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PubMed1992

Assembly of mutant subunits of the nicotinic acetylcholine receptor lacking the conserved disulfide loop structure.

Each subunit of the nicotinic acetylcholine receptor (AChR) contains two conserved cysteine residues, which are known to form a disulfide bond, in the N-terminal extracellular domain. The role of this retained structural feature in the biogenesis of the AChR was studied by expressing site-directed mutant alpha and beta subunits together with other normal subunits from Torpedo californica AChR in Xenopus oocytes. Mutation of the cysteines at position 128 or 142 in the alpha subunit, or in the beta subunit, did not prevent subunit assembly. All Cys128 and Cys142 mutants of the alpha and beta subunits were able to associate with coexpressed other normal subunits, although associational efficiency of the mutant alpha subunits with the delta subunit was reduced. Functional studies of the mutant AChR complexes showed that the mutations in the alpha subunit abolished detectable 125I-alpha-bungarotoxin (alpha-BuTX) binding in whole oocytes, whereas the mutations in the beta subunit resulted in decreased total binding of 125I-alpha-BuTX and no detectable surface 125I-alpha-BuTX binding. Additionally, all mutant subunits, when co-expressed with the other normal subunits in oocytes, produced small acetylcholine-activated membrane currents, suggesting incorporation of only small numbers of functional mutant AChRs into the plasma membrane. The functional acetylcholine-gated ion channel formed with mutant alpha subunits, but not mutant beta subunits, could not be blocked by alpha-BuTX. Thus, a disulfide bond between Cys128 and Cys142 of the AChR alpha or beta subunits is not needed for acetylcholine-binding. However, this disulfide bond on the alpha subunit is necessary for formation of the alpha-BuTX-binding site. These results also suggest that the most significant effect caused by disrupting the conserved disulfide loop structure is intracellular retention of most of the assembled AChR complexes.

Animals↗
PubMed2005

[Soluble expression and characterization of disulfide bond-rich subdomains of membrane protein p185 in Escherichia coli].

Transmembrane protein p185 (the product of Her2/c-erbB-2 gene) is a member of the epidermal growth factor receptor (EGFR) family. Its overexpression was found in about 30% of breast cancer. It is essential to obtain soluble extracellular domain (ECD) of p185, especially disulfide bond rich domains, for identifying the epitopes of anti-p185 antibodies and researching the interrelationship between the antigen and antibody. The disulfide bond rich domain I-II and domain IV of p185 ECD were amplified from plasmid pBabe/erbB-2 by PCR respectively. These two fragments were inserted into pGEX/4T-1 vector, transfected into E. coli Origami B (DE3) pLysS and expressed inductively by low concentration of IPTG and low temperature overnight. After the pressure lysis of cells, the supernatants were analyzed by SDS-PAGE and the result demonstrated that this GST-fusion protein was expressed solubly in the amount of 10-15 mg/L. By the ELISA, Western blot and other immunological assays, the fusion proteins and their GST cut-off derivates both showed binding activities with several anti-p185 antibodies respectively. These results indicated that it was a feasible and effectual method to express disulfide bond rich domain I-II and domain IV of p185 ECD and this method may also be used to express other disulfide bond rich proteins.

Antibodies, Monoclonal↗
PubMed1991

Purification and characterization of a new isozyme of thiol:protein-disulfide oxidoreductase from rat hepatic microsomes. Relationship of this isozyme to cytosolic phosphatidylinositol-specific phospholipase C form 1A.

