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M Ascoli

Publications and source records attributed to M Ascoli.

106 records · Page 6Linked to original sources
PubMed1978

Inhibition of the degradation of receptor-bound human choriogonadotropin by lysosomotropic agents, protease inhibitors, and metabolic inhibitors.

A previous report from this laboratory showed that binding of iodine-labeled human choriogonadotropin to Leydig tumor cells is not a reversible process (Ascoli, M., and Puett, D. (1978) J. Biol. Chem. 253, 4892--4899). Most of the cell-bound hormone was found to be degraded to 3'-monoiodotyrosine before being released from the cells, and the degradation process could be inhibited by the lysosomotropic agents NH4Cl, chloroquine, and Triton WR-1339. It is reported herein that the degradation of receptor-bound human choriogonadotropin is an energy-dependent process, which can be inhibited by compounds that interfere with glycolysis or oxidative phosphorylation (e.g. NaF, NaN3, NaCN, and 2-deoxyglucose). Hormone degradation is also inhibited by some protease inhibitors such as the chloromethyl ketones of lysine and phenylalanine, but not by specific trypsin inhibitors (e.g. p-aminobenzamidine and p-tosyl-L-arginine methyl ester). With the exception of NH4Cl, it was found that the compounds which inhibit hormone degradation also inhibit hormone-stimulated steroidogenesis. However, the present results involving dose dependency, and those given in the following paper (Ascoli, M. (1978) J. Biol. Chem. 253, 7839--7843), indicate that these two phenomena are not related.

Ammonium Chloride↗
PubMed1978

Demonstration of a direct effect of inhibitors of the degradation of receptor-bound human choriogonadotropin on the steroidogenic pathway.

Results presented in the previous paper (Ascoli, M., and Puett, D. (1978) J. Biol. Chem. 253, 7832--7838) show that the degradation of receptor-bound 125I-labeled human choriogonadotropin can be inhibited by chloroquine, protease inhibitors, and metabolic inhibitors. These compounds were also shown to inhibit gonadotropin-stimulated steroidogenesis. It is reported herein that these inhibitors also block the stimulation of steroidogenesis by both cholera toxin and 8-Br-adenosine 3':5'-monophosphate, thus showing that they are not specific for the hormonal stimuli. These results, taken together with previous observations that show that NH2Cl can block hormone degradation without inhibiting hormone-stimulated steroidogenesis, strongly suggest that the degradation of choriogonadotropin is not required for its stimulatory action on progesterone production.

Ammonium Chloride↗
PubMed1978

Gonadotropin binding and stimulation of steroidogenesis in Leydig tumor cells.

Testicular tumors are generally characterized by a loss of responsiveness to gonadotropins. The M5480 Leydig cell tumor is unusual, if not unique, in that it responds to human choriogonadotropin and to lutropin via increased steroidogenesis. This report describes the identification of two variants of the original M5480 tumor that have altered steroid output both in the basal state and in response to human choriogonadotropin. One of the tumors produces mainly progesterone, which is stimulated by the choriogonadotropin; the other tumor produces about equal amounts of progesterone and testosterone, and the secretion of both is stimulated by the choriogonadotropin. The dissociation constant describing the interaction between Leydig tumor cells and (125)I-labeled human choriogonadotropin is between 3 and 5x10(-11) M. This agrees with values reported for normal Leydig cells, although the tumor cells appear to have fewer receptors. The differences noted in the two tumors and normal Leydig cells may have arisen from alterations in gene regulation, or in mutations, involving one or more enzymes in the pathway in which progesterone is converted to testosterone. Under the experimental conditions used, all the tumors studied (seven generations) responded to the choriogonadotropin both in binding and in the resultant stimulation of steroidogenesis. This property, together with the characteristic that a homogeneous cell population can be obtained without enzymatic treatment, should qualify the M5480 Leydig cell tumor(s) as a model system for further studies on the mechanism of action of gonadotropin, on hormone receptors, and on hormonally responsive tumors.

