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1.
Transformation of calf uterine progesterone receptor: analysis of the process when receptor is bound to progesterone and RU38486 总被引:2,自引:0,他引:2
Effects of different transforming agents were examined on the sedimentation characteristics of calf uterine progesterone receptor (PR) bound to the synthetic progestin [3H]R5020 or the known progesterone antagonist [3H]RU38486 (RU486). [3H]R5020-receptor complexes [progesterone-receptor complexes (PRc)] sedimented as fast migrating 8S moieties in 8-30% linear glycerol gradients containing 0.15 M KCl and 20 mM Na2MoO4. Incubation of cytosol containing [3H]PRc at 23 degrees C for 10-60 min, or at 0 degrees C with 0.15-0.3 M KCl or 1-10 mM ATP, caused a gradual transformation of PRc to a slow sedimenting 4S form. This 8S to 4S transformation was molybdate sensitive. In contrast, the [3H]RU486-receptor complex exhibited only the 8S form. Treatment with all three activation agents caused a decrease in the 8S form but no concomitant transformation of the [3H]RU486-receptor complex into the 4S form. PR in the calf uterine cytosol incubated at 23 or at 0 degrees C with 0.3 M KCl or 10 mM ATP could be subsequently complexed with [3H]R5020 to yield the 4S form of PR. However, the cytosol PR transformed in the absence of any added ligand failed to bind [3H]RU486. Heat treatment of both [3H]R5020- and [3H]RU486-receptor complexes caused an increase in DNA-cellulose binding, although the extent of this binding was lower when RU486 was bound to receptors. An aqueous two-phase partitioning analysis revealed a significant change in the surface properties of PR following both binding to ligand and subsequent transformation. The partition coefficient (Kobsd) of the heat-transformed [3H]R5020-receptor complex increased about 5-fold over that observed with PR at 0 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
2.
Effects of aurintricarboxylic acid (ATA) were examined on the DNA binding properties of rat liver glucocorticoid-receptor complex. The DNA-cellulose binding capacity of the glucocorticoid-receptor complex was completely abolished by a pretreatment of receptor preparation with 0.1-0.5 mM ATA at 4 degrees C. The half-maximal inhibition (i.d.50) in the DNA binding of [3H]triamcinolone acetonide-receptor complex [( 3H]TARc) was observed at 130- and 40 microM ATA depending upon whether the inhibitor was added prior to or following the receptor activation. The entire DNA-cellulose bound [3H]TARc could be extracted in a concentration-dependent manner by incubation with 2-100 microns ATA. The [3H]TARc remained intact under the above conditions, the receptor in both control and ATA-treated preparations sedimented in the same region in salt-containing 5-20% sucrose gradients. The action of ATA appeared to be on the receptor and not on DNA-cellulose. The DNA-binding capacity of ATA-treated receptor preparations could be recovered upon exhaustive dialysis. The treatment with ATA did not appear to change the ionic behavior of heat activated GRc; the receptor in both control and the ATA-treated preparations showed similar elution profiles. Therefore, ATA appears to alter the binding to and dissociation of glucocorticoid-receptor complex from DNA. The use of ATA should offer a good chemical probe for analysis of the DNA binding domain(s) of the glucocorticoid receptor. 相似文献
3.
A specific ultrastructural stain for arylsulfatase A activity in human cultured fibroblasts 总被引:1,自引:0,他引:1
A staining reaction was developed to specifically detect arylsulfatase A activity in the presence of arylsulfatases B and C. Nitrocatechol, generated by all arylsulfatases from the substrate p-nitrocatechol sulfate, can be coupled to produce Hatchett 's brown which reacts with 3,3'-diaminobenzidine to yield an osmiophilic polymer visible under the electron microscope. The reaction was made specific for arylsulfatase A by inhibiting arylsulfatase C activity with low pH and arylsulfatase B activity with pyrophosphate. The specificity was confirmed both by electrophoretic analysis and by patient fibroblasts deficient only in arylsulfatase A activity. Under optimal conditions for preserving structural integrity and enzyme activity, enzyme reaction deposits were found mainly around vesicles. Some of these vesicles were large and heterogeneous (48-330 nm in diameter), distributed randomly within the cytoplasm, but most of the positive-reacting vesicles were uniform in size (86 +/- 18 nm in diameter) and distributed in a peripheral zone about 0.1-0.5 micron wide. These periplasmic vesicles might be partly fused with each other or with the plasma membrane. In conclusion, a specific stain for arylsulfatase A activity suitable for light and electron microscopy and the optimal conditions for structural and enzymatic preservations were developed. Although this enzyme has been considered to be lysosomal in origin, most of the activity was detected in periplasmic vesicles near the cell surface. 相似文献
4.
