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)

We have previously reported a bioassay for human plasma ACTH based upon trypsin dispersed guinea-pig adrenal cells which was sensitive to 100 ng/L ACTH in unextracted human plasma when measured against human pituitary ACTH (1-39) standard 74/555. We now present a bioassay of increased sensitivity (12 ng/L) which incorporates three major changes. The trypsin/trypsin inhibitor step in the cell dispersion protocol has been replaced with collagenase, donor calf serum (3%) has been incorporated into the standard curve and ACTH has been extracted from human plasma and dilutions of standard hormone by a sephacryl bound monoclonal antibody (2A3) directed towards the 25-39 sequence. The extracted standard curve has a detection limit of 6 ng/L and the cells can tolerate up to 50% plasma equivalent concentration. Thus, the improved assay has a detection limit of 12 ng/L ACTH in plasma. The assay can now measure bioactive plasma ACTH levels reliably in the normal range.
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PMID:ACTH adrenal cell bioassay: improved sensitivity (12 ng/L) achieved by immunoextraction of ACTH from human plasma by a monoclonal antibody. 215 72

The effect of chymotrypsin on aldosterone biosynthesis by dispersed rabbit adrenal capsular cells was examined. Bovine alpha-chymotrypsin at concentrations of 10(-7) to 10(-5) M stimulated aldosterone production, and human chymotrypsin had an even stronger stimulatory effect. Bovine trypsin had no effect on aldosterone production by adrenal cells. Chymotrypsin treatment did not change the sensitivity of the adrenal cells to ACTH or angiotensin II. These results suggest the existence of unique chymotrypsin-susceptible sites on rabbit adrenal capsular cells, the digestion of which results in stimulation of aldosterone biosynthesis.
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PMID:Proteolytic activation of aldosterone biosynthesis in rabbit adrenal capsular cells by alpha-chymotrypsin. 255 36

The influence of proteinase inhibitors on the lipotropic effect of somatotropic (STH), adrenocorticotropic (ACTH) and beta-lipotropic (LPH) hormones in adipose tissue was studied in vitro. The effect of STH was found to be completely dependent on the activity of tissue serine proteinases of trypsin and chymotrypsin types. The effect of LPH partly depended on serine proteinases of chymotrypsin type, whereas that of ACTH--on chymotrypsin and carboxylic proteinases. The effects of all the three hormones were also manifested during lysosomal proteolysis. The protease-dependent inhibition was specific for polypeptide hormones and was unobserved in the lipotropic effect of adrenaline. The inhibiting effect of serine proteinase inhibitors on hormones pretreated with blood plasma or proteinases was much weaker than on untreated hormones. In adipose tissue the early insulin-like effect of STH, unlike the late lipotropic effect, was independent of proteolysis. It was assumed that primary proteolysis plays a role in the activation of polypeptide hormones which is necessary for the manifestation of the lipotropic action.
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PMID:[The role of proteolytic processes in the stimulation of lipolysis in the adipose tissue by somatotropin, adrenocorticotropin and beta-lipotropin]. 282 47

The secretion of aldosterone and its responses to stimulation have been studied in rat adrenal zona glomerulosa tissue incubated as intact capsules or as collagenase-dispersed cell suspensions, and in intact perfused rat adrenal glands. Several differences are apparent in the functions of the various preparations. Aldosterone secretion rates are similar in incubated intact capsules and in the perfused gland. Relative to corticosterone, lower yields of aldosterone are obtained in dispersed glomerulosa cell in vitro. This may be related to the loss in the dispersed cells of a pool of tissue steroid (aldosterone or a precursor) which is revealed only in intact tissue incubations by trypsin stimulation of aldosterone secretion. Trypsin-released aldosterone is increased by prior dietary sodium restriction. In addition, differences occur in the responses of dispersed cells and perfused glands to stimulation. Perfused glands from animals on a normal diet are less sensitive to stimulation by ACTH or alpha-MSH, but more sensitive than dispersed cells to angiotensin II amide. In the perfused gland, sensitivity of response (lowest effective concentration) to all three stimulants is increased by prior dietary sodium restriction, in contrast to dispersed cells in which increased sensitivity has been reported only to alpha-MSH. The perfused gland is particularly sensitive to angiotensin II amide, and a bolus administration of 1 amol gives significant stimulation in glands from animals on low sodium intake. Electrical (field) stimulation or dopamine administration at 10(-6) mol/l (which is ineffective in dispersed cells) both depress aldosterone secretion by the perfused gland. The data suggest that the sequestered pool of steroid is utilized in the perfused gland for aldosterone secretion. They furthermore suggest that in the intact gland there are mechanisms, which involve neural components, for intraglandular regulation of aldosterone secretion, which are lost in dispersed cells in vitro. Such mechanisms may be involved in sensitivity increases in sodium depletion.
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PMID:Control of aldosterone secretion in zona glomerulosa cell suspensions and in the perfused adrenal gland of the rat. 282 12

