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)

Proenkephalin-derived peptides, in common with other prohormones, are associated with membranes of microsomes and secretory granules in the bovine adrenal medulla. Post-translational processing of the precursor molecule varies depending upon the tissue. The relationship between post-translational events in different tissues was examined by studying the membrane association of endogenous proenkephalin-derived peptides in the crude microsomal fraction of rat adrenal medulla, brain striatum and heart ventricle. [Met]-Enkephalin and synenkephalin (proenkephalin(1-70)) immunoreactivities were quantified by radioimmunoassay after sequential enzymatic digestion with trypsin and carboxypeptidase B. Between 60 and 75% of total immunoreactive peptides present in intact microsomes of the three tissues were associated with membranes and specifically released with 2 M KSCN (pH 7.4). Analysis of the chromatographic profile of materials present in the soluble and associated fractions produced the following results. In the three tissues the materials associated with microsomal membranes corresponded to peptides larger than 3-5 kDa and displayed synenkephalin and [Met]-enkephalin immunoreactivity. Adrenal and heart microsomes showed a continuous pattern of membrane-associated proenkephalin-derived peptides of high, intermediate and low molecular weights containing the synenkephalin and [Met]-enkephalin sequences. These tissues, however, presented quantitative differences, as the highest concentrations belonged to materials larger and smaller than 12.5 kDa in adrenal and heart microsomes respectively. On the other hand, brain striatal microsomes displayed a discontinuous pattern of associated materials, with the absence of some products of high and intermediate molecular weight. Only in the soluble fraction of striatal microsomes were peptides detected of high and intermediate molecular weight containing the [Met]-enkephalin but not the synenkephalin sequence.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Differential association of endogenous proenkephalin-derived peptides with membranes of microsomes from rat striatum, adrenal medulla and heart ventricle. 224 89

Synenkephalin (SYN), the nonopioid amino-terminal portion of proenkephalin (PRO), is stable and well conserved in mammals and therefore a promising marker for PRO systems. We immunized rabbits with synthetic [Tyr63]SYN(63-70)-octapeptide, coupled by glutaraldehyde to bovine serum albumin. In radioimmunoassay (RIA) using antiserum no. 681, [Tyr63]SYN(63-70)-octapeptide as standard, and 125I-[Tyr63]SYN(63-70)-octapeptide as tracer, the IC50 was approximately 51 fmol/100-microliters sample at equilibrium or 12 fmol/100 microliters in disequilibrium, and the sensitivity was approximately 3 fmol/100 microliters. Cross-reactivity of the assay was 100% with [Cys63]SYN(63-70)-octapeptide and with bovine adrenal 8.6-kilodalton peptide digested with trypsin and carboxypeptidase B, but less than 0.1% with transforming growth factor-alpha, less than or equal to 2 x 10(-6) with Leu-Leu-Ala [SYN(68-70)-tripeptide], and much less than 10(-6) with all other peptides tested. Therefore in RIA this antiserum is specific for the free carboxyl terminus of SYN. Because the peptide detected after enzyme digestion is the complete SYN(63-70)-octapeptide, we refer to the RIA as an assay for SYN(63-70). Tissue extracts were made in 1 M acetic acid, dried, reconstituted in Tris-CaCl2, and digested sequentially with trypsin plus carboxypeptidase B. Extracts from bovine corpus striatum gave SYN(63-70) RIA dilution curves parallel to the standard curve both before and after digestion. Digestion increased the amount of immunoreactive SYN(63-70) in striatum by a factor of 1.5-2.0. The ratio of total immunoreactive [Met5]enkephalin to total immunoreactive SYN(63-70) (after sequential digestion) was approximately 6:1. At least 90% of the immunoreactive SYN(63-70) in extracts of bovine caudate nucleus eluted from Sephadex G-100 with an apparent molecular weight equal to that of bovine PRO(1-77). Using the new RIA we were able to detect and characterize SYN processing for the first time in extracts of whole rat brain, human globus pallidus, and human pheochromocytoma. Results in these tissues were similar to those in cattle, in that most stored SYN had been processed to a free carboxyl terminus. Since the C-terminal octapeptide of SYN is practically identical in all known mammalian PRO, antiserum no. 681 should be useful for detecting, measuring, and purifying SYN from various mammals, including human beings.
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PMID:Detection of synenkephalin, the amino-terminal portion of proenkephalin, by antisera directed against its carboxyl terminus. 229 45

