共查询到20条相似文献,搜索用时 31 毫秒
1.
Bapat KN Venkatesh M Sarma HD Pillai MR Sainis KB 《Indian journal of experimental biology》2001,39(6):526-532
Myocardial infarction (MI) can be monitored using several protein markers including human cardiac myosin (HCM). Monoclonal antibodies were raised against HCM by hybridoma technique. Antimyosin antibody producing clones were identified by ELISA and monoclonality was established by limiting dilution. The antibodies were purified, isotyped and their cross reactions with myosin from other species were estimated. All the clones showed negligible cross reaction with rabbit myosin, but reacted with bovine skeletal myosin to different extents (40-100%). The most avid antibody Mab 4G4 which also strongly reacted with rat cardiac myosin, was labelled with 125I using different oxidising agents such as iodogen, chloramine-T and lactoperoxidase. More than 95% pure radiolabelled antibody could be obtained by gel filtration. The immunoreactivity was retained. Mab 4G4 was also labelled with 99mTc using stannous tartrate as the reducing agent. Radiolabelling yield was approximately 60%, the purity was >95%. Both the radiolabelled preparations were tested for biodistribution in rats--both normal and those with induced MI. Approximately 0.7 % of the injected activity/g was found in the infarcted region and the accumulation of activity in the infarcted heart was 1.5 times that in the normal heart. A very high percentage of activity (80%) accumulated in the thyroid. With further optimisation of labelling and use of F(ab')2 fragments, better delineation of the infarct sites may become possible. 相似文献
2.
Localization of macrophage agglutination factor activity to the gelatin-binding domain of fibronectin 总被引:2,自引:0,他引:2
H P Godfrey C V Angadi R A Wolstencroft C Bianco 《Journal of immunology (Baltimore, Md. : 1950)》1984,133(3):1417-1423
We previously proposed that macrophage agglutination factor (MAggF, a T cell-derived guinea pig lymphokine) is a fibronectin (FN). We now show MAggF binding to gelatin and to peritoneal macrophages is mediated by domains similar to corresponding domains of plasma FN. MAggF activity in lymphokine concentrates prepared by two different methods differed nearly 10-fold in m.w. on gel filtration chromatography. Despite this difference, MAggF dose-activity curves of both preparations were parallel, and MAggF in both preparations bound reversibly to gelatin and to monoclonal anti-guinea pig FN immunoadsorbents. MAggF activity in one preparation was inhibited by the addition of soluble monoclonal antibody specific for the gelatin-binding domain of human FN; inhibitory activity of this antibody was blocked by purified guinea pig plasma FN or partially purified MAggF from the other preparation. Measured MAggF activity of both preparations was reduced in a dose-dependent manner by pretreatment of indicator macrophages with monoclonal anti-human monocyte FN receptor antibody or F(ab')2 fragments or with guinea pig plasma FN. Neither anti-FN receptor antibody nor plasma FN interacted directly with MAggF. Indirect immunofluorescence studies confirmed the presence of uncomplexed plasma membrane receptors for FN on indicator macrophages in MAggF-responsive populations that were able to bind added FN. Our identification of MAggF as lymphokine FN provides a basis for future biochemical analysis of delayed hypersensitivity inflammatory reactions. 相似文献
3.
The concentrations and molecular forms of urinary and plasma gastrin from normal subjects were studied by radioimmunoassays using two region-specific antisera. Urinary concentration of NH2-terminal big gastrin (G-34) immunoreactivity was several hundred times as great as that of COOH-terminal gastrin immunoreactivity. Fractionation of urine extract showed a broad giant peak of NH2-terminal G-34 immunoreactivity (gastrin fragments "U") eluting in a later position than G-34(1-17) by Sephadex G-50 column chromatography. HPLC revealed that urinary NH2-terminal G-34 immunoreactivity was composed of four fragments including G-34(1-8), G-34(1-9), and G-34(1-10). Sephadex G-50 column chromatography of plasma extract revealed two or three peaks of NH2-terminal G-34 immunoreactivity, and a major peak eluted in the same position as urinary gastrin fragments "U". These results and data on renal clearances suggest that most of all gastrin fragments "U" in plasma are excreted in urine without renal reabsorption, whereas almost all of plasma COOH-terminal gastrin peptides including G-34 and little gastrin (G-17) are removed and metabolized in the kidney. 相似文献
4.
