The reductive cleavage of myeloperoxidase in half, producing enzymically active hemi-myeloperoxidase

Reduction and alkylation of human myeloperoxidase under nondenaturing conditions results in the cleavage of this enzyme. Sedimentation equilibrium data is presented which shows that the molecular weight of the cleavage product (78,000 +/- 2,000) is half that of the native enzyme (153,000 +/- 4,000). We conclude that the cleavage product is the half-enzyme hemi-myeloperoxidase. Hemi-myeloperoxidase retains both heme groups and contains both subunit types (Mr = 57,500 and 14,000) in the same ratio as native myeloperoxidase. The two halves of native myeloperoxidase are apparently not dependent upon one another for peroxidatic activity, as the specific activity of the half-enzyme is the same as that of the native enzyme. Analytical ultracentrifugation studies show native myeloperoxidase has a sedimentation coefficient of 8.0 and an axial ratio of 5:1, while hemi-myeloperoxidase has a sedimentation coefficient of 4.3 and an axial ratio of 10:1. When [3H]iodoacetic acid was used to prepare hemi-myeloperoxidase, the label incorporated with a stoichiometry of 1.2 [3H]carboxymethyl groups per hemi-myeloperoxidase, with 90% of this label associated with the heavy subunit. From these observations we conclude that native myeloperoxidase contains two heavy-light protomers, which are joined along their long axes by a single disulfide bond between the heavy subunits. Selective reduction of this disulfide bond by the use of nondenaturing conditions results in the formation of hemi-myeloperoxidase, a catalytically active heavy-light protomer of native myeloperoxidase.     

Repair of alkylated DNA in Escherichia coli. Physical properties of O6-methylguanine-DNA methyltransferase

An inducible methyltransferase of Escherichia coli acts on O6-methylguanine in DNA by conveying the methyl group to one of its own cysteine residues. The protein has now been purified to apparent homogeneity from a constitutively expressing strain. The homogeneous methyltransferase exhibits no DNA glycosylase or endonuclease activity on alkylated DNA. Further, the methyltransferase activity is strikingly resistant to heat inactivation under reducing conditions. The protein has Mr = 18,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while the sedimentation coefficient and Stokes radius of the native enzyme yield Mr = 18,400. The amino acid composition of the purified protein shows 4 to 5 cysteine residues/transferase molecule. The methylated, inactive form of the transferase has an unaltered molecular weight.     

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.     

The sodium channel from rat brain. Separation and characterization of subunits

Procedures are described for separation of the alpha, beta 1, and beta 2 subunits of the voltage-sensitive sodium channel from rat brain by gel filtration in sodium dodecyl sulfate (SDS) before and after reduction of intersubunit disulfide bonds or by preparative SDS-gel electrophoresis. Partial proteolytic maps of the SDS-denatured subunits indicate that they are nonidentical polypeptides. They are all heavily glycosylated and contain complex carbohydrate chains that bind wheat germ agglutinin. The apparent molecular weights of the separated subunits were estimated by gradient SDS-gel electrophoresis, by Ferguson analysis of migration in SDS gels of fixed acrylamide concentration, or by gel filtration in SDS or guanidine hydrochloride. For the alpha subunit, SDS-gel electrophoresis under various conditions gives an average Mr of 260,000. Gel filtration methods give anomalously low values. Removal of carbohydrate by sequential treatment with neuraminidase and endoglycosidase F results in a sharp protein band with apparent Mr = 220,000, suggesting that 15% of the mass of the native alpha subunit is carbohydrate. Electrophoretic and gel filtration methods yield consistent molecular weight estimates for the beta subunits. The average values are: beta 1, Mr = 36,000, and beta 2, Mr = 33,000. Deglycosylation by treatment with endoglycosidase F, trifluoromethanesulfonic acid, or HF yields sharp protein bands with apparent Mr = 23,000 and 21,000 for the beta 1 and beta 2 subunits, respectively, suggesting that 36% of the mass of the native beta 1 and beta 2 subunits is carbohydrate.     

