Localization of thiol and disulfide groups in guinea pig spermatozoa during maturation and capacitation using bimane fluorescent labels

The distribution of thiols and disulfides in the guinea pig spermatozoon during maturation and capacitation was studied using both membrane-permeable (mBBr) and impermeable (qBBr) forms of bromobimane, a specific fluorescent probe for thiol groups. In conjunction with the disulfide (SS)-reducing agent dithiothreitol (DTT) and the thiol-alkylating agent N-ethylmaleimide (NEM), quantitative spectrofluorometric measurements of the relative amounts of total thiol (SH) versus SS were performed on cauda epididymal spermatozoa. Under conditions labeling 70% of the reactive thiols, the ratio total SS/SH was 2.4/1.0. Contamination by other cell types prevented similar measurements on spermatozoa at earlier stages of epididymal maturation; thus, the qualitative localization of SH and SS groups in these and in capacitated spermatozoa was visualized using fluorescence microscopy. As spermatozoa moved from the testis to the caput epididymidis, there was a slight apparent increase in staining both on the surface and internally in all regions. Thereafter, surface and internal staining decreased by the time spermatozoa reached the cauda epididymidis. Fluorescence patterns were unaltered under short-term (1 h) capacitation conditions in calcium-free modified Tyrode's medium containing lysophosphatidyl choline and after induction of the acrosome reaction with 2 mM calcium. However, long-term capacitation (16-18 h) in calcium-free modified Tyrode's medium resulted in a loss of detectable SH in the head and acrosome. Regardless of the stage examined, sperm tails contained the greatest relative amount of SH, followed by the head and the acrosome. In addition, there was always more SH detectable internally than on the surface. DTT pretreatment caused a dramatic increase in staining in all regions, both surface and internal, consistent with the quantitative estimates of the SS/SH ratio.     

The disulphide bonds of human and rabbit γ-globulins

1. The reaction of the disulphide bonds of the predominant species of human and rabbit gamma-globulins (the 7s gamma-globulins) with sulphite was studied in the presence and absence of denaturing agents and heavy-metal reagents. 2. The total number of bonds reacting/mol. of mol.wt. 160000 was approx. 18 for human and 20 for rabbit gamma-globulin. 3. Six S.S bonds/mol. of human and 6.5 S.S bonds/mol. of rabbit gamma-globulin reacted with sulphite alone at pH6. These appeared to include all the interchain S.S bonds. 4. The number of free SH groups was less than 0.2/mol. of human and less than 0.3/mol. of rabbit gamma-globulin.     

Analysis of free sulfhydryl groups and disulfide bonds in Na+,K+-ATPase

The content of free SH groups and disulfide bonds in the purified pig kidney Na+,K+-ATPase was determined by ammetric titration with silver nitrate. In the native enzyme, most of the free SH groups are masked due to their location in the polypeptide chain regions poorly accessible to SH reagents. Denaturation with 5% SDS and 8 M urea makes these regions accessible thus revealing 22 free SH groups/mol of the protein. After complete blocking of free SH groups with silver ions, 8 SH groups/mol of the protein are being released upon sulfitolysis which indicates the presence of four disulfide bonds in the enzyme. At least one disulfide bridge is located in the alpha-subunit whereas the beta-subunit contains three disulfide bonds.     

Sulphydryl and Disulphide Levels in Protein Fractions from Hydrated and Dry Leaves of Resurrection Plants

Significant changes in sulphydryl (‘SH’) and disulphide(‘SS’) levels during air-drying in leaves of ‘resurrection’plants (whose protoplasm survives dehydration) stemmed mainlyfrom protein turnover effects. No significant changes were foundin the SH, SS levels in leaves of the desiccation sensitivespecies Sporobolus pyramidalis following air-drying. The three tolerant species studied differed in the directionof change. Some data were consistent with Levitt's SH, SS hypothesis:increases in protein-SS levels in Sporobolus stapfianus (desiccationtolerant) were consistent with a stabilization of new proteinby SS bonds; lower reactivity of protein-SH in the tolerantspecies Talbotia elegans (which on the other hand has decreasedprotein-SS) is consistent with a second mechanism of decreasingprotein denaturation proposed in Levitt's hypothesis. Evidence of some conversion of SH to SS in the soluble proteinsof Xerophyta viscosa (a tolerant species) would on Levitt'shypothesis indicate an injurious process. Some degree of proteindenaturation might be indicated by partial inactivation of thesoluble enzyme ribulose bisphosphate carboxylase in this species,and loss of some soluble isoenzymes (peroxidase and alkalinephosphatase). An apparent lack of SH conversion to SS in thesensitive species Sporobolus pyramidalis was not consistentwith the SH, SS hypothesis. Resurrection plants, Sporobolus pyramidalis, Sporobolus stapfianus, Talbotia elegans, Xerophyta viscosa, drought resistance, desiccation tolerance, protein turnover, sulphydryl groups     

