Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effects of various chemical and enzymatic treatments on the biological activity of porcine luteinizing hormone-releasing hormone (LH-RH) are described. This experiment was performed before the elucidation of the structure of LH-RH. LH-RH activity was abolished by the following endopeptidases: chymotrypsin, subtilisin, papain, and thermolysin, but not by pepsin or trypsin. Exopeptidases did not affect LH-RH activity, but a purified preparation of pyrolidone carbosylpeptidase did. The amino acid sequence of LH-RH/FSH-RH was established to be (pyro)Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Amine. This decapeptide lacks both the Amine terminus and the COOH terminus. Its Amine-terminal dipeptide sequence,(pyro)Glu-His, is similar to that of tyrotropin-releasing hormone. The lack of inactivation by the exopeptidases is in good agreement with these findings. Treatment with various chemical reagents showed that tyrosine, histidine, tryptophan, and arginine in LH-RH are important for its biological activity. Nitrous acid and Edman degradation did not inactivate LH-RH. These results are also in agreement with the determined structure of LH-RH. This hormone showed a high follicle-stimulating hormone-releasing hormone (FSH-RH) activity. The inactivation of LH-RH was always accompanied by a loss of FSH-RH activity. These experiments also shed some light on the structure-activity relationship of this hormone.
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PMID:Studies on the properties of hypothalamic luteinizing hormone-releasing hormone. 494 14

The Sertoli cell is thought to play a significant role in the hormonal regulation of spermatogenesis within the rat testis. Little, however, is known about Sertoli cell function in man, largely because of the difficulties associated with the isolation of pure cell populations from human tissue. We have now developed a rapid and reproducible technique for establishing a human Sertoli cell monolayer culture. This has involved mechanical separation of the tissue, sequential trypsin and collagenase enzyme digestion, and final disruption of tubules by passage through a wire mesh grid. Using this technique, primary cultures can be maintained for up to 45 days. Ultrastructural studies of these cells have demonstrated the presence of the perinucleolar spheres, cell to cell junctional complexes, abundant lipid droplets, and smooth endoplasmic reticulum, all characteristic of Sertoli cells. Furthermore, biochemical markers of animal Sertoli cells, androgen-binding protein and gamma-glutamyl transpeptidase, have also been identified in these human cells. Concentrated media electrophoresed on nondenaturing gels containing 2 nM [3H]dihydrotestosterone produced a single peak of bound activity which coelectrophoresed with rat androgen-binding protein. This binding activity persisted despite media changes, thus ruling out contamination by serum binding proteins; fresh media lacked demonstrable binding activity. Using a colorimetric assay, these cells were also found to contain significant gamma-glutamyl transpeptidase activity compared to human foreskin fibroblasts and Leydig cells. Enzyme activity increased in a characteristic dose-response fashion in the presence of FSH (0.05-0.5 microgram/ml) and dibutyryl cAMP (0.1-1 microgram/ml), but not with LH or testosterone. These data offer the first demonstration of human Sertoli cells in monolayer culture and their production of a marker specifically regulated by FSH.
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PMID:Characterization of human Sertoli cells in vitro. 612 20

A newly discovered small peptide purified from rat follicular fluid stimulates the pituitary to release FSH and LH in vitro as well as in vivo. Dialysates of crude acid extracts of ovarian follicular tissue and fluid from rats pretreated with PMS gonadotropin stimulate the secretion of both LH and FSH, but not PRL, GH, or TSH, in a pituitary monolayer culture system. This stimulating factor, named gonadocrinin for operational facility, is smaller than 3500 daltons; its biological activity disappears after treatment with trypsin. Gonadocrinin is not recognized by two-antisera binding the decapeptide LRF even though D-Phe2,D-Trp6-LR, an LRF analog antagonist, competitively inhibits the activity of ovarian gonadocrinin. Cultured rat granulosa cells also secret substances with gonadocrinin activity in vitro, indicating that the granulosa cells probably are in vivo the source of gonadocrinin. A crude preparation of gonadocrinin given iv to rats on the second day of diestrus induced secretion of LH comparable to that produced by a 250-ng LRF injection. Gonadocrinin has chemical characteristics different from those of LRF. When purified gonadocrinin or LRF was applied to an identical isocratic high pressure liquid chromatography system, LRF was eluted at a position different from that of gonadocrinin, indicating that, chemically, gonadocrinin is not identical to the hypothalamic decapeptide, LRF.
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PMID:Gonadocrinins: peptides in ovarian follicular fluid stimulating the secretion of pituitary gonadotropins. 616 33

