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1.
The interaction between chick embryo fibroblasts and A1-specific blood group Dolichos biflorus lectin has been studied at various stages of embryo development. The site number ((0.26 plus or minus 0.03)-10-6 sites/cell) remains the same during development whereas the affinity constant apparently decreases from 8-day cells onwards. The effects of cell number, temperature and time course on the Dolichos binding to fibroblasts were not age dependent. Competitive binding experiments revealed that Dolichos receptor sites were distinct from binding sites fo Robina pseudoacacia lectin and concanavalin A, but partially related to binding sites of Ricinus lectin. Thymidine incorporation by fibroblasts in the presence of Dolichos lectin was age dependent. It was inhibited in 6-day cells and weakly stimulated in 16-day cells, but not modified in 12-day cells. Dolichos lectin effects on embryo fibroblasts were very specific because both binding to cells and effect on thymidine incorporation were blocked by N-acetylgalactosamine, the determinant of Dolichos lectin, as well as by Dolichos antiserum.  相似文献   

2.
(1) A quantitative study has been made of the binding of ouabain to the (Na+ + K+)-ATPase in homogenates prepared from brain tissue of the hawk moth, Manduca sexta. The results have been compared to those obtained in bovine brain microsomes. (2) The insect brain (Na+ + K+)-ATPase will bind ouabain either in the presence of Mg2+ and Pi, (‘Mg2+, Pi’ conditions) or in the presence of Na+, Mg2+, and an adenine nucleotide (‘nucleotide’ conditions) as is the case for the bovine brain (Na+ + K+)-ATPase. The binding conditions did not alter the total number of receptor sites measured at high ouabain concentrations in either tissue. (3) Potassium ion decreases the affinity (increases the KD) of ouabain to the M. sexta brain (Na+ + K+)-ATPase under both binding conditions. However, ouabain binding is more sensitive to K+ inhibition under the nucleotide conditions. In bovine brain ouabain binding is equally sensitive to K+ inhibition under the both conditions. (4) The enzyme-ouabain complex has a rate of dissociation that is 10-fold faster in the M. sexta preparation than in the bovine brain preparation. Because of this, the M. sexta (Na+ + K+)-ATPase has a higher KD for ouabain binding and is less sensitive to inhibition by ouabain than the bovine brain enzyme. (5) This data supports the hypothesis that two different conformational states of the M. sexta (Na+ + K+)-ATPase can bind ouabain.  相似文献   

3.
Evidence is presented for the presence of a lectin on Streptococcussanguis with specificity towards the major acidic oligosaccharide of human salivary mucin. Based upon hemagglutination inhibition studies, the strongest inhibitor was NeuAcα2,3Galβ1,3GalNAcol ? NeuAcα2,3Galβ1,4Glc ? NeuAc > Gal. Interactions were not heat sensitive or charge dependent, and were not affected by the presence of bacterial cell associated neuraminidase. The lectin could be extracted from Streptococcussanguis with lithium 3,5-diiodosalicylate (LIS). Incubation of LIS extracts with carbohydrate ligands demonstrated that the specificity of binding was NeuAcα2,3Galβ1,3[3H-]GalNAcol ? Galβ1,3[3H-]GalNAcol.  相似文献   

4.
Seedlings carrying mutations in regulatory genes for protochlorophyll(ide) synthesis accumulate protochlorophyll(ide) in darkness in amounts exceeding the wildtype level. Thus, +/tig-d12 and tig-b24tig-b24accumulate 2-fold, tig-o34tig-o34 5- to 6-fold, and tig-d12tig-d12 15-fold more protochlorophyll(ide) than the wild type.The amount of photoconvertible protochlorophyll(ide) accumulated in darkness is the same in all genotypes, despite the large differences in total protochlorophyll(ide) content, indicating a constant number of photoconversion sites.When briefly illuminated leaves are returned to darkness, regeneration of active protochlorophyll(ide) from the pool of inactive protochlorophyll(ide) takes place in wild-type and mutant leaves. Compared to the wild type, the rate of protochlorophyll(ide) activation during 4- and 10-min dark periods is higher in +/tig-d12, tig-b24tig-b24, and tig-o34tig-o34, but lower in tig-d12tig-d12.There was no indication that the accumulation of protochlorophyll(ide) influences the conversion sites of the protochlorophyll(ide) holochrome, as the kinetics of photoconversion of initially active protochlorophyll(ide) in leaves with the genotypes +/+, +/tig-o34, and tig-o34tig-o34 are similar or identical.  相似文献   