Enzymatic Phosphorylation of Soybean Proteins by Protein Kinase

Soybean proteins were subjected to phosphorylation with cyclic adenosine monophosphate- dependent protein kinase (A-kinase). As a result, acidic subunits of the 11S fraction were found to be phosphorylated by A-kinase. To estimate the effect of the phosphorylation, 11S acidic subunits were isolated and subjected to A-kinase phosphorylation. The optimal enzyme amount and Mg^{2 +} concentration for the phosphorylation of 11S acidic subunits were determined to be 1.5U/ml and 1.6 mm, respectively. The rate of phosphorylation was 2mol/mol acidic subunits (MW 38,000) under the above conditions. The protein structures of 11S acidic subunits, as determined from UV and CD spectra, were slightly affected by the enzymatic phosphorylation.     

Soybean nodule sucrose synthase (nodulin-100): further analysis of its phosphorylation using recombinant and authentic root-nodule enzymes.

Sucrose synthase (SS) is a known phosphoserine-containing enzyme in legume root nodules and various other plant "sink" tissues. In order to begin to investigate the possible physiological significance of this posttranslational modification, we have cloned a full-length soybean nodule SS (nodulin-100) cDNA and overexpressed it in Escherichia coli. Authentic nodule SS and recombinant wild-type and mutant forms of the enzyme were purified and characterized. We document that a conserved serine near the N-terminus (Ser(11)) is the primary phosphorylation site for a nodule Ca(2+)-dependent protein kinase (CDPK) in vitro. Related tryptic digestion and mass spectral analyses indicated that this target residue was also phosphorylated in planta in authentic nodulin-100. In addition, a secondary phosphorylation site(s) in recombinant nodule SS was implicated given that all active mutant enzyme forms (S11A, S11D, S11C, and N-terminal truncation between Ala(2) and Arg(13)) were phosphorylated, albeit weakly, by the CDPK. This secondary site(s) likely resides between Glu(14) and Met(193) as evidenced by CNBr cleavage and phosphopeptide mapping. Phosphorylation of the recombinant and authentic nodule Ser(11) enzymes in vitro by the nodule CDPK had no major effect on the sucrose-cleavage activity and/or kinetic properties. However, phosphorylation decreased the apparent surface hydrophobicity of the recombinant wild-type enzyme, suggesting that this covalent modification could potentially play some role in the documented partitioning of nodulin-100 between the nodule symbiosome/plasma membranes and cytosol in planta.     

Contribution of intra- and intermolecular hydrogen bonds to the conformational stability of human lysozyme(,).

In globular proteins, there are intermolecular hydrogen bonds between protein and water molecules, and between water molecules, which are bound with the proteins, in addition to intramolecular hydrogen bonds. To estimate the contribution of these hydrogen bonds to the conformational stability of a protein, the thermodynamic parameters for denaturation and the crystal structures of five Thr to Val and five Thr to Ala mutant human lysozymes were determined. The denaturation Gibbs energy (DeltaG) of Thr to Val and Thr to Ala mutant proteins was changed from 4.0 to -5.6 kJ/mol and from 1.6 to -6.3 kJ/mol, respectively, compared with that of the wild-type protein. The contribution of hydrogen bonds to the stability (DeltaDeltaG(HB)) of the Thr and other mutant human lysozymes previously reported was extracted from the observed stability changes (DeltaDeltaG) with correction for changes in hydrophobicity and side chain conformational entropy between the wild-type and mutant structures. The estimation of the DeltaDeltaG(HB) values of all mutant proteins after removal of hydrogen bonds, including protein-water hydrogen bonds, indicates a favorable contribution of the intra- and intermolecular hydrogen bonds to the protein stability. The net contribution of an intramolecular hydrogen bond (DeltaG(HB[pp])), an intermolecular one between protein and ordered water molecules (DeltaG(HB[pw])), and an intermolecular one between ordered water molecules (DeltaG(HB[ww])) could be estimated to be 8. 5, 5.2, and 5.0 kJ/mol, respectively, for a 3 A long hydrogen bond. This result shows the different contributions to protein stability of intra- and intermolecular hydrogen bonds. The entropic cost due to the introduction of a water molecule (DeltaG(H)()2(O)) could be also estimated to be about 8 kJ/mol.     

