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1.
We describe the application of immunoaffinity extraction and mass spectrometry to the analysis of Ty1 Gag protein in lysates of Saccharomyces cerevisiae. A magnetic bead-conjugated monoclonal antibody was used to achieve selective extraction, the specificity of which was established by matrix-assisted laser desorption/ionization mass spectrometric (MS) analysis of an extract of the lysate of cells overexpressing the Ty1 Gag protein. MS analysis of similar extracts of lysates following tryptic hydrolysis confirmed selective extraction of the epitope-containing peptide fragment. Sufficient sensitivity was achieved to allow the application of this approach to the analysis of lysates of wild-type cells. Furthermore, the sequence of the epitope-containing peptide was confirmed by electrospray-tandem MS. To our knowledge, this constitutes the first report of the application of immunoaffinity extraction and tandem MS analysis to the characterization of an antigen recovered from a complex cellular system. 相似文献
2.
The Ni(II) complex of the tripeptide NH2-glycine-glycine-histidine-COOH (GGH) mediates efficient protein-protein cross-linking in the presence of oxidants such as oxone and monoperoxyphthalic acid (MMPP). Here we demonstrate that GGH fused to the amino terminus of a protein can still support cross-linking. The tripeptide was expressed at the amino terminus of ecotin, a dimeric macromolecular serine protease inhibitor found in the periplasm of Escherichia coli. In the presence of Ni(OAc)2 and MMPP, GGH-ecotin is cross-linked to give a species that has an apparent molecular mass of a GGH-ecotin dimer with no observable protein degradation. The cross-linking reaction occurs between two ecotin proteins in a dimer complex. Furthermore, GGH-ecotin can be cross-linked to a serine protease target, trypsin, and the reaction is specific for proteins that interact with ecotin. The cross-linking reaction has been carried out on small peptides, and the reaction products have been analyzed by matrix-assisted laser desorption/ionization mass spectrometry. The target of the reaction is tyrosine, and the product is bityrosyl cross-links. The yield of the cross-linking is on the order of 15%. However, the reaction efficiency can be increased 4-fold by a single amino acid substitution in the carboxy terminus of ecotin that places an engineered tyrosine within 5 A of a naturally occurring tyrosine. This cross-linking methodology allows for the protein cross-linking reagent to be encoded for at the DNA level, thus circumventing the need for posttranslational modification. 相似文献
3.
Y Abe M Odaka F Inagaki I Lax J Schlessinger D Kohda 《Canadian Metallurgical Quarterly》1998,273(18):11150-11157
The extracellular domain of the human epidermal growth factor receptor (sEGFR) consists of 621 amino acid residues, including 50 cysteines. The connections of the 25 disulfide bonds in the recombinant sEGFR protein, obtained from Chinese hamster ovary cells, have been determined using N-terminal sequencing and matrix-assisted laser desorption/ionization mass spectroscopy. We identified a basic repeat of eight cysteines with a 1-3, 2-4, 5-6, and 7-8 disulfide pairing pattern in the two cysteine-rich regions of sEGFR. By comparison to other cysteine-rich motifs, it was concluded that the cysteine-rich repeat of sEGFR belongs to the laminin-type EGR-like (LE) structural motif. Three-dimensional structure models of the two cysteine-rich regions have been built, based on the three-dimensional structures of the LE domains from the laminin gamma1 chain and secondary structure predictions for the EGF receptor. 相似文献
4.
