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Mouse neuroblastoma x rat glioma hybrid NG108-15 and mouse neuroblastoma x embryonic hamster brain NCB20 cells were transfected with a construct containing a human 2 adrenoceptor cDNA under the control of the actin promoter. Clones were selected on the basis of resistance to geneticin sulphate and those expressing a range of levels of the receptor expanded for further study. Membranes from a clone of NG108-15 cells expressing high levels of the receptor (N22) but not one expressing only low levels of the receptor (N17) exhibited a markedly elevated adenylyl cyclase activity when measured in the presence of Mg2+ compared to wild type cells. This was not due to elevated levels of the adenylyl cyclase catalytic moiety however as there was no difference in these membranes when the adenylyl cyclase activity was measured in the presence of Mn2+. The elevated basal activity was partially reversed by addition of the -adrenoceptor antagonist propranolol. Agonist activation of N22 but not N17 cells led to a large selective down-regulation of cellular Gs levels which was independent of the generation of cyclic AMP. Isoprenaline stimulation of adenylyl cyclase activity and of the specific high affinity binding of [3H] forskolin was achieved with substantially greater potency (some 30 fold) in N22 cell membranes than in N17. By contrast agonist activation of the endogenously expressed IP prostanoid receptor caused stimulation of adenylyl cyclase and stimulation of high affinity [3H] forskolin binding which was equipotent in each of N22, N17 and wild type NG108-15 cells. Agonist activation of the IP prostanoid receptor caused an equivalent degree of Gs down-regulation in each cell type. Expression of an epitope tagged variant of Gs in NG108-15 cells resulted in prostanoid agonist-induced down-regulation of this polypeptide in a manner indistinguishable from that of wild type Gs. Isolation of clones of NCB20 cells expressing high levels of the 2 adrenoceptor also resulted in a specific agonist-induced down-regulation of Gs.  相似文献   

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EF-1a binds aminoacyl-tRNA to the ribosome with the hydrolysis of GTP; the complex facilitates the exchange of GDP for GTP to initiate another round of elongation. To examine the subunit structure of EF-1 and phosphorylation by protein kinase CKII, recombinant , , and subunits from rabbit were expressed in E. coli and the subunits were reconstituted into partial and complete complexes and analyzed by gel filtration. To determine the availability of the and subunits for phosphorylation by CKII, the subunits and the reconstituted complexes were examined as substrates for CKII. Formation of the nucleotide exchange complex increased the rate of phosphorylation of the subunit and reduced the Km, while addition of to or the complex inhibited phosphorylation by CKII. However, a had little effect on phosphorylation of . Thus, the and subunits in EF-1 were differentially phosphorylated by CKII, in that phosphorylation of was altered by association with other subunits, while the site on was always available for phosphorylation by CKII. From the availability of the subunits for phosphorylation by CKII and the composition of the reconstituted partial and complete complexes, a model for the subunit structure of EF-1 consisting of (22)2 is proposed and discussed.  相似文献   

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The carbohydrate-binding specificity ofPseudomonas aeruginosa lectin I (PA-I) in iodinated or biotinylated form was studied. A large number of glycosphingolipids, as well as some glycoproteins and neoglycoproteins were used as ligands. Also, inhibition by free saccharides of PA-I binding to glycosphingolipids was tested. It was found that the lectin binds most strongly to terminal and nonsubstituted Gal3Gal- or Gal4Gal-structures.Abbreviations PA-I Pseudomonas aeruginosa lectin I - Cer ceramide - lactosylceramide Gal4GlcCer - iso globotriaosylcerami Gal3Gal4GlcCer - globotriaosylceramide Gal4Gal4GlcCer - globoside or globotetraosylceramide GalNAc3Gal4Gal4GlcCer - Forssman glycolipid GalNAc3GalNAc3Gal4Gal4GlcCer - P1 glycolipid Gal4Gal4GlcNAc3Gal4GlcCer - lactoneotetraosylceramide Gal4GlcNAc3Gal4GlcCer - B5 glycolipid Gal3Gal4GlcNAc3Gal4GlcCer - gangliotetraosylceramide Gal3GalNAc4Gal4GlcCer - GM1 Gal3GalNAc4(NeuAc3)Gal4GlcCer - RBC red blood cells - BSA bovine serum albumin - PBS phosphate-buffered saline - SDS sodium dodecyl sulfate - TLC thin-layer chromatography - HPLC high pressure liquid chromatography - MS mass spectrometry - FAB fast-atom bombardment - EI electron impact  相似文献   

