Biomedical subjects

G Litwack

Publications and source records attributed to G Litwack.

At least 91 records · Page 5Linked to original sources

PubMed1986

Effects of bovine pancreatic ribonuclease A, S protein, and S peptide on activation of purified rat hepatic glucocorticoid-receptor complexes.

Bovine pancreatic ribonuclease (RNase) A and S protein (enzymatically inactive proteolytic fragment of RNase A which contains RNA binding site) stimulate the activation, as evidenced by increasing DNA-cellulose binding, of highly purified rat hepatic glucocorticoid-receptor complexes. These effects are dose dependent with maximal stimulation of DNA-cellulose binding being detected at approximately 500 micrograms (50 units of RNase A/mL). RNase A and S protein do not enhance DNA-cellulose binding via their ability to interact directly with DNA or to increase nonspecific binding of receptors to cellulose. Neither S peptide (enzymatically inactive proteolytic fragment which lacks RNA binding site) nor cytochrome c, a nonspecific basic DNA binding protein, mimics these effects. RNase A and S protein do not stimulate the conformational change which is associated with activation and is reflected in a shift in the elution profile of receptor complexes from DEAE-cellulose. In contrast, these two proteins interact with previously heat-activated receptor complexes to further enhance their DNA-cellulose binding capacity and thus mimic the effects of an endogenous heat-stable cytoplasmic protein(s) which also function(s) during step 2 of in vitro activation [Schmidt, T. J., Miller-Diener, A., Webb, M. L., & Litwack, G. (1985) J. Biol. Chem. 260, 16255-16262]. Preadsorption of RNase A and S protein to an RNase affinity resin containing an inhibitory RNA analogue, or trypsin digestion of the RNA binding site within S protein, eliminates the subsequent ability of these two proteins to stimulate DNA-cellulose binding of the purified receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenalectomy↗

PubMed1986

Characterization of the rat colonic aldosterone receptor and its activation process.

Aldosterone increases sodium absorption, short circuit current, and transmural potential difference in rat colon. We studied the rat colonic aldosterone receptor using the synthetic glucocorticoid, 11 beta, 17 beta-dihydroxy-17 alpha-propynylandrosta-1,4,6-triene-3-one, to prevent binding to the glucocorticoid receptor. Specific aldosterone binding was found in proximal and distal colon. Heating to 25 degrees C decreased binding within 15 min, but the protease inhibitor, phenylmethylsulfonyl fluoride, stabilized binding. Binding was highest in terminal distal colon. Competitive binding assay showed aldosterone specificity compared to other competitors was greater at 30 than at 4 degrees C, suggesting temperature-sensitive changes in receptor specificity. Scatchard analysis revealed a straight line with a KD of 2.5 nM at 0 degrees C and 4.1 nM at 30 degrees C. Bmax was higher in distal than in proximal colon (30 degrees C, 156 +/- 33 versus 65 +/- 9 fmol/mg protein) and increased by 36% in proximal and 180% in distal colon at 30 degrees C compared to 0 degrees C. DEAE-cellulose chromatography of unactivated receptor demonstrated a single peak eluting at 200-250 mM KCl. Heat, ATP, and gel filtration did not activate the receptor, whereas increasing cytosolic salt concentration to 300 mM KCl, raising the pH to 8, or adding EGTA and EDTA caused increased DNA-cellulose binding and a new peak eluting at 30-80 mM KCl on DEAE-cellulose chromatography. There is a specific aldosterone receptor in colon with increasing number of binding sites from proximal to most distal segments paralleling aldosterone's physiological effects. Absence of receptor activation with heat, gel filtration, or ATP suggests differences between activation of the aldosterone receptor and other steroid hormone receptors.

Aldosterone↗

PubMed1986

The nuclear matrix is the site of glucocorticoid receptor complex action in the nucleus.

Binding of highly purified glucocorticoid receptor complexes to nuclear matrix was evaluated. Extraction of purified nuclei with 2M potassium chloride and brief deoxyribonuclease digestion leaves a matrix structure containing 1% of nuclear DNA and 6-12% of nuclear proteins. The nuclear matrix retained two binding sites for receptor complexes, a high affinity, low capacity site and a low affinity, high capacity site. These sites have affinities and capacities consistent with those reported for binding of these complexes to intact nuclei. More extensive deoxyribonuclease treatment of the matrix resulted in a marked reduction of high affinity complex binding. Furthermore, the DNA binding form of the receptor complex but not the unactivated receptor complex bound to DNA fibers anchored to nuclear matrix as visualized by 18 nm gold particle receptor complexes. The data suggest that the nuclear matrix is the major site for coordinating glucocorticoid hormone action in the nucleus.