This study characterizes disulfide cross-links between fragments of a well defined tryptic preparation of Na,K-ATPase, 19-kDa membranes solubilized with C12E10 in conditions preserving an intact complex of fragments and Rb occlusion (Or, E., Goldshleger, R., Tal, D. M., and Karlish, S. J. D. (1996) Biochemistry 35, 6853-6864). Upon solubilization, cross-links form spontaneously between the beta subunit, 19- and 11.7-kDa fragments of the alpha subunit, containing trans-membrane segments M7-M10 and M1/M2, respectively. Treatment with Cu2+-phenanthroline (CuP) improves efficiency of cross-linking. Sequencing and immunoblot analysis have shown that the cross-linked products consist of a mixture of beta-19 kDa dimers ( approximately 65%) and beta-19 kDa-11.7 kDa trimers ( approximately 35%). The alpha-beta cross-link has been located within the 19-kDa fragment to a 6.5-kDa chymotryptic fragment containing M8, indicating that betaCys44 is cross-linked to either Cys911 or Cys930. In addition, an internal cross-link between M9 and M10, Cys964-Cys983, has been found by sequencing tryptic fragments of the cross-linked product. The M1/M2-M7/M10 cross-link has not been identified directly. However, we propose that Cys983 in M10 is cross-linked either to Cys104 in M1 or internally to Cys964 in M9. Based on this study, cross-linking induced by o-phthalaldehyde (Or, E., Goldshleger, R., and Karlish, S. J. D. (1998) Biochemistry 37, 8197-8207), and information from the literature, we propose an approximate spatial organization of trans-membrane segments of the alpha and beta subunits. 相似文献
5.
NA Naryshkin MA Farrow MG Ivanovskaya TS Oretskaya ZA Shabarova MJ Gait 《Canadian Metallurgical Quarterly》1997,36(12):3496-3505
A chemical ligation procedure has been developed for the synthesis of oligoribonucleotides carrying a trisubstituted pyrophosphate (tsp) linkage in place of a single phosphodiester. Good yields of tsp were obtained when a single 2'-deoxynucleoside 5' to the tsp was used in the ligation reaction. A tsp linkage was found to be reasonably stable to hydrolysis but cleaved by treatment with ethylenediamine or lysine to give phosphoamidate adducts. A model human immunodeficiency virus type 1 (HIV-1) TAR RNA duplex containing an activated tsp was able to bind to HIV-1 Tat protein with only 3-fold reduced affinity and to a Tat peptide (residues 37-72) with identical affinity compared to that of an unmodified duplex. Tsps incorporated at sites previously identified as being in close proximity to Tat protein were able to cross-link to Tat peptide (37-72) to form a covalent phosphoamidate conjugate. Endopeptidase cleavage followed by MALDI-TOF (matrix-assisted laser desorption ionization time of flight) mass spectrometric analysis provided strong evidence that a TAR duplex containing a tsp replacing the phosphate at 38-39 had reacted specifically with Lys51 in the basic region of Tat peptide (37-72). The new chemical cross-linking method may be generally useful for identifying lysines in close proximity to phosphates in basic RNA-binding domains of proteins. 相似文献
6.
The specific formylation of initiator methionyl-tRNA by methionyl-tRNA formyltransferase (MTF) is important for initiation of protein synthesis in Escherichia coli. In attempts to identify regions of MTF that come close to the 3'-end of the tRNA, we oxidized 32P-3'-end-labeled E. coli initiator methionine tRNA with sodium metaperiodate and cross-linked it to MTF. The cross-linked MTF was separated from uncross-linked MTF by DEAE-cellulose chromatography, and the tRNA in the cross-linked MTF was hydrolyzed with nuclease P1 and RNase T1, leaving behind an oxidized fragment of [32P]AMP attached to MTF. Trypsin digestion of the cross-linked MTF followed by high pressure liquid chromatography of the digest yielded two peaks of radioactive peptides, I* and II*. These peptides were characterized by N- and/or C-terminal sequencing and by matrix-assisted laser desorption ionization mass spectroscopy. Peptide I* contained amino acids Gln186-Lys210 with Lys207 as the site of the cross-link. Peptide II*, a partial digestion product, contained amino acids Gln186-Arg214 also with Lys207 as the site of the cross-link. The molecular masses of peptides I* and II* indicate that the final product of the cross-linking reaction between the periodate-oxidized AMP moiety of the tRNA and Lys207 is most likely a morpholino derivative rather than a reduced Schiff's base. 相似文献
7.
