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1.
Cultured hepatocytes from adult rats stimulated with combinations of growth factors enter into S phase but do not undergo multiple rounds of DNA synthesis nor mitosis. We have examined the potential of an introduced oncogene to induce alterations in the DNA synthetic activity of the cultured hepatocytes in response to epidermal growth factor (EGF). Overexpression of c-myc did not initiate significant DNA synthesis in rat hepatocyte cultures alone, although it cooperated with added EGF to super-induce thymidine incorporation into DNA. From our results, it is suggested that EGF is also necessary to initiate hepatocyte DNA synthesis probably by inducing a battery of cell cycle-related genes if incubated with c-myc transfected cultures for only 5 hours. Hepatocyte polypeptides reacting with anti-MYC antisera were found to migrate between 55-67 KDa in SDS-PAGE; only the 64-67 KDa species were found to be phosphorylated, and the observed size heterogeneity may be due to proteolytic degradation or may reflect presently unknown posttranslational modifications. 相似文献
2.
Anisakis simplex larvae were cultured in vitro in medium containing 35S-methionine for ten days. The medium and the larval tissues were analysed for biosynthetically labelled polypeptide by sodium dodecyl sulphate polyacrylamide gel electrophoresis and autoradiography. Immunoprecipitates with positive and negative human antisera were similarly analysed, using Staphylococcus aureus to absorb immuno-complexes. ES products of Anisakis larvae contained many polypeptides with molecular weights of less than 200 K. 180 KDa and 40 KDa polypeptides in ES products reacted with IgG in Anisakis-infected human sera. Somatic extracts also contained many polypeptides with molecular weights of less than 200 K. One of these polypeptides with a molecular weight of 130 K reacted with IgG in Anisakis-infected human sera. These polypeptides did not react with other nematode-infected human sera. 相似文献
3.
Munir Abdullah Laura L. Tres Hiroshi Ueda Ping-Chuan Hu Abraham L. Kierszenbaum 《Molecular and cellular biochemistry》1988,81(2):165-176
A high performance liquid chromatographic procedure has been used for the purification of rat Sertoli cell secretory protein S70 and S45-S35 heterodimeric protein to determine their role during spermatogenesis. These two proteins display binding affinity for each other and appear antigenically related. We have observed that: 1. S70 and S45-S35 heterodimeric protein coelute during purification, 2. polyclonal antiserum raised against protein S70 recognizes common antigenic determinants in polypeptides S45 and S35, the disulfide-linked components of the heterodimeric protein, and 3. a monoclonal antibody that recognizes polypeptide S35 but does not crossreact with either protein S70 or polypeptide S45, immunoprecipitates the S70/S45-S35 heterodimeric protein complex. In immunofluorescent experiments, antisera raised against protein S70 and polypeptide components of S45-S35 heterodimeric protein immunoreact with two major sperm intracellular structures: the acrosome and periaxonemal outer dense fibers of sperm tail. Immunoreactivity was not detected on the sperm plasma membrane surface of unfixed, living sperm. Outer dense fibers extracted from sperm tails by a combined treatment with cetylthrimethylammonium bromide and 2-mercaptoethanol, yielded a characteristic polypeptide pattern. In immunoblotting experiments sperm tail polypeptides were recognized by polyclonal antisera raised against Sertoli cell secretory proteins. We conclude that Sertoli cell secretory proteins S70 and S45-S35 heterodimeric protein are antigenically related to each other and to keratin-like polypeptides from sperm tail. 相似文献
4.
Purified goat sperm plasma membrane was used as antigen to raise the antibody in rabbit. Using this antisera four groups of antigenic membrane polypeptides are determined in caput and cauda epididymal sperm. The immunoresponsiveness of the polypeptides in caput and cauda sperm differs significantly. In case of cauda epididymal sperm, the polypeptides of region A (96KDa, 82KDa, 78KDa, 68KDa) and region D (24KDa, 20KDa, 18KDa) are highly immunoresponsive whereas in case of caput epididymal sperm the same antisera recognized the polypeptides of region B, C and D. By surface labelling with lactoperoxidase iodination and subsequent immunoprecipitation in the iodinated cell extract we demonstrate eight of these above polypeptides (96KDa, 82KDa, 68KDa, 50KDa, 29KDa, 24KDa, 20KDa and 18KDa) as surface antigen. The 96KDa, 82KDa and 68KDa surface polypeptides are highly immunoresponsive than the other lower molecular weight surface antigens in cauda epididymal goat spermatozoa. 相似文献
5.
