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)

A polyclonal antibody monospecific for an intracellular epitope of the atrial natriuretic factor (ANF)-R1 receptor was produced. The receptor protein (200 pmoles) was purified to homogeneity from bovine adrenal zona glomerulosa (BAZG), reduced, alkylated and digested with trypsin. The tryptic fragments were purified by reverse-phase h.p.l.c. on a C18 column. Based on the sequence of one of these fragments, a peptide was chemically synthesized, coupled to thyroglobulin and injected into rabbits. The antibody obtained was shown to be specific for the R1-type as no receptor was detected in bovine red blood cells (RBC) (which are devoid of ANF receptors) and in NIH-3T3 cell membranes (where only the R2-type is expressed). Several other tissues were screened and comparison of the immunoreactive receptor density estimates with those obtained by ANF binding yielded a correlation coefficient (r2) of 0.965. The minimal detectable dose was typically 3 fmoles/tube and the ED50 of the RIA was 30 fmoles/tube. Cyanogen bromide digestion of the receptor was essential for antigenic detection, indicating that the epitope is probably hindered due to the tertiary structure of the native protein. Moreover, location of the epitope in the kinase homology domain of the receptor, combined with partial tryptic digestion, suggests that the proteolysis-sensitive region of the receptor is located between the transmembrane-spanning domain and the amino acid 586. This method of production of antibodies should be useful to precisely map the amino acids involved in various functions of the receptor.
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PMID:Production of polyclonal antibody to the bovine adrenal atrial natriuretic factor-R1 receptor. 128 Dec 31

Coculture of endothelial cells with atrial cells (R. A. Lew and A. J. Baertschi. Biochem. Biophys. Res. Commun. 163: 701-709, 1989) increased atrial natriuretic factor (ANF) release to 205 +/- 15% (n = 33 experiments) of basal secretion (2.02 +/- 0.33 ng/ml). Stimulation of ANF release by endothelial cells was significantly reduced (P < 0.05) by addition of the calcium channel antagonist nicardipine (Nic, 100 nM; by 69 +/- 4%), the guanylate cyclase activator sodium nitroprusside (SNP, 1 microM; by 97 +/- 27%), or acetylcholine (ACh, 10 microM; by 55 +/- 13%). Endothelial cell-conditioned medium elicited a 62 +/- 10% (n = 10) increase in ANF release. Rat and porcine endothelin (0.1-100 nM) each elicited a dose-dependent increase in ANF release [up to 84 +/- 14% (n = 18) over baseline]. The activity of conditioned medium was not affected by heat or trypsin treatment, but was significantly reduced by addition of Nic or SNP and was attenuated by ACh. Stimulation of ANF by 1 nM synthetic rat or porcine endothelin was also unaffected by heat or trypsin but was significantly reduced by Nic, SNP, and ACh. Addition of endothelin-specific antiserum abolished the ANF stimulatory activity of endothelial cell-conditioned medium. Neither inhibition of superoxide anion by superoxide dismutase nor inhibition of endothelium-derived nitric oxide production by NG-monomethyl-L-arginine affected the ANF release from coculture. Thus endothelial cells release a heat-stable, diffusible ANF stimulatory factor, which is not endothelium-derived relaxing factor or superoxide anion but is biologically and immunologically similar to endothelin.
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PMID:Endothelium-dependent ANF secretion in vitro. 141 54

Atrial natriuretic factor (ANF-R1) receptor is a 130-kDa protein that contains a cytoplasmic guanylate cyclase domain. We report that ATP interacts in an allosteric manner with the ANF-R1 receptor, resulting in reduced ANF binding and enhanced ANF-stimulated guanylate cyclase activity. The modulatory properties of various nucleotides indicate a preference for the adenine family with a rank order of potency of ATP greater than App(NH)p greater than or equal to ADP greater than or equal to AMP while cyclic and guanine nucleotides except GTP are inactive. The negative modulation by ATP of ANF binding is specific for the ANF-R1 receptor subtype since the amount of ANF bound by the guanylate cyclase uncoupled ANF-R2 subtype is increased in the presence of ATP. Furthermore, the effects of ATP on ANF-R1 receptor binding function are still observed with the affinity-purified ANF-R1 receptor, suggesting an allosteric binding site for ATP on the ANF-R1 receptor. In intact membranes, limited proteolysis of the ANF-R1 receptor with trypsin dose-dependently prevents the ATP-induced decrease in ANF binding concomitantly with the formation of a membrane-associated ANF-binding fragment of 70 kDa. These results confirm the direct modulatory role of ATP on hormone binding activity of ANF-R1 receptor and suggest that the nucleotide regulatory binding site is located in the intracellular domain vicinal to the protease-sensitive region.
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PMID:Allosteric modulation by ATP of the bovine adrenal natriuretic factor R1 receptor functions. 165 83

