Specific Disulfide Bond Research Articles

Multiplicity and Species Differences of Carboxylesterase Isozymes in Mammals and Humans.

Liver microsomal carboxylesterases (E. C. 3.1.1.1) function in the hydrolysis of a wide variety of endogenous and xenobiotic compounds, and play an important role in drug and lipid metabolism in many mammalian species. Multiple isozymes of liver microsomal carboxylesterases exist in various animal species, and the enzyme is involved in the metabolic activation of ester and amide-type prodrugs. In the present study, twenty-seven carboxylesterase isozymes were purified to electrophoretic homogeneity from liver microsomes of ten mammalian species and humans, and their physical, enzymological and immunological properties were compared with each other. And also we tried to compare the primary structure of carboxylesterase isozymes in various animal species and humans using cDNA cloning and analysis. The carboxylesterase isozymes from various species examined here showed considerable similarities in physicochemical and immunochemical properties, but not similar in substrate specificity. The deduced amino acid sequence of the clone possessed many structural characteristics that are highly conserved among rat RLl, RH1, RHlec, RS1, RS2, mouse ML1, MH1, MS1, guinea pig GP, beagle dog D1 and human HU1, including active site sequence (GESAGG, NKQEXG, GDHXD), and four cysteines that may be involved in the specific disulfide bond. It is well known that proteins that are retained in the endoplasmic reticulum (ER) lumen contain the retention signal at their carboxy terminal of the tetrapeptide (KDEL-COOH). The seven carboxylesterase clones (RL1, RH1, RHlec, MH1, GP, Dl and HUI) also contained an ER-retention signal of carboxylesterase (HXEL), and three clones (RS1, RS2, MS1) did not contain this peptide. When clone was expressed in COST and V79 cells, the plasmid-coded protein was retained. The cells expressing carboxylesterase is very high activity towards xenobiotic ester and amide. Site-specific mutation of the three amino acid residues of catalytic triad (Ser203 to Thr203, Glu335 to Ala 335, or His 448 to A1a448) greatly reduced the carboxylesterase activity. In conclusion, liver microsomal carboxylesterase in mammals and humans are closely involved in drug and lipid metabolism in the endoplasmic reticulum, and it is noteworthy that the isozymes from various species examined here showed considerable similarities in amino acid sequences, but not similar in substrate specificity. These reasons may be due to the variances of substrate binding site.

Identification of Cysteine Pairs within the Amino-terminal 5% of Apolipoprotein B Essential for Hepatic Lipoprotein Assembly and Secretion

There is growing evidence that the amino-terminal globular domain of apolipoprotein B (apoB) is essential for lipoprotein particle formation in the hepatic endoplasmic reticulum. To identify the structural requirements for its function in lipoprotein assembly, cysteine (Cys) pairs required to form the seven disulfide bonds within the amino-terminal 21% of apoB were replaced in groups or individually by serine. Substitution of Cys pairs required for formation of disulfide bonds 1-3 or 4-7 (numbered from amino to carboxyl terminus) completely blocked the secretion of apoB28 in transfected HepG2 cells. To identify the specific disulfide bonds required for secretion, Cys pairs were mutated individually. Substitution of Cys pairs required for disulfide bonds 1, 3, 5, 6, or 7 had little or no impact on apoB28 secretion or buoyant density. In contrast, individual substitution of Cys pair 2 (amino acid residues 51 and 70) or 4 (218 and 234) severely inhibited apoB28 secretion and its capacity to undergo intracellular assembly with lipid. The same assembly and secretion defects were observed when these mutations were expressed as part of apoB50. These studies provide direct evidence that the ability of the internal lipophilic regions of apoB to engage in the recruitment and sequestration of lipid during translation is critically dependent upon a structural configuration contained within or affected by the amino-terminal 5% of the protein.

Analysis of the mechanism of lipoprotein(a) assembly.

