Regulation of nuclear uptake of "activated" receptor-glucocorticoid complex by pyrophosphate

The desialylated human chorionic gonadotropin α- and β-subunits were combined with their native complementary subunits and the thyrotropic activities of the recombinants were compared to those of native and desialylated human chorionic gonadotropin using human thyroid membranes. All the combined forms interacted with the thyrotropin receptor-adenylate cyclase system, but only those with sialic acid residues present on the α-subunit were able to activate the enzyme. These data support the concept that the α-subunit contains the domain through which this hormone activates adenylate cyclase.     

An ATP-stimulated factor that enhances the nuclear binding of "activated" receptor-glucocorticoid complex

A macromolecular material that enhances the translocation, or binding, of already "activated" receptor-glucocorticoid complex to nuclei in the presence of 5 mM ATP was separated from the cytosol of rat liver by DEAE-cellulose column chromatography with about 0.025 M NaCl. The molecular weight of the material was about 93,000 +/- 4,900, as determined by agarose gel filtration. After incubation at 60 degrees C for 15 min, this material still had activity to increase the nuclear binding, but on boiling for 15 min it lost its activity.     

Characterization of the rat liver glucocorticoid receptor purified by DNA-cellulose and ligand affinity chromatography

Two rapid and high yield purification methods for the rat liver glucocorticoid receptor based on differential DNA affinity (method A) and ligand affinity (method B) chromatography are described. In method A, the amount of receptor in rat liver cytosol that can be activated and subsequently eluted from a DNA-cellulose column has been increased to 80% by introducing a second heat activation step. Using this method, 1.5 nmol of 25% pure glucocorticoid receptor can be routinely obtained per day from 15-20 rat livers. Method B yields about 2.2 nmol of 60% pure receptor with an overall yield of congruent to 60%. The quality of these purifications has been controlled by affinity labeling. In each case, more than 95% of purified binding activity represented the intact 92,000 +/- 400-Da glucocorticoid receptor polypeptide as shown by sodium dodecyl sulfate-gel electrophoresis and fluorography. No difference in the labeling pattern was observed using either [3H]triamcinolone acetonide (photoaffinity labeling) or [3H]dexamethasone 21-mesylate (electrophilic labeling). The electrophilic labeling step was performed in the cytosol prior to purification by method A to compare the labeled components thus purified with those obtained when the photoaffinity labeling was performed after the purification. Using this approach, distinct breakdown products of the glucocorticoid receptor were revealed, co-purifying during DNA affinity chromatography. Cross-linked receptor obtained by method A has been further purified to homogeneity by preparative sodium dodecyl sulfate-gel electrophoresis and successfully used as immunogen to raise glucocorticoid receptor antibodies in rabbits. These antibodies raised against glucocorticoid receptor, as well as those previously obtained using affinity chromatography-purified receptor, react with the receptor molecules irrespective of their method of purification. Glucocorticoid receptors purified by methods A and B have been analyzed for specific DNA-binding properties by the nitrocellulose filter binding assay.     

Evidence for nuclear and DNA binding forms of the receptor-glucocorticoid complex from hepatoma tissue culture cells

Binding to DNA associated with cellulose has been used to investigate the receptor-glucorticoid complex isolated from a line of rat hepatoma tissue culture cells. The amount of activated complex that bound to DNA was approximately half that which bound to nuclei. Additional results suggest the existence of two forms of the activated glucocorticoid receptor-steroid complex in about equal amounts: one form binds only to nuclei and the other binds to DNA and nuclei. The two forms also differ in their stability, with the DNA/nuclei binding form being relatively labile. The binding of either form to the appropriate acceptor is reduced by cytosol inhibitors by the same mechanism.     

Evidence for nuclear and DNA binding forms of the receptor-glucocorticoid complex from hepatoma tissue culture cells.

