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61.
F M Finn  C J Stehle  K Hofmann 《Biochemistry》1985,24(8):1960-1965
Biotinylated photoaffinity derivatives of adrenocorticotropin (ACTH) are potentially useful tools for the identification of ACTH receptors. The hormone can be attached covalently to its receptor by photoactivation, and the presence of biotin in the molecule facilitates isolation of the solubilized hormone-receptor complex on columns of immobilized succinoylavidin (Suc-avidin). Six photoprobes of ACTH1-24 have been prepared by reacting ACTH1-24, [25-biocytin]ACTH1-25 amide, and [25-dethiobiocytin]ACTH1-25 amide with either 4- or 5-azido-2-nitrophenylsulfenyl (4-NAPS and 5-NAPS, respectively) chlorides in acetic acid. The homogeneity of the photoprobes was carefully monitored by thin-layer chromatography and amino acid analyses of acid hydrolysates. The presence of underivatized starting material in the photoprobes was critically scrutinized by high-pressure liquid chromatography and was estimated to be less than 0.5%. Both the 4- and 5-NAPS derivatives stimulated maximal steroidogenesis (as compared with ACTH1-24) in calf adrenal cortical cells. However, the potencies of the two isomers differed significantly. The ED50 for steroidogenesis with 5-NAPS-ACTH1-24 was 100-fold greater than the standard (ACTH1-24) while that for 4-NAPS-ACTH1-24 was only approximately 7 times greater. Although 4-NAPS-ACTH1-24 was capable of stimulating maximal adenosine cyclic 3',5'-phosphate (cAMP) production, the 5-NAPS derivative was usually not. The level of stimulation with the 5-NAPS derivative varied considerably from cell preparation to cell preparation. ACTH1-24-induced cAMP production was inhibited by 5-NAPS-ACTH1-24 or 5-NAPS-[25-dethiobiocytin]ACTH1-25 amide.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   
62.
A detailed three-dimensional model of the collagenous part of C1q was derived by model building and computer-aided energy refinement calculations. The proposed structure is based on the collagen-like (-Gly-Xaa-Yaa-) repeating sequence of 78 to 81 residues in the N-terminal regions of the constituent A, B and C chains, on the mode of disulphide linkage between the 18 chains of C1q, and on its electron microscopically derived gross structure. It is demonstrated that the interruptions of the repeating sequence about half-way along the length of the collagenous regions (Gly36-Ile37-Arg38-Thr39 in the A chain and Ala36-Ile37-Hy138 in the C chain) do not lead to a disruption of the triple helical conformation but rather to a bend of about 60 degrees in an otherwise continuous triple helix. These features are consistent with a flexibility comparable with that of regular triple helices and with the observed low proteolytic susceptibility of the kink region. The azimuthal orientation of the kink is defined approximately by ArgA38 being located in the cap of the knee. Because of this extra residue between two glycine residues, a bad contact that would arise between the methyl group of AlaC36 and the peptide carbonyl of IleA37 in a straight triple helix is relaxed. The model features also a cluster of hydrophobic contacts between large hydrophobic side-chains in the interaction edges between the six collagen triple helices aligned with their about 10 nm long N-terminal regions in the fibril-like endpiece of C1q. The azimuthal orientations of the triple helices were derived by energy calculations of side-chain interactions previously applied to fibre-forming collagens. Independently, the same orientations and interaction edges were derived from the azimuthal orientation of the kink and the electron microscopically observed orientations of the triple helical arms that emerge from the endpiece, and which carry the C-terminal globular binding domains. The structural model has a number of implications for the assembly of the first component of complement from C1q and the zymogen complex C1r2C1s2 and possible mechanisms of its activation.  相似文献   
63.
