Yvonne A. Lefebvre

Diet fat alters the structure and function of the nuclear envelope: Modulation of membrane fatty acid composition, NTPase activity and binding of triiodothyronine

Mice were fed a diet either high or low in P/S ratio to determine the effect of altering dietary lipid on the fatty acid composition of liver nuclear envelopes and thereby on functions of the nuclear envelope. Mice fed the high P/S diet exhibited higher levels of C18:2 omega 6 and unsaturates in liver nuclear envelopes, higher specific activity of NTPase and specific binding for L-triiodothyronine at 15 degrees C and 22 degrees C compared with the low P/S diet fed group. These observations indicate that diet-induced differences in the fatty acid composition of nuclear envelope lipid affects functions of the nuclear envelope.

Identification of an actively phosphorylated component of adrenal medulla chromaffin granules

Abstract Adrenal chromaffin granules incubated with ATP rapidly incorporate the terminal phosphate into diphosphoinositide, resulting in a net increase in the amount of this lipid. The formation of diphosphoinositide has been demonstrated by thin-layer, paper and ionexchange chromatography. The formation of a phosphorylated non-lipid component has been confirmed.

Multiple thyroid hormone binding sites on rat liver nuclear envelopes

Nuclear envelopes relatively free of plasma membrane contamination were isolated from the male rat liver. Equilibrium binding of T3 to nuclear envelopes occurred after incubation for 3 h at 20 degrees C. Scatchard analysis revealed two classes of binding sites; a high affinity site having a KD of 1.8 nM with a maximum binding capacity of 14.5 pmol/mg protein and a low affinity site having a KD of 152.1 nM with a maximum binding capacity of 346.8 pmol/mg protein. No degradation of the radioligand occurred during incubation with the nuclear envelope. T4, rT3 and Triac competed effectively for the binding of T3 to the high affinity site whereas only T4 competed well for binding to the lower affinity site. The binding site was protease sensitive but not salt extractable. Multiple T3 binding sites having similar affinities have been reported on plasma membranes. An intriguing possibility is that membrane binding sites may be involved in translocation of thyroid hormone across membrane barriers.

Effect of sulfhydryl and disulfide agents on 3β and 17β-hydroxysteroid dehydrogenase and on steroid uptake ofPseudomonas testosteroni

Abstract Uptake of testosterone and the activity of the 3β and 17β-hydroxysteroid dehydrogenase of membrane vesicles ofPseudomonas testosteroni were similarly inhibited by p-hydroxymercuribenzoate (PHMB), N-ethylmaleimide, ZnSO4 and CuSO4. The inhibition of 3β and 17β-hydroxysteroid dehydrogenase by PHMB could be reversed with dithiothreitol, whereas the inhibition of testosterone uptake could not be reversed. The transport process was inhibited by dithiothreitol, cysteine and glutathione. These results indicated that the 3β and 17β-hydroxysteroid dehydrogenase was involved in steroid uptake by membrane vesicles ofP. testosteroni and, a disulfide bond present on a transport component, distinct from dehydrogenase activity, was also required.

Membrane-bound dehydrogenases of Pseudomonas testosteroni

Abstract During uptake of testosterone by membrane vesicles prepared from Pseudomonas testosteroni , NADH produced by 3β- and 17β-hydroxy steroid dehydrogenase is generated inside the vesicles. The membrane vesicles have been shown to possess an NADH dehydrogenase which appears to be located on the inner face of vesicle membranes and of whole cell cytoplasmic membranes. This suggests that the orientation of the membrane vesicles is the same as the cells from which they were derived. A membrane-bound 1-ene-dehydrogenase activity has also been demonstrated. 1-ene-Dehydrogenase activity of membrane vesicles was demonstrable both in the presence and absence of NAD + . In the absence of NAD + , testosterone was converted to 1-dehydrotestosterone. In the presence of NAD + testosterone was converted initially to androstenedione by 3β- and 17β-hydroxysteroid dehydrogenase activity and subsequently dehydrogenated to 1,4-androstadiendione.

The effects of specific inhibitors and an antiserum of 3β and 17β-hydroxysteroid dehydrogenase on steroid uptake in Pseudomonas testosteroni

Uptake of testosterone by membrane vesicles and membrane bound 3β and 17β-hydroxysteroid dehydrogenase activity of Pseudomonas testosteroni were inhibited by diethylstilbestrol, estradiol-17α, hydroxymethylenesteroid and cyanoketosteroid. An antibody raised against purified 3β and 17β-hydroxy-steroid dehydrogenase inhibited the activity of the membrane vesicle enzyme and uptake of testosterone. Accessibility of the antibody to the enzyme on the membrane vesicles indicated that the enzyme is located on the outer surface of the vesicle membrane.