Modification of arginine residues in bradykinin, [1-5]-bradykinin, splenopentin and two synthetic pentapeptides with acetylacetone (pentane-2,4-dione) significantly increases the relative abundance of sequence-specific fragment ions produced by matrix-assisted laser desorption/ionization (MALDI). The fragmentation efficiency as measured by post-source decay in a reflectron time-of-flight mass spectrometer increases by a factor of 2-3.5. Peptide bonds adjacent to modified residues are more susceptible to cleavage than in the non-derivatized peptide ions. The increased lability of these bonds gives rise to more complete sequence information. In addition, the relative abundances of sequence-specific fragment ions are enhanced. This strategy makes it possible to obtain valuable structural information from arginine-containing peptides that otherwise do not fragment well. 相似文献
8.
In Azotobacter vinelandii, deletion of the fdxA gene, which encodes ferredoxin I (FdI), leads to activation of the expression of the fpr gene, which encodes NADPH-ferredoxin reductase (FPR). In order to investigate the relationship of these two proteins further, the interactions of the two purified proteins have been examined. AvFdI forms a specific 1:1 cross-linked complex with AvFPR through ionic interactions formed between the Lys residues of FPR and Asp/Glu residues of FdI. The Lys in FPR has been identified as Lys258, a residue that forms a salt bridge with one of the phosphate oxygens of FAD in the absence of FdI. UV-Vis and circular dichroism data show that on binding FdI, the spectrum of the FPR flavin is hyperchromatic and red-shifted, confirming the interaction region close to the FAD. Cytochrome c reductase assays and electron paramagnetic resonance data show that electron transfer between the two proteins is pH-dependent and that the [3Fe-4S]+ cluster of FdI is specifically reduced by NADPH via FPR, suggesting that the [3Fe-4S] cluster is near FAD in the complex. To further investigate the FPR:FdI interaction, the electrostatic potentials for each protein were calculated. Strongly negative regions around the [3Fe-4S] cluster of FdI are electrostatically complementary with a strongly positive region overlaying the FAD of FPR, centered on Lys258. These proposed interactions of FdI with FPR are consistent with cross-linking, peptide mapping, spectroscopic, and electron transfer data and strongly support the suggestion that the two proteins are physiological redox partners. 相似文献
9.
GH Zhou GA Luo Y Zhou KY Zhou XD Zhang LQ Huang 《Canadian Metallurgical Quarterly》1998,19(13):2348-2355
High performance capillary electrophoresis (HPCE), high performance liquid chromatography (HPLC), matrix-assisted laser desorption/ionization - time of flight - mass spectrometry (MALDI-TOF-MS), on-line CE-electrospray ionization-mass spectrometry (CE-ESI-MS) and on-line LC-ESI-MS have been employed to characterize a heterogeneous glycoprotein, recombinant human erythropoietin (rHuEPO) expressed from Chinese hamster ovary (CHO) cells. The analysis was demonstrated through two specific levels of detail: the intact protein and tryptic digests of the protein. Six glycoforms of rHuEPO were separated by HPCE; seventeen tryptic fragments in a total of 21 nonglycosylated and glycosylated peptides were characterized; the O-linked glycopeptides were analyzed directly by CE-ESI-MS and LC-ESI-MS. In particular, four glycans of O-acetylation of sialic acid were identified in the O-linked glycosylated fragments. The molecular weight of rHuEPO was accurately determined by MALDI-TOF-MS. 相似文献
10.
The dominant Chinese hamster ovary cell glycosylation mutant, LEC18, was selected for resistance to pea lectin (Pisum sativum agglutinin (PSA)). Lectin binding studies show that LEC18 cells express altered cell surface carbohydrates with markedly reduced binding to 125I-PSA and increased binding to 125I-labeled Datura stramonium agglutinin (DSA) compared with parental cells. Desialylated [3H]Glc-labeled LEC18 cellular glycopeptides that did not bind to concanavalin A-Sepharose exhibited an increased proportion of species that were bound to DSA-agarose. Most of these glycopeptides bound to ricin-agarose and were unique to LEC18 cells. This fraction was purified from approximately 10(10) cells and shown by 1H NMR spectroscopy and methylation linkage analysis to contain novel N-linked structures. Digestion of these glycopeptides with mixtures of beta-D-galactosidases and N-acetyl-beta-D-glucosaminidases gave core glycopeptides that, in contrast to cores from parental cells, were mainly not bound to concanavalin A-Sepharose or to PSA-agarose. 1H NMR spectroscopy, matrix-assisted laser desorption ionization/time of flight mass spectrometry, electrospray mass spectrometry, and collision-activated dissociation mass spectrometry showed that the LEC18 core glycopeptides contained a new GlcNAc residue that substitutes the core GlcNAc residues. Methylation linkage analysis of the parent compound provided evidence that the GlcNAc is linked at O-6 to give the following novel, N-linked core structure. [formula: see text] 相似文献
11.
