Androgen receptor binding to nuclear matrix in vitro and its inhibition by 8S androgen receptor promoting factor

The partially purified 4.5S [3H]dihydrotestosterone receptor binds to nuclear matrix isolated from rat Dunning prostate tumor with properties similar to those reported for androgen receptor binding in intact nuclei [Colvard, D.S., & Wilson, E.M. (1984) Biochemistry (preceding paper in this issue)] in that it requires Zn2+ and mercaptoethanol, is saturable, and is temperature dependent and of high affinity (Ka approximately 10(13) M-1). On a milligrams of DNA equivalent basis, the extent of matrix binding of androgen receptor (700 fmol of receptor bound/mg of matrix protein) is similar to that of intact nuclei, corresponding to approximately 1400 sites/nucleus. Association rate constants (ka) for 4.5S androgen receptor binding to matrix at 0, 15, and 25 degrees C are 2.7 X 10(5), 1.2 X 10(6), and 2.4 X 10(6) M-1 min-1, respectively, indicating an energy of activation of 15 kcal/mol. Up to 50% of matrix-bound receptor is extractable in buffer containing 3 mM ethylenediaminetetraacetic acid plus either 0.4 M KCl or 5 mM pyridoxal 5'-phosphate. A protein fraction designated 8S androgen receptor promoting factor that promotes conversion of the 4.5S androgen receptor to 8 S [Colvard, D. S., & Wilson, E. M. (1981) Endocrinology (Baltimore) 109, 496-504] has been further purified and found to inhibit the binding of the 4.5S androgen receptor to isolated nuclei and nuclear matrix in a concentration-dependent manner. The results support the hypothesis that the 8S steroid receptor is a complex of the activated 4.5S androgen receptor with a non-steroid binding protein that renders the receptor incapable of binding in nuclei.     

Binding properties of androgen receptors. Evidence for identical receptors in rat testis, epididymis, and prostate.

Androgen receptors in crude and partially purified 105,000 X g supernatant fractions from rat testis, epididymis, and prostate were studied in vitro using a charcoal adsorption assay and sucrose gradient centrifugation. Androgen metabolism was eliminated during receptor purification allowing determination of the kinetics of [3H]-androgen-receptor complex formation. In all three tissues, receptors were found to have essentially identical capabilities to bind androgen, with the affinity for [3H] dihydrotestosterone being somewhat higher than for [3H] testosterone. Equilibrium dissociation constants for [3H] dihydrotestosterone and [3H] testosterone (KD = 2 to 5 X 10(-10) M) were estimated from independently determined rates of association (ka congruent to 6 X 10(7) M-1 h-1 for [3H] dihydrotestosterone and 2 X 10(8) M-1 h-1 for [3H] testosterone) and dissociation (t 1/2 congruent to 40 hr for [3H] dihydrotestosterone and 15 h [3H] testosterone). Evaluation of the effect of temperature on androgen receptor binding of [3H]testosterone allowed estimation of several thermodynamic parameters, including activation energies of association and dissociation (delta H congruent to 14 kcal/mol), the apparent free energy (delta G congruent to -12 kcal/mol), enthalpy (delta H congruent to -2.5 kcal/mol), and entropy (delta S congruent to 35 cal col-1 K-1). Optimum receptor binding occurred at a pH of 8. Receptor stability was greatly enhanced when bound with androgen. Receptor specificity for testosterone and dihydrotestosterone was demonstrated by competitive binding assays. The potent synthetic androgen, 7 alpha, 17 alpha-dimethyl-19-nortestosterone, inhibited binding of [3H] testosterone or [3H] dihydrotesterone nearly as well as testosterone and dihydrotestosterone while larger amounts of 5 alpha-androstane-3alpha, 17 beta-diol and nonandrogenic steroids were required. Sedimentation coefficients of androgen receptors in all unfractionated supernatants were 4 and 5 to 8 S. Differences in sedimentation coefficients were observed following (NH4)2SO4 precipitation which did not influence the binding properties of the receptors. These results, together with measurements of3alpha/beta-hydroxysteroid oxidoreductase activity in vitro, suggest that organ differences in receptor binding of [3H] dihydrotestosterone and [3H] testosterone in vivo result from relative differences in intracellular concentrations of these androgens rather than from differences in receptor affinities.     