Purification of RNA polymerase II from chicken myeloblastosis (leukemia) cells to homogeneity and subsequent structural analysis of the purified enzyme revealed that the enzyme contained seven polypeptides with molecular masses ranging from 27 KDa to 220 KDa. Inclusion of protease inhibitors in the buffer system during purification significantly increased the molar ratio of the largest (220 KDa) polypeptide to the second largest (180 KDa) polypeptide. However, proteolytic conversion of the 220 KDa to 180 KDa polypeptide did not inhibit the DNA binding activity of the enzyme. The enzyme, after dissociation into subunits in a SDS-polyacrylamide gel containing urea was blotted onto a nitrocellulose filter. The filter was incubated with 32P-labeled calf thymus DNA and both the 220 KDa and 180 KDa polypeptides of the enzyme bind DNA, suggesting that the DNA-binding site of the enzyme resides on the 180 KDa polypeptide of the largest subunit. 相似文献
6.
Abraham L. Kierszenbaum Munir Abdullah Hiroshi Ueda Laura L. Tres 《Molecular and cellular biochemistry》1989,86(2):171-179
We have previously reported that (a) polyclonal antisera raised against rat Sertoli cell secretory protein S70 and S45-S35 heterodimeric protein recognize outer dense fiber polypeptides from rat sperm tail, and (b) protein S70 is antigenically related to polypeptides S45 and S35, the disulfide-linked components of the heterodimeric protein. We now report that polyclonal antisera generated against three different outer dense fiber polypeptides recognize (a) the putative antigen of the sperm tail and (b) Sertoli cell secretory protein S70 and its antigenically-related polypeptides. Immunogold electron microscopy shows that outer dense fibers of epididymal sperm crossreact with anti-S70 serum as well as with an antiserum raised against the polypeptide D complex of extracted outer dense fibers. Electron microscopy demonstrates that outer dense fibers consist of filamentous, coil-coiled units aligned side-by-side with each other. Results of this study strengthen the antigenic homology between Sertoli cell secretory proteins and outer dense fiber polypeptides of the sperm tail. 相似文献
7.
We have studied the polypeptides associated with the expression of the transforming region of the Ad5 genome by immunoprecipitating antigens (using the double antibody and protein A-Sepharose techniques) from cells infected with wild-type (wt) Ad5 or transformation-defective host range (hr) mutants and from cells transformed by Ad5. Three different antisera were used: P antiserum specific for early viral products (Russell et al., 1967) and two different hamster tumor antisera. Immunoprecipitation of antigens from wt-infected KB cells followed by SDS-polyacrylamide gel electrophoresis of precipitated proteins revealed that a major polypeptide having a molecular weight of approximately 58,000 was detected with all three antisera and with both the double antibody and the protein A-Sepharose techniques, while P antiserum also precipitated polypeptides of molecular weights 72,000, 67,000 and 44,000, which probably represent the DNA binding protein and related polypeptides, respectively. With the double antibody technique, in addition to the proteins mentioned above, P antiserum and the hamster tumor antisera precipitated a 10,500 dalton polypeptide which was not detected when the protein A-Sepharose procedure was used. Using either the double antibody or the protein A-Sepharose technique, we found that hr mutants from complementation group II failed to induce the synthesis of the 58,000 dalton protein, whereas mutants from complementation group I produced normal or near normal amounts. Using the double antibody technique, we found that the 10,500 dalton protein was absent or made in reduced amounts by group I mutants. A 58,000 dalton protein was detected in a number of different Ad5-transformed cell lines, including the 293 human line, the 14b hamster line and several transformed rat cell lines. This observation and the fact that transformation negative group II mutants fail to induce the synthesis of a 58,000 dalton polypeptide suggest that this protein is one of the Ad5-specific products necessary for cell transformation. 相似文献
8.
9.
Identification of the in vitro translation products of adenovirus mRNA by immunoprecipitation. 总被引:3,自引:3,他引:0
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Adenovirus type 2 mRNA was translated in S30 extracts from Ehrlich ascites and wheat embryo cells. The in vitro products were identified by sodium dodecyl sulfate-gel electrophoresis after immunoprecipitation with specific antisera in the presence of urea. Seven virion polypeptides could be identified by immunoprecipitation. Three of these appear to be precursors to polypeptides of the virion. mRNA isolated late in adenovirus infection was separated into three size classes by zonal sedimentation. Material sedimenting at 26S was translated into polypeptides corresponding to the largest virion polypeptides II to IV, a 22S fraction corresponding to polypeptide V, and smaller polypeptides and a 15S fraction corresponding to polypeptide IX. A significant amount of polypeptide IX was also synthesized by the 26S and 22S RNA. 相似文献
10.
