Sequence of the cysteinyl-containing peptides of 4-aminobutyrate aminotransferase. Identification of sulfhydryl residues involved in intersubunit linkage

Three cysteine-containing tryptic peptides were isolated and sequenced from mitochondrial 4-aminobutyrate aminotransferase using DABIA (4-dimethylaminoazobenzene-4-iodoacetamide) as specific labeling reagent for sulfhydryl groups. The enzyme is a dimer made up of two identical subunits, but four out of the six cysteinyl residues/dimer form disulfide bonds when treated with iodosobenzoate to yield inactive enzyme species. To identify the cysteinyl residues undergoing reversible oxidation/reduction, the S-DABIA-labeling patterns of the fully reduced (active) and fully oxidized (inactive) forms of the enzyme were compared. Tryptic digests of the reduced enzyme contained three labeled peptides. If the enzyme was treated with iodosobenzoate prior to reaction with DABIA and tryptic digestion, only one labeled peptide was detected and identified (peptide I), indicating that the two missing cysteinyl-containing peptides (peptides II, III) have been oxidized. The sulfhydryl groups undergoing oxidation/reduction were found to be intersubunit, based on SDS/polyacrylamide gel electrophoresis results. The loss of catalytic activity of 4-aminobutyrate aminotransferase by oxidation of sulfhydryl residues is related to constraints imposed at the subunit interface by the insertion of disulfide bonds.     

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.     

Mung bean nuclease I. Physical, chemical, and catalytic properties.

A simplified purification procedure for mung bean nuclease has been developed yielding a stable enzyme that is homogeneous in regards to shape and size. The nuclease is a glycoprotein consisting of 29% carbohydrate by weight. It has a molecular weight of 39 000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme contains 1 sulfhydryl group and 3 disulfide bonds per molecule. It has a high content (12.6 mol %) of aromatic residues. Approximately 70% of the enzyme molecules contain a peptide bond cleavage at a single region in the protein. The two polypeptides, 25 000 and 15 000 daltons, are covalently linked by a disulfide bond(s). Both the cleaved and intact forms of the enzyme are equally active in the hydrolysis of the phosphate ester linkages in either DNA, RNA, or adenosine 3'-monophophate. The enzymatic activity of mung bean nuclease can be stabilized at pH 5 in the presence of 0.1 mM zinc acetate, 1.0 mM cysteine, and 0.001% Triton X-100. The enzyme can be inactivated and reactivated by the removal and readdition of Zn2+ or sulfhydryl compounds.     

Identification of fifteen neighboring protein pairs in the Escherichia coli 50 S ribosomal subunit crosslinked with 2-iminothiolane.

The 50 S ribosomal subunit of Escherichia coli was allowed to react with 2-iminothiolane under conditions in which amidine-linked sulfhydryl derivatives were formed between lysine ?-amino groups in ribosomal proteins and the heterocyclic thioimidate. Crosslinking between sulfhydryl groups close enough to form intermolecular disulfide bonds was promoted by oxidation of the modified ribosomal subunits. Disulfide-linked dimers were partially purified by extraction of the oxidized subunits with lithium chloride and electrophoresis of the salt-extracted fractions in polyacrylamide/urea gels at pH 5.5. Crosslinked protein dimers were separated by polyacrylamide/sodium dodecyl sulfate diagonal gel electrophoresis. Fifteen protein dimers were identified. Many of them involve proteins implicated in functional sites of the 50 S subunit and in ribosome assembly. The crosslinking results show the proximity of many of these proteins at these active centers, and extend the neighborhood by demonstrating the presence of additional proteins.     

Chemical investigations on pig kidney aminoacylase.

1. Preparations of purified pig kidney aminoacylase (N-Acylamino-acid amidohydrolase, EC 3.5.1.14) were obtained by Sephadex and DEAE-cellulose chromatography in homogeneous form as judged by polyacrylamide gel electrophoresis and immunoelectrophoresis. 2. The apparent molecular weight of the enzyme, determined by gel filtration, was about 86 000. After treatment with mercaptoethanol, performic acid or sodium dodecyl sulphate a band with an apparent molecular weight of approximately 43 000 was observed in polyacrylamide gels containing sodium dodecyl sulphate. Thus pig kidney aminoacylase seems to be composed of two subunits. 3. The amino acid composition of the enzyme was determined. Aminoacylase contains 772 amino acids, which corresponds to a molecular weight of 85 500. 12 tryptophan and 12 half-cystine residues were found. 4. Each subunit of the enzyme contains two -SH groups of different reactivity and two disulfide bonds one of which is easily cleaved by -SH compounds, the second only by performic acid oxidation. 5. Chemical modification of two -SH groups abolishes the catalytic activity of aminoacylase. Cleavage of two disulfide bonds also inactivates the enzyme. It is suggested that the enzyme has two active sites each containing an essential -SH group and disulfide bond. One active site is assumed to be part of each subunit.     