The bovine cardiac sarcolemmal binding sites for the dihydropyridine nimodipine and the phenylalkylamine (-)-desmethoxyverapamil were studied. The density of the nimodipine and (-)-desmethoxyverapamil binding sites increased 8.3-fold and 3.4-fold with the sarcolemma. The binding sites for both compounds were destroyed by trypsin. Nimodipine bound in the presence of 1 mM free calcium to a high-affinity and a low-affinity site with apparent Kd values of 0.35 +/- 0.09 nM (n = 9) and 33 +/- 6.0 nM (n = 9) and with apparent densities of 0.3 +/- 0.05 pmol/mg (n = 9) and 8.2 +/- 1.0 pmol/mg (n = 9). The binding to the high-affinity site was abolished by 1 mM EGTA. The binding sites were specific for dihydropyridines. The (-)-isomers of several phenylalkylamines inhibited nimodipine binding by an apparent allosteric mechanism. (-)-Desmethoxyverapamil bound in the presence of 5 mM EGTA to a high-affinity and a low-affinity site with apparent Kd values of 1.4 +/- 0.3 nM (n = 6) and 171 +/- 26 nM (n = 6) and with apparent densities of 0.16 +/- 0.02 pmol/mg (n = 6) and 13.6 +/- 2.7 pmol/mg (n = 6). The binding to both sites was inhibited by calcium with a half-maximal concentration of 4.3 mM. The binding sites were specific for the other phenylalkylamines and had a higher affinity for the (-)-isomers than for the (+)-isomers. Nimodipine inhibited the binding of (-)-desmethoxyverapamil by an apparent allosteric mechanism. d-cis-Diltiazem inhibited non-competitively the binding of (-)-[3H]desmethoxyverapamil with a Ki of 3.7 microM. Diltiazem up to concentrations of 10 microM did not affect the amount of nimodipine bound at equilibrium at 20 degrees C. However, but in agreement with this result, diltiazem decreased threefold at 20 degrees C the dissociation and association rates for the high-affinity nimodipine receptor. These rates were only marginally affected at 4 degrees C and 37 degrees C. d-cis-Diltiazem reversed in a competitive manner the inhibition of nimodipine binding elicited by the addition of (-)-desmethoxyverapamil with a Ka value of 1.6 microM. The amount of nimodipine bound was inhibited by 50% by the adenosine uptake inhibitors nitrobenzylthioinosine and hexobendine with apparent median inhibitory concentrations of 1 nM and 3 nM, respectively. Nitrobenzylthioinosine completely abolished binding of nimodipine to the low-affinity site, but did not affect binding to the high-affinity site.(ABSTRACT TRUNCATED AT 400 WORDS)  相似文献   
64.
Detection of sugar-binding proteins in membrane-depleted nuclei   总被引:1,自引:0,他引:1  
Nuclear sugar-binding proteins were detected in membrane-depleted nuclei isolated from hamster BHK cells and mouse L 1210 leukemia cells by means of fluorescein-labelled neoglycoproteins. In fluorescence microscopy, the fluorescence was seen throughout the nucleus but was generally brighter over the nucleoli than over the rest of the nucleus. Flow cytofluorometry analysis demonstrated the presence of nuclear sugar-binding proteins for synthetic glycoproteins associated with different sugar residues. Among the nine neoglycoproteins used, four neoglycoproteins (namely alpha-rhamnosylated, alpha-glucosylated, N-acetyl-beta-glucosaminylated and alpha-mannosylated-6P-serum albumin) strongly labelled nuclei. Various controls strongly argue for the specificity of the nuclear labelling. The possibility that some of the sugar-binding proteins might correspond to endogenous nuclear lectins is considered.  相似文献   
65.
The organization of the intranuclear elements observed in histone-depleted (2 M NaCl-extracted) HeLa cell nuclei was investigated by means of electron microscopy and two-dimensional gel electrophoresis. This work was mainly aimed at verifying whether or not an intranuclear skeleton or matrix existed, which could explain the stable attachment of RNA to the residual nuclear structure after high-salt extraction, and its three-dimensional organization. We compared the ultrastructure and the polypeptide composition of RNA-containing and RNA-depleted (RNase-treated) nuclear residues, and we visualized intermediate stages of RNase action on the intranuclear material. We showed that this material was made of two types (fibrillar and granular) of salt-resistant RNP components equally sensitive to RNase when the enzyme was used prior to high-salt extraction. At least in our material and under our experimental conditions, no intranuclear matrix could be distinguished from the residual RNP material. Our results further suggest that formation of such a matrix is a path-dependent phenomenon.  相似文献   
66.
Abstract The chemical structure of the lipid A moiety of the lipopolysaccharide of the type strain of Plesiomonas shigelloides was elucidated. It consists of a β-(1 → 6)-linked glucosamine disaccharide carrying phosphate groups at C-1 of the reducing and at C-4' of the non-reducing glucosamine. It contains a total of 6 residues of fatty acids, 2 amide-linked and 4 ester-linked. The amino groups of the backbone disaccharide are N -acylated by substituted 3-hydroxyacyl residues: at the reducing glucosamine by 3-O-(14:0)14:0; and at the non-reducing glucosamine by 3-O-(12:0)14:0.
