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1.
Human insulin was glycated under hyperglycemic reducing conditions and a novel diglycated form (M(r) 6135.1 Da) was purified by RP-HPLC. Endoproteinase Glu-C digestion combined with mass spectrometry and automated Edman degradation localized glycation to Gly(1) and Phe(1) of the insulin A- and B-chains, respectively. Intraperitoneal (i.p.) administration of diglycated insulin to mice alone or in combination with glucose (7 nmol/kg) resulted in a 43-61% and 11-34% reduction in glucose lowering activity, respectively, compared with native insulin. Consistent with these findings, diglycated insulin (10(-9) to 10(-7) mol/liter) was 22-38% less effective (P < 0.001) than native insulin in stimulating glucose uptake, glucose oxidation and glycogen production in isolated mouse abdominal muscle.  相似文献   

2.
This study evaluates the nature of glycated human insulin formed following exposure to hyperglycemic conditions in vitro. Glycated insulin was purified by RP-HPLC and its molecular mass (5971.3 Da) determined by plasma desorption mass spectrometry (MS). The difference in mass (163.7 Da) from nonglycated insulin (5807.6 Da) corresponds to a single reduced glucose (glucitol) residue. Following reduction of insulin disulfide bridges, MS confirmed that the B-chain was glycated. Enzymatic digestions with trypsin, endoproteinase Glu-C, and thermolysin, followed by RP-HPLC and identification of fragments by MS, localized glycation to the B-chain (1–5) region. Electrospray tandem MS identified the site of glycation as the B-chain NH2-terminal Phe1 residue. This was confirmed by automated Edman degradation with glycated human insulin.  相似文献   

3.
The intracellular concentration of the 27-kDa mammalian heat shock protein, HSP27, increases several-fold after heat and other metabolic stresses and is closely associated with the acquisition of thermotolerance. Posttranslational modifications may also affect the function of HSP27. Heat shock of HeLa cell cultures, or treatment with arsenite, phorbol ester, or tumor necrosis factor, caused a rapid phosphorylation of preexisting HSP27 and the appearance of three phosphorylated isoforms, HSP27 B, C, and D. Digestion with trypsin and fractionation of the peptides by reverse phase high performance liquid chromatography revealed three 32P-labeled phosphopeptides. Microsequence analysis identified peak I as Ala76-Leu77-Ser78-Arg79 and peak II as Gln80-Leu81-Ser82-Ser83-Gly84-Val85- Ser86-Glu87-Ile88-Arg89; peak III contained the undigested peptide pair Ala76-Arg89. Ser82 was the major site and Ser78 the minor site of phosphorylation. Mutant proteins with Ser78 or Ser82 altered to glycine or Ser78-Ser82 double mutants were phosphorylated to reduced extents in vivo after heat or arsenite treatment. Ser78 and Ser82 (and Ser15) occur in the sequence motif RXXS, which is recognized by ribosomal protein S6 kinase II. Mitogenic stimulation of serum-deprived, Go-arrested Chinese hamster cells with serum, thrombin, or fibroblast growth factor also stimulated phosphorylation of HSP27 Ser78 and Ser82, and mitogenic stimulation and heat shock activated protein kinase activities that phosphorylated HSP27 and protein S6 in vitro. These results suggest that HSP27 may exert phosphorylation-activated functions linked with growth signaling pathways in unstressed cells. A homeostatic function at this level could protect cells from adverse effects of signal transduction systems which may be activated inappropriately during stress.  相似文献   

4.
The structure of a 43,000 Da aggregate generated from bovine lens alpha-crystallin polypeptides of 20,000 Da using Fe2+ catalyzed oxidation, was studied by sequence analysis of a 30,000 Da proteolytic fragment. Three polypeptide components were simultaneously sequenced in the electroblotted 30,000 Da fragment, corresponding to Phe114-Ser130... of the alpha A chain, and His 111-Ser135... and Ser 35-Leu49... of the alpha B chain. The relative proportions of the components suggests that the three polypeptides are present in equimolar amounts. It is concluded that the 30,000 Da fragment and, therefore, the 43,000 Da aggregate is constructed of both the A and B polypeptide chains covalently cross-linked with non-reducible bonds. At least one of these cross-links is present towards the carboxy-terminus of the A and B chains after Phe114 and His111, respectively.  相似文献   

