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)

Human proenkephalin generated by means of a recombinant vaccinia virus expression vector was used as the substrate for a putative processing enzyme obtained from bovine adrenal chromaffin granules. The adrenal enzyme successfully cleaved proenkephalin to generate low mol wt enkephalins as well as other enkephalin-containing intermediates. Radioactively labeled proenkephalin prepared with this system was also cleaved; however, under identical conditions bovine proinsulin was not cleaved. These results provide support for the notion that the adrenal trypsin-like enzyme is involved in the processing of proenkephalin in vivo and demonstrate the usefulness of protein substrates prepared by expression vector systems in testing the reactivity and specificity of proposed prohormone processing enzymes in vitro.
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PMID:Cleavage of proenkephalin by a chromaffin granule processing enzyme. 229

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

The effect of chronic neuroleptic treatment, using haloperidol or clozapine, on immunoreactive dynorphin peptide and substance P levels in basal ganglia of rats was examined. The drugs were administered i.p. in daily doses for 10 days (haloperidol 1 mg/kg and clozapine 10 mg/kg). Dynorphin A, dynorphin B and substance P were measured in substantia nigra, striatum, globus pallidus and hypothalamus using specific radioimmunoassays. The most prominent effects were observed with with clozapine which increased levels of all measured peptides in substantia nigra. Haloperidol only affected nigral substance P levels which declined, while nigral dynorphin peptide levels remained unchanged. In striatum, haloperidol slightly reduced dynorphin peptides while substance P was unaffected. Clozapine increased striatal substance P but the dynorphin peptides were not affected. Minor changes in dynorphin peptides found in globus pallidus and hypothalamus were not statistically reliable. Substance P was not changed in these structures after either of the two drugs. High molecular weight fragments (greater than or equal to 5,000) from the dynorphin precursor, proenkephalin B, were measured in substantia nigra and striatum using trypsin digestion and subsequent analysis of generated Leu-enkephalin-Arg6. These high molecular weight fragments were found to be affected in the same manner as the dynorphin peptides. This study indicates that the two types of neuroleptic drugs have different modes of interaction on peptide systems in basal ganglia of rats. Dynorphin peptides and substance P were also differentially affected.
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PMID:Chronic haloperidol and clozapine differentially affect dynorphin peptides and substance P in basal ganglia of the rat. 242 23

A putative processing enzyme for proenkephalin, with activity directed toward basic residues, was purified over 2000-fold from washed bovine adrenal medullary chromaffin granule membranes. The molecular mass of this membrane-bound adrenal trypsin-like enzyme (mATLE) is 31 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the enzyme is extremely basic, binding to carboxymethyl-Sephadex at pH 8.5. The pH optimum of mATLE using t-butoxycarbonyl-Glu-Lys-Lys-aminomethylcoumarin as a substrate is 8.5-8.7, and its Km value for this substrate is 2.2 mM. mATLE activity was inhibited by soybean trypsin inhibitor, lima bean trypsin inhibitor, and aprotinin but not by metal chelators or thiol-directed reagents. Sequencing of cleavage products released from Peptide B revealed that the enzyme preferentially cleaves between and following the paired basic residues at positions 23 and 24 of Peptide B (thus generating [Met-enkephalin]-Arg-Phe and Arg-[Met-enkephalin]-Arg-Phe). Dynorphin A was cleaved following a single lysine at position 11 but not at the paired arginine site. Our results suggest that mATLE is a trypsin-like serine protease with the specificity appropriate to that of a proenkephalin processing enzyme.
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PMID:A putative processing enzyme for proenkephalin in bovine adrenal chromaffin granule membranes. Purification and properties. 276 80

