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 investigated the possible role of the fetal pituitary and ACTH in the control of the synthesis and post-translational processing of the enkephalin precursor, proenkephalin A (proEnk A), in the fetal sheep adrenal gland in late gestation. Fetal hypophysectomy (n = 8) or sham operations (n = 4) were performed between 109 and 118 days of gestation. At 138-139 days, either ACTH(1-24) (10.5 micrograms/0.24 ml saline per h, n = 4) was infused intravenously for 72 h into hypophysectomized fetal sheep or 0.9% (w/v) NaCl alone (0.24 ml/h, n = 4) was infused for 72 h into hypophysectomized fetal sheep and sham-operated animals. At the end of the infusion the pregnant ewe was killed and left or right adrenal glands (n = 12) were collected from the fetal sheep that were intact and given saline (Intact + sal; n = 4), hypophysectomized and given saline (Hx + sal; n = 4) and hypophysectomized and given ACTH (Hx + ACTH; n = 4). Each adrenal was homogenized in acid (acetic acid (1 mol/l)/HCl (20 mmol/l)/2-mercaptoethanol (0.2%)). After centrifugation, the supernatant was loaded onto a Sephadex G-75 column (2.0 x 50 cm), eluted at 80 ml/24 h and fractions were collected (5 ml, n = 42). An aliquot of each fraction (2 ml) was dried down prior to enzymatic digestion (trypsin/carboxypeptidase B) and oxidation with H2O2, and assay for methionine-O-enkephalin (immunoreactive Met-O-Enk).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The effect of fetal hypophysectomy with or without ACTH replacement on the molecular weight profile of enkephalin-containing peptides in the adrenal medulla of the fetal sheep. 132 54

Proenkephalin A (PEA) gene was found to be expressed in primary, secondary and tertiary cultures of rat fibroblasts. The 1.4 kb PEA mRNA was detected by Northern blot analysis. The same cultures do not express detectable amounts of proenkephalin B (prodynorphin) or (POMC) mRNAs. Acidic cell extracts were purified on a C18 octadecyl Amprep column and analysed with a specific methionine enkephalin radioimmunoassay to detect whether PEA mRNA is translated. A significant amount of enkephalin immunoreactivity (178-185 fmol/mg protein) was observed upon trypsin and carboxypeptidase B digestion of fibroblast cell extracts, whereas only 3-5% of this amount was free enkephalin. It is therefore indicated that the PEA mRNA expressed in fibroblasts is indeed translated to the proenkephalin precursor protein, but the cells accumulate only a small quantity of the processed pentapeptides. The implication of these observations to the possible developmental role of PEA in various non-neuronal cells, including mesodermal lineages, is discussed.
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PMID:Expression of proenkephalin A mRNA and enkephalin-containing peptides in cultured fibroblasts. 169 63

Previous immunochemical investigations have demonstrated various opioid peptides in the pancreas. However, controversies exist related to the cellular localization of these peptides in the endocrine pancreas. Therefore, the guinea pig endocrine pancreas was immunohistochemically investigated for the presence of opioid peptides derived from pro-dynorphin, pro-enkephalin or pro-opiomelano-cortin. Immunoreactivities were demonstrated on serial semithin sections by the peroxidase anti-peroxidase technique. In routinely immunostained sections, immunoreactivities for dynorphin A and alpha-neo-endorphin were localized in pancreatic enterochromaffin cells, but not in islet cells. Immunoreactivity for Met-enkephalin was confined exclusively to B-cells and was localized only in some secretory granules. However, pre-treatment of semi-thin sections with trypsin and carboxypeptidase B led to a marked increase of Met-enkephalin immunoreactivity in B-cells. In addition, immunoreactivities for Met-enkephalin-Arg-Gly-Leu and bovine adrenal medulla dodecapeptide could be demonstrated in B- and A-cells, and beta-endorphin immunoreactivity was localized in A-cells. In no case, however, were immunoreactivities detected for bovine adrenal medulla docosapeptide, peptide F, corticotropin, melanotropin or dynorphin 1-32. The immunohistochemical findings indicate that opioids of different peptide families are present in the guinea pig endocrine pancreas. Since several opioid peptides of the corresponding pro-hormones could be demonstrated in the reference organs but not in the pancreas, it is concluded that the biosynthetic pathways of the respective precursors are different from those in the adrenal medulla or in the pituitary.
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PMID:Immunohistochemistry of opioid peptides in the guinea pig endocrine pancreas. 197 Sep 50

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

Immunohistochemical analysis of the pituitary of the holostean fish, Amia calva, indicated that enkephalin-related immunoreactivity was restricted to the pars nervosa, and was not detected in other regions of the pituitary. Fractionation of acid extracts of posterior pituitaries by reverse phase HPLC followed by RIA analysis indicated the presence of immunoreactive Met-enkephalin and Leu-enkephalin. No immunoreactive forms were detected with RIAs specific for either Met-enkephalin-RF or Met-enkephalin-RGL. The molar ratio of Met- to Leu-enkephalin in this terminal field was 3:1 (n = 4). HPLC fractions were also digested with trypsin and carboxypeptidase B to test for C-terminally extended forms of Met-enkephalin. A novel modified form of Met-enkephalin was detected. Extracts of the posterior pituitary, forebrain, midbrain, hypothalamus and hindbrain were screened with RIAs specific for the Pro-dynorphin end products, alpha-neo-endorphin, dynorphin A(1-17), dynorphin A(1-8) and dynorphin B(1-13). The results of these analyses were negative. Collectively, these data suggest that a Pro-enkephalin-like molecule is present in holostean fish. The holostean enkephalin precursor contains at least Met-enkephalin and Leu-enkephalin. However, Pro-dynorphin-related end products with antigenic determinants similar to mammalian dynorphin A(1-17), dynorphin A(1-8), dynorphin B(1-13) and alpha-neo-endorphin could not be detected in the brain or pituitary of this species.
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PMID:Detection of Met-enkephalin and Leu-enkephalin in the posterior pituitary of the holostean fish, Amia calva. 260 57