5.
An analysis of the repeat structure of the highly repetitive sequence, component α DNA of the African green monkey, shows that the DNA contains restriction sites for EcoRI, EcoRI1, HindIII and HaeIII. All four restriction enzyme activities indicate a basic repeat length of 176 ± 4 base-pairs. In addition to primary EcoRI1 and HindIII sites, about 59% of the repeat sequences contain secondary EcoRI1 sites and about 36% of the repeat sequences contain secondary HindIII sites. The secondary sites are located less than 176 base-pairs from the primary sites and their cleavage yields several complex series of minor, intermediate segments in gels of the partial EcoRI1 or HindIII digests. Cleavage at the secondary sites yields segments shorter than the unit monomer in the limit digests. The sites for EcoRI, EcoRI1, HindIII and HaeIII have been mapped within the repeat unit.Treatment of the monkey nuclei with micrococcal nuclease at 2 °C and in the presence of 80 mm-NaCl reveals two distinct populations of nucleosomes. One population contains bulk DNA sequences, and after cleavage with micrococcal nuclease this population yields heterogeneous segments of DNA spanning 180 to 200 base-pairs in length. The other population contains component α sequences and after cleavage with micrococcal nuclease yields homogeneous segments of component α DNA that are exact multiples of the basic sequence repeat unit of 176 base-pairs. Thus, the cleavage by micrococcal nuclease of nucleosomal arrays containing component α sequences is as regular and precise as the cleavage of the purified DNA by the restriction enzymes. The resolution of the two distinct subsets of nucleosomes in the monkey nuclei is dependent upon the conditions of ionic strength and temperature employed during the nuclear isolation and the micrococcal nuclease digestion.These observations are consistent with a phase relation between the component α repeat sequences and the associated nucleosomal proteins (Musich et al., 1977b). They are also in accord with the hypothesis that the subunit structure of constitutive heterochromatin modulates or determines the repeat sequence structure and hence, the evolution of many highly repetitive mammalian DNAs (Maio et al., 1977).  相似文献   

6.
Mutations at the bithoraxoid (bxd) and postbithorax (pbx) loci cause a transformation of posterior haltere to posterior wing. It has previously been shown that pbx and pbxUbx101 cause this transformation by affecting the maintenance (or cell heredity function) of determination so that the transformed cells are indistinguishable from normal wing cells, and have no “memory” of having been part of a haltere disk (Adler, 1978a). I report here that Tp(3) bxd100Ubx101 and bxd1, pbx, ew both cause the transformation of posterior haltere to posterior wing in the same way as pbx. On the other hand, bxd1, bxd1Ubx101, bxd51j, bxd51jUbx101, and bxd51jred pbx cause this same transformation of posterior haltere to posterior wing by interfering with the expression of the determined state so that the developmental information of posterior haltere is “misread” as posterior wing. The transformed cells in these disks retain the memory of having been part of a haltere disk; that is, these posterior cells that would secrete wing cuticle during metamorphosis regenerate anterior haltere structures. Thus it appears clear that it is possible to uncouple the expression and cell heredity functions of determination in the haltere disk of Drosophila.  相似文献   

7.
(1) H+/electron acceptor ratios have been determined with the oxidant pulse method for cells of denitrifying Paracoccus denitrificans oxidizing endogenous substrates during reduction of O2, NO?2 or N2O. Under optimal H+-translocation conditions, the ratios H+O, H+N2O, H+NO?2 for reduction to N2 and H+NO?2 for reduction to N2O were 6.0–6.3, 4.02, 5.79 and 3.37, respectively. (2) With ascorbate/N,N,N′,N′-tetramethyl-p-phenylenediamine as exogenous substrate, addition of NO?2 or N2O to an anaerobic cell suspension resulted in rapid alkalinization of the outer bulk medium. H+N2O, H+NO?2 for reduction to N2 and H+NO?2 for reduction to N2O were ?0.84, ?2.33 and ?1.90, respectively. (3) The H+oxidant ratios, mentioned in item 2, were not altered in the presence of valinomycinK+ and the triphenylmethylphosphonium cation. (4) A simplified scheme of electron transport to O2, NO?2 and N2O is presented which shows a periplasmic orientation of the nitrite reductase as well as the nitrous oxide reductase. Electrons destined for NO?2, N2O or O2 pass two H+-translocating sites. The H+electron acceptor ratios predicted by this scheme are in good agreement with the experimental values.  相似文献   