Neutralization and binding of heparin by S protein/vitronectin in the inhibition of factor Xa by antithrombin III. Involvement of an inducible heparin-binding domain of S protein/vitronectin

The interference of the heparin-neutralizing plasma component S protein (vitronectin) (Mr = 78,000) with heparin-catalyzed inhibition of coagulation factor Xa by antithrombin III was investigated in plasma and in a purified system. In plasma, S protein effectively counteracted the anticoagulant activity of heparin, since factor Xa inhibition was markedly reduced in comparison to heparinized plasma deficient in S protein. Using purified components in the presence of heparin, S protein induced a concentration-dependent reduction of the inhibition rate of factor Xa by antithrombin III. This resulted in a decrease of the apparent pseudo-first order rate constant by more than 10-fold at a physiological ratio of antithrombin III to S protein. S protein not only counteracted the anticoagulant activity of commercial heparin but also of low molecular weight forms of heparin (mean Mr of 4,500). The heparin-neutralizing activity of S protein was found to be mainly expressed in the range 0.2-10 micrograms/ml of high Mr as well as low Mr heparin. S protein and high affinity heparin reacted with apparent 1:1 stoichiometry to form a complex with a dissociation constant KD = 1 X 10(-8) M as determined by a functional assay. As deduced from dot-blot analysis, direct interaction of radiolabeled heparin with S protein revealed a dissociation constant KD = 4 X 10(-8) M. Heparin binding as well as heparin neutralization by S protein increased significantly when reduced/carboxymethylated or guanidine-treated S protein was employed indicating the existence of a partly buried heparin-binding domain in native S protein. Radiolabeled heparin bound to the native protein molecule as well as to a BrCN fragment (Mr = 12,000) containing the heparin-binding domain as demonstrated by direct binding on nitrocellulose replicas of sodium dodecyl sulfate-polyacrylamide gels. Kinetic analysis revealed that the heparin neutralization activity of S protein in the inhibition of factor Xa by antithrombin III could be mimicked by a synthetic tridecapeptide from the amino-terminal portion of the heparin-binding domain. These data provide evidence that the heparin-binding domain of S protein appears to be unique in binding to heparin and thereby neutralizing its anticoagulant activity in the inhibition of coagulation factors by antithrombin III. The induction of heparin binding and neutralization may be considered a possible physiological mechanism initiated by conformational alteration of the S protein molecule.(ABSTRACT TRUNCATED AT 400 WORDS)     

Insulin-like growth factor II binding to the type I somatomedin receptor. Evidence for two high affinity binding sites

We have previously shown that the antireceptor antibody alpha IR-3 inhibits binding of 125I-somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) to the 130-kDa alpha subunit of the type I receptor in human placental membranes, but does not block 125I-insulin-like growth factor II (IGF-II) binding to a similar 130-kDa complex in these membranes. To determine whether the 130-kDa 125I-IGF-II binding complex represents a homologous receptor or whether 125I-IGF-II binds to the type I receptor at a site that is not blocked by alpha IR-3, type I receptors were purified by affinity chromatography on Sepharose linked alpha IR-3. The purified receptors bound both 125I-Sm-C/IGF-I and 125I-IGF-II avidly (KD = 2.0 X 10(-10) M and 3.0 X 10(-10) M, respectively). The maximal inhibition of 125I-Sm-C/IGF-I binding by the antibody, however, was 62% while only 15% of 125I-IGF-II binding was inhibited by alpha IR-3. In the presence of 500 nM alpha IR-3, Sm-C/IGF-I bound with lower affinity (KD = 6.5 X 10(-10) M) than IGF-II (KD = 4.5 X 10(-10) M) and IGF-II was the more potent inhibitor of 125I-Sm-C/IGF-I binding. These findings suggest that the type I receptor contains two different binding sites. The site designated IA has highest affinity for Sm-C/IGF-I and is blocked by alpha IR-3. Site IB has higher affinity for IGF-II than for Sm-C/IGF-I and is not blocked by alpha IR-3.     

