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111.
Futoshi Aranishi Kenji Hara Kiyoshi Osatomi Tadashi Ishihara 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》1996,114(4):371-376
Cathepsin B was purified from the crude extract of carp (Cyprinus carpio) hepato-pancreas by the method involving ammonium sulfate fractionation and five sequential chromatographies monitored the activity with Z-Arg-Arg-MCA as a substrate, and the specific activity increased about 11,400 fold with a 2% recovery. Although the homogeneity of the purified cathepsin B was established on Native-PAGE, it migrated as two bands of 29,000 and 25,000 molecular weights by the single and heavy chains on SDS-PAGE, respectively. The monospecific antibody against the homogeneous cathepsin B was purified by the affinity chromatography on cathepsin B-Sepharose 4B, and did not immunologically react with rat cathepsin B, carp cathepsins H and L but only with carp cathepsin B by immunoelectrophoretic blot analysis. As the result of the tissue and liver distributions of cathepsin B, the remarkable immunological reactivities in the extracts of spleen, kidney and hepato-pancreas in carp and those of pacific cod, yellow fin tuna, skip jack tuna and common mackerel in pisces were detected with the anti-carp hepato-pancreas cathepsin B at molecular weight of nearby 29,000 or 25,000. 相似文献
112.
Thomas Hiltonen Jan Karlsson Kristin Palmqvist Adrian K. Clarke Göran Samuelsson 《Planta》1995,195(3):345-351
An intracellular carbonic anhydrase (CA; EC 4.2.1.1) was purified and characterised from the unicellular green alga Coccomyxa sp. Initial studies showed that cultured Coccomyxa cells contain an intracellular CA activity around 100 times higher than that measured in high-CO2-grown cells of Chlamydomonas reinhardtii CW 92. Purification of a protein extract containing the CA activity was carried out using ammonium-sulphate precipitation followed by anion-exchange chromatography. Proteins were then separated by native (non-dissociating) polyacrylamide gel electrophoresis, with each individual protein band excised and assayed for CA activity. Measurements revealed CA activity associated with two discrete protein bands with similar molecular masses of 80 +5 kDa. Dissociation by denaturing polyacrylamide gel electrophoresis showed that both proteins contained a single polypeptide of 26 kDa, suggesting that each 80-kDa native protein was a homogeneous trimer. Isoelectric focusing of the 80-kDa proteins also produced a single protein band at a pH of 6.5. Inhibition studies on the purified CA extract showed that 50% inhibition of CA activity was obtained using 1 M azetazolamide. Polyclonal antibodies against the 26-kDa CA were produced and shown to have a high specific binding to a single polypeptide in soluble protein extracts from Coccomyxa cells. The same antiserum, however, failed to cross-react with soluble proteins isolated from two different species of green algae, Chlamydomonas reinhardtii and Chlorella vulgaris. Correspondingly, antisera directed against pea chloroplastic CA, extracellular CA from C. reinhardtii and human CAII, showed no cross-hybridisation to the 26-kDa polypeptide in Coccomyxa. The 26-kDa protein was confirmed as being a CA by N-terminal sequencing of two internal polypeptide fragments and alignment of these sequences with that of previously identified CA proteins from several different species.Abbreviations CA
carbonic anhydrase
- CCM
CO2-concentrating mechanism
- IEF
isoelectric focusing
- Rubisco
ribulose-l,5-bisphosphate carboxylase/oxygenase
We would like to thank Drs. Cecilia Forsman, Inga-Maj Johansson and Nalle Jonsson for their valuable advice concerning the isolation of CA. This work was supported by the Swedish Natural Research Council and Seth M. Kempes Memorial foundation. 相似文献
113.
Alexander A. Kortt Robin E. Guthrie Mark G. Hinds Barbara E. Power Neva Ivancic J. Bruce Caldwell L. Clem Gruen Raymond S. Norton Peter J. Hudson 《Journal of Protein Chemistry》1995,14(3):167-178
The VH domain of anti-influenza neuraminidase antibody NC41, with and without a C-terminal hydrophilic marker peptide (FLAGTM), has been expressed in high yield (15–27 mg/L) inEscherichia coli. Both forms were secreted into the periplasm where they formed insoluble aggregates which were solubilized quantitatively with 2 M guanidine hydrochloride and purified to homogeneity by ion-exchange chromatography. The VH-FLAG was composed of three isoforms (pI values of 4.6, 4.9, and 5.3) and the VH molecule was composed of two isoforms with pI values of 5.1 and 6.7; the difference between the VH isoforms was shown to be due to cyclization of the N-terminal glutamine residue in the pI 5.1 isoform. At 20°C and concentrations of 5–10mg/ml the VH domain dimerized in solution and then partly precipitated, resulting in the broadening of resonances in its1H NMR spectrum. Reagents such as CHAPS,n-octylglucoside, and ethylene glycol, which presumably mask the exposed hydrophobic interface of the VH molecule, prevented dimerization of the VH and permitted good-quality NMR spectra on isotope-labeled protein to be obtained. 相似文献
114.
