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:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The proteolytic degradation of porcine zinc insulin by alpha-chymotrypsin was previously found to depend markedly on the state of insulin aggregation (Pharm. Res. 9:864-869, 1992). In this study, the effect of bile salt-unsaturated fatty acid mixed micelles on alpha-chymotryptic degradation of insulin was further characterized. The incorporation of linoleic acid has greatly accelerated insulin degradation with the apparent first order rate constant being linearly related to the concentration of linoleic acid. At a 10 mM linoleic acid concentration solubilized in 10 mM sodium glycocholate, the proteolytic degradation rate constant increased by 16 times, which could not be explained solely by the mechanism of insulin oligomer dissociation. Further, this effect is significantly reduced when the free carboxylic group of linoleic acid is methylated. The catalytic role of mixed micelles on chemical degradation of insulin was found to depend on the concentration of linoleic acid incorporated. When solubilized in the form of mixed micelles, linoleic acid chemically catalyzes peptide bond cleavage in a concentration-dependent manner.
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PMID:Chemical and alpha-chymotrypsin-mediated proteolytic degradation of insulin in bile salt-unsaturated fatty acid mixed micellar systems. 829 Apr 78

Purified human serum butyrylcholinesterase, which exhibits cholinesterase, aryl acylamidase, and peptidase activities, was cross-reacted with two different monoclonal antibodies raised against human serum butyrylcholinesterase. All three activities were immunoprecipitable at different dilutions of the two monoclonal antibodies. At the highest concentration of the antibodies used, nearly 100% of all three activities were precipitated, and could be recovered to 90-95% in the immunoprecipitate. The peptidase activity exhibited by the purified butyrylcholinesterase was further characterized using both Phe-Leu and Leu-enkephalin as substrates. The pH optimum of the peptidase was in the range of 7.5-9.5 and the divalent cations Co2+, Mn2+, and Zn2+ stimulated its activity. EDTA and other metal complexing agents inhibited its activity. Thiol agents and -SH group modifiers had no effect. The serine protease inhibitors, diisopropylfluorophosphate and phenyl methyl sulfonyl fluoride, did not inhibit. When histidine residues in the enzyme were modified by diethylpyrocarbonate, the peptidase activity was not affected, but the stimulatory effect of Co2+, Mn2+, and Zn2+ disappeared, suggesting the involvement of histidine residues in metal ion binding. These general characteristics of the peptidase activity were also exhibited by a 50 kD fragment obtained by limited alpha-chymotrypsin digestion of purified butyrylcholinesterase. Under all assay conditions, the peptidase released the two amino acids, leucine and phenylalanine, from the carboxy terminus of Leu-enkephalin as verified by paper chromatography and HPLC analysis. The results suggested that the peptidase behaved like a serine, cysteine, thiol-independent metallopeptidase.
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PMID:The peptidase activity of human serum butyrylcholinesterase: studies using monoclonal antibodies and characterization of the peptidase. 842 27

Various surfactants were investigated to compare their effects on insulin dissociation, alpha-chymotryptic degradation, and rat enteral absorption. With a circular dichroism technique, sodium dodecyl sulfate (SDS) at a 5 mM concentration was found to completely dissociate porcine-zinc insulin hexamers (0.5 mg/ml) into monomers. The catalytic activity of alpha-chymotrypsin (0.5 microM) was also abolished by 5 mM SDS. When insulin was injected into the distal jejunum/proximal ileum segment of the rat, 5 mM SDS greatly enhanced its pharmacological availability, from a negligible value to 2.8%. Being a cationic surfactant, hexadecyl trimethylammonium bromide (CTAB) also efficiently dissociated insulin hexamers at concentrations of 1-5 mM. However, extensive charge-charge interaction was observed below a CTAB concentration of 0.6 mM, leading to insulin precipitation at a molar CTAB:insulin ratio of 1:1 to 2:1. An alpha-chymotryptic degradation study also revealed near-complete dissociation of insulin hexamers at 1 mM CTAB. Above 1 mM, however, CTAB acted as an enzyme inhibitor, most likely by means of charge repulsion. Enteral absorption studies showed a much lower pharmacological availability, only 0.29%. Nonionic surfactants such as Tween 80 and polyoxyethylene 9 lauryl ether were ineffective in dissociating insulin hexamers. Tween 80, at 5 mM, neither significantly altered the alpha-chymotryptic degradation pattern nor enhanced the enteral absorption of insulin. The relative effectiveness of different species of bile salts on insulin hexamer dissociation appeared to be similar. Sodium glycocholate at a 30 mM concentration also significantly increased insulin pharmacological availability, to 2.3%. A morphological study did not reveal any significant alteration of the rat intestinal mucosal integrity after exposure to 5 mM SDS for 30 min.2+ transport.
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PMID:Differential effects of anionic, cationic, nonionic, and physiologic surfactants on the dissociation, alpha-chymotryptic degradation, and enteral absorption of insulin hexamers. 845 72

