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:3.4.21.9 (enterokinase)
675 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A limited reduction of the disulfide bonds of bovine enterokinase (enteropeptidase, EC 3.4.21.9) was accomplished with 50 mM dithioerythritol, at pH 9.0, and at 4 degrees C. The conditions separated the heavy and light subunits quantitatively with improved reliability when compared to the conditions used previously (Savithri, H. S., and Light, A. (1980) Biochim. Biophys. Res. Commun, 94, 360-365). Pancreatic trypsin inhibitor was added to the reaction to ensure that the yield of the heavy subunit was equal to that of the catalytic subunit (light subunit). Otherwise the heavy subunit was subject to extensive degradation. The subunits were alkylated with iodoacetate and then resolved on Sephadex G-150. Amino acid analyses and the incorporation of [14C]carboxymethyl groups showed that 3.1 carboxymethylcysteine residues were in the catalytic subunit and 8.9 in the heavy subunit. The catalytic subunit had normal catalytic activity toward N-benzoyl-L-arginine ethyl ester, enhanced activity toward N-tosyl-L-arginine methyl ester and N-tosyl-L-lysine methyl ester, and lower activity toward N-benzoyl-DL-arginine p-nitroanilide. The catalytic subunit retained the restricted specificity of intact enterokinase, but the rate of activation of trypsinogen was much slower. It is likely that the limited reduction of the disulfide bonds of the catalytic subunit altered the interaction of protein substrates with the specificity site.
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PMID:The preparation and properties of the catalytic subunit of bovine enterokinase. 638 10

Two trypsin assay methods for the estimation of this enzyme in duodenal fluid from children have been compared. Assay results for a fluorometric method based on the use of N-carbobenzoxy-diglycyl-L-arginyl-2-naphthylamide hydrochloride (GANA) as the trypsin substrate were found to correlate well (r = 0.91, P less than 0.001) with those obtained with a much less sensitive titrimetric assay which used benzoylarginine ethylester hydrochloride (BAEE) as substrate. The higher sensitivity of the fluorometric assay has allowed accurate determination of trypsin activity in 10 microliter aliquots of duodenal fluid. This low volume requirement makes the assay suitable for studies on infants of all ages and conserves duodenal fluid for use in other investigations often warranted during the assessment of childhood malabsorption. The fluorometric assay has also been used to monitor the separation of enteropeptidase from trypsin(ogen) by chromatography on Sephacryl S-200 in samples of duodenal fluid from two children. Different proteolytic pathway deficiencies were confirmed in these children.
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PMID:Fluorometric microassay of trypsin and enteropeptidase in children--comparison with a titrimetic assay. 639 83

The influence of bile salts on the mucosal surface of rat jejunum was tested with an in vivo technique of segmental perfusion. Sodium taurocholate and chenodesoxycholate were applied in a concentration of 3 mmol/l. The release of 5 brush border membrane enzymes, 5 cytosolic, 1 mitochondrial, and 2 lysosomal enzymes during a perfusion time of 150 min as well as morphological alterations after bile salt treatment were investigated. Among the membrane enzymes, due to their superficial localization, the solubilization of enteropeptidase and alpha-1,4-glucosidase was highest both in the control perfusion and in the presence of bile salts. At the same time, cytoplasmic enzyme activities were liberated extensively whereas lysosomal and mitochondrial enzymes were scarcely detectable. This disproves any serious injury of the enterocytes. Electronmicroscopic results supported this suggestion. After administration of taurocholate (in physiological concentration), only an occasional diminution of the glycocalyx was observed and even chenodeoxycholate (in an unphysiological concentration) caused only negligible destructions of intestinal brush borders. Investigations with ruthenium red to contrast the glycocalyx showed a partially unchanged structure. Microvesiculation from the microvilli was observed in many electron microscopic photographs. That is a possibility for the release of membrane-bound and cytosolic enzymes without destruction of enterocytes.
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PMID:[Biochemical and morphologic studies on the effect of bile acids on the epithelium of the rat jejunum]. 643 37

