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T A Bramley

Publications and source records attributed to T A Bramley.

At least 55 records · Page 3Linked to original sources
PubMed1991

Characterization of a factor(s) from partially purified human gonadotrophin preparations which inhibit(s) the binding of radiolabelled human LH and human chorionic gonadotrophin to Candida albicans.

We have shown previously that partially purified human chorionic gonadotrophin (hCG) preparations inhibited the specific binding of 125I-labelled hLH or hCG to Candida albicans membranes at much lower concentrations than did highly purified hLH or hCG preparations. We now describe the characterization and partial purification of a heat-labile glycoprotein from commercially available gonadotrophin preparations. The factor strongly inhibited LH binding to Candida membranes, but not to sheep or pig luteal LH receptors. This material had a molecular weight of 16,000-21,000 daltons, bound strongly to CM-Sepharose at physiological pH, and could be resolved completely from hCG and from epidermal growth factor-like factors present in commercial gonadotrophin preparations. Its activity was not attenuated by a range of inhibitors specific for the four major classes of proteolytic enzymes, nor did it inhibit hormone binding by causing degradation of 125I-labelled hLH or hCG tracers. Factors which inhibited hLH binding to Candida membranes were also present in partially purified human urinary and equine serum gonadotrophin preparations and in placental extracts, but were not detected in highly purified CG of hLH preparations. The properties of this factor were similar to those described for beta-core protein, a cleavage product of the beta subunit of hCG which is a contaminant of commercial gonadotrophin preparations. Highly purified beta-core protein inhibited 125I-labelled hLH binding to Candida membranes, but not to sheep luteal binding sites. Preparations of hCG depleted of inhibitor activity could stimulate adenylate cyclase activity in Candida membranes almost five fold. In contrast, partially purified inhibitor preparations strongly inhibited basal adenylate cyclase activity (to 18% of control levels). These observations suggest that endogenous LH-like factors, perhaps similar to beta-core proteins of hCG, may play a role in the regulation of morphogenesis in Candida species.

Adenylyl Cyclases↗
PubMed1991

Binding sites for LH in Candida albicans: comparison with the mammalian corpus luteum LH receptor.

We have described recently the presence of binding sites for human LH (hLH) and human chorionic gonadotrophin (hCG) in microsomal and cytosol fractions prepared from the dimorphic, pathogenic fungus, Candida albicans. We have now compared the properties of Candida LH/hCG-binding sites with those of the ovine luteal LH receptor. Sheep luteal LH-binding sites were associated with luteal membranes, and little or no binding activity was present in cytosol fractions. In contrast, significant LH/hCG-binding activity was present in Candida cytosol. Moreover, there were marked differences in sensitivity to inhibition by metal ions, association and dissociation rates, and affinity constants between sheep and Candida LH-binding sites. Scatchard plots of 125I-labelled hLH binding to sheep luteal receptors demonstrated a single high-affinity component (association constant (Ka) 0.3 litres/pmol) which was displaceable by hCG. In contrast, Scatchard plots of binding to Candida microsomes and cytosol fractions demonstrated two components, one with high affinity (Ka 0.18 litres/pmol) and low capacity and a second site with lower affinity (Ka 4 litres/nmol) and high capacity, both of which were displaceable by unlabelled hCG. Gel permeation chromatography of cytosol demonstrated two distinct peaks of LH-binding activity with approximate molecular weights of greater than 1,000,000 and 30,000-50,000. Scatchard plots of 125I-labelled hLH binding to the higher molecular weight peak demonstrated a single, high-affinity LH-binding site (Ka 0.18 litres/pmol), whereas the lower molecular weight fraction contained both high- (Ka 0.17 litres/pmol) and low-affinity (Ka 4 litres/nmol) LH-binding sites. Both partially purified and highly purified hCG and hLH preparations displaced binding of 125I-labelled hLH and hCG to sheep luteal LH receptors at similar concentrations. 125I-labelled hLH/hCG binding to Candida membranes was also displaceable by low levels (ng) of partially purified hCG preparations, but much higher levels (micrograms) of highly purified hLH and hCG were required. This paradoxical observation suggested the presence of radiolabelled contaminants, or damaged forms induced during radioiodination of hormone tracers, which can bind more strongly to Candida membranes than unlabelled hCG and hLH but which do not bind to sheep LH receptors. However, no evidence for hLH tracer contaminants with differential binding to Candida and sheep luteal receptors was obtained following gel exclusion chromatography or fractionation on Concanavalin A-Sepharose. (Although three distinct 125I-labelled hLH fractions were resolved on Concanavalin A-Sepharose, presumably reflecting differences in their carbohydrate compositions, all three tracer peaks bound equivalently to both Candida membranes and ovine luteal LH receptors).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Specific binding sites for 125I-labelled human luteinizing hormone (hLH) in microsomal fractions from Drosophila.