S Lesage J Bouhnik J P Richoux T Baussant F Gauthier K Eager P Corvol F Alhenc-Gelas 《European journal of biochemistry》1992,204(2):501-508
A panel of 16 monoclonal antibodies (mAb) were produced against rat T-kininogen to characterize this family of proteins. These mAbs bound 125I-T-kininogen by radioimmunoassay as well as reacting strongly with immobilized T-kininogen in an enzyme-linked immunosorbent assay (ELISA). The reactivity of these antibodies with proteolytic fragments of T-kininogen demonstrated the recognition of several different epitopes. One antibody was specific for the domain 1 of the heavy chain and/or the light chain, twelve antibodies were specific for domain 2 and three antibodies were specific for domain 3. All monoclonal antibodies recognized the two forms of T-kininogen encoded by the two different T-kininogen genes, TI and TII kininogen, except antibody TK 16-3.1 which uniquely reacted with TII kininogen. Two antibodies recognizing domain 2 cross-reacted with the high-molecular-mass kininogen (H-kininogen), whereas all the other monoclonal antibodies were specific to T-kininogen and did not recognize the heavy chain of H-kininogen. None of the antibodies tested altered the thiol protease inhibitory activity of T-kininogen, its partial proteolysis by rat mast cell chymase or the hydrolysis of H-kininogen by rat urinary kallikrein. The use of these antibodies in the development of sensitive ELISA to measure T-kininogen levels in plasma, urine, liver microsomes and hepatocytes is described. Two different forms of T-kininogen were distinguished by these monoclonal antibodies in Western blotting using rat plasma. The localization of T-kininogen was defined using these monoclonal antibodies by immunohistochemistry in rat liver hepatocytes and rat kidney. 相似文献
5.
A Slomiany K Okazaki S Tamura B L Slomiany 《Archives of biochemistry and biophysics》1991,286(2):383-388
Human and rat intestinal mucin was purified by equilibrium density gradient centrifugation and Sepharose 2B chromatography according to M. Mantle, D. Mantle, and A. Allen (1981, Biochem. J. 195, 277-285) and analyzed using mucin, DNA, and fibronectin-specific antibodies in dot-blot, ELISA, and Western blotting. The 118-kDa component of the mucins and the 118-kDa fragment of fibronectin from the same source displayed affinity for concanavalin A and immunoreacted with fibronectin antibodies. The amino acid and carbohydrate compositions of the 118-kDa peptide electroeluted by gel electrophoresis of mucin and fibronectin preparations were identical within each pair of glycopeptides and closely resembled the "link protein component" of human and rat intestinal mucin preparations of R. E. F. Fahim, R. D. Specian, G. G. Forstner, and J. F. Forstner (1987, Biochem. J. 243, 631-640) and M. Mantle and G. Stewart (1989, Biochem. J. 259, 631-640). We therefore conclude that the "link protein" claimed to be an integral part of mucus glycoproteins in actuality is the 118-kDa fragment of fibronectin. 相似文献
6.
In vitro synthesis of a putative precursor to the mitochondrial enzyme, carbamyl phosphate synthetase. 总被引:4,自引:0,他引:4
A simple and rapid procedure is described for purification of carbamyl phosphate synthetase from the matrix fraction of rat liver mitochondria. Antibodies to the enzyme were raised in sheep and purified from antiserum by affinity chromatography on enzyme-bound Sepharose columns. When membrane-free polyribosomes, isolated from a cytosolic fraction of rat liver, were incubated in a messenger-dependent rabbit reticulocyte protein-synthesizing system in the presence of [35S]methionine, the purified antibody precipitated a product of translation representing 0.2% of total trichloroacetic acid-insoluble radioactivity. It demonstrated mobility characteristics in sodium dodecyl sulfate-polyacrylamide gels expected for a polypeptide of molecular mass approximately 5500 daltons larger than the mature mitochondrial form of the enzyme (160,000 daltons). Proteolysis of both the mature and presumptive in vitro precursor forms of the enzyme yielded respective sets of peptide fragments which gave similar patterns upon gel electrophoresis. Excess mitochondrial enzyme effectively competed with the in vitro product for interaction with anti-carbamyl phosphate synthetase antibody. 相似文献
7.
