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 glutamic acid-specific protease has been purified to homogeneity from Bacillus licheniformis ATCC 14580 utilizing Phe-Leu-D-Glu-OMe-Sepharose affinity chromatography and crystallized. The molecular weight of the protease was estimated to be approximately 25,000 by SDS-polyacrylamide gel electrophoresis. This protease, which we propose to call BLase (glutamic acid-specific protease from B. licheniformis ATCC 14580), was characterized enzymatically. Using human parathyroid hormone (13-34) and p-nitroanilides of peptidyl glutamic acid and aspartic acid, we found a marked difference between BLase and V8 protease, EC 3.4.21.9, although both proteases showed higher reactivity for glutamyl bonds than for aspartyl bonds. Diisopropyl fluorophosphate and benzyloxycarbonyl Leu-Glu chloromethyl ketone completely inhibited BLase, whereas EDTA reversibly inactivated the enzyme. The findings clearly indicate that BLase can be classified as a serine protease. To elucidate the complete primary structure and precursor of BLase, its gene was cloned from the genomic DNA of B. licheniformis ATCC 14580, and the nucleotide sequence was determined. Taking the amino-terminal amino acid sequence of the purified BLase into consideration, the clones encode a mature peptide of 222 amino acids, which follows a prepropeptide of 94 residues. The recombinant BLase was expressed in Bacillus subtilis and purified to homogeneity. Its key physical and chemical characteristics were the same as those of the wild-type enzyme. BLase was confirmed to be a protease specific for glutamic acid, and the primary structure deduced from the cDNA sequence was found to be identical with that of a glutamic acid-specific endopeptidase isolated from Alcalase (Svendsen, I., and Breddam, K. (1992) Eur. J. Biochem. 204, 165-171), being different from V8 protease and the Glu-specific protease of Streptomyces griseus which consist of 268 and 188 amino acids, respectively.
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PMID:Purification, characterization, cloning, and expression of a glutamic acid-specific protease from Bacillus licheniformis ATCC 14580. 142 18

An endoproteolytic activity that specifically cleaves CCK 33, producing CCK 8, has been purified from a rat brain synaptosome preparation. The purification, which included anion exchange, chromatofocusing, hydroxyapatite, and gel filtration chromatography, resulted in a greater than 3000-fold increase in specific activity. This neutral endoprotease (pH optimum 8) exists as a 90-kDa species, which can be dissociated into active 40-kDa species. The enzyme is a non-trypsin serine protease, which is inhibited by diisopropyl-fluorophosphate and p-aminobenzamidine but not by soybean trypsin inhibitor, phenylmethylsulfonyl fluoride, aprotinin, or a number of thiol or metalloprotease inhibitors. It is highly substrate-specific and cleaves neither trypsin, enteropeptidase, kallikrein substrates, nor analogues of mono- or dibasic cleavage sites of prohormones other than pro-CCK. The endoprotease will not cleave CCK 12 desulfate or CCK (20-29), although these peptides contain common sequences with CCK-33. The protease does cleave [Glu27]CCK (20-29), a peptide in which the glutamate mimics the negative charge normally present on tyrosine sulfate. This suggests that the negative charge at position 27 is important in substrate recognition. The enzyme will also cleave CCK 33 and CCK (1-21) on the carboxyl-terminal side of a single lysine residue in position 11. The subcellular location and specificity of this endoprotease make it a good candidate for a CCK-processing protease.
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PMID:Characterization of a cholecystokinin 8-generating endoprotease purified from rat brain synaptosomes. 152 68

Bovine enterokinase (enteropeptidase) is a serine protease and functions as the physiological activator of trypsinogen. The enzyme has a heavy chain (115 kD) covalently linked to a light or catalytic subunit (35 kD). The amino acid composition showed that the light chain has nine half-cystine residues (four as intramolecular disulfides) and that one half-cystine was in a disulfide link between the light and heavy subunits. The amino-terminal 27 residues of the S-vinylpyridyl derivative of the light chain were determined by gas-phase Edman degradation. The sequence has homologies with other serine proteases containing one or two chains. The homologies suggest that the catalytic subunit has the same three-dimensional structure and, therefore, the same mechanism of enzymatic action as pancreatic chymotrypsin, trypsin, and elastase. The presence of the conserved amino-terminal activation peptide sequence (IVGG) shows that enterokinase must have a zymogen precursor and that the two-chain enzyme arises from limited proteolysis during posttranslational processing.
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PMID:The amino-terminal sequence of the catalytic subunit of bovine enterokinase. 179 6

The serine protease enterokinase is the physiological activator of trypsinogen and has a specificity for the sequence (Asp)4-Lys-Ile. The enzyme consists of two subunits linked by a disulfide bond. The heavy chain achors enterokinase in the intestinal brush border membrane and the light chain is the catalytic subunit, which has the same mechanism of action as trypsin and chymotrypsin. Many properties of enterokinase resemble blood-clotting enzymes, suggesting that enterokinase lies on the same phylogenetic branch as the blood-clotting proteins.
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PMID:Enterokinase (enteropeptidase): comparative aspects. 265 18