We examined the effects of several in vitro experimental systems on the apparent potencies of putative secretagogues for stimulating ACTH release from rat anterior pituitary cells. Cells were prepared by trypsin digestion and gentle mechanical dispersion. Aliquots of the same cell preparations were tested in 1) a microperifusion system immediately after dispersion (day 0), 2) the same microperifusion system after 4 days of static suspension culture on a layer of Sephadex G-10 gel particles (day 4), 3) a static suspension system after 4 days of static suspension culture, and 4) a static monolayer system after 4 days of monolayer culture. Ovine CRF stimulated release of similar amounts of ACTH in all of the systems on days 0 and 4, except in one experiment, in which the response was less on day 4. Arginine vasopressin (AVP), oxytocin, and angiotensin II all appeared to be more potent in day 4 than in day 0 cells in the perifusion system, and the synergism of AVP with ovine CRF was also increased. Dioctanoylglycerol, which directly activates protein kinase-C, and forskolin, which directly activates adenylate cyclase, both stimulated greater release in day 4 cells. The mechanism(s) responsible for the difference in the responses of day 0 and day 4 cells is unknown. Epinephrine had only a small effect in the microperifusion system, but both epinephrine and norepinephrine had potencies comparable to AVP in the static suspension and monolayer systems. This was not due to prolonged exposure to the catecholamines, suggesting that these agents may act on other anterior pituitary cells to release metabolic products that secondarily stimulate the corticotrophs to release ACTH. The same situation appears to be true for atrial natriuretic factor. Gastrin-releasing peptide, its bioactive COOH-terminal half, which was active in a rat urinary bladder smooth muscle assay, its amphibian analog, bombesin, and cholecystokinin (26-33) were devoid of ACTH-releasing activity in all of the systems, in contrast to the findings of others. Since 4-day culture of dispersed cells improved most of their responses and diminished none, we postulate that they may more closely resemble normal pituitary cells in function, and since cellular metabolites are unlikely to accumulate in the interstitial fluid of the pituitary gland, we propose that the secretory functions of cells in perifusion systems may more closely resemble those in the pituitary gland in situ than they do in static incubation systems.
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PMID:Effects of several in vitro systems on the potencies of putative adrenocorticotropin secretagogues on rat anterior pituitary cells. 283 88

A human plasma CRH-binding protein (CRH-BP) was identified and characterized by chemical cross-linking of 125I-Tyr-hCRH to human plasma using disuccinimidyl suberate. The apparent mol wt of the cross-linked complex determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography was approximately 43,000. The mol wt was slightly lower in the nonreduced state, suggesting the presence of intramolecular disulfide bonds. Subtracting the mol wt of 125I-Tyr-CRH, the BP appeared to have a mol wt of approximately 38,000. Binding was specific since the appearance of the 43,000 dalton band was not affected by unlabeled ACTH, vasopressin, serum albumin, or gamma-globulin, but was inhibited by unlabeled hCRH dose dependently. Pretreatment of plasma with 0.1 mol/L HCl, 0.01 mol/L NaOH, 10 mmol/L dithiothreitol, or trypsin before cross-linking abolished its ability to bind 125I-Tyr-hCRH. Rat, rabbit, or goat plasma or human cerebrospinal fluid did not bind 125I-Tyr-CRH. It is unlikely that CRH-BP is a CRH receptor, because the estimated mol wt of the CRH-BP is smaller than the reported size of CRH receptors, and the CRH-BP did not bind to ovine CRH. The binding of 125I-Tyr-CRH to CRH-BP decreased in the third trimester of pregnancy, when plasma CRH levels were markedly elevated. However, after dissociating endogenous CRH from the CRH-BP, the binding was almost the same as in nonpregnant subjects. In addition, CRH-BP inhibited CRH-induced ACTH secretion from cultured rat anterior pituitary cells. We conclude that most of the increased plasma CRH found in pregnant women is bound to CRH-BP, and so is inactive, therefore plasma ACTH levels do not increase to above the normal range.
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PMID:Characterization of corticotropin-releasing hormone binding protein in human plasma by chemical cross-linking and its binding during pregnancy. 284 56