Ultrastructural localization of glycinin and beta-conglycinin in Glycine max (soybean) cv. Maple Arrow by the immunogold method

beta-Conglycinin (7 S globulin) and glycinin (11 S globulin) are the major reserve proteins of soybean. They were localized by the protein A immunogold method in thin sections of Glycine max (soybean) cv. Maple Arrow. In cotyledons, both globulins were simultaneously present in all protein bodies. Statistical analysis of marking intensities indicated no correlation between globulin concentration and size of protein bodies. The immunogold method failed to detect either globulin in the embryonic axis and in cotyledons of four-day-old seedlings. Similar observations were made with cotyledons of two soy varieties lacking either the lectin or the Kunitz trypsin inhibitor. In another variety (T-102) lacking the lectin, the 7 S globulin could not be detected.     

Tryptophan and cystein residues of the acetylcholine receptors of Torpedo species. Relationship to binding of cholinergic ligands.

Several methods were used to analyze for tryptophan in the acetylcholine (ACh) receptors purified from the electric organs of the electric rays, Torpedo californica and Torpedo marmorata. The best value of tryptophan was 2.4 mol %. When excited at 290 nm, both receptors fluoresced with a maximum at 336, but there was no change in the fluorescence emission spectra upon binding of carbamylcholine, d-tubocurarine, ACh, or decamethonium. The free SH content of the Torpedo receptors varied in different preparations, and was highest in that purified from fresh T. californica using deaerated solutions and dialysis under nitrogen, and lowest in that prepared from the aged lyophilized membranes of T. marmorata. The maximum free SH content was 20 nmol/mg of protein or 0.22 mol %, equal to at most 18% of the total cysteic acid residues. Reaction of either 33% or of all the SH residues with p-chloromercuribenzoate reduced maximum ACh binding to the pure receptor prepared from fresh T. californica by only 23%.     

Formation of Pseudoglycinins from Intermediary Subunits of Glycinin and Their Gel Properties and Network Structure

Intermediary subunits of soybean 11S globulin (glycinin) designated as IS I, IS II and IS III were isolated by DEAE-Sephadex column chromatography. Pseudoglycinins composed of one of the intermediary subunits alone were reconstituted. The pseudoglycinins were similar to the native glycinin as to molecular size, subunit structure and secondary structure. The turbidity and hardness of the heat-induced gels formed from pseudoglycinins were different from those derived from the native glycinin, depending on the constituent intermediary subunits. The results indicate that IS II is closely related to the generation of the gel turbidity and IS III plays an important role in increasing the gel hardness. The hardness of the gel seems to be determined by both the length and extent of branching of the constituent strands of the gel network structure.     

Separation and characterization of oat globulin polypeptides

The storage globulin of oat seeds was separated into its acidic (α) and basic (β) polypeptides by ion-exchange chromatography in 6 m urea and further characterized by several electrophoretic techniques. Molecular weights of the α and β polypeptides were 32,500–37,500 and 22,000–24,000, respectively. The unreduced protein existed as disulfide-linked αβ species of molecular weight 53,000–58,000. Isoelectric points were approximately 5.9–7.2 (α) and 8.7–9.2 (β). Two-dimensional electrophoresis showed considerable heterogeneity within both groups of polypeptides. More complete amino acid analyses of the globulin and its polypeptides are presented along with a proposed structure of the native protein based on previous and present data. Similarities were noted between the oat globulin and the legumin (11 S) class of storage proteins in certain legumes.     