Binding sites for alpha-bungarotoxin and the noncompetitive inhibitor phencyclidine on a synthetic peptide comprising residues 172-227 of the alpha-subunit of the nicotinic acetylcholine receptor.

The binding of the competitive antagonist alpha-bungarotoxin (alpha-Btx) and the noncompetitive inhibitor phencyclidine (PCP) to a synthetic peptide comprising residues 172-227 of the alpha-subunit of the Torpedo acetylcholine receptor has been characterized. 125I-alpha-Btx bound to the 172-227 peptide in a solid-phase assay and was competed by alpha-Btx (IC50 = 5.0 x 10(-8) M), d-tubocurarine (IC50 = 5.9 X 10(-5)M), and NaCl (IC50 = 7.9 x 10(-2)M). In the presence of 0.02% sodium dodecyl sulfate, 125I-alpha-Btx bound to the 56-residue peptide with a KD of 3.5 nM, as determined by equilibrium saturation binding studies. Because alpha-Btx binds to a peptide comprising residues 173-204 with the same affinity and does not bind to a peptide comprising residues 205-227, the competitive antagonist and hence agonist binding site lies between residues 173 and 204. After photoaffinity labeling, [3H]PCP was bound to the 172-227 peptide. [3H]PCP binding was inhibited by chlorpromazine (IC50 = 6.3 x 10(-5)M), tetracaine (IC50 = 4.2 x 10(-6)M), and dibucaine (IC50 = 2.7 x 10(-4)M). Equilibrium saturation binding studies in the presence of 0.02% sodium dodecyl sulfate showed that [3H]PCP bound at two sites, a major site of high affinity with an apparent KD of 0.4 microM and a minor low-affinity site with an apparent KD of 4.6 microM. High -affinity binding occurred at a single site on peptide 205-227 (KD = 0.27 microM) and was competed by chlorpromazine but not by alpha-Btx.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rat Brain S100b Protein: Purification, Characterization, and Ion Binding Properties. A Comparison with Bovine S100b Protein

We purified to homogeneity rat brain S100b protein, which constitutes about 90% of the soluble S100 protein fraction. Purified rat S100b protein comigrates with bovine S100b protein in nondenaturant system electrophoresis but differs in its amino acid composition and in its electrophoretic mobility in urea-sodium dodecyl sulfate-polyacrylamide gel with bovine S100b protein. The properties of the Ca2+ and Zn2+ binding sites on rat S100b protein were investigated by flow dialysis and by fluorometric titration, and the conformation of rat S100b in its metal-free form as well as in the presence of Ca2+ or Zn2+ was studied. The results were compared with those obtained for the bovine S100b protein. In the absence of KCl, rat brain S100b protein is characterized by two high-affinity Ca2+ binding sites with a KD of 2 X 10(-5) M and four lower affinity sites with KD about 10(-4) M. The calcium binding properties of rat S100b protein differ from bovine S100b only by the number of low-affinity calcium binding sites whereas similar Ca2+-induced conformational changes were observed for both proteins. In the presence of 120 mM KCl rat brain S100b protein bound two Zn2+-ions/mol of protein with a KD of 10(-7) M and four other with lower affinity (KD approximately equal to 10(-6) M). The occupancy of the two high-affinity Zn2+ binding sites was responsible for most of the Zn2+-induced conformational changes in the rat S100b protein. No increase in the tyrosine fluorescence quantum yield after Zn2+ binding to rat S100b was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of [3H]methyltrienolone to androgen receptor in rat liver

