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W R Moyle

Publications and source records attributed to W R Moyle.

At least 55 records · Page 3Linked to original sources
PubMed1983

Further characterization of cooperative interactions of monoclonal antibodies.

We reported previously that mixtures of some monoclonal antibodies directed against the glycoprotein hormone human chorionic gonadotropin (hCG) had a higher affinity for the antigen than either monoclonal antibody separately. The synergistic interaction could no longer be detected when one of the antibodies was replaced with its F(ab) fragment. This cooperative interaction has now been further characterized. One-half of 10 possible pairs prepared from five IgG1 monoclonal antibodies against hCG result in a synergistic interaction. The addition of an IgG2b monoclonal antibody to one of the IgG1 monoclonal antibodies also induces a cooperative interaction, which shows that the effect is not subclass restricted. Cooperative interactions between antibodies are also not restricted to solution conditions; adsorption of one antibody to a solid support appears to increase the cooperative effect. Indeed, one pair of antibodies that failed to bind hCG synergistically in solution did so when one antibody was bound to a solid surface. The liquid phase antibody also has an effect on the specificity of the solid phase antibody. The sensitivity of the solid phase assay system has enabled us to develop a rapid method of determining if two monoclonal antibodies can bind to an antigen simultaneously. A quantitative theoretical model has been devised that successfully predicts the cooperative behavior observed between antibodies and should be useful in devising conditions that result in sensitive solid phase radioimmunoassays.

Antibodies, Monoclonal↗
PubMed1982

Use of monoclonal antibodies to subunits of human chorionic gonadotropin to examine the orientation of the hormone in its complex with receptor.

Monoclonal antibodies were prepared against the alpha and beta subunits of human chorionic gonadotropin (hCG). Although all were selected on the basis of their ability to bind the intact hormone, each also bound one of the two subunits but not both. Using a solid phase double antibody system to measure the relative binding to sites on the surface of hCG, we observed that four of the five antibodies bound to different sites on the molecule. This information was correlated with the ability of each antibody to inhibit the biological activity of hCG. Of the five antibodies tested for their ability to inhibit hCG-induced stimulation of rat testes steroidogenesis in vitro, two proved to be potent inhibitors, whereas the other three had almost no effect. This inhibition of steroidogenesis was highly correlated with the ability of the antibodies to inhibit hCG binding to testes homogenates. Thus, we have begun to derive a scheme that describes the relative binding positions of individual monoclonal antibodies and receptor on hCG. The purified monoclonal antibodies were iodinated and employed to evaluate which antigenic sites on hCG remained free in hCG-receptor complexes. The data indicated that portions of the beta subunit in hCG-receptor complexes were buried (i.e., failed to bind radiolabeled antibody), whereas other portions remained exposed (i.e., they bound radiolabeled antibody). Those antibodies that interacted with portions of hCG that became inaccessible in the receptor complex also blocked the biological actions of hCG, whereas those that interacted with exposed sites had little or no effect on activity. Although we did not find antibodies to the alpha subunit that would bind to the hormone-receptor complex, we found that one of the two antibodies specific to alpha subunit epitopes blocked the actions of the hormone. Both antigenic determinants on the alpha subunit appeared to be lost after the hCG-receptor complex had formed. These studies suggest that each hCG subunit participates in the hormone-receptor complex and that portions of the beta subunit project from the surface of the receptor.

Animals↗
PubMed1982

Acute inhibition of corticosteroidogenesis by inhibitors of calmodulin action.

