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 properties of angiotensin II receptors were studied in isolated rat anterior pituitary cells prepared by trypsin digestion. Angiotensin II bound in a time- and temperature-dependent manner to pituitary cells, with Kd of 4.1 x 10(-9) M. The heptapeptide, des-Asp1-angiotensin II, had only one-tenth of the affinity of the octapeptide (Ki = 5.5 x 10(-8) M). These two peptides displayed a similar potency ratio in their ability to stimulate ACTH release from pituitary cells. These results indicate that angiotensin II may play a regulatory role in controlling ACTH secretion from the pituitary gland.
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PMID:Binding and activation properties of angiotensin II in dispersed rat anterior pituitary cells. 627 51

When deplasticized Epon sections were treated with endo- and/or exopeptidases prior to incubation with antibodies, the neuropeptide immuno-reactivity of secretory nerves was often altered in a predictable way. Cleavage of neurosecretory material in octopus nerves by trypsin and carboxypeptidase-B enhanced enkephalin-like immunoreactivity, while Molluscan neuropeptide-like immunoreactivity was prevented by tryptic cleavage. The enzyme effects indicated the occurrence of a heptapeptide (Tyr-Gly-Gly-Phe-Met/Leu-Arg-Phe) that contains both the enkephalin and the Molluscan neuropeptide sequence. Vasopressin terminals of the rat neurohypophysis, which presumably contain enkephalin precursor sequences, exhibited enkephalin-like immunostaining after tryptic cleavage. ACTH/beta-endorphin cells of the rat intermediate pituitary, which synthesize the enkephalin sequence at the N-terminus of Beta-endorphin, exhibited enkephalin=like immunoreactivity when sections were treated with alpha-chymotrypsin or trypsin, but not after incubation with leucine-aminopeptidase or carboxypeptidase-B. Enkephalin-like immunostaining could not be induced in any way in ACTH/beta-endorphin cells of the anterior pituitary. Enzymatic cleavage may give additional information in immunocytochemical localization studies on neuropeptide sequences in secretory nerves and hormonal granules.
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PMID:Enzymatic cleavage prior to antibody incubation as a method for neuropeptide immunocytochemistry. 628 42

A mammalian isolated adrenal cell system was validated as a bioassay for goldfish ACTH; the log dose-response curve for the goldfish hormone is parallel to that for synthetic mammalian ACTH1-24, and the two ACTHs induce the same maximum rate of corticosterone production. Using this assay, it was observed that (1) there is a marked and consistent biphasic change in pituitary ACTH content as related to the length of time the fish are held in laboratory aquaria, and (2) the absolute and relative (to tissue wt) ACTH content of the rostral pars distalis is considerably greater than that of the proximal portion of the gland. Using a trypsin technique, isolated pituitary cells were prepared from both (rostral and proximal) portions of the gland; bioassay data indicate that cell suspensions prepared from the rostral pars distalis are enriched with respect to corticotrophs. The implications of these findings with regard to formulating an advantageous in vitro system for studying ACTH secretion are discussed.
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PMID:Preparation of enriched populations of corticotrophs from goldfish rostral pars distalis. 629 60

The influence of N-ethylmaleimide and trypsin was studied on stimulatory and inhibitory regulations of the hamster adipocyte adenylate cyclase. Treatment of intact adipocytes or adipocyte ghosts with N-ethylmaleimide decreased basal and forskolin-stimulated adenylate cyclase activities. In the pretreated membrane preparations, inhibition of the enzyme by GTP and by stable GTP analogues was abolished. Concomitantly, activation of the adenylate cyclase by NaCl and its inhibition by the antilipolytic agents, prostaglandin E1 and nicotinic acid, were obliterated. In contrast, adenylate cyclase stimulation by ACTH and stable GTP analogues was not impaired but rather increased. Similarly, the NaCl-induced attenuation of the ACTH-stimulated enzyme activity was increased by the N-ethylmaleimide treatment. Limited proteolysis of hamster adipocyte ghosts with trypsin also obliterated GTP and prostaglandin E1-induced inhibitions and NaCl-induced activation of the adenylate cyclase. In contrast, adenylate cyclase activity stimulated by isoproterenol was increased after trypsin treatment. The data suggest that the activity of the adenylate cyclase is regulated via two distinct guanine nucleotide sites and that treatment with N-ethylmaleimide and limited proteolysis with trypsin functionally eliminates the regulatory site mediating adenylate cyclase inhibition, leading to a state where the enzyme activity is regulated only via the stimulatory site. The differential effects of these treatments on NaCl-induced activation and attenuation of the adenylate cyclase suggest that sodium acts on both regulatory sites in an inhibitory manner, and that by the functional elimination of the inhibitory site, only the sodium-induced attenuation of the adenylate cyclase via the stimulatory site is observed.
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PMID:Inactivation of the guanine nucleotide regulatory site mediating inhibition of the adenylate cyclase in hamster adipocytes. 630 Jun 99