5.
To study disulphide bridge formation by Streptomyces lividans TK 24 in secreted single chain precursors of insulin a fusion protein (PTF 1) was investigated consisting of monkey proinsulin and the aminoterminal sequence Asp1 to Gly43 of the alpha-amylase inhibitor tendamistat from Streptomyces tendae. The purified soluble protein PTF 1 has a molecular mass of 14.4 kDa. The primary structure was elucidated after digestion with lysyl endopeptidase and fragment analysis. In this system, disulphide bond formation occurs in a way that the first cysteine in proinsulin is linked to the next following cysteine in the amino-acid chain resulting in a non-natural folding of the insulin part of the fusion protein. Re-folding of PTF 1 by reduction and re-oxidation followed by proteolytic digestions led to insulins which are identical to authentic material. The ease of correct disulphide bond formation in solution and incorrect processing during secretion suggests involvement of yet unknown factors leading to an unfavourable folding of proinsulin.  相似文献   

6.
The gamma-aminobutyric acid, type A (GABA(A)), receptor is the target for numerous therapeutic compounds. In the present study, the Gln(28)-Leu(296), Gln(28)-Arg(276), Gln(28)-Arg(248), and Gln(28)-Glu(165) (numbering of bovine precursor protein) segments of its alpha(1) subunit were overexpressed in Escherichia coli, along with Cys(166)-Leu(296) produced previously, for structural analysis by circular dichroism and ligand binding studies by fluorescence spectroscopy. Results showed that the protein segments were rich in beta-sheet structures. Binding of the fluorescent benzodiazepine Bodipy-FL Ro-1986 was evident from fluorescence resonance energy transfer and fluorescence anisotropy measurements. The binding affinity was in the micromolar range. The binding was attributable more to Cys(166)-Leu(296) than to Gln(28)-Glu(165) and was inhibited by known central benzodiazepine site ligands. Three point mutations, Y187A, T234A, and Y237A, were found to perturb protein secondary structures. Studies with the single Trp mutants W198Y and W273Y indicated that Trp(273) was closer to the binding site than Trp(198).  相似文献   

7.
Digestion of methylamine-treated alpha 2-macroglobulin (alpha 2M X MA) with catalytic amounts of papain at pH 4.5 has been investigated. Cleavage of Lys(1313)-Glu resulted in two major products, which could be separated by gel chromatography: a large disulfide bridged fragment set nearly the size of intact alpha 2M X MA, and an 18 kDa fragment, constituting the carboxy-terminal domain of alpha 2M. This domain contained the receptor recognition site, exposed as a result of cleavage of the internal beta-cysteinyl-gamma-glutamyl thiol esters in alpha 2M. Compared with alpha 2M-trypsin complex the apparent affinity for binding to rat hepatocyte receptors was 0.1 and 2% at 4 and 37 degrees C, respectively. The receptor-binding domain presumably forms a compact globular beta-barrel-type structure, stable at pH 2.5-9.0. Chemical modification experiments suggest that receptor binding is contributed by a determinant formed by the precise folding of the polypeptide chain.  相似文献   

8.
9.
In this study we investigated the molecular mechanism by which the Orp1 (Gpx3) protein in Saccharomyces cerevisiae senses and reacts with hydrogen peroxide. Upon exposure to H(2)O(2) Orp1(Cys36) forms a disulfide-bonded complex with the C-terminal domain of the Yap1 protein (Yap1-cCRD). We used 4-nitrobenzo-2-oxa-1,3-diazole to identify a cysteine sulfenic acid (Cys-SOH) modification that forms on Cys(36) of Orp1(Cys36) upon exposure to H(2)O(2). Under similar conditions, neither Cys(82) of Orp1(Cys82) nor Cys(598) of Yap1 forms Cys-SOH. A homology-based molecular model of Orp1 suggests that the structure of the active site of Orp1 is similar to that found in mammalian selenocysteine glutathione peroxidases. Proposed active site residues Gln(70) and Trp(125) form a catalytic triad with Cys(36) in the Orp1 molecular model. The remainder of the active site pocket is formed by Phe(38), Asn(126), and Phe(127), which are evolutionarily conserved residues. We made Q70A and W125A mutants and tested the ability of these mutants to form Cys-SOH in response to H(2)O(2). Both mutants were unable to form Cys-SOH and did not form a H(2)O(2)-inducible disulfide-bonded complex with Yap1-cCRD. The pK(a) of Cys(36) was determined to be 5.1, which is 3.2 pH units lower than that of a free cysteine (8.3). In contrast, Orp1 Cys(82) (the resolving cysteine) has a pK(a) value of 8.3. The pK(a) of Cys(36) in the Q70A and W125A mutants is also 8.3, demonstrating the importance of these residues in modulating the nucleophilic character of Cys(36). Finally, we show that S. cerevisiae strains with ORP1 Q70A and W125A mutations are less tolerant to H(2)O(2) than those containing wild-type ORP1. The results of our study suggest that attempts to identify novel redox-regulated proteins and signal transduction pathways should focus on characterization of low pK(a) cysteines.  相似文献   