Microsomal fractions prepared from Drosophila flies and larvae bound 125I-labelled human luteinizing hormone (hLH) in a displaceable manner. Binding increased linearly with increasing microsome concentration, and was dependent on the pH and metal ion concentration of the medium, and on the temperature and duration of incubation. Scatchard plots of 125I-hLH binding demonstrated the presence of two distinct binding sites, one with high affinity and low capacity, the other with low affinity and high capacity. 125I-hLH binding was inhibited in a dose-dependent fashion by partially purified human gonadotropin preparations, but not by other proteins, hormones and peptides. Highly purified human chorionic gonadotropin (hCG) or hLH preparations were much less effective at inhibiting 125I-hLH binding to Drosophila microsomes than partially purified gonadotropins. This was due to the presence of a contaminant present in partially purified commercial hCG preparations. Chromatography of crude hCG preparations clearly resolved the LH-binding inhibitor from hCG. Moreover, whereas hCG (and epidermal growth factor-like factors present in crude hCG preparations) were not bound to CM-Sepharose, the Drosophila LH-binding inhibitor was adsorbed strongly. Fractionation of 125I-hLH tracer on concanavalin A-Sepharose suggested that Drosophila binding sites did not preferentially bind a subfraction of the hormone tracer. These data suggest that high affinity binding sites for hLH/hCG-like molecules are present in Drosophila microsomes.

Animals↗
PubMed1990

Specific, high-affinity binding sites for human luteinizing hormone (hLH) and human chorionic gonadotrophin (hCG) in Candida species.

The presence of specific binding sites for [125I]-labelled hLH and hCG is described in Candida species. Binding was present in three strains of Candida albicans, and in Candida tropicalis, and was greatest in microsomes, though binding was also present in cytosol fractions. hLH and hCG mutually competed for these binding sites. Other hormones did not bind and did not compete for hLH binding sites. Scatchard plots showed two classes of binding sites, one with high affinity, low capacity and the other with lower affinity, high capacity binding in both microsomes and cytosol. This is the first report of specific binding sites for mammalian peptide hormones in a yeast.

Animals↗
PubMed1990

Receptor-mediated elevation of adenylate cyclase by luteinizing hormone in Candida albicans.

Human luteinizing hormone (hLH) and the GTP analogue guanosine 5'-O-(3-thio)triphosphate stimulated morphogenesis in the dimorphic fungal pathogen Candida albicans. hLH bound specifically to subcellular fractions from C. albicans and stimulated adenylate cyclase activity in C. albicans microsomes. We provide the first demonstration of guanine-nucleotide-binding proteins (G-proteins) in C. albicans, and propose that the stimulation of C. albicans morphogenesis by hLH is mediated by a receptor-coupled adenylate cyclase system involving G-proteins.

Adenylyl Cyclases↗
PubMed1989

The binding of steroids to myometrium and leiomyomata (fibroids) in women treated with the gonadotrophin-releasing hormone agonist Zoladex (ICI 118630).

