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R J Ryan

Publications and source records attributed to R J Ryan.

At least 91 records · Page 5Linked to original sources
PubMed1978

Interations of gonadotropins with corpus luteum membranes. VII. Association of hCG-binding and adenylate cyclase activities with rabbit corpus luteum plasma-membranes.

Previous studies with rat luteal cell plasma membranes indicated a dissociation of hCG binding sites and adenylate cyclase activity. The hormone binding sites were associated with a light membrane (d 1.13) fraction derived from the microvillous surface while the cyclase enzyme was associated with a heavy membrane (d 1.17) fraction derived from the basolateral surface of the cell. In this study of plasma membranes from 8-day pseudopregnant rabbit corpora lutea both hCG binding and adenylate cyclase activity were associated with a light membrane (d 1.13) fraction. No heavy membrane material was detected.

Adenylyl Cyclases↗
PubMed1978

Structure of human luteinizing hormone alpha subunit.

Structural studies have substantiated the concept that the glycoprotein hormones consist of a "common" alpha subunit and "hormone-specific" beta subunit. Despite this, consensus is still lacking concerning certain portions of the amino acid sequences, including alignment of residues 81--82 in the alpha subunit. We have carried out sequence analysis of the alpha subunit of human luteinizing hormone (hLH) to clarify the assignment of these residues and to examine further the nature of the amino-terminal heterogeneity found among the different alpha subunits. Our structure showed residues 80--84 to be -His-Cys-Ser-Thy-Cys-, consistent with findings of others for human follicle stimulating hormone (hFSH) and human chorionic gonadotropin (hCG). The extensive degree of heterogeneity found in the amino terminal region of hFSH and hCG is present to only a minor extent in hLH. The differences in the pattern of amino-terminal heterogeneity among the various hormones may result from differences in the nature of cleavage of subunit from a larger intracellular precursor peptide.

Amino Acid Sequence↗
PubMed1978

Studies on the isolation and chemical-physical properties of porcine follicle-stimulating hormone.

Methods are described for isolating highly purified FSH from porcine pituitary glands. The biological activity of pure material was 22 NIH-FSH-P2 U/mg, as judged by the ovarian weight augmentation bioassay. Virtually no immunoreactive LH was indicated by RIA, and only one component was detected by both gel electrophoresis and immunoprecipitation. Amino acid and carbohydrate analyses of the highly purified FSH indicated the presence of 1-tryptophan, a high content of aspartic and glutamic acids, and 6% sialic acid. Molecular weights of untreated and guanidine-treated porcine FSH were estimated from hydrodynamic measurements of Stokes radii and sedimentation coefficients. A highly specific and sensitive RIA was also developed for this hormone.

Amino Acids↗
PubMed1978

Interactions of gonadotropins with corpus luteum membranes. I. Properties and distributions of some marker enzyme activities after subcellular fractionation of the superovulated rat ovary.

The properties of a number of enzyme activities of the superovulated rat ovary have been studied to establish optimal assay conditions and specific assay procedures for each activity. The activities were chosen on the basis of their extensive use in other tissues of the rat as marker enzymes for the major cell organelles. Homogenates of superovulated rat ovaries were subjected to fractionation by differential rate centrifugation, and sedimentation profiles were constructed for each marker enzyme activity. The various subcellular fractions were also monitored by electron microscopy. The enrichment of fractions with particular organelles by electron microscopy, and enrichment of the appropriate organelle marker enzyme activities correlated well. Sedimentation profiles of a number of plasma membrane marker enzymes demonstrated a marked discrepancy between hCG-binding activity, and 5'-nucleotidase-, alkaline phosphatase-, and Mg2+-dependent ATP-ase on the one hand, and basal, hCG-stimulated, and fluoride-stimulated adenylate cyclase activities on the other hand. Fractions enriched in hCG-binding and adenylate cyclase activities were subjected to further fractionation on discontinuous sucrose density gradients. The distributions of the various plasma membrane markers again indicated a partial dissociations between hCG-binding and adenylate cyclase activities of luteinized rat ovaries, suggesting the existence of two distinct major plasma membrane populations, with different buoyant densities, marker enzyme profiles and adenylate cyclase and hormone-binding levels.