10.
The glycated form of a basic recombinant humanized monoclonal antibody (rhuMAb) was separated and quantitated by boronate affinity chromatography using optimized shielding reagents. Characterization on the isolated glycated material by peptide mapping analysis, using liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectrometry (MS/MS) sequencing techniques, identified eight reactive lysine primary amine sites. The glycation reaction extent was similar among the various reactive sites, ranging from approximately 1 to 12%, and a single histidine residue separated the most and least reactive sites. Boronate chromatography run in a linear gradient mode separated monoglycated rhuMAb from higher order glycated species and indicated that the majority ( approximately 90%) of glycated rhuMAb is monoglycated. Low-level glycation on a heavy chain lysine located within a complementarity-determining region (CDR) did not significantly affect binding activity in potency measurements. The glycated forms also behaved as slightly more acidic than the nonglycated antibody in charge-based separation techniques, observable by capillary isoelectric focusing (cIEF) and ion exchange chromatography (IEC). The boronate column has significantly increased retention of aggregated rhuMAb material under separation conditions optimized for the monomer form. Recombinant protein glycation initially occurred during production in mammalian cell culture, where feed sugar and protein concentrations contribute to the total overall glycation on this antibody product.  相似文献   

11.
We have reported that prothrombin (1 microm) is able to replace high molecular weight kininogen (45 nm) as a cofactor for the specific binding of factor XI to the platelet (Baglia, F. A., and Walsh, P. N. (1998) Biochemistry 37, 2271-2281). We have also determined that prothrombin fragment 2 binds to the Apple 1 domain of factor XI at or near the site where high molecular weight kininogen binds. A region of 31 amino acids derived from high molecular weight kininogen (HK31-mer) can also bind to factor XI (Tait, J. F., and Fujikawa, K. (1987) J. Biol. Chem. 262, 11651-11656). We therefore investigated the role of prothrombin fragment 2 and HK31-mer as cofactors in the binding of factor XI to activated platelets. Our experiments demonstrated that prothrombin fragment 2 (1 microm) or the HK31-mer (8 microm) are able to replace high molecular weight kininogen (45 nm) or prothrombin (1 microm) as cofactors for the binding of factor XI to the platelet. To localize the platelet binding site on factor XI, we used mutant full-length recombinant factor XI molecules in which the platelet binding site in the Apple 3 domain was altered by alanine scanning mutagenesis. The recombinant factor XI with alanine substitutions at positions Ser(248), Arg(250), Lys(255), Leu(257), Phe(260), or Gln(263) were defective in their ability to bind to activated platelets. Thus, the interaction of factor XI with platelets is mediated by the amino acid residues Ser(248), Arg(250), Lys(255), Leu(257), Phe(260), and Gln(263) within the Apple 3 domain.  相似文献   

12.
A 74-bp DNA sequence coding for the pre sequence of human preproinsulin and containing EcoRI termini was synthesized by the chemical enzymatic method, joined with previously synthesized proinsulin DNA, and cloned in the M 13mp8 vector. A clone pNB82 -121 was identified by DNA sequence which confirmed the correct orientation of the pre sequence to the proinsulin DNA. The EcoRI site at the junction of pre- and proinsulin DNA was eliminated by removing a triplet ATT using a synthetic 19-mer primer. To simplify preproinsulin isolation and to study its expression in the M 13 system, a 25-bp affinity leader sequence coding for (glu)7 was inserted at the remaining EcoRI site; this put the preproinsulin DNA in a correct reading frame with the AUG initiation codon of beta-galactosidase. Preproinsulin was expressed under lac promoter control as analyzed by a radioimmunoassay (RIA) against C-peptide.  相似文献   