Antibodies raised against rat plasma apoVLDL and a purified fraction of arginine-rich peptides (ARP) were labeled with Na125I and were shown to bind to polyribosomes isolated from rat liver. Antibody fractions enriched by selective affinity chromatography exhibited increased levels of binding to polysomes. Anti-apoVLDL immunoreactivity was further resolved into anti-ARP and anti apoB components, each reactive with a distinct polysome population. Binding was specific for rat polysomes, and was directed toward nascent polypeptide chains. About 2% of normal rat liver polysomes were recovered by indirect immunoprecipitation with anti-apoVLDL. Ribonucleic acid (RNA) extracted from this immunoprecipitate contained species with polyadenylate (poly[A] sequences characteristic of eukaryotic messenger RNA (mRNA). These species, purified by affinity chromatography on poly(U)-Sepharose, stimulated the in vitro synthesis of immunoprecipitable apoVLDL-like proteins by about 17-fold when compared to unfractionated rat liver mRNA. Most of the in vitro translation products precipitated by purified anti-ARP migrated identically on polyacrylamide gel electrophoresis with unlabeled purified ARP. Some implications of these findings with respect to plasma VLDL biosynthesis are discussed. 相似文献
8.
M A Montesano M S Lima R Correa-Oliveira G Gazzinelli D G Colley 《Journal of immunology (Baltimore, Md. : 1950)》1989,142(7):2501-2506
Antibodies were purified from pooled sera from patients with different clinical forms of schistosomiasis mansoni on immunoaffinity columns of schistosome soluble egg Ag (SEA). As previously reported, T lymphocytes in PBMC preparations from schistosomiasis patients (but not control subjects who have never been infected) proliferate when cultured in the presence of certain of these anti-SEA purified antibodies. We now show that PBMC from most patients with chronic schistosomiasis, regardless of the clinical form of their infection, respond to anti-SEA antibodies from sera of asymptomatic (intestinal) or hepatointestinal patients. In stark contrast, none responds to anti-SEA antibodies purified from sera of acute or hepatosplenic patients. All of these multiclonal anti-SEA antibody preparations were active in anti-SEA ELISA assays and gave comparable patterns of reactivity with SEA upon immunoblotting analysis. Immunization of rabbits with some of these anti-SEA antibody preparations, followed by absorption of the rabbit antisera on absorbents of normal Ig, produced specific anti-Id reagents. Use of these reagents in competitive ELISA systems demonstrated that the Id in stimulatory and nonstimulatory anti-SEA antibody preparations differ with regard to the proportion of the serologically defined Id expressed by each. It appears possible to screen patients' plasmas for the presence of shared Id by use of suitable Id/anti-Id competitive ELISA assays. Taken together these data indicate that only certain Id-positive preparations are stimulatory to patients' PBMC, and the expression of these T cell stimulatory, immunoregulatory Id on anti-SEA antibodies correlates with the clinical form of a patient's infection. 相似文献
9.
B Sarkadi A Enyedi J T Penniston A K Verma L Dux E Molnár G Gárdos 《Biochimica et biophysica acta》1988,939(1):40-46
Calcium pumps of various plasma membrane, endoplasmic reticulum and sarcoplasmic reticulum preparations were visualized by simultaneous immunoblotting and autoradiography of the 32P-labelled phosphoenzymes. The pump proteins and their fragments produced by a proteolytic pretreatment of the membranes were selectively phosphorylated by [gamma-32P]ATP, separated on an acidic SDS-polyacrylamide gel, blotted onto nitrocellulose and reacted with polyclonal antibodies raised against the purified human erythrocyte and rat skeletal muscle sarcoplasmic reticulum calcium pumps, respectively. The immuno-reaction was detected by peroxidase staining, while the phosphoproteins were shown by autoradiography of the same blot. An antibody against the erythrocyte calcium pump, reacting on the blot with the 140 kDa erythrocyte calcium pump and its 80 kDa proteolytic fragment, did not show a cross-reaction with the calcium pump of similar molecular mass in rat synaptosome membranes or with any of the endoplasmic- or sarcoplasmic-type calcium pumps. An anti-sarcoplasmic reticulum calcium pump antibody cross reacted with several sarcoplasmic and endoplasmic calcium pump proteins and their proteolytic fragments but with none of the plasma membrane pumps. This sensitive double-labelling method can be applied to study structural relationships and molecular alterations in various ion pump proteins. 相似文献
10.
