共查询到20条相似文献,搜索用时 31 毫秒
1.
Samel M Vija H Subbi J Siigur J 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2003,135(4):575-582
We have previously shown that Vipera berus berus venom contains several factor X activating enzymes. In the present study we have investigated one of them. The enzyme was separated from venom by gel filtration on Sephadex G-100 superfine and chromatography on agarose HPS-7 and phenyl-agarose. The enzyme is a glycosylated metalloproteinase containing hexoses, hexosamines and neuraminic acid. The purified factor X activating enzyme consists of two equal chains (59 kDa). The specificity studies have shown that enzyme is nonspecific factor X activating proteinase hydrolysing also proteins such as azocasein, gelatin and fibrinogen. The enzyme hydrolyses oxidized insulin B-chain at the positions Ala14–Leu15 and Tyr16–Leu17 but it is inactive on fibrin, plasminogen and prothrombin. We used 8–10 amino acid residues containing peptides, which reproduce the sequence around the cleavage sites in factor X, factor IX and fibrinogen, as potential substrates for enzyme. Cleavage products of peptide hydrolysis were determined by MALDI-TOF MS. The peptide Asn–Asn–Leu–Thr–Arg–Ile–Val–Gly–Gly—factor X fragment was cleaved by enzyme at positions Leu3–Thr4 and Arg5–Ile6. The fibrinogen peptide fragment Glu–Tyr–His–Thr–Glu–Lys–Leu–Val–Thr–Ser was hydrolysed at position Lys6–Leu7. 相似文献
2.
Siigur E Aaspõllu A Trummal K Tõnismägi K Tammiste I Kalkkinen N Siigur J 《Biochimica et biophysica acta》2004,1702(1):41-51
Vipera lebetina venom contains specific coagulant Factor X activator (VLFXA) that cleaves the Arg52-Ile53 bond in the heavy chain of human factor X. VLFXA is a glycoprotein that is composed of a heavy chain (HC) and two light chains (LC) linked by disulfide bonds. The complete amino acid sequences of the three chains of the factor X activator from V. lebetina snake venom are deduced from the nucleotide sequences of cDNAs encoding these chains. The full-length cDNA (2347 bp) sequence of the HC encodes an open reading frame (ORF) of 612 amino acids that includes signal peptide, propeptide and mature metalloproteinase with disintegrin-like and cysteine-rich domains. The light chain LC1 contains 123 and LC2 135 amino acid residues. Both light chains belong to the class of C-type lectin-like proteins. The N-termini of VLFXA chains and inner sequences of peptide fragments detected by liquid chromatography-electrospray ionization tandem mass spectrometry (LC MS/MS) from protein sequence are 100% identical to the sequences deduced from the cDNA. The molecular masses of tryptic fragments of VLFXA chains analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) also confirm the protein sequences deduced from the cDNAs. These are the first cloned factor X activator heavy and light chains. We demonstrate that the heavy and light chains are synthesized from different genes. 相似文献
3.
Gerry A. F. Nicolaes Paul E. Bock Kenneth Segers Karin C. A. A. Wildhagen Bj?rn Dahlb?ck Jan Rosing 《The Journal of biological chemistry》2010,285(30):22890-22900
Activated protein C (APC) down-regulates thrombin formation through proteolytic inactivation of factor Va (FVa) by cleavage at Arg506 and Arg306 and of factor VIIIa (FVIIIa) by cleavage at Arg336 and Arg562. To study substrate recognition by APC, active site-mutated APC (APC(S360A)) was used, which lacks proteolytic activity but exhibits anticoagulant activity. Experiments in model systems and in plasma show that APC(S360A), and not its zymogen protein C(S360A), expresses anticoagulant activities by competing with activated coagulation factors X and IX for binding to FVa and FVIIIa, respectively. APC(S360A) bound to FVa with a KD of 0.11 ± 0.05 nm and competed with active site-labeled Oregon Green activated coagulation factor X for binding to FVa. The binding of APC(S360A) to FVa was not affected by protein S but was inhibited by prothrombin. APC(S360A) binding to FVa was critically dependent upon the presence of Arg506 and not Arg306 and additionally required an active site accessible to substrates. Inhibition of FVIIIa activity by APC(S360A) was >100-fold less efficient than inhibition of FVa. Our results show that despite exosite interactions near the Arg506 cleavage site, binding of APC(S360A) to FVa is almost completely dependent on Arg506 interacting with APC(S360A) to form a nonproductive Michaelis complex. Because docking of APC to FVa and FVIIIa constitutes the first step in the inactivation of the cofactors, we hypothesize that the observed anticoagulant activity may be important for in vivo regulation of thrombin formation. 相似文献
4.