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Summary Effect of temperature-sensitive, assembly-defective mutations in Escherichia coli RNA polymerase (rpoB) or subunit gene (rpoC) was investigated on the expression of wild-type rpoB +C+operon, which was introduced by infection of a lambda transducing phage drif + (rpoB +)-6 after UV-irradiation of the mutant cells. In rpoB2·rpoB7 strain which accumulates assembly-intermediates, free , 2 complex and premature core, the expression of rpoB +C+operon measured by the rate of subunit synthesis was considerably inhibited whereas that of EF(translation elongation factor)-Tu, ribosomal proteins L1 and L7/L12, and some -coded proteins remained unaffected. On the other hand, the expression was enhanced specifically for only rpoB +C+operon in either rpoC4 or rpoC1 mutants, which are defective in the association of 2 complex and subunit or the activation of premature core enzyme, respectively. Upon preincubation of the mutant cells at 42° C prior to phage infection, during which assembly intermediates degraded rapidly, the rate of subunit synthesis relative to other phage-corded proteins increased remarkably in rpoB2·rpoB7 mutant as well as in rpoC4 and rpoC1 mutants. These observations strongly suggested the autogenous regulation for at least (rpoB +C+) operon by some trans-active diffusible protein complexes built of RNA polymerase subunits. Nature of the regulatory molecules is discussed.Paper VI in this series is Saitoh and Ishihama (1977)  相似文献   

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In this investigation, an overlapping set of synthetic peptides spanning the entire primary structures of the -subunit of bovine and human thyrotropin, bTSH and hTSH respectively, have been prepared to aid the delineation of the amino acid sequence regions involved in two spatially related epitopes of bTSH. These peptides were then evaluated for their ability to inhibit the binding of two anti-hTSH monoclonal antibodies, designated mAb279 and mAb299, to radiolabeled I125-bTSH using competitive radioimmunoassay procedures. Synthetic peptides related to the sequence region b/hTSH[56–68] were found to specifically inhibit the binding of I125-bTSH to mAb299, whilst having no effect on the binding of mAb279. In previous studies we have shown that mAb279 and mAb299 recognise epitopic sites located within the receptor-binding site of the TSH -subunit. This investigation has therefore permitted identification of a contribution to the receptor binding site from the TSH[56–68] sequence, which forms part of the L3 loop region of the TSH -subunit that is held in close proximity to the L1 loop region and the C-terminus of the TSH - subunit by the disulphide bonds TSH[Cys16- Cys67] and TSH[Cys19-Cys105]. This finding is in agreement with previous investigations which have shown that TSH[Tyr59] and TSH[Tyr74] are also associated with the mAb299 epitope site, as well as contributing to the receptor binding region of the TSH -subunit.  相似文献   

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1,4 galactosyltransferase 1 ( 1,4GT1) synthesizes Gal 14GlcNAc groups in N-linked sugar chains of animal glycoproteins, which have been demonstrated to play an important role in many biological events, including sperm-egg interaction, cell migration and mammalian embryonic development. In this study, the mRNA level of 1,4GT1 was found to increase greatly during the 7721 hepatocarcinoma cells apoptosis induced by cycloheximide. Ricinus Communis Agglutinin-I staining indicated generous increase of Gal 14GlcNAc groups during apoptosis. Further study showed that the 7721 hepatocarcinoma cells transiently transfected with 1,4GT1 were more susceptible to the apoptosis induced by cycloheximide. The increased susceptibility was in accordance to the transfection concentration of 1,4GT1, which also led to the increased Gal 14GlcNAc groups on the transfected cell surface. All the observations suggested that 1,4GT1 and Gal 14GlcNAc groups might be associated with the apoptosis of human hepatocarcinoma cells.  相似文献   