Thiol:protein-disulfide oxidoreductase catalyzes the GSH reduction of protein disulfides to sulfhydryls. Chromatography of solubilized hepatic microsomes on Mono Q yielded two peaks, Q-2 and Q-5, which contained all the thiol:protein-disulfide oxidoreductase activity. These were further purified by chromatofocusing giving specific activities of 14.4 and 45.9 nmol/mg of protein/min, respectively with purifications of 45.0- and 143.6-fold. Amino acids 1-18 of Q-5 were the same as previously reported for Thiol:protein-disulfide oxidoreductase (Edman, J. C., Ellis, L., Blacher, R. W., Roth, R. A., and Rutter, W. J. (1985) Nature 317, 267-270), except amino acid 1 was leucine instead of aspartate and amino acid 6 was asparagine instead of glutamate. The N-terminal amino acid sequence of Q-2 differed markedly from Q-5 but Q-2 showed 100% identity at amino acids 25-54, 258-269, 285-310, 347-350, 412-419, and 434-463 for the reported sequence of rat, hepatic, cytosolic phosphatidylinositol-specific phospholipase C form 1a (PLC) (Bennett, C. F., Balcarek, J. M., Varrichio, A., and Crooke, S. T. (1988) Nature 334, 268-270). PLC activity was found in the elution from the Mono Q column, but none was found in purified Q-2 or Q-5. Antibodies to Q-5 reacted with Q-2, but anti-Q-2 did not react with Q-5. Anti-Q-2 antibody showed immunoreactivity with 55- and 60-kDa microsomal proteins, whereas Q-5 antibody reacted with a number of microsomal proteins. Although Q-2 was immunoreactive with a polyclonal antibody to guinea pig, uterine cytosolic PLC, partially purified PLCs from rat liver cytosol did not react to this antibody. Our data would suggest that the published sequence for PLC form 1a may actually be the sequence for Q-2.

Amino Acid Sequence↗
PubMed1991

Reduction of disulfide bonds within lysosomes is a key step in antigen processing.

Reduction of disulfide bonds is a key step in antigen processing both to allow the unfolding of protein antigens, increasing the access of proteolytic processing enzymes, and to expose free Cys residues within linear peptide epitopes recognized by T cells. We show here that reduction and alkylation of Ag (hen egg lysozyme and ribonuclease A) vastly increased their proteolysis (by specific enzymes or lysosomal fractions) and the production of specific immunogenic peptides that bound to class II MHC molecules recognized by T hybridoma cells. We also show that the lysosome is the vesicular compartment that mediates protein disulfide reduction. We coupled [125I]tyrosine to 131I-alpha 2-macroglobulin or [131I] transferrin via a reducible disulfide linker. Removal of [125I]tyrosine from the alpha 2-macroglobulin conjugate was initiated only after 15 to 20 min of uptake by macrophages, suggesting that reduction occurred late in the endocytic pathway. No reduction of transferrin conjugates was seen, indicating that early, recycling endosomes did not contain reducing activity. Subcellular fractionation showed that the disulfide bonds were reduced only in heavy density (lysosome) fractions and remained intact in fractions of light density (endosomes and plasma membrane). These results indicate the importance of lysosomes in the biochemical processing of protein Ag presented to T cells.

Animals↗
PubMed1991

Agonist binding site of Torpedo electric tissue nicotinic acetylcholine receptor. A negatively charged region of the delta subunit within 0.9 nm of the alpha subunit binding site disulfide.

The positively charged quaternary ammonium group of agonists of the nicotinic acetylcholine (ACh) receptor binds to a negative subsite at most about 1 nm from a readily reducible disulfide. This disulfide is formed by alpha Cys192 and Cys193 (Kao and Karlin, 1986). In order to identify Asp or Glu residues that may contribute to the negative subsite, we synthesized S-(2-[3H]glycylamidoethyl)dithio-2-pyridine. Purified ACh receptor from Torpedo californica was mildly reduced and reacted with S-(2-[3H]glycylamidoethyl)dithio-2-pyridine. The predominant product was a mixed disulfide between the 3H-N-glycylcysteamine moiety and alpha Cys192 or Cys193. In the extended conformation of [3H] N-glycylcysteamine, the distance from the glycyl amino group to the cysteamine thio group is 0.9 nm. Thus, the amino group of disulfide-linked [3H]N-glycylcysteamine could react with carboxyls within 0.9 nm of Cys192/Cys193. To promote amide bond formation between the tethered amino group and receptor carboxyls, we added 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide. The predominant sites of amide coupling were on the delta subunit, in CNBr fragment 4 (delta 164-257). This reaction was inhibited by ACh. Only the first 61 residues of delta CNBr 4 are predicted to be extracellular, and there are 11 Asp or Gly residues in this region. One or more of these residues is likely to contribute to the binding of ACh.