To identify the possible role of calmodulin in ACTH function, we tested the ability of chlorpromazine (CP) and other calmodulin antagonists to inhibit steroidogenesis of isolated adrenocortical cells of the rat. CP reversibly inhibited maximal ACTH-induced corticosterone (B) production. The presence of the drug did not alter the ED50 of ACTH stimulation (3.2 X 10(3) pg/ml), suggesting that it inhibited ACTH-induced steroidogenesis in a noncompetitive manner. The CP concentration required for half-maximal inhibition was 8.2 microM, a value close to the dissociation constant of the CP-calmodulin complex (5.3 microM). Concentrations greater than 40 microM resulted in complete inhibition. Similar concentrations of CP inhibited ACTH-induced cAMP accumulation in a dose-dependent manner, indicating an effect of the drug on early events in ACTH action. In addition, CP also apparently acted at a site distal to the point of cAMP formation, as shown by the finding that it inhibited cAMP-induced B production. CP inhibition of ACTH-induced B production was independent of the Ca2+ concentration, suggesting that the drug did not compete with Ca2+ directly. Concentrations of CP greater than 20 microM inhibited protein synthesis as measured by leucine incorporation into cellular proteins. Thus, although the inhibitory effect of high concentrations of CP on steroidogenesis might be explained by an effect on protein synthesis, the inhibition seen at 10 microM appeared to be independent of protein synthesis. Other antagonists of calmodulin action inhibited maximal ACTH-induced B production with the following relative potencies: trifluoperazine greater than CP greater than haloperidol greater than chlordiazepoxide. This order is similar to that reported for inhibition of calmodulin-activated phosphodiesterase and for binding to calmodulin. These findings suggest that calmodulin may modulate the effect of ACTH on steroidogenesis at multiple sites.

Adrenal Cortex↗
PubMed1982

Mixing two monoclonal antibodies yields enhanced affinity for antigen.

We observed that mixing monoclonal antibodies directed against various epitopes of human chorionic gonadotropin (hCG) can increase the sensitivity of antigen-binding assays. Depending on the antibody pair chosen, the affinity of the mixture was as much as 10-fold higher than either of the monoclonal antibodies assayed separately. Because hCG does not have any repeating sequences, these results do not reflect a change in avidity of an individual antibody. The increased avidity of the mixture can be detected in both solid-phase assays and liquid-phase double antibody radioimmunoassays. This was not a general property of all monoclonal antibodies raised against hCG. Certain antibodies, apparently reacting with the same region of the antigen molecule, do not bind simultaneously and do not result in enhanced affinity when mixed. In addition, certain other pairs of antibodies do not bind cooperatively even though they can bind simultaneously. The mechanism for the increase in affinity depends on the formation of a multicomponent complex. If one of the antibodies of a pair that results in enhanced affinity upon mixing is replaced by its F(ab) fragment, the enhancement is no longer detectable, indicating it is unlikely the enhancement is due to an allosteric effect. Although the F(ab')2 fragment shows some enhancement when mixed with another antibody, it is not as effective as the intact antibody.

Animals↗
PubMed1980

hCG-Leydig cell functional binding kinetics: threshold and nonlinearity in response.

Functional kinetic methods, developed to measure the interaction of human chorionic gonadotropin (hCG) with rat Leydig-cell receptors, appear to be useful tools for correlating response with receptor occupancy. In the functional procedures, hCG was allowed to bind to the cells (period I), the free hormone was removed by washing and/or antiserum treatment (period II), and the response of the cells was measured at 37 degrees C (period III). Once initiated, the response to hCG was stable throughout period III. Assuming a one-to-one relationship between occupancy and response during period III, we estimated the rate of association to be 10(8) M-1/min at 37 degrees C with an activation energy of 14-17 kcal/mol. Removal of sialic acid from hCG increased this rate; removal of other carbohydrate residues decreased it. Similar values for the kinetics of binding were observed when either steroidogenesis or cyclic AMP accumulation was measured, suggesting that the same receptor may mediate both processes. Use of either functional or direct (i.e., 125I-labeled hCG) methods to estimate response as a function of occupancy gave equal results, suggesting that most binding sites were coupled to a response. Response was nonlinearly coupled to occupany. Threshold amounts of hormone-receptor complex (0.1% total receptors testosterone synthesis; 2.7% total receptors cyclic AMP accumulation) were required to induce any response. Increased stimulation required progressively larger increments of receptor occupancy. The threshold was inversely proportional to the efficacy of the hCG derivative used and was reduced by the presence of isobutylmethylxanthine.

Animals↗
PubMed1978

Action of PMSG and asialo-PMSG on rat Leydig and granulosa cells.

