Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.1 (chymotrypsin)
 10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The acetylcholine receptor from Torpedo californica electroplax was purified approximately 100-fold by affinity chromatography on alpha-neurotoxin-Sepharose 6B. Four putative subunits (alpha, beta, gamma, delta) of apparent molecular weights of 43,000, 52,000, 58,000, and 63,000 were found when the purified material was analyzed by sodium dodecyl sulfate (NaDodSO4) gel electrophoresis. In some preparations, however, the amount of the gamma polypeptide was small. The presence of N-ethylmaleimide throughout the purification procedure greatly enhanced the amount of the gamma chain. To investigate the possibility that the putative subunits may be structurally related, they were isolated by preparative NaDodSO4 gel electrophoresis and subjected to peptide mapping analyses. The patterns of fragments generated by Staphylococcus aureus V8 protease, papain, or chymotrypsin were different for each of the polypeptides. Thus, it is unlikely that they are derivatives of each other.
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PMID:Comparison of the subunits of Torpedo californica acetylcholine receptor by peptide mapping. 42 Jul 86

A colourimetric enzyme-linked sandwich assay has been developed to investigate the binding of human platelets to fibrinogen. The presence of platelets bound to fibrinogen-coated plastic can easily be detected and quantitated. Platelets treated with chymotrypsin to expose the fibrinogen receptor, are fixed with paraformaldehyde, and stored frozen. The detection sandwich consists of a mouse monoclonal antibody directed against the human platelet CD9 antigen, and a rabbit anti-mouse immunoglobulin conjugated to the enzyme alkaline phosphatase. The cleavage of the phosphatase substrate p-nitrophenyl phosphate can be monitored colourimetrically. The data presented provide evidence that this method is capable of detecting platelet-fibrinogen binding in a physiologically relevant manner. The binding is inhibited by EDTA or excess fibrinogen. The fibrinogen alpha and gamma chain peptides, RGDS and LGGAKQAGDV, and the snake venom echistatin are also inhibitory with IC50 values of 135 microM, 1.8 mM and 100 nM respectively.
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PMID:A colourimetric enzyme-linked sandwich assay for the detection of human platelets bound to a fibrinogen-coated surface. 189 64

In summary: Incubation of platelets with ADP or proteolytic enzymes (chymotrypsin or pronase) results in an exposure of two classes of specific binding sites on platelet surface: low and high affinity fibrinogen receptors. Fibrinogen interaction with these receptors results in platelet aggregation. High affinity fibrinogen receptors are not exposed on thrombasthenic platelets stimulated by ADP but are rendered available on chymotrypsin-treated thrombasthenic platelets; low affinity receptors cannot be exposed by ADP or chymotrypsin on these platelets. Availability of high affinity fibrinogen receptors on thrombasthenic platelets may depend on the residual glycoprotein IIIa. Fibrinogen receptors appear to be associated with glycoproteins IIb, IIIa and a 66,000 Mr platelet membrane component that is exposed during proteolysis of platelet membranes. Some of the platelet-binding sites on the fibrinogen molecule appear to be associated with the COOH-terminal portion of the gamma chain (gamma 374-411). Additional binding sites may also be located in the COOH-terminal portion of the A alpha chain. The conformation of the fibrinogen molecule may be important in its interaction with platelets. Platelet aggregation may result from bridging platelets by fibrinogen molecule in the presence of bivalent cations. In conclusion, platelet interaction with fibrinogen is a complex process involving different binding sites of the fibrinogen molecule. Our own data and review of literature suggest that platelet-interaction with fibrinogen is of major significance in hemostasis.
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PMID:Fibrinogen interaction with platelet receptors. 630 5