Subunit structure and amino acid composition of xylose isomerase from Streptomyces albus.

The subunit structure and amino acid composition of xylose isomerase from Streptomyces albus have been examined. A native molecular weight of 165,000 determined by sedimentation equilibrium was reduced to 43,000 when the protein was treated with 6 M guanidine hydrochloride. No further reduction in molecular weight was observed when potential disulfide bridges of xylose isomerase were reduced and alkylated, indicating that the protein was devoid of interchain disulfide bonds. NH2-terminal analysis using [3H]dansyl chloride showed 0.86 residues of methionine per Mr equals 41,500 unit. Analysis of the native protein with an automated protein sequenator revealed the presence of only one degradable polypeptide chain. Fractionation of the soluble tryptic peptides of S-[14C]carboxymethyl xylose isomerase by ion exchange chromatography and one-dimensional paper electrophoresis yielded 37 to 43 peptides. When the acid-insoluble tryptic peptides were dissolved and analyzed using gel filtration techniques, and additional four peptides were found. A unique radioactive tryptic peptide containing S-carboxymethylcysteine was found among the soluble peptides, confirming cysteine as the limiting amino acid residue in the amino acid composition of xylose isomerase. On the basis of its lysine and arginine content, the number of tryptic peptides is consistent with the hypothesis that the native xylose isomerase is a tetramer of four very similar or identical subunits of Mr equals 41,500, associated by noncovalent bonds.     

Interchain disulfide bond formation in types I and II procollagen. Evidence for a protein disulfide isomerase catalyzing bond formation

The assembly of reduced pro-alpha chains of type I and type II procollagen into the native triple-helical molecule was examined in vitro in the presence and absence of pure protein disulfide isomerase. The data clearly indicates that protein disulfide isomerase is able to accelerate the formation of native interchain disulfide bonds in these procollagens. It takes about 6 min after disulfide bonding before triple-helical molecules exist, while the time required to produce triple-helical type I procollagen in the presence of protein disulfide isomerase is 9.4 min and that for type II procollagen 17.2 min. These values agree with those obtained for type I and II procollagen in vivo suggesting that protein disulfide isomerase is also an enzyme catalyzing interchain disulfide bond formation in procollagen in vivo. The formation of native disulfide bonds can proceed without any enzyme catalysis but then requires the presence of reduced and oxidized glutathione. Bonding is rather slow in such a case, however, resulting in a delay in the formation of the triple helix.     

Murein hydrolase (N-acetyl-muramyl-l-alanine amidase) in human serum

An enzyme was identified in human serum which unlike lysozyme cleaved the amide bond between N-acetyl-muramic acid and l-alanine of the peptide side chain of the rigid layer (murein) of Escherichia coli. The N-acetylmuramyl-l-alanine amidase released all of the peptide side chains including those to which the lipoprotein is bound. A portion of the peptide side chains of the Micrococcus lysodeikticus murein was also hydrolysed from the polysaccharide chains. E. coli, M. lysodeikticus, Bacillus subtilis and Staphylococcus aureus were not killed by the amidase. Treatment of E. coli with EDTA or osmotic shock rendered the cells sensitive to the amidase and they were killed. Possible biological functions of the amidase are discussed.The enzyme was separated from lysozyme in human serum. Gel permeation chromatography indicated a molecular weight of the active enzyme of 82,000 while gel electrophoresis in the presence of sodium dodecyl sulfate revealed a molecular weight of 75,000. Thus, the enzyme probably consists of a single polypeptide chain. Incubation with neuraminidase rendered the amidase more basic suggesting the release of sialic acid residues. The modified glycoprotein disclosed an increased activity to murein. Enzyme activity was inhibited by p-chloromercuribenzene sulfonate and ethyleneglycol-bis(2-aminomethyl) tetraacetate (EGTA) at 1 and 0.2 mM concentration, respectively, whereas EDTA up to 5 mM was without effect. The amidase was also inactivated by agents that reduce disulfide bridges.     

