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

---

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)
...
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

A putative proenkephalin-cleaving enzyme (PCE) extracted from bovine adrenal chromaffin granules was purified with soybean trypsin inhibitor high-performance affinity chromatography. The 12,600-fold purified enzyme was maximally active at pH 8.0. The enzyme was completely inhibited with lima bean trypsin inhibitor (0.1 mg/ml), soybean trypsin inhibitor (0.1 mg/ml), and p-(chloromercuri)benzenesulfonic acid (1.0 mM), indicating PCE is a serine protease with cysteine residues likely to be involved in its structure or activity. It exhibited significant autoproteolysis without specific substrates present. The substrate specificity and kinetic constants with the enkephalin-containing (EC) peptides Leu-9 and proenkephalin Peptides B, E, and F as substrates were studied. The cleavage patterns were substantially different than with trypsin digestion. PCE specifically recognized the paired basic amino acid residues and predominantly cleaved the peptide bonds between Lys and Arg sites and peptide bonds after Lys-Lys and Arg-Arg sites. Different Km and Vmax values for the different Lys-Arg sites indicate sequences in addition to the paired basic residues can affect enzyme activity. Also, the lower Km and Vmax of Peptide E suggest a higher affinity for this peptide but much slower cleavage. The C-terminally located Lys-Arg site appears responsible for this high affinity. Based on these observations, we propose the following: (a) the primary structure of these peptides contains enough information to be processed correctly by PCE and (b) PCE may be regulated by pH and Peptide E to prevent extensive processing of the intermediate EC peptides which are the major opioid peptides found in the adrenal chromaffin granules.
...
PMID:Purification and characterization of a putative proenkephalin cleaving enzyme. 163 49

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.
...
PMID:Expression of proenkephalin A mRNA and enkephalin-containing peptides in cultured fibroblasts. 169 63

Proteinases capable of cleaving proenkephalin into smaller peptides have been identified in bovine adrenal chromaffin granules using [35S]methionine-labeled recombinant rat proenkephalin as a selective substrate in sodium dodecyl sulfate-polyacrylamide gel electrophoresis proteinase radiozymography. This technique was used for the screening of subcellular fractions, general characterization of pH optima, and the mechanistic characterization of proteinases with both reversible and irreversible inhibitors. Two enzymes with approximate molecular masses of 76 and 30 kDa were shown to be localized to the highest-density fractions of chromaffin granules by sucrose density gradient fractionation. Both were enriched in a 1 M NaCl wash of purified chromaffin granule membranes, were active at high pH, and were characterized as serine proteinases based on inhibition by soybean trypsin inhibitor. The 30-kDa enzyme was also inhibited by diisopropyl fluorophosphate, D-Phe-Pro-Arg-CH2Cl, and D-Val-Phe-Lys-CH2Cl and appeared to be the previously described adrenal trypsin-like enzyme. A third enzyme, of 66 kDa, was also associated with the 1 M NaCl wash of purified chromaffin granule membranes but was not localized exclusively to chromaffin granules in sucrose gradients. This proteinase was found to be Ca2+ activated and inhibited by EDTA but not diisopropyl fluorophosphate, soybean trypsin inhibitor, p-chloromercuriphenylsulfonic acid, 1,10-phenanthroline, or pepstatin.
...
PMID:Characterization of proenkephalin-cleaving proteinases in bovine adrenal chromaffin granules using [35S]proenkephalin copolymerized into sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 172 4

The processing of proenkephalin was studied using [35S]methionine pulse-chase techniques in primary cultures of bovine adrenal medullary chromaffin cells. Following radiolabeling, proenkephalin-derived peptides were extracted from the cells and separated by reverse-phase HPLC. Fractions containing proenkephalin fragments were digested with trypsin and carboxypeptidase B to liberate Met-enkephalin sequences and subjected to a second HPLC step to demonstrate association of radiolabel with Met-enkephalin. Processing of proenkephalin is complete within 2 h of synthesis, suggesting completion at or soon after incorporation into storage vesicles. Pretreatment of the cells with nicotine, histamine, or vasoactive intestinal peptide to enhance the rate of proenkephalin synthesis failed to alter the time course of processing and had minimal effects on the distribution of products formed. Addition of tetrabenazine, an inhibitor of catecholamine uptake into chromaffin vesicles, during radiolabeling and a 6-h chase period caused enhanced proenkephalin processing. These results suggest that the full range of proenkephalin fragments normally found in the adrenal medulla (up to 23.3 kDa) represents final processing products of the tissue and that termination of processing may depend on the co-storage of catecholamines.
...
PMID:Processing of proenkephalin in adrenal chromaffin cells. 186 Nov 55