Binding to DNA associated with cellulose has been used to investigate the receptor-glucocorticoid complex isolated from a line of rat hepatoma tissue culture cells. The amount of activated complex that bound to DNA was approximately half that which bound to nuclei. Additional results suggest the existence of two forms of the activated glucocorticoid receptor-steroid complex in about equal amounts: one form binds only to nuclei and the other binds to DNA and nuclei. The two forms also differ in their stability, with the DNA/nuclei binging form being relatively labile. The binding of either form to the appropriate acceptor is reduced by cytosol inhibitors by the same mechanism.     

Properties of activated chromatin from rat liver shortly after partial hepatectomy

The properties of rat liver chromatin 1, 2 and 6 hours after partial hepatectomy have been studied by means of cytochemical and biochemical methods. An increase in the accessibility of DNA to low molecular weight ligands, RNA--polymerase and RNAse I and also of the distances between nucleosomes and their heterogeneity in length on electron -- microscopic photographs has been found. Analysis of the isotherms of adsorption has revealed an increase in the number of binding sites for ethidium bromide on DNA and accordingly a decrease in the extent of the filling of the template with protein in activated chromatin. Two hours after partial hepatectomy rat liver chromatin does not differ in all parameters studied from control chromatin. Limited digestion of chromatin with DNAse I almost fully eliminates the difference between the fractions of activated and control chromatin in the number of binding sites for the ligands to the fractions resistent in these conditions to nuclease. A suggestion that the changes in the properties of chromatin upon activation are due to the change in the character of chromatin proteins interaction with DNA are discussed.     

Characterization of the purified activated glucocorticoid receptor from rat liver cytosol

The activated glucocorticoid receptor (GR) from rat liver cytosol was purified by sequential chromatography on DNA-cellulose and DEAE-Sepharose. Analysis by sodium dodecyl sulfate-gel electrophoresis demonstrated a main band with Mr = 94,000 (94K band). Two minor bands with Mr = 79,000 (79K band) and 72,000 (72K band) were also seen in this preparation. Photoaffinity labeling showed that the hormone is bound to the 94K and 79K components but not to the 72K component. Immunoblotting using antibodies raised against the 94K protein demonstrated cross-reactivity between the 94K and 79K components but not with the 72K species. The 72K species could be partially separated from the 94K and 79K components by density gradient centrifugation. Limited proteolysis of the purified GR with trypsin or alpha-chymotrypsin led to degradation of the 94K and 79K components and appearance of a 39K fragment which still retained the hormone and could be bound to DNA-cellulose. The 72K component was not affected by digestion with trypsin or alpha-chymotrypsin. However, chromatography on DNA-cellulose of the alpha-chymotrypsin-treated GR resulted in elution of the 72K component in the flow-through of the column while the 39K fragment was retained on the column and eluted with 0.18 M NaCl. In the control experiment where no alpha-chymotrypsin treatment was performed, the 72K component could not be detected in the flow-through fraction but was eluted together with the 94K and 79K components at 0.18 M NaCl. These results suggest that the 72K protein might be bound to the 94K and/or 79K component. The 39K fragment did not bind antibodies raised against the 94K protein. The 39K fragment was further degraded by trypsin but not by alpha-chymotrypsin to a 27K and a 25K fragment while both still retained the ligand. These data obtained with limited proteolysis of the purified GR are in agreement with previous findings on proteolysis of the GR in crude cytosol (Wrange, O., and Gustafsson, J.-A. (1978) J. Biol. Chem. 253, 856-865; Carlstedt-Duke, J., Okret, S., Wrange, O., and Gustafsson, J.-A. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 4260-4264).     

The Mg,Ca, EDTA and ATP dependence of Na binding by rat liver microsomes

The role of ionic interactions in the adenosinetriphosphate (ATP) dependent Na binding by rat liver microsomes was investigated. In the concentration range of 0 to 20 mM, Mg and Ca are demonstrated to compete strongly against Na for microsome binding sites. In the presence of Ca, the nonbiological complexing agent ethylenediaminetetraacetate (EDTA) produced a marked increase in Na binding accompanied by a concomitant decrease in Ca binding. Under similar conditions ATP, which is a weaker complexing agent than EDTA, produced quantitatively smaller but qualitatively similar changes in binding. The data show that the effect of ATP on Na binding is not dependent upon the formation of a hypothetical Na binding intermediate in the hydrolysis of ATP as other investigators have postulated. Rather, the effect of ATP is demonstrated to depend upon the presence of unhydrolyzed ATP and its ability to complex divalent cations, and thereby to reduce divalent cation competition against monovalent cations for membrane binding sites.     