We have assessed the ability of a battery of purified recombinant apolipoprotein(a) (r-apo(a)) derivatives to bind to immobilized low-density lipoprotein (LDL) by ELISA. Removal of the apo(a) kringle IV type 8 and type 9 sequences dramatically reduced apo(a) binding to LDL. The binding of apo(a) to LDL was effectively inhibited by arginine, lysine, the lysine analogue epsilon-aminocaproic acid and proline; comparable inhibition was observed using the 17K and KIV5-8 r-apo(a) derivatives, suggesting a direct role for sequences contained in the latter species in mediating the initial non-covalent interactions which precede specific disulfide bond formation. We also determined that r-apo(a) binds directly to a synthetic apoB peptide spanning amino acid residues 3732-3745; this interaction appeared to be mediated by sequences present in apo(a) kringle IV types 8 and 9, and could be inhibited by arginine, lysine and proline. The results of this study indicate that the efficiency of Lp(a) assembly is a direct function of the initial non-covalent interactions between apo(a) and LDL; in addition, these studies suggest that Cys3734 in apoB mediates covalent linkage with apo(a) by virtue of the ability of the apoB sequences surrounding this residue to directly interact with apo(a) KIV type 9.

Alteration of N-linked oligosaccharide structures of human chorionic gonadotropin beta-subunit by disruption of disulfide bonds.

The human chorionic gonadotropin beta-subunit (hCGbeta) is a glycoprotein in which 12 cysteine residues pair to form six intramolecular disulfide bonds. In order to elucidate the effect of each disulfide bond on glycosylation of the molecule, we analysed structures of asparagine-linked oligosaccharides of various recombinant hCGbeta produced in Chinese hamster ovary (CHO) cells: wild-type hCGbeta (betaWT) and mutants in which any one of the six intramolecular disulfide bonds had been disrupted by site-directed mutagenesis. SDS-PAGE analysis of betaWT and these mutants before and after digestion with endoglycosidase F and H revealed structural changes in the oligosaccharide moieties of some mutants. In addition, structural analysis of oligosaccharides obtained from metabolically labeled betaWT and a mutant showed that the mutant contained additional high mannose type oligosaccharides. These results suggest that elimination of a specific disulfide bond, resulting in a change in the protein conformation, disturbs the normal assembly of the mature complex type oligosaccharides in the hCGbeta molecule.

Specific Disulfide Bond in α‐Lactalbumin Infiuences Heat‐ Induced Gelation of α‐Lactalbumin‐Ovalbumin‐Mixed Gels

ABSTRACTThe gel strength of ovalbumin mixed with α‐lactalbumin (α‐La) was determined after heating at 80°C for 15 min at pH 7.0 Gel strength of the mixture of 4%α‐La and 4% ovalbumin was two times that of 8% ovalbumin. Modified α‐La at Cys6‐Cys120 (3 SSα‐La) had low enhancement effects on ovalbumin gelation. Competitive ELISA using monoclonal antibody to α‐La showed decreased binding reactivities after heating α‐La with ovalbumin at specific concentrations. The decrease of total SH groups in the gel mixed with 3SS α‐La was much less than when mixed with α‐La. A disulfide bond of Cys6‐Cys120 in α‐La contributed to interactions between ovalbumin and α‐La in heat‐induced gels.

Multiplicity of Drug Metabolizing Enzyme Isoforms in Relation to their Functions.

Liver microsomal carboxylesterases (CES) function in the hydrolysis of a wide variety of endogenous and xenobiotic compounds, and play an important role in drug and lipid metabolism in many mammalian species. For past three decades, we have undertaken the diverse studies on CES from the view points of enzymology and drug metabolism. First, we have purified twenty-seven CES isozymes to electrophoretic homogeneity from liver microsomes of ten mammalian species and humans, and their physical, enzymological and immunological properties were compared each other. The CES isozymes from various species examined showed considerable similarities in physicochemical and immunological properties, but not similar in substrate specificities. Ramda Zap II library from mouse liver, and λ gt 11 libraries from human and rat liver were screened with antibodies raised to a purified liver CES isozymes. The deduced amino acid sequence of the clone possessed many structural characteristics that are highly conserved among rat RL1, RH1, RHlec, RS1 and RS2, mouse ML1, MH1, MS1 and human HU1, including active site sequence (GESAGG, NKQEXG, GDHXD), and four cysteines which may be involved in the specific disulfide bond. It is well known that proteins which are retained in the endoplasmic reticulum lumen contain the retention signal at their carboxyl-terminal of the tetrapeptide. The five CES clones (RL1, RH1, RHlec, MH1 and HU1) also contained ER-retention signal (HXEL). When clone was expressed in COS cells, the plasmid-coded protein was retained. The cells expressing CES were found to have very highly activity towards xenobiotic esters and amides. In conclusion, liver microsomal CES in mammalian and humans are closely involved in drug and lipid metabolism in the endoplasmic reticulum, and it is noteworthy that the isozymes from various species examined showed considerable similarities in amino acid sequences, but not similar in substrate specificities. These reasons may be, at least in part, due to the variances of substrate binding site.

