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)

The structural and functional domains of Escherichia coli carbamoyl phosphate synthetase (CPS) have been identified by limited proteolysis. Incubation of CPS with several proteases, including trypsin, chymotrypsin, subtilisin and endoproteinase Asp-N, under native conditions, causes a time-dependent loss of enzymatic activity and the generation of a common fragmentation pattern. Amino-terminal sequencing studies demonstrated that the initial cleavage event by trypsin occurred at the carboxy-terminal end of the large subunit. The ultimate fragments produced in most of the proteolysis studies, 35- and 45-kDa peptides, were derived from areas corresponding to the putative ATP binding regions. Substrate protection studies showed that the addition of ligands did not affect the final fragmentation pattern of the protein. However, ornithine and UMP were found to significantly reduce the rate of inactivation by inhibition of proteolytic cleavage. MgATP and IMP provided modest protection whereas bicarbonate and glutamine showed no overall effect on proteolysis. Limited proteolysis by endoproteinase Asp-N resulted in the production of a fragment (or multiple fragments) which contained enzymatic activity but had lost all regulation by the allosteric ligands, UMP and ornithine. The small subunit has been shown to be protected from proteolysis by the large subunit. Proteolysis of the isolated small subunit resulted in the generation of a stable 31-kDa species which contained 10% of the original glutaminase activity. These studies demonstrate that a portion of the C-terminal end of the large subunit can be excised without entirely destroying the ability of CPS to catalyze the formation of carbamoyl phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mapping the structural domains of E. coli carbamoyl phosphate synthetase using limited proteolysis. 764 1

This study demonstrates the effects of conditioned media from transformed neonatal rat retinal pigment epithelial cells (tnrRPE-CM) in a culture system consisting of neonatal rat retinal explants. For this study, retinal explants from postnatal day 2 (PN2) normal rats were cultured for over 3 weeks on a poly-D-L-ornithine-coated surface in RPE-CM only, 10% serum, or a serum-free defined media, and then examined by phase-contrast and scanning electron microscopy and immunocytochemistry. After 2 days in vitro, long ganglion cell-like neurites projected from retinal explants grown in tnrRPE-CM. These neurites increased in number and length with prolonged time in culture. In addition, by 5 days, round cells were observed adjacent to neonatal explants grown in tnrRPE-CM. By day 10, these round cells had increased in number and were seen along the neurites, in massive clusters immediately adjacent to these explants and dispersed throughout the culture-plate surface. Media conditioned by primary cultures of normal neonatal rat RPE cells caused a similar, but less robust, cellular response in retinal explants when compared to tnrRPE-CM. At 10 days, retinal explants grown in 10% serum showed only a few short processes, but no round cells, while those explants grown in defined media appeared to be degenerating. The round migrating cells are classified as retinal progenitor cells since they immunostained for opsin and interphotoreceptor retinoid-binding protein (IRBP), two photoreceptor cell markers, and a few for cellular retinaldehyde binding protein (CRALBP), a Muller cell marker. Neurite outgrowth and retinal progenitor cell production from explants were eliminated when the tnrRPE-CM was subjected to trypsin or heat treatment, indicating that the factor(s) responsible for promoting these cellular events was most likely proteinaceous. Growth factors, including basic fibroblast growth factor, were unable to generate long neurite outgrowth or progenitor cell production as observed in RPE-CM-supplemented explant cultures. We report that CM from cultures of primary and transformed neonatal rat RPE cells promoted ganglion cell-like neurites and the production of migrating retinal progenitor cells that primarily expressed photoreceptor-specific markers, from neonatal rat retinal explants. This evidence further confirms the important role of RPE in retinal development. The production of large numbers of progenitor cells by an RPE-secreted factor(s) may have important implications for possible therapeutic approaches to help correct retinal disease states by replacing lost cells through transplantation technology.
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PMID:Effects of retinal pigment epithelial cell-secreted factors on neonatal rat retinal explant progenitor cells. 879 43

A novel 9 kD protein inhibitor of trypsin and related proteinases was purified from culture filtrate of Yersinia pseudotuberculosis by ultrafiltration, affinity chromatography, and gel filtration. The protein is stable at pH from 1 to 9. The inhibitor activity dramatically decreases at temperature above 37 degrees C. The purified inhibitor significantly suppresses activities of an endogenous trypsin-related proteinase and trypsin but does not affect chymotrypsin, pepsin, and papain. One molecule of yersinia inhibitor binds two molecules of endogenous trypsin-related proteinase and about one trypsin molecule. The Ki for yersinia proteinase is 1.7.10(-7) and Ki for trypsin is 2.4.10(-7). Amino acid composition of the inhibitor is characterized by the presence of beta-aminobutyric acid and ornithine and relatively high contents of alanine and arginine whereas cysteine, histidine, and proline are absent.
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PMID:[Trypsin and microbial serine proteinase inhibitors isolated from Yersinia pseudotuberculosis]. 901 Dec 35