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A partially purified preparation of 1,3-fucosyltransferase(s) from human milk was used to [14C]fucosylate oligosac-charides containing Gal1-4GlcNAc units. Substitution ofN-acetyllactosamine at position 3 with a -linkedN-acetylglucosamine enhanced the reactivity of the acceptor, whereas similar substitution at position 6 was inhibitory. Thus, the trisaccharide GlcNAcl-6Gal1-4GlcNAc (5), the branched tetrasaccharide GlcNAc1-3(GlcNAc1-6)Gal1-4GlcNAc (11) and the triply branched decasaccharide GlcNAc1-3(GlcNAc1-6)Gall-4GlcNAc1-3[GlcNAc1-3(GlcNAc1-6)Gal1-4GlcNAc1-6]Gal1-4GlcNAc (26) gave remarkably poor yields of 1,3-fucosylated products in comparison to GlcNAc1-3Gal1-4GlcNAc (3). 1,4-Galactosyl derivatives of5 and11, however, gave good yields of 1,3-fucosylated products, but the fucosylation was restricted to the distalN-acetyllactosamine units of Gal1-4GlcNAc1-6Gal1-4GlcNAc (16), Gal1-4GlcNAc1-3(Gal1-4GlcNAc1-6)Gal1-4GlcNAc (18) and also in Gal1-3Gal1-4GlcNAc1-3(Gal1-3Gal1-4GlcNAc1-6)Gal1-4GlcNAc (22). Immobilized wheat germ agglutinin (WGA), possessing high affinity for16 [1], revealed no affinity for the fucosylated derivative Gal1-4(Fuc1-3)GlcNAc1-6Gal1-4GlcNAc (17). The isomeric heptasaccharides Gal1-4(Fuc1-3)GlcNAc1-3(Gal1-4GlcNAc1-6)Gal1-4GlcNAc (19) and Gal1-4GlcNAc1-3[Gal1-4(Fuc1-3)GlcNAc1-6]Gal1-4GlcNAc (20) were readily separated from each other on WGA-agarose, and so were the isomeric nonasaccharides Gal1-3Gal1-4(Fuc1-3)GlcNAc1-3(Gal1-3Gal1-4GlcNAc1-6)Gal1-4GlcNAc (23) and Gal1-3Gal1-4GlcNAc1-3[Gal1-3Gal1-4(Fuc1-3)GlcNAc1-6]Gal1-4GlcNAc (24).  相似文献   

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Incubation of synthetic Man\1-4GlcNAc-OMe, GalNAc1-4GlcNAc-OMe, Glc1-4GlcNAc-OMe, and GlcNAc1-4GlcNac-OMe with CMP-Neu5Ac and rat liver Gal1-4GlcNAc (2-6)-sialyltransferase resulted in the formation of Neu5Ac2-6Man1-4GlcNAc-OMe, Neu5Ac2-6GalNAc1-4GlcNAc-OMe, Neu5Ac2-6Glc1-4GlcNAc-OMe and Neu5Ac2-6GlcNAc1-4GlcNAc-OMe, respectively. Under conditions which led to quantitative conversion of Gal1-4GlcNAc-OEt into Neu5Ac2-6Gal1-4GlcNAc-OEt, the aforementioned products were obtained in yields of 4%, 48%, 16% and 8%, respectively. HPLC on Partisil 10 SAX was used to isolate the various sialyltrisaccharides, and identification was carried out using 1- and 2-dimensional 500-MHz1H-NMR spectroscopy.Abbreviations 2D 2-dimensional - CMP cytidine 5-monophosphate - CMP-Neu5Ac cytidine 5-monophospho--N-acetylneuraminic acid - COSY correlation spectroscopy - DQF double quantum filtered - HOHAHA homonuclear Hartmann-Hahn - MLEV composite pulse devised by M. Levitt - Neu5Ac N-acetylneuraminic acid - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid  相似文献   