Kinetic properties of human dopamine sulfotransferase (SULT1A3) expressed in prokaryotic and eukaryotic systems: comparison with the recombinant enzyme purified from Escherichia coli.

Sulfation, catalyzed by members of the sulfotransferase enzyme family, is a major metabolic pathway which modulates the biological activity of numerous endogenous and xenobiotic chemicals. A number of these enzymes have been expressed in prokaryotic and eukaryotic systems to produce protein for biochemical and physical characterization. However, the effective use of heterologous expression systems to produce recombinant enzymes for such purposes depends upon the expressed protein faithfully representing the "native" protein. For human sulfotransferases, little attention has been paid to this despite the widespread use of recombinant enzymes. Here we have validated a number of heterologous expression systems for producing the human dopamine-metabolizing sulfotransferase SULT1A3, including Escherichia coli, Saccharomyces cerevisiae, COS-7, and V79 cells, by comparison of Km values of the recombinant enzyme in cell extracts with enzyme present in human platelets and with recombinant enzyme purified to homogeneity following E. coli expression. This is the first report of heterologous expression of a cytosolic sulfotransferase in yeast. Expression of SULT1A3 was achieved in all cell types, and the Km for dopamine under the conditions applied was approximately 1 microM in all heterologous systems studied, which compared favorably with the value determined with human platelets. We also determined the subunit and native molecular weights of the purified recombinant enzyme by SDS-PAGE, electrospray ionization mass spectrometry, dynamic light scattering, and sedimentation analysis. The enzyme purified following expression in E. coli existed as a homodimer with Mr approximately 68,000 as determined by light scattering and sedimentation analysis. Mass spectrometry revealed two species with experimentally determined masses of 34,272 and 34,348 which correspond to the native protein with either one or two 2-mercaptoethanol adducts. We conclude that the enzyme expressed in prokaryotic and eukaryotic heterologous systems, and also purified from E. coli, equates to that which is found in human tissue preparations.     

Structural characterization of insulin receptors. II. Subunit composition of receptors from turkey erythrocytes

In the preceding paper (Aiyer, R. A. (1983) J. Biol. Chem. 258, 14992-14999), the hydrodynamic properties of insulin receptors from turkey erythrocyte plasma membranes solubilized in nondenaturing detergents (Triton X-100 and sodium deoxycholate) were characterized. Two specific insulin-binding species are observed after velocity sedimentation in linear sucrose density gradients: peak II whose protein molecular weight (Mp) is 180,000 +/- 45,000 and its disulfide-linked dimer, peak I (Mp, 355,000 +/- 65,000). This paper describes the subunit composition of these species determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Insulin receptors were covalently attached to [125I]iodoinsulin with disuccinimidyl suberate. After solubilization in Triton X-100 or deoxycholate, peaks I and II were separated by sedimentation and subjected to SDS-PAGE; the constituent polypeptides were then identified by autoradiography. Under reducing conditions, both peaks I and II yield a major band of apparent molecular weight (Mapp) of 135,000; this band most likely represents the insulin-binding subunit (alpha). Minor bands of lower molecular weight are also seen whose significance is not entirely obvious. Under nonreducing conditions, peak I yields bands at Mapp = 230,000 and at greater than 240,000, while peak II yields bands at Mapp = 120,000 and 200,000. When these bands were cut out of the gel and subjected to SDS-PAGE following reduction with 10% beta-mercaptoethanol, all of them produced a single band that migrated with Mapp = 135,000. These results indicate that the alpha subunit is linked by disulfide bonds to at least one more subunit (beta). It is also apparent that the alpha subunit travels with higher mobility (Mapp = 120,000) under nonreducing conditions, suggesting the presence of intrachain disulfide bonds. Thus, peak II has a minimum subunit composition of alpha beta, where alpha is the insulin-binding subunit with a minimum Mr = 120,000-135,000 and beta has a minimum Mr = 80,000-90,000. And peak I, the disulfide-linked dimer of peak II, has a minimum subunit composition of alpha 2 beta 2. These results were further confirmed by cross-linking of protein subunits with glutaraldehyde, an (alpha, omega)-dialdehyde that reacts with amino groups. Within the limits of error, these molecular weights are in agreement with those estimated from the hydrodynamic properties of the detergent-solubilized, native receptor species reported in the preceding paper.     

