Interaction of steroids with the nuclear envelope

1986 ◽  
Vol 64 (6) ◽  
pp. 594-600 ◽  
Author(s):  
Y. A. Lefebvre ◽  
J. T. Venkatraman ◽  
E. J. Golsteyn ◽  
G. M. Howell
Keyword(s):  
Nuclear Envelope ◽  
Binding Site ◽  
Rat Liver ◽  
Mammary Carcinoma ◽  
Binding Sites ◽  
Ventral Prostate ◽  
Mouse Mammary Carcinoma ◽  
Rat Ventral Prostate ◽  
Affinity Labelling ◽  
Steroid Binding

Three approaches have been taken to determine the molecular mechanism by which steroid hormones traverse the nuclear envelope on their way to the genome. The first approach involved characterization of steroid binding to nuclear envelope preparations. We have characterized androgen binding to nuclear envelopes isolated from the rat ventral prostate, the rat liver, and androgen-responsive and androgen-unresponsive cell lines of the Shionogi mouse mammary carcinoma and glucocorticoid binding to rat liver. Relatively high affinity binding sites for steroids have been identified on nuclear envelopes. Importantly, the number and specificity of the sites correlates with the responsiveness of the tissue to the steroid. In the second approach, we have undertaken to identify the steroid binding site directly. As the characteristics of the rat ventral prostate site resembled those of the nuclear androgen receptor, we have begun purifying that receptor and have found fast protein liquid chromatography to be very effective. By affinity labelling studies, the dexamethasone binding site on the rat liver nuclear envelope has been identified as a peptide of molecular weight of approximately 90 000. The third approach we have used is to identify androgen-dependent peptides in nuclear envelope preparations. In both the rat ventral prostate and an androgen-responsive cell line of the Shionogi mouse mammary carcinoma, we have identified abundant androgen-dependent peptides. The relationship of these peptides to the binding sites identified by the first two approaches and their role in steroid transport is being investigated.

scholarly journals Binding of dihydrotestosterone to a nuclear-envelope fraction from the male rat liver

1982 ◽  
Vol 202 (1) ◽  
pp. 225-230 ◽  
Author(s):  
Y A Lefebvre ◽  
S J Morante
Keyword(s):  
Heat Treatment ◽  
Nuclear Envelope ◽  
Binding Site ◽  
Rat Liver ◽  
Binding Sites ◽  
Specific Binding ◽  
Male Rat ◽  
Scatchard Analysis ◽  
Female Rat ◽  
Similar Class

Intact nuclear ‘ghosts’ containing small amounts of DNA were obtained from rat liver. Incubation of radiolabelled dihydrotestosterone with isolated nuclear-envelope fraction from male rat liver resulted in specific binding of the dihydrotestosterone to the membranes. Optimal binding occurred at 20 degrees C after 20h incubation. Storage for 2 weeks at -80 degrees C resulted in little loss of specific binding. Scatchard analysis revealed a class of binding sites with a KD of 23.2 nM. Pronase and heat treatment destroyed the binding site. Androgens and glucocorticoids competed for labelled dihydrotestosterone binding to the ghosts, whereas oestrogens did not compete. Castration 24h before preparation of ghosts did not alter the binding site, and a similar class of binding sites was identified on female rat liver nuclear envelopes.

scholarly journals Binding of androgens to a nuclear-envelope fraction from the rat ventral prostate

1980 ◽  
Vol 186 (3) ◽  
pp. 641-647 ◽  
Author(s):  
Y A Lefebvre ◽  
Z Novosad
Keyword(s):  
Nuclear Envelope ◽  
Binding Sites ◽  
Specific Binding ◽  
Ventral Prostate ◽  
Scatchard Analysis ◽  
Rat Ventral Prostate ◽  
Membrane Isolation ◽  
Triton X ◽  
Androgen Binding ◽  
Plasma Membrane Isolation