Animals↗

PubMed1986

Steroid receptor activation: the glucocorticoid receptor as a model system.

The glucocorticoid receptor has been used as a model for steroid receptor activation. Because of recent evidence for the essentially nuclear location of the unoccupied receptors of 1,25-dihydroxycholecalciferol and 17 beta-estradiol, the significance of the activation mechanism converting unactivated receptor complexes to DNA-binding forms is unclear for some receptors. Up to now the weight of evidence favors a cytoplasmic location of the unactivated glucocorticoid receptor. In this article we describe studies on the nature of the activation mechanism and of regulatory factors.

Animals↗

PubMed1986

Decreased glucocorticoid binding and receptor activation in brain of genetically diabetic (MDB/MDB) mice.

Binding of [3H]triamcinolone acetonide (TA) to cytosolic receptors and subsequent in vitro activation of glucocorticoid-receptor complexes were studied in whole brain and liver from misty diabetic mice (mdb/mdb) and their control littermates (??/++). Binding was specific for glucocorticoid receptor (GcR) since the specific glucocorticoid, RU26988, was used to compete with [3H]TA for binding. Reduced [3H]TA binding was observed in whole brain and liver in diabetic animals when compared to control animals. Within the brain, binding was significantly (P less than 0.05) decreased in cortex, hippocampus, and hypothalamus. No significant differences in binding were found in the striatum or "midbrain". GcR binding was similar in diabetic and control animals until 2 months of age when overt diabetic symptoms appeared and the GcR binding was lower in diabetic animals. Though GcR from mdb/mdb brain cytosol could be thermally activated, the extent of activation was significantly (P less than 0.05) less than that for controls. These data indicate that GcR in liver and brain cytosol are decreased in mdb/mdb mice and that the GcR available for binding in mdb/mdb brain cytosol appears less capable of undergoing activation and binding to DNA-cellulose than GcR from control brain cytosol. Decreased GcR activation in brain cytosol from mdb/mdb mice was associated with increased dissociation of [3H]TA from the GcR. These results suggest that the decreased negative feedback previously observed in diabetic animals may be due to decreased binding of hormones and a decreased level of activation of hormone bound receptor complexes.

Androstanols↗

PubMed1986

A monoclonal antibody specific for the Yc subunit of rat liver glutathione S-transferase B.

A monoclonal antibody against rat liver glutathione S-transferase B, which consists of subunits Ya (Mr 27,000) and Yc (Mr 29,500), was produced in a mouse hybridoma system. The monoclonal antibody, BE6Yc1, was of the immunoglobulin G1 class and specifically recognized glutathione S-transferase B showing no cross-reactivity against the dimeric form of Ya, ligandin, in enzyme-linked immunosorbent assay. A glutathione S-transferase B-BE6Yc1 monoclonal antibody complex was identified from the zymogram pattern on starch gel electrophoresis. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis of rat liver glutathione S-transferase B, followed by Western blotting with BE6Yc1 antibody, a single polypeptide with a molecular weight of 29,500, identical to the Yc subunit of rat liver glutathione S-transferase B, was identified. The antibody showed no cross-reactivity with the Ya subunit with a molecular weight of 27,000 of either glutathione S-transferase B or ligandin. These results show that there are at least two distinct epitopes in the Ya and the Yc subunit. The monoclonal antibody did not block the catalytic activity of glutathione S-transferase B toward cumene hydroperoxide and possibly also 1-chloro-2,4-dinitrobenzene. Quantitative differences in expression of the Yc subunit of glutathione S-transferases were demonstrated in extracts of various normal tissues with the BE6Yc1 antibody by Western blot analysis. The Yc subunit was found to be present at much lower levels in kidney, small intestine, spleen, and lung than in liver and testis.

Animals↗

PubMed1985

Thermal activation of the purified rat hepatic glucocorticoid receptor. Evidence for a two-step mechanism.