Two residues of 3-hydroxytetradecanoic acid are linked to C-3 and C-3' of the glucosamine residues; the hydroxy groups of these ester-linked 3-hydroxytetradecanoic acids are unsubstituted. In free lipid A, the hydroxyl groups at C-4 and C-6' are unsubstituted, indicating that the 2-keto-3-deoxyoctonic acid (KDO) is linked to C-6' of the non-reducing glucosamine, as was shown with enterobacterial lipid A. The taxonomical significance of these structural details is discussed.  相似文献   
67.
68.
Co-isolated residual nuclear shells and residual nucleoli from membrane-depleted rat liver nuclei were spread according to Kleinschmidt's method. Comparison of the spread residual structures isolated from nuclear shells and spread pore complex-lamina isolated from nuclear envelopes showed that these residual structures are morphologically identical. Furthermore, our nuclear shell isolation procedure allowed visualization of DNA strands bound to a granular component of the lamina. The fragmentation of nuclear shells allowed us to obtain well-spread nucleolar remnants, in which we observed DNA strands anchored on a residual nucleolar network attached to the lamina. The different molecular features revealed by the spreading of residual nucleolar structures suggest that both non-transcribing nucleolar DNA and active ribosomal genes are linked to the nucleolar network. Although the exact nature of this network remains to be defined, the results of the present study strongly suggest that the DNA molecules of the chromosomes bearing ribosomal genes have many sites of attachment to a non-chromatin nucleolar network which can be referred to as a nucleolar skeletal complex.  相似文献   
69.
We cloned ras-related sequences from goldfish genomic libraries constructed as recombinants using the lambda phage. Restriction enzyme mapping of the clones obtained revealed three kinds of ras-related sequences among approximately 350,000 genomic clones. One of these clones was partially sequenced. Comparison with the nucleotide sequences of mammalian ras genes showed that the determined sequences covered the predicted amino acid coding regions and parts of the intervening regions. The predicted amino acid sequences of the cloned ras-related goldfish gene suggested that the coding region is localized separately in DNA, and that its exon-intron boundaries are exactly the same as those of corresponding mammalian genes. The nucleotide and amino acid sequences of the goldfish ras-related gene may have extensive homologies to mammalian p 21 protein. Among the three mammalian ras proteins, the predicted amino acid sequence of the sequenced ras-related goldfish clone is most closely homologous (96%) to the Kirsten ras protein. Differences in the predicted amino acid sequence were greatest in the sequence predicted from the fourth exon; fewer differences were found in the sequence from the third exon, and only slight or no differences were found in the sequence predicted for the first and second exons. The 12th and 61st amino acids from the N-terminal of the protein, which are thought to be critical positions for GTP binding and catalysis, are both conserved in the goldfish protein.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   
70.
Previous results from this laboratory have shown that very low chronic doses of gamma radiation can stimulate proliferation of the Cyanobacterium Synechococcus lividus. This modification of cell proliferation occurred during the first doubling. In this paper, we have compared the metabolism of cells cultivated in a normal environment or under chronic irradiation. Incubation of the cells in a new medium induced a high superoxide dismutase (EC 1.15.1.1, SOD) activity at the 18th hour and a degradation of phycocyanin, thus demonstrating that cells were submitted to a photooxidative stress. This increase in superoxide dismutase activity was followed by concomittant peaks of glutathione reductase (EC 1.6.4.2, GR) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49, G6P-DH) at the 24th hour. Irradiated cultures at a dose of 53.5 mGray/year show an earlier and higher peak of SOD, GR, and G6P-DH. In a second stage, cultures showed an earlier onset of photosynthesis under irradiation, as evidenced by an increase in pigment content and an enhancement of glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13, GAP-DH). These results show that the radiostimulation is related to the activation of enzymes protecting against peroxides that were induced under oxidative circumstances and to the activation of a glucose catabolism via the oxidative pentose phosphate pathway.Abbreviations mGy milli-Gray - SOD superoxide dismutase - G6P-DH glucose-6-phosphate dehydrogenase - GAP-DH glycer-aldehyde-3-phosphate dehydrogenase - GSSG oxidized glutathione  相似文献   
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