A nuclear-envelope fraction was isolated from the rat ventral prostate which is virtually free of DNA and contains little RNA or plasma membrane. Isolation of this nuclear-envelope fraction after incubation of purified nuclei with radioactive dihydrotestosterone results in labelling of the membrane. More binding of dihydrotestosterone is observed after incubations at 22 degrees C for 17 h than at 4 degrees C for 17 h or at 22 degrees C for 60 min. Scatchard analysis revealed a class of binding sites with KD 8.4 nM. Dihydrotesterone and testosterone were almost equally effective as competitors of labelled dihydrotestosterone binding on the purified nuclear-envelope fraction, whereas diethylstilboestrol was less effective and dexamethasone did not compete well. When the outer membrane of the nuclei was removed with Triton X-100, a 24% decrease in specific binding of androgens was observed. Castration 24 h before preparation of nuclei resulted in loss of the androgen binding to the membrane.

scholarly journals Effects of dl-2-bromopalmitoyl-CoA and bromoacetyl-CoA in rat liver and heart mitochondria. Inhibition of carnitine palmitoyltransferase and displacement of [14C]malonyl-CoA from mitochondrial binding sites

1985 ◽  
Vol 230 (1) ◽  
pp. 169-179 ◽  
Author(s):  
M R Edwards ◽  
M I Bird ◽  
E D Saggerson
Keyword(s):  
Binding Site ◽  
Rat Liver ◽  
Binding Sites ◽  
Liver Mitochondria ◽  
Carnitine Palmitoyltransferase ◽  
Heart Mitochondria ◽  
Nitrobenzoic Acid ◽  
Malonyl Coa ◽  
Tissue Differences ◽  
The Relationship

The overt form of carnitine palmitoyltransferase (CPT1) in rat liver and heart mitochondria was inhibited by DL-2-bromopalmitoyl-CoA and bromoacetyl-CoA. S-Methanesulphonyl-CoA inhibited liver CPT1. The inhibitory potency of DL-2-bromopalmitoyl-CoA was 17 times greater with liver than with heart CPT1. Inhibition of CPT1 by DL-2-bromopalmitoyl-CoA was unaffected by 5,5′-dithiobis-(2-nitrobenzoic acid) or (in liver) by starvation. In experiments in which DL-2-bromopalmitoyl-CoA displaced [14C]malonyl-CoA bound to liver mitochondria, the KD (competing) was 25 times the IC50 for inhibition of CPT1 providing evidence that the malonyl-CoA-binding site is unlikely to be the same as the acyl-CoA substrate site. Bromoacetyl-CoA inhibition of CPT1 was more potent in heart than in liver mitochondria and was diminished by 5,5′-dithiobis-(2-nitrobenzoic acid) or (in liver) by starvation. Bromoacetyl-CoA displaced bound [14C]malonyl-CoA from heart and liver mitochondria. In heart mitochondria this displacement was competitive with malonyl-CoA and was considerably facilitated by L-carnitine. In liver mitochondria this synergism between carnitine and bromoacetyl-CoA was not observed. It is suggested that bromoacetyl-CoA interacts with the malonyl-CoA-binding site of CPT1. L-Carnitine also facilitated the displacement by DL-2-bromopalmitoyl-CoA of [14C]malonyl-CoA from heart, but not from liver, mitochondria. DL-2-Bromopalmitoyl-CoA and bromoacetyl-CoA also inhibited overt carnitine octanoyl-transferase in liver and heart mitochondria. These findings are discussed in relation to inter-tissue differences in (a) the response of CPT1 activity to various inhibitors and (b) the relationship between high-affinity malonyl-CoA-binding sites and those sites for binding of L-carnitine and acyl-CoA substrates.

scholarly journals Affinity labelling of the binding site of rabbit antibody. Evidence for the involvement of the hypervariable regions of the heavy chain

1974 ◽  
Vol 139 (1) ◽  
pp. 135-149 ◽  
Author(s):  
Christopher E. Fisher ◽  
Elizabeth M. Press
Keyword(s):  
Binding Site ◽  
Heavy Chain ◽  
Binding Sites ◽  
Light Chains ◽  
Rabbit Antibody ◽  
Heavy Chains ◽  
Group 4 ◽  
Hypervariable Regions ◽  
Affinity Labelling ◽  
Antibody Complex

The binding sites of rabbit antibodies with affinity for the haptenic group 4-azido-2-nitrophenyl-lysine have been specifically labelled by photolysis of the hapten–antibody complex. The extent of covalent labelling was 0.5–0.9mol of hapten bound/mol of antibody and, by using an immunoadsorbent, antibody with 1.3mol of hapten/mol was obtained. The antibody was specifically labelled in the binding site and the ratio of labelling of heavy and light chains was in the range 3.3–5.0. The labelled heavy chains were cleaved by CNBr treatment and after reduction and alkylation of the intrachain bonds, were digested with trypsin. Evidence is presented that two regions of the heavy chain, positions 29–34 and 95–114, together contain about 80% of the label on the heavy chain; these two regions respectively include two of the hypervariable regions of rabbit heavy chain.

