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1.
Decorsin. A potent glycoprotein IIb-IIIa antagonist and platelet aggregation inhibitor from the leech Macrobdella decora 总被引:3,自引:0,他引:3
J L Seymour W J Henzel B Nevins J T Stults R A Lazarus 《The Journal of biological chemistry》1990,265(17):10143-10147
The discovery, purification, and characterization of decorsin, a protein isolated from the North American leech Macrobdella decora, are described. Decorsin acts as an antagonist of platelet glycoprotein IIb-IIIa (GPIIb-IIIa), and is a potent inhibitor of platelet aggregation. The protein was purified to apparent homogeneity from crude whole leech extracts by treatment with trifluoroacetic acid followed by GPIIb-IIIa affinity chromatography and C18 reverse-phase high performance liquid chromatography. Decorsin was also isolated from a solution of leech ingestate by treatment with trifluoroacetic acid followed by C18 reverse-phase high performance liquid chromatography. The primary sequence of decorsin indicates that the protein is 39 amino acids long and contains 6 cysteine and 6 proline residues, as well as the sequence Arg-Gly-Asp, (RGD), a proposed recognition site of many adhesion proteins. A molecular mass of 4379 was obtained by fast atom bombardment mass spectrometry and is consistent with the mass calculated from the observed sequence. Evidence for an N-3 isoform, lacking the first 3 amino-terminal residues is also presented. Both decorsin and the N-3 isoform inhibit GP IIb-IIIa binding to immobilized fibrinogen with an IC50 of approximately 1.5 nM. Human platelet aggregation induced by ADP is inhibited by decorsin with an IC50 of approximately 500 nM; complete inhibition was observed at less than or equal to 1 microM. Based on overall sequence homology, decorsin does not belong to the family of GPIIb-IIIa protein antagonists that is found in snake venoms (Dennis, M. S., Henzel, W. J., Pitti, R. M., Lipari, M. T., Napier, M. A., Deisher, T. A., Bunting, S., and Lazarus, R. A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 2471-2475); however the carboxyl-terminal RGD-containing region from residues 27 to 38 of decorsin is approximately 60% homologous with the corresponding region of the snake venom proteins, suggesting that high affinity binding of these proteins to GPIIb-IIIa is defined by this epitope. 相似文献
2.
R S McDowell M S Dennis A Louie M Shuster M G Mulkerrin R A Lazarus 《Biochemistry》1992,31(20):4766-4772
The purification, complete amino acid sequence, functional activity, and structural modeling are described for mambin, a platelet glycoprotein GP IIb-IIIa antagonist and potent inhibitor of platelet aggregation from the venom of the Elapidae snake Dendroaspis jamesonii (Jameson's mamba). Mambin is 59 residues in length and contains four disulfide linkages and an RGD amino acid sequence found in protein ligands that bind to GP IIb-IIIa. Mambin inhibits ADP-induced platelet aggregation (IC50 = 172 +/- 22 nM) and inhibits the binding of purified platelet fibrinogen receptor GP IIb-IIIa to immobilized fibrinogen (IC50 = 3.1 +/- 0.8 nM). Mambin has very little sequence similarity to the Viperidae family of platelet aggregation inhibitors, except for the RGD-containing region in the protein. However, mambin does have ca. 47% similarity to the short-chain postsynaptic neurotoxins found in other Elapidae venoms, which do not contain the RGD sequence and do not act as GP IIb-IIIa antagonists. On the basis of its circular dichroism spectrum, mambin has a beta-sheet structure characteristic of the neurotoxins. Molecular modeling of the mambin sequence onto the erabutoxin b structure predicts a very similar structure within the entire protein except for the loop containing the RGD sequence. Mambin may therefore represent a genetic hybrid of neurotoxic and hemotoxic proteins found in snake venoms. 相似文献
3.