Keire DA Vincent Wu S Diehl DL Chew P Ho FJ Davis MT Lee TD Shively JE Walsh JH Reeve JR 《Regulatory peptides》2003,113(1-3):115-124
Details of prohormone processing patterns are revealed by purification and characterization of molecular forms stored in the tissues where the hormones are expressed. Molecular forms of rat gastrin were purified from antral extracts by gel permeation, anion exchange, and reverse-phase HPLC. Amidated and glycine-extended gastrins were detected with specific antisera and their structures determined by mass spectrometry. In rats, the only form shorter than gastrin-17 observed contained 16 amino acids. These data suggest that two enzymes process the amino terminus of gastrin-17. Pyrrolidone carboxylic acid peptidase removes the amino terminal pyrrolidone carboxylic acid (pyroGlu), forming gastrin-16. In mammals other than rat, gastrin-16 is then cleaved by dipeptidyl peptidase IV to form gastrin-14. In rat, this reaction does not take place because of proline residues Pro(2)-Pro(3)- in gastrin-16. Gastrin-16 is found in sulfated and nonsulfated forms and comprises 28% of the total gastrin immunoreactivity. Glycine-extended forms of gastrin-16 and gastrin-17 comprises 45% of the total gastrin immunoreactivity. The sulfated forms of gastrin-16 and gastrin-17 bind to the CCK-B receptor transfected into CHO cells with 10-fold higher affinity than the nonsulfated forms of these peptides. Therefore, processing of rat progastrin may modulate the expression of gastrin biological activity. 相似文献
11.
Choline kinase was purified from rat kidney to apparent homogeneity with respect to both native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme showed a minimum molecular weight of 42,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On the other hand, the molecular size of 75,000-80,000 was estimated through Sephadex G-150 gel filtration, indicating that the enzyme in rat kidney exists most likely in a dimeric form. Specific antibody was raised in rabbit against the highly purified rat kidney choline kinase protein, then immunochemical cross-reactivity was investigated between rabbit antiserum and choline kinase preparations from various rat tissues. The antiserum inhibited choline kinase activity almost completely in the crude preparation not only from kidney but also from lung, intestine, and normal untreated liver cytosol, but it could inhibit only partially the activity from either 3-methylcholanthrene- or carbon tetrachloride-induced rat liver cytosol. The overall results demonstrated that, although choline kinase protein appears to exist in multiple forms in rat tissues, most of them are immunochemically identical, and that either 3-methylcholanthrene- or carbon tetrachloride-inducible form(s) of choline kinase in rat liver could be quite different from a form or forms existing in normal untreated rat liver cytosol. 相似文献
12.
B C Zhu S F Fisher H Pande J Calaycay J E Shively R A Laine 《The Journal of biological chemistry》1984,259(6):3962-3970
Human placental fibronectin was isolated from fresh term placenta by urea extraction and purified by gelatin affinity chromatography. A 44-kDa chymotryptic fragment, also purified by gelatin affinity chromatography, gave a broad, diffuse band on polyacrylamide gel electrophoresis, whereas the analogous 43-kDa fragment from human plasma fibronectin migrated as a defined, narrow band. Upon extended treatment with endo-beta-galactosidase from Escherichia freundii, the 44-kDa chymotryptic gelatin-binding fragment from placental fibronectin changed its behavior on gel electrophoresis and migrated as a narrower, more defined band. The carbohydrates on human placental fibronectin contained a large percentage of polylactosamine structures, part of which occurred on the gelatin-binding fragment, comprising almost twice as much carbohydrate as plasma fibronectin. NH2-terminal amino acid sequence analysis of the chymotryptic gelatin-binding fragments from both fibronectins showed the first 21 residues to be identical. Tryptic and chymotryptic peptide maps of the gelatin-binding fragment from placental fibronectin, however, showed differences including several protease-resistant domains not found in the analogous fragment from plasma fibronectin. Intact placental fibronectin contains 20,000 Da of carbohydrate, whereas plasma fibronectin contains 11,000 Da. Placental fibronectin is more protease-resistant than plasma fibronectin, possibly due to the additional carbohydrate. Polyclonal antibodies against either fibronectin completely cross-react with amniotic fluid fibronectin, placental fibronectin, and plasma fibronectin upon Ouchterlony immunodiffusion. Human fibronectins of putatively the same polypeptide structure are, therefore, glycosylated in a dramatically different fashion, depending on the tissue of expression. If the patterns of glycosylation comprise the only difference in the glycoprotein, this may confer the characteristic protease resistance found for each of the fibronectins. 相似文献
13.
