobenzoate (NTSB) in the presence of excess sodium sulfite. The synthesis, purification, and characterization of NTSB are described. The assay is rapid, requiring 3-5 min for oligopeptides and 20 min for proteins, and is as sensitive and quantitative as the sulfhydryl group assay employing 5,5'-dithiobis(2-nitrobenzoic acid) (Ellman's reagent). It can be used for the analysis of as little as 10(8) mol of disulfide bonds, with an error of +/- 3%.     

Food Processing Characteristics of Soybean Proteins

Relation between sulfhydryl groups in soybean proteins and the physical properties of tofu was studied. Changes in the amount of sulfhydryl groups by heating or treatment with urea were more rapid in 11S protein as compared with 7S protein. Moreover, by changing the amount of sulfhydryl groups in proteins by N-ethylmaleimide, 2-mercapto-ethanol and dithiothreitol, the physical properties of tofu from 11S protein were more significantly effected than that from 7S protein. Namely, tofu-gel from 11S. protein got harder and stronger as the amount of sulfhydryl groups increased.

The results may suggest that tofu prepared from IIS protein has more disulfide bonds in its gel than that from 7S protein.     

Distribution of free sulfhydryl and disulfide groups among platelet membrane proteins.

Reactive sulfhydryl and disulfide groups were identified in platelet membrane proteins resolved by sodium dodecyl sulfate-polycrylamide gel electrophoresis. Platelet membranes treated with N-ethyl(1-14C)maleimide, phenyl(203Hg)mercuric acetate and p-chloro(203Hg)mercuribenzoate showed similar patterns of distribution of sulfhydryl groups among the sodium dodecyl sulfate-solubilized membrane proteins. Four major and two minor polypeptides ranging in molecular weight from greater than 200 000 to 20 000 were found to have reactive SH groups. Reduction of membrane proteins by sulfite coupled with subsequent mercaptide formation of the resultant monothiols led to the identification of four polypeptides with disulfide bonds. Reaction of platelet membranes with 14C-labeled 5,5'-dithio-bis(2-nitrobenzoic acid) resulted changes in the distribution profile of the solubilized membrane proteins suggestive of a polymerization process dependent upon, 5,5'-dithio-bis(2-nitrobenzoic acid)-induced intermolecular disulfide interchange.     

Disulfide bond formation during the folding of influenza virus hemagglutinin

To study the importance of individual sulfhydryl residues during the folding and assembly in vivo of influenza virus hemagglutinin (HA), we have constructed and expressed a series of mutant HA proteins in which cysteines involved in three disulfide bonds have been substituted by serine residues. Investigations of the structure and intracellular transport of the mutant proteins indicate that (a) cysteine residues in the ectodomain are essential both for efficient folding of HA and for stabilization of the folded molecule; (b) cysteine residues in the globular portion of the ectodomain are likely to form native disulfide bonds rapidly and directly, without involvement of intermediate, nonnative linkages; and (c) cysteine residues in the stalk portion of the ectodomain also appear not to form intermediate disulfide bonds, even though they have the opportunity to do so, being separated from their correct partners by hundreds of amino acids including two or more other sulfhydryl residues. We propose a role for the cellular protein BiP in shielding the cysteine residues of the stalk domain during the folding process, thus preventing them from forming intermediate, nonnative disulfide bonds.     

The structure of hepadnaviral core antigens. Identification of free thiols and determination of the disulfide bonding pattern.

A set of wild-type and mutant human, woodchuck, and duck hepatitis viral core proteins have been prepared and used to study the free thiol groups and the disulfide bonding pattern present within the core particle. Human (HBcAg) and woodchuck (WHcAg) core proteins contain 4 cysteine residues, whereas duck (DHcAg) core protein contains a single cysteine residue. Each of the cysteines of HBcAg has been eliminated, either singly or in combinations, by a two-step mutagenesis procedure. All of the proteins were shown to have very similar physical and immunochemical properties. All assemble into essentially identical core particle structures. Therefore disulfide bonds are not essential for core particle formation. No intra-chain disulfide bonds occur. Cys107 is a free thiol buried within the particle structure, whereas Cys48 is present partly as a free sulfhydryl which is exposed at the surface of the particle. Cys61 is always and Cys48 is partly involved in interchain disulfide bonds with the identical residues of another monomer, whereas Cys183 is always involved in a disulfide bond with the Cys183 of a different monomer. WHcAg has the same pattern of bonding, whereas DHcAg lacks any disulfide bonds, and the single free sulfhydryl, Cys153 which is equivalent to Cys107 of HBcAg, is buried.     