The amino acid sequence of antistasin. A potent inhibitor of factor Xa reveals a repeated internal structure 总被引:7,自引:0,他引:7
E Nutt T Gasic J Rodkey G J Gasic J W Jacobs P A Friedman E Simpson 《The Journal of biological chemistry》1988,263(21):10162-10167
Antistasin is a 15-kDa protein from the salivary glands of the Mexican leech, Haementeria officinalis, which manifests anticoagulant activity by inhibiting factor Xa. Previous work demonstrating the presence of this activity in salivary gland extracts and its partial purification has been reported (Tuszynski, G. P., Gasic, T. B, and Gasic, G.J. (1987) J. Biol. Chem. 262, 9718-9723). The present study includes further purification to homogeneity of antistasin and its subsequent fragmentation and complete amino acid sequence determination. The protein, which possesses 119 amino acid residues, is blocked at its amino terminus by the presence of a pyroglutamic acid residue and has an unusually high cysteine content, with 20 cysteine residues. The primary structure of antistasin shows no homology to hirudin, a 65-residue anticoagulant protein from the medicinal leech, Hirudo medicinalis. Of great interest is the finding of significant internal homology within antistasin where a 2-fold internal repeated structure is observed. At least four isoforms of antistasin have been identified in leech salivary gland extracts by high performance liquid chromatography analysis, and partial amino acid sequence analysis of these isoforms indicates they differ by 1 or 2 amino acid residues. 相似文献
4.
Krezel AM Ulmer JS Wagner G Lazarus RA 《Protein science : a publication of the Protein Society》2000,9(8):1428-1438
5.
Ca2+-dependent binding of a synthetic Arg-Gly-Asp (RGD) peptide to a single site on the purified platelet glycoprotein IIb-IIIa complex 总被引:3,自引:0,他引:3
B Steiner D Cousot A Trzeciak D Gillessen P Hadváry 《The Journal of biological chemistry》1989,264(22):13102-13108
The platelet glycoprotein IIb-IIIa complex (GP IIb-IIIa) is a member of the integrin receptor family that recognizes adhesive proteins containing the Arg-Gly-Asp (RGD) sequence. In the present study the binding characteristics of the synthetic hexapeptide Tyr-Asn-Arg-Gly-Asp-Ser (YNRGDS, a sequence present in the fibrinogen alpha-chain at position 570-575) to purified GP IIb-IIIa were determined by equilibrium dialysis. The binding of 125I-YNRGDS to GP IIb-IIIa was specific, saturable, and reversible. The apparent dissociation constant was 1.0 +/- 0.2 microM, and the maximal binding capacity was 0.92 +/- 0.02 mol of 125I-YNRGDS/mol of GP IIb-IIIa, indicating that GP IIb-IIIa contains a single binding site for RGD peptides. The binding of 125I-YNRGDS to purified GP IIb-IIIa showed many of the characteristics of fibrinogen binding to activated platelets: the binding was inhibited by fibrinogen, by the monoclonal antibody A2A9, and by the dodecapeptide from the C terminus of the fibrinogen gamma-chain. In addition, the binding of 125I-YNRGDS to GP IIb-IIIa was divalent cation-dependent. Our data suggest that two divalent cation binding sites must be occupied for YNRGDS to bind: one site is specific for calcium and is saturated at 1 microM free Ca2+, whereas the other site is less specific and reaches saturation at millimolar concentrations of either Ca2+ or Mg2+. The results of the present study support the hypothesis that the RGD domains within the adhesive proteins are responsible for their binding to GP IIb-IIIa. 相似文献
6.