Met-enkephalin and related proenkephalin A-derived peptides circulate in plasma at picomolar concentration as free, native pentapeptide and at nanomolar concentration in cryptic forms. We have optimized conditions for measurement of immunoreactive Met-enkephalin in plasma and for generation by trypsin and carboxypeptidase B of much greater amounts of total peptidase-derivable Met-enkephalin in plasma of rats, dogs, and humans. Free Met-enkephalin (11 pM) is constituted by native pentapeptide and its sulfoxide. Characterization of plasma total Met-enkephalin derived by peptidic hydrolysis revealed a small amount (38 pM) of Met-enkephalin associated with peptides of molecular mass less than 30,000 D, and probably derived from proenkephalin A, but much larger amounts of Met-enkephalin associated with albumin (1.2 nM) and with a globulin-sized protein (2.8 nM). Thus, plasma protein precursors for peptidase-derivable Met-enkephalin differ structurally and chemically from proenkephalin A. Met-enkephalin generated from plasma by peptidic hydrolysis showed naloxone-reversible bioactivity comparable to synthetic Met-enkephalin. Prolonged exposure of adult, male rats to restraint stress produced biphasic plasma responses, with peaks occurring at 30 s and 30 min in both free native and total peptidase-derivable Met-enkephalin. Repeated daily exposure to this 30-min stress resulted in adaptive loss of responses of both forms to acute restraint. Initial plasma responses of Met-enkephalin paralleled those of epinephrine and norepinephrine, but subsequently showed divergence of response. In conclusion, Met-enkephalin circulates in several forms, some of which may be derived from proteins other than proenkephalin A, and plasma levels of both free native, and peptidase-derivable Met-enkephalin are modulated physiologically.
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PMID:Plasma native and peptidase-derivable Met-enkephalin responses to restraint stress in rats. Adaptation to repeated restraint. 231 29

Opioid and tachykinin neuropeptides, which were derived from two biological sources (intact, and released from their corresponding precursors by the action of human cerebrospinal fluid (CSF) neuropeptidases), were characterized in human CSF by using a combination of post-high-performance liquid chromatographic (HPLC) detection techniques. Peptides were separated using gradient and isocratic reversed-phase HPLC. Radioimmunoassay measured immunoreactivity corresponding to several different individual neuropeptides including methionine enkephalin, leucine enkephalin, substance P and beta-endorphin. Commercial enzymes (trypsin, carboxypeptidase B) were used to release methionine- and leucine-enkephalin from precursors. Human CSF also served as a source of endogenous neuropeptidases. Mass spectrometry produced fragment ions that corroborated the amino acid sequence of methionine enkephalin and of substance P derived from both sources (intact, from precursors). These results demonstrated the presence of endogenous intact neuropeptides, several different neuropeptide-containing precursors and appropriate precursor-processing enzymes in human CSF for precursors of methionine enkephalin, leucine enkephalin, beta-endorphin1-31 and substance P.
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PMID:Opioid and tachykinin peptides, and their precursors and precursor-processing enzymes, in human cerebrospinal fluid. 232 43

The amino acid sequences of trypsin inhibitors I and II from the hemolymph of a solitary ascidian, Halocynthia roretzi, were determined after reduction and S-pyridylethylation. The results indicated that inhibitor I consists of a single polypeptide chain with 55 amino acid residues and four intramolecular disulfide bridges, whereas inhibitor II is composed of two polypeptide chains corresponding to a form derived from inhibitor I by cleavage at the Lys16-Met17 bond. Lys16 may be the reactive-site residue of these inhibitors, because carboxypeptidase B treatment destroys most of the inhibitory activity of inhibitor II but not that of inhibitor I.
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PMID:Primary structure of ascidian trypsin inhibitors in the hemolymph of a solitary ascidian, Halocynthia roretzi. 234 75

Enkephalins, endogenous opioid pentapeptides which are found in high concentration in normal chromaffin tissue, may play a role in blood pressure regulation. We therefore examined the presence and actions of enkephalins in pheochromocytoma in a rat model. Transplantable norepinephrine-rich tumors, which gave rise to significant blood pressure elevations, contained measurable immunoreactive enkephalins as determined by specific radioimmunoassays for leucine-enkephalin and methionine-enkephalin. Enkephalin immunoreactivity paralleled the enkephalin assay standard curves and was not abolished by boiling or by protease inhibitors (EDTA, PMSF). Authenticity of the immunoreactive enkephalins was confirmed by reverse-phase high pressure liquid chromatography. The amount of enkephalin immunoreactivity present initially in these tumors was greatly augmented by the prohormone activators trypsin or trypsin plus carboxypeptidase B, suggesting that most of the immunoreactive enkephalin was present in higher molecular weight precursor form. Enkephalin determinations on human pheochromocytoma and catecholamine measurements in both rat and human pheochromocytoma, demonstrated certain similarities and differences in enkephalin and catecholamine content between rat and human tumors. Total tumor enkephalins correlated (r = 0.91, p less than 0.05) with total tumor catecholamines in rat pheochromocytoma, suggesting co-regulation of synthesis of these 2 chromaffin tissue substances. Physiologic studies, in which intravenous leucine-enkephalin and the opioid antagonist nalaxone were administered to pheochromocytoma-implanted rats and sham-operated controls, failed to uncover an opioid peptide influence upon blood pressure in this animal model of pheochromocytoma.
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PMID:Enkephalins in pheochromocytoma: studies in a rat model. 240 93