D H Lawson H G Thomas R G Roy D S Gordon R K Chawla D W Nixon A Richmond 《Journal of cellular biochemistry》1987,34(3):169-185
Autostimulatory growth factors may contribute to the ability of malignant cells to escape normal growth controls. We have previously shown that Hs0294 human malignant melanoma cells release into culture medium an acid-soluble, heat-stable, trypsin-sensitive, autostimulatory monolayer mitogen which can be purified from acetic acid extracts of conditioned medium by gel filtration, reverse-phase high-performance liquid chromatography, and preparative electrophoresis. The majority of this melanoma growth-stimulatory activity (MGSA) resides in a 16-Kd moiety, though bioactivity is also associated with 24-26 and less than 14-Kd forms of MGSA (Richmond and Thomas: J Cell Physiol 129:375, 1986). In order to further characterize this growth factor, monoclonal antibodies were prepared against a partially purified preparation of the autostimulatory melanoma mitogen. Monoclonal antibody clones were selected based on supernate inhibition of 3H-thymidine incorporation in serum-free Hs0294 melanoma cultures. One of these, termed FB2AH7, slows, but does not completely block, the growth of Hs0294 cells in serum-free medium in a dose-dependent manner. This antibody does not slow the growth of normal rat kidney fibroblasts, which neither produce nor require this mitogen, in either serum-free medium or medium containing 0.8% calf serum. This monoclonal antibody also blocks the mitogenic effects of partially purified preparations of this melanoma growth stimulatory activity (MGSA) on both Hs0294 cells and normal rat kidney fibroblasts. The FB2AH7 antibody has been demonstrated to bind MGSA by Western blot and by immunoprecipitation procedures. Western blot analysis of reverse-phase high-performance liquid chromatography purified growth factor demonstrated that FB2AH7 antibody binds to the 16-Kd and approximately 13-14-Kd forms of MGSA. FB2AH7 antibody can be used in immunoprecipitation experiments to bind the approximately 13-16-Kd forms of MGSA. The specificity of the binding of FB2AH7 antibody for MGSA but not other growth factors has been demonstrated in a modified dot blot assay. These data thus support the hypothesis that MGSA is an autostimulatory melanoma mitogen distinct from other growth factors. 相似文献
14.
M Katoh H Inagaki K Kurosawa-Ohsawa M Katsuura S Tanaka 《Biochemical and biophysical research communications》1990,167(3):1065-1072
A sensitive enzyme-linked immunosorbent assay (ELISA) system for human transforming growth factor alpha (TGF alpha) was developed in combination with polyclonal and monoclonal antibodies. Employing this assay system, we detected TGF alpha like activity in normal human plasma as well as in cancer patients' urine and plasma. These TGF alpha were analyzed by chromatography, immunoreactivity, and EGF-TGF alpha receptor binding assay and found to be identical to authentic human TGF alpha. The presence of TGF alpha circulating in normal adult plasma suggests a new role of TGF alpha in the human body. 相似文献
15.