Mechanistic investigation of capability of enzymatically synthesized polycysteine to cross-link proteins

BackgroundPreviously, we had reported that α-chymotrypsin–catalyzed polymerization of l-cysteine ethyl ester in a frozen buffer provided poly-l-cysteine (PLCys) in good yield, of which degree of polymerization had been determined to be 6–11. Almost all of SH groups in PLCys were in free forms. Such a multi-thiol peptide may cross-link proteins through thiol/disulfide (SH/SS) exchange reactions, considering the knowledge that other synthetic multi-thiol additives changes properties of protein materials.MethodsThis study explored the capability of PLCys to cross-link proteins using lysozyme as a model protein which has four disulfide bonds but no free SH group. The protein was incubated with PLCys at neutral pH and at below 70 °C to avoid PLCys-independent, β-elimination-mediated cross-linkings. Protein polymerization was analyzed by SDS-PAGE and SEC. PLCys peptides involved in the protein polymer, which were released by reduction with dithiothreitol, were analyzed by RP-HPLC.ConclusionsAddition of urea and thermal treatment at 60 °C caused PLCys-induced lysozyme polymerization. Compared with free cysteine, a higher level of PLCys was required for the polymerization probably due to its low water solubility. RP-HPLC analyses suggested that PLCys played a role in the protein polymerization as a cross-linker.General significanceEnzymatically synthesized PLCys shows promise as a peptidic cross-linker for the production of protein polymers with novel physiochemical properties and functionalities.     

Ultrastructural localization of glycinin and β-conglycinin in Glycine max (soybean) cv. Maple Arrow by the immunogold method

Summary -Conglycinin (7S globulin) and glycinin (11S globulin) are the major reserve proteins of soybean. They were localized by the protein A immunogold method in thin sections of glycine max (soybean) cv. Maple Arrow. In cotyledons, both globulins were simultaneously present in all protein bodies. Statistical analysis of marking intensities indicated no correlation between globulin concentration and size of protein bodies. The immunogold method failed to detect either globulin in the embryonic axis and in cotyledons of four-day-old seedlings. Similar observations were made with cotyledons of two soy varieties lacking either the lectin or the Kunitz trypsin inhibitor. In another variety (T-102) lacking the lectin, the 7S globulin could not be detected.     

Sulfhydryl and Disulfide Patterns Associated with Vernalization of Wheat

Analyses of plumules from seedlings of a winter wheat variety (Elgin) and a spring wheat variety (Red Bobs) indicated that SH and SS contents based on protein levels were similar in the whiter and spring wheat tissues grown at 20 C. Boll) varieties, when grown nt 3 C, contained greater concentrations of SH and SS groups, based on protein levels, than those present in the tissues grown at 20 C. The spring wheat tissues at 3 C maintained relatively constant levels of SH and SS during the growth period. Sulfhydryl (SH) and SS groups based on fresh weights were present in greater quantities in the tissues of the spring wheat grown at 20 C than in the winter wheat tissues grown at 20 C. The high SH and SS concentrations present in the warm-grown spring wheat tissues were due to a high concentration of protein extracted from that tissue. The SH and SS contents per unit fresh weight were quite similar in the cold-grown tissues of both varieties during the growth period. Both varieties at both temperatures showed a similar decrease in SH and SS contents, based on fresh weights, as maturation progressed. The spring wheats grown at 3 C and 20 C and the winter wheat grown at 3 C all showed similar characteristics and differed considerably from the winter wheat grown at 20 C. These data were positively correlated with the flowering potential of the wheat varieties.     

Completely buried, non-ion-paired glutamic acid contributes favorably to the conformational stability of pyrrolidone carboxyl peptidases from hyperthermophiles