The study of deficiencies of small intestinal brush-border hydrolases increased our knowledge about the specific functions of hydrolases in the digestion of smaller molecules on the microvillus surface of the absorptive cells. The sucrase-isomaltase (SI) complex has been shown to be synthesized as a precursor (pro-sucrase-isomaltase) which is then incorporated into the membrane. The hydrophobic N-terminal end of the molecule is anchored in the lipid bilayer. In SI deficiency the molecular base of the disease is still not clear. Absence of SI activity could be due to complete lack of precursor synthesis or to structural changes within the N-terminal end of the SI-complex. Deficiencies of peptide hydrolases have not been reported with the exception of enteropeptidase (EP). Here a congenital deficiency of the enzyme was observed as the primary defect in enzyme synthesis within the enterocytes and as a secondary defect due to exocrine pancreatic insufficiency. In contrast to the primary EP deficiency, the activity of EP can be restored in the cases of exocrine pancreatic insufficiency by treatment with pancreatic extracts. Primary lactase deficiency exists in various forms. Besides congenital lactase deficiency, the late onset or adult type of lactase deficiency has been observed. The latter occurs in many different ethnic groups around the world. Here, using gel electrophoresis and immunoelectrophoresis, the lack of enzyme activity could be shown to be a primary defect in enzyme protein synthesis. In man and in the rat, two different lactases have been identified. In contrast to adult lactase, fetal lactase contains sialic acid at the end of carbohydrate side chains.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Clinical significance of enzymatic deficiencies in the gastrointestinal tract with particular reference to lactase deficiency. 643 79

We have isolated and identified a new zymogen in human pancreatic tissue and fluid. It is secreted as a minor component of pancreatic juice and resembles the two known trypsinogen variants in many properties. Its electrophoretic mobility and isoelectric pH lie between those of the cationic and anionic trypsinogen variants, and we propose the name "mesotrypsinogen" for the new enzyme precursor. It is activated by enteropeptidase or trypsin, and the free enzyme possesses a substrate specificity similar to that of the trypsins. Its pH optimum is at 8.2, and it appears to require Ca2+ for full enzymatic activity. The molecular weight of the new enzyme is approximately 25,000, similar to that of the known trypsin variants. Its stability resembles that of anionic trypsin extending over a pH range of 4-8.5. Activity is lost gradually at pH 2. The enzyme is inactivated rapidly by diisopropylfluorophosphate, but in contrast to the trypsins, it reacts only slowly with tosyllysine chloromethylketone. Immunologically, it is different from the cationic trypsin variant with which it does not cross-react. The most remarkable property of mesotrypsin is its almost total resistance to biological trypsin inhibitors, such as pancreatic trypsin inhibitor, soybean, lima bean, ovomucoid inhibitor, alpha 1-antitrypsin, etc. It is capable of activating trypsinogen in the presence of excess pancreatic trypsin inhibitor and thus inducing activation of other pancreatic zymogens, but it also possesses the ability to degrade trypsinogen rapidly to inert products. The physiological or pathophysiological role of this unique enzyme remains to be explored.
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PMID:Mesotrypsin: a new inhibitor-resistant protease from a zymogen in human pancreatic tissue and fluid. 669 68

The technique of segmental perfusion was applied in vivo to study the release of enzymes from the mucosa cells under the influence of bile salts. Five brush border membrane enzymes, five cytosolic, one mitochondrial and two lysosomal enzymes were measured. Spontaneous release (Ringer solution), due to their superficial localization, was greatest for enteropeptidase and alpha-glucosidase. 3 and 10 mmol/l sodium taurocholate, and 0.5 and 3.0 mmol/l chenodeoxycholate were used. Surprisingly, the majority of cytosolic enzymes was released to a greater extent than membrane enzymes. On the other hand, lysosomal or mitochondrial enzymes were low or absent in the perfusion medium, a finding that excludes a serious injury to the mucosa cells. It must be inferred, therefore, that cytosolic enzymes are regularly externalized during the digestive process, the glycocalyx perhaps being a common matrix for pancreatic, membrane-associated and cytosolic enzymes.
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PMID:Release of enzymes from rat jejunal mucosa by bile salts. 685 14