Amino Acid Sequence↗
PubMed1991

Role of a disulfide bond in the thermal stability of the LamB protein trimer in Escherichia coli outer membrane.

In order to understand the unusual heat resistance of LamB protein (the outer membrane component of the maltose transport system in Escherichia coli and its receptor for bacteriophage lambda), we investigated the role of its 2 cysteinyl residues. Our studies show that Cys22 and Cys38 form an intrasubunit disulfide bond which contributes to the heat stability of the LamB protein trimer. Physical evidence for the disulfide was obtained by using site-directed mutagenesis to convert Asn36 to Met, which allowed cyanogen bromide cleavage between the 2 cysteines. Upon reduction one of the N36M fragments migrated as two pieces, resolved by two-dimensional polyacrylamide gel electrophoresis. Other mutagenized LamB proteins, in which 1 or both Cys residues were converted to Ser, exhibited a sharp loss of thermal stability. In contrast to wild-type LamB protein trimer, which does not dissociate to monomers even after 60 min at 100 degrees C, only 10-15% of the mutant LamB proteins remain trimeric after boiling 10 min. The disulfide bond in LamB protein is not required for its transport function, since both mutagenized LamB protein and N-ethylmaleimide-labeled LamB protein exhibit normal uptake of sugars in proteoliposomes. Finally, the disulfide bond must not be between subunits of the LamB trimer since reversible dissociation of trimer is achieved by low pH or denaturants in the absence of reducing agent.

Amino Acid Sequence↗
PubMed1991

Mimicking the active site of protein disulfide-isomerase by substitution of proline 34 in Escherichia coli thioredoxin.

To mimic the active sites (Trp-Cys-Gly-His-Cys) contained in two thioredoxin-like domains of the eukaryotic enzyme protein disulfide-isomerase (PDI, EC 5.3.4.1), the Pro-34 residue of Escherichia coli thioredoxin (Trx) was replaced by His using site-directed mutagenesis. The mutant P34H Trx was isolated in high yield and was stable. The equilibrium between Trx and NADPH in the thioredoxin reductase (TR)-catalyzed reaction revealed that the redox potential (E'o) or P34H Trx at pH 7.0 was -235 mV as compared with -270 mV for wild type (wt) Trx. The higher E'o value made P34H Trx more similar to PDI and contributed to prominent changes in Trx functions, e.g. improved activity with TR and slower reduction of protein disulfides. Compared to wt Trx, the P34H oxidized Trx was about twice as good a substrate for TR from E. coli and four times as efficient with calf thymus TR. A novel fluorimetric assay permitted direct recording of the reaction between insulin disulfide(s) and reduced Trx. At pH 8 and 15 degrees C, second-order rate constants for wt Trx of 2 x 10(4) M-1 s-1 and for P34H Trx of 3 x 10(3) M-1 s-1 were obtained, and a different equilibrium was observed consistent with differences in E'o values. Also when the reduction mechanism of insulin was examined using NADPH and TR, P34H Trx behaved differently from wt Trx or PDI. P34H Trx may be useful as an analogue of PDI for disulfide formation in vivo and in vitro.

Amino Acid Sequence↗
PubMed1991

The integrity of the cysteine 186-cysteine 209 bond of the second disulfide loop of tissue factor is required for binding of factor VII.