Hepatic associated metabolic disorders represent 5% of the indications for orthotopic liver transplantation (OLTX) according to the European Liver Transplant Registry. We studied the outcome of this group at our institution after OLTX and combined liver/kidney transplantation. Between September 1988 and January 1997, 837 OLTXs were performed in 735 patients. Patient survival and graft function at 1 yr were 91.3 and 86%, respectively. Thirty-nine OLTXs were performed in 38 patients (15 female/23 male, median age +/- SD: 35 +/- 14 yr, range 4-60 yr) due to liver associated metabolic disorders (4.7%). Indications included Wilson's disease (n = 14), alpha-1-anti-trypsin-deficiency (n = 7), hemochromatosis (n = 4), erythropoetic protoporphyria (n = 4), cystic fibrosis (n = 2), Crigler-Najjar syndrome type I (n = 1), glycogenosis type I (n = 1), ornithine-transcarbomylase-deficiency (n = 1). In addition 4 patients suffering from primary hyperoxaluria type I received combined liver/kidney grafts. Survival rate the 1 yr after OLTX and combined OLTX/NTX was 91.8%. Twenty patients received cyclosporin A (55%) and 17 patients tacrolimus (45%) as primary immunosuppression. The mean follow-up was 28.6 months (range 4-73 months). Two patients with hemochromatosis died 1 and 3 months after OLTX, respectively, from Aspergillus sepsis followed by multiorgan-failure. One patient died of malignant lymphoma 5 months after transplantation. One patient required retransplantation 2 months after OLTX following arterial thrombosis and ischemic type biliary lesion. One year after OLTX, all patients demonstrated good graft function, liver grafts (ALT 17.9 +/- 13.6 IU/L, bilirubin 0.8 +/- 0.3.mg/dl, thromboplastin time 94 +/- 15%), and combined liver/kidney grafts (creatinine 2.4 +/- 1.4 mg/dl). OLTX, respectively combined OLTX/NTX, represent a successful therapy for hepatic associated metabolic disorders. Survival rates and graft function are similar to those in liver graft recipients for established indications at our institution. OLTX seems to be an excellent treatment for hepatic based therapy resistant neurological disorders.
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PMID:Orthotopic liver transplantation for hepatic associated metabolic disorders. 964 15

The degradation of the cross-linked cationic poly(amino acid)-glutaraldehyde (GA) hydrogels by two kinds of proteolytic enzymes, trypsin and Aspergillus Protease Type XXIII, and by seven species of soil filamentous fungi has been investigated using homo- and copolypeptides of lysine (Lys) and ornithine (Orn). Trypsin degraded the hydrogels prepared from poly(Lys) and copoly(Lys Orn)s but not poly(Orn), while Aspergillus protease degraded all of them. Degradation time of hydrogels by the two proteases became longer with increasing Orn content in the gel. Seven species of soil filamentous fungi were cultured with hydrogels on Czapeck medium to evaluate the degree of microbial degradation of the hydrogels, and the three species of the fungi, Aspergillus oryzae, Penicillium citrinum and Curvularia sp., were grown in culture with an accompanying degradation of the gel matrix, while the other four species, Mucor sp., Rhizopus sp., Cladosporium sp., and Trichoderma sp., were not. The degree of degradation of gel matrix with growth of the three fungi became lower with increasing Orn content in the gel matrix. The results might offer some clues to the applications for the controlled biodegradation of cationic poly(amino acid) hydrogel by introduction of Orn, suggesting that unnatural amino acid resists hydrolysis by proteases or microorganisms.
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PMID:Biodegradation of ornithine-containing polylysine hydrogels. 985 86

Human liver ornithine carbamoyltransferase undergoes absorbance changes in the UV region upon formation of the carbamoylphosphate-norvaline-enzyme ternary complex. The UV changes are similar in the presence of carbamoylphosphate alone, whilst they are lower in the presence of ornithine or norvaline alone. The extent of the UV changes correlates with the enzyme susceptibility to proteolytic degradation. The free native enzyme is completely and rapidly hydrolyzed by trypsin, whilst it is partially protected upon carbamoylphosphate binding. The extent of protection increases for the carbamoylphosphate-norvaline-enzyme ternary complex. These results strongly suggest that the binding of the first substrate, i.e. carbamoylphosphate, to human ornithine carbamoyltransferase induces a large protein isomerization, which regards the polar domain plus a part of equatorial domain of each subunit.
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PMID:Evidence of carbamoylphosphate induced conformational changes upon binding to human ornithine carbamoyltransferase. 1041 Feb 42

Native and wild-type recombinant human liver arginases (EC 3.5.3.1) were photoinactivated by Rose bengal, and protection was afforded by the competitive inhibitor l-lysine. The dissociation constant for the enzyme-protector complex was essentially equal to the corresponding K(i) value. Upon mutation of His141 by phenylalanine, the enzyme activity was reduced to 6-10% of wild-type activity, with no changes in K(m) for arginine or K(i) for l-lysine or l-ornithine. The subunit composition of active enzyme was not altered by mutation, but the mutant H141F was markedly more sensitive to trypsin inactivation and completely insensitive to inactivation by diethyl pyrocarbonate (DEPC) and photoinactivation. Species with histidine groups blocked with DEPC were also insensitive to photoinactivation. We conclude that His141, which is the target for both inactivating procedures, is not involved in substrate binding, but plays a critical, albeit not essential role in the hydrolysis of enzyme-bound substrate.
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PMID:Chemical modification and site-directed mutagenesis of human liver arginase: evidence that the imidazole group of histidine-141 is not involved in substrate binding. 1054 6