Since uterine leiomyomata (fibroids) are not found in conditions where oestradiol is either absent or present only in low concentrations, oestradiol is considered to be an important factor in the control of fibroid growth. To investigate whether this is due to a direct effect on the tissue, oestradiol and progestogen receptors were measured in tissue removed at hysterectomy from 12 normally cycling women and 13 women who had received the gonadotrophin-releasing hormone (GnRH) agonist Zoladex (ICI 118630) as a subcutaneous depot given at monthly intervals for 3 months preoperatively and a third group of three women who had received the antioestrogen tamoxifen (20 mg daily) for 3 months before surgery. Both unoccupied oestradiol receptors (measured by separating bound from free hormone with dextran-coated charcoal; DCC) and 'total' receptor populations (as measured by an enzyme immunoassay) were measured in each fibroid and adjoining myometrium. There was significantly more binding of both oestradiol and progestogen to fibroid than to myometrium in both the control (P less than 0.01) and agonist-treated groups (P less than 0.05). Oestradiol binding to fibroids in women treated with Zoladex exceeded that in the normally cycling women (P less than 0.05) which in turn exceeded that in the tamoxifen-treated group (P less than 0.05). However, the binding of progestogen, measured by DCC showed the reverse trend. These results may be explained by the low circulating oestradiol concentration in the GnRH agonist-treated women leading to low receptor occupancy.

Buserelin↗
PubMed1988

Subcellular fractionation of the ovine corpus luteum: association of progesterone with ovine luteal membranes?

Sheep corpus luteum homogenates were fractionated by centrifugation on continuous sucrose density gradients, with or without digitonin, and gradient fractions were assayed for progesterone, and for a range of intracellular organelle and plasma-membrane markers. Digitonin had little effect on the density distributions of mitochondrial, rough endoplasmic reticulum (RER) and Golgi-endoplasmic reticulum-lysosomal (GERL) membranes. However, digitonin did disrupt lysosomal membranes, leading to release of acid hydrolases, and induced a decrease in buoyant density of NADH-cytochrome c reductase, a putative smooth endoplasmic reticulum (SER) marker. Oxytocin-containing granules were clearly resolved from other organelles accumulating in a sharp peak (density, 1.20 g/cm3). Luteal cell-surface membrane marker activities equilibrated at similar buoyant densities in control gradients, and pretreatment with digitonin induced a marked increase in their buoyant densities. The majority of the progesterone of the sheep corpus luteum equilibrated at a buoyant density of 1.10 g/cm3 in control gradients, and was highly perturbed by digitonin. These fractions also accumulated [3H]progesterone. The buoyant density profile of progesterone in both control and digitonin-treated gradients most closely resembled that of sheep luteal lactogenic receptor, a putative plasma-membrane marker.

Animals↗
PubMed1988

Subcellular fractionation of the porcine corpus luteum: sequestration of progesterone in a unique particulate fraction.

Homogenates of porcine corpus luteum were subjected to fractionation by differential-rate centrifugation or sucrose density gradient fractionation, with or without pretreatment with digitonin. Fractions of each gradient were assayed for a number of markers characteristic of the major intracellular organelles and cell-surface membranes, and for progesterone content. The majority of the progesterone content of homogenates of porcine corpus luteum was associated with a low-density particulate fraction which equilibrated at a buoyant density of 1.07-1.09 g/cm3. Pretreatment with digitonin increased the buoyant density of the progesterone-enriched fraction markedly (to 1.13-1.15 g/cm3) without causing release of steroid. The density distributions of progesterone content in control and digitonin-treated luteal gradient fractions were quite distinct from those of the major intracellular organelles and luteal cell-surface membranes. However, NADH-cytochrome C reductase activity (but not other endoplasmic reticulum markers) was also enriched in this fraction. The results suggest that most of the progesterone of the porcine corpus luteum is associated with a unique particulate fraction which is enriched in digitonin-reactive lipids and NADH-cytochrome C reductase activity.

Animals↗
PubMed1987

Specific binding sites for LH/chorionic gonadotrophin, low-density lipoprotein, prolactin and FSH in homogenates of human corpus luteum. I: Validation of methods.