Noriko Murakami Therese M. Quattrociocchi Shaun P. Healy Virinder K. Moudgil 《Archives of biochemistry and biophysics》1982,214(1):326-334
Effects of sodium tungstate on the nuclear uptake of rat liver cytosolic glucocorticoidreceptor complex were examined at pH 7. The nuclear uptake of heat-activated [3H]triamcinolone acetonide-receptor complex was blocked completely in the presence of 1 mm tungstate. A preincubation of nuclear preparation with tungstate (>0.1 mm) blocked the subsequent uptake of [3H]triamcinolone acetonide-receptor complex. When the tungstate-treated nuclear preparation was washed with 0.3 M KCl, its [3H]triamcinolone acetonide-receptor complex binding capacity recovered to 50% of that of control samples with no tungstate treatment. A preincubation of chromatin with tungstate yielded similar results. The nuclear-bound [3H]triamcinolone acetonide-receptor complex, formed either by an in vivo administration of [3H]triamcinolone acetonide or by an in vitro incubation of glucocorticoid-receptor complex with isolated nuclei, was extracted by tungstate in a concentration-dependent manner. The majority of nuclear-bound [3H]triamcinolone acetonide could be extracted with 0.1 and 1 mm tungstate from in vitro- and in vivo-labeled nuclei, respectively. The tungstate-extracted steroid-receptor complexes sedimented in 4–5 S and 3.3–3.5 S region in 10 mm KCl- and 0.3 mm KCl-containing sucrose gradients, respectively. Tungstate treatment caused an irreversible loss of the nuclear binding capacity of [3H]triamcinolone acetonide-receptor complex which could not be recovered after dialysis. These studies indicate that tungstate affects both glucocorticoidreceptor complex and certain nuclear or chromatin proteins. 相似文献
5.
Inactivation of avian progesterone receptor binding to ATP-Sepharose by pyridoxal 5'-phosphate 总被引:1,自引:0,他引:1
H Nishigori V K Moudgil D Toft 《Biochemical and biophysical research communications》1978,80(1):112-118
The affinity of progesterone receptor from hen oviduct for ATP-Sepharose was diminished by preincubation with pyridoxal 5′-phosphate. This effect was specific for pyridoxal 5′-phosphate since the related compounds, pyridoxal, pyridoxine, pyridoxamine and pyridoxamine 5′-phosphate, were not effectors. The inactivation was easily reversed by the addition of the primary amine, Tris. However, in the presence of the reducing agent NaBH4, the inhibitory effect of pyridoxal 5′-phosphate was irreversible. The results suggest that pyridoxal 5′-phosphate forms a Schiff base with a critical amino group, presumably at the nucleotide binding site of the progesterone receptor. 相似文献
6.
Introduction
Autoimmune inflammation is a characteristic feature of rheumatoid arthritis (RA) and other autoimmune diseases. In the natural course of human autoimmune diseases, it is rather difficult to pinpoint the precise timing of the initial event that triggers the cascade of pathogenic events that later culminate into clinically overt disease. Therefore, it is a challenge to examine the early preclinical events in these disorders. Animal models are an invaluable resource in this regard. Furthermore, considering the complex nature of the pathogenic immune events in arthritis, microarray analysis offers a versatile tool to define the dynamic patterns of gene expression during the disease course. 相似文献7.