Inhibition of human cytomegalovirus UL80 protease by specific intramolecular disulfide bond formation.

A symmetrically substituted disulfide compound, CL13933, was identified as a potent inhibitor of human cytomegalovirus UL80 protease. Two types of inhibited protease were observed, depending on inhibitor concentration. At high concentrations, CL13933 formed a covalent adduct with the protease on Cys residues. At lower concentrations, this compound induced specific intramolecular disulfide formation between Cys84 and Cys87, and between Cys138 and Cys161. In contrast, Cys202 did not form disulfide bonds. Inhibition was reversed upon reduction of the protease. Each of the five cysteines of the UL80 protease was individually mutated to Ala. Each of the mutant proteases retained enzymatic activity, but mutants C138A and C161A were resistant to inhibition by CL13933, suggesting that disulfide bond formation between Cys138 and Cys161 is responsible for inhibition. This disulfide is apparently not induced by air oxidation. Examination of the CL13933 loading patterns of wild type and the five mutant proteases by mass spectrometry revealed that residues Cys87, Cys138, and Cys161 react with CL13933, and that the disulfide pair partner of each (Cys84, Cys161, and Cys138, respectively) is able to displace the compound via thiol-disulfide exchange. The possible significance of these reactive thiols in the protease is discussed.

Rotation of subunits during catalysis by Escherichia coli F1-ATPase.

During oxidative and photo-phosphorylation, F0F1-ATP synthases couple the movement of protons down an electrochemical gradient to the synthesis of ATP. One proposed mechanistic feature that has remained speculative is that this coupling process requires the rotation of subunits within F0F1. Guided by a recent, high-resolution structure for bovine F1 [Abrahams, J. P., Leslie, A. G., Lutter, R. & Walker, J. E. (1994) Nature (London) 370, 621-628], we have developed a critical test for rotation of the central gamma subunit relative to the three catalytic beta subunits in soluble F1 from Escherichia coli. In the bovine F1 structure, a specific point of contact between the gamma subunit and one of the three catalytic beta subunits includes positioning of the homolog of E. coli gamma-subunit C87 (gamma C87) close to the beta-subunit 380DELSEED386 sequence. A beta D380C mutation allowed us to induce formation of a specific disulfide bond between beta and gamma C87 in soluble E. coli F1. Formation of the crosslink inactivated beta D380C-F1, and reduction restored full activity. Using a dissociation/reassembly approach with crosslinked beta D380C-F1, we incorporated radiolabeled beta subunits into the two noncrosslinked beta-subunit positions of F1. After reduction of the initial nonradioactive beta-gamma crosslink, only exposure to conditions for catalytic turnover results in similar reactivities of unlabeled and radiolabeled beta subunits with gamma C87 upon reoxidation. The results demonstrate that gamma subunit rotates relative to the beta subunits during catalysis.

Thymus peptides limits myocardium injury during stress reply at the mature rats

Mutant forms of kinin B1 receptor (B1R) and analogs of the full agonist des-Arg9-bradykinin (DABK) were investigated aiming to verify the importance of selected receptor residues and of each agonist-peptide residue in the specific binding and activation. Linked by a specific disulfide bond (Cys100–Cys650), the N-terminal (Nt) and the EC3 loop C-terminal (Ct) segments of angiotensin II (AngII) receptor 1 (AT1R) have been identified to form an extracellular site for binding the agonist Nt segment (Asp1 and Arg2 residues). Asp712 residue at the receptor EC3 loop binds the peptide Arg2 residue. By homology, a similar site might be considered for DABK binding to B1R since this receptor contains the same structural elements for composing the site in AT1R, namely the disulfide bond and the EC3 loop Asp712 residue. DABK, Alan-DABK analogs (n = Ala1-, Ala2-, Ala3-, Ala4-, Ala5-, Ala6-, Ala7-, Ala8-DABK), and other analogs were selected to binding wild-type, Asp712Ala and Cys100Ser mutated B1R receptors. The results obtained suggested that the same bimodal scheme adopted for AngII-AT1R system may be applied to DABK binding to B1R. The most crucial similarity in the two cases is that the Nt segments of peptides equally bind to the homologous Asp712 residue of both AT1R and B1R extracellular sites. Confirming this preliminary supposition, mutation of residues located at the B1R extracellular site as EC3 loop Asp712 and Cys100 caused the same modifications in biological assays observed in AT1R submitted to homologous mutations, such as significant weakening of agonist binding and reduction of post-receptor-activation processes. These findings provided enough support for defining a site that determines the specific binding of DABK to B1R receptors.