Studies of Sertoli cell structure, maturation, and function have been aided by the use of in vitro systems. Although numerous papers have appeared that utilize the Sertoli cell culture model, few papers have dealt with the characterization of these cells under various culture environments. Recently, it has been reported that the addition of serum to the culture medium prevents induction of long cytoplasmic appendages in cultured Sertoli cells that have been treated with FSH, TSH, or c-AMP. The purpose of this investigation was to determine which serum components, obtained by gel filtration, are capable of inhibiting the morphological response induced by FSH, TSH, or c-AMP. Sertoli cell-enriched cultures were prepared using collagenase and trypsin digestion, each followed by gravity sedimentation. Untreated cells grown on plastic or glass substrates assumed an epithelioid appearance after several days. Cells treated with FSH, TSH, or c-AMP formed long cytoplasmic appendages after 1-2 days. This response was prevented or reversed by the addition of fetal calf serum (10%), crystallized bovine serum albumin (0.25%-2%), or purified albumin obtained by gel filtration of whole serum (0.25%). It was also found that fractions that elute between the void volume and the initial albumin fractions (molecular weights of approximately 50,000 and greater) mimic the hormone-induced response after only 10-12 hours. The results of this investigation indicate that albumin is the primary serum component responsible for inhibiting morphological alterations induced by FSH, TSH, and c-AMP. Furthermore, it is apparent that the production of long filamentous cytoplasmic appendages in Sertoli cells can be induced by a wide variety of substances.
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PMID:Effects of serum components on the morphology of Sertoli cells in culture. 625 34

The properties of adult rhesus monkey testicular FSH receptor was investigated in these experiments. The interaction of 125I-labeled human FSH with a monkey testicular particulate fraction is a time- and temperature dependent phenomenon. Equilibrium of hormone-receptor interaction occurred by about 4-6 at 37 or 34 C, was slow at 25 C, and was extremely slow at 4 C. Maximum binding occurred at pH 7-7.5, with a requirement of 5-10 mM MgCl2 or CaCl2. The half-life of the receptor with exposed sites for hormone interaction was temperature related (1 h at 37 C, 1.5 h at 34 C, 6 h at 25 C, and 36 h at 4 C). Occupancy of these sites by the labeled hormone rendered the receptor more stable. The hormone-receptor complex was highly stable, as shown by the fact that excess unlabeled hormone was unable to displace the already bound labeled hormone from the receptor. Conditions unfavorable for hormone-receptor interaction, such as pH 5.0 or pH 10 or high salt concentration (0.5 M MgCl2), induced the maximum dissociation of the preformed hormone-receptor complex. The primate testis FSH receptor was inactivated by trypsin, phospholipase C, and reducing agents, but it was not influenced by nucleases. Neuraminidase treatment of the particulate receptor may have enhanced its ability to bind labeled human FSH.
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PMID:Studies on primate gonadotropin receptors: characterization of the rhesus monkey testicular follicle-stimulating hormone receptors. 629 May 23

To evaluate the role of nonsteroidal, follicular fluid proteins in folliculogenesis, the 10-55% saturated ammonium sulfate fraction of pooled human follicular fluid was dialyzed against 0.025 M Tris/HCl (pH 7.5) using 10,000 molecular weight exclusion membranes, then passed through agarose immobilized textile dye. Activity was determined by test fraction inhibition of human menopausal gonadotropin (2 U human LH/FSH . day), induced ovarian weight, and serum estradiol increase in hypophysectomized, diethylstilbesterol-treated, 25-day-old female rats. Specific inhibition (89 +/- 6.8% SEM) of ovarian weight increase was found in the material (2 ml) eluted from an Orange A column with KCl (1.5 M, pH 6.8). Inhibitory activity of the Orange A-bound material, which eluted through a standardized Sephadex G-50 column, corresponded to a molecular weight of 13,000-25,000. Isoelectric focusing on a Sephadex G-15 support bed or ampholyte displacement chromatography of Orange A bound material demonstrated inhibitory activity at pH 3.5-4.5 and 6.5-7.0. No demonstrable activity was found in similar fractions eluted through a Concanavalin A-Sepharose 4B column before or after addition of alpha-methyl-D-mannoside (2 M, pH 7). When active fractions were heated (56 C, 1 h) or exposed to trypsin (10 mg/100 ml), activity was lost. When aliquots of the saturated ammonium sulfate-extracted, dialyzed, Orange A-bound eluent were separated by high performance liquid chromatography using gel exclusion columns, activity in the bioassay was recovered in the 13,000-35,000 molecular weight range. Although confirmatory data await further studies, it is tempting to speculate that this protein(s) may be an important inter- and/or intraovarian regulator of follicular response to gonadotropins.
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PMID:Identification of proteins in pooled human follicular fluid which suppress follicular response to gonadotropins. 640 Nov 85

Treatment of ovine pituitary cell cultures with an acetone powder of porcine follicular fluid (APPFF; 50 micrograms/ml) decreased FSH secretion 60%, did not alter basal LH secretion, but increased by 2- to 3-fold the effectiveness of LHRH or D-Lys6-LHRH (10(-8) M) in releasing LH. Chromatography of APPFF on Matrex Gel Red A (MGRA) yielded a protein fraction (MGRA-IV) in which both FSH-inhibiting and LHRH-enhancing activities were enriched 8-fold. Both activities were destroyed by trypsin, but both were highly resistant to heat. The apparent mol wt of the active substance(s) was greater than 10,000. The LHRH-enhancing effect of MGRA-IV was reversible and declined, with an apparent half-life of 7 h, when MGRA-IV treatment was discontinued. There was too little estrogen in either APPFF or MGRA-IV to account for any of the activities. These results demonstrate that porcine follicular fluid contains LHRH-enhancing activity along with classical inhibin activity and that both activities may be linked in one molecule. These dual activities may be important in a number of species.
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PMID:Porcine ovarian inhibin preparations sensitize cultured ovine gonadotrophs to luteinizing hormone-releasing hormone. 643 Jun 75