Characterization of the cyanogen bromide fragments of the beta chain of human haptoglobin 总被引:1,自引:0,他引:1
A Kurosky RE Hay H Kim B Touchstone MA Rasco BH Bowman 《Canadian Metallurgical Quarterly》1976,15(24):5326-5336
Characterization of the cyanogen bromide (CNBr) fragments of the beta chain of human haptoglobin revealed five major fragments resulting from cleavage of four methionyl residues. The fragments were isolated by gel filtration in guanidine-HCl on Sepharose 6B and Bio-Gel P10 and P60. Compositional analyses of the five cyanogen bromide fragments accounted for 248-253 amino acid residues in agreement with the number of residues determined for the intact beta chain. Most of the carbohydrate was attached to CNBr II. Automated amino-terminal sequence analysis and carboxyl-terminal hydrolysis with carboxypeptidase of the haptoglobin beta chain and cyanogen bromide fragments identified 139 residues, or about 55% of the beta-chain molecule. The placement of the fragments within the beta-chain molecule was established by sequence analysis of whole beta chain and a plasmin cleavage fragment. The position of CNBr V was confirmed by the absence of homoserine or homoserine lactone. Cyanogen bromide reaction of intact haptoglobin 1-1 resulted in the isolation of a beta-chain fragment, CNBr III, covalently attached to the intact alpha1 chain by a single disulfide bond. The beta chain was shown to have primary structural similarities to the chymotrypsin family of serin eproteases. Partial sequence analysis of CNBr V established the region which is comparable to the serine-195 active-site region: /Asp-Thr-Cys-Tyr-Gly-Asp-Ala-Gly-Ser-Ala-Phe/ (residues 189-199, chymotrypsinogen A numbering). The active-site serine-195 is replaced by alanine; however, the specificity residue of the trypsin-like enzymes, Asp-189, is preserved. Several minor cyanogen bromide cleavage products were also identified in yields of up to 15%. These minor cleavage products give evidence that tryptophanyl residues in proteins, or glycoproteins, are also susceptible to cyanogen bromide cleavage. 相似文献
12.
The present study addresses the site occupancy and the site-specific carbohydrate microheterogeneity of N-linked oligosaccharides in recombinant human factor VIII, expressed in Chinese hamster ovary cells. The four factor VIIIa polypeptides, formed upon incubation with human thrombin, were isolated and separately subjected to proteolysis with trypsin. These tryptic digests were analyzed by reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry. Selected ion monitoring of diagnostic carbohydrate ions was utilized to identify glycopeptide-containing chromatographic peaks. Oligomannose and complex carbohydrates were detected at the glycosylation sites of the 50 and the 73 kDa polypeptides, while all the oligosaccharides identified on the B-domain were complex-type structures. Only the 43 kDa polypeptide was found nonglycosylated. These studies established a biantennary core-fucosylated carbohydrate as the major substituent, consistent with the conclusions of the analyses on the entire N-linked carbohydrate pool (Kumar, H. P. M.; Hague, C.; Haley, T.; Starr, C. M.; Besman, M. J.; Lundblad, R.; Baker, D. Biotechnol. Appl. Biochem. 1996, 24, 207-216.). In addition, this mass spectrometric investigation revealed the presence of a complex nonfucosylated oligosaccharide not reported previously for this glycoprotein. 相似文献
13.