2] Thiol/disulfide exchange equilibria and disulfidebond stability

Publisher Summary Disulfide-bond formation is a versatile oxidation used biologically in diverse processes, such as enzyme catalysis, protection against oxidative damage, stabilization of extracellular proteins, and regulation of biological activity. Because disulfide formation is a reversible process, disulfide-bond stability often plays an important role in the biological utility of disulfide bonds. In turn, the ability to form and break a specific disulfide bond under appropriate biological conditions depends on the nature of the oxidant or reductant, the disulfide stability, the kinetics of the forward and reverse reactions, and the nature and redox state of the environment in which the reaction occurs. The stability of disulfide bonds in small molecules and proteins spans an enormous range, a factor of approximately 1011, corresponding to a free-energy difference of about 15 kcal/mol or a redox potential difference of 0.33 V. This chapter outlines the importance of reversible thiol/disulfide exchange and discusses some practical considerations in measuring disulfide bond stability.

Specific disulfide cleavage is required for ubiquitin conjugation and degradation of lysozyme.

Both ubiquitin conjugation and ubiquitin-dependent degradation of chicken egg white lysozyme in a reticulocyte lysate depend on the presence of a reducing agent. We present evidence that the reduction of a specific disulfide bond, namely that at Cys6-Cys127, facilitates ubiquitination and is a prerequisite to the formation of a multiubiquitin chain on one of at least four chain initiation sites on lysozyme. The Cys6-Cys127 disulfide bond in lysozyme can be specifically reduced, and the modified protein can be isolated after carboxymethylation of the 2 resulting cysteines. This modified lysozyme no longer requires the presence of a reducing agent for ubiquitin conjugation and degradation. Inhibition of ubiquitination by the dipeptide Lys-Ala revealed that this modified lysozyme, like the unmodified protein, is recognized via the binding of the ubiquitin protein ligase, E3, to the substrate's N-terminal lysyl residue. Both the rate and the extent of ubiquitin-lysozyme conjugation, however, are significantly higher with this modified substrate. Likewise, ubiquitin-dependent degradation of 6,127-reduced/carboxymethylated lysozyme was 2-4-fold faster than degradation of the unmodified counterpart. These results are consistent with an interpretation that the modified lysozyme mimics an intermediate formed at the rate-limiting step of the degradation of lysozyme in the reticulocyte lysate. Reduction of the Cys6-Cys127 disulfide bond is expected to unhinge the N-terminal region of lysozyme, and we propose that the recognition of this otherwise stable protein by the ubiquitin pathway is due to facilitated binding of E3 that results from such a conformational transition.

Subunit interaction sites between the regulatory and catalytic subunits of cAMP-dependent protein kinase. Identification of a specific interchain disulfide bond.

The catalytic (C) subunit and the type II regulatory (RII) subunit of cAMP-dependent protein kinase can be cross-linked by interchain disulfide bonding. This disulfide bond can be catalyzed by cupric phenanthroline and also can be generated by a disulfide interchange using either RII-subunit or C-subunit that has been modified with either 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) or N-4(azidophenylthio)phthalimide (APTP). When the 2 cysteine residues of the C-subunit are reacted with DTNB prior to incubation with the RII-subunit, interchain disulfide bonding occurs. Similar observations are seen with C-subunit that had been modified with APTP. Interchain disulfide bonds also form when the RII-subunit is modified with DTNB prior to incubation with the C-subunit. The presence of cAMP facilitates this cross-linking while autophosphorylation of the RII-subunit retards the rate at which the interchain disulfide bond forms. Interchain disulfide bonds also form spontaneously when the RII-subunit and the C-subunit are dialyzed at pH 8.0 in the absence of reducing agents. The specific amino acid residues that participate in intersubunit disulfide bonding have been identified as Cys-97 in the RII-subunit and Cys-199 in the C-subunit. Based on the sequence homologies of the RII-subunit with other kinase substrates and on the proximity of Cys-97 to the catalytic site, a model is proposed in which the autophosphorylation site of the RII-subunit occupies the substrate-binding site in the holoenzyme. The model also proposes that this same site may be occupied by the region flanking Cys-199 in the C-subunit when the C-subunit is dissociated.