Bone sialoprotein (BSP), a small (approximately 80,000 M(r)) integrin binding, RGD-containing bone matrix glycoprotein, has been purified in milligram quantities from the serum-free medium of the rat osteosarcoma cell line UMR-106-BSP using nondenaturing conditions. Routine protein purification without serine protease inhibitors or reducing agents consistently resulted in three major fragments. The largest fragment (E1) started at amino acid 117 and did not bind to antibodies made to the RGD region of the protein. Furthermore, the smallest fragment (E3), was shown by sequencing to contain the RGD region of the protein. Digestion of intact BSP with highly purified chymotrypsin also resulted in a large fragment (C1) with properties nearly identical to those of E1. The large, non-RGD-containing fragments, E1 and C1, as well as the intact BSP, supported attachment by normal human bone cells and human skin fibroblasts in vitro. Attachment to the intact BSP was totally blocked by 0.4 mM GRGDS peptide. Both preparations of skin fibroblasts and approximately half of the preparations of normal human bone cells, however, also would not attach to the E1 and C1 fragments in the presence of 0.4 mM GRGDS peptide. In contrast, half of the bone cell preparations had significant attachment activity to E1 (> 50%) and C1 (> 25%) in the presence of 0.4 mM GRGDS peptide. These data suggest that cleavage of the BSP results in either (1) the exposure of a previously unavailable or cryptic cell attachment site or (2) a conformational change that increases the affinity of the complex between a non-RGD-encoded binding region of the E1 and C1 fragments and at least one receptor. The possible homology of the second, non-RGD-suppressible site of BSP with the second cell attachment site on the gamma chain of fibrinogen is discussed.
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PMID:Purification and fragmentation of nondenatured bone sialoprotein: evidence for a cryptic, RGD-resistant cell attachment domain. 821 61

Evidence is emerging for the regulation of platelet function at sites of vascular injury or thrombosis by multiple platelet recognition sites in fibrinogen. This study examined the interaction of platelets with immobilized fibrinogen degradation products, fragments D and E. A 60 kDa D fragment (D60) and 30 kDa fragment E supported the adhesion of activated platelets in a static system, despite the absence of gamma chain 400-411 dodecapeptide and RGD sequences. Moreover, platelet adhesion to these fragments was incompletely inhibited by EDTA. In the absence of divalent cations, ADP-stimulated platelet adhesion to fragments D60 or E constituted 31 +/- 12% and 33 +/- 10% (mean +/- SD,n = 23) of adhesion to intact fibrinogen in the presence of divalent cations, respectively. This EDTA-resistant adhesion was distinctly modulated by thrombin which preferentially supported platelet adhesion to fragment E, and chymotrypsin which selectively supported platelet adhesion to fragment D60. Furthermore, two potent inhibitors of fibrinogen binding, the 10E5 monoclonal antibody directed against the GPIIb-IIIa complex and the RGDF peptide, inhibited EDTA-resistant platelet adhesion to fragment D60 but not to fragment E. These data suggest the presence of novel, non-RGD, non-dodecapeptide containing platelet recognition sequences in both fibrinogen D and E domains which support divalent cation dependent and independent platelet adhesion via potentially unique binding mechanisms.
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PMID:Platelet adhesion to late fibrinogen degradation products. 873 44

In this study we purified a fibrinolytic enzyme from the culture supernatant of Flammulina velutipes mycelia by ion exchange and gel filtration chromatographies, it was designated as F. velutipes protease (FVP-I). This purification protocol resulted in 18.52-fold purification of the enzyme at a final yield of 0.69%. The molecular mass of the purified enzyme was estimated to be 37 kDa by SDS-PAGE, fibrin-zymography and size exclusion by FPLC. This protease effectively hydrolyzed fibrin, preferentially digesting alpha-chain over beta-and gamma-gamma chain. Optimal protease activity was found to occur at a pH of 6.0 and a temperature of 20 to 30 degrees C. The protease activity was inhibited by Cu2+, Fe2+ and Fe3+ ions, but was found to be enhanced by Mn2+ and Mg2+ ions. Furthermore, FVP-I activity was potently inhibited by EDTA and EGTA, and it was found to exhibit a higher specificity for chromogenic substrate S-2586 for chymotrypsin, indicating that the enzyme is a chymotrypsin-like metalloprotease. The first 20 amino acid residues of the N-terminal sequence of FVP-I were LTYRVIPITKQAVTEGTELL. They had a high degree of homology with hypothetical protein CC1G_11771, GeneBank Accession no. EAU86463.
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PMID:Purification and characterization of a fibrinolytic protease from a culture supernatant of Flammulina velutipes mycelia. 1782 81