The nonconsecutive disulfide bond of Escherichia coli phytase (AppA) renders it dependent on the protein-disulfide isomerase, DsbC

The formation of protein disulfide bonds in the Escherichia coli periplasm by the enzyme DsbA is an inaccurate process. Many eukaryotic proteins with nonconsecutive disulfide bonds expressed in E. coli require an additional protein for proper folding, the disulfide bond isomerase DsbC. Here we report studies on a native E. coli periplasmic acid phosphatase, phytase (AppA), which contains three consecutive and one nonconsecutive disulfide bonds. We show that AppA requires DsbC for its folding. However, the activity of an AppA mutant lacking its nonconsecutive disulfide bond is DsbC-independent. An AppA homolog, Agp, a periplasmic acid phosphatase with similar structure, lacks the nonconsecutive disulfide bond but has the three consecutive disulfide bonds found in AppA. The consecutively disulfide-bonded Agp is not dependent on DsbC but is rendered dependent by engineering into it the conserved nonconsecutive disulfide bond of AppA. Taken together, these results provide support for the proposal that proteins with nonconsecutive disulfide bonds require DsbC for full activity and that disulfide bonds are formed predominantly during translocation across the cytoplasmic membrane.     

Isolation and characterization of the major androgen-dependent glycoprotein of canine prostatic fluid

Canine prostatic fluid, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing conditions, is characterized by the presence of a single diffuse band (Mr approximately 30,000) which accounts for over 90% of the total protein. The biosynthesis of this protein is under androgen control. Castration results in the disappearance of this protein, whereas its presence in the prostate can be maintained in the castrated animal with exogenous androgen. Analysis of the native protein by isoelectric focusing revealed the presence of 10-13 charged variants with pI values in the range of 6.5 to 8.4. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions revealed that each isoform is constructed of two dissimilar polypeptide subunits covalently linked through disulfide bonds. One subunit has a molecular weight of 15,000 (H chain); the second subunit (L chain) has a variable molecular weight in the 12,000-14,000 range. The H and L subunits have been purified by preparative isoelectric focusing and chemically characterized. Based on tryptic peptide mapping, NH2-terminal analysis, amino acid and carbohydrate composition, the H and L subunits are structurally unrelated and consequently appear to be unique gene products. Furthermore, the L subunit is glycosylated which potentially accounts for its size heterogeneity. Quantitative NH2-terminal analysis indicated that the H and L subunits are present in the native molecule in a ratio of 2:1, suggesting that the native molecule is a trimer with an apparent molecular weight of 43,000. Based on electrophoretic data, the glycoprotein also constitutes the major fraction of the soluble protein in canine prostatic tissue; its presence is organ specific. This glycoprotein should prove useful as a marker for prostatic function under varying hormonal and environmental conditions.     

Purification and properties of a coagulant thrombin-like enzyme from the venom of Bothrops leucurus