13.
This study describes the isolation of angiotensin I converting enzyme and antioxidative peptides from head protein hydrolysate of red scorpionfish (Scorpaena notata) prepared by treatment with a protease from the fungus Penicillium digitatum. After ultrafiltration, three peptides were isolated by a two-step procedure: size exclusion chromatography on a Toyopearl HW-40 followed by reversed-phase high performance liquid chromatography (RP-HPLC) with a high purification yield of 2.22 mg of peptide/g of initial protein. Active peptides were then identified by nanoscale liquid chromatography coupled to tandem mass spectrometry (nanoLC/MS–MS), corresponding to the following sequences: Gln–Gln–Pro–His–Ser–Arg–Ser–Lys–Gly–Phe–Pro–Gly–Pro (1424.724 Da), Gly–Gln–Lys–Ser–Val–Pro–Glu–Val–Arg (1000.565 Da) and Val–Glu–Gly–Lys–Ser–Pro–Asn–Val (830.448 Da). Peptides D-I, E-I and F-I showed high angiotensin-I converting enzyme inhibitory activity with an IC50 values of 0.98, 1.69 and 1.44 µM, respectively as well as a synergistic antioxidant activity between the different fractions. Thus, we have demonstrated that underutilized wastes can be valorized by production of peptides that can be used as potential therapeutic compounds active against oxidative stress and hypertension.  相似文献   

14.
Hirudin, a thrombin-specific inhibitor, comprises a compact amino-terminal core domain (residues 1-52) and a disordered acidic carboxyl-terminal tail (residues 53-65). An array of core fragments were prepared from intact recombinant hirudin by deletion of various lengths of its carboxyl-terminal tail on selective enzymatic cleavage. Hir1-56 and Hir1-53 were produced by pepsin digestion at Phe56-Glu57 and Asp53-Gly54. Hir1-52 was generated by Asp-N cleavage at Asn52-Asp53. Hir1-49 was prepared by cleavage of Gln49-Ser50 by chymotrypsin, elastase, and thermolysin. In addition, Hir1-62 (containing part of the carboxyl-terminal tail) was derived from Hir1-65 by selective removal of the three carboxyl-terminal amino acids using carboxypeptidase A. Hirudin amino-terminal core fragments were stable at extreme pH (1.47 and 12.6), high temperature (95 degrees C), and resistant to attack by various proteinases. For instance, following 24-h incubation with an equal weight of pepsin, the covalent structure of Hir1-52 remained intact and its anticoagulant activity unaffected. Unlike intact hirudin (Hir1-65) the inhibitory potency of which is a consequence of concerted binding of its amino-terminal and carboxyl-terminal domains to the active site and the fibrinogen recognition site of thrombin, the core fragments block only the active site of thrombin with binding constants of 19 nM (Hir1-56), 35 nM (Hir1-52), and 72 nM (Hir1-49). As an anticoagulant Hir1-56 is about 2-, 4-, and 30-fold more potent (on a molar basis) than Hir1-52, Hir1-49, and Hir1-43, respectively. Hir1-56 was also about 15-fold more effective than the most potent carboxyl-terminal fragment of hirudin, sulfated-Hir54-65, although they bind to independent sites on thrombin. The potential advantages of hirudin core fragments as antithrombotic agents are discussed in this report.  相似文献   