Enterokinase is a serine protease of the duodenal brush border membrane that cleaves trypsinogen and produces active trypsin, thereby leading to the activation of many pancreatic digestive enzymes. Overlapping cDNA clones that encode the complete human enterokinase amino acid sequence were isolated from a human intestine cDNA library. Starting from the first ATG codon, the composite 3696 nt cDNA sequence contains an open reading frame of 3057 nt that encodes a 784 amino acid heavy chain followed by a 235 amino acid light chain; the two chains are linked by at least one disulfide bond. The heavy chain contains a potential N-terminal myristoylation site, a potential signal anchor sequence near the amino terminus, and six structural motifs that are found in otherwise unrelated proteins. These domains resemble motifs of the LDL receptor (two copies), complement component Clr (two copies), the metalloprotease meprin (one copy), and the macrophage scavenger receptor (one copy). The enterokinase light chain is homologous to the trypsin-like serine proteinases. These structural features are conserved among human, bovine, and porcine enterokinase. By Northern blotting, a 4.4 kb enterokinase mRNA was detected only in small intestine. The enterokinase gene was localized to human chromosome 21q21 by fluorescence in situ hybridization.
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PMID:cDNA sequence and chromosomal localization of human enterokinase, the proteolytic activator of trypsinogen. 771 57

Enterokinase (enteropeptidase) is a heterodimeric serine protease that is responsible for the physiological activation of trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the sequence (Asp)4-Lys. In this paper, we report the cloning and functional expression of a cDNA encoding the catalytic domain (light chain) of bovine enterokinase. The nucleotide sequence of this cloned cDNA predicts a 235-amino acid polypeptide that shares a high degree of homology with a variety of mammalian serine proteases involved in digestion, coagulation, and fibrinolysis. We have developed a novel expression method for the enzyme which utilizes the secretory leader and propeptide of the mammalian serine protease PACE fused to the enterokinase light chain amino terminus. Efficient cleavage of the paired dibasic amino acid cleaving enzyme (PACE) propeptide was achieved by coexpression with human PACE or yeast KEX2. The mature product migrates at 43,000 Da on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, comparable to light chain derived from bovine duodena, and exhibited high levels of activity in cleaving the enterokinase-specific fluorogenic substrate Gly-(Asp)4-Lys-beta-naphthylamide. The recombinant single-chain form of enterokinase was also capable of activating trypsinogen, indicating that the specificity of the enzyme for its natural substrate is retained even in the absence of the noncatalytic enterokinase heavy chain.
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PMID:Cloning and functional expression of a cDNA encoding the catalytic subunit of bovine enterokinase. 822 55

A cDNA encoding a precursor for a novel serine protease (neurosin) was cloned from a cDNA library prepared from a human colon adenocarcinoma cell line, COLO 201. The sequence consisted of 155 bp 5' non-coding region and a 732 bp open reading frame which was followed by a 551 bp 3' non-coding region. The predicted protein consists of 244 amino acids which is possibly processed to an active enzyme of 223 amino acids that shows some similarity (< 30%) to other members of serine protease family. As found in other trypsin-like proteases, the enzyme contains the catalytic triad which is characterized as the essential amino acid residues for the activity. Northern blot analyses of the mRNA showed the strongest expression in brain followed by a lower but significant one in spleen. A construct of cDNA encoding chimeric protein that carries pro-sequence of trypsin II and putative mature neurosin starting from Leu22 was transfected to COS-1 cells. Successful production of the active neurosin was shown after treating the supernatant of the culture of the transfectants with enterokinase.
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PMID:Molecular cloning of a novel trypsin-like serine protease (neurosin) preferentially expressed in brain. 900 50

Duodenase, a serine protease from bovine duodenum mucosa, was located in endoplasmic reticulum, the Golgi secretory granules of epithelial cells and ducts of Brunner's glands by the A-gold immunocytochemical method. Duodenase exhibits trypsin-like and chymotrypsin-like specificities with a preference for substrates having lysine at the P1 and proline at the P2 positions. The kinetic constants for the hydrolysis of 21 potential duodenase substrates are reported. The best substrates were found to be alpha-N-tosylglycylprolyllysine 4-nitroanilide (k[cat]/Km of 35000 M[-1] s[-1]), alpha-N-succinylthreonylprolyllysine 4-nitroanilide (k[cat]/Km of 18000 M[-1] s[-1]) and alpha-N-serylprolyllysine 4-nitroanilide (k[cat]/Km of 2600 m[-1] s[-1]), all of which contain the P1-P3 sequence of the enteropeptidase zymogen/activation site. On the basis of its catalytic properties and sites of localization, duodenase has been postulated to be an activator of the enteropeptidase precursor. A tetradecapeptide (LVTQEVSPKIVGGS) having the P9-P5'sequence of the cleavage site of zymogen activation of bovine proenteropeptidase was synthesized, and kinetic parameters of its hydrolysis by duodenase were determined (Km of 87 microM; k[cat] of 1.4 s[-1]; k[cat]/Km of 16000 M[-1] s[-1]). Crystals of duodenase frozen in a stream of liquid nitrogen diffracted synchrotron X-rays to 0.2-nm resolution.
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PMID:Subcellular localization, substrate specificity and crystallization of duodenase, a potential activator of enteropeptidase. 937 Mar 74