A specific disulfide bond associated with the activity of human urokinase. Its topological identification and reductive cleavage followed by kinetic changes in enzymatic reaction and affinity labeling.

A single SH group in the B chain (33 kDa), generated by the specific reduction of the single interchain SS bond of human urinary urokinase, was alkylated (UK X B) with iodoacetamide to prevent a spontaneous SH-SS interchange. An SS bond in UK X B was exclusively alkylated with iodoacetamide (R X CAM-UK X B) after reduction with dithiothreitol in 0.3 M guanidine X HCl in the presence of the competitive inhibitor N alpha-benzoyl-L- argininamide with concomitant loss of 65-68% of the esterolytic activity towards N-acetyl-glycyl-L-lysine methyl ester. This specific SS bond was located at Cys194 - Cys222 whose SS loop contained the active-site Ser198 , as determined by amino acid analyses and identification of the N and C termini of the tryptic digest. Transformation of UK X B into R X CAM-UK X B induced no shift of the optimal pH in the bell-shaped pH/activity profile; pH values for 50% activity were similar (pH 9.7) for 10-min alkalization of the enzyme but different between UK X B (pH 9.4) and R X CAM-UK X B (pH 8.8) for 18-h alkalization. An unaltered Km value and a decline by 64% in kcat in the esterolytic activity indicate that the pretransition Michaelis complex is formed without degeneration of the primary substrate-binding site, but the catalytic pathway thereafter has deteriorated. In affinity labeling with dansyl chloride or N alpha-tosyl-L-lysine chloromethylketone, which interrupted the catalysis at the latest at a stage involving the abortive acyl intermediate, the second-order rate constant for UK X B was lowered to 28% or 35% for R X CAM-UK X B, respectively, but the labeling yields were similar. The results indicate that indispensable structural elements, such as the catalytic triad and oxyanion hole, are maintained but a local conformation, which is necessary for efficient transition to the acyl intermediate and/or for resistance against alkaline inactivation, is destabilized with Cys194 - Cys222 scission.

Activation of the frog sartorius acetylcholine receptor by a covalently attached group

The frog sartorius motor endplate was treated with the specific disulfide bond reducing agent dithiothreitol and subsequently exposed to a covalently reacting compound (the nitrophenyl ester of p-carboxyphenyltrimethylammonium iodide, NPTMB) known to activate the dithiothreitol-reduced acetylcholine receptor in Electrophorus electroplax. NPTMB causes a maximum depolarization of about 35 mV when applied to the dithiothreitol-treated sartorious motor endplate. It is ineffective on postjunctional membrane prior to disulfide bond reduction and on extrajunctional regions, reduced or unreduced. High concentrations of a competitive antagonist such as (+)-tubocurarine prevent reaction between NPTMB and the reduced receptor and cause a repolarization of the membrane when applied to the already-depolarized preparation. We conclude that in frog muscle, as in electroplax, the attached activator bridges the acetylcholine binding site of the reduced receptor between a sulfhydryl group, to which it is covalently bound, and a negative subsite, with which it forms a reversible ionic band.

Cleavage of One Specific Disulfide Bond in Papain

Abstract Conditions leading to cleavage of all of the disulfide bridges in ribonuclease and several other proteins (0.32 m 2-mercaptoethanol in 8 m urea) caused in papain only partial reduction of the disulfide bonds. Electrophoretic studies indicated that alkylation of the partially reduced derivative yielded a unique molecular species (3-RCM papain) in which one specific disulfide bond had been split, rather than a mixture of unreduced and open chain papain. On the other hand, reduction of papain in 6 m guanidine hydrochloride, followed by alkylation, yielded a completely open polypeptide chain (7-RCM papain). Alkylation of native papain after reduction in aqueous solution resulted in the sole blocking of the active sulfhydryl group (1-RCM papain). Immunological interactions with antipapain serum indicated identity between 1-RCM papain and native papain, whereas 7-RCM papain was completely unreactive. 3-RCM papain cross-reacted with 60% of the antibodies. The single cleaved disulfide bond in 3-RCM papain was identified by isolation and analysis of the peptides containing carboxymethylcysteine, obtained from a tryptic digest. It was shown to be the bond connecting the cysteine residues in positions 43 and 152 in the amino acid sequence of papain. It was also shown that during the reduction of native papain in 8 m urea autodigestion takes place. This phenomenon was avoided when 1-RCM papain was reduced instead of the native enzyme; in both cases the product of reduction and alkylation was 3-RCM papain.