15.
16.
Protein glycation through Maillard reaction (MR) is a fundamental reaction both in foods and in the human body. The first step of the reaction is the formation of Amadori product (AP) that is converted into intermediate and advanced MR products during reaction development. Although the MR is not an enzymatic reaction, a certain degree of specificity in the glycation site has been observed. In the present study, we have monitored the glycation of different lysine-containing dipeptides to evaluate the influence on the NH(2) reactivity of the neighboring amino acid.Lysine dipeptides were reacted with glucose, galactose, lactose and maltose. The formation and identification of glycated compounds were monitored by mass spectrometry (MALDI-TOF and ESI-MS/MS) and by HPLC of their Fmoc derivatives. MS/MS analysis showed that the glucose APs formed on dipeptides have a characteristic fragmentation pattern: the fragment at [M - 84](+) due to the formation of pyrylium and furylium ion is mainly present in the monoglucosylated form, while the [M - 162](+) and the [M - 324](+) are more evident in the fragmentation pattern of the diglucosylated forms.The nature of the vicinal amino acids strongly affects lysine reactivity towards the different carbohydrates: the presence of hydrophobic residues such as Ile, Leu, Phe strongly increases lysine reactivity. Contrasting results were obtained with basic residues. The Lys-Arg dipeptide was among the most reactive while the Lys-Lys was not.  相似文献   

17.
The 1.8-A resolution structure of the ATP-Mg(2+)-Ca(2+)-pyruvate quinary complex of Escherichia coli phosphoenolpyruvate carboxykinase (PCK) is isomorphous to the published complex ATP-Mg(2+)-Mn(2+)-pyruvate-PCK, except for the Ca(2+) and Mn(2+) binding sites. Ca(2+) was formerly implicated as a possible allosteric regulator of PCK, binding at the active site and at a surface activating site (Glu508 and Glu511). This report found that Ca(2+) bound only at the active site, indicating that there is likely no surface allosteric site. (45)Ca(2+) bound to PCK with a K(d) of 85 micro M and n of 0.92. Glu508Gln Glu511Gln mutant PCK had normal activation by Ca(2+). Separate roles of Mg(2+), which binds the nucleotide, and Ca(2+), which bridges the nucleotide and the anionic substrate, are implied, and the catalytic mechanism of PCK is better explained by studies of the Ca(2+)-bound structure. Partial trypsin digestion abolishes Ca(2+) activation (desensitizes PCK). N-terminal sequencing identified sensitive sites, i.e., Arg2 and Arg396. Arg2Ser, Arg396Ser, and Arg2Ser Arg396Ser (double mutant) PCKs altered the kinetics of desensitization. C-terminal residues 397 to 540 were removed by trypsin when wild-type PCK was completely desensitized. Phe409 and Phe413 interact with residues in the Ca(2+) binding site, probably stabilizing the C terminus. Phe409Ala, DeltaPhe409, Phe413Ala, Delta397-521 (deletion of residues 397 to 521), Arg396(TAA) (stop codon), and Asp269Glu (Ca(2+) site) mutations failed to desensitize PCK and, with the exception of Phe409Ala, appeared to have defects in the synthesis or assembly of PCK, suggesting that the structure of the C-terminal domain is important in these processes.  相似文献   

18.
The CMY-2 beta-lactamase, a plasmid determined class C cephalosporinase, was shown to be susceptible to inhibition by tazobactam (K(i)=40 microM). The reaction product(s) of CMY-2 beta-lactamase with the beta-lactamase inhibitor tazobactam were analyzed by electrospray ionization/mass spectrometry (ESI/MS) to characterize the prominent intermediates of the inactivation pathway. The ESI/MS determined mass of CMY-2 beta-lactamase was 39851+/-3 Da. After inactivating CMY-2 beta-lactamase with excess tazobactam, a single species, M(r)=39931+/-3.0, was detected. Comparison of the peptide maps from tryptic digestion of the native enzyme and the inactivated beta-lactamase followed by LC/MS identified two 22 amino acid peptides containing the active site Ser64 modified by a fragment of tazobactam. These two peptides were increased in mass by 70 and 88 Da, respectively. UV difference spectra following inactivation revealed the presence of a new species with a 302 nm lambda(max). Based upon the increase in molecular mass of the tazobactam inactivated CMY-2 beta-lactamase, we propose that during the inactivation of this beta-lactamase by tazobactam an imine is formed. Tautomerization forms the spectrally observed enamine. Hydrolysis generates the covalently attached malonyl semialdehyde, its hydrate, or an enol. This work provides information on the mass of a stable enzyme intermediate of a class C beta-lactamase inactivated by tazobactam and, for the first time, unequivocal evidence that a cross-linked species is not required for apparent inactivation.  相似文献   