Intramolecular disulfide bonds are required for folding hydrolase B into a catalytically active conformation but not for maintaining it during catalysis

Carboxylesterases represent a large class of hydrolytic enzymes that are involved in lipid metabolism, pharmacological determination, and detoxication of organophosphorus pesticides. These enzymes have several notable structural features including two intramolecular disulfide bonds. This study was undertaken to test the hypothesis that the disulfide bonds are required during catalysis by stabilizing the catalytically active conformation. Hydrolase B, a rat liver microsomal carboxylesterase, was reduced by dithiothreitol, electrophoretically separated and assayed for hydrolysis. Contrary to the hypothesis, reduced hydrolase B was as active as the native enzyme on the hydrolysis of 1-naphthylacetate, and sulfhydryl alkylation following reduction caused no changes in the hydrolytic activity. Interestingly, substitution of a disulfide bond-forming cysteine with an alanine caused marked reduction or complete loss of the catalytic activity, suggesting that disulfide bond formation plays a role in the biosynthetic process of hydrolase B. In support of this notion, refolding experiments restored a significant amount of hydrolytic activity when hydrolase B was unfolded with urea alone. In contrast, little activity was restored when unfolding was performed in the presence of reducing agent dithiothreitol. These results suggest that formation of the disulfide bonds plays a critical role in folding hydrolase B into the catalytically active conformation, and that the disulfide bonds play little role or function redundantly in maintaining this conformation during catalysis.     

Mastication effort estimated by electromyography for cooked rice of differing water content

The objective of this study was to quantify the mastication effort for cooked rice. We analyzed mastication patterns while normal subjects ate a spoonful of cooked rice that had been prepared by cooking with different amounts of water (1.5, 2.0, 3.0, and 4.0 times the water to rice weight). The rice samples were served with the same weight, same volume and same solid content, and electromyography (EMG) of the masticatory muscles was measured. The texture of the four cooked rice samples was instrumentally analyzed by the two-bite method. The number of chews, masticatory time, and jaw-closing muscle activities per chew evaluated by EMG were higher in the rice sample cooked with least water, which exhibited a high firmness value in the instrumental test. Rice cooked with 4.0 times the amount of water exhibited the longest jaw-opening duration, which was related to the adhesiveness value in the instrumental test. The ratio of jaw-opening muscle activity to the preceding jaw-closing muscle activity was lower for the rice containing least water, this corresponding to the area ratio (balance degree) in the instrumental test. Softer rice containing more water reduced the total mastication effort until swallowing because it required a shorter mastication time. It was not difficult for the softer rice with high density to be ingested in greater weight, decreasing the mastication effort for a certain amount.     

2,2'-Bispyridyl disulfide rapidly induces intramolecular disulfide bonds in peptides.

A linear peptide containing two reduced cysteine residues can be rapidly converted to its oxidized cyclic form containing an intramolecular disulfide bond by adding an excess of 2,2'-bispyridyl disulfide (2,2'-dipyridyl disulfide or 2,2'-dithiodipyridine) to conventional buffer solutions. The reactants and products are easily separated by reverse-phase chromatography. This reaction will find wide application in forming intramolecular disulfide bonds because of its selectivity for free sulfhydryl groups, quickness, safety, and applicability under acidic conditions.     

