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 mouse anterior pituitary tumor cell line, AtT-20, targets secretory proteins into two distinct intracellular pathways. When the DNA that encodes trypsinogen is introduced into AtT-20 cells, the protein is sorted into the regulated secretory pathway as efficiently as the endogenous peptide hormone ACTH. In this study we have used double-label immunoelectron microscopy to demonstrate that trypsinogen colocalizes in the same secretory granules as ACTH. In vitro mutagenesis was used to test whether the information for targeting trypsinogen to the secretory granules resides at the amino (NH2) terminus of the protein. Mutations were made in the DNA that encodes trypsinogen, and the mutant proteins were expressed in AtT-20 cells to determine whether intracellular targeting could be altered. Replacing the trypsinogen signal peptide with that of the kappa-immunoglobulin light chain, a constitutively secreted protein, does not alter targeting to the regulated secretory pathway. In addition, deletion of the NH2-terminal "pro" sequence of trypsinogen has virtually no effect on protein targeting. However, this deletion does affect the signal peptidase cleavage site, and as a result the enzymatic activity of the truncated trypsin protein is abolished. We conclude that neither the signal peptide nor the 12 NH2-terminal amino acids of trypsinogen are essential for sorting to the regulated secretory pathway of AtT-20 cells.
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PMID:In vitro mutagenesis of trypsinogen: role of the amino terminus in intracellular protein targeting to secretory granules. 304 Jul 70

Immunoreactive CRH was detected in extracts of human term placentae [5.2 +/- 0.8 (+/- SE) pmol/g wet wt; n = 9]. Molecular sieve chromatography revealed three size classes of immunoreactive CRH. The major species eluted with the Kav of synthetic rat CRH; the minor species had apparent mol wt (MW) of 18,000 and 8,000. A placental CRH-(1-41)-sized peptide was isolated by fractional acetone precipitation, molecular sieve chromatography, and sequential reverse phase high performance liquid chromatography steps. This peptide had the same chromatographic behavior as did rat CRH in all high performance liquid chromatographic isolation steps as well as the same UV absorbance to immunoreactive CRH ratio after the final purification step. Purified placental CRH stimulated ACTH release from anterior pituitary tissue in a dose-dependent manner and was equipotent with synthetic rat CRH. Partial sequencing indicated that 32 amino acids of this peptide are identical to those of rat and human CRH (sequence deduced from genomic sequence), and comparative peptide mapping with rat CRH provided further evidence that the placental CRH-like peptide is very homologous if not identical to CRH. The high mol wt placental CRH fractions also were partially purified by acetone precipitation, immune affinity chromatography, and gel filtration. Neither of these materials [big form (MW, 18,000) or intermediate form (MWr, 8,000)] stimulated ACTH release from rat pituitary tissue in vitro. Limited trypsin digestion of the highest MW CRH, followed by gel filtration analysis, resulted in conversion to the smaller [8,000 MW-sized and CRH-(1-41)-sized] forms. The detection of a CRH-like peptide in placenta together with our previous demonstration of plasma immunoreactive CRH in pregnant women suggest that the placenta synthesizes and secretes CRH into the maternal circulation.
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PMID:Isolation and characterization of a corticotropin-releasing hormone-like peptide from human placenta. 326 20

Cultured Y-l mouse adrenal tumor cells treated with ACTH (0.5 U/ml) rounded, formed filopodia and numerous thin microvilli, and produced steroids. Rounding, filopodia and bleb formation occurred for trypsin (0.01%), and hyaluronidase (0.1%), treated cells; but neither affected control or ACTH-stimulated steroidogenesis. Neuraminidase treatment (20 mU/ml) caused rounding, thin microvilli, bleb formation, slightly increased steroid production and prevented subsequent ACTH effects. Neuraminidase appeared to alter a carbohydrate-containing ACTH receptor preventing ACTH binding. We conclude rounding and steroidogenesis are not always associated.
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PMID:Rounding and steroidogenesis of enzyme- and ACTH-treated Y-l mouse adrenal tumor cells. 608 55