We have previously demonstrated an increase in plasma met-enkephalin levels during the pain attacks in episodic cluster headache. The present study was undertaken in order to clarify the source of the plasma met-enkephalin increase. Recent evidence has shown that peripheral blood polymorphonuclear cells contain peptides derived from the proenkephalin A system, which can be released by specific stimuli. We studied neutrophil met-enkephalin containing peptides (NMECP) in 27 episodic cluster headache patients: 24 in a cluster period (6 of them during a pain attack), and 3 in the remission period. Neutrophil met-enkephalin containing peptide levels (after sequential enzymatic digestion with trypsin and carboxypeptidase B) were determined by radioimmunoassay with specific antiserum. Neutrophil peptide concentration (pmol/mg prot) was lower (p less than 0.01) in patients during the pain attack (14.4 +/- 0.36) than after their pain had subsided (36.7 +/- 0.31) and lower than in the remission period patients (35.8 +/- 0.4). We conclude that neutrophil met-enkephalin containing peptides decrease during pain in episodic cluster headache, and that they may be involved in the concomitant plasma met-enkephalin increase.
...
PMID:Changes in neutrophil met-enkephalin containing peptides in episodic cluster headache. 188 84

The synthesis of proenkephalin was assessed in primary cultures of bovine adrenal medullary chromaffin cells by incubation of the cells with [35S]methionine, digestion of proenkephalin-derived peptides with trypsin and carboxy-peptidase B, and quantitation of radioactivity incorporated into Met-enkephalin following reversed-phase HPLC. Nicotine, histamine, and vasoactive intestinal peptide each enhanced the rate of proenkephalin synthesis approximately 10-fold when examined between 16 and 32 h after the drug or hormone addition. Inclusion of nifedipine (1 microM) partially blocked the stimulatory effect of nicotine, but not that of vasoactive intestinal peptide or histamine, or proenkephalin synthesis. Theophylline, tetrabenazine, and angiotensin II also increased the rate of proenkephalin synthesis (three- to eight-fold). These increases in the apparent rate of proenkephalin synthesis were not attributable to altered [35S]methionine specific radioactivity or rates of turnover and did not reflect similar increases in total protein synthesis. The half-life for turnover of Met-enkephalin sequences was 3-4 days in the cultured chromaffin cell. These studies directly show that proenkephalin synthesis is the primary regulatory step in control of chromaffin cell opioid peptide content.
...
PMID:Regulation of proenkephalin synthesis in adrenal medullary chromaffin cells. 199 99

A putative prohormone-processing enzyme complex with specificity toward basic residues was partially purified from whole bovine pituitary glands. The complex is basic, binding to S-Sepharose at pH 8.2. The pH optimum of the enzyme is around 8.0. The enzyme is capable of cleaving proenkephalin and is present in at least three forms with relative molecular masses of about 36,000, 58,000, and 90,000 Da. The proteinase complex is inhibited by soybean trypsin inhibitor, limabean trypsin inhibitor, and aprotinin, but not by inhibitors of thiol proteinases or metal chelators. Our results indicate that this proteinase is a trypsin-like serine esterase with properties appropriate to that of a prohormone-processing enzyme.
...
PMID:Partial purification and characterization of a putative prohormone-processing enzyme complex from bovine pituitary. 201 54

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)
...
PMID:Differential association of endogenous proenkephalin-derived peptides with membranes of microsomes from rat striatum, adrenal medulla and heart ventricle. 224 89

A novel method is described for the zymographic analysis of proteinases in sodium dodecyl sulfate-polyacrylamide gels containing copolymerized radiolabeled protein substrates such as [35S]methionine-labeled proenkephalin or 125I-labeled proinsulin. After electrophoresis the enzyme is reactivated and cleaves the radiolabeled in situ substrate into smaller peptides. These small peptides are able to diffuse out of the gel, leaving clear areas against a dark background when visualized by autoradiography. The technique can be used to detect as little as 200 fg of trypsin using only 50 ng (1.25 microCi) of [35S]proenkephalin. Soluble- and membrane-bound adrenal trypsin-like enzyme were isolated from bovine adrenal chromaffin granules. Both proteinases cleaved [35S]methionine-labeled proenkephalin but not 125I-labeled proinsulin. Moreover, both had a Mr of approximately 30,000. The potential of this technique for general use is discussed. An additional method using the synthetic fluorogenic substrate t-butoxycarbonyl Glu-Lys-Lys aminomethylcoumarin is also described.
...
PMID:Electrophoretic analysis of proteinases in sodium dodecyl sulfate-polyacrylamide gels containing copolymerized radiolabeled protein substrates: application to proenkephalin processing enzymes. 228 41


1 2 3 4 Next >>

---