Chemical properties and amino acid composition of beta1-bungarotoxin from the venom of Bungarus multicinctus (Formosan banded krait)

beta-Bungarotoxin purified from the venom of Bungarus multicinctus (Formosan banded krait) contained no carbohydrate and behaved as a homogeneous protein on polyacrylamide gel electrophoresis at pH 4.1 and SDS-polyacrylamide gel electrophoresis without 2-mercaptoethanol treatment. Its molecular weight and isoelectric point were estimated to be about 21,000 by gel filtration and about 9.5 by isoelectric focusing, respectively. The toxin treated with the reducing agent was split into two polypeptide chains as revealed by SDS-polyacrylamide gel electrophoresis and their molecular weights were calculated to be about 13,000 and 7,000. The two polypeptide chains (the large one named the A chain and the small one the B chain) were isolated by gel filtration after reduction of disulfide bonds in the toxin followed by alkylation. The A chain contained 120 amino acid residues including 13 half-cystines and the B chain 60 residues including 7 half-cystines. The two chains were supposed to link by disulfide bond(s) in the intact toxin which contained no free sulfhydryl groups. The N-terminal residues of the A and B chains were asparagine and arginine and the C-terminal ones were glutamine and proline, respectively, in accordance with the results of the terminal analyses of the intact toxin.     

Free sulfhydryl in recombinant monoclonal antibodies

Monoclonal antibody (mAb) therapy applications have been growing rapidly in recent years. Like other recombinant protein drugs, therapeutic mAb's need to be well characterized to ensure their structural and functional integrity. IgG mAb's are composed of two heavy and two light chains covalently linked by interchain disulfide bonds. Each domain of the heavy or light chain contains one additional disulfide bond. Native IgG mAb's, with completely formed disulfide bonds, should not bear any free sulfhydryl. This report describes detection and quantification of free sulfhydryl in recombinant mAb's produced in Chinese hamster ovary (CHO) cells using a fluorescent technique. The method utilizes the fluorescent probe N-(1-pyrenyl)maleimide (NPM). The purified mAb's appear to be homogeneous under native conditions with approximately 0.02 mol of free sulfhydryl per mole of protein. Upon denaturation, minor species related to the mAb's are observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the free sulfhydryl level is determined to be approximately 0.1 mol/mol of protein. These results suggest that a small portion of these recombinant mAb's lack in intermolecular disulfide bonds but remain noncovalently associated under native conditions. The formation of the free sulfhydryl containing mAb species is likely to occur during the culture process and/or protein folding process in the endoplasmic reticulum (ER).     

Determination of sulfhydryl groups and disulfide bonds in a protein by polyacrylamide gel electrophoresis

A general method by polyacrylamide gel electrophoresis for the determination of sulfhydryls and disulfides in a protein was developed. The method included a two-step alkylation procedure: the first step consisted of alkylation of the sulfhydryl groups with iodoacetic acid in the presence and absence of 8 M urea; the second step consisted of alkylation of the disulfide groups with iodoacetamide after reduction with a thiol. By high-pH urea gel electrophoresis, all the half-cystine residues in a protein could be categorized into three states: reactive sulfhydryls, nonreactive sulfhydryls, and disulfide bonded. The particular advantage of the method is that the states of half-cystines in different protein species can be analyzed independently both in isolated protein and in biological translation systems.     