The synthetic androgen methyltrienolone is superior to testosterone and androstenedione for the measurement of androgen receptor in tissues where the native ligands are metabolized into inactive derivatives. [3H]Methyltrienolone binds with a high affinity to androgen receptor in cytosol prepared from male rat livers, as the Scatchard analysis revealed that the Kd value was 3.3 X 10(-8) M and the number of binding sites was 35.5 fmol/mg protein. Since methyltrienolone also binds glucocorticoid receptor which exists in rat liver, the apparent binding of androgen receptor is faulty when measured in the presence of glucocorticoid receptor. The binding of methyltrienolone to glucocorticoid receptor can be blocked by the presence of a 100-fold molar excess of unlabeled synthetic glucocorticoid, triamcinolone acetonide, without interfering in its binding to androgen receptor, because triamcinolone does not bind to androgen receptor. Triamcinolone-blocked cytosol exhibited that the Kd value was 2.5 X 10(-8) M and the number of binding sites was 26.3 fmol/mg protein, indicating a reduction to 3/4 of that in the untreated cytosol. The profile of glycerol gradient centrifugation indicated that [3H]methyltrienolone-bound receptor migrated in the 8-9 S region in both untreated and triamcinolone-blocked cytosols, but the 8-9 S peak in triamcinolone-blocked cytosol was reduced to about 3/4 of that of untreated cytosol.     

Binding of human blood-coagulation Factors IXa and X to phospholipid membranes.

A simple centrifugation technique has been developed to study the interaction of human coagulation Factors IXa and X with phospholipid membranes. In the presence of Ca2+, equimolar phosphatidylserine/phosphatidylcholine membranes form tight complexes with Factor X (KD = 2.8 X 10(-8) M); the KD is independent of the phospholipid concentration. Binding sites are available for about 2 mmol of Factor X/mol of phospholipid. Factor IXa has a slightly higher affinity for the phospholipid membrane (KD = 1.2 X 10(-8)M), and competes with Factor X for binding. The experimentally observed competition between Factor X and Factor IXa is in agreement with a model that describes the binding of two distinct ligands to a single class of independent binding sites.     

Characterization of a cyclic AMP-resistant Chinese hamster ovary cell mutant containing both wild-type and mutant species of type I regulatory subunit of cyclic AMP-dependent protein kinase

We have characterized a cyclic AMP-resistant Chinese hamster ovary (CHO) cell mutant in which one of two major species of type I regulatory subunit (RI) of cyclic AMP-dependent protein kinase is altered. Wild-type CHO cell extracts contain two cyclic AMP-dependent protein kinase activities. As shown by DEAE-cellulose chromatography, there is a peak of type I protein kinase activity in mutant extracts, but the type II protein kinase activity is considerably reduced even though free type II regulatory subunit (RII) is present. The type I kinase from the mutant has an altered RI (RI*) whose KD for the binding of 8-N3[32P] cAMP (KD = 1.3 X 10(-5) M) is increased by more than 200-fold compared to RI from the wild-type enzyme (KD = 5.5 X 10(-8) M). No differences were found between the catalytic subunits from the wild-type and mutant type I kinases. A large portion of RI in mutant and wild-type extracts is present in the free form. The RI* derived from mutant type I protein kinase shows altered labeling by 8-N3[32P]cAMP (KD = 1.3 X 10(-5) M) whereas the free RI from the mutant is labeled normally by the photoaffinity label (KD = 7.2 X 10(-8) M), suggesting that the RI* which binds to the catalytic subunit is functionally different from the free form of RI. The decreased amount of type II kinase activity in the mutant appears to be due to competition of RI* with RII for binding to the catalytic subunit. Translation of mRNA from wild-type CHO cells results in the synthesis of two different charge forms of RI, providing biochemical confirmation of two different species of RI in CHO cells. Additional biochemical evidence based on isoelectric focusing behavior of 8-N3[32P]cAMP-labeled RI species and [35S]methionine-labeled RI from mutant and wild-type extracts confirms the charge heterogeneity of RI species in CHO cells. These genetic and biochemical data taken together are consistent with the conclusion that there are at least two different species of RI present in CHO cells and that one of these species is altered in the mutant analyzed in this work.     