The specific binding of 125I-labelled human chorionic gonadotrophin (hCG), human low-density lipoprotein (hLDL), human FSH (hFSH) and human prolactin (hPRL) to homogenates of human corpus luteum tissue was measured. Specific binding of 125I-labelled hCG was dependent on the temperature and duration of incubation, was inhibited by divalent metal ions or chelating agents, and increased linearly with homogenate concentration. Recovery of bound hormone was more effective using Millipore filtration or polyethylene glycol precipitation compared with centrifugation alone. Binding of 125I-labelled hCG was inhibited specifically by low levels of hCG and human LH (hLH) but not by ovine LH or bovine LH. Incubation of human luteal tissue with ice-cold citrate buffer (pH 3) released more than 90% of specifically bound 125I-labelled hCG within 5 min. This treatment inactivated LH receptors, but did not affect the immunoactivity of hLH released, enabling the measurement of released hormone by radioimmunoassay. Scatchard plots of binding of 125I-labelled LDL to human corpus luteum demonstrated a single class of binding sites. Binding was saturable, increased linearly with increasing concentration of homogenate, and was displaceable by low concentrations of unlabelled LDL. Binding of 125I-labelled hPRL to human luteal homogenates was increased by Mg2+ and was specific for lactogenic hormones (human prolactin, human growth hormone and ovine prolactin). Binding of 125I-labelled hFSH was not dependent on divalent metal ion concentration (in marked contrast to hFSH binding to immature pig granulosa cell receptors) and was displaced by hFSH preparations but not by hPRL, ovine LH or hCG at 1 microgram/ml. These results establish optimal conditions and hormone specificities for the measurement of human luteal gonadotrophin and LDL receptors, and methods for the estimation of hLH/hCG endogenously bound to human corpus luteum tissue.

Animals↗
PubMed1987

Specific binding sites for gonadotrophin-releasing hormone, LH/chorionic gonadotrophin, low-density lipoprotein, prolactin and FSH in homogenates of human corpus luteum. II: Concentrations throughout the luteal phase of the menstrual cycle and early pregnancy.

Corpora lutea were obtained from 52 women undergoing laparotomy during the luteal phase of the menstrual cycle. In addition, stromal, thecal and granulosa cell preparations were obtained from seven women undergoing ovariectomy during the late follicular-preovulatory phase of the cycle. The specific binding of a 125I-labelled gonadotrophin-releasing hormone (GnRH) agonist [D-Ser(But)6] GnRH(1-9)-ethylamide (buserelin) and of human chorionic gonadotrophin (hCG), human FSH (hFSH), human prolactin (hPRL) and human low-density lipoprotein (hLDL) to tissue homogenates was measured under optimal conditions. Bound LH/hCG was estimated by elution with acid-citrate buffer, followed by radioimmunoassay of released hormone. Binding of GnRH agonist, though variable, was highest in mid-luteal corpus luteum and high binding was also present in three out of four corpora lutea of pregnancy. Binding of LH/hCG increased significantly with luteinization, reaching maximal levels in the mid-luteal phase before falling significantly. Occupancy of LH receptors by bound LH was relatively constant throughout the luteal phase (10.7-35.3%), but occupancy increased to greater than 90% in corpora lutea from early pregnancy. Binding of hFSH was variable, with only five out of 50 corpora lutea having binding greater than 10 pg/micrograms DNA. Similarly, hPRL binding varied markedly with only six out of 44 having binding greater than 50 pg/micrograms DNA. Binding of LDL was highest in the early- to mid-luteal phases of the cycle. In corpora lutea from all stages of the menstrual cycle (excluding corpora albicantia), GnRH agonist binding was highly correlated with the levels of unoccupied and occupied LH receptors (P less than 0.001; n = 49 and n = 48 respectively) and with LDL receptors (P less than 0.002; n = 49). Binding of GnRH agonist was also correlated with PRL binding (P less than 0.05; n = 21) but not with FSH receptors (P greater than 0.4; n = 25). In addition, LDL binding was associated with PRL (P less than 0.005; n = 21) and FSH receptors (P less than 0.05; n = 25) and with endogenously bound LH (P less than 0.03; n = 48), but not with unoccupied LH receptors (P = 0.8; n = 49). Moreover, in corpora lutea from the mid-luteal phase, there was a strong association between GnRH agonist binding and LDL receptors (P less than 0.02; n = 23). The correlations between GnRH agonist binding and a number of important indices of luteal function suggest a physiological role for GnRH-like factors in the human corpus luteum.