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We have developed a new method for the large scale preparation of pyridylaminated (PA-) oligosaccharides from glycoproteins. Phenol/chloroform extration was adapted for the removal of protein and excess 2-aminopyridine, improving the efficiency of preparation. From a 2.5 g sample of human apo-transferrin, 25–30 mol of agalacto biantennary PA-oligosaccharide could be obtained. By increasing the concentration of PA-oligosaccharide substrate, we were able to detect a very low level ofN-acetylglucosaminlytransferase IV activity in CHO cell extracts.Abbreviations PA 2-aminopyridine - SDS sodium dodecyl sulfate - GlcNAc N-acetylglucosamine - GnT N-acetylglucosaminyltransferase - Gn,Gn-bi-PA GlcNAc1-2Man1-3(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc-2-aminopyridine - Gn,Gn,Gn-tri-PA GlcNAc1-2(GlcNAc1-4)Man1-3(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc-2-aminopyridine - Gn,Gn,Gn-trí-PA GlcNAc1-2Man1-3({GlcNAc1-2(GlcNAc1-6)Man1-6})Man1-4GlcNac1-4GlcNAc-2-aminopyridine - Gn,(Gn),Gn-bi-PA GlcNAc1-2Man1-3(GlcNAc1-4)(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc-2-aminopyridine  相似文献   

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K99 lectin fromEscherichia coli was purified and biotinylated via its carboxyl groups using biocytin hydrazide and a water soluble carbodiimide. Biotinylation of two out of the nine carboxyl groups was sufficient to permit detection of the lectin by avidin and did not cause any loss of the haemagglutinating activity. It was demonstrated that the biotinylated K99 lectin retained other important properties of native K99 and that it will probably become a very sensitive detecting reagent. Indeed, it was able to bind to HeLa cells, as do intact bacteria carrying K99 fimbriae, and also to recognizeN-glycolyl-neuraminyl-lactosyl-ceramide in an overlay binding assay. Abbreviations: NeuAc,N-acetylneuraminic acid; NeuGc,N-glycolylneuraminic acid; PBS, phosphate buffered saline (0.9% NaCl containing 150mm sodium phosphate, pH 7.2); LPS, lipopolysaccharide; BCHZ, biocytin hydrazide; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; BSA, bovine serum albumin; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle medium. For the gangliosides, trivial names and structures are given according to the recommendations in [43]. NeuAc2-3Gal1-4Glc1-1Cer (NeuAc-GM3); NeuGc2-3Gal1-4Glc1-1Cer (NeuGc-GM3); GalNAc1-4(NeuAc2-3)Gal1-4Glc1-1Cer (GM2); NeuAc2-8NeuAc2-3Gal1-4Glc1-1Cer (GD3); Gal1-3GalNAc1-4(NeuAc2-3)Gal1-4Glc1-1Cer (GM1); NeuAc2-3Gal1-3GalNAc1-4(NeuAc2-3)Gal1-4Glc1-1Cer (GD1a); Gal1-3GalNAc1-4(NeuAc2-8NeuAc2-3)Gal1-4Gle1-1Cer (GD1b); NeuAc2-3Gal1-3GalNAc1-4(NeuAc2-8NeuAc2-3) Gal1.-4Glc1-1Cer (GT1b). NeuGc2-3Gal1-4GleNAc1-4Gal1-4Glc1-1Cer (NeuGc-SPG).  相似文献   