The biotransformation of adrenocorticotropin (ACTH-(1-39)) by brain synaptic membranes has been studied. Peptide fragments of ACTH-(1-39) which were formed during in vitro incubation of the peptide with membrane preparations were isolated by high pressure liquid chromatography and characterized by determination of amino acid composition and NH2- terminal residue. At pH 7.4, ACTH-(1-38) was found as the main metabolite, together with ACTH-(7-21) and ACTH-(7-20). In addition, a series of secondary products was identified. At pH 6.2, ACTH-(1-38), ACTH-(1-37), and ACTH-(1-36) were exclusively formed, while at pH 8.5, ACTH-(1-39) was converted into ACTH-(1-16), ACTH-(17-39), ACTH-(22-39), and ACTH-(3-15). Time course experiments demonstrated the action of a carboxypeptidase activity and a trypsin-like endopeptidase on ACTH-(1-39) as predominant proteolytic events. The carboxypeptidase was optimally active at pH values of 5.7 or below. These enzymes play an essential role in the stepwise conversion of ACTH-(1-39) in brain. It is suggested that they are involved in the modulation of the central activities of ACTH fragments in the brain.
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PMID:Proteolysis of adrenocorticotropin in brain. Characterization of cleavage sites by peptidases in synaptic membranes and formation of peptide fragments. 630 63

ACTH activity in glacial acetic acid extracts of normal rat tissues was studied by both ACTH RIA and a sensitive in vitro bioassay. ACTH immunoactivity was found in all tissues: brain, 278 +/- 54 (mean +/- SE; picograms per mg protein); stomach, 59 +/- 4; kidney, 47 +/- 3; colon, 40 +/- 4; small intestine, 37 +/- 4; liver, 18 +/- 2; and heart, 16 +/- 3. Tissue ACTH showed parallelism with ACTH standard in the RIA. No correlation existed between tissue ACTH and plasma ACTH in normal rats. Dexamethasone treatment (0.4 mg/day for 5 days) suppressed plasma ACTH, but did not affect tissue ACTH levels. When tissue extracts were passed through Sephadex G-75-SF columns, ACTH immunoactivity was exclusively eluted in the portion of bigger molecular weight than ACTH standard, except in the brain. Based on this column chromatography, the molecular weight of the main peak of activity was estimated as 26,000. Tissue ACTH-like material contained no detectable biological activity (less than 2 pg/100 ng tissue). However, biological activity was easily detectable after exposure of the tissue extracts to trypsin. When studied by immunoassay and bioassay, this 26,000 mol wt ACTH was digested and cleaved to 4,500 mol wt and biologically active ACTH with trypsin treatment. Tissue ACTH immunoactivity does not seem to be the result of artifacts: 1) extracts were adjusted to pH 8.0 and a common osmolality (150 mosmol/liter) before assay; 2) protein contents in RIA tubes were only 0.1-1.6 mg; 3) tissue extracts incubated with [125I]iodo-ACTH for 48 h produced less than 5% damage of labeled hormone, as assessed by moderate excess of antibody binding; 4) enzyme inhibitors did not modify tissue ACTH levels; and 5) ACTH immunoactivity of tissue extracts was absorbed by anti-ACTH immunocolumns. We conclude that high molecular weight ACTH-like materials are widespread in normal rat extrapituitary tissues and are probably a precursor form of 4500 mol wt ACTH.
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PMID:Widespread presence of large molecular weight adrenocorticotropin-like substances in normal rat extrapituitary tissues. 630 59