Platelet receptor recognition domains on the alpha chain of human fibrinogen: structure-function analysis 总被引:3,自引:0,他引:3
We have previously shown that the alpha chain of human fibrinogen interacts directly with ADP-activated human platelets [Hawiger, J., Timmons, S., Kloczewiak, M., Strong, D. D., & Doolittle, R. F. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2068]. Now, we report that platelet receptor recognition domains are localized on two CNBr fragments of the human fibrinogen alpha chain. They encompass residues 92-147 and 518-584, which inhibit 125I-fibrinogen binding to ADP-stimulated platelets. The inhibitory CNBr fragment alpha 92-147 contains the RGD sequence at residues 95-97. Synthetic peptides encompassing this sequence were inhibitory while peptide 99-113 lacking the RGD sequence was inactive. The synthetic peptide RGDF, corresponding to residues alpha 95-98, inhibited the binding of 125I-fibrinogen to ADP-treated platelets (IC50 = 2 microM). However, the peptides containing sequence RGDF, with residues preceding Arg95 or following Phe98, were less inhibitory. It appears that the sequence alpha 95-98 constitutes a platelet receptor recognition domain which is constrained by flanking residues. The second inhibitory CNBr fragment, alpha 518-584, also contains the sequence RGD at positions 572-574. Synthetic peptides overlapping this sequence were inhibitory, while peptides lacking the sequence RGDS were not reactive. Thus, another platelet reactive site on the alpha chain encompasses residues 572-575 containing sequence RGDS. In conclusion, the platelet receptor recognition domains on the human fibrinogen alpha chain in the amino-terminal and in the carboxy-terminal zones contain the ubiquitous cell recognition sequence RGD shared with other known adhesive proteins.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
7.
Adhesive properties of the beta 3 integrins: comparison of GP IIb-IIIa and the vitronectin receptor individually expressed in human melanoma cells
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N Kieffer L A Fitzgerald D Wolf D A Cheresh D R Phillips 《The Journal of cell biology》1991,113(2):451-461
Glycoprotein IIb-IIIa (alpha IIb beta 3) and the vitronectin receptor (alpha v beta 3), two integrins that share the common beta 3 subunit, have been reported to function as promiscuous receptors for the RGD-containing adhesive proteins fibrinogen, vitronectin, fibronectin, von Willebrand factor, and thrombospondin. The present study was designed to establish a cell system for the expression of either GP IIb-IIIa or the vitronectin receptor in an otherwise identical cellular environment and to compare the adhesive properties of these two integrins with those of native GP IIb-IIIa and the vitronectin receptor constitutively expressed in HEL cells or platelets. M21 human melanoma cells lack GP IIb-IIIa and use the vitronectin receptor to attach to vitronectin, fibrinogen, fibronectin, and von Willebrand factor. To study the functional properties of GP IIb-IIIa in these cells, we transfected GP IIb into M21-L cells, a variant of M21 cells (Cheresh, D.A., and R.C. Spiro. 1987. J. Biol. Chem. 262:17703-17711), which lack the expression of functional alpha v and are therefore unable to attach to vitronectin, fibrinogen, and von Willebrand factor. Transfectants expressing GP IIb were isolated by immunomagnetic beads and surface expression of the GP IIb-IIIa complex was documented by FACS analysis and immunoprecipitation experiments performed with 125I-labeled M21-L/GP IIb cells. Comparative functional studies demonstrated that GP IIb-IIIa expressed in M21-L/GPIIb cells as well as native GP IIb-IIIa constitutively expressed in HEL-5J20 cells (an HEL variant lacking alpha v beta 3) mediated cell attachment to immobilized fibrinogen, but not to vitronectin or von Willebrand factor, whereas the vitronectin receptor expressed in M21 cells and HEL-AD1 cells (an HEL variant expressing alpha v beta 3) mediated cell attachment to fibrinogen, vitronectin, and von Willebrand factor. Similarly, PGl2-treated resting platelets attached to immobilized fibrinogen but not to vitronectin or von Willebrand factor, and this attachment could be inhibited by mAb A2A9 (directed against a functional site on the GP IIb-IIIa complex). However, in contrast to platelets, which adhered to vitronectin and von Willebrand factor after stimulation by thrombin or PMA, activation of the protein kinase C pathway in M21-L/GP IIb or HEL cells did not induce cell adhesion to vitronectin or von Willebrand factor.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
8.