One novel venom factor was isolated and purified from the venom of Taiwan habu (Trimeresurus mucrosquamatus) using two consecutive anion-exchange and gel-filtration chromatographies followed by cation-exchange HPLC. Further characterization of the purified protein indicated that it lacks the proteolytic activity toward fibrinogen molecules, suggesting that this protein factor does not belong to the familes of metalloproteinases and thrombin-like serine proteases commonly found in the crude venoms of various crotalid snakes. The purified protein exists as a native dimeric protein of 26 kDa, consisting of two closely similar subunits of 16 and 13 kDa, held together by disulfide linkage. N-Terminal sequence analysis revealed that both chains are homologous to each other at the N-terminal fragment and also similar to the factors IX/X-binding protein isolated fromTrimeresurus flavoviridis and botrocetin fromBothrops jararaca. This study points to the existence of one new two-chain venom factor without fibrinogenase activity from Taiwan habu, which, in contrast to botrocetin, promotes platelet agglutination even in the absence of von Willebrand factor. Unlike factors IX/X-binding proteins, it did not show affinity to coagulation factors IX and X in the presence of Ca2+ ion. It also shows no inhibition on thrombin, in contrast with bothrojaracin, a thrombin inhibitor isolated fromBothrops jararaca venom. We have therefore named this novel venom factortrimecetin to distinguish it from some structurally related venom factors present in various crotalid and viperid snakes. 相似文献
5.
Yipeng Geng Ingrid M. Verhamme Amanda Messer Mao-fu Sun Stephen B. Smith S. Paul Bajaj David Gailani 《The Journal of biological chemistry》2012,287(45):38200-38209
During blood coagulation, the protease factor XIa (fXIa) activates factor IX (fIX). We describe a new mechanism for this process. FIX is cleaved initially after Arg145 to form fIXα, and then after Arg180 to form the protease fIXaβ. FIXα is released from fXIa, and must rebind for cleavage after Arg180 to occur. Catalytic efficiency of cleavage after Arg180 is 7-fold greater than for cleavage after Arg145, limiting fIXα accumulation. FXIa contains four apple domains (A1–A4) and a catalytic domain. Exosite(s) on fXIa are required for fIX binding, however, there is lack of consensus on their location(s), with sites on the A2, A3, and catalytic domains described. Replacing the A3 domain with the prekallikrein A3 domain increases Km for fIX cleavage after Arg145 and Arg180 25- and ≥90-fold, respectively, and markedly decreases kcat for cleavage after Arg180. Similar results were obtained with the isolated fXIa catalytic domain, or fXIa in the absence of Ca2+. Forms of fXIa lacking the A3 domain exhibit 15-fold lower catalytic efficiency for cleavage after Arg180 than for cleavage after Arg145, resulting in fIXα accumulation. Replacing the A2 domain does not affect fIX activation. The results demonstrate that fXIa activates fIX by an exosite- and Ca2+-mediated release-rebind mechanism in which efficiency of the second cleavage is enhanced by conformational changes resulting from the first cleavage. Initial binding of fIX and fIXα requires an exosite on the fXIa A3 domain, but not the A2 or catalytic domain. 相似文献
6.