Atrial natriuretic peptide (ANP) is released from the atria and acts to regulate blood volume and pressure. The release of ANP appears to be stimulated by atrial distension, initiated by stretch on the cardiocytes. The purpose of the present study was to develop an assay that would allow for the detection of ANP release from single, isolated cells in the absence of distension. Using the reverse hemolytic plaque assay and antibody raised against human-alpha ANP, the release of ANP was detected from trypsin dissociated rat atrial cells. The specificity of the assay was demonstrated by a 67% reduction in ANP plaque forming cells detected following preabsorption of the anti-sera with rat-alpha ANP. The assay also proved efficient in monitoring changes in ANP secreting cell populations, where an acute treatment with dexamethasone resulted in a doubling of the percentage of atrial cardiocytes detected within a 4 hour antibody incubation. Finally, the assay established that about 52% of the dispersed atrial cardiocytes release ANP. The establishment of a plaque assay for ANP release should assist in addressing questions concerning what hormones may regulate ANP secretion directly and also allow for the determination of ANP secreting cell population dynamics.
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PMID:Release of atrial natriuretic peptide from individual rat cardiocytes. 214 40

The 21 amino-acids endothelium-derived peptide, endothelin, recently isolated by Yanagisawa et al. (Nature 1988; 33, 411-5) possesses potent vasoconstrictive properties in vivo and in vitro. In the present study, we investigated the binding of endothelin on cultured rat aortic smooth muscle cells using 125I-iodotyrosyl-endothelin labelled by the chloramine T method. 125I-endothelin bound to a single class of hight affinity binding sites in vascular smooth muscle cells. After 2 hours incubation at 37 degrees C, dissociation constant (Kd) was 1.2 +/- 0.3 nM and binding capacity (Bmax) was 59 +/- 11 fmol/10(6) cells (n = 5). 125I-endothelin was displaced by unlabelled endothelin with a inhibition constant (Ki) of 0.2 nM, whereas an absence of competition was observed with 1 microM of vasoactive substances such as angiotensin II, arg-vasopressin, atrial natriuretic factor, histamine, epinephrine and norepinephrine, and with the calcium entry blocks nifedipine, diltiazem and D 600. 125I-endothelin binding was not reversible by addition of unlabelled endothelin (1 microM) and not dissociable by acetic acid (10 mM) or trypsin (0.1 p. 100) treatment of the cells. Furthermore, preincubation of vascular smooth muscle cells with endothelin (1 nM) at 37 degrees C induced a rapid down-regulation of endothelin binding capacity by about 50 p. 100. These data indicate that specific endothelin bindind sites are present in smooth muscle cells, and suggest a tight binding or a rapid captation of endothelin into the cell membrane leading to contractile events.
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PMID:[Presence of a specific binding site of endothelin on cultured smooth muscle cells]. 251 Jun 58

We have studied the structure and function of the membrane atrial natriuretic factor R1 (ANF-R1) receptor using limited proteolysis and exoglycosidase treatment. Limited digestion with trypsin of the receptor from bovine adrenal zona glomerulosa membranes resulted in the conversion of the native 130-kDa receptor into a single membrane-associated ANF-binding proteolytic fragment of 70 kDa. The 70-kDa fragment bound ANF with enhanced binding affinity but retained intact ANF-R1 pharmacological specificity and was still sensitive to modulation by amiloride. Trypsin treatment of the membranes produced a dual effect on ANF binding. Low concentrations of trypsin (less than or equal to 25 micrograms/mg of protein) increased ANF binding while higher concentrations dose dependently reduced the binding of the hormone. The increase of ANF-binding activity was associated with the formation of the 70-kDa fragment while the loss of ANF binding paralleled the degradation of the 70-kDa fragment. Low concentrations of trypsin drastically decreased the ANF-sensitive guanylate cyclase activity of the membrane fraction. This loss of catalytic activity strongly correlated with the formation of the 70-kDa tryptic fragment. We also evaluated the effect of ANF binding on the susceptibility of the receptor to proteolytic cleavage. The occupied receptor exhibited a greater sensitivity to trypsin digestion than the unoccupied protein, consistent with the hypothesis that hormone binding induces an important conformational change in the receptor structure. On the other hand, the 70-kDa fragment was much more resistant to proteolysis when occupied by ANF, suggesting that the ANF-binding domain forms a very compact structure.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Topographical characterization of the domain structure of the bovine adrenal atrial natriuretic factor R1 receptor. 255 56