The binding free energies of four inhibitors to bovine beta-trypsin are calculated. The inhibitors use either ornithine, lysine, or arginine to bind to the S1 specificity site. The electrostatic contribution to binding free energy is calculated by solving the finite difference Poisson-Boltzmann equation, the contribution of nonpolar interactions is calculated using a free energy-surface area relationship and the loss of conformational entropy is estimated both for trypsin and ligand side chains. Binding free energy values are of a reasonable magnitude and the relative affinity of the four inhibitors for trypsin is correctly predicted. Electrostatic interactions are found to oppose binding in all cases. However, in the case of ornithine- and lysine-based inhibitors, the salt bridge formed between their charged group and the partially buried carboxylate of Asp189 is found to stabilize the complex. Our analysis reveals how the molecular architecture of the trypsin binding site results in highly specific recognition of substrates and inhibitors. Specifically, partially burying Asp189 in the inhibitor-free enzyme decreases the penalty for desolvation of this group upon complexation. Water molecules trapped in the binding interface further stabilize the buried ion pair, resulting in a favorable electrostatic contribution of the ion pair formed with ornithine and lysine side chains. Moreover, all side chains that form the trypsin specificity site are partially buried, and hence, relatively immobile in the inhibitor-free state, thus reducing the entropic cost of complexation. The implications of the results for the general problem of recognition and binding are considered. A novel finding in this regard is that like charged molecules can have electrostatic contributions to binding that are more favorable than oppositely charged molecules due to enhanced interactions with the solvent in the highly charged complex that is formed.
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PMID:Structural determinants of trypsin affinity and specificity for cationic inhibitors. 1063 77

Kinetics for the hydrolysis of the chromogenic active-site titrant N alpha-(N,N-dimethylcarbamoyl)-alpha-azaornithine p-nitrophenyl ester (Dmc-azaOrn-ONp) catalysed by bovine beta-trypsin, bovine alpha-thrombin, bovine Factor Xa, human alpha-thrombin, human Factor Xa, human Lys77-plasmin, human urinary kallikrein, Mr 33 000 and Mr 54 000 species of human urokinase, porcine pancreatic beta-kallikrein-A and -B and Ancrod (the coagulating serine proteinase from the Malayan pit viper Agkistrodon rhodostoma venom) have been obtained between pH 6.0 and 8.0, at 21.0 degrees C, and analysed in parallel with those for the enzymatic cleavage of N alpha-(N,N-dimethylcarbamoyl)-alpha-azalysine p-nitrophenyl ester (Dmc-azaLys-ONp). The enzyme kinetics are consistent with the minimum three-step catalytic mechanism of serine proteinases, the rate-limiting step being represented by the deacylation process. Bovine beta-trypsin kinetics are modulated by the acid-base equilibrium of the His57 catalytic residue (pKa approximately 6.9). Dmc-azaOrn-ONp and Dmc-azaLys-ONp bind stoichiometrically to the serine proteinase active site, and allow the reliable determination of the active enzyme concentration between 1.0 x 10-6 M and 3.0 x 10-4 M. The affinity and the reactivity for Dmc-azaOrn-ONp (expressed by Ks and k+2/Ks, respectively) of the serine proteinases considered are much lower than those for Dmc-azaLys-ONp. The very different affinity and reactivity properties for Dmc-azaOrn-ONp and Dmc-azaLys-ONp have been related to the different size of the ornithine/lysine side chains, and to the ensuing different positioning of the active-site titrants upon binding to the enzyme catalytic centre (i.e. to P1-S1 recognition). These data represent the first detailed comparative investigation on the catalytic properties of serine proteinases towards an ornithine derivative (i. e. Dmc-azaOrn-ONp).
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PMID:Serine proteinase inhibition by the active site titrant N alpha-(N, N-dimethylcarbamoyl)-alpha-azaornithine p-nitrophenyl ester. A comparative study. 1067 36

Two analogues of the 29 amino acid sequence of human growth hormone-releasing hormone, namely [Nle27]hGH-RH(1-29)-NH2 and [Orn(12,21),Nle27]hGH-RH(1-29)-NH2, have been synthesized and subjected to digestion by trypsin. The course of degradation was followed using RP-HPLC and ESI-MS. Several intermediates and final products of degradation were identified and conclusions regarding the rate of cleavages at different positions occupied by Lys and Arg residues were drawn. The analogue containing ornithine was found to be less susceptible to hydrolysis by trypsin: the 12-13 and 21-22 peptide bonds were completely resistant to the cleavage. The results show that by replacing Lys with Orn, a possibility exists to design new peptides, which could be more stable in biological fluids.
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PMID:Tryptic hydrolysis of hGH-RH(1-29)-NH2 analogues containing Lys or Orn in positions 12 and 21. 1129 53


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