Characterization of a protein C activator from Agkistrodon contortrix contortrix venom 总被引:2,自引:0,他引:2
W Kisiel S Kondo K J Smith B A McMullen L F Smith 《The Journal of biological chemistry》1987,262(26):12607-12613
The protease from Southern Copperhead venom that activates protein C was purified to homogeneity by sulfopropyl (SP)-Sephadex C-50 ion-exchange chromatography, Sephadex G-150 gel filtration, and Mono-S fast protein liquid chromatography. The purified enzyme is a glycoprotein containing 16% carbohydrate, and migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular mass of 40,000 kDa. The enzyme is composed of a single polypeptide chain possessing an NH2-terminal sequence of Val-Ile-Gly-Gly-Asp-Glu-Cys-Asn-Ile-Asn-Glu-His. The purified venom protein C activator hydrolyzed several tripeptide p-nitroanilides. The amidolytic and proteolytic activities of the enzyme were readily inhibited by phenylmethanesulfonyl fluoride, p-amidinophenylmethanesulfonyl fluoride, chloromethyl ketones, and human antithrombin III. Covalent binding of diisopropyl fluorophosphate to the enzyme was confirmed using a tritium-labeled preparation of the inhibitor. The venom protease readily activated human and bovine protein C at 1:1000 enzyme:substrate weight ratio. The protease also cleaved human prothrombin, factor X, factor IX, factor VII, and fibrinogen. Prothrombin coagulant activity decreased upon incubation with the venom protease, and the rate of this reaction was reduced in the presence of calcium. Factor X and factor IX coagulant activity increased upon incubation with the venom protease in the presence of calcium, and decreased in the absence of calcium. Human factor VII clotting activity decreased slightly upon incubation with the venom protease. Although the venom protease did not clot human fibrinogen, it nonetheless cleaved the A alpha chain of fibrinogen, and this cleavage appeared to be associated with a measurable increase in the clottability of the protease-treated fibrinogen by thrombin. These data demonstrate that the protein C activator from Southern Copperhead venom is a typical serine protease with a relatively broad specificity. 相似文献
7.
S. -H. Chiou K. -F. Huang L. -P. Chow A. Tsugita S. -H. Wu 《Journal of Protein Chemistry》1996,15(7):667-674
One novel venom factor was isolated and purified from the venom of Taiwan habu (Trimeresurus mucrosquamatus) using two consecutive anion-exchange and gel-filtration chromatographies followed by cation-exchange HPLC. Further characterization of the purified protein indicated that it lacks the proteolytic activity toward fibrinogen molecules, suggesting that this protein factor does not belong to the familes of metalloproteinases and thrombin-like serine proteases commonly found in the crude venoms of various crotalid snakes. The purified protein exists as a native dimeric protein of 26 kDa, consisting of two closely similar subunits of 16 and 13 kDa, held together by disulfide linkage. N-Terminal sequence analysis revealed that both chains are homologous to each other at the N-terminal fragment and also similar to the factors IX/X-binding protein isolated fromTrimeresurus flavoviridis and botrocetin fromBothrops jararaca. This study points to the existence of one new two-chain venom factor without fibrinogenase activity from Taiwan habu, which, in contrast to botrocetin, promotes platelet agglutination even in the absence of von Willebrand factor. Unlike factors IX/X-binding proteins, it did not show affinity to coagulation factors IX and X in the presence of Ca2+ ion. It also shows no inhibition on thrombin, in contrast with bothrojaracin, a thrombin inhibitor isolated fromBothrops jararaca venom. We have therefore named this novel venom factortrimecetin to distinguish it from some structurally related venom factors present in various crotalid and viperid snakes. 相似文献
8.
Aoi Inosaki Akikazu Yasuda Tetsuro Shinada Yasufumi Ohfune Hideharu Numata Sakiko Shiga 《Comparative biochemistry and physiology. Part A, Molecular & integrative physiology》2010,155(2):190-199
Neuropeptides in neurosecretory cells of the pars intercerebralis (PI) and pars lateralis (PL) in the brain, and those in the corpus cardiacum–hypocerebral ganglion complex (CC-HG) and corpus allatum (CA) were examined by mass spectrometry and immunocytochemistry in adult females of the blowfly, Protophormia terraenovae. By using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and electrospray ionization quadrupole orthogonal acceleration time-of-flight mass spectrometry (ESI-Q-Tof MS) and MS/MS, 4 peptides (including myosuppressin and SIFamide) were detected in the PI, 12 peptides (including [Arg7]-corazonin and [Arg7]-corazonin3–11) in the PL, 13 peptides (including myosuppressin, [Arg7]-corazonin and [Arg7]-corazonin3–11) in the CC-HG, and 6 peptides in the CA. MALDI-TOF MS analysis of each tissue or organ was made in about 20 flies under diapause-inducing (LD 12:12 at 20 °C) and diapause-averting conditions (LD 18:6 at 25 °C). These molecular ion peaks did not distinctively differ between diapause-inducing and diapause-averting conditions. A peptide with an m/z value at 1395.1 was purified from 240 brains and the 2nd–10th amino acids were sequenced as –YRKPPFNGS–, corresponding to a partial sequence of SIFamide. Only two pairs of somata in the PI were immunoreactive to antisera against SIFamide, which were local neurons widely extending fibers throughout the brain neuropils. 相似文献
9.