Mytilus pedal ganglia extract was treated sequentially with TPCK-trypsin and carboxypeptidase B. The treated sample and an untreated sample were purified separately with Sep-Pak C18 and subjected to HPLC. Fractions with Rt's corresponding to the Met-, Leu-enkephalin and Met-enkephalin-Arg6-Phe7 were assayed for enkephalin activities by displacement studies using pedal ganglia membrane and 3H-DAMA. The data showed the activities in the regions of Met-, Leu-enkephalin and Met-enkephalin-Arg6-Phe7 of the treated sample increased over that of the untreated sample by 2.3, 2.5 and 1.6 folds respectively. These results provided strong evidence for the presence of enkephalin precursor material in Mytilus.
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PMID:Presence of enkephalin precursor in molluscan neural tissue extract. 312 41

Enkephalins, endogenous opioid pentapeptides, are found in normal chromaffin tissue and may influence blood pressure regulation. We studied the subcellular localization and precursor-product status of enkephalin immunoreactivity in 11 human phaeochromocytomas (seven adrenal, four extra-adrenal). Enkephalin immunoreactivity was found in all phaeochromocytomas, it paralleled radio-immunoassay standard curves and was not destroyed by boiling or protease inhibitors i.e. ethylenediaminetetraacetic acid (EDTA) and phenylmethylsulfonyl fluoride (PMSF). Sucrose gradients localized enkephalin immunoreactivity to chromaffin granules (55 +/- 17% of total immunoreactivity; n = 6). In vitro granule lysis released 81% of the enkephalins and 91% of the catecholamines. Thus, phaeochromocytoma enkephalins are present in the soluble core of chromaffin granules, along with catecholamines. Enkephalin immunoreactivity was not contained in purified chromogranin A, either before or after trypsin cleavage. High pressure liquid chromatography (HPLC) elution of enkephalin immunoreactivity matched that of synthetic methionine-enkephalin, leucine-enkephalin, and methionine-sulfoxide-enkephalin standards. Enkephalin immunoreactivity was augmented by trypsin alone and by trypsin plus carboxypeptidase B (by 352 +/- 56%), suggesting that the majority of the enkephalins were present in higher molecular weight precursor form. Sephacryl S-200 gel filtration of chromaffin granule lysate revealed a trypsin-augmented putative human enkephalin precursor with a molecular weight of 2000-4000 daltons as well as product enkephalins. Enkephalin concentration in phaeochromocytoma closely paralleled the epinephrine, but not the norepinephrine content of the tumours. However, it was not statistically different in adrenal versus extra-adrenal tumours. Thus, these peptides are contained in high molecular weight form in the soluble core of catecholamine storage vesicles, predominantly epinephrine vesicles.
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PMID:Enkephalins in human phaeochromocytomas: localization in immunoreactive, high molecular weight form to the soluble core of chromaffin granules. 336 Nov 17

To detect the presence of a mammalian-like enkephalin precursor in suboesophageal ganglia of Squilla mantis, an arthropod shown to be sensitive in vivo to opiates [8], protein acid extracts were fractionated by gel filtration into three large pools: A(Mr greater than 65,000), B(10,000 less than Mr less than 65,000) and C(Mr less than 10,000). Only the low molecular weight pool, pool C, showed opioid-like activity when assayed by displacing labeled D-Ala2-D-Leu5-enkephalin from rat brain membranes. After trypsin and carboxypeptidase B proteolysis, pool A remained inactive, while pool B turned out to be active and was shown to inhibit the twitch response of electrically stimulated guinea-pig ileum. After HPLC fractionation of proteolyzed pool B, most of the opioid-like activity was found to be associated with a fraction showing an elution volume different from that of opioid peptide standards. Furthermore, no fraction showed immunoreactivity with anti-Met-enkephalin antibodies. The results suggest that native opioid-like peptides are present in Squilla mantis and are most likely released from higher molecular weight precursor(s).
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PMID:Native opioid-like peptides in Squilla mantis ganglia. 383 68

Adrenal chromaffin granules contain at least 10 peptides, ranging in size from 3 to 5 kilodaltons, that yield, upon digestion with trypsin, peptides that show specific binding to opiate receptors. All are distinctly different from beta-endorphin. Two of these peptides have been purified to homogeneity and subjected to chemical analysis. One is apparently a [Met]enkephalin precursor containing two copies of the [Met]enkephalin sequence. The other peptide contains both [Leu]enkephalin and [Met]enkephalin sequences and is presumably a common precursor of the two forms of enkephalin.
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PMID:Probable precursors of [Leu]enkephalin and [Met]enkephalin in adrenal medulla: peptides of 3-5 kilodaltons. 624

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


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