摘要:

AbstractIn a previous study we showed that antibodies directed to restricted sequences in the hinge domain ofα-type triiodothyronine (T3) receptor (TR) (aminoacids 150-166, 172-191, 144-162) selectively recognized and immunoprecipitatedα-type TR in tissues. Furthermore, antibodies to peptide 172-191 (anti-α172) strongly impaired T3 binding to natural TRα. To get more precise informations on the ability of these antibodies to recognize the TR, alter TR properties and/or detect conformational changes in TR, TRαl was produced in E. coli as a non-mutated, non-fusion protein from a rat c-erb Aα1 cDNA inserted intopTrc99A vector. The recombinant c-Erb Aαl protein, after solubilization with 5 M guanidine and progressive refolding, presented the main characteristics of TRα: unique or largely dominant band of 46 KDa in Western blots with the different anti-c-Erb Aαantibodies; binding to DNA and to T3. Binding to DNA was markedly attenuated by anti-α144 but not by anti-α150 and anti-α172. Binding to T3 was modified by anti-α150 and -α172 with different characteristics whether recombinant TR was previously bound to T3 or not, and with marked differences in comparative studies with natural TR. When liganded to T3, recombinant and natural TRαl present the same pattern of interaction with both antibodies: immunoprecipitation without any dissociation of T3 by anti-α150; marked dissociation of bound T3 by anti-α172. By contrast unliganded recombinant and natural TR are oppositely altered by these antibodies in their ability to bind T3: strong impairment restricted to anti-α172 for natural TR, and to anti-α150 for recombinant TR. Anti-α144 did not interfere. These results lay emphasis on: 1/ the existence and biological relevance of different conformational states of TRαhinge domain, particularly whether TR is liganded or not to T3 and whether it is in a nuclear environment or bacterially-produced; 2/ an important role of the C-terminal part of hinge domain for efficient hormone binding, this involving a region that overlaps theα150 andα172 sequences.

ISSN:1079-9893    

DOI:10.3109/10799899509079902

出版商:Taylor&Francis    

年代:1995

数据来源: Taylor

摘要:

AbstractA modified Chloramine T labelling procedure was used to iodinate mEGF in order to perform radio-receptor assays. The reaction was conducted at 4°C with lµg Chloramine T only. The tracer obtained was characterized by its maximal binding, specific activity and binding properties compared with the native peptide. Fast Liquid Protein Chromatography was performed to analyse the homogeneity of the preparation and membrane extracts from A431 cells were used to purify the tracer. The modified Chloramine T procedure was compared with two other methods: the classical Chloramine T iodination and the labelling procedure using Enzymobeads.The modified Chloramine T procedure is reproducible, provides labelled mEGF with high binding capacity (65 to 80% with canine placental membrane extracts) and high specific activity (351±107µCi/µg mEGF) and seems to preserve the binding properties of the native peptide.

ISSN:1079-9893    

DOI:10.3109/10799899509079903

出版商:Taylor&Francis    

年代:1995

数据来源: Taylor

摘要:

AbstractWe have identified the receptor for epithelial type transforming growth factor (TGFe). TGFe, a member of the epithelin/granulin family of proteins, is present primarily in tissues of epithelial origin. It is a powerful mitogen for epithelial and fibroblastic cells. TGFe, iodinated using an immobilized glucose oxidase-lactoperoxidase method, was chemically crosslinked to receptors on membranes isolated from SW-13 adrenal carcinoma cells by the crosslinker disuccinimidyl suberate (DSS). The receptor appears to be a protein which migrates at an apparent molecular weight of approximately 170-175 kDa under reducing and nonreducing conditions in SDS-polyacrylamide gels.

ISSN:1079-9893    

DOI:10.3109/10799899509079904

出版商:Taylor&Francis    

年代:1995

数据来源: Taylor

摘要:

AbstractThe effects of MgCl2on the binding of the serotonin 1a (5HT1a) receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) to bovine hippocampal membranes were investigated. MgCl2was found to enhance both [3H]8-OH-DPAT binding and guanine nucleotide modulation of high affinity binding. The effect of Mg2+on promoting high affinity agonist binding was due to a 3.3 fold decrease in the dissociation constant rather than an increase in the number of binding sites. Mg2+acted to increase the efficacy of the nonhydrolyzable GTP analog 5′guanylylimidodiphosphate (GppNHp) in promoting the interconversion of high and low affinity states. Addition of MgCl2significantly increased the sensitivity of [3H]8-OH-DPAT binding to GppNHp, decreasing the concentration needed for half-maximal inhibition of binding from greater than 50µM to 300 nM. Our findings that Mg2+enhances high affinity [3H]8-OH-DPAT binding without change in number of binding sites and that guanine nucleotide modulation of binding can occur in the absence of Mg2+suggests that ternary complex formation between receptor, ligand and G-protein can occur in the absence of Mg2+.

ISSN:1079-9893    

DOI:10.3109/10799899509079905

出版商:Taylor&Francis    

年代:1995

数据来源: Taylor

摘要:

AbstractThe neuropeptide, eclosion hormone (EH), triggers ecdysis behavior at the end of each molt inManduca sexta. Previous studies have shown that the action of EH is mediated by an increase in cGMP and is associated with the phosphorylation of two proteins, named the EGPs. The ability of insects to respond to EH is developmentally regulated with sensitivity being first seen at about 8 hr prior to the normal time of ecdysis. The EGPs are also first detectable in the CNS at 8 hr prior to ecdysis, suggesting that it is their synthesis which determines EH sensitivity.The protein synthesis inhibitor, cycloheximide was used to study the development of the events leading to pupal ecdysis inManduca. The results of these experiments suggest that protein synthesis is necessary about 10 hr before ecdysis for both the development of EH sensitivity and for the appearance of the EGPs.

ISSN:1079-9893    

DOI:10.3109/10799899509079906

出版商:Taylor&Francis    

年代:1995

数据来源: Taylor

ISSN:1079-9893    

DOI:10.3109/10799899509079901

出版商:Taylor&Francis    

年代:1995

数据来源: Taylor