流行性出血热免疫球蛋白的研制 总被引:1,自引:0,他引:1
陈冬星 《微生物学免疫学进展》1994,22(3):1-6
本文首次报告采用纯化灭活流行性出血热(EHF)Ⅰ型疫苗免疫健康献血员,采集EHF抗体高滴度的血浆,用低温乙醇法及盐析法分离纯化三批EHF免疫球蛋白。结果表明:(1)采用0,1,3,4(月)或0,1,4,5(月)免疫程序,疫苗剂量1~2ml(0.15~0.30mg蛋白),受免献血员血清平均抗体滴度可达1∶406(ELISA)或1∶112(RPHI)。(2)通过生化检定,三批制品的电泳纯度为97.05%,96.84%,99.26%;IgG单体和二聚体含量为89.55%,91.30%和98.21%。(3)用空斑抑制中和试验及免疫印染试验证明所纯化的免疫球蛋白具有抗EHF病毒特异性。(4)三批EHF免疫球蛋白的效价测定,结果为ELISA滴度≥1∶512,RPHI滴度≥1∶1024,PRNT(中和抗体)滴度1∶40。按16%蛋白计,EHF免疫球蛋白的效价可达原料血浆的10倍以上。(5)无菌、安全、毒性及热原质试验检定结果,全部通过《中国生物制品规程》要求。 相似文献
16.
The adhesive glycoprotein fibronectin has been isolated from fresh hamster plasma by affinity chromatography on gelatin coupled to Sepharose beads by the method of Engvall & Ruoslahti [Int. J. Cancer (1979) 20, 1-5]. Polyacrylamide-gel electrophoresis of material heated in sodium dodecyl sulphate and 2-mercaptoethanol shows two prominent polypeptide subunits of approx. mol.wts. 215 000 and 200 000, with variable amounts of lower-molecular-weight fragments. The unexpected polypeptide heterogeneity of different preparations of hamster fibronectins and bovine serum fibronectin is shown to be partly an artefact and is generated during isolation and storage of purified fibronectin. Treatment of each hamster fibronectin subunit or a smaller fragment of approx. mol.wt. 140 000 with thermolysin or trypsin after radioiodination produces similar patterns of tyrpsine-containing peptides, indicating similar primary amino-acid sequences. Antibodies raised against the major subunits of hamster plasma fibronectin were coupled to Sepharose beads and used in conjunction with gelatin affinity chromatography to isolate fibronectins extracted with urea from baby-hamster kidney (BHK) cells and present in the long-term culture medium of these cells. The cell and medium fibronectins are similar to hamster plasma fibronectin in amino-acid and carbohydrate composition and also produce very similar peptide 'maps'. We conclude that the various forms of hamster fibronectins are structurally analogous in agreement with indistinguishable biological properties in mediating the substance adhesion of BKH cells [Pena & Hughes (1978) Cell Biol. Int. Rep. 3, 339-344]. 相似文献
17.
Pugia MJ Jortani SA Basu M Sommer R Kuo HH Murphy S Williamson D Vranish J Boyle PJ Budzinski D Valdes R Basu SC 《Glycoconjugate journal》2007,24(1):5-15
Urinary trypsin inhibitors (uTi) suppress serine proteases during inflammation. After liberation from proinhibitors (P-alpha-I
and I-alpha-I) by the white blood cell (WBC) response, uTi readily pass through the kidneys into urine. A key uTi, bikunin,
is attached to O-linked and N-linked glycoconjugates. Recently, uTi inhibitors, called uristatins, were found to lack the
O-linked glycoconjugates. Monoclonal antibodies were produced using purified uristatin and screened for binding differences
to uristatin, bikunin, P-α-I, and I-α-I. Antibody-binding patterns were characterized using immunoaffinity binding onto protein-chip
surfaces and analysis by Surface Enhanced Laser Desorption/Ionization mass spectrometry (SELDI), using specimens from patients
and from purified uTi standards. Antibodies were developed and used in an enzyme-linked immunosorbent assay (ELISA) method
for uTi measurement in urine and plasma specimens. ELISA was performed on specimens from normal, presumed healthy, controls
and from patients who had been screened for inflammation using a high sensitivity C-reactive protein (CRP) test and a complete
blood count (CBC). Polyclonal antibody against uTi showed cross-reactivity with the Tamm–Horsfall protein (THP) and with proinhibitors.