8.
The binding of the crustacean selective protein neurotoxin, toxin B-IV, from the nemertine Cerebratulus lacteus to lobster axonal vesicles has been studied. A highly radioactive, pharmacologically active derivative of toxin B-IV has been prepared by reaction with Bolton-Hunter reagent. Saturation binding and competition of 125I-labeled toxin B-IV by native toxin B-IV have shown specific binding of 125I-labeled toxin B-IV to a single class of binding sites with a dissociation constant of 5–20 nM and a binding site capacity, corrected for vesicle sidedness, of 6–9 pmol per mg membrane protein. This compares to a value of 3.8 pmol [3H]saxitoxin bound per mg in the same tissue. Analysis of the kinetics of toxin B-IV association (k+1=7.3·105M?1·s?1) and dissociation (k? 1=2·10?3s?1) shows a nearly identical Kd of about 3 nM. There is no competition of toxin B-IV binding by purified toxin from Leiurus quinquestriatus venom while Centruroides sculpturatus Ewing toxin I appears to cause a small enhancement of toxin B-IV binding.  相似文献   

9.
Dispersed acini from dog pancreas were used to examine the ability of dopamine to increase cyclic AMP cellular content and the binding of [3H]dopamine. Cyclic AMP accumulation caused by dopamine was detected at 1·10?8 M and was half-maximal at 7.9±3.4·10?7M. The increase at 1·10?5 M, (7.5-fold) was equal to the half-maximal increase caused by secretin at 1·10?9 M. Haloperidol, a dopaminergic receptor antagonist inhibited cyclic AMP accumulation caused by dopamine. The IC50 value for haloperidol, calculated from the inhibition of cyclic AMP increase caused by 1·10?5 M dopamine was 2.3±0.9·10?6M. Haloperidol did not alter basal or secretin-stimulated cyclic AMP content. [3H]Dopamine binding was studied on the same batch of cells as cyclic AMP accumulation. At 37°C, it was rapid, reversible, saturable and stereospecific. The Kd value for high affinity binding sites was 0.43±0.1·10?7M and 4.7±1.6·10?7M for low affinity binding sites. The concentration of drugs necessary to inhibit specific binding of dopamine by 50% was 1.2±0.4·10/t-7M noradrenaline, 2·10/t-7 M epinine, 4.1±1.8·10/t-6M fluphenazine, 8.0±1.6·10/t-6M haloperidol, 4.2±1.2·10?6Mcis-flupenthixol, 2.7±0.4·10?5Mtrans-flupenthixol, >1·10?5M apomorphine, sulpiride, naloxone and isoproterenol.  相似文献   

10.
11.
Purified chloroplasts from leaves of Spinacia oleracea L. (spinach) incorporated glycerol 3-phosphate into diacylglycerol, monoacylglycerol, phosphatidylglycerol, phosphatidic acid, and lysophosphatidic acid. The omission of ATP or CTP, CoA or illumination decreased the incorporation markedly. The fraction of incorporated glycerol 3-phosphate found in phosphatidylglycerol was greatly reduced by the omission of bicarbonate, acetate, and ATP, or in darkness, low-osmolarity medium, or high magnesium ion concentration (10 mM). Incorporation of glycerol 3-phosphate into lipid and specifically into phosphatidylglycerol was optimal at a Mg2+CTP ratio of 1, whereas the optimal ratio for Mg2+ATP was closer to 2. The Mg2+CTP gave lower total incorporation but a higher fraction of incorporation in phosphatidylglycerol. Triton X-100 inhibited incorporation of glycerol 3-phosphate into lipid, especially into phosphatidylglycerol.  相似文献   

12.
M1 cells, which are cell line cells established from myeloid leukemia cells of the SL strain mouse, can differentiate from blast cells (M1?) to mature macrophages (M1+) within 48 hr, when they are cultured with conditioned medium (CM) obtained from murine embryonic fibroblasts. While M1? cells have no phagocytic activity nor Fc receptor (FcR), M1+ cells possess both characteristics. The appearance of FcR is temperature-dependent and inhibited by a metabolic inhibitor, cycloheximide. FcR on M1+ cells is resistant to trypsin and pronase. M1+ cells improve the viability of macrophage-depleted SL splenic lymphocytes and restore the in vitro secondary plaque forming cell response of macrophage-depleted spleen cells to particulate and soluble antigens. M1? cells lack this macrophage-substituting capacity. Mm1 cells, mutant cells from M1 cells, having FcR and higher phagocytic activity than M1+ cells, are also devoid of this capacity.  相似文献   