The structural basis of function of tissue factor (TF), the cell surface receptor and cofactor for the serine protease factor VIIa, cannot be inferred from the primary sequence. The functional significance of the two disulfide bonded loops in the surface domain of TF has been analyzed using site-directed mutagenesis to selectively preclude covalent stabilization of these loops by pairwise substitution of serine residues for cysteines. Mutant TF lacking either the amino (TFS49S57) or carboxyl (TFS186S209) disulfide bond were expressed on the surface of cells consistent with proper processing. Each reacted with a panel of monoclonal antibodies further suggesting proper global folding of the mutant proteins. TFS186S209 exhibited a selective decrease in reactivity with an antibody directed against one epitope locus in the carboxyl aspect of the surface domain of TF. Whereas TFS49S57 was functionally comparable to the wild type protein, TFS186S209 was functionally 30-40-fold less effective, and the affinity of factor VIIa binding to this mutant was indirectly estimated to be diminished 20-fold. These data suggest that the Cys186-Cys209 disulfide bond is required to maintain conformation and implicate the disulfide loop or adjacent structures in the carboxyl half of the surface domain of TF in receptor function.

Animals↗
PubMed1991

Identification of the disulfide-linked homodimer of apolipoprotein E3 in plasma. Impact on receptor binding activity.

The nature of disulfide-linked structures of apolipoprotein (apo) E3 in the plasma of E3/3 subjects was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis performed under nonreducing conditions followed by immunoblotting with apoE-specific antibodies. In addition to the expected presence of the heterodimer apoE3-A-II and monomeric apoE3, a band with an apparent Mr approximately 100,000 was also observed in plasma that had been treated with sulfhydryl-trapping reagents. This band and apoE3-A-II were both eliminated by disulfide reduction, which produced a corresponding increase in monomeric apoE3. Both bands were absent in plasma from a subject with the E4/4 phenotype. In spite of its apparent molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the high molecular weight band was demonstrated to represent the disulfide-linked homodimer of apoE3. It was isolated from purified apoE3 preparations that had undergone oxygen-mediated dimerization and shown to elute from a Sephacryl S-300 column in a position with the expected molecular weight of a homodimer. The apoE3 dimer displayed a preference for high density lipoproteins, as determined by agarose chromatography of E3/3 plasma but was stripped from high density lipoproteins by ultracentrifugation. Quantitation of the relative ratios of homodimer, apoE3-A-II, and monomer in the plasma of 22 normolipidemic E3/3 subjects by immunoblotting revealed that the disulfide-linked structures accounted for the majority (approximately 55%) of plasma apoE. Both the homodimer and apoE3-A-II displayed a reduced ability to compete with low density lipoproteins for fibroblast low density lipoprotein receptors (20 and 30% of monomeric apoE3 binding activity, respectively). These results raise the possibility that the amount or availability of receptor-active apoE3 in E3/3 subjects may be rate limiting for metabolic events involving the low density lipoprotein receptor.

Apolipoproteins E↗
PubMed1990

A fraction of CD3 epsilon subunits exists as disulfide-linked dimers in both human and murine T lymphocytes.

In a T cell antigen receptor complex (TCR), the clonotypic disulfide-linked Ti heterodimer is noncovalently associated with the invariant CD3 polypeptides. The latter are composed of three monomeric subunits (gamma, delta, epsilon) and either a disulfide-linked homodimer (zeta zeta) or a disulfide-linked heterodimer (zeta eta). The exact stoichiometry of the Ti-CD3 subunits in a given complex is still largely unknown. Here, we report the presence of a CD3 epsilon dimer in a fraction of the TCR. When TCRs from both human and murine T lymphocytes were immunoprecipitated with monoclonal antibodies against either CD3 epsilon or Ti, a 40-kDa disulfide-linked dimer was coprecipitated with the other TCR subunits from digitonin lysates. Amino acid sequence analysis of peptides obtained by in situ CNBr cleavage of the 20-kDa product blotted to polyvinyl difluoride membranes from reducing/nonreducing two-dimensional gels identified human CD3 epsilon. Assuming this CD3 epsilon to derive from a homodimer, then either some TCRs contain more than one CD3 epsilon chain or several TCRs are covalently associated with one another via their CD3 epsilon subunits. Although it has been suggested that a putative TCR association with CD2 exists under similar conditions to those utilized to detect CD3 epsilon dimers, the CD2 molecule was not coimmunoprecipitated with the TCR by any of a series of anti-CD3 epsilon monoclonal antibodies. In conjunction with the fact that CD2 and the TCR do not colocalize during conjugate formation between T cells and antigen-presenting cells (Koyasu, S., Lawton, T., Novick, D., Recny, M. A., Siliciano, R. F., Wallner, B. P., and Reinherz, E. L. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2603-2607), we conclude that CD2 and the TCR are not physically associated on the T cell surface.