10.
Eduardo S. Kitano Thalita C. Garcia Milene C. Menezes Alexandre K. Tashima André Zelanis Solange M.T. Serrano 《Biochimie》2013
Snake venom serine proteinases (SVSPs) may affect hemostatic pathways by specifically activating components involved in coagulation, fibrinolysis and platelet aggregation or by unspecific proteolytic degradation. In this study, we purified and characterized an SVSP from Bothrops cotiara venom, named cotiarinase, which generated thrombin upon incubation with prothrombin. Cotiarinase was isolated by a two-step procedure including gel-filtration and cation-exchange chromatographies and showed a single protein band with a molecular mass of 29 kDa by SDS-polyacrylamide gel electrophoresis under reducing conditions. Identification of cotiarinase by mass spectrometric analysis revealed peptides that matched sequences of viperid SVSPs. Cotiarinase did not show fibrinogen-clotting, platelet-aggregating, fibrinogenolytic and factor X activating activities. Upon incubation with prothrombin the generation of thrombin was detected using the peptide substrate d-Phe-Pip-Arg-pNA. Moreover, mass spectrometric identification of prothrombin fragments generated by cotiarinase in the absence of co-factors (phospholipids, factor Va, factor Xa and Ca2+ ions), indicated the limited proteolysis of this protein to release prothrombin 1, fragment 1 and thrombin. Cotiarinase is a novel SVSP that acts on prothrombin to release active thrombin that does not match any group of the current classification of snake venom prothrombin activators. 相似文献
11.
Maitreyee Sharma Janaki Krishnamurthy Iyer Norrapat Shih Munmi Majumder Venkata Satish Kumar Mattaparthi Rupak Mukhopadhyay Robin Doley 《PloS one》2016,11(4)
In the present study a major protein has been purified from the venom of Indian Daboia russelii russelii using gel filtration, ion exchange and Rp-HPLC techniques. The purified protein, named daboxin P accounts for ~24% of the total protein of the crude venom and has a molecular mass of 13.597 kDa. It exhibits strong anticoagulant and phospholipase A2 activity but is devoid of any cytotoxic effect on the tested normal or cancerous cell lines. Its primary structure was deduced by N-terminal sequencing and chemical cleavage using Edman degradation and tandem mass spectrometry. It is composed of 121 amino acids with 14 cysteine residues and catalytically active His48 -Asp49 pair. The secondary structure of daboxin P constitutes 42.73% of α-helix and 12.36% of β-sheet. It is found to be stable at acidic (pH 3.0) and neutral pH (pH 7.0) and has a Tm value of 71.59 ± 0.46°C. Daboxin P exhibits anticoagulant effect under in-vitro and in-vivo conditions. It does not inhibit the catalytic activity of the serine proteases but inhibits the activation of factor X to factor Xa by the tenase complexes both in the presence and absence of phospholipids. It also inhibits the tenase complexes when active site residue (His48) was alkylated suggesting its non-enzymatic mode of anticoagulant activity. Moreover, it also inhibits prothrombinase complex when pre-incubated with factor Xa prior to factor Va addition. Fluorescence emission spectroscopy and affinity chromatography suggest the probable interaction of daboxin P with factor X and factor Xa. Molecular docking analysis reveals the interaction of the Ca+2 binding loop; helix C; anticoagulant region and C-terminal region of daboxin P with the heavy chain of factor Xa. This is the first report of a phospholipase A2 enzyme from Indian viper venom which targets both factor X and factor Xa for its anticoagulant activity. 相似文献
12.