Highly purified pregnant mares serum gonadotropin (PMSG) was nearly as potent as ovine luteinizing hormone (LH) and human follicle stimulating hormone (hFSH) when bioassayed in vitro in systems known to respond primarily to LH or FSH. An analogue of human chorionic gonadotropin treated with neuraminidase, galactosidase, beta-N-acetylglucosaminidase, and mannosidase (hCG) inhibited the stimulatory effects of hCG, LH, and PMGS on cAMP accumulation in rat Leydig cells but did not inhibit the stimulatory effects of FSH or PMSG on cAMP accumulation in ovarian granulosa cells obtained from immature rats fed diethylstillbestrol. Thus PMSG appeared to form functional complexes with both LH and FSH receptors and may be unique among mammalian gonadotropins. Treatment of PMGS with neuraminidase increased its potency nearly tenfold in vitro apparently by increasing its affinity for both LH and FSH receptors. Although the kinetics of PMSG binding were not investigated with radiolabeled materials, indirect functional binding studies are described that suggest that hCG more rapidly forms stable hormone-receptor complexes than PMSG, asialo-PMSG, FSH, and LH when all hormones are incubated under the same conditions.

Animals↗
PubMed1977

New method of quantifying ligand binding based on measurement of an induced response.

A new general method is proposed for quantifying ligand-receptor interactions using the biological response induced by the ligand as an index of ligand binding. With this method the binding of human chorionic gonadotropin (hCG), several hCG derivatives, and luteinizing hormone (LH) to rat Leydig cells was measured by analysis of the ability of these materials to stimulate testosterone formation. As applied here, hormone dose-response curves were generated in the presence of increasing numbers of cells incubated in vitro in a successful attempt to alter the concentrations of bound and free hormone in the incubation mixture. Measurements of testosterone synthesis as a function of the total amounts of hormone and numbers of cells enabled us to evaluate the concentrations of both bound and free hormone at any constant fractional response (i.e. quarter-, half-, or three-quarter-maximal). We were thus able to measure hormone binding at the extremely low hormone concentrations (1 pM) within the steroidogenic dose-response range under conditions that would not have been possible using currently available radioiodinated hCG preparations. The results obrained confirmed the presence of spare functional receptors. Specific quantitative results are discussed in the text.

Animals↗
PubMed1976

Role of histone kinases as mediators of corticotropin-induced steroidogenesis.

In an attempt to determine the role of protein (histone) kinases as mediators of corticotropin-induced corticosterone formation, the ability of homogenates, prepared from adrenals treated with various doses of corticotropin to catalyse the phosphorylation of calf thymus histones was measured. Although corticotropin promoted an increase in histone kinase activity, much more of the hormone was required to induce this response than to stimulate steroidogenesis maximally. In addition, a derivative, nitrophenylsulphenyl-corticotropin, which inhibits the stimulatory effect of corticotropin on cyclic AMP accumulation, stimulated corticosterone synthesis without altering histone kinase activity. Very high doses of nitrophenylsulphenyl-corticotropin were capable of stimulating histone kinase activity. In contrast, when dibutyryl cyclic AMP was used to stimulate steroidogenesis under the same conditions, any dose of the nucleotide which increased adrenal corticosteroid content also increased histone kinase activity. Assuming that histones serve as useful substrates for measurement of total adrenal protein kinase activity, the role of protein kinases as mediators of steroidogenesis is not supported by these studies.

Adrenal Glands↗
PubMed1975

Role of carbohydrate of human chorionic gonadotropin in the mechanism of hormone action.