The polypeptides of the subunits of 70S ribosomes isolated from rye (Secale cereale L.) leaf chloroplasts were analyzed by two-dimensional polyacrylamide gel electrophoresis. The 50S subunit contained approx. 33 polypeptides in the range of relative molecular mass (Mr) 13000–36000, the 30S subunit contained approx. 25 polypeptides in the range of Mr 13000–40500. Antisera raised against the individual isolated ribosomal subunits detected approx. 17 polypeptides of the 50S and 10 polypeptides of the 30S subunit in the immunoblotting assay. By immunoblotting with these antisera the major antigenic ribosomal polypeptides (r-proteins) of the chloroplasts were clearly and specifically visualized also in separations of leaf extracts or soluble chloroplast supernatants. In extracts from rye leaves grown at 32° C, a temperature which is non-permissive for 70S-ribosome formation, or in supernatants from ribosome-deficient isolated plastids, six plastidic r-proteins were visualized by immunoblotting with the anti-50S-serum and two to four plastidic r-proteins were detected by immunoblotting with the anti-30S-serum, while other r-proteins that reacted with our antisera were missing. Those plastidic r-proteins that were present in 70S-ribosome-deficient leaves must represent individual unassembled ribosomal polypeptides that were synthesized on cytoplasmic 80S ribosomes. For the biogenesis of chloroplast ribosomes the mechanism of coordinate regulation appear to be less strict than those known for the biogenesis of bacterial ribosomes, thus allowing a marked accumulation of several unassembled ribosomal polypeptides of cytoplasmic origin.Abbreviations L
polypeptide of large ribosomal subunit
- Mr
relative molecular mass
- r-protein
ribosomal polypeptide
- S
polypeptide of small ribosomal subunit
- SDS
sodium dodecyl sulfate 相似文献
11.
Immunological Reactivity of Antisera Prepared Against the Sodium Dodecyl Sulfate-Treated Structural Polypeptides of Adenovirus-Associated Virus 总被引:7,自引:6,他引:1
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The preparation of antisera to the three purified sodium dodecyl sulfate (SDS)-treated polypeptide components (VP1, VP2, VP3) of adenovirus-associated virus (AAV) type 3H is described. In immunofluorescence tests (FA), these antisera stained heat-stable antigens with distinct morphologies in cells co-infected with either adenovirus or herpes simplex virus. Kinetic studies of antigen formation showed that VP1 antiserum first stained the cytoplasm (14 hr) and later (by 18 hr) stained both cytoplasmic and intranuclear areas. VP2 antiserum stained only discrete intranuclear areas, and VP3 antiserum stained nearly the entire nucleus. All three VP antigens appeared at about the 14th hr postinfection, about 2 hr prior to the appearance of whole virion antigen. The VP antisera cross-reacted in FA with AAV types 1 and 2 (all at one-eighth of the homologous titer), but did not react with other parvoviruses, i.e., rat virus, hemadsorbing enteric virus of calves, minute virus of mice, or H-1 virus. These non-neutralizing antisera reacted specifically with SDS-treated AAV virion antigens in complement fixation and immunodiffusion tests, and antiserum prepared against SDS-treated helper adenovirus structural polypeptides reacted with adenovirus polypeptide antigens. All antisera to SDS-treated polypeptides were specific for new antigens revealed on the dissociated peptides and did not react with whole virions, whereas whole-virion antisera did not cross-react with the polypeptide antigens. These findings suggest that antigens unique to the polypeptides of AAV are revealed by SDS treatment and that these antigens can be detected in cells prior to the folding of the polypeptides into the molecular configuration they possess as virion subunits. These results also indicate that at least one AAV polypeptide component is synthesized in the cell cytoplasm. 相似文献
12.