Sequence-specific nuclear magnetic resonances assignments have been obtained for the protons of kistrin. Kistrin is a small naturally occurring snake venom protein that inhibits platelet aggregation by blocking the interaction of fibrinogen with the membrane-bound glycoprotein IIb-IIIa (GP IIb-IIIa), a receptor from the integrin family. Kistrin has an Arg-Gly-Asp sequence which is believed to form an adhesion recognition sequence that is essential for activity. Therefore, the interaction between kistrin and GP IIb-IIIa may provide important information on the motif used by integrins to recognize their target proteins which bear RGD sequences. Kistrin consists of 68 residues and contains six intramolecular disulfide bonds. Although one-third of the amide protons are protected from exchange with the solvent, there appears to be little or no regular secondary structure. The large number of NOE's between residues separated by two and three positions in the sequence indicates that the protein contains a large number of tightly packed loops. Along with the sequential assignments, this paper also discusses the construction and use of computerized data bases for manipulating NMR results. A strategy for computer-assisted sequential resonance using these data bases is also presented. 相似文献
9.
Xingzhen Lao Jingxiao Bao Tingting Yu Qingqing Li Heng Zheng 《Journal of molecular modeling》2016,22(11):281
Decorsin is an antagonist of platelet glycoprotein integrin αIIbβ3 on platelets; the protein is 39 amino acids long with three disulfide bridges in its tertiary structure. To demonstrate decorsin’s mechanism of action, we applied the computational virtual technique and platelet aggregation inhibition assay, which showed that the flanking amino-acid residues of the Arg–Gly–Asp (RGD) motif play an important role in platelet aggregation. The computational simulations revealed that the RGD motif mainly contributes to the stability of the complex when decorsion interacts with integrin αIIbβ3. However, the C-terminal residues, such as 34A→W and 35D→R, was also found to possibly play a key role in their binding structures. Moreover, we produced a decorsin analog (A34W plus D35R decorsin), in which the 34A (alanine) and 35D (aspartic acid) residues were respectively substituted by W (tryptophan) and R (arginine). This isoform was then recombinantly expressed in Escherichia coli. Intriguingly, this mutant type showed higher anti-platelet aggregation activity than the wildtype. Our study may further contribute to finding decorsin mutants with higher anti-platelet aggregation activity. 相似文献
10.
The binding interactions between platelet fibrinogen receptor, glycoprotein (GP) IIb-IIIa, and kistrin, a snake venom disintegrin protein that contains the adhesion site recognition sequence Arg-Gly-Asp (RGD) and potently inhibits platelet aggregation, have been investigated by site-directed mutagenesis of a synthetic kistrin gene. Kistrin was expressed as a fusion protein in Escherichia coli under control of the alkaline phosphatase promoter. This construction included the stII signal sequence to direct secretion to the periplasmic space and one synthetic (Z) domain of Staphylococcal protein A to allow affinity purification using IgG Sepharose. Kistrin was cleaved from the Z-domain by site-specific proteolysis using a mutant subtilisin BPN' and purified by reverse-phase HPLC. This approach facilitated the rapid purification of a set of 43 alanine replacement mutants whose relative affinity for GP IIb-IIIa was measured by competition with immobilized kistrin and by inhibition of platelet aggregation in human platelet-rich plasma. Alanine replacements at R49, G50, and D51 led to weaker inhibitors of platelet aggregation by 90-fold, 2-fold, and >200-fold, respectively. The conservative D51E mutant was still >100-fold less potent whereas R49K had a minor effect (1.8-fold), implying the critical nature of the aspartate for high affinity binding. However, mutations outside of the RGD region led to proteins indistinguishable from kistrin, suggesting no substantial secondary binding interactions. Furthermore, reduced kistrin is not active. We therefore propose that a favorable conformation of the RGD region alone is responsible for the high affinity binding of kistrin to GP IIb-IIIa. © 1993 Wiley-Liss, Inc. 相似文献
11.