Human liver manganese superoxide dismutase. Purification and crystallization, subunit association and sulfhydryl reactivity

Manganese superoxide dismutase (Mn-SOD) has been purified with a high yield (320 mg) from human liver (2 kg) and crystallized. Low-angle laser light scattering of the enzyme has shown that native enzyme is a tetrametic form. Four of the eight cysteine residues in the tetramer reacted with 5,5'-dithiobis(2-nitrobenzoic acid) or with iodoacetamide. The others were only reactive in protein heated with SDS or urea after reduction with dithiothreitol or 2-mercaptoethanol. The reactive sulfhydryl group was found to be located at Cys196 by amino acid sequence analysis of Nbs2-reactive peptides isolated by activated thiol-Sepharose covalent chromatography. Incubation of Mn-SOD in 1% SDS for 2 or 3 days at 25 degrees C or 5 min at 100 degrees C gave material showing two prominent components on polyacrylamide gel electrophoresis in the presence of 0.1% SDS. The major component had a molecular mass of 23 kDa; the other, 25 kDa. Reduction of the protein by dithiothreitol or 2-mercaptoethanol heated in SDS produced only the 25-kDa monomer species. Essentially, no thiol groups were detected in the 23-kDa form, in which two cysteine residues appear to have been oxidized to form an intrasubunit disulfide. This indicates that Cys196 has a reactive sulfhydryl and appears to be a likely candidate for a mixed disulfide formation in vivo.     

The nature of the multiple forms of bovine thiol:protein disulfide oxidoreductase

Preparations of thiol"protein disulfide oxidoreductase from bovine liver were shown to be homogeneous by polyacrylamide gel electrophoresis, sedimentation equilibrium centrifugation and NH2-terminal analysis (Carmichael et al., 1977). When the enzyme was subjected to prolonged storage at -20 degrees, freeze-thawing, or heating at 60 degrees, at least one new protein species was observed using polyacrylamide gel electrophoresis. The new protein results from dimerization of the enzyme. The dmier consisted of two monomers held together by an intermolecular disulfide bond. The formation of this dimer can be reversed and partially prevented by thiols.     

A study of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. Improved purification, relative molecular mass, and amino acid composition.

The purification procedure for p-hydroxybenzoate hydroxylase has been modified by replacement of the DEAE-cellulose (DE-32) column in the original procedure by a Sephadex--Cibacron-blue affinity column. In this way the yield of enzyme could be improved from 16% to about 40--50%. Preparative gel chromatography indicated that the enzyme does not exist as a monomeric species as earlier believed but mainly as a dimer. Sodium dodecyl sulfate gel electrophoresis of purified enzyme revealed a minimum relative molecular mass (Mr) of 43000--45000. Analytical gel chromatography, sedimentation equilibrium and sedimentation velocity experiments showed that the enzyme exists in solution mainly as a dimer but also in higher-order quaternary structures (presumably tetramer and hexamer). Temperature dependence of the distribution of the oligomers suggests that the association is of hydrophobic nature. The amino acid composition of the enzyme is also presented. The enzyme contains no disulfide but five sulfhydryl groups. In the native state of the enzyme only one sulfhydryl group is accessible to N-ethylmaleimide or 5,5'-dithiobis(2-nitrobenzoic acid). The iso-electric point of the enzyme was found to be 5.8.     

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.     

AUT凝胶电泳在牛胰核糖核酸酶折叠研究中的应用

目的:牛胰核糖核酸酶是一种用于蛋白折叠研究的经典模式蛋白,在折叠研究过程中主要使用高效液相色谱用于分离检测不同阶段的蛋白折叠中间体。高效液相色谱具有自动化、分离效果好、样品可回收等优点,同时也存在检测通量较低、仪器设备较为昂贵等不足。AUT凝胶电泳简便、快捷、检测通量较高,本文尝试将其应用于牛胰核糖核酸酶的折叠研究。方法:使用AUT凝胶电泳、酶活性检测、质谱对牛胰核糖核酸酶还原变性过程及产生的折叠中间体进行检测;通过高效液相色谱和质谱对折叠中间单体进行分离检测,并分别进行AUT凝胶电泳检测以解析各折叠中间单体在电泳中的条带位置;通过AUT凝胶电泳和酶切后质谱鉴定各折叠中间单体的二硫键配对方式。结果:AUT凝胶电泳可以有效区分不同条件下的牛胰核糖核酸酶还原变性过程,检测结果与酶活性、质谱结果相符,并可以很好地区分牛胰核糖核酸酶还原变性过程折叠中间体。高效液相色谱将牛胰核糖核酸酶还原变性过程折叠中间体分离为13个色谱峰,并与AUT凝胶电泳中的11个条带位置进行匹配。确认牛胰核糖核酸酶还原变性过程折叠中间单体的二硫键配对方式,并与AUT凝胶电泳条带进行匹配,Cys58-Cys110和Cys26-Cys84构象熵减作用强于Cys40-Cys95和Cys65-Cys72。结论:AUT凝胶电泳适用于检测牛胰核糖核酸酶折叠中间体,可以与高效液相色谱、质谱等检测技术相互补充,共同应用于牛胰核糖核酸酶的折叠研究。     

Purification and properties of streptococcal nicotinamide adenine dinucleotide glycohydrolase.