There are many convincing arguments to accept the existence of inhibin. This hormone is produced inside the seminiferous tubules by the Sertoli cells in males and by the granulosa cells of the follicule in females. The biological, immunological and chemical characteristics of testicular and ovarian inhibin are identical so that it could be speculated the same molecule is secreted by both organs. This hormone is not a knownsteroid but is a protein substance. Thus, its biological activity is destroyed by trypsin and pepsin digestion and by heating at 60 degrees for 30 minutes. Furthermore, immunization with inhibin from rete testis fluid induces antibodies capable of neutralizing endogenous inhibin of adult male and female rats. This polypeptide hormone is not identical neither to ABP nor to a fragment of gonadotrophins. The molecular weight is not yet exactly defined and the possibility exists that two forms of inhibin are present in RTF: one of high (greater than 10,000 Daltons) and the other of low molecular weight. The high M.W. species could be a polymer or alternatively the combination of native inhibin and a carrier substance or unique precursor molecule. Inhibin preparations selectively depress the synthesis and the release of FSH in pituitary cell culture. The threshold dose to affect the LH production is higher than that active on FSH secretion. Furthermore, they reduce LH-RH content of hypothalamus maintained in organ culture. In animals, inhibin induced effects are depending on both hypothalamus and pituitary actions according to the functions of these two structures. In that sense, apparently contradictory results are obtained in short and long term castrated animals. Inhibin does not modify TSH, GH and prolactin in vivo and in vitro. This substance displays an inhibition on the synthesis of DNA in the testis of pubertal male rats and depresses the maturation of follicle in female.
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PMID:[Inhibin: new gonadal hormone (author's transl)]. 677 85

A purified basic protein from bull seminal plasma having inhibin activity was labeled with 125I and tested for binding with ovine pituitary membrane fractions. The bound radioactivity could be eluted under acidic conditions and shown to rebind to fresh pituitary membranes. The properties of the eluted labeled inhibin were similar to the unlabeled fraction. The eluted labeled inhibin exhibited specific binding to the membranes, which was displaceable in a dose-dependent manner by an unlabeled active fraction. Only those fractions in the purification scheme which had inhibin activity also competed for the binding. A bovine follicular fluid fraction with molecular weight greater than 10 000 and inhibin activity also displaced the bound radioactivity from the membranes. Other purified hormones such as ovine FSH, LH or their subunits, prolactin or bovine serum albumin, dialyzed serum or unrelated basic macromolecules such as lysozyme, polylysine, histones, had no influence on the binding of labeled inhibin to ovine pituitary membranes. Synthetic LH-RH also failed to displace the labeled inhibin from the membranes. The binding was sensitive to heat and trypsin treatments. The data are consistent with the direct action of inhibin on the pituitary and demonstrate the existence of binding sites for the active fraction in this target.
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PMID:Binding of an inhibin-like protein from bull seminal plasma to ovine pituitary membranes. 678 37

The production of inhibin by cultures of Sertoli cells from 21-day-old rats was assessed by the use of an in vitro bioassay using rat pituitary cells in culture. Sertoli cell culture media (SCCM) caused a dose-dependent suppression of the pituitary cell FSH content which was parallel with that of an ovine testis lymph preparation used as an inhibin standard. SCCM also caused a dose-dependent inhibition of FSH secreted by pituitary cells in response to 10 nM GnRH stimulation. The FSH-inhibitory activity in SCCM was destroyed by heat or trypsin digestion and could not be attributable to the steroid content of the medium, since ether extraction caused no change in the inhibitory activity. The inhibin activity in SCCM was not due to cytotoxicity in the bioassay, since the LH cell content was unchanged and the media produced no change in the release of 51Cr from labelled pituitary cells, a parameter which has been shown to be a useful test of cytotoxicity. Sertoli cell cultures produced inhibin for the 8-day duration of the cultures. The amount of inhibin produced was proportional to the number of Sertoli cells initially plated. If foetal calf serum was included for more than the initial 48 h, the spent medium caused toxic effects in the pituitary cells as evidenced by an increase in 51Cr release from 51Cr-labelled pituitary cells. Similar toxic effects were found if the lyophilized spent media contained cellular debris. A dose-dependent increase in inhibin activity was observed in the presence of graded doses of FSH (0.05-5 micrograms/ml NIH-FSH-S13).
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PMID:Inhibin production by Sertoli cell cultures. 681 56


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