Monoclonal antibodies specific for each of the two toxin-binding sites of Torpedo acetylcholine receptor

We have isolated and characterized 12 monoclonal antibodies (mAbs) that block the binding of alpha-bungarotoxin (alpha-BuTx) to the acetylcholine receptor (AChR) of Torpedo californica. Two of the mAbs block alpha-BuTx binding completely; the other 10 inhibit only about 50% of the binding. The mAbs that partially inhibit alpha-BuTx binding can be divided into two groups by examination of the additive effect of pairs of mAbs on toxin binding, and by analysis of competition between mAbs for binding to the AChR. These two groups of mAbs, which we have termed A and B, appear to recognize different toxin-binding sites on the same receptor. A and B mAbs were used to determine the kinetic and pharmacological properties of the two sites. The site recognized by A mAbs binds alpha-BuTx with a forward rate constant of 0.98 X 10(5) M-1 s-1, d-tubocurarine (dTC) with a KD of (6.8 +/- 0.3) X 10(-8) M, and pancuronium with a KD of (1.9 +/- 1.0) X 10(-9) M. The site recognized by B mAbs binds alpha-BuTx with a forward rate constant of 9.3 X 10(5) M-1 s-1, dTC with a KD of (4.6 +/- 0.3) X 10(-6) M, and pancuronium with a KD of (9.3 +/- 0.8) X 10(-6) M. Binding of A and B mAbs to the AChR was variably inhibited by nicotinic cholinergic agonists and antagonists, and by alpha-conotoxin. The observed pattern of inhibition is consistent with the relative affinity of the two sites for antagonists as given above but also indicates that the mAbs recognize a diversity of epitopes within each site.     

6-Nitro-L-tryptophan: a novel spectroscopic probe of trp aporepressor and human serum albumin

The binding of 6-nitro-L-tryptophan to trp aporepressor and human serum albumin has been examined by visible difference spectroscopy and circular dichroism. 6-Nitro-L-tryptophan, prepared by nitration of L-tryptophan with nitric acid in glacial acetic acid, exhibits a visible and near-uv absorption spectrum with lambda max at about 330 nm (epsilon = 7 X 10(3) M-1 cm-1) and a shoulder near 380 nm in H2O. In the presence of trp aporepressor, the visible absorption intensity is sharply diminished. Visible difference spectral titration data give KD = 1.27 X 10(-4) M and n = 0.95 per subunit at 25 degrees C. While 6-nitro-L-tryptophan exhibits no significant circular dichroism between 300 and 500 nm, the complex with trp aporepressor exhibits strong circular dichroism signals, with a negative maximum at 386 nm (delta epsilon = -7.5 M-1 cm-1) and a positive maximum at 310 nm (delta epsilon = +6 M-1 cm-1). Circular dichroism titration data give KD = 1.69 X 10(-4) M and n = 0.90 per subunit at 25 degrees C. The KD values determined spectroscopically are in excellent agreement with that determined by equilibrium dialysis, KD = 1.5 X 10(-4) M at 25 degrees C. In the presence of human serum albumin, the spectrum of 6-nitro-L-tryptophan exhibits a blue shift and an increase in absorption intensity; similar changes are observed in solvents of low dielectric contrast such as 80% aqueous dioxane. Visible difference spectral titration data give KD = 8.0 X 10(-5) M and n = 0.95 for human serum albumin. The complex of 6-nitro-L-tryptophan with human serum albumin exhibits a strong positive circular dichroism maximum at 380 nm (delta epsilon = +9.8 M-1 cm-1) with a shoulder at 310-320 nm. Circular dichroism titration data give KD = 6.4 X 10(-5) M and n = 0.83, in good agreement with the visible difference spectral results. Taken together, our results demonstrate the utility of 6-nitro-L-tryptophan as a spectroscopic probe for tryptophan-binding proteins.     

Purification of human C4b-binding protein and formation of its complex with vitamin K-dependent protein S.