Thermal "activation" or "transformation" of rat hepatic [6,7-3H]triamcinolone acetonide (TA)-receptor complexes purified in the unactivated state to near homogeneity (Grandics, P., Miller, A., Schmidt, T. J., Mittman, D., and Litwack, G. (1984) J. Biol. Chem. 259, 3173-3180) has been further investigated. The data generated in reconstitution experiments demonstrate that warming (25 degrees C for 30 min) of the purified unactivated complexes promotes their activation as judged by an increase in DNA-cellulose binding, but to a lower extent than that observed after warming of glucocorticoid-receptor complexes in crude cytosols. However, maximal DNA-cellulose binding capacity can be detected in reconstituted systems (also heated at 25 degrees C for 30 min) consisting of purified unactivated [3H]TA-receptor complexes and a cytoplasmic "stimulator(s)." This cytoplasmic factor(s), which does not copurify with the receptor, is heat-stable (90 degrees C for 30 min), excluded from Sephadex G-25, and trypsin-sensitive and stimulates DNA-cellulose binding in a dose-dependent manner. The ability of Na2MoO4 to block thermal activation of the highly purified receptor complexes suggests that this transition metal anion interacts directly with the receptor protein itself. The fact that the cytoplasmic stimulator(s) enhances DNA-cellulose binding of the [3H]TA-receptor complexes without increasing the proportion of those complexes eluted in the activated (low salt) position from DEAE-cellulose is consistent with a proposed two-step model of in vitro activation. During the Na2MoO4-sensitive Step 1, elevated temperature (25 degrees C for 30 min) may directly alter the conformation of the purified receptor complexes (i.e. subunit dissociation or disaggregation), resulting in the appropriate shift in the elution profile of the [3H]TA-receptor complexes on DEAE-cellulose but only in a minimal (approximately 2-3-fold) increase in the binding of these complexes to DNA-cellulose. During the Na2MoO4-insensitive and temperature-independent Step 2, a heat-stable cytoplasmic protein(s) may interact with these thermally activated [3H]TA-receptor complexes and enhance their ability to bind to DNA-cellulose without further increasing the percentage of those complexes which elute from DEAE-cellulose in the activated position. In crude cytosols these two steps would presumably occur simultaneously, and addition of Na2MoO4 prior to warming would block Step 1 and hence Step 2 would not occur.

Adrenalectomy↗

PubMed1985

Purification, characterization, and activation of the glucocorticoid-receptor complex from rat kidney cortex.

The unactivated molybdate-stabilized glucocorticoid receptor (GcR) was purified from rat kidney cortex cytosol (RKcC) by using a modification of the procedure previously described by this laboratory for rat hepatic receptor. The purification includes affinity chromatography, gel filtration, and ion-exchange chromatography. The final preparation (approximately 1000-fold pure as determined from specific radioactivity) was used in subsequent physicochemical and functional analyses. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a single heavily Coomassie-stained band at 90 kilodaltons. Density gradient ultracentrifugation indicated a sedimentation coefficient of 10.5 +/- 0.05 S (n = 2). Chromatography on an analytical gel filtration column produced a Stokes radius (Rs) of 6.4 +/- 0.07 nm (n = 5). The Rs was unchanged when the molybdate-stabilized GcR was analyzed in the presence of 400 mM KCl or when analyzed in the unpurified (cytosolic) state. In contrast, the hepatic GcR was observed to exist as a larger form in cytosol (7.7 +/- 0.2 nm). Following purification, or upon gel filtration analysis under hypertonic conditions, the Rs was similar to that of the unpurified RKcC GcR. Following removal of molybdate from RKcC GcR and thermal activation (25 degrees C/30 min), DNA-cellulose binding increased 1.5-2-fold over the unheated control. Addition of RKcC or hepatic cytosol (endogenous receptors thermally denatured at 90 degrees C/30 min or presaturated with 10(-7) M radioinert ligand) during thermal activation increased DNA-cellulose binding an additional 2-6-fold beyond the heated control.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1985

Mebendazole and insulin secretion from isolated rat islets.

In a preliminary communication we reported that mebendazole, a vermicide, decreased plasma glucose and free fatty acid concentrations and increased plasma C peptide concentrations in both type II diabetic patients. Therefore, we suggested that mebendazole was an insulin secretagogue. However, these were uncontrolled studies, and improved metabolic control in these patients due to spontaneous remission rather than drug-induced insulin secretion was a possibility. To investigate the direct effect of mebendazole on insulin secretion we used intact islets isolated from normal rat pancreata. Mebendazole in concentrations as low as 10 to 20 mumol/L caused a twofold to threefold increase in acute-phase insulin release from isolated perifused rat islets. This heightened insulin release occurred in the presence of glucose-stimulated insulin secretion.

Animals↗

PubMed1985

Protein kinase activity associated with the purified rat hepatic glucocorticoid receptor.