A new inhibitory peptide binding exosite on the protease domain of coagulation Factor VIIa (FVIIa) has been identified. A novel series of peptide inhibitors of FVIIa, termed the "A-series" peptides, identified from peptide phage libraries and exemplified by peptide A-183 [Dennis, M. S., Roberge, M., Quan, C., and Lazarus, R. A. (2001) Biochemistry 40, 9513-9521], specifically bind at a site that is distinct from both the active site and the exosite of another recently described peptide inhibitor of FVIIa, E-76 [Dennis, M. S., Eigenbrot, C., Skelton, N. J., Ultsch, M. H., Santell, L., Dwyer, M. A., O'Connell, M. P., and Lazarus, R. A. (2000) Nature 404, 465-4701. Peptide A-183 prolonged TF-dependent clotting in human, but not rabbit plasma. Thus, a panel of human FVIIa mutants, containing 70 of the 76 rabbit sequence differences in the protease domain, localized the binding site to residues in the 60s loop and the C-terminus. The location of the exosite was refined by a series of FVIIa alanine mutants, which showed that proximal residues Trp 61 and Leu 251 were critical for binding. Kinetic and equilibrium binding constants for zymogen FVII, FVIIa and TF x FVIIa were determined using immobilized N-terminal biotinylated A-183 by surface plasmon resonance. No peptide binding to nine other human serine proteases was observed. Key residues on the peptide were determined from binding to FVIIa and inhibition of FX activation using a series of alanine mutants of A-183 fused to the Z domain of protein A. Analysis of the mutagenesis data is presented in the context of a crystal structure of A-183 in complex with a version of zymogen FVII [Eigenbrot, C., Kirchhofer, D., Dennis, M. S., Santell, L., Lazarus, R. A., Stamos, J., and Ultsch, M. H. (2001) Structure 9, 627-636]. The shape and proximity of this exosite to the active site may lend itself towards the design of new anticoagulants that inhibit FVIIa. 相似文献
12.
A novel platelet aggregation inhibitor, sal-C, was purified to homogeneity from the venom of Korean snake (Agkistrodon halys brevicaudus). Several lines of experimental evidence clearly indicated that sal-C inhibits not only the collagen-induced platelet aggregation, but also the aggregation mediated by the cell surface glycoprotein IIb-IIIa (GP IIb-IIIa). We have isolated the cDNA encoding sal-C from the cDNA library of the snake venom gland and analyzed its complete nucleotide sequence. Sal-C is a single-chain polypeptide composed of 212 amino acids including 24 cysteines. The deduced polypeptide sequence of sal-C demonstrated considerable homology to previously described protein species of the collagen-induced platelet aggregation inhibitor family. Sal-C does not have the Arg-Gly-Asp (RGD) motif, but contains the Ser-Glu-Cys-Asp sequence. Interestingly, sal-C was found to inhibit GP IIb-IIIa binding to immobilized fibrinogen which is antagonized by the typical RGD motif of disintegrins. 相似文献
13.
We have designed a new binding assay based on crossed immunoelectrophoresis that allowed us to test for the relative capacities of platelet membrane glycoprotein IIb-IIIa (GP IIb-IIIa), and glycoprotein IV (GP IV) to bind purified Arg-Gly-Asp (RGD)-containing adhesive proteins. Preformed immune complexes were made by reacting a platelet lysate with murine monoclonal antibodies to GP IV (OKM5 and FA6-152) or to GP IIb-IIIa (AP-2). Upon two-dimensional electrophoretic separation in agarose gels and immunoprecipitation by a polyclonal antibody to mouse IgG, the immobilized complexes containing the desired antigen were further probed with purified 125I-labeled TSP or fibrinogen. Under these conditions, immobilized GP IV was found to specifically bind TSP, whereas it was unreactive with fibrinogen. By contrast, immobilized GP IIb-IIIa demonstrated fibrinogen binding capacity but did not demonstrate any reactivity toward TSP. These observations suggest that the overall structure of the adhesive protein may determine the accessibility of the RGD sequence to its binding site on GP IIb-IIIa. 相似文献
14.