Highly purified streptococcal nicotinamide adenine dinucleotide glycohydrolase (NADase) was obtained by utilizing disodium tetrathionate to protect the enzyme by blocking the sulfhydryl groups of streptococcal proteinase. This was followed by two-step ion-exchange chromatography. The pure enzyme, demonstrated as a single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, had a specific activity of 11,200 NADase units per mg of protein and was devoid of hemolytic activity. NADase had a molecular weight of about 55,000 as determined by gel filtration, by summation of amino acid residues, and by sodium dodecyl sulfate/gel electrophoresis. The purified enzyme had optimal activity at pH 7.3 and at 40 C and a calculated Km of 5.1 times 10- minus 4 mM. It was inhibited by alpha-iodoacetamide.     

Concerted action of cosolvents, chaotropic anions and thioredoxin on chloroplast fructose-1,6-bisphosphatase. Reactivity to iodoacetamide

The incubation of chloroplast fructose-1,6-bisphosphatase with both dithiothreitol and protein denaturants made sulfhydryl groups available for reaction with [1-14C]iodoacetamide (10-12 mol iodoacetamide incorporated/mol enzyme). Digestion of S-carboxyamidomethylated enzyme with trypsin and polyacrylamide gel electrophoresis, in the presence of sodium dodecylsulfate, yielded two 14C-labeled fragments whose apparent molecular mass were 10 kDa and 16 kDa. In the absence of either dithiothreitol or protein denaturants the incorporation of iodoacetamide to the enzyme was lower than 4 mol. When chloroplast fructose-1,6-bisphosphatase was initially incubated with dithiothreitol (2.5 mM) and (a) high concentrations of both fructose 1,6-bisphosphate (4 mM) and Ca2+ (0.3 mM) or (b) low concentrations of both fructose 1,6-bisphosphate (0.8 mM) and Ca2+ (0.05 mM) in the presence of either 2-propanol (15%, by vol.), trichloroacetate (0.15 M) or chloroplast thioredoxin-f (0.5 microM) and subsequently subjected to proteolysis and electrophoresis, S-carboxyamidomethylated tryptic fragments had similar molecular masses. Thus, conditions that stimulated the specific activity of chloroplast fructose-1,6-bisphosphatase caused conformational changes which favoured both the reduction of disulfide bridges and the exposure of sulfhydryl groups. In this aspect, thioredoxin exerted structural and kinetic effects similar to compounds not involved in redox reactions (organic solvents, chaotropic anions). These results indicated that the modification of hydrophobic (intramolecular) interactions in chloroplast fructose-1,6-bisphosphatase constituted the underlying mechanism in light-activation by the ferredoxin-thioredoxin system.     

Guanidoacetate methyltransferase from rat liver: Purification,properties, and evidence for the involvement of sulfhydryl groups for activity

Guanidoacetate methyltransferase (EC 2.1.1.2) has been purified about 800-fold from rat liver. The purified preparation shows a single protein band on polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The molecular weight of the enzyme is estimated to be 25,000 and 26,000 by Sephadex gel molecular-exclusion chromatography and by electrophoresis in polyacrylamide gradient gel, respectively. The sodium dodecyl sulfate-denatured enzyme also has a molecular weight of 26,000; thus, the enzyme is a monomeric protein. Guanidoacetate methyltransferase as isolated is catalytically inactive, but is readily reactivated by incubation with a thiol. The reactivated enzyme, which contains 3 mol of sulfhydryl groups/mol of enzyme, is again inactivated by oxidized glutathione. This inactivation is accompanied by the disappearance of two sulfhydryl residues. The relationship between the loss of enzyme activity and the number of residues disappeared indicates that the integrity of these sulfhydryl residues is critical for activity. The oxidized enzyme fails to bind the substrate S-adenosylmethionine as evidenced by the equilibrium dialysis study. Alkylation of the nonoxidizable sulfhydryl by N-ethylmaleimide shows that this residue is also essential for activity. UV absorption, fluorescence, and CD spectra show no difference between the reduced and oxidized enzymes, but the former is more susceptible to proteolytic attack by trypsin. The enzyme has an isoelectric pH of 5.3, and is most active at pH 9.0. From the CD spectrum, an α helix content of 15% is calculated. The K_m values for guanidoacetate and S-adenosylmethionine are 97.5 and 6.73 μm, respectively, at pH 8.0 and 37 °C.     