C4b-binding protein was purified from human plasma in high yield by a simple procedure involving barium citrate adsorption and two subsequent chromatographic steps. Approx. 80% of plasma C4b-binding protein was adsorbed on the barium citrate, presumably because of its complex-formation with vitamin K-dependent protein S. The purified C4b-binding protein had a molecular weight of 570 000, as determined by ultracentrifugation, and was composed of about eight subunits (Mr approx. 70 000). Uncomplexed plasma C4b-binding protein was purified from the supernatant after barium citrate adsorption. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in non-reducing conditions and on agarose-gel electrophoresis it appeared as a doublet, indicating two forms differing slightly from each other in molecular weight and net charge. The protein band with the higher molecular weight in the doublet corresponded to the C4b-binding protein purified from the barium citrate eluate. Complex-formation between protein S and C4b-binding protein was studied in plasma, and in a system with purified components, by an agarose-gel electrophoresis technique. Protein S was found to form a 1:1 complex with the higher-molecular-weight form of C4b-binding protein, whereas the lower-molecular-weight form of C4b-binding protein did not bind protein S. The KD for the C4b-binding protein-protein S interaction in a system with purified components was approx. 0.9 X 10(-7) M. Rates of association and dissociation at 37 degrees C were low, namely about 1 X 10(3) M-1 . S-1 and 1.8 X 10(-4)-4.5 X 10(-4) S-1 respectively. In human plasma free protein S and free higher-molecular-weight C4b-binding protein were in equilibrium with the C4b-binding protein-protein S complex. Approx. 40% of both proteins existed as free proteins. From equilibrium data in plasma a KD of about 0.7 X 10(-7) M was calculated for the C4b-binding protein-protein S interaction.     

Binding of the rat liver 7-8 S dexamethasone receptor to deoxyribonucleic acid

The 7-8 S form of the [3H]dexamethasone (9 alpha-fluoro-11 beta,17,21-trihydroxy-16 alpha-methylpregna-1,4-diene-3, 20-dione) receptor from rat liver cytosol can be converted to the 3-4 S form by RNase treatment or high salt, suggesting a salt-sensitive association between the receptor protein and RNA. In DNA-cellulose column assays, the gradient-purified 3-4 S form bound DNA more efficiently than the 7-8 S form, though the 7-8 S form was also capable of binding to DNA-cellulose to a significant extent. Activated 7-8 S dexamethasone receptor could be released from its association with soluble DNA by treatment with DNase I. Sucrose gradient analysis showed that the released receptor sedimented as the 7-8 S form and was sensitive to RNase treatment, which induced a conversion to the 3-4 S form. Activated RNase-generated 3-4 S receptor again displayed a higher degree of binding to soluble DNA and was recovered in the 3-4 S form following DNase extraction. The fact that the 3-4 S form bound immobilized or soluble DNA more efficiently suggests that the associated RNA of the 7-8 S form interferes directly or indirectly with the receptor association with DNA. The observation that the receptor binds to DNA in its 7-8 S form suggests that the receptor complex is capable of binding RNA and DNA concurrently.     

Interaction of noncompetitive inhibitors with the acetylcholine receptor. The site specificity and spectroscopic properties of ethidium binding

The spectroscopic properties and specificity of binding of a fluorescent quaternary amine, ethidium, with acetylcholine receptor-enriched membranes from Torpedo californica have been examined. Competition binding with [3H]phencyclidine in the presence of carbamylcholine showed that ethidium binds with high affinity to a noncompetitive inhibitor site (KD = 3.6 X 10(-7) M). However, in the presence of alpha-toxin, ethidium's affinity is substantially lower (KD approximately 1 X 10(-3) M). Ethidium was also found to enhance [3H]acetylcholine binding with a KD characteristic of ethidium binding to a high-affinity noncompetitive inhibitor site. These findings indicate that ethidium binds to an allosteric site which is regulated by agonist binding and can convert the agonist sites from low to high affinity. Fluorescence titrations of ethidium in the presence of carbamylcholine yielded a similar KD (2.5 X 10(-7) M) and showed an ethidium stoichiometry of one site/acetylcholine receptor monomer. Ethidium was completely displaced by noncompetitive inhibitors such as phencyclidine, histrionicotoxin, and dibucaine. The enhanced fluorescence lifetime of the bound species showed that the increased fluorescence intensity reflects a 13-fold increase in quantum yield for the complex compared to ethidium in buffer. Fractional dissociation of ethidium with phencyclidine produced a double-exponential fluorescence decay rate with lifetime components characteristic of ethidium free in solution and bound to the receptor. These data argue that the alterations in ethidium fluorescence elicited by other ligands is due to a change in the fraction of specifically bound ethidium rather than a change in quantum yield of a pre-existing ethidium-acetylcholine receptor complex. The extent of polarization indicates that bound ethidium is strongly immobilized. The magnitude of the quantum yield enhancement and the shifts of excitation and emission maxima of bound ethidium suggest that its binding site is within a hydrophobic domain with limited accessibility to the aqueous phase.     