The application of a new synthetic substrate to the direct determination of enteropeptidase is described. The substrate Gly-(L-Asp)4-L-Lys-2-naphthylamide contains the amino acid sequence of the activation peptides of trypsinogen linked via an amide bond to the fluorophore 2-naphthylamine. The sequence of amino acids is responsible for the specificity and substrate recognition of the enteropeptidase-catalyzed activation of trypsinogen. Interference in the assay by trypsin is prevented by the addition of soybean trypsin inhibitor to the substrate solution. The fluorimetric determination of the liberated 2-naphthylamine allows the direct observation of the reaction kinetics. For the hyrolysis of the synthetic substrate by purified enteropeptidase the pH optimum was 8.2 and the Km 0.17 mmol/l. The new substrate was used to determine the distribution of enteropeptidase along the rat small intestine and also to measure enteropeptidase activity in human intestinal biopsies.
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PMID:The application of a new synthetic substrate to the determination of enteropeptidase in rat small intestine and human intestinal biopsies. 698 11

Histidine 57 of the catalytic triad of trypsin was replaced with alanine to determine whether the resulting variant would be capable of substrate-assisted catalysis [Carter, P., & Wells, J. A. (1987) Science 237, 394-9]. A 2.5-fold increase in kcat/Km was observed on tri- or tetrapeptide substrates containing p-nitroanilide leaving groups and histidine at P2. In contrast, hydrolysis of peptide substrates extending from P6 to P6' is improved 70-300-fold by histidine in the P2 or P1' position. This preference creates new protease specificities for sequences HR decreases, R decreases H, HK decreases, and K decreases H. The ability of histidine from either the P2 or the P1' position of substrate to participate in catalysis emphasizes the considerable variability of proteolytically active orientations which can be assumed by the catalytic triad. Trypsin H57A is able to hydrolyze fully folded ornithine decarboxylase with complete specificity at a site containing the sequence HRH. Trypsin H57A was compared to enteropeptidase in its ability to cleave a propeptide from trypsinogen. Trypsin H57A cleaved the propeptide of a variant trypsinogen containing an introduced FPVDDDHR cleavage site only 100-fold slower than enteropeptidase cleaved trypsinogen. The selective cleavage of folded proteins suggests that trypsin H57A can be used for specific peptide and protein cleavage. The extension of substrate-assisted catalysis to the chymotrypsin family of proteolytic enzymes indicates that it may be possible to apply this strategy to a wide range of serine proteases and thereby develop various unique specificities for peptide and protein hydrolysis.
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PMID:Trypsin specificity increased through substrate-assisted catalysis. 754 82

Cleavage of different chimeric proteins after specific linker (Asp)4Lys by the highly purified enteropeptidase was investigated, proteins being were accumulated in inclusion bodies or secreted from the cell. Kinetic constants for enzymatic hydrolysis were obtained, indicating that the substrate binding depended mainly on the affinity to the linker peptide (Asp)4Lys. Conditions for the efficient cleavage of recombinant proteins with enteropeptidase are formulated.
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PMID:[Enteropeptidase and its use for cleavage of chimeric proteins]. 782 15

Guanylate kinase (EC 2.7.4.8), catalysing the reaction GMP+ATP = GDP+ADP, was purified to homogeneity from bovine retina. Primary structure of the enzyme was determined by parallel analyses of amino acid sequences of its peptides and nucleotide sequence of the corresponding cDNA. It is shown that the bovine retinal guanylate kinase like the analogous enzyme from yeast Saccharomyces cerevisiae contains a characteristic glycine-rich motif, involved in ATP binding. All of the amino acids, involved in GMP binding in the yeast enzyme, are conserved or conservatively substituted in the bovine retinal guanylate kinase. The bovine retinal enzyme was expressed in E. coli as a fusion protein. Data are presented on the purification of the fusion protein, its digestion by enteropeptidase, purification of the recombinant enzyme and its functional characteristics.
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PMID:[Guanylate kinase from bovine retina: isolation, primary structure, and expression in E. coli]. 791 63


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