Construction and characterization of trifunctional single-chain urokinase-type plasminogen activators. 总被引:2,自引:0,他引:2
Qun Bi Xiaodong Cen Yixiu Huang Shenggeng Zhu 《European journal of biochemistry》2002,269(6):1708-1713
Two chimeric proteins have been constructed. One consists of four parts: a portion of the low molecular mass single-chain urokinase-type plasminogen activator (scu-PA-32K, residues 144-411), a 15-mer linker sequence, the C-terminal amino-acid sequence (residues 53-65) of hirudin (Hir), and an RGD sequence derived from the leech protein decorsin, i.e. scu-PA(32 k)-linker-Hir (residues 53-65)-RGD peptide. The other comprises two main segments: scu-PA(32 k) and hirudin into which RGDSP is inserted between its residues 33 and 34, i.e. hirudin (residues 1-33)-RGDSP-hirudin (residues 34-65)-scu-PA(32 k). These two chimeric genes were expressed in Escherichia coli, and the products were purified by Zn2+-chelating Sepharose 4B chromatography and benzamidine Sepharose 6B chromatography. Our results suggested that these two chimeric proteins not only had plasminogen-dependent fibrinolytic activity, but also possessed platelet aggregation inhibitory activity and antithrombin activity. 相似文献
15.
The leukocyte integrin complement receptor type III (CR3, CD11b/CD18) binds the C3 cleavage product iC3b. Many other integrins bind their ligands via an Arg-Gly-Asp (RGD) triplet. Both the RGD-containing C3 peptide 1390TRYRGDQDATMS1401 (pro-C3 numbering) and the RGD-like fibrinogen peptide GGAKQAGDV, which binds to the platelet integrin glycoprotein IIb-IIIa, were shown to inhibit the iC3b-CR3 interaction, suggesting that this binding is also RGD-mediated (Wright, S.D., Weitz, J.I., Huang, A. J., Levin, S.M., Silverstein, S.C., and Loike, J.D. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7734-7738). However, unlike other integrin-ligand interactions, that of CR3 and iC3b is unaffected by the hexapeptide GRGDSP, and substitutions in the RGD triplet of C3 from other species appear to be tolerated. It was, therefore, proposed (Grossberger, D., Marcuz, A., du Pasquier, L., and Lambris, J.D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 1323-1327) that the highly conserved DATMS portion of the inhibitory C3 peptide may have been responsible for its binding. To address these inconsistencies and directly assess the role of the 1390-1401 segment within the complete iC3b molecule in mediating binding to CR3, a human C3 cDNA was altered by site-directed mutagenesis and the expressed recombinant proteins were examined in a CR3-specific assay. Replacement of RGD by AAA did not abolish rosetting of the corresponding iC3b-coated erythrocytes to human CR3-bearing leukocytes. In addition, mutant iC3b molecules in which the positively charged R1391 (corresponding to K in the fibrinogen peptide) and the highly conserved 1397DATMS sequence were replaced by Q and NAAMA respectively, were still bound by CR3. We conclude that the iC3b-CR3 interaction is not mediated by the RGD triplet or its neighboring residues. 相似文献
16.