F Hofmann C Busch U Prepens I Just K Aktories 《Canadian Metallurgical Quarterly》1997,272(17):11074-11078
Clostridium difficile toxin B that is one of the largest cytotoxins (270 kDa) known acts on Rho subfamily proteins by monoglucosylation (Just, I., Selzer, J., Wilm, M., von Eichel-Streiber, C., Mann, M., and Aktories, K. (1995) Nature 375, 500-503). By deletion analysis we identified the enzyme and cytotoxic activity of the toxin to be located at the N terminus of the holotoxin. A 63-kDa fragment of toxin B covering the first 546 amino acid residues glucosylated Rho, Rac, and Cdc42, but not Ras, by using UDP-glucose as a cosubstrate. As known for the holotoxin, glucosylation by the toxin fragment was favored with the GDP-bound form of the low molecular mass GTPases. Microinjection of the toxin fragment into NIH-3T3 cells induced rounding up of cells and redistribution of the actin cytoskeleton. In contrast, a toxin fragment encompassing the first 516 amino acid residues was at least 1000-fold less active than toxin fragment 1-546 and cytotoxically inactive. The data give direct evidence for location of the enzyme activity of C. difficile toxin B at the N-terminal 546 amino acids residues and indicate a functionally and/or structurally important role of the region from amino acid residues 516 through 546 for enzyme and cytotoxic activities. 相似文献
14.
Biopolymer sequencing with mass spectrometry has become increasingly important and accessible with the development of matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). Here we examine the use of sequential digestion for the rapid identification of proteolytic fragments, in turn highlighting the general utility of enzymatic MALDI ladder sequencing and ESI tandem mass spectrometry. Analyses were performed on oligonucleotides ranging in size from 2 to 50 residues, on peptides ranging in size from 7 to 44 residues and on viral coat proteins. MALDI ladder sequencing using exonuclease digestion generated a uniform distribution of ions and provided complete sequence information on the oligonucleotides 2-30 nucleic acid residues long. Only partial sequence information was obtained on the longer oligonucleotides. C-terminal peptide ladder sequencing typically provided information from 4 to 7 amino acids into the peptide. Sequential digestion, or endoprotease followed by exoprotease exposure, was also successfully applied to a trypsin digest of viral proteins. Analysis of ladder sequenced peptides by LCMS generated less information than in the MALDI-MS analysis and ESI-MS2 normally provided partial sequence information on both the small oligonucleotides and peptides. In general, MALDI ladder sequencing offered information on a broader mass range of biopolymers than ESI-MS2 and was relatively straightforward to interpret, especially for oligonucleotides. 相似文献
15.
C Wiesmann HW Christinger AG Cochran BC Cunningham WJ Fairbrother CJ Keenan G Meng AM de Vos 《Canadian Metallurgical Quarterly》1998,37(51):17765-17772
Vascular endothelial growth factor (VEGF) is a specific and potent angiogenic factor and, therefore, a prime therapeutic target for the development of antagonists for the treatment of cancer. As a first step toward this goal, phage display was used to generate peptides that bind to the receptor-binding domain (residues 8-109) of VEGF and compete with receptor [Fairbrother, W. J., Christinger, H. W., Cochran, A. G., Fuh, G., Keenan, C. J., Quan, C., Shriver, S. K., Tom, J. Y. K., Wells, J. A., and Cunningham, B. C. (1999) Biochemistry 38, 17754-17764]. The crystal structure of VEGF in complex with one of these peptides was solved and refined to a resolution of 1.9 A. The 20-mer peptide is unstructured in solution and adopts a largely extended conformation when bound to VEGF. Residues 3-8 form a beta-strand which pairs with strand beta6 of VEGF via six hydrogen bonds. The C-terminal four residues of the peptide point away from the growth factor, consistent with NMR data indicating that these residues are flexible in the complex in solution. In contrast, shortening the N-terminus of the peptide leads to decreased binding affinities. Truncation studies show that the peptide can be reduced to 14 residues with only moderate effect on binding affinity. However, because of the extended conformation and the scarcity of specific side-chain interactions with VEGF, the peptide is not a promising lead for small-molecule development. The interface between the peptide and VEGF contains a subset of the residues recognized by a neutralizing Fab fragment and overlaps partially with the binding site for the Flt-1 receptor. The location of the peptide-binding site and the hydrophilic character of the interactions with VEGF resemble more the binding mode of the Fab fragment than that of the receptor. 相似文献
16.
