Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A coagulogen in Limulus lysate has been purified to apparent homogeneity as judged by sodium dodecyl sulfate-gel electrophoresis. The purified coagulogen identified by its ability to clot with either trypsin or the purified Limulus clotting enzyme (Tai, J.Y., and Liu, T. Y. (1976) Fed. Proc. 35, 1486) has a molecular weight of 24,500. It consists of a single polypeptide chain of about 220 amino acids with glycine and serine as its NH2- and COOH-terminal residues; respectively. When acted upon by the Limulus clotting enzyme, the coagulogen releases a soluble C-peptide of about 45 amino acids and an insoluble coagulin of about 170 amino acids. The latter interacts in a noncovalent fashion to form the clot. Amino acid analyses together with the results of NH2- and COOH-terminal analyses suggest that the clot formation involves the cleavage of an--Arg--Lys--bond. Trypsin acts on the coagulogen to cause clotting by splitting the same peptide bond.
 ...
PMID:Studies on Limulus amoebocyte lysate II. Purification of the coagulogen and the mechanism of clotting. 87 16

Proclotting enzyme is an intracellular serine protease zymogen closely associated with an endotoxin-sensitive hemolymph coagulation system in limulus. Its active form, clotting enzyme, catalyzes conversion of coagulogen to insoluble coagulin gel. We present here the cDNA and amino acid sequences, disulfide locations, and subcellular localization of proclotting enzyme. The isolated cDNA for proclotting enzyme consists of 1,501 base pairs. The open reading frame of 1,125 base pairs encodes a sequence comprising 29 amino acid residues of prepro-sequence and 346 residues of the mature protein with a molecular mass of 38,194 Da. Three potential glycosylation sites for N-linked carbohydrate chains were confirmed to be glycosylated. Moreover, the zymogen contains six O-linked carbohydrate chains in the amino-terminal light chain generated after activation. The cleavage site that accompanies activation catalyzed by trypsin-like active factor B, proved to be an Arg-Ile bond. The resulting carboxyl-terminal heavy chain is composed of a typical serine protease domain, with a sequence similar to that of human coagulation factor XIa (34.5%) or factor Xa (34.1%). The light chain has a unique disulfide-knotted domain which shows no significant homology with any other known proteins. Thus, this proclotting enzyme has a mammalian serine protease domain and a structural domain not heretofore identified in coagulation and complement factors. Immunohistochemical studies showed that the proclotting enzyme is localized in large granules of hemocytes.
 ...
PMID:Proclotting enzyme from horseshoe crab hemocytes. cDNA cloning, disulfide locations, and subcellular localization. 226 34

A low molecular weight protein protease inhibitor was purified from Japanese horseshoe crab (Tachypleus tridentatus) hemocytes. It consisted of a single polypeptide with a total of 61 amino acid residues. This protease inhibitor inhibited stoichiometrically the amidase activity of trypsin (Ki = 4.60 X 10(-10) M), and also had inhibitory effects on alpha-chymotrypsin (Ki = 5.54 X 10(-9) M), elastase (Ki = 7.20 X 10(-8) M), plasmin, and plasma kallikrein. However, it had no effect on T. tridentatus clotting enzyme and factor C, mammalian blood coagulation factors (activated protein C, factor Xa and alpha-thrombin), papain, and thermolysin. The complete amino acid sequence of this inhibitor was determined and its sequence was compared with those of bovine pancreatic trypsin inhibitor (BPTI) and other Kunitz-type inhibitors. It was found that the amino acid sequence of this inhibitor has a high homology of 47 and 43% with those of sea anemone inhibitor 5-II and BPTI, respectively. Thus, this protease inhibitor appeared to be one of the typical Kunitz-type protease inhibitors.
 ...
PMID:Purification and amino acid sequence of Kunitz-type protease inhibitor found in the hemocytes of horseshoe crab (Tachypleus tridentatus). 330 64

