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)

Acetylcholinesterase (AChE, EC 3.1.1.7) purified from the electric organ of eel possesses a protease activity resembling that of a neuropeptide processing enzyme. To examine whether any mammalian AChEs possess a similar protease activity, the enzyme was purified, 110,000-fold from foetal bovine serum. Purified serum AChE cleaved 2 synthetic peptide substrates in a manner resembling the combined actions of trypsin-like and carboxypeptidase B-like enzymes. A synthetic fragment of preproenkephalin A (residues 97-107) containing a complete methionine-enkephalin sequence was cleaved by serum AChE to yield free methionine-enkephalin. The carboxypeptidase action of AChE was weakly stimulated by the presence of 100 microM CoCl2 suggesting the requirement of a metal ion for complete activity. The results support the hypothesis that in many tissues AChE may act as a neuropeptide processing enzyme.
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PMID:Serum acetylcholinesterase possesses trypsin-like and carboxypeptidase B-like activity. 322 17

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

The effects of hypophysectomy (HPX) and dexamethasone (DEX) on the levels of Met5-enkephalin (ME), ME precursors, and the abundance of proenkephalin (pEK) mRNA, were examined in the adrenal medulla (AM) and superior cervical ganglia (SCG). To assess possible changes in enkephalin processing, both cryptic (after trypsin and carboxypeptidase B digestions) and native (without enzyme digestions) ME-like immunoreactivity (ME-LI) was measured. Three weeks after HPX the proportion of pEK mRNA to the total RNA content in the AM was not significantly changed when compared to sham-operated (SO) animals. Total (native + cryptic) ME-LI was decreased by 45% in the AM of HPX rats. This decrease was paralleled by a 58% depletion of AM proteins. Cryptic ME-LI was also reduced by 43%. In contrast, native ME-LI was not altered after HPX, indicating enhanced processing of ME precursors. Treatment with DEX (5 daily injections--1 mg/kg, i.p.) increased the relative abundance of pEK mRNA (+27%) and total ME-LI in the AM of HPX group, but not in SO group. Native ME-LI, cryptic ME-LI, and their ratio were not significantly affected by DEX in the AM of HPX or SO rats. In SCG, the relative abundance of pEK mRNA decreased by 25% after hypophysectomy. Total and cryptic ME-LI in the SCG of HPX rats were not changed when compared to SO rats. In contrast, HPX reduced native ME-LI suggesting decreased processing of ME precursors. Similarly, as in AM, DEX produced increase in the SCG pEK mRNA only in HPX (+68%) and not in the SO rats. In SCG, DEX produced decreases in total ME-LI which could be attributed to an increased enkephalin release. An overall reduction of cryptic ME-LI was also observed after DEX, whereas native ME-LI remained unchanged suggesting increased processing of enkephalins. Our findings indicate that the pituitary adrenocortical axis controls the relative proportions of ME to its precursors, and that this control involves both glucocorticoid-dependent (SCG) and glucocorticoid-independent (AM) mechanisms. In contrast, our studies do not suggest specific control of pEK synthesis by the pituitary adrenocortical axis. The pituitary adrenocortical axis may also influence the relative contents of ME and catecholamines in the AM and SCG. The ratio of ME/catecholamines increased after HPX (AM and SCG) and after DEX (SCG). Such regulation may contribute to the control of co-transmitter output in the sympathoadrenal system.
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PMID:Roles of the pituitary-adrenocortical axis in control of the native and cryptic enkephalin levels and proenkephalin mRNA in the sympathoadrenal system of the rat. 340 3

Human plasma carboxypeptidase N was purified to homogeneity and its active and inactive subunits were separated. By introducing a novel technique, both forms of the active subunit (Mr = 55,000 and Mr = 48,000) were isolated. N-terminal sequencing of the active subunit of human carboxypeptidase N revealed significant homology with the N-terminal sequence of bovine carboxypeptidase H (43% identity) and to a lesser extent with carboxypeptidase A (29% identity) or carboxypeptidase B (18% identity). The active subunit of carboxypeptidase N was hydrolyzed with trypsin and 4 of the tryptic peptides were isolated by HPLC and sequenced. The sequences of the four peptides were homologous (39-64% identity) with regions of carboxypeptidase H corresponding to the middle (residues 148-175) and C-terminal portion (residues 321-408). These regions had essentially no homology with carboxypeptidase A or B. These data indicate that carboxypeptidase H and the active subunit of carboxypeptidase N may have diverged from a common ancestral gene.
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PMID:Amino acid sequence of the N-terminus and selected tryptic peptides of the active subunit of human plasma carboxypeptidase N: comparison with other carboxypeptidases. 340 1