Animals↗
PubMed1977

Isolation and recombination of ovine lutropin subunits with complete recovery of biological activity.

Based on the results obtained in the previous paper (Liu, W.-K., Ascoli, M., and Ward, D. N. (1977) J. Biol. Chem. 252, 5274-5279) we have devised a method that permits the isolation of the subunits of ovine lutropin (oLH) in their "native" form. The hormone was dissociated by guanidine HCl, and the subunits separated by salt precipitation (Sairam, M. R., and Li, C. H. (1974) Arch. Biochem. Biophys. 165, 709-714). The present method differs significantly from that of Sairam and Li in that all operations are carried out at pH 5.0 or higher, a condition we have shown to be crucial to maintain the integrity of the isolated subunits. The yield of subunits was 75 to 85%. The isolated subunits exceed 95% purity (i.e. contamination with the other subunit or intact lutropin is less than 5%), migrate as single bands in sodium dodecyl sulfate-polyacrylamide gels, and show only the NH2-terminal heterogeneity expected from that of the native hormone. Recombination of the subunits can be accomplished with 75 to 85% yield (with respect to mass) and with full recovery of biological activity.

Amino Acid Sequence↗
PubMed1977

Renal uptake of lutropin. Studies based on electron microscopic autoradiography and nephrectomy.

Nephrectomy of mature rats was found to result in a significant increase in the circulatory half-life of tritiated ovine lutropin. The interaction of the glycoprotein hormone with the kidneys was studied in a more direct fashion using electron microscopic autoradiography. Evidence is presented showing the transfer of the hormone from microvilli into tubular epithelia (probably via vesicular transport), where radioactivity then becomes associated with lysosomes. This provides direct support for related results based on subcellular fractionation in which renal lysosomal catabolism was suggested as being important in the degradation of tritiated lutropin (M. Ascoli, R. A. Liddle, and D. Puett, Molecular and Cellular Endocrinology 4, 297, 1976). These results add substantial weight to the growing evidence that the kidneys assume a major role in controlling the concentration of circulating macromolecules.

Animals↗
PubMed1977

On the optical activity of ionized tyrosyl residues in ovine lutropin.

The effect of alkali on the circular dichroic (CD) spectra of ovine lutropin and its subunits has been studied. Mild alkaline pH induces the appearance of a new optically active band in the 250-nm region of the spectra of lutropin without any detectable alteration in the secondary structure of the protein. This change is reversible and can be correlated with ionization of 2--3 exposed tyrosyl residues in the intact hormone. In a previous report from this laboratory it was concluded that the three exposed tyrosyl residues are located in the alpha subunit, in positions 21, 92 and 93 [Burleigh, B.D., Liu, W.-K, and Ward, D.M. (1976) J. Biol. Chem. 251, 308--315]. Nitration of these residues lowers the pH at which the intensity of the 250-nm band is maximal. The importance of the tyrosyl residues of lutropin alpha (as opposed to those of lutropin beta) is also supported by the similarity of the effect of alkali on the CD spectra of lutropin and lutropin alpha. Further evidence for this involvement was also obtained by a comparison of the alkali-induced changes of refolded lutropin (alpha + beta recombinant) and the product obtained by recombination of des-(92--96)-lutropin alpha (obtained from carboxypeptidase treatment of the alpha-subunit) and lutropin beta. The results indicate that removal of tyrosines alpha 92 and alpha 93 results in a decrease of the intensity of the 235-nm band of ovine lutropin (at pH7.5) as well as that of the 250-nm band observed under alkaline conditions. It is therefore concluded that the 250-nm band observed in alkaline solutions of lutropin arises (at least partially) from the red shift produced in the short-wavelength optically active band of tyrosines alpha 21, alpha 92, and alpha 93 upon ionization.

Amino Acids↗
PubMed1976

Renal and hepatic lysosomal catabolism of luteinizing hormone.