The polypeptide composition of the mitochondrial NADH: ubiquinone reductase complex from several mammalian species. 总被引:4,自引:1,他引:3
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The polypeptide composition of isolated mitochondrial NADH:ubiquinone reductase (NADH dehydrogenase) is very similar to that of material immunoprecipitated from detergent-solubilized bovine heart submitochondrial particles by antisera to the holoenzyme. The specificity of the antisera for dehydrogenase polypeptides was determined by immunoblotting, which showed that antisera reacting with only a few proteins were able to immunoprecipitate all others in parallel. The polypeptide compositions of rat, rabbit and human NADH dehydrogenase were determined by immunoprecipitation of the enzyme from solubilized submitochondrial particles and proved to be very similar to that of the bovine heart enzyme, particularly in the high-Mr region. Further homologies in these and other species were explored by immunoblotting with antisera to the holoenzyme and monospecific antisera raised against iron-sulphur-protein subunits of the enzyme. 相似文献
13.
Impaired Maturation of Mouse Mammary Tumor Virus Precursor Polypeptides in Lymphoid Leukemia Cells, Producing Intracytoplasmic A Particles and No Extracellular B-Type Virions 总被引:4,自引:4,他引:0
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Roeland Nusse Lex van der Ploeg Luuk van Duijn Rob Michalides Jo Hilgers 《Journal of virology》1979,32(1):251-258
Processing of polypeptides of the mouse mammary tumor virus, a type B retrovirus, was investigated in a transplanted thymic lymphoma cell line of the GR strain (GRSL). This cell line was maintained in vivo in ascites form and in vitro as a suspension culture. GRSL cells produce clusters of intracytoplasmic A particles and are virtually deficient in the production of mature extracellular B-type particles. As control, a mammary tumor cell line of the same mouse strain capable of complete virion synthesis was used. The kinetics of viral polypeptide synthesis were studied by pulse labeling with various isotopes (including (35)S and (32)P), followed by immunoprecipitation of cell lysates with monospecific antisera to the major mouse mammary tumor virus gag and env proteins, p27 and gp52, respectively. Both the primary gag and env precursor polypeptides were synthesized in the GRSL cells, but their conversion into viral proteins was impaired. The major gag precursor, Pr73(gag), was stable over a period of 8 h, and mature viral core polypeptides could not be detected. Also, the highly phosphorylated intermediates in the proteolytic processing of Pr73(gag) in virus-producing cells were absent in GRSL cells. By immunoprecipitation, Pr73(gag) was detected in a GRSL particle fraction with the density of intracytoplasmic A particles. The precursor for envelope proteins, Pr73(env), was turned over without the generation of mature viral envelope components gp52 and gp36. The in vivo-transplanted ascites GRSL cells, however, were shown to express gp52 on the cell surface together with a 73,000-dalton polypeptide, as indicated by cell surface iodination and immunoprecipitation. 相似文献
14.
A monoclonal antibody to dopamine beta-monooxygenase (DBH) has been produced by an in vitro immunization technique. This antibody has been found to react with an epitope common to both soluble DBH (SDBH) and membrane-bound DBH (MDBH). A single CNBr fragment contains this antigenic site. Examination of electrophoretograms of chromaffin granule membrane and lysate by the immunoblot procedure revealed additional complexity. A 58-KDa polypeptide in low abundance was stained which could not be detected with the polyclonal antiserum. Chemical deglycosylation of SDBH produces two new polypeptides of 67 and 58 KDa. The entirety of this data suggests that the 58-KDa band is an unglycosylated form of DBH, proteolytically cleaved after biosynthesis. 相似文献
15.
16.
Michal Lorenz Brygida Bisikirska Beata Hanus-Lorenz Kazimierz Strzalka Aleksander F. Sikorski 《Cell biology international》1995,19(2):83-90
It was found either in Western-blot analysis or in indirect immunofluorescence microscopy that cells of the alga Chlamydomonas reinhardhi contain polypeptides cross-reactng with antibodies directed against red blood cell spectrin. The protein could also be detected by immunoprecipitation with anti-spectrin antibodies. C. reinhardtii cells contain distinct polypeptide chains reacting with antibodies directed against either α- or β- spectrin subunits. This protein was extracted from the cells with low ionic strength solution but was not with nonionic detergent. 相似文献
17.