A thrombin-like enzyme from Bothrops leucurus venom, named leucurobin (leuc), was purified by gel filtration, affinity and ion exchange chromatographies. Physicochemical studies indicated that the purified enzyme is a 35 kDa monomeric glycoprotein on SDS-PAGE under reducing conditions, which decreased to 29 kDa after deglycosylation with N-glycosidase F (PNGase F). The amino acid sequence of leuc was determined by automated sequencing of the intact native protein and peptides produced by digestion of the S-pyridyl-ethylated protein with trypsin. The protein sequence exhibits significant similarities with other serine proteases reported from snake venoms, and contains two potential sites of N-linked glycosylation. The proteinase split off fibrinopeptide A (FPA) rapidly from human fibrinogen; however, only negligible traces of fibrinopeptide B (FPB) were observed. In addition, the enzyme released the N-terminal peptide (Mr=4572) containing the first 42 residues from the Bbeta-chain. Leuc could neither activate factor XIII nor release kinins from heat-treated bovine plasma. Its specific clotting activity was equivalent to 198 NIH thrombin U/mg on human fibrinogen. Kinetic properties of leuc were determined using representative chromogenic substrates. The enzyme evoked the gyroxin syndrome when injected into the tail veins of mice at levels of 0.143 microg/g mouse. The inhibitory effects of PMSF and benzamidine on the amidolytic activity suggest that leuc is a serine proteinase, and inhibition by beta-mercaptoethanol revealed the important role of the disulfide bonds in the stabilization of the native structure. Antibothropic serum, SBTI and EDTA had little or no effect on its amidolytic activity. However, the clotting effect of the enzyme was strongly inhibited by antibothropic serum. A Dixon plot showed that the hydrolysis of Bz-L-Arg-pNA by leuc was competitively inhibited by benzamidine (Ki=1.61+/-0.25 mM).     

Escherichia coli alkaline phosphatase fails to acquire disulfide bonds when retained in the cytoplasm.

The cysteines of the Escherichia coli periplasmic enzyme alkaline phosphatase, which are involved in disulfide bonds in the native enzyme, were found to be fully reduced when the protein was retained in the cytoplasm. Under these circumstances the cysteines remained reduced for at least several minutes after the synthesis of the protein was completed. This contrasted with the normally exported protein, wherein disulfide bonds formed rapidly. Disulfide bond formation accompanied export and processing. The implications of these findings for the inactivity of the enzyme in the cytoplasm are discussed.     

Role of disulfide bonds in conformational stability and folding of 5'-deoxy-5'-methylthioadenosine phosphorylase II from the hyperthermophilic archaeon Sulfolobus solfataricus

Sulfolobus solfataricus 5'-deoxy-5'-melthylthioadenosine phosphorylase II (SsMTAPII), is a hyperthermophilic hexameric protein with two intrasubunit disulfide bonds (C138-C205 and C200-C262) and a CXC motif (C259-C261). To get information on the role played by these covalent links in stability and folding, the conformational stability of SsMTAPII and C262S and C259S/C261S mutants was studied by thermal and guanidinium chloride (GdmCl)-induced unfolding and analyzed by fluorescence spectroscopy, circular dichroism, and SDS-PAGE. No thermal unfolding transition of SsMTAPII can be obtained under nonreducing conditions, while in the presence of the reducing agent Tris-(2-carboxyethyl) phosphine (TCEP), a Tm of 100°C can be measured demonstrating the involvement of disulfide bridges in enzyme thermostability. Different from the wild-type, C262S and C259S/C261S show complete thermal denaturation curves with sigmoidal transitions centered at 102°C and 99°C respectively. Under reducing conditions these values decrease by 4°C and 8°C respectively, highlighting the important role exerted by the CXC disulfide on enzyme thermostability. The contribution of disulfide bonds to the conformational stability of SsMTAPII was further assessed by GdmCl-induced unfolding experiments carried out under reducing and nonreducing conditions. Thermal unfolding was found to be reversible if the protein was heated in the presence of TCEP up to 90°C but irreversible above this temperature because of aggregation. In analogy, only chemical unfolding carried out in the presence of reducing agents resulted in a reversible process suggesting that disulfide bonds play a role in enzyme denaturation. Thermal and chemical unfolding of SsMTAPII occur with dissociation of the native hexameric state into denatured monomers, as indicated by SDS-PAGE.     