Following an intravenous injection of tritiated ovine lutenizing hormone (LH) into mature male rats, the liver and kidneys accumulate a significant portion of the non-excreted hormone. The subcellular distribution of total radioactivity in both tissues was found to be similar to that of beta-galactosidase, a lysosomal enzyme marker. Moreover, the subcellular fraction with the highest relative specific activity of beta-galactosidase exhibited the highest degradation rate of endogenous hormone under in vitro conditions. Based on these and other observations, it is concluded that the intracellular catabolism of LH by these tissues is due to lysosomal enzymes. An analysis of the radioactive degradation products produced by a lysosomal-rich subcellular fraction showed the presence of free amino acids and oligopeptides. Thus, the uptake and degradation of the hormone by these tissues appear to occur by endocytosis followed by lysosomal catabolism. This phenomenon may represent a regulatory role in the control of (circulating) hormone concenttrations.

Animals↗
PubMed1976

Biotransformations of pituitary luteinizing hormone in serum and urine. I. Association with serum components.

In the effort to elucidate the nature of luteinizing hormone (LH) in the circulation, studies in adult male rats have been conducted using a highly purified and well-characterized tritiated and methylated ovine pituitary LH, a derivative which retains full biological activity. Following an intravenous injection of the radioactive hormone, serum chromatograms (molecular exclusion chromatography) are characterized by three radioactive components. The first one, a rapidly formed high molecular weight heterogeneous fraction, involves the non-covalent interaction of the hormone with one or more circulating proteins. These high molecular weight complexes are cleared rather slowly from the circulation, relative to the non-associated hormone. A second component co-chromatographs with control hormone and is rapidly cleared from the circulation. These two fractions retain full biological activity judged by their ability to stimulate testosterone production in Leydig cell suspensions. A third component is observed in the circulation about 15 minutes after injection and was shown to represent tritiated amino acids. The lag period for the appearance of the labeled amino acids in the circulation correlates with the kinetics of hepatic and renal uptake and subsequent lysosomal catabolism of [3H]methylated-LH. The high molecular weight fraction may represent a physiologically important circulatory storage form for LH by conferring a relatively long circulatory half-life. This appears to be brought about mainly by reduced urinary excretion, presumably due to its high molecular weight.

Animals↗
PubMed1976

Biotransformations of pituitary luteinizing hormone in serum and urine. II. Evidence for reduced potency following urinary excretion.

The renal clearance of tritiated and methylated ovine pituitary luteinizing hormone (LH) has been determined in mature male rats, and the urinary product has been characterized with regard to molecular size, charge, and heterogeneity. [3H]Methylated-LH was found to have a renal clearance of 0.17+/-0.03 ml/min/100 g body weight, compared to a value of 0.99+/-0.18 for tritiated inulin. This indicates that LH is not secreted and that much of the filtered hormone is reabsorbed from the tubular lumen. Evidence is presented which shows that urinary ovine [3H]methylated-LH is not extensively degraded; yet, the biological potency is only 10-20% that of pituitary and serum LH. The large reduction in potency is a result of excretion since the in vitro incubation of LH with urine does not produce the same effect. The biotransformation occurring during excretion results in charge heterogeneity of LH; this may arise from either inhibitor binding or enzymic alterations.

Animals↗
PubMed1975

The metabolism of luteinizing hormone. Plasma clearance, urinary excretion, and tissue uptake.

The kinetics of plasma clearance, tissue uptake, and urinary excretion of tritiated ovine pituitary luteinizing hormone in adult male rats are reported. Most of the intravenously injected tritiated gonadotropin is cleared from circulation with a half-life of five minutes, and this is independent of the injected amount of hormone over a wide dose range. It was found that the hormone is rapidly removed from circulation by the kidneys, probably by glomerular filtration, and excreted in the urine. The radioactivity present in the urine is associated with material of the same molecular size as the native hormone and, moreover, the urinary hormone retains a significant amount of biological activity. A small amount of the hormone is catabolized by the kidney and liver, and our data suggest that this occurs in the cortex and hepatocytes, respectively.

Animals↗