Screening of anti-uTi monoclonal antibodies (Mab) revealed antibodies that did not cross-react with either of the above, thus
providing a tool to measure both uristatin and bikunin in urine with Mab 3G5 and in plasma with Mab 5D11. The monoclonal antibody
5D11 cross-reacts with specific N-linked glycoconjugates of uristatin present in plasma. In ca 96% of healthy adults, uTi
were present at <12 mg/l in urine and <4 mg/l in plasma. We also found that patients with an inflammation and a CRP of >2.0 mg/l
had higher urinary concentrations of uTi than the control population in every subject. Free uristatin and bikunin pass readily
into urine and are primarily bound to heavy chains that constitute the proinhibitor form in plasma. 相似文献
18.
《Cell biology international reports》1978,2(4):339-344
Highly purified preparations of fibronectin from bovine or hamster plasma and conditioned medium or urea extracts of BHK cells facilitate spreading of BHK cells to plastic surfaces. The different forms of fibronectin have very similar specific activities. 相似文献
19.
Cryptic chemotactic activity of fibronectin for human monocytes resides in the 120-kDa fibroblastic cell-binding fragment 总被引:20,自引:0,他引:20
R A Clark N E Wikner D E Doherty D A Norris 《The Journal of biological chemistry》1988,263(24):12115-12123
Monocytes and lymphocytes form a second wave of infiltrating blood leukocytes in areas of tissue injury. The mechanisms for monocyte accumulation at these sites are not completely understood. Recently, however, fragments from extracellular matrix proteins including collagen, elastin, and fibronectin have been shown to induce monocyte chemotaxis. In this report we demonstrate that chemotactic activity for human monocytes is expressed when a 120-kDa fragment containing the RGDS cell-binding peptide is released from intact fibronectin or from larger fibronectin fragments. Monocytes, either from mononuclear cell Ficoll-Hypaque preparations (10-20% monocytes, 89-90% lymphocytes) or from elutriation preparations (95% monocytes, 5% lymphocytes), but not lymphocytes, migrated toward 120-kDa fragment preparations (10(-7) M) in blind-end chambers when the cells were separated from the chemoattractant by a 5-micron pore polycarbonate filter either alone or overlying a 0.45-micron pore nitrocellulose filter. Neutrophils migrated toward zymosan-activated serum but not toward 10(-5)-10(-8) M concentrations of the 120-kDa fragment. Intact fibronectin had no chemotactic activity for human monocytes. Fibronectin was isolated from citrated human plasma by sequential gelatin-Sepharose affinity and DEAE ion-exchange chromatography in the presence of buffers containing 1 mM phenylmethylsulfonyl fluoride to prevent fragmentation. Controlled enzymatic digestion with thermolysin cleaved fibronectin into 30 kDa fibrin, 45 kDa collagen, and 150/160-kDa cell and heparin domains. Upon prolonged digestion, purified 150/160-kDa fragments were cleaved into 120-kDa cell and 30/40-kDa heparin-binding fragments. Even though the intact fibronectin molecule, the 150/160-kDa fragments, and the 120-kDa fragment, have cell binding activity for Chinese hamster ovary fibroblasts, only the 120-kDa fragment expressed chemotactic activity for human monocytes. Thus, the 120-kDa fibroblastic cell-binding fragment contains a cryptic site for monocyte chemotaxis which is expressed upon enzymatic cleavage of fibronectin. 相似文献
20.
Proteolysis of the calcium-dependent protease inhibitor by myocardial calcium-dependent protease 总被引:2,自引:0,他引:2
Bovine heart peak II calcium-dependent protease was capable of hydrolyzing its specific inhibitor protein at high molar ratios of protease to inhibitor. The proteolysis was inhibited by leupeptin and required millimolar calcium. Thus, it appeared to be attributable to the calcium-dependent protease and not to possible contaminating proteases in the purified preparations of inhibitor or calcium-dependent protease. Incubation of the purified inhibitor with the calcium-dependent protease produced a discrete pattern of inhibitor fragments on Western blots developed with an inhibitor-specific monoclonal antibody. Traces of similar or identical lower molecular weight immunoreactive material could be observed in Western blots of bovine heart extracts, and the immunoreactivity present as these lower molecular weight forms could be increased by incubation of the extracts with calcium ion. These results suggest that the inhibitor can be proteolyzed to low molecular weight forms which can be detected in cardiac tissue extracts, and that calcium-dependent protease(s) may be responsible for this phenomenon. 相似文献