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

---

Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Chorionic villi excised from freshly delivered human term placentae and small endothelial cell aggregates were released from them by the sequential use of collagenase and trypsin. The endothelial cells were further isolated by rosetting with magnetic polystyrene beads which were coated with QB END/40, the endothelial-specific monoclonal antibody (mAb) to thrombomodulin. Cell rosettes were plated on gelatin coated Petri dishes. The cells initially grew as discrete colonies but reached confluence within 7 days. The monolayers were sub-cultured five times, and grew to confluence each time. All the cells were immunoreactive to the endothelial markers von Willebrand factor, QB-End/40 and Ulex europaeus-1 lectin. They did not show immunoreactivity to trophoblast markers (mAbs ED341 and ED235). The isolated cells could also incorporate acetylated low-density lipoprotein. Most of the cells possessed an elongated morphology, though some were slightly spread and polygonal in shape. The cell monolayers did not resemble the typical cobblestone appearance of endothelial cells isolated from large vessels. Ultrastructurally, most of the cells resembled placental microvascular cells in shape and frequency of caveolae; undifferentiated cell-cell contacts and extracellular matrix material was observed. Human placental microvascular endothelial cells may offer an in vitro model which complements the use of the perfused term placental lobule in studies of microvascular permeability.
...
PMID:Isolation of endothelial cells from human term placental villi using immunomagnetic beads. 793 93

Rat liver cells (the C-9 cell line) are stimulated to metabolize arachidonic acid by alpha-thrombin, its receptor polypeptide, gamma-thrombin, and trypsin. Prostaglandin (PG) I2 synthesis stimulated by alpha-thrombin is inhibited by dansylarginine N-(3-ethyl-1,5-pentanediyl) amide (DAPA), by hirudin, by the synthetic tyrosine-sulfated dodecapeptide corresponding to residues 53-64 of hirudin (hirugen), by the Tyr(SO3H)63-hirudin fragment 54-65 and by rabbit lung thrombomodulin. Stimulation of arachidonic acid metabolism by the receptor octapeptide, SFLLRNPN, is not affected by DAPA or hirudin. gamma-Thrombin stimulates arachidonic acid metabolism but at 300 to 400-fold higher concentrations. Trypsin stimulates arachidonic acid metabolism. Trypsin's proteolytic activity is required--its ability to stimulate is abolished if it is incubated with Na-p-tosyl-L-lysine chloromethyl ketone (TLCK) or bovine pancreatic trypsin inhibitor. Prior treatment of the rat liver cells with alpha-thrombin blocks subsequent stimulation by alpha-thrombin, but not by trypsin, whereas prior treatment with trypsin blocks subsequent stimulation by trypsin, but not the activity stimulated by alpha-thrombin. Prior treatment of the cells with the serine-proteases, chymotrypsin, pancreatic or neutrophil elastase and thrombocytin from Bothrups atrox venom, block alpha-thrombin's activation of PGI2 production, but not the activity stimulated by trypsin. These findings indicate that alpha-thrombin and trypsin stimulate PGI2 production via different receptors.
...
PMID:Alpha-thrombin and trypsin use different receptors to stimulate arachidonic acid metabolism. 793 15

The pulmonary vasculature is of great physiological/pathological significance. We have isolated and cultured microvessel endothelial cells (HuLEC) from lung tissue obtained from lung transplant recipients by modification of published methods. Pure cultures of HuLEC were isolated by mechanical disaggregation of the tissue prior to sequential dispase and trypsin digestion to obtain microvessel fragments. Magnetic beads (Dynabeads) coated with Ulex europaeus agglutinin-1 were then used to enhance the purity of cultures at the first passage. HuLEC formed contact-inhibited "cobblestone" monolayers on gelatin and fibronectin substrates and capillary-like "tubes" on Matrigel and accumulated acetylated low-density lipoprotein. Immunofluorescent characterization of these cells revealed the presence of von Willebrand Factor, angiotensin-converting enzyme, and thrombomodulin and the expression of antigens for the endothelial cell-specific monoclonal antibodies EN4, PAL-E, and H4-7/33. The endothelial origin of these cells was confirmed by the demonstration of the cell adhesion molecules, platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31), and E-selectin (endothelial leukocyte adhesion molecule-1/ELAM-1) upon stimulation with TNF alpha. These cells should provide a useful tool for studying various aspects of pathology and biology of the pulmonary microvasculature in vitro.
...
PMID:Human lung microvessel endothelial cells: isolation, culture, and characterization. 841 55