Cellulosome-like complexes were identified in the broth and sonic extracts of cellobiose-and cellulose-grown cells ofBacteroides cellulosolvens. The extracellular fractions contained three to four major polypeptides and several minor polypeptide bands that were localized in two major gel filtration peaks indicating average molecular weights of about 700 kDa and >10 MDa. A relatively large molecular weight component (Mr 230 kDa) was found to contain carbohydrate, but no apparent enzymatic activity of its own could be detected. The cell sonicate displayed a more complicated polypeptide profile, and glycosylated polypeptides were larger (ca. 310 and 290 kDa) than that of the extracellular fraction. The 230-kDa extracellular component interacted strongly with the GSI isolectin fromGriffonia simplicifolia, exhibited immunochemical cross-reactivity with the S1 subunit of the cellulosome fromClostridium thermocellum, and displayed anomalous pH- and salt-dependent migratory behavior in SDS-PAGE. Taken together, this evidence strongly suggests a structural similarity between the glycoconjugates of these two distinct cellulolytic bacteria. A major 84-kDa polypeptide was identified as a xylanase, and a 50-kDa polypeptide displayed endoglucanase activity. Additional biochemical and cytochemical evidence indicated that cellulosome-like cellulolytic complexes are associated with the cell surface in this bacterium. 相似文献
18.
P M Alliel A Dautigny J Lamy J N Lamy P Jollès 《European journal of biochemistry》1983,134(3):407-414
Translation of Androctonus australis poly(A)-RNA in vitro led to a number of polypeptides products (8-10) of 70-73 kDa analyzed by two-dimensional gel electrophoresis and identified by immunoprecipitation with an anti-(dissociated hemocyanin) antiserum. The translated hemocyanin polypeptides have the same physico-chemical characteristics as authentic hemocyanin subunits. Subunits Aa 2 and Aa 4 have been identified with monospecific antisera characterized (a) by their capability of reacting with their homologous subunit and (b) by their inability of binding to cross-reacting subunits. Each polypeptide chain is coded by a different messenger without significant post-translational events. Hemocyanin could be detected among the translation products of the poly(A)-RNA isolated from the cuticle under the carapace. 相似文献
19.
Immunological relation between 14 S dynein and 30 S dynein from the cilia of Tetrahymena pyriformis.
The immunological relation between 14 S dynein and 30 S dynein obtained from Tetrahymena cilia was investigated by using antisera specific for each dynein subunit or some dynein subunits separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Although 14 and 30 S dynein main subunits have different electrophoretic mobilities, our immunodiffusion tests showed that there exists a close immunological relation between them. At least three immunologically different polypeptides designated polypeptides A, B and C are included in the 30 S dynein main band which has been recognized as a single component by electrophoresis, and that the polypeptides designated A',B' and C' are included in the 14 S dynein main bands. Polypeptides A and A',B and B', or C and C' appeared to have a certain common antigenic determinant(s). Polypeptide C of 30 S dynein was shown to possess a certain antigenic determinant(s) specific for 30 S dynein, besides the determinant common with that of polypeptide C' of 14S dynein. The second main component of 30 S dynein proved to be a specific polypeptide of 30 S dynein but not to be a degraded product of the main polypedtide. All antisera reacted with native dynein molecules to some extent, but did not inhibit dynein ATPase (ATP phosphohydrase, EC 3.6.1.3) activity significantly. 相似文献
20.
J. Muñoz-Maines J. Randall Slemmon Mitradas M. Panicker Noel Neighbor Paul M. Salvaterra 《Journal of neurochemistry》1988,50(1):167-175
A fusion protein containing a Drosophila choline acetyltransferase (ChAT) cDNA insert was purified from a lambda gtll lysate of Escherichia coli. The cDNA insert, which contained a 728-amino acid coding region for ChAT, was used for immunizing rabbits. Three different antisera were produced that could recognize native Drosophila ChAT with low titer. In addition, all three antisera stained enzyme polypeptides using the Western blot technique at high titers. The antisera recognized ChAT polypeptides with molecular masses of 67 and 54 kilodaltons in Western blots of partially purified enzyme; these polypeptides had previously been identified using monoclonal anti-ChAT antibodies and are the major components of completely purified enzyme. It was surprising that when these antisera were used to stain Western blots of Drosophila head homogenates, the major immunoreactive band had a molecular mass of 75 kilodaltons. The relationship of this 75-kilodalton polypeptide to ChAT activity was investigated by fractionating fresh fly head homogenates using rapid HPLC gel filtration chromatography. Analysis of column fractions for enzyme activity and immunoreactive polypeptides indicated that the 75- and 67-kilodalton polypeptides can be resolved and are both enzymatically active. In addition, a correlation was observed between the relative immunostaining intensities of both the 75- and 67-kilodalton bands and ChAT activity when supernatants from fresh fly head homogenates were autolyzed at 37 degrees C. Our results indicate that ChAT is present in fresh Drosophila heads primarily as an active enzyme with a molecular mass of 75 kilodaltons.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献