The Mr 94,000 steroid binding component of rat hepatic glucocorticoid receptor purified 5000-fold under-goes calcium-stimulated phosphorylation in vitro by [gamma-32P]ATP. Exogenous histones can be phosphorylated by this preparation without calcium. Calmodulin did not stimulate phosphorylation of the glucocorticoid receptor beyond that obtained with calcium alone. Although the specific calmodulin inhibitor calmidazolium had no effect, trifluoperazine and chlorpromazine, nonspecific calmodulin inhibitors, abolished the calcium-dependent phosphorylation of receptor. EGTA blocks the effect of calcium; magnesium cannot substitute for calcium. Cyclic nucleotides (cAMP or cGMP) do not stimulate phosphorylation of the receptor in the absence of calcium. Phosphorylation of the glucocorticoid receptor is steroid dependent. Triamcinolone acetonide elicited activation and phosphorylation of receptor in the presence of calcium, whereas the antagonists progesterone, cortexolone, and beta-lapachone did not. Sodium molybdate, which blocks the thermal activation step, inhibits phosphorylation of the receptor. The activated form of the glucocorticoid receptor is required for phosphorylation to occur. The ATP analogues 8-azido-ATP or fluorosulfonylbenzoyl adenosine, inhibit phosphorylation of the Mr 94,000 component, implying the presence of an ATP binding site inherent to the receptor.

Adenosine Triphosphate↗

PubMed1984

Beta-lapachone, a specific competitive inhibitor of ligand binding to the glucocorticoid receptor.

Beta-Lapachone, a derivative of 1,2-naphthoquinone, inhibits the specific binding of [6,7-3H]triamcinolone acetonide (TA) to unbound hepatic and thymic glucocorticoid receptors in a dose-dependent manner with 50% of the maximal inhibition in thymus cytosol achieved at a final concentration of 5-10 microM. Preincubation of cytosol with 10 mM Na2MoO4, which stabilizes unbound receptors, potentiates the subsequent beta-lapachone-mediated inhibitory activity, while preincubation with 1 or 10 mM dithiothreitol blocks the subsequent inhibition of [6,7-3H]TA binding. A double reciprocal plot indicates that beta-lapachone is a competitive inhibitor of [6,7-3H]TA binding with an apparent Ki of approximately 6 microM. The ability of beta-lapachone to displace prebound [6,7-3H]TA and the ability of elevated concentrations of [6,7-3H]TA to reverse the beta-lapachone-mediated inhibition are totally consistent with this kinetic interpretation. The ability of beta-lapachone to interact directly with the ligand-binding site is confirmed by the fact that this compound can block the binding of [6,7-3H]TA to highly purified unactivated hepatic glucocorticoid receptors. Although beta-lapachone may interact specifically with receptor sulfhydryl groups, this compound is not a general oxidizing agent which inactivates the essential free sulfhydryl groups at the glucocorticoid-binding site. Beta-Lapachone does not affect activation of [6,7-3H]TA-receptor complexes nor does it itself act like a glucocorticoid and facilitate receptor activation (transformation). Interestingly, this compound does not affect the ligand-binding sites of estrogen, progesterone, androgen, or mineralocorticoid receptors or serum transcortin. Thus, beta-lapachone can be utilized as a specific probe for the ligand-binding site of the glucocorticoid receptor.

Adrenalectomy↗

PubMed1984

Phosphorylation in vivo of rat hepatic glucocorticoid receptor.

Rat liver glucocorticoid receptors were labeled in vivo with [32P]orthophosphate. In the last two fractionation procedures leading to purified, molybdate-stabilized, unactivated receptor complex, bound [32P] coeluted with peaks of bound [3H]triamcinolone acetonide. SDS-gel electrophoresis revealed [32P] labeled 90K and 24K bands. The lower molecular weight band is heavily phosphorylated and it could be either a component of the unactivated receptor or a degradation product.

Animals↗

PubMed1984

Purification of the unactivated glucocorticoid receptor and its subsequent in vitro activation.