Metazoan origin selection: origin recognition complex chromatin binding is regulated by CDC6 recruitment and ATP hydrolysis

Using a plasmid competition assay, we have measured the stability of origin recognition complex (ORC) associated with sperm chromatin under physiological conditions. Under conditions in which pre-RCs are formed, both ORC and CDC6 dissociate from sperm chromatin with a relatively fast t(1/2) of 15 min. ORC dissociation from chromatin is regulated through the recruitment of CDC6 and MCM proteins as well as ATP hydrolysis. The t(1/2) for ORC alone in the absence of Cdc6 is 40 min and increases 8-fold to >2 h when Cdc6 is present. Strikingly, the presence of a non-hydrolyzable ATP derivative, ATPgammaS, not only increases both ORC and CDC6 t(1/2) but also inhibits the loading of MCM. The very stable association of ORC and Cdc6 with chromatin in this sequence-independent replication system suggests that origin selection in metazoans cannot be strictly dependent on the interaction of ORCs with specific DNA binding sequences.     

Identification and regulation of a rat liver cDNA encoding farnesyl pyrophosphate synthetase

CR39 is a cholesterol-repressible rat liver cDNA previously isolated by differential hybridization (Clarke, C.F., Tanaka, R.D., Svenson, K., Wamsley, M., Fogelman, A.M., and Edwards, P.A. (1987) Mol. Cell. Biol. 7, 3138-3146). To precisely identify the function of CR39 a fusion protein was constructed that contained the amino-terminal region of the bacterial protein anthranilate synthetase fused to the full length CR39 polypeptide. Affinity purified antisera directed against the fusion protein inactivated rat liver cytosolic prenyltransferase activity in vitro. In addition, affinity purified antisera made to purified chicken prenyltransferase cross-reacted with the fusion protein containing CR39. Rat hepatic prenyltransferase activity and enzyme mass were quantitated in animals fed diets or drugs known to alter endogenous cholesterol biosynthesis. Rats fed a diet supplemented with cholestyramine and mevinolin showed a 3.5-fold increase in activity and a 5.0-fold increase in mass of cytosolic prenyltransferase. A diet supplemented with cholesterol resulted in approximately a 4.0-fold decrease in hepatic enzyme activity and a 10-fold decrease in enzyme mass. Under these same dietary regimens the mass of prenyltransferase in the testes remained unchanged. We conclude that CR39 encodes the prenyltransferase of cholesterol biosynthesis, farnesyl pyrophosphate synthetase. Furthermore, in the liver this enzyme shows coordinate regulation with two other enzymes, 3-hydroxy-3-methylglutaryl-CoA reductase and 3-hydroxy-3-methylglutaryl-CoA synthase, in response to cholesterol feeding and hypocholesterolemic drugs.     

Characterization of DNA binding protein from rat liver chromatin which decreases during growth

A nuclear nonhistone protein which decreases in chromatin during growth (Yeoman, L. C., et al. (1975) Cancer Res. 35, 1249) has been isolated in high purity from the chromatin of normal rat liver nuclei by gel electrophoresis and column chromatography. This protein, designated BA (Yeoman, L.C., et al. (1973) Biochem Biophys. Res. Commun. 53, 1067), has a molecular weight of 31 000, an acidic to basic amino acid composition ratio of 0.9, and contains one tryptophan residue per molecule. Hydrazinolysis indicated protein BA has a lysine carboxyl terminus; however, the amino terminal is blocked as no reaction occurred with dansyl chloride. Maps of tryptic peptides of protein BA contained 46 spots. Protein BA binding to various DNAs was examined by the nitrocellulose filter assay. Binding was slightly enhanced by 2mM Mn2+ion; Mg2+, however, decreased binding. Binding was optimal at neutral pH and an ionic strength of 0.2 M [NaCl]. Equilibrium competition binding studies indicated a binding preference of protein BA for dA-dT rich DNA.     