Pyrrolidone carboxyl peptidases (PCPs) from hyperthermophiles have a structurally conserved and completely buried Glu192 in the hydrophobic core; in contrast, the corresponding residue in the mesophile protein is a hydrophobic residue, Ile. Does the buried ionizable residue contribute to stabilization or destabilization of hyperthermophile PCPs? To elucidate the role of the buried glutamic acid in stabilizing PCP from hyperthermophiles, we constructed five Glu192 mutants of PCP-0SH (C142S/C188S, Cys-free double mutant of PCP) from Pyrococcus furiosus and examined their thermal and pH-induced unfolding and crystal structures and compared them with those of PCP-0SH. The stabilities of apolar (E192A/I/V) and polar (E192D/Q) mutants were less than PCP-0SH at acidic pH values. In the alkaline region, the mutant proteins, except for E192D, were more stable than PCP-0SH. The thermal stability data and theoretical calculations indicated an apparent pKa value > or = 7.3 for Glu192. Present results confirmed that the protonated Glu192 in PCP-0SH forms strong hydrogen bonds with the carbonyl oxygen and peptide nitrogen of Pro168. New intermolecular hydrogen bonds in the E --> A/D mutants were formed by a water molecule introduced into the cavity created around position 192, whereas the hydrogen bonds disappeared in the E --> I/V mutants. Structure-based empirical stability of mutant proteins was in good agreement with the experimental results. The results indicated that (1) completely buried Glu192 contributes to the stabilization of PCP-0SH because of the formation of strong intramolecular hydrogen bonds and (2) the hydrogen bonds by the nonionized and buried Glu can contribute more than the burial of hydrophobic groups to the conformational stability of proteins.     

The effect of interaction between human urokinase and its competitive inhibitor, N alpha-benzoyl-L-arginine amide, on reduction of a specific SS bond related to enzymatic activity

The 55- (H-UK) and 36-kDa forms (L-UK) of human urinary urokinase lost most of esterase activity toward acetyl-glycyl-L-lysine methyl ester upon reductive cleavage of 3 SS bonds with dithiothreitol in the presence of the competitive inhibitor, N alpha-benzoyl-L-arginine amide (BAA), bound to polyacrylyl azide with C16N3-arm (PAA) at 0.3 M guanidine, a threshold point of the native state where a protein-denaturating transition began. One of the 3 SS bonds was protected from reduction, with an unaltered activity, under the similar conditions except for replacement of BAA-PAA conjugate by glycine-PAA conjugate. This "specific" SS bond was reduced and, after the other SH groups produced were blocked with iodoacetamide (IAM), selectively reoxidized, which resulted in complete reactivation. The intact B-chain isolated from H-UK was completely inactivated when its specific SS bond was reduced and selectively alkylated with IAM after the other SH groups were reversibly blocked with 5, 5'-dithiobis (2-nitrobenzoic acid), which was finally removed. The results indicate that a single specific SS bond is essential for retaining a conformation necessary to activity exhibition.     

Crystal structure of soybean proglycinin A1aB1b homotrimer

Soybean glycinin is a member of the 11 S globulin family. The crystal structure of proglycinin was determined by X-ray crystallography at 2.8 A resolution with an R-factor of 0.199 and a free R-factor of 0.250. A trimer molecule was found in an asymmetric unit of crystals. The trimer model contains three A1aB1b subunits and comprises 1128 amino acid residues and 34 water molecules. The constituent protomers of the homo-trimeric protein are arranged around a 3-fold symmetry axis with dimensions of 95 Ax95 Ax40 A. The protomer model is composed of five fragments which correspond roughly to conserved regions based on the sequence alignment of various 11 S globulins. The core of the protomer consists of two jelly-roll beta-barrels and two extended helix domains. This structure of proglycinin is similar to those of canavalin and phaseolin belonging to the 7 S globulin family, strongly supporting the hypothesis that both 7 S and 11 S globulins are derived from a common ancestor. The inter and intra-chain disulfide bonds conserved in the 11 S globulin family are clearly observed. It is found that the face with the inter-chain disulfide bond (IE face) contains more hydrophobic residues than that with the intra-chain disulfide bond. This suggests that a mature hexamer is formed by the interaction between the IE faces after processing.     