13.
The association constant, KA, for myosin subfragment-1 binding to actin was measured as a function of ionic strength [KCl, LiCl, and tetramethylammonium chloride (TMAC)]and temperature by the method of time-resolved fluorescence depolarization. The following thermodynamic values were obtained from solutions of 0.20 × 10?6m S-1, 1.00 × 10?6m actin in 0.15 m KCl, pH 7.0, at 25 °C: ΔG ° = ?39 ± 1 kJ M?1, ΔH0 = 44 ± 2 kJ M?1 and ΔS0 = 0.28 ± 0.01 kJ M?10K?1. For measurements in KCl (0.05 to 0.60 m), In Ka = ?8.36 (KCl)12. Thus, the binding is endothermic and strongly inhibited by high ionic strength. When KCl was replaced by LiCl or TMAC the ionic effects on the binding were cation specific. The nature of actin-(S-1) binding in the rigor state is discussed in terms of these results.  相似文献   

14.
Diploid embryos which are homozygous for the t12 mutation die at the morula stage. In the current studies, ova from heterozygous (+t12) females were fertilized in vitro with spermatozoa from +t12 males. The fertilized ova were immediately placed into media containing cytochalasin B to prevent second polar body formation, producing +/+/+, +/+/t12, +/t12/t12, and t12/t12/t12 embryos. The subsequent development of these triploid embryos was compared with that of diploid +/+, +t12, and t12t12 embryos developing from ova which were also fertilized in vitro with spermatozoa from +t12 males but which were not treated with cytochalasin B immediately following gamete coincubation. The data show that those triploid embryos which possess a wild-type allele and two mutant alleles are phenotypically wild type while those possessing three mutant alleles are not phenotypically distinguishable from their diploid (t12t12) counterparts. Like t12t12 embryos, t12/t12/t12 embryos die at the morula stage, prior to blastocoelic cavity formation.  相似文献   

15.
Two analogs of N-acetylmannosamine, 2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-d-mannopyranose (Ac4-NAcMan) and the 2-trifluoroacetamido derivative (Ac4F3-NAcMan), were synthesized as potential inhibitors of the formation of sialic acid-containing glycoconjugates and were examined for their ability to modify the incorporation of N-[3H]acetylmannosamine into cellular glycoconjugates of Friend murine erythroleukemia cells. Ac4F3-NAcMan and Ac4-NAcMan inhibited cellular replication in suspension culture at concentrations of 0.02 and 0.08 mM, respectively. The cytotoxicity of Ac4-NAcMan was relatively reversible, whereas that produced by Ac4F3-NAcMan was not, as judged by measurement of the cloning efficiencies of cells exposed to these agents. The analogs inhibited incorporation of N-[3H]acetylmannosamine into ethanol-soluble and -insoluble materials. Separation of ethanol-soluble metabolites by HPLC demonstrated that Ac4F3-NAcMan caused accumulation of radioactivity from N-[3H]acetylmannosamine in CMP-N-acetylneuraminic acid (CMP-NeuNAc) equal to the decrease in macromolecular-bound 3H caused by this agent. In contrast, similar exposure to Ac4-NAcMan produced a large increase in the amount of radioactivity in ethanol-soluble N-acetylneuraminic acid while decreasing the amount of label from N-[3H]acetylmannosamine in cellular CMP-NeuNAc, suggesting that the analogs differ in their biochemical sites of action. Treatment of cells with either analog increased the amount of neuraminidase-hydrolyzable sialic acid-like material on the cell surface; this appeared to be due to the incorporation of the analogs into cellular glycoconjugates, since incubation of cells with 3H-labeled analogs resulted in the appearance of radioactivity in cellular ethanol-insoluble and neuraminidase-hydrolyzable material. Incubation of cells with Ac4-NAcMan labeled with 14C in the 4-O-acetyl group further demonstrated that incorporation occurred with approx. 50% retention of this substituent. Thus, both the amount and the nature of the surface sialic acid constituents of treated cells were altered, suggesting that these or similar analogs could potentially be used to modify cellular membrane function.  相似文献   