The distribution of cryptic forms (larger enkephalin-containing peptides) in neostriatum, hypothalamus, spinal cord T3-L1 and neurointermediate lobe of pituitary were determined by radioimmunoassay. Optimal conditions for enzymic hydrolysis of the cryptic enkephalins by trypsin and carboxypeptidase B were established. The proportion of total Met- and Leu-enkephalin represented by native pentapeptide varied markedly among these central nervous system regions. Also, the distributions of native and cryptic Met-enkephalin were distinct from that of Leu-enkephalin. Chromatographic separation by HPLC of immunoreactive Met-enkephalin peptides revealed only two peaks corresponding to Met-enkephalin and Met-enkephalin sulfoxide in rather equal amounts. Hydrolysis of cryptic Met-enkephalin also produced only two HPLC-separable peaks of immunoreactive Met-enkephalin, again corresponding to Met-enkephalin and Met-enkephalin sulfoxide. Bioactivity of cryptic striatal Met-enkephalin after hydrolysis was demonstrated by antinociception and catalepsy in rats following its intracerebroventricular injection. Repeated short-term administration of nicotine, 0.1 mg/kg IP six times at 30 min intervals, produced significant increases in native and cryptic Met-enkephalin in striatum, consistent with an increase in neuronal release of Met-enkephalin together with increases in synthesis and processing of proenkephalin A in this brain region. This regimen of nicotine also decreased levels of native Met-enkephalin and of both native and cryptic Leu-enkephalin in neurointermediate lobe, consistent with nicotine-induced release of both proenkephalin A- and prodynorphin-derived peptides from neurointermediate lobe.
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PMID:Nicotine-induced alterations in brain regional concentrations of native and cryptic Met- and Leu-enkephalin. 344 43

The processing of preprocholecystokinin in human pituitary extracts was investigated using gel and ion-exchange chromatography monitored by sequence-specific radioimmunoassays before and after incubation with trypsin, carboxypeptidase B, and arylsulfatase. Whereas the neural lobe contained only the bioactive alpha-carboxyamidated cholecystokinin (CCK) peptides (32 pmol/g), of which CCK-8 predominated, the anterior lobe contained substantial amounts of three large nonamidated procholecystokinin fragments (95 pmol/g; Mrs, 9000, 7000, and 5000) and small amounts of alpha-amidated CCK (8.3 pmol/g). The latter occurred only in the following large molecular forms: component I, CCK-58, and traces of CCK-33. Corticotrophic tumors processed the large forms to small CCK-8-like forms as are found in the brain and in the gut. The results show that a hormone gene, although translated, is expressed only to a limited extent as mature, active peptide outside the principal production region(s). Thus the processing of CCK to small alpha-amidated peptides in the less-differentiated tumor tissue supports the hypothesis that differentiation of endocrine cells may be sustained also at the posttranslational level.
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PMID:Preprocholecystokinin processing in the normal human anterior pituitary. 347 48

Acetylcholinesterase was purified by passage through 3 affinity columns. The enzyme so purified was found to be homogeneous by electrophoresis and the peptidase and AChE activities co-eluted from a high pressure liquid chromatography column. The purified AChE degraded the chromogranins, the soluble proteins from the adrenal chromaffin granules, at a rate of nearly 8 micrograms/microgram AChE/h. The rate was fastest with the largest chromogranins, but proteins across the whole molecular weight spectrum were hydrolyzed. Immunoassay of extracts after incubation with AChE showed that enkephalin-like material had been produced. Incubations were also done with chromogranins that had been fractionated by size exclusion chromatography. The AChE degraded protein in all fractions and generated enkephalin-like immunoreactive material in fractions where it was produced by sequential treatment with trypsin and carboxypeptidase B. It seems likely, therefore, that AChE can hydrolyze some of the enkephalin precursors that are sensitive to trypsin and carboxypeptidase B, but the one-step nature of its action suggests a mode of action with fewer restrictions. It is concluded that AChE can hydrolyze proteins of widely differing sizes and the data add to the evidence that AChE is able to hydrolyze enkephalin precursors resulting in the generation of immunoreactive peptide.
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PMID:Acetylcholinesterase generates enkephalin-like immunoreactivity when it degrades the soluble proteins (chromogranins) from adrenal chromaffin granules. 352 46