Adenylyl Cyclases↗
PubMed1978

Interactions of gonadotropins with corpus luteum membranes. II. The identification of two distinct surface membrane fractions from superovulated rat ovaries.

Fractions enriched in hCG-binding activity were prepared by differential rate centrifugation of superovulated rat ovarian homogenates and were applied to continuous sucrose density gradients (20-55%). After centrifugation at 63,000 x gav for 3.5 h, fractions of each gradient were collected and assayed for a range of marker enzyme activities characteristic of surface membranes and subcellular organelles. Mitochondria, lysosomes, and rough and smooth endoplasmic reticulum membranes accumulated in the gradient between 38-41% sucrose (1.165-1.180 g/cm3). Nuclei passed through the gradient. However, the various surface membrane markers concentrated in two distinct regions of the gradient. Alkaline phosphatase, phosphodiesterase, (Na+ + K+)ATPase I, and hCG-binding activity concentrated at 29-32% sucrose (1.120-1.135 g/cm3), whereas 5'-nucleotidase, Mg2+-dependent ATPase, and adenylate cyclase activities (and minor peaks of hCG-binding and phosphodiesterase activities) were enriched at 36-38% sucrose (1.16-1.17 g/cm3). A second ATPase, [(Na+ + K+)ATPase II], was also observed in this region of the gradient, which could be distinguished from (Na+ + K+)ATPase I of the light membrane fraction by its sensitivity to the Ca2+-chelating agent, ethylene glycol bis-(aminoethyl)tetraacetic acid (EGTA). The kinetics of binding of radioiodinated hCG to the gonadotropin receptors of the light and heavy membrane fractions were very similar. It is suggested that fractionation of superovulated rat ovaries yields two distinct populations of surface membrane material which have distinct densities and marker enzyme profiles. Furthermore, in contrast to the heavy membrane fraction, light membranes seem to possess considerable amounts of hCG receptor activity but very little adenylate cyclase.

Animals↗
PubMed1978

Effect of chronic administration of estrogen, androgen, or both on serum levels of gonadotropins in adult men.

Ethinyl estradiol (50 micrograms/day) or fluoxymesterone (10 or 20 mg/day), chosen because each is orally active and because fluoxymesterone is probably not converted to an estrogen, were given alone and in combination to adult men over several weeks. Measurements were made of serum FSH, LH, testosterone, and estradiol. The estrogen given alone suppressed serum FSH while the androgen given alone did not; however, the androgen may have enhanced the suppressive effect of the estrogen on the serum FSH. Neither steroid alone changed the serum LH but both together suppressed it. The estrogen alone decreased the serum testosterone, an effect probably mediated by the concomitant fall in serum FSH and a resulting decrease in sensitivity to the constant level of LH; a direct effect of estrogen on the testis seems less likely. The doses of estrogen and androgen used probably had a biologic effect equal to or somewhat above that of endogenously produced estrogen and androgen and thus reflected the maximum physiological effects of the endogenous steroids. Thus, in the chronic physiological control of FSH and LH in adult men, these data indicate that (1) testosterone alone, as an androgen, has little effect on FSH or LH, (2) estradiol (or total estrogen) has a greater suppressive effect on FSH than on LH and by its effect on FSH may indirectly regulate the secretion of testosterone, and (3) testosterone and estradiol together may be involved in the regulation of both FSH and LH.

Adult↗
PubMed1977

Acute effects of intravenous infusion of 17beta-estradiol and 17alpha-hydroxyprogesterone on gonadotropin release.