Ribonuclease isolated from yeast (Saccharomyces carlsbergensis): Characterization and properties

The homogeneity of a purified ribonuclease from brewers' yeast was determined by velocity sedimentation and polyacrylamide gel electrophoresis techniques. The velocity sedimentation pattern gave a single peak with a S_app 3.46 and polyacrylamide gel electrophoresis showed one major band. The absorption spectrum of the enzyme showed maximum absorption at 277–278 nm and minimum at 252 nm. The enzyme was relatively stable to extreme pH values and high temperature. Both NaCl and KCl increased the enzyme activity whereas enzyme was inhibited by divalent metal ions. The inhibition of the enzyme was increased in the order of Ca²⁺ > Mg²⁺ > Fe²⁺Cu²⁺. Chemical modification studies of the enzyme showed that tryptophan residues and disulfide bonds were required for enzyme activity.     

Deoxyribose 5-phosphate aldolase of Bacillus cereus: purification and properties.

Deoxyribose 5-phosphate aldolase was purified 41 times from Bacillus cereus induced by growth on deoxyribonucleosides. The purification procedure includes ammonium sulphate fractionation, gel filtration on Sephadex G-100, ion-exchange chromatography on DEAE-Sephacel and preparative electrophoresis on 10% polyacrylamide gel. The enzyme is stable above pH 6.5, but is rapidly inactivated by sulfhydryl reagents. Being insensitive to EDTA, it may be considered as a Class I aldolase. Among a number of compounds tested (including some carboxylic acids, free and phosphorylated pentoses, nucleotides and nucleosides), none has been found to affect the enzyme activity. The enzyme appears to be dimeric, with a subunit Mr of 23,600. A Km of 4.4 x 10(-4) M was calculated for dRib 5-P.     

Purification and characterization of ribulose-5-phosphate kinase from spinach

An efficient purification procedure utilizing affinity chromatography is described for spinach ribulose-5-phosphate kinase, a light-regulated chloroplastic enzyme. Gel filtration and polyacrylamide gel electrophoresis of the purified enzyme reveal a dimeric structure of 44,000 Mr subunits. Chemical crosslinking with dimethyl suberimidate confirms the presence of two subunits per molecule of native kinase, which are shown to be identical by partial NH2-terminal sequencing. Based on sulfhydryl titrations and on amino acid analyses, each subunit contains four to five cysteinyl residues. The observed slow loss of activity during spontaneous oxidation in air-saturated buffer correlates with the intramolecular oxidation of two sulfhydryl groups, presumably those involved in thioredoxin-mediated regulation.     

Characterization and substrate specificity of fumarylacetoacetate fumarylhydrolase.

The molecular weight of fumarylacetoacetate fumarylhydrolase (EC 3.7.1.2) is 86 000 +/- 10 000, as determined by gel filtration. The enzyme appears to be a dimer with a monomer molecular weight of 38 000 - 43 000, as determined by gel electrophoresis, gel filtration in guanidine-hydrochloride, and ultracentrifugation. The subunits appear to be identical, as only one band is seen in gel electrophoresis, only one protein peak is detected in gel filtration in guanidine-hydrochloride, and only one amino-terminal amino acid (proline) is detected. Three free sulfhydryl groups per denatured monomer are detected by reaction with 5,5'-dithiobis(2-nitrobenzoic acid), while for the active enzyme only two sulfhydryl groups react with this reagent, The extinction coefficients at 260 and 280 nm, the amino acid composition, and the isoelectric point (6.7) of the enzyme are also reported. The enzyme catalyzes the hydrolysis of six 2,4-diketo acids and three 3,5-diketo acids tested. The Km of the substrates is similar but V varies by a factor of 120. The pH optimum is 7.3. The enzyme did not catalyze the hydrolysis of a number of esters tested.