Modulation of 5-Hydroxytryptamine1A Receptor Density by Nonhydrolyzable GTP Analogues

Co-incubation of rat cortical membranes with 10(-4) M GTP results in a competitive inhibition of 5-hydroxytryptamine1A (5-HT1A) receptor binding sites labeled by [3H]8-hydroxy-2-(di-n-propylamino)tetralin [( 3H]8-OH-DPAT). Preincubation of cortical membranes with 10(-4) M GTP does not significantly change either KD or Bmax values, indicating that the effect of GTP is reversible. By contrast, GTP gamma S and 5'-guanylylimidodiphosphate (GppNHp) are nonhydrolyzable analogues of GTP which lengthen the time course of guanine nucleotide activation of guanine nucleotide binding proteins (G proteins) and thereby alter G protein-receptor interactions. These nonhydrolyzable GTP analogues were used to characterize the effects of persistent alterations in G proteins on [3H]8-OH-DPAT binding to 5-HT1A receptors. Co-incubation of rat cortical membranes with either 10(-4) M GTP gamma S or GppNHp results in a decrease in both the affinity and apparent density of 5-HT1A binding sites. Co-incubation with the nonhydrolyzable nucleotides reduces the affinity of [3H]8-OH-DPAT binding by 65-70% and lowers the density of the binding site by 53-61%. Similarly, preincubation of membranes with a 10(-4) M concentration of either GTP gamma S or GppNHp significantly increases the KD value and reduces the Bmax value of [3H]8-OH-DPAT binding. These results indicate that GTP gamma S and GppNHp induce persistent changes in 5-HT1A receptor-G protein interactions that are reflected as a decrease in the density of binding sites labeled by [3H]8-OH-DPAT.     

Effects of proteases and protease inhibitors on the 4.5 S and 8 S androgen receptor.

The size of androgen receptors from rat ventral and dorsal prostate, dorsal prostate (Dunning) tumor, testis, epididymis, and seminal vesicle was determined using Sephadex G-200 chromatogrpahy and sucrose gradient centrifugation. The protease inhibitor diisopropyl fluorophosphate (DFP) was used to minimize receptor breakdown. An 8-9 S, 85 to 106 A receptor (Mr = 280,000 to 365,000; f/fo = 1.9 to 2.4) observed in unfractionated cytosol prepared in low ionic strength buffer with or without DFP is in equilibrium with a 4.5-5 S, 58 A form (Mr = 117,000; f/fo = 1.8) observed at salt concentrations greater than 0.1 M KCl. Receptor partially purified using (NH4)2SO4 or phosphocellulose chromatography in the absence of DFP was present as smaller fragments of 3.6 S, 37 A and 3.0 S, 23 A. Similar fragments could be generated from the 4.5 S or 8 S receptor by mild trypsin treatment. In addition, ventral prostate contains a DFP-insensitive enzyme which specifically converts the 4.5 S, 58 A receptor to the 3.6 S 37 A fragment. The DFP-insensitive enzyme is partially inhibited by rabbit bile and appears similar to the enzyme seminin, a secretory protein of human prostate. Androgen receptor isolated in the presence of DFP from nuclei labeled in vivo is predominantly 4.5 S, 58 A, with smaller forms (37 and 23 A) appearing in the absence of DFP. The 4.5 S, 58 A nuclear receptors were also in equilibrium with a large 8 S form. Receptor breakdown by DFP-insensitive and sensitive proteases appears to be an in vitro phenomenon. Furthermore, the size of the androgen receptor is not significantly changed during receptor migration from cytoplasm to nucleus.     