Amino Acid Sequence↗
PubMed1990

Assembly of functional rhodopsin requires a disulfide bond between cysteine residues 110 and 187.

Cysteine residues 110 and 187 are essential for the formation of the correct bovine rhodopsin structure (Karnik, S. S., Sakmar, T. P., Chen, H.-B., and Khorana, H. G. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8459-8463). We now show that the sulfhydryl groups of these 2 cysteine residues interact to form a disulfide bond. Rhodopsin mutants containing cysteine----serine substitutions were prepared as follows. In one mutant, CysVII, all the 10 cysteine residues of rhodopsin were replaced by serines. A second mutant, CysVIII, contained only C110 and C185; a third mutant, CysIX, contained only C185 and C187 while the fourth mutant, CysX, contained only C110 and C187. Only mutant CysX formed functional rhodopsin. Mutants CysVIII and CysIX reacted with [3H]iodoacetic acid showing the presence of free sulfhydryl groups while mutant CysX was inert to this reagent. CysX reacted with cyanide ion to form a thiocyanate derivative showing the presence of a disulfide bond. The C110-C187 disulfide bond is buried in rhodopsin because reactions with disulfide reducing agents and cyanide ion require prior treatment with denaturants.

Amino Acid Sequence↗
PubMed1990

Alleles at the PRB3 locus coding for a disulfide-bonded human salivary proline-rich glycoprotein (Gl 8) and a null in an Ashkenazi Jew.

From electrophoretic analysis, we identified in the saliva of an Ashkenazi Jew a disulfide-bonded major glycoprotein variant (Gl 8) that is a product of the proline-rich protein (PRP) locus PRB3. A previous study of this variant protein misidentified it as Pa 2 and as a product of a different PRP locus. The other PRB3 allele in this individual is an apparent null. To identify the mutations, we sequenced the tandemly repetitious exon 3 (the major protein-coding portions) of both alleles. A CGT----TGT (Arg----Cys) mutation was found in one allele (PRB3Scys), which accounts for the disulfide-bonded and peroxidase-modifying properties of Gl 8. A single nucleotide insertion was found in the other allele (PRB3Mnull) that leads to a frameshift with a premature termination codon that causes an apparent lack of gene expression. Null alleles are frequent at PRP loci coding for basic and glycosylated PRPs, and the mechanism described might explain other null phenotypes among PRPs. From nucleotide comparisons, a model of intragenic unequal crossing-over is proposed to explain, in part, the generation of the PRB3Mnull allele. The Gl 8 protein variant is found in Ashkenazi Jews (gene frequency around .008) but not in the general white, black, or Japanese populations. It is interesting that products of different PRP genes, Gl 8 from PRB3 and Pa 1 from PRH1, are both disulfide bonded and probably modify salivary peroxidase (part of an important intraoral antibacterial system) through formation of disulfide-bonded heterodimers.

Alleles↗
PubMed1975

The interdomain disulfide bond of a homogeneous rabbit pneumococcal antibody light chain.