A proclotting enzyme associated with the hemolymph coagulation system of limulus (Tachypleus tridentatus) was highly purified from the hemocyte lysate. The first step of purification was performed by chromatography of the lysate on a pyrogen-free dextran sulfate-Sepharose CL-6B column, which was essential for separation of the proclotting enzyme from its activator, named factor B. The following steps consisted of column chromatographies on DEAE-Sepharose CL-6B, Sephadex G-150, benzamidine-CH-Sepharose and Sephacryl S-300. Through these procedures, 1.4 mg of the purified material was obtained from 630 ml of the lysate and approximately 300-fold purification was achieved. The preparation gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the presence and absence of 2-mercaptoethanol. The single-chain proclotting enzyme was a glycoprotein with an apparent molecular weight of 54,000, and no gamma-carboxyglutamic acid was detected. The proclotting enzyme was converted to its active form by purified factor B or by trypsin. The resulting clotting enzyme had a molecular weight of 54,000, consisting of a heavy chain of Mr = 31,000 and a light chain of Mr = 25,000. The serine active site of the clotting enzyme was found in the heavy chain. The chemical analyses of the isolated heavy and light chains indicated that the activation of the proclotting enzyme to its active form by factor B or trypsin is induced by a limited proteolysis, yielding two chains bridged by a disulfide linkage(s).
 ...
PMID:Intracellular proclotting enzyme in limulus (Tachypleus tridentatus) hemocytes: its purification and properties. 403 Jul 38

A clotting enzyme, associated with the endotoxin-mediated activation of the cellularly based coagulation system of the American horseshoe crab (Limulus polyphemus), was considerably purified by a modification of the method employed to purify the corresponding enzyme from the Japanese horseshoe crab (Tachypleus tridentatus) (Nakamura, et al., 1982). This enzyme was inhibited by DFP, benzamidine, p-aminobenzamidine, antithrombin III, soybean trypsin inhibitor, and antipain, suggesting that it is a trypsin-type serine protease. The enzyme demonstrated amidolytic activity to Ac-Ile-Glu-Gly-Arg-pNA (S-2423) and related synthetic substrates (S-2222, S-2422, S-2337, and Boc-Leu-Gly-Arg-pNA) but not to other substrates (S-2160, S-2238, S-2251, S-2444, S-2266, and S-2302), indicating specificity similar to mammalian blood coagulation Factor Xa. These properties of the Limulus enzyme were identical with those of the corresponding Tachypleus enzyme. The structure and function of the enzymes in these two species probably have been highly conserved during the past few hundred million years of their evolution.
 ...
PMID:Properties of the clotting enzyme responsible for endotoxin-mediated Limulus coagulation. 637 72

We have reported the cloning from mouse genomic DNA of a fragment encoding a G-protein-coupled receptor related to the receptor for the blood clotting enzyme thrombin. Like the thrombin receptor this receptor is activated by proteolytic cleavage of its extracellular amino terminus. Because the physiological agonist at the receptor was unknown, we provisionally named it proteinase-activated receptor 2 (PAR-2). Here we present a PAR-2 cDNA of 2729 nucleotides that differs from the published genomic sequence at the 5' end, including a part of the protein coding region. The differences do not affect the peptide sequence of the activating proteinase cleavage site proper, but may include amino acid residues important for enzyme-substrate recognition. Analysis of the PAR-2 gene structure showed that the cDNA 5' end is derived from a separate exon located about 10 kilobases away from the 3' exon. Results from a primer extension experiment indicate that transcription starts at a unique site around nucleotide -203 respective to the translation initiation ATG. Chinese hamster ovary cells transfected with either the PAR-2 cDNA or a construct made from the published PAR-2 genomic sequence responded with intracellular calcium mobilization to stimulation with 1 nM trypsin, 10 microM PAR-2-activating peptide (SLIGRL), or 1 microM thrombin receptor-activating peptide (SFLLRN). Untransfected cells responded only to stimulation with thrombin receptor activating peptide. Only transcripts corresponding to the PAR-2 cDNA could be detected in three mouse tissues examined.
 ...
PMID:The mouse proteinase-activated receptor-2 cDNA and gene. Molecular cloning and functional expression. 789 Jul 26

Hirudin is the most potent and specific inhibitor of the blood-clotting enzyme thrombin so far known. Several hirudin variants were isolated mostly from Hirudo medicinalis and shown to be polypeptide chains of approximately 7 kDa with three internal disulfide bridges. In this study, limited proteolysis has been used to probe aspects of the structure and dynamics of a hirudin variant HM2 isolated from Hirudinaria manillensis. Proteolysis of the polypeptide chain of 64-amino-acid residues of hirudin HM2 by protease from Staphylococcus aureus V8, trypsin, thermolysin and subtilisin occurs at region 41-49 of the chain. The N-terminal fragments 1-41 and 1-47 were isolated to homogeneity and shown to maintain inhibitory action on thrombin, though much lower than the intact protein. The results were interpreted on the basis of a proposed three-dimensional structure of hirudin HM2 deduced by protein modelling the known structure of hirudin variant HV1 from Hirudo medicinalis (75% sequence similarity between HM2 and HV1). Both proteolysis experiments and protein modelling provide evidence for the existence in hirudin HM2 of a N-terminal well-structured domain (core) and a C-terminal flexible polypeptide segment. Determination of the accessible surface area of the three-dimensional model of hirudin HM2 showed that the sites of preferential cleavages are at the surface of the polypeptide molecule.
 ...
PMID:Probing the structure of hirudin from Hirudinaria manillensis by limited proteolysis. Isolation, characterization and thrombin-inhibitory properties of N-terminal fragments. 800 50