19.
Platelet glycoprotein Ib (GpIb) mediates interaction with both von Willebrand factor and thrombin. Thrombin binds to GpIb via its heparin-binding site (HBS) (De Candia, E., De Cristofaro, R., De Marco, L., Mazzucato, M., Picozzi, M., and Landolfi, R. (1997) Thromb. Haemostasis 77, 735-740; De Cristofaro, R., De Candia, E., Croce, G., Morosetti, R., and Landolfi, R. (1998) Biochem. J. 332, 643-650). To identify the thrombin-binding domain on GpIbalpha, we examined the effect of GpIbalpha(1-282), a GpIbalpha fragment released by the cobra venom mocarhagin on the heparin-catalyzed rate of thrombin inhibition by antithrombin III (AT). GpIbalpha(1-282) inhibited the reaction in a dose-dependent and competitive fashion. In contrast, the GpIbalpha(1-271) fragment, produced by exposing GpIbalpha(1-282) to carboxypeptidase Y, had no effect on thrombin inhibition by the heparin-AT complex. Measurements of the apparent equilibrium constant of the GpIbalpha(1-282) binding to thrombin as a function of different salts (NaCl and tetramethyl-ammonium chloride) concentration (0.1-0.2 M) indicated a large salt dependence (Gamma(+/-) = -4.5), similar to that pertaining to the heparin binding to thrombin. The importance of thrombin HBS in its interaction with GpIbalpha was confirmed using DNA aptamers, which specifically bind to either HBS (HD22) or the fibrinogen recognition site of thrombin (HD1). HD22, but not HD1, inhibited thrombin binding to GpIbalpha(1-282). Furthermore, the proteolytic derivative gamma(T)-thrombin, which lacks the fibrinogen recognition site, binds to GpIbalpha via its intact HBS in a reaction that is inhibited by HD22. Neither alpha- nor gamma(T)-thrombin bound to GpIbalpha(1-271), suggesting that the Asp(272)-Glu(282) region of GpIbalpha may act as a "heparin-like" ligand for the thrombin HBS, thereby inhibiting heparin binding to thrombin. It was also demonstrated that intact platelets may dose-dependently inhibit the heparin-catalyzed thrombin inhibition by AT at enzyme concentrations <5 nM. Altogether, these findings show that thrombin HBS binds to the region of GpIbalpha involving the Asp(272)-Glu(282) segment, protecting the enzyme from the inactivation by the heparin-AT system.  相似文献   

20.
Two immunodominant gametocyte antigens from Eimeria maxima with M(r) 56 kDa and M(r) 82 kDa have been identified previously as potential candidates for inclusion in a recombinant subunit vaccine against coccidiosis in poultry. Here, these proteins have been biochemically characterised, immunolocalised within the parasite, and sequences for their amino termini determined. These antigens co-purify by affinity chromatography suggesting an interaction with each other. However, separation of the proteins by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in the absence of beta-mercaptoethanol did not reveal the presence of inter-chain disulphide bonds. The true masses of the 56 and 82 kDa antigens are 52450 and 62450 Da, respectively, as determined by mass spectrometry. TX-114 separations suggested that they exist, in part, as soluble proteins within the parasite, and immunolocalisation studies indicated that they were found in the wall forming bodies of macrogametocytes. Separation of the proteins by 2D SDS-PAGE revealed that they are acidic in nature and heterogeneous in charge. Cleavage by neuraminidase and O-glycosidase indicated that the presence of O-linked glycans contributed to some of the charge microheterogeneity of both proteins. The absence of these O-glycans however, did not abolish antibody recognition, suggesting that the development of a recombinant subunit vaccine is possible. A more extensive investigation of the carbohydrate moieties of these proteins revealed that they also possess glucose, fucose, mannose and galactose. There was no evidence for the presence of N-linked glycans. The 56 and 82 kDa antigens were separated from a mixture of proteins in a crude gametocyte lysate by 2D SDS-PAGE, the proteins isolated, and the N-terminus amino acid sequence determined. They showed no homology to each other at the N-terminus, or to any other previously characterised protein. Characterisation of these novel proteins has provided further insights into the molecular mechanisms of gametocyte differentiation in E. maxima.  相似文献   

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