The wealth of structural information now available for thrombin, its precursors, its substrates, and its inhibitors allows a rationalization of its many roles. alpha-thrombin is a rather rigid molecule, binding to its target molecules with little conformational change. Comparison of alpha-thrombin with related trypsin-like serine proteinases reveals an unusually deep and narrow active site cleft, formed by loop insertions characteristic of thrombin. This canyon structure is one of the prime causes for the narrow specificity of thrombin. The observed modularity of thrombin allows a diversity in this specificity; its "mix-and-match" nature is exemplified by its interactions with macromolecules (Fig. 20). The apposition of the active site to a hydrophobic pocket (the apolar binding site) on one side and a basic patch (the fibrinogen recognition exosite) on the other allows for a fine tuning of enzymatic activity, as seen for fibrinogen. Thrombin receptor appears to use the same sites, but in a different way. Protein C seems only able to interact with thrombin if the recognition exosite is occupied by thrombomodulin. These two sites are also optimally used by hirudin, allowing the very tight binding observed; thrombin inhibition is effected by blocking access to the active site. On the other hand, antithrombin III makes little use of the recognition exosite; instead, its interactions are tightened with the help of heparin, which binds to a second basic site (the heparin binding site). Thrombin's modularity is a result of the conjunction of amino acid residues of like properties, such as charge or hydrophobicity. The charge distribution plays a role, not only in the binding of oppositely charged moieties of interacting molecules, but also in selection and preorientation of them. Nonproteolytic cellular properties are attributed to 1) the rigid insertion loop at Tyr60A, and 2) a partially inaccessible RGD sequence. The former can interact with cells in the native form; the latter would appear to be presented only in an (at least partially) unfolded state. The membrane binding properties of prothrombin can be understood from the ordered arrangement of calcium ions on binding to the Gla domain. Kringle F2 binds to thrombin at the heparin binding site through charge complementarity; a conformational change appears to occur on binding. The observed rigidity of the thrombin molecule in its complexes makes thrombin ideal for structure based drug design. Thrombin can be inhibited either at the active site or at the fibrinogen recognition exosite, or both.(ABSTRACT TRUNCATED AT 400 WORDS)
...
PMID:A player of many parts: the spotlight falls on thrombin's structure. 846 68

Endothelial cells were isolated from human full-term placenta by perfusion with trypsin solution via the umbilical cord vein. Human placental endothelial cells (HPEC) were successfully grown and kept in culture. HPEC exhibited endothelial characteristics as judged by morphology of confluent monolayers, staining with low density lipoprotein, binding of Ulex europaeus I lectin, and immunostaining against von Willebrand factor, alpha-thrombomodulin, VE-cadherin and a series of integrins. Different growth requirements and particular morphological characteristics indicated the different vascular origin of HUVEC and HPEC.
...
PMID:Isolation and cultivation of endothelial cells derived from human placenta. 898 Sep 11

Thrombin undergoes allosteric modulation by thrombomodulin (TM) that results in a shift in macromolecular specificity, blocking fibrinogen clotting while enhancing protein C activation. The TM enhancement of protein C activation involves both an 8-fold decrease in Km and a 200-fold increase in kcat. Although TM-mediated conformational changes in thrombin have been detected by many techniques, the nature of these changes remains obscure. Access to the active center of thrombin is relatively restricted due to the presence of a large insertion loop at residue 60 (chymotrypsin numbering) that has been implicated in modeling studies as being responsible for poor inhibition by BPTI. Thrombin and the E192Q mutant, which binds BPTI much more tightly than thrombin, are both inhibited very slowly by BPTI. TM increases the rate of thrombin or thrombin E192Q inhibition by BPTI approximately 10-fold. When analyzed as slow tight binding inhibition, the TM effect on thrombin E192Q inhibition by BPTI is primarily on the first, reversible step in the reaction. Structural studies of the thrombin E192Q-BPTI complex have previously shown that the 60 loop lies over the BPTI, a position which requires 8 A movement at the apex of the 60 loop, and that BPTI is found in the same canonical orientation as in the trypsin complex. It follows that TM enhancement of the initial interaction of thrombin results in a conformation that favors interactions with BPTI, probably involving motion of the 60 loop.
...
PMID:Thrombomodulin increases the rate of thrombin inhibition by BPTI. 942 93

Thrombomodulin (TM) is an anticoagulant glycoprotein on the surface of endothelial cell that directly inhibits the procoagulant activities of thrombin, and the TM-thrombin complex accelerates thrombin-catalyzed activation of protein C. Soluble TM in urine has no glycosaminoglycan (GAG) chain which accelerates the anticoagulant activities. Therefore, we expressed recombinant GAG-modified urinary thrombomodulin (GAG-UTM) in C127 cells. The glycosylation sites were determined by amino acid sequence analysis of peptides digested with trypsin after S-carboxymethylation. The structures of N-linked oligosaccharides were estimated by two-dimensional sugar mapping of pyridylaminated oligosaccharides that were treated with exoglycosidase. The disaccharide composition analysis of the GAG chain was performed by HPLC using digestion with chondroitinase ABC, ACII and B. Consequently, it was revealed that the N-linked oligosaccharides were assigned to Asn29, Asn98, Asn364, Asn391; those structures were estimated biantennary, 2-6 branched triantennary and 2-4 branched triantennary complex type oligosaccharides that were linked by fucose at the ratio of 1.0:0.5:0.1, respectively. Moreover, the attachment site of the GAG chain was assigned to Ser472. It was then estimated that the GAG chain contained chondroitin-4-sulfate and dermatan sulfate, which were repeated approximately 30 times. In this paper, the GAG attachment site and structural characteristics of GAG-UTM, were confirmed. Moreover, structures of the N-linked oligosaccharides of GAG-UTM are described for the first time.
...
PMID:The glycosylation sites and structural characteristics of oligosaccharides on recombinant human thrombomodulin. 959 55