A Limulus intracellular coagulation inhibitor, designated LICI, was isolated from hemocytes of the Japanese horseshoe crab (Tachypleus tridentatus), using three steps of chromatography, including dextran sulfate-Sepharose CL-6B, Sephacryl S-200, and Mono S. LICI is a single-chain glycoprotein with an apparent M(r) = 48,000 estimated by SDS-polyacrylamide gel electrophoresis. It blocks the amidolytic activities of Limulus lipopolysaccharide-sensitive serine protease, factor C, by forming a covalent 1:1 complex with the protease. The second-order rate constant for inhibition of factor C was 2.5 x 10(6) M-1 s-1 at 37 degrees C. LICI also inhibited human alpha-thrombin, rat salivary kallikrein, bovine plasmin, and trypsin but not Limulus clotting enzyme, Limulus factor B, bovine factor Xa, human factor XIa, human tissue plasminogen activator, human urokinase, chymotrypsin, elastase, and papain. Glycosaminoglycans such as heparin and heparan sulfate had no effect on the inhibitory activity. A cDNA coding for LICI was isolated from a hemocyte cDNA library. The open reading frame of the 1,257-base pair cDNA codes for the mature protein of 394 amino acids, of which 223 residues were confirmed by amino acid sequence analysis. LICI shows significant sequence identities to members of the serpin superfamily, such as human plasminogen activator inhibitor type 2 (40%) and human monocyte/neutrophil elastase inhibitor (39%). LICI contains a putative reactive site, -Arg-Ser-, at the corresponding position present in several inhibitors of the serpin superfamily. The subcellular localization, determined using an anti-LICI polyclonal antibody, indicated that LICI colocates with the Limulus serine protease zymogens in large granules in the hemocyte.
 ...
PMID:A Limulus intracellular coagulation inhibitor with characteristics of the serpin superfamily. Purification, characterization, and cDNA cloning. 827 48

Moulting fluid of pharate adult tobacco hornworm moths, Manduca sexta, contains a novel cuticle-degrading proteinase, designated as MFP-1. The enzyme has been purified using heparin affinity chromatography and partially characterized. Before purification MFP-1 is associated with a large complex having an apparent native molecular mass > 669 kDa. After purification MFP-1 has a molecular mass of 41 kDa. The pI of the enzyme is 5.54. MFP-1 can be classified as generally trypsin-like on the basis of its substrate specificity and inhibition by soybean trypsin inhibitor. The enzyme's preferred substrate, Tos-Gly-Pro-Arg-pNA, its inhibition by hirudin, and its affinity for heparin, all indicate that MFP-1 has some characteristics in common with the vertebrate blood-clotting enzyme thrombin. MFP-1 is probably a serine protease, since it is inhibited by both DFP and PMSF (specific inhibitors of serine proteinases). However, the enzyme was also inhibited by a number of agents that affect cysteine proteinases. Purified MFP-1 degrades Manduca cuticle in vitro. We suggest that the enzyme may act as the first step in the degradation of the cuticle during the moulting process.
 ...
PMID:A cuticle-degrading proteinase from the moulting fluid of the tobacco hornworm, Manduca sexta. 835 21

Thrombin, the blood-clotting enzyme, is a serine proteinase with trypsin-like specificity and is able to cleave Arg-Xaa peptide bonds but only in a very limited number of substrates (and sites therein). For the prevention and treatment of thrombosis the control of thrombin activity is a key target, and a variety of synthetic inhibitors have been introduced recently, all of which have a positive charge at the P1 site. We report the synthesis of the first example of a new class of inhibitor containing a neutral side chain at the P1 site, the peptide benzyloxycarbonyl-D-Phe-Pro- methoxypropylboroglycine. The peptide is a potent inhibitor of thrombin [Ki (limiting) = 7 nM] and is highly selective for its target enzyme in respect of other serine proteinases. This may be expected to confer considerable advantage in terms of specificity of action and reduced toxicity over conventional, positively charged, inhibitors.
 ...
PMID:Benzyloxycarbonyl-D-Phe-Pro-methoxypropylboroglycine: a novel inhibitor of thrombin with high selectivity containing a neutral side chain at the P1 position. 845 16


1 2 Next >>