Degradation of coagulation proteins by an enzyme from Malayan pit viper (Akistrodon rhodostoma) venom 总被引:2,自引:0,他引:2
Three hydrolases from the crude venom of the Malayan pit viper (Akistrodon rhodostoma) can be differentiated. The first, which we designate ARH alpha, is the well-known fibrinogenolytic enzyme ancrod. The second, ARH beta, which has not been described previously, is identified by its electrophoretic mobility after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), by its ability to hydrolyze H-D-phenylalanyl-L-piperyl-L-arginyl-rho-nitroanilide, and by inhibition of its activity by diisopropyl phosphorofluoridate. The third, ARH gamma, also previously not described, has been purified by using gel permeation and ion-exchange chromatography and preparative PAGE. Chemical, electrophoretic, and hydrodynamic data indicate that it is a single-chain, nonglobular glycoprotein with a molecular weight of 25,600. ARH gamma catalyzes the degradation of several plasma vitamin K dependent coagulation factors, including factor IX, factor X, prothrombin, and protein C. The products are electrophoretically similar to factor IXa beta, factor Xa, thrombin, and activated protein C, respectively. However, these products contain little or no enzymatic activity. ARH gamma-degraded factor IX, factor X, prothrombin, and protein C can be subsequently activated by factor XIa, Russell's viper venom X coagulant protein, crude taipan snake venom, and thrombin, respectively. The N-terminal sequence of the peptides resulting from the ARH gamma digest of porcine factor IX shows that at least three bonds are hydrolyzed: (1) at position 152, seven residues from the Arg145-Ala146 factor XIa cleavage site; (2) at position 167 within the factor IX activation peptide; and (3) at position 177, three residues from the Arg180-Val181 factor XIa cleavage site. The degradation of factor IX by ARH gamma is not affected by several serine protease inhibitors. ARH gamma catalyzes the degradation of both the heavy and light chains of porcine factor VIII which results in the inability of thrombin to activate factor VIII. ARH gamma also catalyzes the degradation of porcine antithrombin III which abolishes its ability to inhibit thrombin. These findings may have relevance to studies of hemostatic derangements following envenomation by this snake. Additionally, several novel coagulation factor derivatives have been generated for structure-function studies. 相似文献
13.
Longguang Jiang Emil Oldenburg Tobias Kromann-Hansen Peng Xu Jan K. Jensen Peter A. Andreasen Mingdong Huang 《Biochimica et Biophysica Acta (BBA)/General Subjects》2018,1862(9):2017-2023
Some peptide sequences can behave as either substrates or inhibitors of serine proteases. Working with a cyclic peptidic inhibitor of the serine protease urokinase-type plasminogen activator (uPA), we have now demonstrated a new mechanism for an inhibitor-to-substrate switch. The peptide, CSWRGLENHAAC (upain-2), is a competitive inhibitor of human uPA, but is also slowly converted to a substrate in which the bond between Arg4 and Gly5 (the P1-P1′ bond) is cleaved. Substituting the P2 residue Trp3 to an Ala or substituting the P1 Arg4 residue with 4-guanidino-phenylalanine strongly increased the substrate cleavage rate. We studied the structural basis for the inhibitor-to-substrate switch by determining the crystal structures of the various peptide variants in complex with the catalytic domain of uPA. While the slowly cleaved peptides bound clearly in inhibitory mode, with the oxyanion hole blocked by the side chain of the P3′ residue Glu7, peptides behaving essentially as substrates with a much accelerated rate of cleavage was observed to be bound to the enzyme in substrate mode. Our analysis reveals that the inhibitor-to-substrate switch was associated with a 7?Å translocation of the P2 residue, and we conclude that the inhibitor-to-substrate switch of upain-2 is a result of a major conformational change in the enzyme-bound state of the peptide. This conclusion is in contrast to findings with so-called standard mechanism inhibitors in which the inhibitor-to-substrate switch is linked to minor conformational changes in the backbone of the inhibitory peptide stretch. 相似文献
14.
Mucha Piotr Rekowski Piotr Szyk Agnieszka Kupryszewski Gotfryd Giel-Pietraszuk Małgorzata Barciszewski Jan 《International journal of peptide research and therapeutics》1998,5(5-6):345-348
The Tat wild-type fragment of sequence Arg49-Lys-Lys-Arg52-Arg-Gln-Arg-Arg-Arg57-NH2 (labelled as Tat1) and three analogues of this fragment with the substitution Arg52 → D-Arg52 (labelled as Tat2) or L-citrulline (Cit) (labelled as Tat3) or L-ornithine (Orn) (labelled as Tat4) were synthesized to study Tat-TAR RNA HIV-1 (27-nucleotide fragment of sequence 5′-AGAUCUGAGCCUGGAGCUCUCU-3′) interactions by circular dichroism. α-helical structure was the most readily adopted by the Tat3 analogue with Arg52 → Cit substitution. All the peptides investigated caused conformational changes in the TAR structure. The most dramatic changes were observed for the Tat2-TAR complex. 相似文献
15.