Cecilia Ballaré Marcela Barrio Paula Portela Jose Mordoh 《Cancer immunology, immunotherapy : CII》1995,41(1):15-22
FC-2.15 is a murine IgM monoclonal antibody (mAb) that recognizes a cell-surface antigen (Ag2.15) expressed in most tumor-proliferating cells of human breast carcinomas and other neoplasias. In this study the cytotoxic ability of mAb FC-2.15, its cell-surface binding properties and endocytosis in Ag2.15-expressing (Ag2.15+) cells were investigated. A51Cr-release assay was used to test the FC-2.15-mediated cytotoxicity. When human serum was used as source of complement, FC-2.15 exerted a strong cytotoxic effect against human Ag2.15+ cells such as MCF-7 (breast cancer cell line), primary breast carcinoma cells, polymorphonuclear leukocytes and chronic myeloid leukemia cells. The mAb concentration range was 1–50 g/ml. Cytotoxicity was completely abolished when complement was inactivated. Only 3.8±2.9% of MCF-7 cells survived the treatment with FC-2.15 in the presence of human serum. A flow-cytometry assay was performed to study the Ag2.15 expression of the surviving cells and they were found to be Ag2.15–. FC-2.15 did not mediate antibody-dependent cell cytotoxicity when different effector cells were used. Scatchard analysis with125I-FC-2.15 on MCF-7 cells demonstrated an affinity constant of 6.9×107 M–1 and 2.8×106 antigenic sites/cell.125I-FC-2.15 was internalized to cytoplasmic vesicles reaching a maximum of 27% after 6 h incubation, followed by the release of labeled degradation products to the supernatant. FC-2.15 appears to exert its cytotoxic effect mainly in the presence of human complement, it reacts with intermediate affinity with a high-density surface antigen, and it is slowly internalized by Ag2.15+ cells. 相似文献
115.
O. Loiseau J. Cottin R. Robert G. Tronchin C. Mahaza J.M. Senet 《FEMS immunology and medical microbiology》1995,11(3):219-230
Abstract Monoclonal antibodies were obtained by the classic hybridoma technique with lymphocytes of BALB/c mice immunized with formalin killed Listeria monocytogenes cells. Among 1000 hybridomas issued from the fusion, four monoclonal antibodies (mAbs A6 A E4, C10 A F7, G4 A D6, G7 A D5) gave interesting results. By Western-blot analysis with various soluble extracts of different Listeria species, the four mAbs reacted with two major antigens of 38 and 41 kDa, with all Listeria species tested. The mAb A6 A E4 is an IgG2b with κ light chains and reacted only with Listeria antigens without any cross reaction with other organisms tested by ELISA, dot-blotting and Western-blotting. With the same conditions, the three other mAbs reacted with Listeria and with other genus extracts, particularly with Streptococcus and Enterococcus . mAb A6 A E4-reactive antigens are proteins, and glycoprotein immunoassay indicated that the epitope is devoid of carbohydrate moiety. This mAb A6 A E4-reactive protein was neither expressed on cell surface nor released outside the bacteria; immunogold electron microscopy showed that these antigens were localized in the cytoplasma area. 相似文献
116.