PH Tsatsos K Reynolds EF Nickels DY He CA Yu RB Gennis 《Canadian Metallurgical Quarterly》1998,37(28):9884-9888
The cytochrome bo3 ubiquinol oxidase contains at least one and possibly two binding sites for ubiquinol/ubiquinone. Previous studies used the photoreactive affinity label 3-[3H]azido-2-methyl-5-methoxy-6-geranyl-1,4-benzoquinone (azido-Q), a substrate analogue, to demonstrate that subunit II contributes to at least one of the quinol binding sites. In the current work, mass spectroscopy is used to identify a peptide within subunit II that is photolabeled by the azido-Q. Purified cytochrome bo3 was photolabeled as previously described using azido-Q that was not tritiated (i.e., not radiolabeled). Subunit II was then isolated from an SDS-PAGE gel and proteolyzed in situ with trypsin. The resulting peptides were eluted from the gel and then identified using matrix-assisted laser desorption ionization mass spectrometry. The resulting mass spectrum was compared to that obtained by analysis of subunit II that had not been exposed to the photolabel. Using the amino acid sequence, each peak in the mass spectrum of the unlabeled subunit II could be assigned to an expected trypsin fragment. Two additional peaks were observed in the mass spectrum of the photolabeled subunit with m/z 1931.9 and 2287.7. Subtraction of the mass of azido-Q from the peak at m/z 1931.9 results in a mass equivalent to that of a peptide consisting of amino acids 165-178. The assignment of the peak at m/z 2287.7 cannot be made unequivocally and may correspond either to the covalent attachment of azido-Q to peptide 254-270 or to a peptide resulting from incomplete proteolysis. The labeled peptide, 165-178, is within the water-soluble domain of subunit II, whose X-ray structure is known. This peptide is located near the site where CuA is located in the homologous cytochrome c oxidases and can be placed near the interface between subunits I and II. 相似文献
17.
Acidic peptides have been studied by negative ion postsource decay (PSD) matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The peptides contained from 5 to 16 residues and were chosen on the basis of their patterns of the acidic residues. Using typical MALDI sample preparation techniques employing an acidic matrix, gastrin I (1-14), and epidermal mitosis inhibiting pentapeptide yielded much larger deprotonated ion signals, [M - H]-, than protonated ions, [M + H]+. This may be due to their absence of basic residues, coupled with their arrays of acidic residues. The PSD fragmentation of the peptide negative ions showed that an array of acidic residues, as in gastrin I (1-14), yielded simple spectra containing mainly backbone cleavage ions from the C-terminus. Hirudin (54-65), which contains two sets of two consecutive Glu residues, and fibrinopeptide A and fibrinopeptide B, with isolated acidic residues, also showed backbone cleavages as common fragment ions. In addition, the two sets of isolated consecutive amino acid residues in Cys(Bzl)84-CD4 (81-92) and hirudin (54-56) yielded internal ions from the cleavages at the (O=C)-NH bond between the acidic residues. Also observed were ions with unique side chain losses, such as the loss of C6H4O from a tyrosine residue and SCH2C6H5 and CH2C6H5 from a benzylated cysteine residue. Compared to the positive mode, the negative-ion PSD yielded fewer fragments which usually involved only one type of backbone cleavage (e.g., [Yn - H2O]-). These simple spectra aided interpretation. Overall, the acidic peptides studied yielded negative ion PSD spectra that were useful for peptide sequencing. 相似文献
18.