Rabbit light chain 3315, prepared from a homogeneous antipneumococcal antibody, was subjected to hydrolysis by pepsin without prior reduction and alkylation of the intrachain disulfide bonds. Gel filtration of the hydrolysate on Sephadex G-10, G-15, and G-25 and ion exchange chromatography on SP-Sephadex yielded several disulfide bridge peptides. These were fully reduced and alkulated and sequenced by Edman degradation. The peptides were located in the light chain sequence determined in independent studies from our laboratory. The half-cystine residues in this KB rabbit chain are located at positions 23, 80, 88, 134, 171, 194, and 214. The extra disulfide bridge extends between residues 80 and 171, thus joining the variable and constant domains. This is consistent with x-ray diffraction crystallographic studies showing that the corresponding residues in human light chains are separated by a distance compatible with disulfide bond formation.

Amino Acid Sequence↗
PubMed1989

Elimination of disulfide bonds affects assembly and secretion of the human chorionic gonadotropin beta subunit.

Human chorionic gonadotropin (hCG) consists of two noncovalently joined alpha and beta subunits similar to the other glycoprotein hormones. To study the function of the individual disulfide bonds in subunit assembly and secretion, site-directed mutagenesis was used to convert the 12 cysteine (Cys) residues in the beta subunit of hCG to either alanine or serine. Both cysteines of proposed disulfide pairs were also mutated. These mutant hCG beta genes were transfected alone or together with the wild-type alpha gene into Chinese hamster ovary cells. Only 3-10% assembly could be achieved with derivatives containing single Cys mutations at positions 26, 110, 72, and 90, whereas no assembly was detected with the other 8 mutants. However, double mutations of pairs 26-110 or 23-72 showed increased dimer formation (11 and 36%, respectively). The secretion rate of individual mutants varied significantly. Whereas the Cys-23 and 72 mutants were secreted normally (t1/2 = 140-190 min), the Cys-26 mutant was secreted faster (t1/2 = 70 min), and the other 9 mutants were secreted slower (t1/2 = 280-440 min); mutations of both Cys at 26 and 110 caused much faster secretion (t1/2 = 34 min). Although the secretion rate of these mutants differed, they were quantitatively recovered in the medium except for mutant Cys-88, Cys-23-72, and Cys-34-88 (40, 55, and 10% secreted, respectively). Thus, interruption of any disulfide bond in the hCG beta subunit alters the structure sufficiently to block dimer formation and in some cases slow secretion, although the stability for most of the mutant hCG beta subunits is not greatly affected. The data indicate that interruption of any hCG beta disulfide bond generates different structural forms that are unable to assemble with the alpha subunit, and that the structural requirements for stability and assembly are different.

Animals↗
PubMed1989

Ubiquitin function studied by disulfide engineering.

Disulfide engineering was used to probe the role of conformational mobility in ubiquitin-mediated proteolysis. Six genes that encode cysteine-containing mutants of ubiquitin were constructed, expressed in Escherichia coli and the proteins purified. Single cysteine-containing mutants and a 4/14 disulfide were active in degradation of a substrate protein in vitro, while the 4/66 disulfide, which cross-links the NH2- and COOH-terminal strands of the protein, was only 20-30% active. The solution structure of the 4/66 mutant was solved: the disulfide is left-handed with no perturbations in the backbone from that of wild type ubiquitin. The results suggest that conformational mobility is required for the activity of ubiquitin in signaling proteolysis.

Computer Simulation↗
PubMed1988

Beta-adrenergic receptors display intramolecular disulfide bridges in situ: analysis by immunoblotting and functional reconstitution.