Gamma-bungarotoxin, a snake venom protein isolated from Bungarus multicinctus, contains 68 amino acids, including 10 cysteine residues and a TAVRGDGP sequence at positions 30-37. The solution structure of gamma-bungarotoxin has been determined by nuclear magnetic resonance (NMR) spectroscopy. The structure is similar to that of the short-chain neurotoxins that contain three loops extending from a disulfide-bridged core. The tripeptide Arg-Gly-Asp (RGD) sequence is located at the apex of the flexible loop and is similar to that of other RGD-containing proteins. However, gamma-bungarotoxin only inhibits platelet aggregations with an IC50 of 34 microM. To understand its weak activity in inhibiting platelet aggregation, we mutated the RGD loop sequences of rhodostomin, a potent platelet aggregation inhibitor, from RIPRGDMP to TAVRGDGP, resulting in a 196-fold decrease in activity. In addition, the average Calpha-to-Calpha distance between R33 and G36 of gamma-bungarotoxin is 6.02 A, i.e., shorter than that of other RGD-containing proteins that range from 6.55 to 7.46 A. These results suggested that the amino acid residues flanking the RGD motif might control the width of the RGD loop. This structural difference may be responsible for its decrease in platelet aggregation inhibition compared with other RGD-containing proteins. 相似文献
17.
Peptide sequence analysis and molecular cloning reveal two calcium pump isoforms in the human erythrocyte membrane 总被引:4,自引:0,他引:4
E E Strehler P James R Fischer R Heim T Vorherr A G Filoteo J T Penniston E Carafoli 《The Journal of biological chemistry》1990,265(5):2835-2842
The sequence of more than 1,000 amino acid residues, derived from two different isoforms, has been determined from peptides generated from purified human erythrocyte membrane Ca2(+)-ATPase (hPMCA). Several of these peptide sequences correspond to the previously reported, cDNA deduced sequence of the "teratoma" Ca2+ pump isoform hPMCA1 (Verma, A. K., Filoteo, A. G., Stanford, D. R., Wieben, E. D., Penniston, J. T., Strehler, E. E., Fischer, R., Heim, R., Vogel, G., Matthews, S., Strehler-Page, M.-A., James, P., Vorherr, T., Krebs, J., and Carafoli, E. (1988) J. Biol. Chem. 263, 14152-14159). The complete primary structure of a novel isoform (hPMCA3) has been determined by molecular cloning and nucleotide sequencing of its corresponding cDNA. This new member of the plasma membrane Ca2+ pump family consists of 1,205 amino acid residues with a calculated Mr of 133,930, and it shows 88% similarity (75% identity) with the previously sequenced pump isoform. Specific probes detect major mRNA species of 5.6 kilobases for hPMCA1, and of 7.5 kilobases for hPMCA3, on Northern blots of human K562 erythroleukemic cell RNA. A large number of peptide sequences match perfectly with only one or the other of these isoforms and all peptides (with 6 exceptions corresponding to a contaminant protein or to a third minor Ca2+ pump isoform) are found in either only one or in both of the isoforms. The two erythrocyte Ca2+ pumps display high sequence divergence in a few localized regions that may determine isoform-specific functional specializations; for example, the putative extracellular loop separating transmembrane domains 1 and 2, the highly negatively charged region previously suggested to be involved in Ca2+ binding, and the site of cAMP-dependent protein kinase phosphorylation. 相似文献
18.
Effect of GPIIb-IIIa complex ligands on calcium ion movement and cytoskeleton organization in activated platelets 总被引:5,自引:0,他引:5
F Sinigaglia A Bisio M Torti C L Balduini G Bertolino C Balduini 《Biochemical and biophysical research communications》1988,154(1):258-264
We studied the influence of the occupancy of the fibrinogen receptor (GP IIb-IIIa complex) on two early aspects of agonist induced platelet activation: the increase of the intracellular Ca2+ concentration and the cytoskeleton reorganization. A monoclonal antibody, a peptide containing the RGD sequence and fibrinogen purified from human plasma were used as GP IIb-IIIa ligands. The obtained results demonstrated that fibrinogen receptor occupancy inhibits Ca2+ movement and cytoskeleton reorganization caused by low thrombin concentration and ADP. 相似文献
19.