C Guidi-Rontani 《Canadian Metallurgical Quarterly》1996,9(7):611-616
In an attempt to introduce a large peptide that is not normally translocated across membranes into the cytosol of eukaryotic cells, we created a new chimeric protein termed CEDH between Pseudomonas aeruginosa exotoxin A (ETA) and a variant enzyme of Mus musculus dihydrofolate reductase (DHFR) with reduced affinity for antifolates, ETA(1-413).DHFR(1-187).ETA(609-613). We have defined, genetically constructed and expressed the chimeric protein in Escherichia coli. We showed that the CEDH chimeric protein, purified to homogeneity on an immunoaffinity resin, confers a methotrexate-resistant phenotype to Chinese hamster ovary cells. Furthermore, the chimeric protein allowed the growth of dihydrofolate reductase-deficient Chinese hamster ovary cells in the absence of hypoxanthine and thymidine. These results demonstrated that the chimeric protein exhibited enzyme activity and possessed the tightly folded native structure, and that the DHFR protein can be selectively internalized and translocated via domains of exotoxin A. These data show that the ETA system is an efficient system for the delivery of a variety of large polypeptides into the cytosol without stress to the target cells, and extends the use of this delivery system to proteins that are not normally translocated across membranes. 相似文献
19.
Cell surface molecules on adherent cells that bind 125I-labeled fibronectin or its 70-kDa N-terminal fragment were identified by cross-linking with factor XIIIa and by photoaffinity labeling. Such cross-linking caused the 70-kDa fragment to become associated irreversibly to cell layers and was greater in cells treated with lysophosphatidic acid, an enhancer of fibronectin assembly and strong modulator of cell shape. Cross-linking of the 70-kDa fragment with factor XIIIa was to molecules that migrated in discontinuous sodium dodecyl sulfate-polyacrylamide gels at the top of the 3.3% stacking gel and near the top of the separating gel. Estimated sizes of these large apparent molecular mass molecules (LAMMs) were >3 MDa and approximately 3 MDa. The label in 70-kDa fragment conjugated with 125I-sulfosuccinimidyl 2-(p-azidosalicylamido)-1, 3'-dithiopropionate was associated with >3-MDa LAMMs without reduction and with approximately 3-MDa LAMMs after reduction and transfer of the cleavable label. The LAMMs were expressed on monolayer cells shortly after adherence, required both 1% Triton X-100 and 2 M urea for efficient extraction, and were susceptible to digestion with trypsin but not to cathepsin D digestion. Complexes of 125I-70-kDa fragment and LAMMs were also susceptible to limited acid digestion and Glu-C protease digestion but were not cleaved by chondroitin lyase or heparitinase. Neither the uncleaved complexes nor the cleavage products were immunoprecipitated with anti-fibronectin antibodies directed toward epitopes outside the 70-kDa region. Thus, cell surface molecules that are either very large or not dissociated in sodium dodecyl sulfate comprise the labile matrix assembly sites for fibronectin. 相似文献
20.
The major envelope protein, p35, of vaccinia virus (VV; strain LIVP) was purified by extraction from virions with the non-ionic detergent Nonidet P-40. The protein was cleaved with CNBr. Four homogeneous peptides were isolated and their N-terminal amino-acid (aa) sequences determined. A computer search of a protein sequence databank revealed complete identity of the determined sequences with aa 44-63, 144-149, 154-165 and 224-238 of ORF H3 of the HindIII-H fragment of the VV genome [Rosel et al., J. Virol. 60 (1989) 436-446]. Earlier, Gordon et al. [Virology 167 (1988) 361-369] determined that the p35 surface protein of VV strain IHD-W is encoded by the H6 gene. Muravlev et al. [Biopolymery i kletka 6 (1990) 83-89 (Russian)] deduced from their data that gene A2 encodes this prominent antigen. Taking into account this ambiguity, we cloned the genes H3, H6 and A2 in expression vectors, prepared the specific antisera against the expression products and conducted the immunochemical analysis of the recombinant and native VV-specific proteins. It has been established that the H6 codes for an early protein that is found only in the infected cell extracts, but is absent in mature virions. The immunodominant protein p35 of VV strain LIVP is encoded by the gene H3. The gene A2 protein product is present mainly in the infected cell extract, but the antiserum against the A2 product shows a rather weak interaction with the 35-kDa fraction of structural VV proteins resolved by electrophoresis. 相似文献