ACTH in vivo induces the formation of several steroidogenic factors in both cytosol and extramitochondrial particulate fractions of rat adrenal. Cycloheximide prevents the formation of these factors. Here we show the presence of a cytosolic steroidogenic component (C1) which is cycloheximide-sensitive and not ACTH-dependent. C1 is able to solubilize an ACTH-dependent steroidogenic factor (C2) from particulate fractions resulting in the release of the rat-limiting constraint of mitochondrial steroidogenesis. The thermolabile and trypsin-resistant factor C1 has an apparent mol.wt of 28,000 Daltons. In contrast, the cycloheximide-sensitive factor C2 from extra-mitochondrial fractions of ACTH-treated rats comigrates on Sephadex G-10 with phospholipids. Endogenous phospholipids isolated from particulate adrenal fractions of ACTH-treated rats or exogenous phospholipids will also stimulate steroidogenesis in vitro. Indeed, cytosolic solubilizing factor C1 enhances the exogenous phospholipid effect 3-4-fold. The results taken together suggest that C1 may be very similar to a well defined phospholipid exchange protein and C2 is itself a phospholipid. Both factors seem to be obligatory for the ACTH-induced steroidogenesis.
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PMID:Rat adrenal cycloheximide-sensitive factors and phospholipids in the control of acute steroidogenesis. 608 29

Five peptides derived from pro-corticotropin/endorphin (pro-ACTH/endorphin), the pituitary corticotroph cell prohormone, were bioassayed with isolated rat adrenocortical cells: alpha- and beta-melanotropin, beta-lipotropin, beta-endorphin, and the amino-terminal region of pro-ACTH/endorphin known as "16k fragment." The effect of each on steroidogenesis was measured at potentially physiological concentrations (0.01-1 nM) in both the absence and presence of varying concentrations of ACTH-(1-24). Of the peptides tested, only 16k fragment, the amino-terminal region of pro-ACTH/endorphin, has a slight but significant potentiating effect on ACTH-(1-24) action. Prior treatment of 16k fragment with trypsin for 30 sec dramatically increases this dose-dependent synergism. Experiments performed in vivo with hypophysectomized female rats indicate that the trypsin digest of 16k fragment stimulates cholesterol ester hydrolase (cholesterol esterase; sterol-ester acylhydrolase, EC 3.1.1.13) activity in the adrenal cortex but fails to activate cholesterol side-chain cleavage. The effect of the trypsinized material can therefore be qualitatively distinguished from that of ACTH-(1-24). When both ACTH-(1-24) and the digest are administered together, a synergistic increase in serum corticosterone concentration results. We propose that a portion of 16k fragment molecule may play a hormonal role in the control of adrenocortical steroidogenesis.
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PMID:Adrenocortical response to corticotropin is potentiated by part of the amino-terminal region of pro-corticotropin/endorphin. 624 28

Basal and modulated secretion of ACTH and lipotropin (LPH) by cultures of trypsin-dispersed cells of a biopsy of a human corticotropic adenoma have been examined. ACTH secretion was detectable throughout the period of culture (13 days) but declined steadily from an initial production rate of 238 +/- 124 ng/3 X 10(5) cells/12 h. The time course of secretion showed a slower phase over the first 4 h, with increases up to 12 h. An extract of rat stalk median eminence caused a significant (P less than 0.005) dose-dependent increase in both ACTH and LPH secretion during 30 min. The patterns of response for ACTH and LPH were very similar; both exhibited a decline in the basal release of peptide subsequent to the period of stimulation. The addition of hydrocortisone (0.2 micrograms/ml) did not suppress basal ACTH secretion during 30 min but significantly (P less than 0.05) inhibited stimulation produced by rat stalk median eminence extract. Arginine vasopressin (dose range, 1-9 ng/ml) significantly (P less than 0.025) stimulated both ACTH and LPH secretion during 30 min. The patterns of response were again very similar. Serotonin (dose range, 0.01-10 micrograms/ml) did not affect ACTH secretion during incubations of 30 min to 4 h. The results obtained with the cell cultures of a human corticotropic cell adenoma concur with in vivo findings of incomplete autonomy of secretion, parallel secretion of ACTH and LPH in response to provocative stimuli, and suppression by corticosteroids. The technique has potential for exploring the cellular mechanisms controlling secretion by human corticotropic adenomas as well as the nature of the hormones produced.
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PMID:Adrenocorticotropin and lipotropin secretion by dispersed cell cultures of a human corticotropic adenoma: effect of hypothalamic extract, arginine vasopressin, hydrocortisone, and serotonin. 625 Nov 5