Purification and characterization of M3 protein expressed on the surface of group A streptococcal type 3 strain C203 总被引:2,自引:0,他引:2
Kyongsu Hong Yoshikazu Komurasaki Hideyuki Kobayashi Hiromichi Ishikawa Kozo Inoue 《FEMS immunology and medical microbiology》1995,12(1):73-82
Abstract Monoclonal antibodies (mAbs) have been produced by immunizing BALB/C mice with whole M+ bacteria in incomplete Freund adjuvant and the resulting mAbs for M3 protein have been selected by an indirect immuno-fluorescent technique using formaldehyde-fixed M+ and M− bacteria. Four mAbs reacted with a 65 kDa protein in an extract obtained from the cell wall of M+ bacteria after treatment with N -acetyl muramidase and lysozyme. The purified 65 kDa protein neutralized the phagocytic activity of rabbit anti-M3 antibody. The N-terminal amino acid sequence of the 65 kDa protein was identical with that of protein generated by the M3 gene which has been previously cloned and sequenced. The evidence indicates that the 65 kDa protein is M3 protein. The M3 protein bound not only human fibrinogen but also human serum albumin (HSA). When the M3 protein was purified by gel-filtration and ion-exchange chromatography in the absence of phenylmethyl sulfonyl fluoride (PMSF), four fragments (35 kDa, 32 kDa, 30 kDa, and 25 kDa) in addition to the intact molecule appeared. N-terminal amino acid sequence analysis showed that 35 kDa and 25 kDa fragments were ANAAD and DARSV, respectively, being identical at positions 1–5 and 198–202 to the M3 gene derived protein. Therefore, the 35 kDa and 25 kDa fragments, which were presumed to be cleavage products, may be derived from the C-terminal part and N-terminal part of the intact molecule, respectively. When the effect of purified M3 protein in the bactericidal activity of normal human blood in the presence of M− bacteria was investigated, the M3 protein was responsible for the organism's resistance to attack by phagocytic cells. 相似文献
117.
Induced and constitutive murine IgG-binding factors (IgG-BFs) have been purified by affinity chromatography from supernatants of T-cells preincubated with or without murine monoclonal IgG1 and IgG2b, respectively. IgG-BF Mr values have been studied by SDS-polyacrylamide gel electrophoresis (PAGE) after treatment with SDS under conditions which do not noticeably alter their immunosuppressive activities on the secondary in vitro IgG antibody response. Suppression was recovered at Mr values of 80000, 40000 and 20000. When induced IgG-BF was tested, the isotype-specific suppressive activity was found only at 40 kDa. The 20-kDa moiety appeared to derive from the 40-kDa component and the material found at 80 kDa exerted non-specific immunosuppressive effects. We conclude therefore that isotype-specific IgG-BF has an apparent Mr of 40000. 相似文献
118.
Monoclonal antibodies (QB01 and 1200) prepared against human proclatin (hPRL) have helped define a variant form of the hormone. This variant is of apparently higher molecular mass (26kDa) than the predominant form of the hormone (24kDa) and its presence does not appear to be species-restricted. The demonstration of the 26 kDa form of the hPRL in fresh pituitary tissue and amniotic fluid suggests it may retain some specific function. 相似文献
119.
Sodium butyrate inhibits the synthesis of the transformation related protein p 53 in 3T6 mouse fibroblasts 总被引:3,自引:0,他引:3
Sodium butyrate, which blocks the cell cycle of many cell types in the G1 phase, strongly inhibits the synthesis of the transformation related, 53 kDa protein in 3T6 fibroblasts but much less so in SV 40 transformed mouse cells. By several criteria, this effect of the fatty acid appears to be indirect; p 53 synthesis takes place several hours after the butyrate-sensitive step in G1. The results are discussed in the light of a putative role of p 53 in growth control. 相似文献
120.
Recognition of sodium- and potassium-dependent adenosine triphosphatase on mouse lymphoid cells by means of a monoclonal antibody 总被引:3,自引:0,他引:3
Summary Previous evidence has established the similarity between (Na++K+)-ATPase (ATP phosphohydrolase, EC.3.6.1.3) and the antigen recognized by the rat antimouse monoclonal antibody anti-BSP-3. This antibody has been used for investigation of the surface expression and biochemical analysis of the enzyme in different mouse lymphoid populations. The BSP-3 determinant is found on almost all thymocytes and concanavalin A-induced thymocytes, to a lesser extent on bone marrow cells and also on a minor population of spleen cells. Spleen cells from athymic mice are negative. The (Na++ K+)-ATPase purified from mouse thymus by affinity chromatography migrates in SDS-polyacrylamide gels in the form of two polypeptide chains of 105000 and 51000 daltons. Chains of the same molecular weight, fractionated on SDS-PAGE from microsomes of mouse thymuses, are shown to react with subunit-specific polyclonal antisera against ATPase in immunoblotting experiments. Immunoprecipitation with anti-BSP-3 from surface iodinated thymocytes yields only the small subunit. Comparison of the chains isolated from thymus and brain shows molecular weight differences in both subunits. These results, and variations in the reactivity pattern of the anti-BSP-3 antibody on several cell types, may indicate a possible heterogeneity of the (Na++K+)ATPase expressed by various tissues and cells. 相似文献