A series of dibasic insulin precursors including proinsulin was expressed and secreted from Saccharomyces cerevisiae. Recombinant plasmids were constructed to encode fusion proteins consisting of a modified mating factor alpha 1 leader sequence and an insulin precursor. The leader sequence serves to direct the fusion protein into the secretory pathway of the cell and to expose it to the Lys-Arg processing enzyme system. The secreted peptides were purified from the fermentation broth and characterized by sequencing and amino acid analysis. Processing at one or both dibasic sequences was shown in proinsulin and in other insulin precursors containing a short spacer peptide in place of the C peptide. In contrast, no processing was observed in the absence of a spacer peptide in the insulin precursor molecule, e.g., B-Lys-Arg-A (where A and B are the A and B chain of human proinsulin, respectively). This type of single-chain insulin precursors isolated from such constructions could be enzymatically converted into insulin by treatment with trypsin and carboxypeptidase B. The above results suggest that the C-peptide region of proinsulin serves to direct the trypsin-like converting enzyme to process at the two dibasic sequences. We propose that in hormone precursors in general the spacer peptides serve to expose dibasic sequences for processing.
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PMID:Secretion and processing of insulin precursors in yeast. 352 91

The effect of reflex splanchnic nerve stimulation on proenkephalin A biosynthesis was investigated in the rat adrenal medulla. Tissue levels of native [Met5]enkephalin-like immunoreactivity (IR) (measured by direct RIA of tissue extracts), cryptic [Met5]enkephalin-like IR (calculated as the increase in [Met5]enkephalin-like IR detected in tissue extracts after sequential digestion with trypsin and carboxypeptidase B), and proenkephalin A mRNA were determined in adrenal medulla from rats sacrificed at various times after a period of insulin-induced hypoglycemia. Two hours of insulin hypoglycemia, which produced intense reflex stimulation of the splanchnic nerves as evidenced by a 55% decrease in the adrenal medulla catecholamine levels, resulted in a 3-fold increase in proenkephalin A mRNA levels in this tissue. The proenkephalin A mRNA levels reached a maximum 15-fold increase over control values 24 hr after this period of hypoglycemic stress and then gradually declined with an approximate half-life of 4 days. Native and cryptic [Met5]enkephalin-like IR had increased 9-fold and 12-fold, respectively, 24 hr after this period of hypoglycemia, and both demonstrated maximum increases of 130-fold and 50-fold, respectively, after 96 hr. Combined pretreatment (i.p. administration) with the ganglionic and muscarinic blocking agents chlorisondamine (5 mg/kg of body weight) and atropine (1 mg/kg) blocked the increase in levels of proenkephalin A mRNA seen in the rat adrenal medulla following insulin hypoglycemia. These data indicate that reflex splanchnic nerve discharge stimulates proenkephalin biosynthesis, probably at the level of gene expression.
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PMID:Reflex splanchnic nerve stimulation increases levels of proenkephalin A mRNA and proenkephalin A-related peptides in the rat adrenal medulla. 353 20

Free enkephalins (enk) and higher molecular weight enkephalin-containing peptides (enk-c-p) are present in the endocrine pancreas of rats, presumably in B cells. To determine whether these opioid peptides show dynamic alterations as insulin content of pancreas changes, we utilized a copper deficient rat model, in which the exocrine pancreas atrophies and the endocrine pancreas is "intact" and insulin (IRI) content increases. Dietary copper deficiency (-C) was produced in weanling male rats for 4 and 7 weeks. The deficient and copper supplemented (+C) groups were further subdivided to receive all dietary carbohydrate as either 62% fructose (F) or 62% starch (S). -CF rats showed the most severe deficiency. After 7 weeks, total units of pancreatic IRI in -CF were 7.5 +CF 2.1, -CS 7.9 and in +CS 2.8 (p less than 0.001). Pancreatic content of Met5- and Leu5-enk was measured in extracts which were purified on C-18 Seppaks with and without prior treatment with trypsin and carboxypeptidase B. -C animals showed progressive, significant increases in pancreatic content of Leu-enk-c-p, with a decrease in free Leu- and Met-enk (p less than 0.02-0.01). The pancreatic findings are compatible with a co-localization of enkephalins and insulin in the endocrine pancreas and are suggestive of co-regulation.
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PMID:Copper deficiency in rats increases pancreatic enkephalin-containing peptides and insulin. 355 Jul 24


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