Postnatal development of lectin binding sites in the rat ventral prostate

1989 ◽  
Vol 21 (11) ◽  
pp. 651-658 ◽  
Author(s):  
Ch. Hauke ◽  
R. Horn ◽  
W. Breuer ◽  
F. Sinowatz
Keyword(s):  
Postnatal Development ◽  
Binding Sites ◽  
Lectin Binding ◽  
Ventral Prostate ◽  
Rat Ventral Prostate ◽  
Lectin Binding Sites

Expression of androgen receptors and prostatic steroid-binding protein during development of the rat ventral prostate

1988 ◽  
Vol 117 (3) ◽  
pp. 361-NP ◽  
Author(s):  
Y. L. Zhang ◽  
Z. X. Zhou ◽  
Y. D. Zhang ◽  
M. G. Parker
Keyword(s):  
Gene Expression ◽  
Androgen Receptors ◽  
Binding Protein ◽  
Transient Increase ◽  
Neonatal Rat ◽  
Ventral Prostate ◽  
Rat Ventral Prostate ◽  
Old Rats ◽  
Steroid Binding ◽  
Steroid Binding Protein

ABSTRACT Prostatic steroid-binding protein (PSBP) mRNAs transcribed from the three genes C1, C2 and C3 were quantitated in neonatal rat ventral prostate by Northern blot analysis. Transcription was initiated at day 14 for C1 and C2 and day 10 for C3, and reached mature levels by day 21 for C1 and C2 and day 28 for C3. The changes of both cytoplasmic and nuclear prostatic androgen receptors in 10- to 150-day-old rats were investigated by radioligand assay and showed a fivefold transient increase between days 10 and 28. Thus there was a good correlation between the onset of PSBP gene expression and the transient increase in androgen receptors; increases in receptor concentration may be a prerequisite for changes in gene expression. J. Endocr. (1988) 117, 361–366

THE ROLE OF THE STEROID-SENSITIVE CATION-DEPENDENT ATPASE IN HUMAN PROSTATIC TISSUE

1972 ◽  
Vol 54 (3) ◽  
pp. 375-385 ◽  
Author(s):  
W. E. FARNSWORTH
Keyword(s):  
Reductase Activity ◽  
Benign Prostatic Hypertrophy ◽  
Enzymic Activity ◽  
Ventral Prostate ◽  
Hormonal Stimulation ◽  
Dependent Atpase ◽  
Rat Ventral Prostate ◽  
Steroid Sensitive ◽  
Steroid Binding

SUMMARY The purposes of this study were: (1) to determine if a (Na+ + K+)-dependent, ouabain-sensitive, androgen-responsive ATPase is present in the microsomal fraction of the human prostate as it is in the rat ventral prostate; (2) to determine the degree and nature of interaction and interdependence of the ATPase with the steroid-binding and steroid-metabolizing activities of the prostate and, also, with the histological characteristics of the gland. The results indicate that ATPase which shows particular responsiveness to 5α-dihydrotestosterone, 5α-androstane-3α,17β-diol and dehydroepiandrosterone is present. The microsomes, which contain this enzymic activity, show relatively high affinity for the active steroids and for oestradiol-17β. The intrinsic steroid-sensitive, cation-dependent ATPase activity varies widely from gland to gland in parallel with the steroid binding and 3α-hydroxysteroid dehydrogenase activity. In comparisons of the enzymatic complements of glands with different histological features, the concentration of 4-en-3-oxosteroid-5α-reductase activity in tissue showing predominantly epithelial hyperplasia was greater than that in normal or carcinomatous glands or glands with stromal hypertrophy. The possible role of the ATPase as a mediator of hormonal stimulation, and the implications of the findings to an understanding of the development of benign prostatic hypertrophy, are discussed.

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