The involvement of the electron transport chain in uptake of testosterone by membrane vesicles of Pseudomonas testosteroni.

Abstract Uptake of testosterone by membrane vesicles of Pseudomonas testosteroni is inhibited when examined under anaerobic conditions and in the presence of the electron transport chain inhibitors, sodium amytal and antimycin A. These results indicated the involvement of the electron transport chain in steroid uptake by membrane vesicles.

Characterization of high affinity and low affinity dexamethasone binding sites on male rat liver nuclear envelopes

Steroids must traverse the nuclear envelope before exerting their action at the chromatin. However, few studies have been done to elucidate the mechanism by which steroids traverse this membrane barrier. As first steps towards investigating the mechanism, we have characterized the binding sites for dexamethasone on male rat liver nuclear envelopes. The nuclear envelopes, prepared in the presence of dithiothreitol, were isolated from purified nuclei after treatment with DNase 1 at high pH. Binding of dexamethasone to the nuclear envelopes was measured after 16 h of incubation at 0-4 degrees C. At pH 7.4, only a single high capacity, low affinity binding site for dexamethasone was identified. However, at pH 8.6, two sites were identified; a low capacity, high affinity site and a high capacity, low affinity site. Adrenalectomy of the animal before preparation of the membranes caused loss of the high affinity site and reduction in the number of the lower affinity sites. Acute dexamethasone treatment of adrenalectomized rats resulted in the reappearance of the high affinity site but long term treatment with dexamethasone was required for complete restoration of the high affinity sites and reappearance of any of the low affinity sites. The steroid specificity of these nuclear envelope binding sites was different from that of the cytosolic glucocorticoid receptor, generally showing broader specificity. However, triamcinolone acetonide, which is a potent competitor for binding to the glucocorticoid receptor, did not complete effectively. The binding sites were sensitive to protease treatment and salt extraction studies revealed that the dexamethasone binding sites do not represent proteins non-specifically bound to the nuclear envelope. The affinity and the hormone responsiveness of the high affinity site are similar to those of the nuclear glucocorticoid receptor. Therefore, the nuclear envelope may be a site of action of glucocorticoids.

Androgen interactions with intact nuclear envelopes from the rat ventral prostate

Intact nuclear envelopes containing nuclear pore complexes have been prepared from the rat ventral prostate. The polypeptide profile of the nuclear envelopes from the rat prostate resembled that of nuclear envelopes prepared from the male rat liver. Isolation of the nuclear envelopes after incubation of purified nuclei with radioactive dihydrotestosterone results in labelling of the membrane. More dihydrotestosterone is bound after incubations at 22 degrees C for 18 h than at 2 degrees C for 18 h or 22 degrees C for 2 h. Scatchard analysis revealed a class of binding sites with an apparent Kd of 46 nM. Dihydrotestosterone, testosterone, cyproterone acetate and methyltrienolone were effective as competitors of labelled dihydrotestosterone binding to the nuclear envelopes, while estradiol did not compete. Castration of the rats 24, 48 and 96 h prior to preparation of nuclei resulted in loss of androgen binding to the membranes. Extraction with 0.6 M NaCl resulted in the loss of 72% of the androgen binding.

Correlation of androgen-responsiveness of Shionogi mouse mammary carcinoma cell lines with binding of dihydrotestosterone to nuclear envelopes

Purified nuclear envelopes have been isolated from an androgen-responsive and two androgen-unresponsive cell lines of the Shionogi mouse mammary carcinoma. The binding of dihydrotestosterone to nuclear envelope fractions isolated from the three variant cell lines is correlated with the androgen-responsiveness of the cell line. Nuclear envelopes prepared from the two androgen-unresponsive cell lines did not bind dihydrotestosterone specifically following incubation with radioactive dihydrotestosterone from 2.5 to 45.0 nM at 20 degrees C for 18 h. Under the same binding conditions, nuclear envelopes prepared from the androgen-responsive cell line demonstrated saturable, specific binding of dihydrotestosterone. Scatchard analysis revealed a class of binding sites with an apparent Kd of 14.2 nM and a maximum binding capacity of 28.7 fmol/mg protein. Proteinase and heat treatments resulted in the complete loss of androgen-binding activity, whereas DNAase treatment resulted in the loss of 38% of the binding activity. The binding sites were specific for dihydrotestosterone. Testosterone was only a weak competitor and estradiol did not compete. Extraction with concentrations of KCl up to 1.0 M did not result in loss of androgen binding.