The finding that incubation of rat adrenal capsules (largely zona glomerulosa) with trypsin reproducibly releases aldosterone and 18-hydroxycorticosterone (18-OH-B) from tightly protein-bound tissue stores leads to the hypothesis that the secretion of these steroids may be under the control of endogenous proteases. Rat adrenal capsule whole tissue and collagenase-dispersed cells were incubated under conditions of stimulation by (1-24)ACTH (10(-7) mol/l), potassium (8.4 X 10(-3) mol/l) or dibutyryl cyclic AMP (dbcAMP) (10(-4) mol/l) with the addition in some flasks of one of the following protease inhibitors at the appropriate concentration for their known actions: N alpha-p-tosyl-L-arginine methyl ester (TAME; 10(-2) mol/l), 2-nitro-4-carboxyphenyl-N,N'-diphenylcarbamate (NCDC; 2 X 10(-6) mol/l), N alpha-benzoyl-L-arginine (BA; 10(-2) mol/l), p-nitrophenyl-N alpha-benzyloxycarbonyl-L-lysinate (CBZ-NL; 2 X 10(-6) mol/l) and soybean trypsin inhibitor (STI; 1 mg/ml). The (1-24)ACTH-stimulated steroid output in dispersed cells was not affected by NCDC, BA or CBZ-NL. However, all of the inhibitors except STI produced selective effects on aldosterone and 18-OH-B production by whole capsule tissue under certain conditions. Thus TAME and NCDC significantly inhibited the dbcAMP-stimulated production of these two steroids (aldosterone values decreased from 328 +/- 35 to 128 +/- 15 and 157 +/- 32 ng/gland pair respectively) and furthermore NCDC elicited the same effect in potassium- or ACTH-stimulated whole tissue (e.g. in K+-stimulated tissue aldosterone decreased from 79 +/- 15 to 40 +/- 7 ng/gland pair). The reverse effect was shown by BA and CBZ-NL in potassium-stimulated whole tissue, and yields of aldosterone and 18-OH-B were significantly enhanced (aldosterone increased from 79 +/- 15 ng/pair to 151 +/- 14 ng in the presence of BA). The high molecular weight inhibitor STI had no effect on potassium-stimulated whole tissue, but enhanced the yield of extractable aldosterone from 9.7 +/- 1.7 to 16.9 +/- 2.3 ng/pair when added to incubations of a cytosol preparation. The results suggest that endogenous proteases in rat adrenal whole capsule tissue play specific roles in the control of aldosterone and 18-OH-B secretion. Some (type 1) whose action is mimicked by trypsin, are inhibited by TAME and NCDC and appear to be involved in the release of these two steroids from their tight (apparently covalent) binding to protein.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Serine proteases selectively control the output of 18-hydroxycorticosterone and aldosterone in stimulated zona glomerulosa tissue of the rat adrenal. 631 38

The present investigation was designed to study the effect of synthetic ACTH on the synthesis of progesterone, 20 alpha-OH-progesterone and adenosine-3', 5'-monophosphate (cAMP) in isolated luteal cells. Experiments were conducted on rats from three groups. Group I: PMS primed immature rats. Group II: PMS plus hCG primed immature rats. Group III: pregnant rats. Luteal cells were isolated by the Kumai Method (7) using a sucrose density gradient after digestion with trypsin and collagenase. Luteal cells were divided into three fractions; S-1, S-2 and S-3. Following an 18 hr-incubation of S-1 cells of day 7 after PMS injection, a significant increase in progesterone release by ACTH lasted from 6 to 18 hr, whereas 20 alpha-OH-progesterone decreased significantly by ACTH from 8 to 18 hr. There were no differences in progesterone and 20 alpha-OH-progesterone releases from S-2 cells of day 7 between media with and without ACTH. Progesterone release without ACTH reached the peak on day 7 in Group I and on day 6 in Group II. ACTH stimulated progesterone and inhibited 20 alpha-OH-progesterone releases in Groups I and II. The maximum effect of ACTH in both Groups was noted on days 6 and 7. There were no differences in the Effective Dose (ED50) and the Inhibitory Dose (ID50) of ACTH between Group I and Group II. In Group III, releases of progesterone and 20 alpha-OH-progesterone from S-1 cells to media with or without ACTH decreased remarkably after 11 days of gestation. On days, 3, 5 and 8 of gestation, a significant increase in progesterone release from S-1 cells was noted by the ACTH addition. 20 alpha-OH-progesterone release from S-1 cells to media with or without ACTH decreased as the pregnancy advanced. Total cAMP of intra- and extra-cellular S-1 cells (Group II) on day 5 after PMS injection was assayed. ACTH induced an increase in intracellular cAMP. The present results indicate the following: (1) In Group I and Group II, progesterone release increased with the age of the luteal cells and ACTH dose. In Group III, ACTH stimulated progesterone release in a dose-dependent manner in early gestation. (2) 20 alpha-OH-progesterone release decreased by ACTH dose in Group I and Group II, and decreased in Group III as the gestation advanced. (3) Intracellular cAMP increased by ACTH in Group II.
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PMID:[Effect of adrenocorticotropin on progesterone, 20alpha-OH- progesterone and adenosine 3',5'-monophosphate in isolated luteal cells from rat ovaries]. 631 5