Tryptic digestion followed by radioimmunoassay for (Leu)enkephalin-Arg6 has been used in this study as a general method to detect the presence of all possible products containing the enkephalin sequence from the opioid peptide prohormone, proenkephalin B. Tissue extracts of human hypothalamus and pituitary were examined. Gel filtration was used to separate the different precursor products according to molecular weight. The elution profile was also monitored with highly sensitive radioimmunoassays for dynorphin A and dynorphin B, respectively. Immunoreactive dynorphin A appeared in three peaks with the approximate molecular weight of 1000, 2000 and 5000. Immunoreactive dynorphin B partly occurred in other peaks, 1500, 5000 and 10 000 dalton. Profiles obtained by measuring immunoreactive (Leu)enkephalin-Arg6 in all fractions from gel filtration after trypsin digestion showed a more complex pattern compared to the profiles of immunoreactive dynorphin A and dynorphin B. The major peaks coincided with dynorphin A and dynorphin B but high levels of immunoreactive (Leu)enkephalin-Arg6 were also generated from higher molecular weight regions (MW greater than 5000).
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PMID:A general procedure for analysis of proenkephalin B derived opioid peptides. 286 27

The chemical nature of peptides in human CSF with the enkephalin core sequence from proenkephalin A and proenkephalin B, was investigated. Direct measurements with radioimmunoassay (RIA) were run on enkephalin, enkephalin hexapeptides, dynorphin A, dynorphin A1-8 and dynorphin B. The hexapeptides occurred in about 3 times higher concentration than the corresponding enkephalins. The only dynorphin RIA which gave positive results was the one for dynorphin A. However, most dynorphin A immunoactive material showed higher apparent molecular weight (MW; 3 and 5 kdalton) than the standard (2 kdalton). To identify and quantitate every possible proenkephalin derived peptide with the enkephalin sequence, chromatographic fractions were treated with trypsin. The products, Leu-enkephalin-Arg6 (from proenkephalin B) and Met-enkephalin-Lys6/Arg6 (from proenkephalin A) were measured by specific RIAs and identified by HPLC. In the higher (greater than 5 kdalton) MW interval, there was about 10-fold higher yield of Met-enkephalyl than Leu-enkephalyl hexapeptides. In the intermediate 1-3 kdalton MW interval, most activity derived from proenkephalin B. Finally, from the low MW region, there was about 5 times more proenkephalin A peptides. The main dynorphin A peak at 5 kdalton was transferred to a major Leu-enkephalin-Arg6 peak by trypsin degradation. The data indicate the presence of a whole family of peptides from the two proenkephalin genes in human CSF. Precursors to the peptides supposed to be the active members in the proenkephalin families occur in relatively high concentrations and may provide good markers for activity in these peptide systems.
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PMID:Enkephalin-containing polypeptides in human cerebrospinal fluid. 287 Jul 78

The endogenous opioid peptides all contain the enkephalin sequence Tyr-Gly-Gly-Phe-Met and Tyr-Gly-Gly-Phe-Leu at their aminoterminus. Three distinct families of these peptides (endorphins, enkephalins and dynorphins) are present in different neuronal pathways within the central nervous system. Molecular genetics have shown that these three families of opioid peptides are derived from three distinct precursors. Pro-opiomelanocortin gives rise to the endorphins, as well as adrenocorticotropic hormone (ACTH) and the melanotropic hormones (MSH's). [Met] enkephalin, [Leu] enkephalin and the related heptapeptide [Met] enkephalin-Arg6-Phe7 and octapeptide [Met] enkephalin-Arg6-Gly7-Leu8 are derived from proenkephalin. The third family is derived from prodynorphin and includes dynorphin A, dynorphin B (also known as rimorphin) and alpha- and beta-neo-endorphin. The structure of the genes coding for these precursors are similar, suggesting the possibility of one common ancestral gene. The most common scheme for enzymatic maturation of precursors proposes the action of a trypsin-like endopeptidase followed by a carboxypeptidase B-like exopeptidase. However, we have provided evidence that this combination of trypsin-like and carboxypeptidase B-like enzymes may not be the only mechanism for liberating enkephalin from low molecular weight enkephalin-containing peptides. Indeed, endo-oligopeptidase A, an enzyme, known to hydrolyze the Phe5-Ser6 bond of bradykinin and the Arg8-Arg9 bond of neurotensin, has been shown to produce, by a single cleavage, [Leu] enkephalin or [Met] enkephalin from small enkephalin-containing peptides, (Camargo et al., 1987, J. Neurochem. 48, 1258-1263).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[Biosynthesis of opioid peptides]. 305 81

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

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


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