Chemical cross-linking of arginyl-glycyl-aspartic acid peptides to an adhesion receptor on platelets 总被引:7,自引:0,他引:7
S E D'Souza M H Ginsberg S C Lam E F Plow 《The Journal of biological chemistry》1988,263(8):3943-3951
A chemical cross-linking approach has been used to characterize the interaction of platelets with small peptides of 7 and 14 residues containing the arginyl-glycyl-aspartic acid (RGD) sequence recognized by a variety of cellular adhesion receptors. The radioiodinated peptides were bound to platelets, and chemical cross-linking was attained by subsequent addition of bifunctional reagents. Three different cross-linking reagents coupled the RGD-containing peptides to platelet membrane glycoprotein IIb-IIIa (GPIIb-IIIa), and both subunits of this platelet membrane glycoprotein became radiolabeled with the RGD peptides. Platelet stimulation with agonists including thrombin, phorbol myristrate acetate, and ADP increased the extent of cross-linking by predominantly enhancing the coupling of the RGD peptides to the GPIIIa subunit. Cross-linking of the labeled RGD peptides to GPIIb and GPIIIa on stimulated and nonstimulated platelets exhibited structural specificity and was inhibited by excess nonlabeled RGD peptides. The interactions were inhibited by nonlabeled RGD peptides and a peptide with an amino acid sequence corresponding to the carboxyl terminus of the gamma chain of fibrinogen but less effectively by an arginyl-glycyl-glutamic acid peptide. Cross-linking of the RGD peptides to GPIIb-IIIa was divalent ion-dependent and, on stimulated platelets, was inhibited by the adhesive proteins fibrinogen and fibronectin, but not by albumin. These results indicate that the RGD-binding sites on platelets reside in close proximity to both subunits of GPIIb-IIIa and that platelet stimulation alters the topography of these sites such that the peptides become more efficiently cross-linked to GPIIIa. 相似文献
20.
Drosophila basement membrane procollagen alpha 1(IV). II. Complete cDNA sequence, genomic structure, and general implications for supramolecular assemblies 总被引:10,自引:0,他引:10
A Drosophila melanogaster gene for a basement membrane procollagen chain was recently identified from the sequence homology of the carboxyl (NC1) end of the polypeptide that it encodes with the corresponding domain of human and murine collagens IV (Blumberg, B., MacKrell, A. J., Olson, P. F., Kurkinen, M., Monson, J. M., Natzle, J. E., and Fessler, J. H. (1987) J. Biol. Chem. 262, 5947-5950). This gene is at chromosome location 25C. Here we report the complete 6-kilobase cDNA sequence coding for a chain of 1775 amino acids, as well as the genomic structure. The gene is composed of nine relatively large exons separated by eight relatively small introns. This organization is different from the multiple small exons separated by large introns reported for mouse and human type IV collagens (Kurkinen, M., Bernard, M. P., Barlow, D. P., and Chow, L. T. (1985) Nature 317, 177-179. Sakurai, Y., Sullivan, M., and Yamada, Y. (1986) J. Biol. Chem. 261, 6654-6657. Soininen, R., Tikka, L., Chow, L., Pihlajaniemi, T., Kurkinen, M., Prockop, D. J., Boyd, C. D., and Tryggvason, K. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 1568-1572). Drosophila and human alpha 1(IV) procollagen chains share not only polypeptide domains near their amino and carboxyl ends for making specialized, intermolecular junctional complexes, but also 11 of 21 sites of imperfections of the collagen triple helix. However, neither the number nor the nature of the amino acids in these imperfections appear to have been conserved. These imperfections of the helical sequence may be important for the supramolecular assembly of basement membrane collagen. The 9 cysteine residues of the Drosophila collagen thread domain are arranged as several variations of a motif found in vertebrate collagens IV only near their amino ends, in their "7 S" junctional domains. The relative positions of these cysteine residues provide numerous opportunities for disulfide bonding between molecules in both parallel and antiparallel arrays. There is a pseudorepeat of one-third of the thread length, and there are numerous possibilities for disulfide-linked microfibrils and networks. We propose that collagen microfibrils, stabilized by disulfide segment junctions, are a versatile ancestral form from which specialized collagen fibers and networks arose. 相似文献