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

Crude extracts of rat atria reduced the basal amount of aldosterone released from rat zona glomerulosa cells and partially inhibited aldosterone stimulation by adrenocorticotropic hormone and angiotensin II. The destruction of this activity by trypsin suggests that the active factor is a peptide, possibly atrial natriuretic factor. These data suggest that atrial natriuretic factor affects sodium excretion by the kidneys both directly and through the inhibition of aldosterone production.
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PMID:Inhibition of aldosterone production by an atrial extract. 632 67

Insulin-degrading enzyme (IDE) is a component of a cytosolic complex that includes multicatalytic proteinase (MCP), the major cytoplasmic proteolytic activity. Insulin, the primary substrate for IDE, inhibits the proteolytic activity of the IDE-MCP complex but not of purified MCP. This provides a regulatory role for IDE in cellular proteolysis and a potential mechanism for intracellular insulin action. To examine the specificity and to explore the mechanisms for the IDE-MCP interaction, we studied the functional interaction of a variety of peptides with the complex. Atrial natriuretic peptide (ANP), relaxin, glucagon, proinsulin, and insulin-like growth factor II (IGF-II) bind to and are degraded by IDE. These peptides have significant inhibitory effects on the chymotrypsin-like and trypsin-like MCP catalytic activities but not the peptidyl-glutamyl hydrolyzing activity. A panel of peptides that are not ligands of IDE had no effect. To explore the potential mechanism for the IDE control of MCP activity, dose response curves for insulin-like growth factor I (IGF-I) and IGF-II effects on MCP chymotrypsin-like activity were determined. IGF-II, which (similar to insulin) is a good substrate for IDE, had a substantial inhibitory effect, whereas IGF-I, which is bound but poorly degraded, had little inhibitory activity on MCP. Proinsulin, another ligand of IDE that is tightly bound but poorly degraded, had a partial effect on MCP activity, but inhibited the full insulin effect. These data suggest a requirement for both the binding and degradation of IDE ligands for the full inhibition of MCP. Insulin-sized degradation products, substrates of IDE, also inhibited MCP activity. Further examination of the insulin effect on MCP included kinetic studies. Insulin produced a noncompetitive inhibition of both the chymotrypsin-like and trypsin-like activities of MCP. These data suggest that the insulin-IDE effect on MCP is due to conformational changes in the IDE-MCP complex and provide an intracellular mechanism of action for insulin.
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PMID:Characterization of the insulin inhibition of the peptidolytic activities of the insulin-degrading enzyme-proteasome complex. 900 Jun 94

We previously reported that atrial natriuretic factor (ANF) stimulates secretin-evoked cAMP efflux through multidrug resistance-associated protein 4 (MRP4) in the exocrine pancreas. Here we sought to establish in vivo whether this mechanism was involved in acute pancreatitis onset in the rat. Rats pretreated with or without probenecid (MRPs general inhibitor) were infused with secretin alone or with ANF. A set of these animals were given repetitive cerulein injections to induce acute pancreatitis. Plasma amylase and intrapancreatic trypsin activities were measured and histological examination of the pancreas performed. Secretin alone activated trypsinogen but induced no pancreatic histological changes. Blockade by probenecid in secretin-treated rats increased trypsin and also induced vacuolization, a hallmark of acute pancreatitis. ANF prevented the secretin response but in the absence of probenecid. In rats with acute pancreatitis, pretreatment with secretin aggravated the disease, but ANF prevented secretin-induced changes. Blockade of MRPs in rats with acute pancreatitis induced trypsinogen activation and larger cytoplasmic vacuoles as well as larger areas of necrosis and edema that were aggravated by secretin but not prevented by ANF. The temporal resolution of intracellular cAMP levels seems critical in the onset of acute pancreatitis, since secretin-evoked cAMP in a context of MRP inhibition makes the pancreas prone to injury in normal rats and aggravates the onset of acute pancreatitis. Present findings support a protective role for ANF mediated by cAMP extrusion through MRP4 and further suggest that the regulation of MRP4 by ANF would be relevant to maintain pancreatic acinar cell homeostasis.
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PMID:Blockade of Multidrug Resistance-Associated Proteins Aggravates Acute Pancreatitis and Blunts Atrial Natriuretic Factor's Beneficial Effect in Rats: Role of MRP4 (ABCC4). 2556 2


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