Corpus Luteum↗
PubMed1987

Receptors for lactogenic hormones in the porcine corpus luteum: properties and luteal phase concentrations.

Homogenates of pig corpora lutea contained specific, high-affinity receptors for ovine prolactin (oPRL) and human GH (hGH). Specific hormone binding was enhanced by divalent metal ions, but only when included in the binding reaction. Divalent metal ions did not act by increasing the recovery of bound hormone by low-speed centrifugation, but appeared to promote the formation of a more stable hormone-receptor complex. Both oPRL and hGH tracers were bound in similar amounts and with similar affinities by pig luteal homogenates and the concentrations of either unlabelled hormone required to displace specific binding of either tracer by 50% were identical. In contrast, 125I-labelled oGH failed to bind to pig luteal homogenates and oGH competed poorly for hGH or oPRL binding. Only hormones with prolactin-like activity competed for 125I-labelled oPRL binding. Specific prolactin binding was low in recently ovulated and early luteal phase corpora lutea, increased significantly in the mid-luteal phase and declined once more in the late luteal phase. Receptor concentrations increased with increasing gestational age.

Animals↗
PubMed1987

Receptors for lactogenic hormones in the ovine corpus luteum. I: A major discrepancy in the specific binding of radiolabelled ovine prolactin and human growth hormone.

The characteristics of the binding of 125I-labelled human GH (hGH) and ovine prolactin (oPRL) were studied in the ovine corpus luteum. Although oPRL is the homologous ligand for sheep lactogenic receptors, its binding was significantly and consistently lower than that of 125I-labelled hGH. This was not due to iodination damage of oPRL since: (1) 125I-labelled oPRL tracers which bound poorly relative to 125I-labelled hGH in the ovine corpus luteum were equipotent in the pig and rat corpus luteum, (2) the differences between 125I-labelled hGH and oPRL binding persisted with tracers of equivalent biopotency and (3) the iodination procedure affected neither oPRL bioactivity in the Nb2 tumour assay nor its binding activity with ovine corpus luteum receptors. Ovine luteal receptors were specific for lactogenic hormones. The specific binding of 125I-labelled hGH or oPRL could be inhibited completely by incubation with either unlabelled hormone, with similar potencies. However, oGH inhibited binding only at much higher concentrations, consistent with its known contamination with oPRL. Moreover, 125I-labelled oGH was not bound specifically to sheep luteal tissue. Fractionation of sheep luteal homogenates on sucrose density gradients (with or without cell-surface membrane perturbation by digitonin) demonstrated that binding of 125I-labelled hGH and 125I-labelled oPRL peaked in the same regions of the gradients, coincident with a number of luteal cell-surface membrane markers. We conclude that the marked discrepancy between the binding of hGH and oPRL tracers by sheep luteal tissue was not due to iodination damage of oPRL, binding of 125I-labelled hGH to somatogenic receptors or differential binding to luteal cell-surface versus intracellular receptors.

Animals↗
PubMed1987

Receptors for lactogenic hormones in the ovine corpus luteum. II: Specific inactivation of ovine prolactin.