Affinity labeling of multiplication stimulating activity receptors in membranes from rat and human tissues

Plasma membranes from rat adipocytes and liver and from human placenta have been labeled by covalent cross-linking to membrane-bound 125I-labeled multiplication stimulating activity (125I-MSA) with three different bishydroxysuccinimide esters: disuccinimidyl suberate, disuccinimidyl succinate, and ethyleneglycolyl bis(succinimidyl succinate). Dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiographic analysis of the 125I-MSA-labeled material in the presence of dithiothreitol reveals one single-labeled protein migrating with an apparent Mr = 255,000 regardless of the kind and concentration of cross-linker used. Electrophoresis in the absence of reductant indicates that the affinity-labeled species is not disulfide-linked to any other protein in the native plasma membrane, but contains internal disulfide bonds that compact its structure. The labeling of the Mr = 255,000 species increases with increasing concentrations of 125I-MSA between 0.3 and 3 nM. Labeling is abolished in a competitive manner by nonradioactive MSA but not by similar concentrations of insulin, proinsulin, or epidermal growth factor in all three tissues examined. The unique labeling of this Mr = 225,000 membrane component and its selective inhibition by MSA suggest that this protein is a plasma membrane receptor for MSA.     

Cell-free expression of disulfide-containing eukaryotic proteins for structural biology

We describe Escherichia?coli based cell-free production of milligram quantities of eukaryotic proteins containing native disulfide bonds. Using a previously described expression system, we systematically investigated the influence of redox potential variation in the reaction mixture and the impact of adding disulfide bond catalysts on soluble protein production. It is then shown that the optimized reaction conditions for native disulfide bond formation can be combined with the use of N-terminal fusion constructs with the GB1 domain for increased expression yields. The resulting cell-free system is suitable for stable-isotope labeling and does not require chemical pretreatment of the cell extract to stabilize the redox potential. For the human doppel protein, the mouse doppel protein and mouse interleukin-22 we obtained 0.3-0.7?mg of purified native protein per milliliter of reaction mixture. Formation of disulfide bonds was validated using the Ellman assay, and native folding of the three proteins was monitored by NMR and CD spectroscopy. Structured digital abstract ? mIL22?and?mIL22?bind?by?nuclear magnetic resonance?(View interaction).     

Proteolysis of the bifunctional methionine-repressible aspartokinase II-homoserine dehydrogenase II of Escherichia coli K12. Production of an active homoserine dehydrogenase fragment.

The dimeric bifunctional enzyme aspartokinase II-homoserine dehydrogenase II (Mr = 2 X 88,000) of Escherichia coli K12 can be cleaved into two nonoverlapping fragments by limited proteolysis with subtilisin. These two fragments can be separated under nondenaturing conditions as dimeric species, which indicates that each fragment has retained some of the association areas involved in the conformation of the native protein. The smaller fragment (Mr = 2 X 24,000) is devoid of aspartokinase and homoserine dehydrogenase activity. The larger fragment (Mr = 2 X 37,000) is endowed with full homoserine dehydrogenase activity. These results show that the polypeptide chains of the native enzyme are organized in two different domains, that both domains participate in building up the native dimeric structure, and that one of these domains only is responsible for homoserine dehydrogenase activity. A model of aspartokinase II-homoserine dehydrogenase II is proposed, which accounts for the present results.     

Covalent structure, disulfide bonding, and identification of reactive surface and active site residues of human prostatic acid phosphatase

The pairing of the half-cysteine residues of human prostatic acid phosphatase was established by proteolytic digestion and analysis of the resulting peptide mixtures by fast atom bombardment mass spectrometry (FAB-MS). An independently derived, full length cDNA clone was used as the basis for the interpretation of the FAB-MS data. The sequence of the native protein is that predicted from the present cDNA sequence, except for the carboxyl-terminal end and some possible post-translational deamidations. Isolated human prostatic acid phosphatase was found to have multiple carboxyl-terminal ends, terminating in Thr, Glu, and Asp, corresponding to residues 349-351 of the 354-residue protein that is predicted from the cDNA sequence after removal of a leader peptide. The protein contains no free sulfhydryl groups. The identical monomer chains of the dimeric native enzyme are found to contain three disulfide bonds, specifically Cys-129 to Cys-340, Cys-183 to Cys-281, and Cys-315 to Cys-319. In view of the conserved positions of cysteines in the homologous human and rat liver lysosomal acid phosphatases, an identical disulfide bonding pattern may be predicted for those proteins. The location of a potential antigenic site was established by selective labeling of proximate tyrosine residues predicted to be on the surface. A conserved RHGXRXP sequence is present in the prostatic, lysosomal, Escherichia coli, and yeast acid phosphatases and is predicted to be of mechanistic significance. In addition, residue Arg-54 is shown to be an active site residue by reaction of the enzyme with phenylglyoxal. Interestingly, this residue is present in a sequence RXRY (R,H) that is also present in lysosomal phosphatase and in recently described protein tyrosine phosphatases.     