The role of the carbohydrate in the stabilizaion and protection of the glycoprotein, pro-opiocortin, from non-specific proteolysis by trypsin and blood proteases was studied in vitro. [3H]Arginine-labeled, glycosylated and non-glycosylated forms of pro-opiocortin were isolated from frog neurointermediate lobes and subjected to proteolysis by trypsin. The non-glycosylated form was degraded by trypsin more rapidly than the glycosylated form. Analysis of the tryptic products after trypsin treatment, showed that the non-glycosylated pro-opiocortin was cleaved to unidentified peptides within 1 min, whereas the glycosylated prohormone yielded 2 products, mol. wt. 23 000 ACTH and mol. wt. 21 000 ACTH, synthesized by the intact neurointermediate lobe. These data provide direct evidence in support of the hypothesis, derived from studies on the intact lobe (Loh and Gainer, 1978, 1979) that the glycosylation of pro-opiocortin is important: (1) to protect it against non-specific proteolysis in situ, and (2) to direct processing by limiting proteolysis. In addition, we demonstrate that glycosylated forms of ACTH are much more stable in blood than non-glycosylated forms.
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PMID:Evidence that glycosylation of pro-opiocortin and ACTH influences their proteolysis by trypsin and blood proteases. 625 21

The amino-terminal region of the common corticotropin/beta-lipotrophin (beta-LPH) precursor has been identified in the AtT-20 mouse tumor cells as a glycopeptide with an apparent molecular weight of 16,000 (the '16K fragment'). A third melanotropin core sequence or gamma-MSH similar to that found in ACTH and beta-LPH was predicted to occur in this glycopeptide from the complementary DNA sequence of mRNA isolated from bovine pituitary intermediate tissue. Recently, the mouse of 16K fragment has been found to have a small but significant potentiation on the corticosteroidogenesis elicited by ACTH in a static cell system, an effect that could be enhanced when the glycopeptide was pretreated with trypsin. This synergism could also be mimicked by synthetic gamma-MSH peptides in vitro and in vivo. We report here the potentiating properties of a naturally occurring human pro-gamma-MSH glycopeptide on the ACTH-induced steroidogenic response of isolated perfused rat and human adrenocortical cells.
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PMID:Circulating human pituitary pro-gamma-melanotropin enhances the adrenal response to ACTH. 626 50

Anterior pituitaries of adrenalectomized and sham operated adult rats were dispersed by trypsin and cultured for 4 and 8 days. Adrenalectomy caused a moderate increase in number of corticotrophs in both zero-time cell suspensions and cultures. There was a pronounced elevation of immunoreactive ACTH content in both cells and media and an enhanced secretory response to stimulation of cultures with stalk-median eminence extract containing cortiocotropin releasing (CRF) activity. Some cells identified as corticotrophs by a specific immunostaining incorporated tritiated thymidine into their nuclei suggesting their ability to enter the cell cycle. The relatively smaller increase in number of ACTH cells and the considerably higher ACTH producing capacity of the corticotrophs after adrenalectomy seem to be inconsistent with the quantal response model of hormone secretion recently introduced by Rodbard.
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PMID:A quantitative approach to trace the corticotrophs in culture after adrenalectomy. 627 27

When Prozime-10 (P-10), a protease extracted from cultured both of Aspergillus melleus, was injected intravenously into anesthetized dogs, plasma ACTH was increased with a latency of 30 min, and this was followed by remarkable elevation of plasma cortisol in many instances. A similar increase in plasma cortisol was elicited after trypsin and alpha-chymotrypsin were injected. Plasma histamine was raised promptly prior to an increase in plasma ACTH after P-10 in every case. However, in certain cases, changes in cortisol occurred simultaneously with ACTH after P-10. Such a rapid elevation of cortisol can be explained, partly, by direct stimulation of the adrenal cortex by histamine.
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PMID:Anti-inflammatory mechanism of prozime-10, a proteolytic enzyme. 627 22


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