Partial purification of the glucocorticoid receptor from rat liver: a rapid, two-step procedure using DNA-cellulose.

Glucocorticoid receptor from rat liver was purified 1800-fold by a rapid two-step procedure using DNA-cellulose. The procedure is based on increasing the affinity of the glucocorticoid-receptor complex for DNA by heating the complex. During a first chromatography step, unheated glucocorticoid-receptor complex is separated from cytosol proteins that bind to DNA-cellulose with high affinity. During a second chromatographic step, heat-treated glucocorticoid-receptor complex is separated from proteins with low affinity for DNA. The partially purified complex is functionally competent in that it is taken up by isolated rat liver nuclei.     

Studies on the nuclear binding of steroid hormone-receptor complex; characteristics of binding of nuclei from fetal rat liver to 3H-dexamethasone-liver cytoplasmic receptor complex

Binding of 3H-dexamethasone (Dex)-rat liver cytoplasmic receptor complex to nuclei from fetal rat livers in vitro exhibited a high-affinity and saturable nature (Kd=1.5 X 10- M, maximal binding sites=470 fmole/mg DNA), and the binding was inhibited competitively by prior injection of Dex in vivo. While binding of 3H-Dex-receptor complex to nuclei from adult rat liver was in low affinity and unsaturable, and injection of Dex prior to the sacrifice of animals did not influence the nuclear binding to 3H-Dex-receptor complex in vitro. Differential salt-extraction with KCl solution of the nuclear bound 3H-Dex receptor complex revealed the presence of salt-extractable and residual forms of bound receptors. The amount of the fraction extracted with 0.3 M KCl reached its maximum at 10 min after the start of incubation, while the 1.0 M KCl-extractable and residual fractions reached their maximum plateaus after 30 min of the incubation. Scatchard analysis revealed that the binding of the receptor complex to the 0.3M and 1.0M KCl fractions was saturable, while the residual fraction did not show any tendency of saturation under the experimental conditions employed in the present study. The results obtained in this work were compared to those which have been reported by other investigators.     

Evidence for binding of metabolically activated 1,2-dibromoethane to chromatin of forestomach and liver

The covalent binding of metabolically activated 1,2-dibromoethane (DBE), a potent carcinogen, to chromatin constituents of forestomach and liver was examined $\text{in vitro}$. Chromatin was prepared from forestomach and liver of B6C3F1 mice and characterized. In order to activate DBE, microsomes and cytosol were isolated from mouse forestomach and liver and incubated with [¹⁴C]-DBE in the presence of a NADPH regenerating system. Results demonstrate that DBE bound covalently to the same extent to protein of microsomes and chromatin isolated from forestomach and liver. On the contrary, DBE bound significantly more to chromatin DNA of forestomach or liver than it did to salmon sperm DNA. It appears from these results that the metabolically activated DBE is more reactive to homologous DNA than exogenous DNA. Fractionation of DBE-bound chromatin protein into histone and nonhistone proteins resulted in higher binding of DBE to non-histone than to histone proteins isolated from forestomach and liver.     

Interaction of acetyl-CoA carboxylase from the rat liver with analogs of activated carbon dioxide and ATP

The interaction of rat liver Ac-CoA-carboxylase with reactive and stable analogs of carbon dioxide and phosphoric acid mixed anhydrides--hypothetic intermediate of the enzyme reaction--has been studied. Carbamoylphosphate showed substrate properties, whereas phosphonacetic acid and beta-oxopropyl-alpha, alpha-diphosphonate inhibited this enzyme (Ki 3.0 and 3.5 mM correspondingly). The analog of another possible intermediate in the reaction of ATP and carbon dioxide, Appp (CH2COOH) also inhibited Ac-CoA-carboxylase (Ki = 0.7 mM).