Carboxypeptidase U (CPU, EC 3.4.17.20) is a recently described basic carboxypeptidase which circulates in plasma as an enzymatically inactive precursor procarboxypeptidase U (proCPU), also known as plasma carboxypeptidase B precursor or thrombin activatable fibrinolysis inhibitor (TAFI). The activation of the zymogen proceeds through a proteolytic cleavage at Arg-92. The active form - CPU - is able to retard the initial phase of fibrinolysis by cleaving C-terminal lysine residues exposed on fibrin partially degraded by the action of plasmin. These C-terminal lysine residues are essential for the high affinity binding of plasminogen to fibrin and the subsequent activation to plasmin. In this report, the activation of purified human proCPU was studied using trypsin and some key proteases of the coagulation and fibrinolytic cascade, i.e., kallikrein, plasmin and thrombin. The most efficient activation is obtained in the presence of thrombin in complex with thrombomodulin. After in vitro activation, CPU is unstable at 37 degrees C (T(1/2)=15 min). Its stability can be improved dramatically using lower temperatures.
...
PMID:Proteolytic activation of purified human procarboxypeptidase U. 1068 74

Thrombin and trypsin activate protease-activated receptors (PARs) that modulate vascular tone. In addition to the PARs, thrombin also binds to thrombomodulin via exosite 1, a domain also involved in the interaction of thrombin with PAR-1 but not PAR-2. The purpose of this study was to determine whether thrombomodulin would alter thrombin-induced vasoconstriction, thought to be mediated predominantly by PAR-1, but not PAR-2, which mediates vascular relaxation. For comparison, thrombomodulin was examined for its effect on both thrombin and trypsin-induced responses. Trypsin was 2000-fold more potent as a relaxant than as a contractile peptide, whereas thrombin was only 7.8-fold more potent as a relaxant than contractile agonist, consistent with activation of PAR-1 predominantly mediating contraction and PAR-2 predominantly mediating relaxation. Although thrombomodulin (10(-7) M) alone did not alter vascular tone or the rate of thrombin-induced vascular responses, thrombomodulin (10(-8) and 10(-7) M) attenuated maximal thrombin (10(-8) and 10(-7) M)-induced vasoconstriction preferentially compared with thrombin-induced relaxation and had no effect on equieffective trypsin-induced responses. The inhibition of thrombin-induced contraction resulted from the interaction of thrombin with thrombomodulin rather than any direct effect of thrombomodulin on tissue PARs. Thus, this study describes a novel vascular action of thrombomodulin to selectively attenuate thrombin-induced vascular contractility. This action of thrombomodulin may serve to protect vasculature from thrombin-induced vasoconstriction during conditions of endothelial injury known to increase plasma and cellular levels of thrombomodulin.
...
PMID:Vascular contraction and relaxation to thrombin and trypsin: thrombomodulin preferentially attenuates thrombin-induced contraction. 1099 91

Carboxypeptidase R (EC 3.4.17.20; CPR) is an unstable basic carboxypeptidase found in fresh serum in addition to carboxypeptidase N (CPN) which is a stable enzyme. CPR in fresh serum is generated from its zymogen (proCPR) during coagulation by trypsin-like enzymes such as thrombin and thrombin/thrombomodulin complexes. Since removal of the C-terminal arginine abrogates the anaphylatoxin activity of C3a and C5a, CPR and CPN are regarded as anaphylatoxin inactivators. We report here that the culture supernatant of activated human neutrophils converts proCPR to CPR. Addition of an elastase specific inhibitor, N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone (MSAAPVCK) to the supernatant of stimulated neutrophils completely inhibited activation of proCPR. On the other hand, a thrombin specific inhibitor, p-Nitrophenyl-p'-amidinophenyl-methanesulfonate hydrochloride (pNP-pAPMS) inhibited only 16% of proCPR activation by the neutrophil supernatant. Furthermore, purified elastase converted proCPR to CPR. Therefore, elastase can activate proCPR directly, or indirectly through activation of some proteases, which have been contaminating in reagents. Release of CPR generating enzymes from neutrophils should play an important role in regulation of excess inflammation.
...
PMID:Elastase from activated human neutrophils activates procarboxypeptidase R. 1200 33


<< Previous 1 2 3 Next >>

---