Spider prey (Araneae) of Trypoxylon (Trypargilum) rogenhoferi (Hymenoptera: Sphecidae) in southeastern Brazil

Fifty five nests and 216 cells of Trypoxylon (Trypargilum) rogenhoferi were obtained from trap-nests (cut bamboo canes) in Santa Carlota Farm (Itaoca Section = IS and Santana Section = SS), Cajuru and on the S?o Paulo University Campus, Ribeir?o Preto (= RP), both in the State of S?o Paulo, Brazil (Sept. 1993-Oct. 1995). The prey (spiders) of 40 cells from IS, 58 from SS and 39 from RP were identified. The greatest nesting frequency occurred during the hot and wet season (September to April). T. rogenhoferi preyed upon individuals of five spider families, with Araneidae (orb-weaver spiders) being the most frequent (99.6%). Alpaida aff. negro (58%) was the most frequently collected species in IS, followed by A. alto (24.8%); in SS (59.6%) and RP (64.7%) the most frequent species was A. veniliae, followed in SS by A. aff. negro (14.9%) and in RP by A. leucogramma (13.5%). The size of reproductive niches, H' = 1.25 (IS), H' = 1.30 (SS) and H' = 1.29 (RP) were not significantly different. There was a positive correlation between the reproductive niche width (H') and evenness.     

The thiol groups of the Folch-Pi protein from bovine white matter. Exposure, reactivity and significance.

The number and the reactivity of accessible thiol groups of the Folch-Pi apoprotein and proteolipid (50% of myelin proteins) were studied, by using a specific thiol-disulphide interchange reaction, in connection with the known solubility of this protein in organic and aqueous solvents. The high reactivity of 2,2'-dipyridyl disulphide towards thiol groups leads to the titration of 4.8 mol of SH groups/mol of protein (Mr 30000) in alkaline and acidic chloroform/methanol (2:1, v/v). Unlike previous findings, this value was consistently found from batch to batch and remained stable with time. In the proteolipid 1 mol of SH groups/mol was not accessible as compared with the apoprotein. In aqueous solvents, a similar number of 4.4 mol of SH groups/mol was also found. For the first time, kinetic studies carried out in chloroform/methanol discriminated between two classes of thiol groups. The reaction of 2 mol of SH groups/mol was characterized by apparent second-order rate constants whose values were 5-10-fold higher than those of the other class. Kinetic studies and cyanylation experiments in aqueous solvents also indicated the high reactivity of these thiol groups with Ellman's reagent. Together with kinetic results, studies on the stoichiometry of the interchange reaction of equimolar solutions of protein and disulphide indicate that these highly reactive thiol groups are near to each other in the amino acid sequence. The location of the thiol groups at the boundary between hydrophilic and hydrophobic domains of the Folch-Pi protein is suggested in connection with their possible structural and biological significance.     

In vitro folding of methionine‐arginine human lyspro‐proinsulin S‐sulfonate—Disulfide formation pathways and factors controlling yield

We investigated the in vitro folding of an oxidized proinsulin (methionine‐arginine human lyspro‐proinsulin S‐sulfonate), using cysteine as a reducing agent at 5°C and high pH (10.5–11). Folding intermediates were detected and characterized by means of matrix‐assisted laser desorption ionization mass spectrometry (MALDI‐MS), reversed‐phase chromatography (RPC), size‐exclusion chromatography, and gel electrophoresis. The folding kinetics and yield depended on the protein and cysteine concentrations. RPC coupled with MALDI‐MS analyses indicated a sequential formation of intermediates with one, two, and three disulfide bonds. The MALDI‐MS analysis of Glu‐C digested, purified intermediates indicated that an intra‐A‐chain disulfide bond formed first among A6, A7, and A11. Various non‐native intra‐A (A20 with A6, A7, or A11), intra‐B (between B7 and B19), and inter‐A‐B disulfide bonds were observed in the intermediates with two disulfide bonds. The intermediates with three disulfide bonds had mainly the non‐native intra‐A and intra‐B bonds. At a cysteine‐to‐proinsulin‐SH ratio of 3.5, all intermediates with the non‐native disulfide bonds were converted to properly folded proinsulin via disulfide bond reshuffling, which was the slowest step. Aggregation via the formation of intermolecular disulfide bonds of early intermediates was the major cause of yield loss. At a higher cysteine‐to‐proinsulin‐SH ratio, some intermediates and folded MR‐KPB‐hPI were reduced to proteins with thiolate anions, which caused unfolding and even more yield loss than what resulted from aggregation of the early intermediates. Reducing protein concentration, while keeping an optimal cysteine‐to‐protein ratio, can improve folding yield significantly. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010