16.
The interaction between chick embryo fibroblasts and various lectins has been studied at different stages of embryo development. There is evidence that Robinia lectin, Dolichos lectin, and Conca navalin A decrease cell number and proportion of cells incorporating [3H] thymidine in case of 8and 10day-old chick embryo fibroblasts, whereas they stimulated the proliferation of 16-dayold embryo cells. No effect was noticed in 12-day cells.
These results suggest that some cell surface changes occur during embryo development. The site number of Dolichos lectin remains the same during embryo development, and the affinity constant decreases. The site number of Robinia lectin and Concanavalin A decreases from the 8th to the 12th day of development, and slowly increases on the 16–day cells, the affinity constant remaining rather constant.
The results indicate that the age–dependent effect of lectin on embryo cells could not be directly related to the number of lectin–binding sites. Competitive binding experiments revealed that Dolichos receptor sites were distincts from binding sites of Robinia lectin and Concanavalin A, and Robina receptor sites distinct from those of Concanavalin A.
Lectin effects on embryo fibroblasts were very specific as determined by inhibitory assays.  相似文献   

17.
The binding sites for the lectins wheat germ agglutinin, Ricinus communis agglutinin and concanavalin A on mouse neuroblastoma cell membranes were identified using SDS-gel electrophoresis in combination with fluorescent lectins. Ricinus communis agglutinin and wheat germ agglutinin were found to bind almost exclusively to a single polypeptide with an apparent molecular weight of 30 000. Concanavalin A labeled over 20 different polypeptides, most with molecular weights greater than 50 000. However, when the neuroblastoma cells were treated with concanavalin A so as to internalize all the concanavalin A binding sites visible at the level of the fluorescent microscope and the purified plasma membranes analyzed for their concanavalin A binding polypeptides, only four of the 20 glycopolypeptides were missing or significantly reduced in amount. Thus, these four high molecular weight concanavalin A-binding polypeptides appear to be the major cell surface receptors for concanavalin A. Binding studies with iodinated concanavalin A indicated that these polypeptides represented the high affinity concanavalin A binding sites Kd = 2 · 10?7M). Low affinity concanavalin A binding sites were present on the cell surface after internalization of high affinity concanavalin A binding sites.  相似文献   

18.
The in vitro reaction of bacteriophage T7-DNA with the radioactive diastereomeric benzo(a)pyrene-diol-epoxides, (±) [3H9, 3H10]-7β,8α-dihydroxy-9α,10β-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, and (±) [3H9, 3H10]-7β,8α-dihydroxy-9β,19β-epoxy-7,8,9,10-tetrahydrobenzo(1)pyrene, was investigated. Chromatographic analysis of digests of the DNA allowed the distinction of characteristic deoxynucleoside adduct peaks for the two benzo(a)pyrene-diol-epoxides. Our results, together with data from the literature, allow the identification of these adducts as mostly N2-(10-7β,8α,9α-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyreney1)deoxyguanosine and N2-(10-7β,8α,9β-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyreney1)deoxyguanosine, respectively. DNA-benzo(a)pyrene adducts with the same chromatographic properties were formed in mouse embryo fibroblasts upon treatment with benzo(a)pyrene.  相似文献   

19.
beta-Endorphin: characteristics of binding sites in the rat brain.   总被引:3,自引:0,他引:3  
Stereospecific binding of human β-endorphin to rat membrane preparations is described for the first time using [3H-Tyr27]-βh-endorphin as the ligand. The binding is time dependent and saturable with respect to βh-endorphin with an apparent dissociation constant of 0.3 nM. Sodium ion (100 mM) elevates this value to 2.5 nM but has no effect on the total number of binding sites present in the membrane preparation. The ability of certain β-endorphin analogs, opiate agonists as well as antagonists to inhibit the binding of βh-endorphin, is presented.  相似文献   

20.
The migration of intestinal epithelial cells from the crypts to the tips of villi is associated with progressive cell differentiation. The changes in Na+-pump levels during migration have been measured in epithelial cells isolated from rabbit small intestine. A significant proportion of ouabain-sensitive (Na++K+)-ATPase in the cell homogenates was latent but could be unmasked by detergent treatment. Highest detergent activation was observed in villus cells. The distribution of pumping sites was also assessed by measuring ouabain binding to intact cells. The kinetics of specific binding was consistent with the interaction of the cardiac glycoside with a single population of binding sites with an apparent Kd of around 10?7 M. Both enzyme assay and ouabain-binding measurements suggest that a 2–3-fold increase in the number of Na+-pumping sites accompanies cell differentiation in rabbit jejunal epithelium. This increase in pumping capacity might be an adaptation of the cells to their absorptive function.  相似文献   

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