The normal midcycle surge of luteinizing hormone (LH), an approximately 10-fold increase, and a smaller surge of follicle-stimulating hormone (FSH) are preceded by peaks in serum estradiol (E2) and 17alpha-hydroxyprogesterone (17-P). The elevation in E2 is thought to be the important trigger mechanism for the LH surge. The possibility that the preovulatory elevation in 17-P may by itself, or combined with E2, be the trigger mechanism was investigated. E2 ans 17-P, alone and combined, were infused to mimic late follicular-phase blood concentrations in postmenopausal women pretreated with E2. This produced 1.6- to 4.6-fold increases in serum LH and lesser increases in serum FSH 24 hours after infusion. Thus, the normal ovulatory surge of gonadotropins could not be reproduced by infusions of 17-P or E2, or both. Infusion with 17-P appeared to stimulate LH release and did not augment or inhibit the effect of E2 on gonadotropin release.

Estradiol↗
PubMed1977

Proteolytic enzyme activation of rat ovarian adenylate cyclase.

Various serine proteases (e.g., trypsin, alpha-chymotrypsin, Pronase, and subtilisin) stimulate adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity in a membrane-enriched fraction of the rat ovary. Maximum stimulation is observed at protease concentrations ranging from 3 to 10 mug/ml. Higher protease concentrations inhibit ovarian adenylate cyclase in a dose-dependent manner. Protease stimulation causes a 6- to 8-fold increase in adenylate cyclase activity, which is comparable to the stimulation observed with human chorionic gonadotropin. Combinations of trypsin plus hormone or trypsin plus NaF stimulate ovarian adenylate cyclase activity to a greater extent than does any one of these alone. The mechanism of protease stimulation of adenylate cyclase involves limited proteolysis because zymogen precursors fail to activate the cyclase as does trypsin pretreated with trypsin inhibitors. Unlike cholera toxin, the serine protease stimulation is immediate (within the first 5 min) and requires no additional factors (e.g., NAD(+)). It is unlikely that protease stimulation of adenylate cyclase results from a proteolytic modification of the hormone receptor on the cell surface, because of the additive effects noted above and because protease stimulation is also observed in ovaries desensitized to hormone that lack this hormone receptor. Results with Lubrol-treated membranes also suggest that proteolytic enzymes do not directly activate the catalytic subunit of the cyclase or unmask new catalytic sites because the protease effect (like hormonal stimulation) is abolished by the detergent, whereas fluoride stimulation is enhanced. Other data suggest that serine protease and chorionic gonadotropin stimulation of adenylate cyclase result from activation of a membrane protease that then regulates adenylate cyclase in the ovary.

Adenylyl Cyclases↗
PubMed1977

Specific gonadotropin binding to Pseudomonas maltophilia.

Binding of 125I-labeled human chorionic gonadotropin to Pseudomonas maltophilia is dependent on time, temperature, and pH and the binding to this procaryotic species is hormone-specific and saturable. The equilibrium dissociation constant is 2.3 X 10(-9) M. There are no cooperative interactions between binding sites (Hill coefficient, 1.05). The number of sites is estimaated as 240 fmol/100 mug of protein. NaCl and KCl, at concentrations from 1 to 10 mM, have no effect on binding. Divalent cations (Mg2+ and Ca2+) and 1 mM EDTA inhibit hormone binding. Binding is destroyed by heat or by treatment with Pronase of alpha-chymotrypsin and is increased by phospholipase C. Binding of the labeled gonadotropin is not observed with other gram-negative organisms--e.g., Escherichia coli, Pseudomonas testosteroni, Pseudomonas aeruginosa, Enterobacter aerogenes, or Enterobacter cloacae.

Binding Sites↗
PubMed1977

Molecular forms of human chorionic gonadotropin in serum, urine, and placental extracts.

The molecular forms of human chorionic gonadotropin (hCG) were assessed in serum, urine, and placental extracts by gel filtration chromatography using radioimmunoassays for hCG, hCGalpha, and hCGbeta and a radioreceptor assay for hCG. The predominant form in all three biologic specimens was native-sized hCG. An excess of free alpha-subunit was also found in all three specimens. A small molecular weight fragment, reactive in the hCGbeta assay, was noted in urine and placental extracts. A large molecular form, reactive in all three radioimmunoassays and in the radioreceptor assay, was found in placental extracts. This large molecular species could not be dissociated by conditions that totally dissociate hCG.

Adult↗