Analyses of intramolecular disulfide bonds in proteins by polyacrylamide gel electrophoresis following two-step alkylation

A method that makes use of polyacrylamide gel electrophoresis was developed for the analysis of intramolecular disulfide bonds in proteins. Proteins with different numbers of cleaved disulfide bonds are alkylated with iodoacetic acid or iodoacetamide as the first step. The disulfide bonds remaining were reduced by excess dithiothreitol, and the newly generated free sulfhydryl groups were alkylated with the reagent not yet used (iodoacetamide, iodoacetic acid, or vinyl-pyridine) as the second step. This treatment made it possible for lysozyme (Mr, 14,000; 4 disulfides), the N-terminal half-molecule of conalbumin (Mr, 36,000; 6 disulfides), the C-terminal half-molecule of conalbumin (Mr, 40,000; 9 disulfides), and whole conalbumin (Mr, 78,000; 15 disulfides) to be separated by acid-urea polyacrylamide gel electrophoresis into distinct bands depending on the number of disulfide bonds cleaved. The method allowed us to determine the total number of disulfide bonds in native proteins and to assess the cleaved levels of disulfide bonds in partially reduced proteins. Two-step alkylation used in combination with radioautography was especially useful for the analysis of disulfide bonds in proteins synthesized in complex biological systems.     

Activation of chicken liver fructose-1,6-bisphosphatase by oxidized glutathione

Treatment of chicken liver fructose-1,6-bisphosphatase with oxidized glutathione (GSSG) leads to an increase in activity. This activation is markedly enhanced if treatment is performed in the presence of AMP or Mn²⁺. The effects of AMP and Mn²⁺ appear to be synergistic. The maximal activation is over 13-fold and is accompanied by the disappearance of 4 sulfhydryl groups per molecule of enzyme. Both fructose 1,6-bisphosphate and fructose 2,6-bisphosphate can largely prevent this activation. Activation can be reversed by dithiothreitol or cysteine. It appears that GSSG activates this enzyme by thiol/disulfide exchanges with the enzyme's specific sulfhydryl groups.     

水稻巯基蛋白酶抑制剂的分子修饰与其对稻瘟病菌的抑制作用

水稻巯基蛋白酶抑制剂（ＣＰＩ）经用二硫苏糖醇,对氯汞苯甲酸和碘乙酸修饰后,对木瓜蛋白酶的抑制活性并无改变;用Ｎ－乙基顺丁烯二酰亚胺与ＣＰＩ反应,可以测出ＣＰＩ分子内有１９个巯基被修饰,被修饰后,抑制活性仍无改变,表明水稻ＣＰＩ的抑制活性不需要巯基参与;应用Ｎ－溴代丁二酰亚胺与ＣＰＩ反应,可测出ＣＰＩ分子内有２个Ｔｒｐ被修饰,修饰后,抑制活性全部丧失,表明Ｔｒｐ是保持抑制活性所必需的基团。水稻巯基蛋白酶抑制剂和丝氨酸蛋白酶抑制剂对稻瘟病菌丝体的生长均有抑制作用,但后者的抑制作用比前者更强,若将两种抑制剂混合使用,则对稻瘟病菌丝体的抑制作用非常强烈;当抑制剂加入量达７２μｇ时,即可产生明显的抑制作用。     

Determination of the Cysteine and Cystine Content of Proteins by Amino Acid Analysis: Application to the Characterization of Disulfide-Coupled Folding Intermediates

A method has been developed for the simultaneous detection of cysteine and cystine in proteins by amino acid analysis. In this method, the sulfhydryl groups of the cysteine residues are first blocked with 2-aminoethyl methanethiosulfonate (AEMTS). This reagent converts all free sulfhydryl groups to mixed disulfides with 2-aminoethanethiol (AET). The isolated blocked protein is subjected to oxidation with performic acid prior to hydrolysis and amino acid analysis. This procedure quantitatively converts the 2-aminoethanethiol blocking groups into taurine, and all cysteine residues (including those involved in disulfide bonds) into cysteic acid. Both of these derivatives are stable and can be recovered quantitatively by amino acid analysis. The speed and specificity with which AEMTS reacts with thiols make this method particularly effective for the characterization of disulfide-coupled folding intermediates.