Recombinant nidogen fragments comprising the globular domains G1 plus G2, the rod-like domain, and the rod connected to the globe G3 were prepared from the culture media of transfected human cell clones. In addition, domains G1 and G2 were separated from each other after cleavage with chymotrypsin. The purified fragments were characterized by N-terminal sequences, electrophoresis, electron microscopy, and radioimmunoassays and the cell clones by Northern hybridization. Transfection with a construct comprising a large part of domain G3 showed high mRNA levels but no secreted protein, indicating a protein folding problem. All these fragments were used as soluble and/or immobilized ligands in binding assays. This demonstrated major binding sites on domain G2 for collagen IV and heparan sulfate proteoglycan. Affinity chromatography on zinc- and cobalt-loaded columns showed binding of domains G2 and G3 and the rod. Protein binding, but not metal binding, was abolished by reduction and alkylation of nidogen. This allowed for the isolation of several zinc-binding tryptic peptides, four from G2, two from the rod, and one from the G3 domain. Most of these short peptides contained several histidines that are likely to mediate binding. Zinc inhibited efficiently G3-mediated nidogen binding to laminin at 4 degrees C (IC50 approximately 5 microM) but less at higher temperatures. Similarly, zinc inhibited binding to collagen IV and proteoglycan at low temperatures but not at high (37 degrees C) temperatures. This indicates a complex modulation of nidogen binding to other basement membrane proteins by some, but not all, transition metals. Whether the particularly striking effects shown for zinc are of biological relevance remains to be established.
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PMID:Mapping of nidogen binding sites for collagen type IV, heparan sulfate proteoglycan, and zinc. 849 53

A Ni(II)-binding serpin, pNiXa, is abundant in Xenopus oocytes and embryos. Kinetic assays show that purified pNiXa strongly inhibits bovine alpha-chymotrypsin (Ki = 3 mM), weakly inhibits porcine elastase (K1 = 0.5 microM), and does not inhibit bovine trypsin. The reversible, slow-binding inhibition of alpha-chymotrypsin by pNiXa is unaffected by Ni(II). Ovochymase in egg exudates is inhibited by pNiXa, but to a limited extent, even at high pNiXa concentrations. An octadecapeptide that models the His-rich domain (-HRHRHEQQGHHDSAKHGH-) of pNiXa forms six-coordinate, octahedral Ni(II)-complexes when the N-terminus is acetylated, and a square-planar Ni(II)-complex when the N-terminus is unblocked. Spectroscopy reveals two distinct types of octahedral Ni(II)-coordination to the N-acetylated octadecapeptide, involving, respectively, 3-4 and 5-6 imidazole nitrogens; the octadecapeptide undergoes partial, reversible precipitation in pH- and Ni(II)-dependent fashion, suggesting an insoluble, Ni(II)-coupled (Hx)n-dimer. Such (Hx)n-peptide interaction is confirmed by an enzyme-linked biotinavidin assay with N-biotin-KHRHRHE-amide and N-acetyl-KHRHRHE-resin beads, which become coupled after adding Ni(II) or Zn(II). H2O2 oxidation of 2'-deoxyguanosine to mutagenic 8-hydroxy-2'-deoxyguanosine is enhanced by the octahedral Ni(II)-octadecapeptide complex, although the effect is more intense with the square-planar Ni(II)-octadecapeptide complex. Immunoperoxidase staining of whole mounts with pNiXa antibody shows that pNiXa is distributed throughout gastrula-stage embryos and is localized during organogenesis in the brain, eye, spinal cord, myotomes, craniofacial tissues, and other sites of Ni(II)-induced anomalies. Patterns of pNiXa staining are similar in controls and Ni(II)-exposed embryos. Binding of Ni(II) to pNiXa may cause embryotoxicity by enhancing oxidative reactions that produce tissue injury and genotoxicity. Although the natural target proteinases for pNiXa inhibition have not been established, pNiXa may be an important regulator of proteolysis during embryonic development.
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PMID:Characterization of pNiXa, a serpin of Xenopus laevis oocytes and embryos, and its histidine-rich, Ni(II)-binding domain. 884 94