Enteropeptidase, also known as enterokinase, initiates the activation of pancreatic hydrolases by cleaving and activating trypsinogen. Enteropeptidase is synthesized as a single-chain protein, whereas purified enteropeptidase contains a approximately 47-kDa serine protease domain (light chain) and a disulfide-linked approximately 120-kDa heavy chain. The heavy chain contains an amino-terminal membrane-spanning segment and several repeated structural motifs of unknown function. To study the role of heavy chain motifs in substrate recognition, secreted variants of recombinant bovine proenteropeptidase were constructed by replacing the transmembrane domain with a signal peptide. Secreted variants containing both the heavy chain (minus the transmembrane domain) and the catalytic light chain (pro-HL-BEK (where BEK is bovine enteropeptidase)) or only the catalytic domain (pro-L-BEK) were expressed in baby hamster kidney cells and purified. Single-chain pro-HL-BEK and pro-L-BEK were zymogens with extremely low catalytic activity, and both were activated readily by trypsin cleavage. Trypsinogen was activated efficiently by purified enteropeptidase from bovine intestine (Km = 5.6 microM and kcat = 4.0 s-1) and by HL-BEK (Km = 5.6 microM and kcat = 2.2 s-1), but not by L-BEK (Km = 133 microM and kcat = 0.1 s-1); HL-BEK cleaved trypsinogen at pH 5.6 with 520-fold greater catalytic efficiency than did L-BEK. Qualitatively similar results were obtained at pH 8.4. In contrast to this striking difference in trypsinogen recognition, the small synthetic substrate Gly-Asp-Asp-Asp-Asp-Lys-beta-naphthylamide was cleaved with similar kinetic parameters by both HL-BEK (Km = 0.27 mM and kcat = 0.07 s-1) and L-BEK (Km = 0.60 mM and kcat = 0.06 s-1). The presence of the heavy chain also influenced the rate of reaction with protease inhibitors. Bovine pancreatic trypsin inhibitor preferred HL-BEK (initial Ki = 99 nM and final Ki* = 1.8 nM) over L-BEK (Ki = 698 nM and Ki* = 6.2 nM). Soybean trypsin inhibitor exhibited a reciprocal pattern, inhibiting L-BEK (Ki* = 1.6 nM), but not HL-BEK. These kinetic data indicate that the enteropeptidase heavy chain has little influence on the recognition of small peptides, but strongly influences macromolecular substrate recognition and inhibitor specificity.
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PMID:Bovine proenteropeptidase is activated by trypsin, and the specificity of enteropeptidase depends on the heavy chain. 939 56

Enterokinase (EK) is a heterodimeric serine protease which plays a key role in initiating the proteolytic digestion cascade in the mammalian duodenum. The enzyme acts by converting trypsinogen to trypsin via a highly specific cleavage following the pentapeptide recognition sequence (Asp)4-Lys. This stringent site specificity gives EK great potential as a fusion protein cleavage reagent. Recently, a cDNA encoding the catalytic (light) chain of bovine enterokinase (EKL) was identified, characterized, and transiently expressed in mammalian COS cells. We report here the production of EKL in Escherichia coli by a novel secretory expression system that utilizes E. coli DsbA protein as an N-terminal fusion partner. The EKL cDNA was fused in-frame to the 3'-end of the coding sequence for DsbA, with the two domains of the fusion protein separated by a linker sequence encoding an enterokinase recognition site. Active, processed recombinant EKL (rEKL) was generated from this fusion protein via an autocatalytic cleavage reaction. The enzymatic properties of the bacterially produced rEKL were indistinguishable from the previously described COS-derived enzyme. Both forms of rEKL were capable of cleaving peptides, polypeptides and trypsinogen with the same specificity exhibited by the native heterodimeric enzyme purified from bovine duodena. Interestingly, rEKL activated trypsinogen poorly relative to the native heterodimeric enzyme, but was superior in its ability to cleave artificial fusion proteins containing the (Asp)4-Lys recognition sequence.
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PMID:Production of recombinant bovine enterokinase catalytic subunit in Escherichia coli using the novel secretory fusion partner DsbA. 963 75


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