The molecular nature of mammalian beta-adrenergic receptors in situ was probed using immunoblotting and functional reconstitution techniques. Membrane proteins of cells replete with beta-adrenergic receptors were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the resolved proteins were transferred to nitrocellulose and then probed with anti-receptor antibodies. When cell membranes were first treated with agents that cleave disulfides of proteins, immunoblots of these membranes revealed intense immunoreactive bands with electrophoretic mobility similar to that of protein standards of Mr 65,000-67,000, comigrating with purified, reduced, and alkylated beta-adrenergic receptors. However, when cell membranes were prepared under anaerobic conditions, solubilized in the presence of agents that alkylate thiols, and denatured in the absence of added thiols, immunoblotting revealed receptor with Mr 55,000, rather than 65,000. This faster electrophoretic mobility is associated with the presence of intramolecular disulfides in the purified receptor and demonstrates that beta-adrenergic receptors possess intramolecular disulfide bridges in situ. Purified receptors that demonstrate this faster mobility (Mr 55,000 under nonreducing conditions) were co-reconstituted into phospholipid vesicles with the stimulatory GTP-binding protein GS and their ability to catalyze the binding of [35S]guanosine-5'-O-(3-thio)triphosphate to GS was measured. Agonist (isoproterenol) as well as thiol increased the receptor-promoted activation of GS. Taken together, these data demonstrate that native beta-adrenergic receptors possess one or more intramolecular disulfide bridges in situ, reduction of which causes functional activation of the receptor.

Animals↗
PubMed1989

Determination of the disulfide array in the human defensin HNP-2. A covalently cyclized peptide.

HNP-2 is a 29-residue peptide present in human neutrophils and is a member of the defensin family of antimicrobial peptides. All defensins contain an invariant disulfide infrastructure comprised of 6 half-cystine residues. The disulfide structure of HNP-2 was determined using a novel method to identify the cross-links involving the amino- and carboxyl-terminal cysteine residues. A derivative of HNP-2 was synthesized by covalent modification of the terminal cysteine residues. This derivative was purified, characterized, and subjected to exhaustive proteolytic digestion. Characterization of purified proteolytic fragments by amino acid analysis and/or sequence analysis identified an oligopeptide containing all 6 cystine residues. This oligopeptide was subjected to a single cycle of Edman degradation to cleave the peptide bond linking 2 adjacent cysteines. Purification and characterization of the Edman reaction products allowed for assignment of the disulfide array in HNP-2, revealing a cystine motif unique to the defensin peptide family. Further, the covalent structure of HNP-2 was found to be cyclic as one disulfide links the amino- and carboxyl-terminal cysteine residues. HNP-2 is the only polypeptide known to possess such a configuration.

Amino Acid Sequence↗
PubMed1986

Essential disulfide and sulfhydryl groups for organic cation transport in renal brush-border membranes.

The disulfide reducing agent, dithiothreitol (DTT) and the sulfhydryl-modifying reagents p-chloromercuribenzenesulfonic acid and N-ethylmaleimide (NEM) were employed to assess the role of disulfide and sulfhydryl groups in organic cation transport. The transport of N1-[3H]methylnicotinamide (NMN), a prototypic organic cation, was examined employing brush-border membrane vesicles isolated from the outer cortex of canine kidneys. DTT inhibited NMN transport reversibly with an IC50 of 250 microM/mg of protein. 5 mM NMN protected against DTT inactivation. The specificity of substrate protection was demonstrated by showing that D-glucose had no effect on the DTT inactivation of NMN transport and conversely that NMN had no effect on the DTT inactivation of D-glucose transport. Disulfide bonds reduced by DTT could be reoxidized by washing with excess buffer or by addition of 0.02% H2O2 thereby restoring NMN transport. p-Chloromercuribenzenesulfonic acid reversibly inactivated NMN transport with an IC50 of 25 microM/mg of protein. 5mM NMN protected against inactivation. NEM irreversibly inactivated transport with an IC50 of 250 microM/mg of protein. The rate of NMN inactivation by NEM followed pseudo-first order reaction kinetics. A replot of the data gave a linear relationship between the apparent rate constants and the NEM concentration with a slope of 1.3. The data are consistent with a simple bimolecular reaction mechanism and imply that one molecule of NEM inactivates 1 sulfhydryl group/active transport unit. The presence of 5 mM NMN affected the rate of NEM (2.5 mM) inactivation: the t1/2 values for inactivation in the presence and absence of substrate were 7.3 and 2.0 min, respectively. The results demonstrate an essential requirement for disulfide and sulfhydryl groups.

Animals↗