The binding properties of the COOH-terminal hemopexin-like domain (C domain) of human gelatinase A (matrix metalloproteinase-2, 72-kDa gelatinase) were investigated to determine whether the C domain has binding affinity for extracellular matrix and basement membrane components. Recombinant C domain (rC domain) (Gly417-Cys631) was expressed in Escherichia coli, and the purified protein, identified using two antipeptide antibodies, was determined by electrospray mass spectrometry to have a mass of 25,925 Da, within 0.1 Da of that predicted. As assessed by microwell substrate binding assays and by column affinity chromatography, the matrix proteins laminin, denatured type I collagen, elastin, SPARC (secreted protein that is acidic and rich in cysteine), tenascin, and MatrigelTM were not bound by the rC domain. Unlike the hemopexin-like domains of collagenase and stromelysin, the rC domain also did not bind native type I collagen. Nor were native or denatured types II, IV, V, and X collagen, or the NC1 domain of type VII collagen bound. However, binding to heparin and fibronectin (Kd, 1.1 x 10(-6) M) could be disrupted by 0.58-0.76 and 0.3 M NaCl, respectively. Using nonoverlapping chymotrypsin-generated fragments of fibronectin, binding sites for the rC domain were found on both the 40-kDa heparin binding and the 120-kDa cell binding fibronectin domains (Kd values, approximately 4-6 x 10(-7) M). The Ca2+ ion, but not the potential structural Zn2+ ion, were found to be essential for maintaining the binding properties of the protein. The apo-form of the rC domain did not bind heparin, and both ethylenediaminetetraacetic acid and the specific Ca2+ ion chelator 1, 2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid, but not the Zn2+ ion chelator 1,10-phenanthroline, eluted the holo form of the rC domain from both heparin-Sepharose and fibronectin. Inductive coupled plasma mass spectrometry also did not detect a Zn2+ ion in the rC domain. In contrast, reduction with 65 mM dithiothreitol did not interfere with heparin binding, further emphasizing the crucial structural role played by the Ca2+ ion. Together, these data demonstrate for the first time that the hemopexin-like domain of gelatinase A has a binding site for fibronectin and heparin, and that Ca2+ ions are important in maintaining the structure and function of the domain.
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PMID:The hemopexin-like domain (C domain) of human gelatinase A (matrix metalloproteinase-2) requires Ca2+ for fibronectin and heparin binding. Binding properties of recombinant gelatinase A C domain to extracellular matrix and basement membrane components. 905 49

We have been developing a novel bioadhesive drug-carrier matrix that protects embedded therapeutic peptides and proteins from degradation by the most abundant intestinal proteases. Increasing amounts of the Bowman-Birk inhibitor (BBI) were thereby covalently linked to chitosan-EDTA. The bioadhesive properties of the resulting polymer-BBI conjugates and their inhibitory effect toward trypsin (EC 3.4.21.4), chymotrypsin (EC 3.4.21.1), elastase (3.4.21.36), carboxypeptidase A (EC 3.4.17.1), and aminopeptidase N (EC 3.4.11.2) were evaluated in vitro. Whereas unmodified chitosan-EDTA exhibited under our experimental conditions an adhesive strength of 54.4 +/- 7.7 mN, it was determined to be 21.0 +/- 3.8 mN for the comparably most adhesive polymer-BBI conjugate (mean +/- SD; n = 5). All polymer-BBI conjugates showed a strong inhibitory activity toward the serine proteases trypsin and chymotrypsin. However, the protective effect toward elastase was markedly lower. Due to the high binding affinity of chitosan-EDTA toward zinc, which represents an essential cofactor for carboxypeptidase A and aminopeptidase N, all polymer-BBI conjugates displayed additionally a strong protective effect toward these exopeptidases. The novel bioadhesive polymer-BBI conjugates described in this study seem to be very useful drug-carrier matrixes in overcoming the enzymatic barrier to orally administered peptide and protein drugs.
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PMID:Intestinal peptide and protein delivery: novel bioadhesive drug-carrier matrix shielding from enzymatic attack. 954 94