N-terminal analysis of the products of hydrolysis of angiotensin, ACTH and the oxidized B chain of insulin after 4 h incubation with trypsin and urokinase reveals a great qualitative similarity in the action of the two enzymes. As expected, the rates of hydrolysis differ significantly and are much higher in the case of trypsin catalysis than in the case of urokinase catalysis. Unexpectedly, however, a decrease in the difference between the catalytic activity of the two enzymes, by increasing the number of Arg and Lys residues present in the substrate, has been observed.
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PMID:Action of urokinase and trypsin on angiotensin, ACTH and the oxidized B chain of insulin. 299 38

Rat gastric antrum, duodenum, pancreas, and spleen were extracted in acetic acid, treated with acetone, and purified on a C-18 cartridge. These extracts, in a dose equivalent to one respective organ, were examined for CRF bioactivity in vitro using rat half pituitaries, with gastric antrum extract showing a significant CRF activity. The antrum extract showed a dose-related CRF activity in vitro using rat pituitary cell culture, and the dose-response curve appeared to be parallel with that of synthetic rat hypothalamic CRF. Subsequent ion-exchange chromatography on a SP-Sephadex column showed that antrum CRF coeluted with basic materials (SP-III fraction), while rat hypothalamic CRF coeluted with weakly basic materials (SP-II fraction). The SP-III fraction was further purified by gel filtration on Sephadex G-50. CRF activity was eluted in two areas: large mol wt fraction (10,000-15,000) and small mol wt fraction (1500-2000). Hypothalamic CRF was eluted between them. The CRF activities of the two fractions were completely abolished by trypsin digestion, suggesting a peptide nature. The large molecular weight fraction exhibited a steeper dose-response curve than the hypothalamic CRF in vitro using cell culture, and the response to a dose equivalent to two antra exceeded the maximum response exhibited by the hypothalamic CRF. However, the fraction failed to increase serum corticosterone when injected in pharmacologically blocked rats. On the other hand, the small molecular weight fraction showed a lesser CRF activity and a similar dose-response curve to that of the hypothalamic CRF as tested in vitro. This fraction significantly stimulated corticosterone secretion in vivo as well. The small molecular weight activity did not appear to be due to other peptides or amines which have been reported as causing ACTH release. Although the physiological roles of the small molecular weight antrum CRF are unknown, it is possible that this CRF plays a role during stress as a tissue CRF.
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PMID:Corticotropin-releasing activity in the rat gastric antrum. 301 62

Bovine parathyroid chromogranin A inhibits the cleavage of Z-Ala-Lys-Arg-AMC by either trypsin or IRCM-serine protease 1 (IRCM-SP1), a putative novel processing enzyme originally isolated from porcine pituitary anterior and neurointermediate lobes. On larger substrates, chromogranin A is a reversible competitive inhibitor of the cleavage at pairs of basic amino acids by IRCM-SP1. The substrates tested included pituitary ACTH and adrenal medulla pro-enkephalin-derived peptides such as the 8.6 kDa synenkephalin-containing precursor and peptide B. Chromogranin A is itself selectively processed by IRCM-SP1, and ACTH was shown to compete for such cleavage. These data suggest that chromogranins as a class of acidic proteins could participate in the tissue-specific processing of pro-hormones.
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PMID:Chromogranin A can act as a reversible processing enzyme inhibitor. Evidence from the inhibition of the IRCM-serine protease 1 cleavage of pro-enkephalin and ACTH at pairs of basic amino acids. 302 46

The critical evaluation of isolation methods for obtaining the adrenocortical cell suspension due to trypsin or collagenase digestion was done. Some collagenase advantages were indicated by morphological observations on the staining smears as well as by ACTH stimulation test. The cytochemical reactions for enzyme activities had the limited applications for those purposes. It also appeared that commonly applied dye exclusion tests were inadequate for characterization of cell suspension. The possible role of the adrenocortical cell debris in the basal corticosterone production was pointed out. The maintenance of the sex dimorphism and the functional differences in the adrenocortical cells isolated from male and female rats have been observed.
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PMID:Cytochemical and functional evaluation of ACTH responsive isolated rat adrenocortical cells. The maintenance of sex differences. 304 Apr 82


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