Hanjiang Yang Felix Christof Wahlmüller Bettina Sarg Margareta Furtmüller Margarethe Geiger 《The Journal of biological chemistry》2015,290(5):3081-3091
Protein C inhibitor (PCI) is a serpin with broad protease reactivity. It binds glycosaminoglycans and certain phospholipids that can modulate its inhibitory activity. PCI can penetrate through cellular membranes via binding to phosphatidylethanolamine. The exact mechanism of PCI internalization and the intracellular role of the serpin are not well understood. Here we showed that testisin, a glycosylphosphatidylinositol-anchored serine protease, cleaved human PCI and mouse PCI (mPCI) at their reactive sites as well as at sites close to their N terminus. This cleavage was observed not only with testisin in solution but also with cell membrane-anchored testisin on U937 cells. The cleavage close to the N terminus released peptides rich in basic amino acids. Synthetic peptides corresponding to the released peptides of human PCI (His1–Arg11) and mPCI (Arg1–Ala18) functioned as cell-penetrating peptides. Because intact mPCI but not testisin-cleaved mPCI was internalized by Jurkat T cells, a truncated mPCI mimicking testisin-cleaved mPCI was created. The truncated mPCI lacking 18 amino acids at the N terminus was not taken up by Jurkat T cells. Therefore our model suggests that testisin or other proteases could regulate the internalization of PCI by removing its N terminus. This may represent one of the mechanisms regulating the intracellular functions of PCI. 相似文献
16.
An l-amino acid oxidase (Akbu-LAAO) was isolated from the venom of Agkistrodon blomhoffii ussurensis snake using DEAE Sephadex A-50 ion-exchange, Sephadex G-75 gel filtration, and high performance liquid chromatographies. The homogeneity and molecular mass of Akbu-LAAO were analyzed by SDS-PAGE and MALDI-TOF spectrometry. The sequences of ten peptides from Akbu-LAAO were established by HPLC-nESI-MS/MS analysis. Protein sequence alignment indicated that i) that Akbu-LAAO is a new snake venom LAAO, and ii) Akbu-LAAO shares homology with several LAAOs from the venoms of Calloselasma rhodost, Agkistrodon halys, Daboia russellii siamensis, and Trimeresurus stejnegeri. Akbu-LAAO is a homodimer with a molecular mass of ∼124.4 kDa. It reacts optimally with its enzymatic substrate, Leu, at pH 4.7 with a Km of 2.1 mM. ICP-AES measurements showed that Akbu-LAAO contains four Zn2+ per dimer that are unessential for the hydrolytic activity of the enzyme. The emission fluorescence intensity of Akbu-LAAO decreases by 61% on removal of Zn2+ indicating that the zinc probably helps maintain the structural integrity of the enzyme. The addition of exogenous metal ions, including Mg2+, Mn2+, Ca2+, Ce3+, Nd3+, Co2+ and Tb3+, increases the l-Leu hydrolytic activity of the enzyme. Akbu-LAAO shows apparent anti-aggregation effects on human and rabbit platelets. It exhibits a strong bacteriostasis effect on Staphylococcus aureus, eighteen fold that of cephalosporin C under the same conditions. Taken together, the biochemical, proteomic, structural and functional characterizations reveal that Akbu-LAAO is a novel LAAO with promise for biotechnological and medical applications. 相似文献
17.
The known 9-amino-acid sequence of the facteur thymique serique (FTS) and the known 49-amino-acid sequence of thymopoietin, in conjunction with background peptide chemistry, raised the possibility that Gln1 of FTS might be linked to Arg49 of thymopoietin in a new 58-amino-acid peptide in tissue. A presumed cleavage between Gln50 and Arg49 adjacent to the Lys48-Arg49 moiety would liberate [H-Gln1]-FTS and thymopoietin; the former fragment would cyclize to FTS by known chemistry. To study this possibility, FTS, and a new dodecapeptide consisting of Val-Lys-Arg linked to the N-terminal of [H-Gln1]-FTS were synthesized on a new solid-phase resin and were found to show comparable immune stimulating activity, in vivo. These data do not negate, but support, the concept of the 58-aminoacid peptide. 相似文献
18.