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Using degenerate primers based on published 2-microglobulin sequences we were able to obtain an expected 111 base pairs (bp) polymerase chain reaction (PCR) fragment from tilapia genomic DNA. The sequence of this fragment showed a high degree of similarity to mouse 2-microglobulin at the protein level. We used these primers in an anchored PCR to obtain a 213 bp PCR fragment from a carp cDNA library. This was then used to clone a full-length 2-microglobulin cDNA from carp. The carp sequence showed the highest similarity to rabbit 2-microglobulin. Both sequences showed strong similarities to all previously published vertebrate 2-microglobulin sequences. The predicted protein secondary structure of both the carp and tilapia clones was almost identical to the corresponding regions of previously known vertebrate 2-microglobulin protein sequences. When either the carp or tilapia probes were used against corresponding northern blots, they hybridized to a message of approximately 800–1000 bases long, which corresponds to the previously published lengths of 2-microglobulin mRNAs. Southern blotting indicated that 2-microglobulin was encoded by a single copy gene in both cases. Phylogenetic analysis indicated that the sequences were related to the 2-microglobulins of higher vertebrates but grouped together in an ancestral position.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers L05536 (carp), L05537 (tilapia).  相似文献   

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The structural requirements for the interaction of the Asn-linked poly-N-acetyllactosamine-type oligosaccharide moieties of glycoproteins with variousN-acetylglucosamine-binding lectins were investigated by means of affinity chromatography on immobilized lectin-Sepharose columns.High molecular weight glycopeptides containing poly-N-acetyllactosamine-type oligosaccharides obtained by Pronase digestion of human erythrocyte ghosts were treated with 0.1 M trifluoroacetic acid at 100°C for 40 min and then several oligosaccharide fragments were purified with an amino-bonded silica column. Among these oligosaccharide fragments, trisaccharide Gal1-4GlcNAc1-6Galol bound to the wheat germ agglutinin (WGA)- and pokeweed mitogen (PWM)-Sepharose columns, and also showed affinity to theDatura stramonium agglutinin (DSA)-,Lycopersicon esculentum (tomato) agglutinin-andSolanum tuberosum (potato) agglutinin-Sepharose columns. Pentasaccharide Gal1-4GlcNAc1-3(Gal1-4GlcNAc1-6)Galol showed weaker affinity to the WGA- and PWM-Sepharose columns, compared to the trisaccharide. Trisaccharide GlcNAc1-3(GlcNAc1-6)Galol showed weak affinity to the WGA-Sepharose column and did not show any affinity to the other lectin-Sepharose columns. Hexasaccharide Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAcol bound only to the DSA-Sepharose column, indicating that only DSA does not require a GlcNAc(1-6)-linkage for interaction.Abbreviations HPLC high performance liquid chromatography - WGA wheat germ agglutinin - PWM pokeweed mitogen - DSA Datura stramonium agglutinin - LEA Lycopersicon esculentum (tomato) agglutinin - STA Solanum tuberosum (potato) agglutinin - EVA Erythrina variegata agglutinin - PBS 10 mM sodium phosphate buffer, pH 7.2, containing 0.15 M NaCl - Galol galactitol - GlcNAcol N-acetylglucosaminitol  相似文献   

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Summary In spite of the generally well-coordinated synthesis of RNA polymerase core enzyme subunits (, and ) in Escherichia coli, a situation was found during the growth transition from exponential to stationary phase in which this coordination was broken (the order of differential repression being ; Kawakami et al. (1979)). The present study indicates that, during a certain period of the growth transition, twice as much subunit is synthesized as subunit and the overproduced subunit accumulates as the assembly intermediate 2 complex, which is rapidly and preferentially degraded.Two independent factors, i.e., carbon source down-shift and oxygen depletion, were examined separately for their influence on the coordinated regulation of the synthesis of RNA polymerase subunits. The depletion of glucose added as a sole carbon source was accompanied by repression of the synthesis of all core enzyme subunits, while under the same conditions the differential rate of subunit synthesis increased. In contrast, the sudden ending of the oxygen supply resulted in specific repression of the synthesis of only and subunits but not of and subunits. The latter result may be explained by the autogenous repression of the rpoBC genes by a temporal increase in the amount of unused cytoplasmic RNA polymerase.Paper XI in this series is Kawakami and Ishihama (1980)  相似文献   

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