Zinc absorption in alcoholism was studied by a combination of zinc tolerance tests in 382 male patients with alcoholism (more than 140 g/day of ethanol) who had alcohol-induced disease of the liver or pancreas. In study 1, the serum zinc level was measured in all patients, and serum zinc and fecal chymotrypsin levels were compared in various disease groups. In study 2, 14 patients with liver cirrhosis (LC), 15 with chronic pancreatitis (CP), 7 with LC + CP, and 7 controls underwent oral zinc tolerance and zinc dipicolinate tolerance tests, zinc absorption and disorders of pancreatic exocrine functions were examined. In study 1, the serum zinc concentration was significantly lower in the CP and LC groups than in the control group, and the fecal chymotrypsin activity was significantly lower in the CP than in the control groups. In study 2, during the oral zinc tolerance test, the serum zinc concentration 3 hours after administration was significantly lower in the LC, CP and LC + CP groups than in the control group. In these groups, the serum zinc concentration was significantly lower in the abnormal fecal chymotrypsin group than in the control group at 2 and 3 hours after administration of zinc sulfate. In the oral zinc dipicolinate tolerance test, the serum zinc levels 2 and 3 hours after administration were significantly elevated in the control and all disease groups; there were no significant differences between the control and each disease group. These results suggest that reduction of pancreatic exocrine functions by alcohol and chronic reduction of synthesis of ligands such as picolinic acid in the liver are involved in the reduction of serum zinc in alcoholism.
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PMID:Evaluation of pancreatic exocrine function and zinc absorption in alcoholism. 965 43

The purpose of the present study was to synthesize and evaluate mucoadhesive polymers, exhibiting a high capacity to bind bivalent cations which are essential co-factors for intestinal proteolytic enzymes. Under the formation of amide bonds, the complexing agent EDTA was covalently bound to the primary amino groups of chitosan. One gram of the resulting conjugate with the lowest amount of remaining free amino groups (0.1 +/- 0.03%; mean +/- SD, n = 3) based on free chitosan as 1.0 was capable of binding 1.4 +/- 0.1 mM calcium, 2.0 +/- 0.1 mM zinc and 1.9 +/- 0.03 mM cobalt (mean +/- SD, n = 3) under intestinal pH-conditions, respectively. Whereas proteolytic activity of the serine proteases trypsin (EC 3.4.21.4), alpha-chymotrypsin (EC 3.4.21.1) and elastase (EC 3.4.21.36) could not be inhibited, proteolytic activity of the zinc proteases carboxypeptidase A (EC 3.4.17.1) and aminopeptidase N (EC 3.4.11.2) was strongly inhibited by the chitosan-EDTA conjugate. Moreover, it displays quick swelling properties in water and basic aqueous solutions. The adhesive force of the conjugate was even higher than of chitosan HCl. However, lowering the percentage of covalently attached EDTA on the polymer, leads to a significantly reduced adhesive force. According to these results, chitosan-EDTA conjugates exhibiting the lowest amount of remaining free amino groups, seem to be a useful tool in overcoming the enzymatic barrier for perorally administered therapeutic peptides.
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PMID:Mucoadhesive polymers as platforms for peroral peptide delivery and absorption: synthesis and evaluation of different chitosan-EDTA conjugates. 968 88

This study was mainly aimed to investigate the efficacy of trypsin:chymotrypsin to elicit anti-oxidant properties. In our earlier studies it was observed that the enzyme preparation exhibited an anti-inflammatory action as there was a remarkable reduction in oedema formation and tissue destruction. This led to further study on the amount of lipid peroxidation products formed and the levels of enzymatic and non-enzymatic anti-oxidants and relative trace element contents of copper, selenium, iron and zinc during administration of the enzyme preparation. Decreased formation of lipid peroxidation products was observed in treated group in comparison with the untreated group. Higher levels of enzymatic anti-oxidants mainly super oxide dismutase, catalase, glutathione peroxidase and glutathione-s-transferase and non-enzymatic antioxidant namely ceruloplasmin persisted for a longer period of time in the treated group than in the untreated group. No statistical significance was observed in non-enzymatic antioxidants viz. ascorbic acid and tocopherol levels in both the groups. Increased serum copper and selenium levels in the treated group could be related to higher levels of the ceruloplasmin and glutathione peroxidase observed in the treated group. The above studies support the finding that treatment with the enzyme preparation reduced tissue destruction leading to decreased formation of free radicals and subsequent effective scavenging of free radicals by the higher levels of enzymatic and non-enzymatic anti-oxidants.
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PMID:The efficacy of trypsin: chymotrypsin preparation in the reduction of oxidative damage during burn injury. 977 92


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