The unactivated, molybdate-stabilized rat hepatic glucocorticoid receptor has been purified approximately 4000-fold, as calculated by specific radioactivity, by affinity chromatography using a deoxycorticosterone-derivatized agarose, gel filtration on Bio-Gel A-1.5m agarose, and DEAE-cellulose chromatography. The final receptor sediments at 9-10 S in low salt (40 mM KCl) glycerol gradients containing molybdate. Elevated salt concentrations up to 1 M KCl reduce the sedimentation coefficient to 8-9 S. The final DEAE-cellulose eluted complexes exhibit a Stokes radius of 7.3 nm, a value similar to that reported for receptors in crude cytosol. From the hydrodynamic parameters an apparent Mr = 303,000 can be calculated for the steroid-receptor complex. Analysis of the receptor-containing fractions from DEAE-cellulose chromatography by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrates the occurrence of a major Mr = 90,000 protein band which closely followed the distribution of bound radioactivity. Two other proteins corresponding to Mr = 41,000 and 40,000 also exhibit the same distribution pattern. Saturation of cytosolic specific binding sites with unlabeled triamcinolone acetonide prior to receptor purification results in the disappearance of these three proteins from the DEAE-cellulose chromatogram. Furthermore, a Mr = 24,000 component, which is eluted from DEAE-cellulose at a salt concentration higher than that of the bound radioactivity peak itself, also disappears. These observations argue that the Mr = 90,000, 41,000, 40,000, and 24,000 components are related as components or degradation products of the unactivated, molybdate-stabilized rat hepatic glucocorticoid-receptor complex. Studies on the in vitro activation of purified steroid-receptor complexes have revealed that Sephadex G-25 gel filtration and warming (25 degrees C for 30 min) enables purified receptors to become activated judged by ability to bind to DNA-cellulose but to a lower extent than observed for receptors in crude tissue homogenates. A DNA-cellulose binding capacity, similar to that shown by crude liver cytosolic receptor under the same conditions, can be conferred on the purified complexes only in a reconstituted system in which crude cytosol has been added. Molybdate is shown to completely inhibit activation induced by gel-filtration and offers significant protection against heat-induced activation both in highly purified and reconstituted systems. The activation inhibitory effect of molybdate has also been confirmed by DEAE-cellulose chromatography.

Animals↗

PubMed1984

Kinetic studies and active site-binding properties of glutathione S-transferase using spin-labeled glutathione, a product analogue.

Kinetic and binding studies with substrates, products, and a spin-labeled product analogue of glutathione (sl-glutathione) have been used to characterize the kinetic mechanism and properties of the catalytic site of the homodimer YaYa of glutathione S-transferase. Product inhibition studies and inhibition by sl-glutathione indicate the random addition of substrates. The kinetically determined dissociation constant for the product S-(2,4-dinitrophenyl)glutathione is approximately 7 microM. A newly described spin-labeled product analogue, S-[[(2,2,5,5,-tetramethyl-1-oxy-3-pyrrolidinyl)-carbamoyl]methyl] glutathione (sl-glutathione), acts as a competitive inhibitor with respect to both substrates (glutathione and 1-Cl-2,4-dinitrobenzene) with a kinetically determined dissociation constant of approximately 40 microM. Analysis of the glutathione S-transferase X sl-glutathione complex by EPR gives a rigid limit spectrum indicative of highly immobilized spin label. Kinetic and EPR results support the proposal that sl-glutathione binds as a bisubstrate or product analogue by occupying both the glutathione and hydrophobic substrate sites. Binding studies of sl-glutathione by EPR give a dissociation constant of 28 microM and a single binding site per homodimer. Displacement of sl-glutathione by substrates and product have been used to directly determine enzyme-ligand dissociation constants. Dissociation constants of 2.1 mM, 17 microM, and 25 microM were obtained for glutathione, 1-Cl-2,4-dinitrobenzene and S-(2,4-dinitrophenyl)glutathione when enzyme was added to a mixture of sl-glutathione and the competing ligand. The dissociation constants for glutathione and 1-Cl-2,4-dinitrobenzene but not for S-(2,4-dinitrophenyl) glutathione were dependent on the order of addition, consistent with the existence of several kinetically stable conformations for the enzyme. The sl-glutathione described here may provide a useful analogue for similar studies with other glutathione S-transferases or other enzymes which bind glutathione.

Amino Acids↗

PubMed1984

Glucocorticoid stimulation of sodium absorption in colon epithelia is mediated by corticosteroid IB receptor.