Tonin, an esteroprotease isolated from rat submaxillary gland, is a serine protease with trypsin- and chymotrypsin-like activity. The substrate specificity of tonin shows that it differs from kallikreins and is definitely not a renin-like enzyme or an angiotensin-converting enzyme. Tonin can produce directly the vasoactive peptide angiotensin II, from angiotensin I, angiotensinogen and the synthetic tetradecapeptide substrate of renin by cleavage of a Phe-His bond. It has also been found to cleave some Phe and Arg bonds in various substrates such as beta-lipotropin (beta-LPH), adrenocorticotropin (ACTH), pro-opiomelanocortin (POMC) and substance P. Here we describe the complete amino acid sequence of rat submaxillary gland, tonin. Comparison of the sequence of 219 amino acids with other serine proteases, particularly kallikreins, gamma-subunit of nerve growth factor (NGF) and the recently described gamma-renin, reveals extensive similarities. More interestingly, it also reveals the substitution of an Asp residue always found in the serine protease active site triad (Asp, His, Ser) by a Leu residue. This unusual substitution does not seem to affect the proteolytic activity of the enzyme.
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PMID:Amino acid sequence of rat submaxillary tonin reveals similarities to serine proteases. 632 14

Recent amino acid sequence data suggest that trypsin-like and carboxypeptidase B-like activities are required for the processing of pituitary prohormones--e.g., pro-opiocortin (pro-adrenocorticotropin/lipotropin) and provasopressin in secretory granules. In this study the existence of a carboxypeptidase B activity in purified secretory granules from anterior, intermediate, and neural lobes of rat pituitary has been examined. A carboxypeptidase B activity that cleaved the COOH-terminal -Lys-Lys-Arg residues from the adrenocorticotropin fragment ACTH-(1-17) (a potential hormone product liberated from pro-opiocortin by a trypsin-like enzyme) was detected in anterior and intermediate lobe granules. A similar carboxypeptidase B activity was also present in purified secretory granules from rat pituitary neural lobes that cleaved the -Lys-Arg residues from [Arg8]vasopressin-Gly-Lys-Arg, a potential product cleaved from provasopressin. Secretory granule carboxypeptidase(s) from the three lobes of the pituitary was shown to cleave 125I-[Met]enkephalin-Arg6 to form 125I-[Met]enkephalin as well. 125I-[Met]Enkephalin was used as a model substrate for the quantitative assay of pituitary carboxypeptidase activity. The carboxypeptidase B in secretory granules from all three lobes was shown to be active at pH 5.5, but not at pH 7.4. Inhibition by the zinc metallocarboxypeptidase inhibitors guanidinopropylsuccinic acid, aminomercaptosuccinic acid, benzylsuccinic acid, 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, and the potato carboxypeptidase B inhibitor, and inhibition by the metal chelators EDTA and 1,10-phenanthroline demonstrate metal ion dependence of the pituitary granule carboxypeptidase activities. However, Co2+ stimulated the secretory granule carboxypeptidase B activities. Thiol protease inhibitors such as Cu2+ and p-chloromercuriphenylsulfonic acid also inhibited the activity. Thus, the secretory granule carboxypeptidase B-like activities in all three lobes of the pituitary appear to be similar thiol-metallopeptidases that differ from other carboxypeptidase activities previously described and may play an exclusive role in hormone biosynthesis in the pituitary.
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PMID:Carboxypeptidase B-like converting enzyme activity in secretory granules of rat pituitary. 632 44


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