The role of the carbohydrate part of human chorionic gonadotropin (hCG) was investigated by measuring the ability of hCG derivatives lacking various sugar residues to bind to rat Leydig cells and stimulate them to synthesize testosterone and cyclic adenosine 3':5'-monophosphate (cyclic AMP). Whereas sequential removal of the sialic acid, galactose, N-acetylglucosamine, and mannose residues led to a progressive increase in the effective dose of the hormone required to stimulate steroidogenesis, it resulted in a marked loss in the ability of the hormone to stimulate cyclic AMP accumulation. Low doses of the glycosidase-treated hormone derivatives were additive with hCG when their ability to stimulate testosterone synthesis was analyzed. Nevertheless, the glycosidase-treated derivatives were potent inhibitors of hCG-induced cyclic AMP accumulation, suggesting that removal of the sugars did not influence binding of the hormone to the cell as much as it reduced the ability of the bound hormone to activate adenyl cyclase. This hypothesis was further supported by our finding that the hCG derivatives were highly effective inhibitors of 125I-hGC binding to the intact cells. Removal of sialic acid and galactose enhanced the inhibition, whereas removal of all the sugar residues only decreased the inhibition slightly. The degree of these effects was comparatively small. The possibility that steroidogenesis and cyclic AMP accumulation are altered independently by hCG stimulation is discussed.

Acetylglucosaminidase↗
PubMed1973

Influence of luteinizing hormone and adenosine 3':5'-cyclic monophosphate on the metabolism of free and esterified cholesterol in mouse Leydig-cell tumours.

1. Male C57B1/6J mice bearing Leydig-cell tumours known to synthesize steroids in response to luteinizing hormone (LH) were given intravenous injections of [1,2-(3)H]cholesterol (50-100muCi per animal). Single-cell suspensions were prepared from the tumours 5-9 days after the injection of [(3)H]cholesterol and were incubated at 37 degrees C in foetal calf serum supplemented with 50mm-Tris-HCl, pH7.4. At various times after the start of incubation cells were collected by filtration of portions of the suspension and their sterols analysed. Within 10min after LH (5mug/ml) or 3':5'-cyclic AMP (20mm) was added to the cell suspensions an increased conversion of ester cholesterol into free cholesterol could be demonstrated. 2. To observe this rapid effect of LH it was necessary to incubate the cells for 60min before addition of hormone. 3. The specific radioactivity of testosterone produced was approximately equal to that of the intracellular cholesterol regardless of the presence or absence of LH. 4. The amount of free cholesterol produced in response to LH was far greater than that needed for steroid synthesis. 5. Free cholesterol, but not esterified cholesterol, was released into the incubation medium linearly with time and this release was unaffected by LH. LH may stimulate steroidogenesis in part by increasing the concentration of free cholesterol within the cell.

Journal Article↗
PubMed1973

Metabolism of free and esterified cholesterol by Leydig-cell tumour mitochondria.

1. Experiments were designed to localize intracellularly the enzymes and sterol substrates required for steroidogenesis in Leydig-cell tumours. Subcellular fractions were prepared by differential centrifugation of tumour homogenates. Both free and esterified cholesterol were associated primarily with the fractions sedimenting at 1400g(av.) and the lipid layer floating on the surface of the isolation tubes; they were not found in the mitochondria, where the conversion of cholesterol into pregnenolone occurred. 2. Hydrolysis of esterified cholesterol was required before it could be oxidized to pregnenolone. 3. An enzyme capable of hydrolysing cholesterol esters was located external to the mitochondria. 4. Mitochondria were subfractionated by allowing them to swell in 0.02m-phosphate buffer (pH7.2) and separating the inner and outer membranes by sedimentation in sucrose gradients. The outer membrane, identified by its content of monoamine oxidase, contained most of the cholesterol associated with the mitochondria. The inner membrane, identified by its content of succinate dehydrogenase, contained the cholesterol side-chain-cleaving enzyme and very little cholesterol. 5. Accumulation of sterols by the mitochondria was studied by incubating this fraction with labelled free and esterified cholesterol suspended in lipid-free bovine serum albumin. Two phases of cholesterol accumulation were observed. The first phase, requiring 10-15min, was independent of the incubation temperature, and was inhibited by the presence of bovine serum albumin in the incubation medium. The second phase of accumulation was independent of the serum albumin content of the medium but was inhibited by low incubation temperature. 6. Esterified cholesterol was not accumulated by the mitochondria after the initial rapid binding phase. 7. The findings suggest that cholesterol was not rapidly accumulated by the mitochondrial fraction in vitro and that mechanisms may be required to facilitate cholesterol transport into mitochondria in intact tumour cells during the periods in which steroidogenesis is stimulated maximally.

Journal Article↗