Phosphorylation of rat liver glucocorticoid receptor 总被引:3,自引:0,他引:3
Rat liver glucocorticoid-receptor complex (GRc) was purified 2000-fold by a combination of methods including (NH4)2SO4-fractionation and phosphocellulose and DNA-cellulose chromatography. The purified glucocorticoid receptor preparation contained a major peptide of Mr = 90,000 and the GRc sedimented as 4 S in 5-20% sucrose gradients. An additional peptide of Mr = 45,000 (45K) was also observed. Some preparations yielded only the Mr = 90,000 (90K) peptide suggesting that the 45K peptide may be a proteolyzed portion of the 90K protein. The purified GRc was incubated with [gamma-32P]ATP in the presence of cAMP-dependent kinase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the above preparation revealed the presence of two 32P-containing bands with apparent Mr = 90,000 and 45,000. The 32P incorporation was dependent on the availability of divalent cation (Mg2+). GRc in cytosol labeled with [3H]dexamethasone mesylate and purified as above co-migrated with 32P-containing bands. GRc was also purified from cytosol obtained from livers of rats injected with [32P]orthophosphate. Both 32P and 3H bands were associated with 90K and 45K peptides. Our results indicate that rat liver glucocorticoid receptor is a phosphoprotein and that both the phosphorylated peptides 90K and 45K also contain the steroid and the DNA binding regions of the glucocorticoid receptor. 相似文献
8.
Conformational epitope on gp120 important in CD4 binding and human immunodeficiency virus type 1 neutralization identified by a human monoclonal antibody. 总被引:16,自引:35,他引:16 下载免费PDF全文
D D Ho J A McKeating X L Li T Moudgil E S Daar N C Sun J E Robinson 《Journal of virology》1991,65(1):489-493
A human monoclonal antibody designated 15e is reactive with the envelope glycoprotein (gp120) of multiple isolates of human immunodeficiency virus type 1 (HIV-1). Antibody 15e also neutralizes HIV-1 with broad specificity and blocks gp120 binding to CD4. Characterization of the 15e epitope shows that it is conformation dependent and is distinct from previously recognized functional domains of gp120, suggesting that this epitope represents a novel site important for HIV-1 neutralization and CD4 binding. These findings have implications for the development of a vaccine for AIDS. 相似文献
9.
We have examined the influence of sulfhydryl (SH)-group modifying agents on the interaction of the rat liver glucocorticoid receptor (GR) with its known agonist triamcinolone acetonide (TA) and the newly synthesized antagonist mifepristone (RU486). In the freshly prepared cytosol, [3H]TA or [3H]RU486 bound to macromolecule(s) which sediment as 8-9 moieties: the binding of either ligand can be competed with radioinert TA or RU486. The presence of 2-10 mM dithiothreitol (DTT), beta-mercaptoethanol (beta-MER), and monothioglycerol (MTG) caused a 2-3 fold increase in the [3H]TA and [3H]RU486 binding to GR. Iodoacetamide (IA) and N-ethylmaleimide (NEM) decreased the agonist binding significantly. In contrast, the [3H]RU486 binding to GR increased by 50 percent in the presence of IA. IA and NEM inhibited the binding of the heat-transformed [3H]TA-receptor complex to DNA-cellulose by 70-90 percent whereas DNA binding of [3H]RU486-bound GR was inhibited only slightly. These results indicate that either a) the interaction of GR with the agonist or antagonist steroid ligands causes differential structural alterations, which are more readily detectable in the presence of SH-modifying agents or b) the agonist and the antagonist interact with distinct steroid binding sites. 相似文献
10.
DNA hybridization analysis of mycobacterial DNA using the 18-kDa protein gene of Mycobacterium leprae 总被引:3,自引:0,他引:3
Abstract DNA hybridization studies using a 611-base pair (bp) probe, encoding the entire 18-kDa protein of Mycobacterium leprae , demonstrated that M. simiae, M. intracellulare, M. kansasii, M. terrae , ADM-2, M. avium, M. scrofulaceum, M. gordonae and M. chelonei appear to posses DNA sequences homologous to the 18-kDa protein gene of M. leprae . RFLP analysis revealed that the restriction sites in the M. leprae 18-kDa gene were not conserved in the putative gene homologs of M. simiae and M. intracellulare . The restriction patterns observed with the 611-bp probe were useful in differentiating M. intracellulare, M. simiae , and M. leprae from each other, as well as in distinguishing strains of M. simiae serovar 1. Finally, the presence of homologous sequences in various mycobacteria did not affect the specificity of a previously described PCR test for detection of M. leprae , based on the M. leprae 18-kDa protein gene. 相似文献