Recognition of 16S rRNA by ribosomal protein S4 from Bacillus stearothermophilus

Protein S4 is essential for bacterial small ribosomal subunit assembly and recognizes the 5' domain (approximately 500 nt) of small subunit rRNA. This study characterizes the thermodynamics of forming the S4-5' domain rRNA complex from a thermophile, Bacillus stearothermophilus, and points out unexpected differences from the homologous Escherichia coli complex. Upon incubation of the protein and RNA at temperatures between 35 and 50 degrees C under ribosome reconstitution conditions [350 mM KCl, 8 mM MgCl2, and 30 mM Tris (pH 7.5)], a complex with an association constant of > or = 10(9) M(-1) was observed, more than an order of magnitude tighter than previously found for the homologous E. coli complex under similar conditions. This high-affinity complex was shown to be stoichiometric, in equilibrium, and formed at rates on the order of magnitude expected for diffusion-controlled reactions ( approximately 10(7) M(-1) x s(-1)), though at low temperatures the complex became kinetically trapped. Heterologous binding experiments with E. coli S4 and 5' domain RNA suggest that it is the B. stearothermophilus S4, not the rRNA, that is activated by higher temperatures; the E. coli S4 is able to bind 5' domain rRNA equally well at 0 and 37 degrees C. Tight complex formation requires a low Mg ion concentration (1-2 mM) and is very sensitive to KCl concentration [- partial differential[log(K)]/partial differential(log[KCl]) = 9.3]. The protein has an unusually strong nonspecific binding affinity of 3-5 x 10(6) M(-1), detected as a binding of one or two additional proteins to the target 5' domain RNA or two to three proteins binding a noncognate 23S rRNA fragment of the approximately same size. This binding is not as sensitive to monovalent ion concentration [- partial differential[log(K)]/partial differential(log[KCl]) = 6.3] as specific binding and does not require Mg ion. These findings are consistent with S4 stabilizing a compact form of the rRNA 5' domain.     

Ribonucleic acid association with androgen receptor from rat submandibular gland

The transformed androgen receptor from rat submandibular gland converts to a faster sedimenting form (6-8S) on a glycerol gradient centrifugation after withdrawal of a transformation-inducing reagent (KCl or ATP). In this report, the association of cytosolic RNA with the transformed androgen receptor was investigated as a possible mechanism of molecular conversion of the androgen receptor. When the transformed and converted androgen receptors were treated with RNase A, these receptors sedimented at 4.5S in a low-salt glycerol gradient. Addition of RNA from rat submandibular gland to the RNase-Sepharose-treated transformed receptor caused a shift of receptor peak from 4.5S to 5.8S. RNA from rat submandibular gland, yeast RNA and E. coli rRNA inhibited DNA-cellulose binding of a RNase-treated transformed receptor in the absence of molybdate. These observations suggest that conversion from the transformed 4S androgen receptor to a 6-8S form resulted from the association of RNA(s) with the transformed receptor.     

Interaction of leucine with the plasma membranes of catfish taste buds

The binding of L-[3H]leucine by the plasma membranes from the catfish Ictalurus nebulosus taste organ was studied. The two types of specific binding centers for amino acid with high (KD = 2,5 X 10(-10) M) and low (KD = 1,04 X 10(-9) M) affinities were found. Concentrations of high and low affinity centers were 11,85 nmol/mg and 26 nmol/mg respectively. The structural rearrangement of the surface layer of the chemoreceptor membrane was revealed by spin label technique using 5-doxylstearic acid at leucine concentrations 10(-4)-10(-9) M.     

Polypeptide halomethyl ketones bind to serine proteases as analogs of the tetrahedral intermediate. X-ray crystallographic comparison of lysine- and phenylalanine-polypeptide chloromethyl ketone-inhibited subtilisin.

1. A detailed study of cytochrome C oxidse activity with Keilin-Hartree particles and purified beef heart enzyme, at low ionic strength and low cytochrome C concentrations, showed biphasic kinetics with apparent Km1 = 5 x 10(-8) M, and apparent Km2 = 0.35 to 1.0 x 10(-6) M. Direct binding studies with purified oxidase, phospholipid-containing as well as phospholipid-depleted, demonstrated two sites of interaction of cytochrome c with the enzyme, with KD2 less than or equal to 10(-7) M, and KD2 = 10(-6) M. 2...