In this paper, we show that the procoagulant action of Bothrops atrox venom is due in part to a protein component that activates prothrombin. The venom prothrombin activator was purified by ion-exchange chromatography and gel filtration. It was separated from a protease by affinity chromatography in a p-aminobenzamidine-CH-Sepharose column. It is a protein of about Mr 70,000, consisting of a single polypeptide chain. We have studied the kinetics of activation of prothrombin under different experimental conditions. The prothrombin activator from B. atrox venom is insensitive to reagents of serine and thiol proteases but is inactivated by ion chelators and by various divalent ions. These results suggest that it is a metalloenzyme. The prothrombin activator from B. atrox venom is inactive on the chromogenic substrates S-2337 and S-2238, and it is selective for prothrombin since it does not act on other blood coagulation factors such as fibrinogen and factor X. We have also studied the pattern of peptide cleavages produced in the human prothrombin molecule during the activation by the activator from B. atrox venom and compared it to that obtained with ecarin, a prothrombin activator from Echis carinatus venom. In the presence of thrombin inhibitors, e.g., hirudin, we found that the activators from B. atrox venom and ecarin act in a similar, or identical, manner by producing a thrombin intermediate, meizothrombin. In the absence of thrombin inhibitors, several peptides are generated, and alpha-thrombin is produced as a consequence of meizothrombin action. 相似文献
19.
Hao Wu Xiaolong Xu Dengke Shen Lili Peng Jiajia Song Yan Zhang 《Journal of biological inorganic chemistry》2011,16(1):69-79
Coagulation factor IX/coagulation factor X binding protein from the venom of Agkistrodon halys Pallas (AHP IX/X-bp) is a unique coagulation factor IX/coagulation factor X binding protein (IX/X-bp). Among all IX/X-bps
identified, only AHP IX/X-bp is a Ca2+- and Zn2+-binding protein. The binding properties of Ca2+ and Zn2+ ions binding to apo-AHP IX/X-bp and their effects on the stability of the protein have been investigated by isothermal titration
calorimetry, fluorescence spectroscopy, and differential scanning calorimetry. The results show that AHP IX/X-bp has two metal
binding sites, one specific for Ca2+ with lower affinity for Zn2+ and one specific for Zn2+ with lower affinity for Ca2+. The bindings of Ca2+ and Zn2+ in the two sites are entropy- and enthalpy-driven. The binding affinity of AHP IX/X-bp for Zn2+ is 1 order of magnitude higher than for Ca2+ for either high-affinity binding or low-affinity binding, which accounts for the existence of one Zn2+ in the purified AHP IX/X-bp. Guanidine hydrochloride (GdnHCl)-induced and thermally induced denaturations of Ca2+–Ca2+-AHP IX/X-bp, Zn2+–Zn2+-AHP IX/X-bp, and Ca2+–Zn2+-AHP IX/X-bp are all a two-state processes with no detectable intermediate state(s), indicating the Ca2+/Zn2+-induced tight packing of the protein. Ca2+ and Zn2+ increase the structural stability of AHP IX/X-bp against GdnHCl or thermal denaturation to a similar extent. Although Ca2+ and Zn2+ have no obvious effect on the secondary structure of AHP IX/X-bp, they induce different rearrangements in local conformation.
The Zn2+-stabilized specific conformation of AHP IX/X-bp may be helpful to its recognition of the structure of coagulation factor
IX. This work suggests that in vitro, Ca2+ plays a structural rather than an active role in the anticoagulation of AHP IX/X-bp, whereas Zn2+ plays both structural and active roles in the anticoagulation. In blood, Ca2+ binds to AHP IX/X-bp and stabilizes its structure, whereas Zn2+ cannot bind to AHP IX/X-bp owing to the low Zn2+ concentration. AHP IX/X-bp prolongs the clotting time in vivo through its binding only with coagulation factor X/activated
coagulation factor X. 相似文献
20.
The fraction between 950 and 4000 Da of the venom of Apis mellifera has been analyzed with MALDI-TOF mass spectrometry and statistical facilities of the ClinProTools™ software. Consistent differences in the composition of this venom fraction were observed between queens and workers while younger and older workers (nurses and guards as well as foragers) differ for the relative percentages of two well known cytolytic peptides, namely Melittin and Apamin. Total in situ body methanol extracts and methanol micro-extractions on the cuticle of various parts of the body of drones and females confirmed that venom peptides are smeared on the body surface of females in a not yet clarified way. The observation that venom peptides have been found also on comb wax rises the hypothesis that the use of venom as antimicrobial agent makes part of the social immunity system of A. mellifera. 相似文献