Studies with RU26988, a synthetic glucocorticoid which does not bind to aldosterone receptors, suggest glucocorticoid-induced colonic cation transport is affected through glucocorticoid-specific receptors. RU26988 produced a 700% increase in sodium absorption and doubled transmural potential difference in proximal and distal colon of adrenalectomized rats. Scatchard analysis suggested a single class of receptors with a KD of approximately 10(-9) M. Competition of unlabeled steroids for [3H]triamcinolone acetonide-binding sites paralleled the steroids' biologic potency as glucocorticoids. Heat treatment (25 degrees C, 30 min) markedly enhanced binding of the glucocorticoid-receptor complexes to DNA-cellulose. The activated receptor from both proximal and distal colon was eluted in the prewash from DEAE-Sephadex A-50 anion exchange columns both in the presence and absence of protease inhibitors and has an estimated molecular weight (Stokes radius) of 33,000-37,000 (25-26 A). These results identify the colonic receptor as glucocorticoid binder IB, a receptor previously identified as the major binder only in kidney cortex. The finding of an apparently unique receptor in the two tissues where glucocorticoids stimulate cation transport suggests that the phenotypic response mediated by glucocorticoids in different tissues might be determined by the structure of the receptor and that glucocorticoid binder IB is the glucocorticoid cation transport receptor.

Adrenalectomy↗

PubMed1984

Evidence that pH induced activation of the rat hepatic glucocorticoid-receptor complex is irreversible.

The possible reversibility of pH induced activation of the glucocorticoid-receptor complex was studied. Generally, this was accomplished by activating rat liver cytosol at pH 8.5 (15 degrees C, 30 min), and then returning it to pH 6.5 for a second incubation (15 degrees C, 30 min). Activation was quantitated by measuring the binding of [3H]triamcinolone acetonide [( 3H]TA)-receptor complexes to DNA-cellulose. When cytosol was incubated at pH 6.5, only 4.1% of the [3H]TA-receptor complexes bound to DNA-cellulose. However, 39.2% of the complexes bound when the cytosol was pH activated. When pH activation was followed by a second incubation at pH 6.5, 47.0% of the steroid-receptor complexes bound. Thus, according to the DNA-cellulose binding assay, pH induced activation was irreversible. In order to visualize both activated and unactivated [3H]TA-receptor complexes during this process, diethylaminoethyl (DEAE)-cellulose chromatography was performed. When cytosol was incubated at pH 6.5, only 19.6% of the [3H]TA-receptor complexes were eluted in the activated form from DEAE-cellulose. However, 67.5% of the complexes were eluted in the activated form when cytosol was pH activated. When pH activation was followed by a second incubation at pH 6.5, 74.9% of the steroid-receptor complexes were eluted in the activated form. Thus, DEAE-cellulose chromatography also showed that pH induced activation was irreversible. This is the first known report that the combination of DNA-cellulose binding and DEAE-cellulose chromatography have been used to study pH induced activation of the glucocorticoid-receptor complex. By these criteria, we conclude that in vitro pH induced activation is irreversible.

Adrenalectomy↗

PubMed1984

Characterization of a monoclonal antibody to ligandin.

A monoclonal antibody has been produced in the mouse system using purified rat liver ligandin as antigen. The antibody is of the immunoglobulin M class and appears to be specific for the Ya subunit of lowest molecular weight which comprises the isoenzymes of glutathione S-transferases in rat liver. Of the series of glutathione S-transferases, the antibody cross-reacted with purified ligandin (YaYa) and, in ion-exchange separations of isoenzymes, gave positive Western blots with fractions containing glutathione S-transferases B and a combination of D and E. Interactions of the immunoglobulin M antibody and the antigen were demonstrated using 125I-ligandin (YaYa) and physical separations in gel filtration experiments. The antibody did not inhibit the glutathione S-transferase activity of ligandin (YaYa) but rather increased the catalytic activity in a dose-dependent fashion up to a molar ratio of antiligandin:ligandin of 24.

Animals↗

PubMed1983

Identification of a macromolecular inhibitor of glucocorticoid-receptor complex activation in rat liver cytosol.

We have identified an endogenous regulator of the glucocorticoid receptor following fractionation of dialyzed rat liver cytosol on DEAE-cellulose. The macromolecular regulator, purified approximately 20-fold as judged by Lowry-reactive material, inhibits activation of glucocorticoid-receptor complexes when assayed by DNA-cellulose binding and by chromatography on DEAE-cellulose minicolumns. In addition the active DEAE-cellulose fraction stabilizes the unoccupied glucocorticoid receptor against heat inactivation. Evidence is presented that the observed inhibition of activation by the active DEAE-cellulose fraction is not due to concentration of cytosolic proteases or RNA. The inhibitory molecule in the active fraction is not stable to heating at 90 degrees C (15 min) and is partially inactivated at 45 degrees C (15-60 min).

Adrenalectomy↗