Purification and biochemical characterization of a soluble human interferon gamma receptor expressed in Escherichia coli

We purified and characterized a soluble human interferon gamma receptor expressed in Escherichia coli. The soluble receptor comprises the amino acids 15-246 of the encoded protein (Aguet, M., Dembic, Z., and Merlin, G. (1988) Cell 55, 273-280) and was purified from large scale fermentations through four chromatographic steps with an overall recovery of 28%. The refolded soluble receptor shows some heterogeneity on nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, where it appears as the major band of 27 kDa molecular mass, accompanied by a few minor bands with molecular masses between 26 and 30 kDa. On reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis it appears as a homogeneous protein of 32 kDa molecular mass. The soluble interferon gamma receptor is an active and stable protein and is recognized by specific antibodies raised against the native receptor. When nonreduced it has the capacity to specifically bind interferon gamma and to compete for the binding of interferon gamma to the cell surface receptor. The observed heterogeneity of the soluble interferon gamma receptor under nonreducing electrophoretic conditions is probably due to different conformational forms resulting from the formation of non-native intramolecular disulfide bonds among the 8 cysteine residues present in the soluble interferon gamma receptor molecule.     

Early intermediates in the PDI-assisted folding of ribonuclease A

The oxidative refolding of ribonuclease A has been investigated in several experimental conditions using a variety of redox systems. All these studies agree that the formation of disulfide bonds during the process occurs through a nonrandom mechanism with a preferential coupling of certain cysteine residues. We have previously demonstrated that in the presence of glutathione the refolding process occurs through the reiteration of two sequential reactions: a mixed disulfide with glutathione is produced first which evolves to form an intramolecular S-S bond. In the same experimental conditions, protein disulfide isomerase (PDI) was shown to catalyze formation and reduction of mixed disulfides with glutathione as well as formation of intramolecular S-S bonds. This paper reports the structural characterization of the one-disulfide intermediate population during the oxidative refolding of Ribonuclease A under the presence of PDI and glutathione with the aim of defining the role of the enzyme at the early stages of the reaction. The one-disulfide intermediate population occurring at the early stages of both the uncatalyzed and the PDI-catalyzed refolding was purified and structurally characterized by proteolytic digestion followed by MALDI-MS and LC/ESIMS analyses. In the uncatalyzed refolding, a total of 12 disulfide bonds out of the 28 theoretical possible cysteine couplings was observed, confirming a nonrandom distribution of native and nonnative disulfide bonds. Under the presence of PDI, only two additional nonnative disulfides were detected. Semiquantitative LC/ESIMS analysis of the distribution of the S-S bridged peptides showed that the most abundant species were equally populated in both the uncatalyzed and the catalyzed process. This paper shows the first structural characterization of the one-disulfide intermediate population formed transiently during the refolding of ribonuclease A in quasi-physiological conditions that mimic those present in the ER lumen. At the early stages of the process, three of the four native disulfides are detected, whereas the Cys26-Cys84 pairing is absent. Most of the nonnative disulfide bonds identified are formed by nearest-neighboring cysteines. The presence of PDI does not significantly alter the distribution of S-S bonds, suggesting that the ensemble of single-disulfide species is formed under thermodynamic control.