The biochemical and pharmacological properties of an endogenous anticonvulsant substance(s) found in rat cerebrospinal fluid (CSF) following seizures are described. CSF taken from donor rats following a single maximal electroshock (MES) seizure caused significant elevations in seizure thresholds in naive recipient rats when intracerebroventricularly injected 15 min prior to exposure to the volatile convulsant flurothyl. Anticonvulsant activity was antagonized by pre-injection in recipients of high doses of naloxone or the selective delta-opioid receptor antagonist ICI 174,864. The anticonvulsant activity was also lost when the CSF was exposed to heat (90 degrees C) or immobilized trypsin. Although unaffected by the peptidase inhibitors thiorphan and bestatin, the anticonvulsant activity was significantly potentiated by a combination of aprotinin and bacitracin. Ultrafiltration of CSF revealed that the anticonvulsant activity passed through membranes with a 10,000 molecular weight cut-off, but was retained by membranes with a 5000 molecular weight cut-off. CSF removed from rats following MES had significantly increased concentrations of beta-endorphin-like, but not dynorphin A, Leu- or Met-enkephalin-like immunoreactivities relative to CSF from sham-treated rats. However, significant increases in Met-enkephalin-like immunoreactivity were measured following exposure of the CSF to the proteolytic enzymes trypsin and carboxypeptidase B, suggesting the seizure-induced presence of a higher molecular weight form of Met-enkephalin not recognized immunologically prior to enzyme exposure. These data reconfirm the anticonvulsant actions of postseizure CSF, and indicate that these effects require mediation through delta-opioid receptors in the recipient rat. These data additionally argue against these effects being mediated by Met-enkephalin, Leu-enkephalin or dynorphin A in the CSF, and suggest instead that anticonvulsant effects are attributable to a heat- and trypsin-sensitive opioid peptide(s) with a molecular weight approximately in the range of 5000-10,000 Da.
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PMID:Characterization of opioid peptide-like anticonvulsant activity in rat cerebrospinal fluid. 245 10

Structural features contributing to the antigenic recognition of the small globular hormone avian pancreatic polypeptide (APP) by a polyclonal antiserum have been defined using a solution phase radioimmunoassay technique. Cross-reactivity studies with PP homologues suggest that the surface residues within the alpha-helix of the peptide may be antigenic, whereas hydrophilicity and atomic mobility predictive methods implicate the molecules beta-turn region. Immunochemical data and circular dichroism measurements on a timed trypsin digest of APP indicate that the secondary structure of the alpha-helix is vital for the molecule's immunological competence. Immunoreactivities of iodinated derivatives of APP, as well as that of peptide fragments of APP and its homologues, support the importance of teritary structure involving the interaction of the polyproline and alpha helices. The highly mobile C-terminal residues 34-36 (His-Arg-Tyr-NH2) have been found by immunological analysis to be unimportant. Arginine residue 33, which has been conserved through vertebrate evolution, is a major antigenic contributor, since a large decrease in immunoreactivity, not accompanied by a significant change in conformation, was observed upon specific removal of this residue by carboxypeptidase B. These results are consistent with a "discontinuous" epitopic model for APP in which Arg-33 and exposed residues in the alpha-helix are principal components of an epitope or epitopes mediated by the secondary and tertiary structures of the molecule.
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PMID:An immunochemical study of avian pancreatic polypeptide: the nature of the principal epitope. 246 36

Mild proteolytic treatment of SW1116 tumor cells with trypsin or plasmin increases their plasmin-binding ability considerably by increasing the number of binding sites without altering their affinity. This mechanism may be operative for increasing the concentration of active plasmin at the surface of tumor cells. C-terminal lysine residues are involved in plasmin binding to cells, since treatment of cells with carboxypeptidase B decreases this binding by 50%.
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PMID:Limited proteolysis of tumor cells increases their plasmin-binding ability. 246 97

Developmental patterns for rat pancreatic opioid peptides and islet hormones were studied from gestational day 20 through adulthood. Fetal tissue was obtained as well as pancreas at birth (day 0), and postnatal days 3, 7, 14, and 21, and 7 weeks. The hormones measured included insulin, glucagon, and somatostatin. The opioids measured were beta-endorphin, Met- and Leu-enkephalins, and the high molecular weight enkephalin precursors. Pancreata were pooled as necessary and extracted (acid alcohol, or hot acetic acid), and opioids were further purified on reversed-phase C-18 (Sep-pak) cartridges. In all instances measurements were made by radioimmunoassays. Precursor peptides were first digested (with trypsin and carboxypeptidase B) prior to immunoassay. All opioids and hormones except the precursors for enkephalins showed a well-defined surge in pancreatic concentration during the first postnatal week. In contrast, the precursors had the highest concentration in the fetus, and by the seventh day of life had decreased by greater than 50%. This progressive decrease may represent maturation of the enkephalin convertase and trypsin-like enzymes in the islets. The opioid and hormonal surges that we have described are similar to the surge in islet concentration of thyroid-releasing hormone (TRH) previously described in neonatal rat islets. It is suggested that these postnatal alterations in opioid and hormone concentration relate to a specific function in the development of the endocrine pancreas.
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PMID:Developmental patterns for pancreatic opioids in the rat. 253 May 76


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