Sheep corpus luteum homogenates and membrane fractions discriminate between 125I-labelled human GH (hGH) and ovine prolactin (oPRL). The present studies were designed to establish whether ovine luteal tissue possessed a prolactin-specific inactivating enzyme. Preincubation of sheep luteal microsomes and cytosol fractions with 125I-labelled hGH had little effect on the ability of the hormone to rebind to pig luteal lactogenic receptors. In contrast, sheep luteal tissue fractions markedly decreased the binding ability of 125I-labelled oPRL. However, despite the profound loss of receptor-binding activity, there was no change in protein-bound radioactivity, nor in the elution profile of 125I-labelled oPRL by gel chromatography on Sephadex G-100. Moreover, the disparity between 125I-labelled hGH and oPRL was not overcome by preincubation of sheep luteal membranes with protease inhibitors of differing specificities. We conclude that the disparity between the binding of hGH and oPRL in ovine tissues was due to the selective inactivation of oPRL. However, the activity responsible did not degrade the hormone extensively, nor was its action blocked by a range of protease inhibitors.

Animals↗
PubMed1987

Receptors for lactogenic hormones in the ovine corpus luteum. III: Inhibition of 125I-labelled human growth hormone binding by a high molecular weight factor in ovine corpus luteum cytosol.

Ovine luteal cytosol fractions inhibited the specific binding of 125I-labelled human GH and ovine prolactin (oPRL) to ovine luteal microsomes in a dose-dependent fashion. Inhibition was dependent on divalent cation concentrations, and was abolished by divalent metal ion chelating agents or by boiling. Inhibition was not due to ionic strength or salt effects on hormone binding, the release of endogenously bound oPRL into the cytosol fraction during tissue disruption and fractionation, or the presence of a soluble (or solubilized) lactogenic receptor in ovine cytosol preparations. Gel chromatography of cytosol fractions gave a molecular weight for the inhibitor of approximately 50,000.

Animals↗
PubMed1986

Subcellular fractionation of the human corpus luteum: distribution of GnRH agonist binding sites.

We have investigated the subcellular localization of GnRH agonist (GnRHA) binding sites in the human corpus luteum. Corpora lutea (CL) were obtained from 13 women undergoing laparotomy during the luteal phase of the menstrual cycle. CL homogenates were subjected to subcellular fractionation by either differential rate centrifugation or by sucrose density gradient fractionation with or without digitonin perturbation. Fractions were then assayed for a number of marker activities characteristic of the major intracellular organelles and cell-surface membranes, and for specific binding of [125I]GnRHA. Specific binding of [125I]GnRH agonist was greatest in regions of the gradient (density, 1.12-1.15 g/cm3) enriched in cell-surface and endoplasmic reticulum membrane markers. Little or no GnRHA binding was associated with fractions enriched in nuclei, mitochondria or lysosomes. Digitonin treatment demonstrated that the majority of GnRHA binding sites were associated with luteal cell plasma-membranes: however, some GnRHA binding was also associated with endoplasmic reticulum. These results indicate that: (i) human luteal GnRHA binding sites were not associated with lysosomal proteases, (ii) a significant fraction (59%) of GnRHA binding sites was localised on the luteal cell-surface membrane. Specific GnRHA binding to both cell-surface and endoplasmic reticulum membranes may indicate an extensive turnover of membrane binding sites.

Binding Sites↗
PubMed1986

Specificity of gonadotrophin-releasing hormone binding sites of the human corpus luteum: comparison with receptors of rat pituitary gland.

The effects of a number of analogues of gonadotrophin-releasing hormone (GnRH) on the binding of a radiolabelled GnRH agonist (GnRH-A; [D-Ser(But)6, des Gly10]GnRH-ethylamide) to homogenates of human corpus luteum (CL) and rat pituitary tissue were compared. Specific binding was inhibited by GnRH and GnRH-like peptides only. Both the C-terminal amide and N-terminal region of the GnRH molecule were required for binding in both tissues. However, amino acid substitutions at position 6 markedly enhanced, and at position 8 markedly reduced, binding potencies in rat pituitary tissue compared with human CL binding sites. These results indicate that GnRH-binding sites of rat pituitary and human luteal tissue have a similar degree of specificity for GnRH-like peptides, and a similar requirement for both N- and C-terminal regions of the peptide, but that differences in specificity related to the mid-chain region of GnRH exist between human luteal and rat pituitary binding sites.

Animals↗