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)

In the fluorescein dilaurate test fluorescein dilaurate is cleaved by the pancreas specific cholesterol ester hydrolase activity and the liberated fluorescein is absorbed and excreted in the urine. Fluorescein recovery is a reflection of exocrine pancreatic function. The test was evaluated in 14 patients with cystic fibrosis and 16 healthy volunteers. The test was well tolerated by patients, was easy to perform, and gave significantly lower values in the patients suffering from cystic fibrosis. The result of the pancreolauryl test was also correlated with the result of the faecal chymotrypsin test in 11 of the patients suffering from cystic fibrosis. A positive correlation was found between the two test results. The test is a practical and reliable index of pancreatic exocrine function and may have a useful role as a screening procedure.
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PMID:Diagnosis of exocrine pancreatic insufficiency in cystic fibrosis by use of fluorescein dilaurate test. 372 27

Fluorescein 5'-isothiocyanate (FITC) has been shown to specifically inactivate the Na+- and K+-stimulated adenosine triphosphatase ((Na,K)-ATPase) at low concentrations (Karlish, S. J. D. (1979) Na+,K+ATPase Structure and Kinetics 115-128). The site of modification of purified dog kidney (Na,K)-ATPase by FITC has been investigated by enzymatic cleavage and fluorescence resonance energy transfer. The binding of FITC, which occurs at a stoichiometry of approximately one site per ATP binding site, causes an ATP-protectable inactivation of ATPase activity suggesting that it is reacting at the ATP hydrolysis site. The FITC reaction site apparently is located near the center of the COOH-terminal 77,000-dalton peptide fragment obtained by chymotryptic cleavage of the alpha subunit. Addition of ouabain to the native enzyme in the presence of chymotrypsin enhances cleavage at this site and releases the fluorescein moiety from the membrane. It is further shown that the distance from the FITC reaction site to the ouabain binding site, as judged by fluorescence resonance energy transfer from anthroyl ouabain to FITC, is approximately 74 A. These results demonstrate that ouabain inhibits the (Na,K)-ATPase by causing a protein conformational change which extends an unusually large distance across the membrane.
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PMID:The active site structure of Na+- and K+-stimulated ATPase. Location of a specific fluorescein isothiocyanate reactive site. 627 7

Fluorescence polarization has been used to study the interaction of human alpha1-protease inhibitor (alpha1 PI; also called alpha1-antitrypsin) with two active site modified chymotrypsins (CT), dehydroalaninyl-195-alpha-CT (AnhCT) and N-methylhistidinyl-57-alpha-CT (MeCT). For the reaction of the fluorescein-labeled AnhCT (FAnhCT) with alpha 1 PI (Pi type MM, the predominant allelic form), a Kassoc of 1.8 x 10(7) M-1 was obtained by Scatchard analysis, which also indicated 1.3 binding sites. An alternate analysis using a direct dissociation plot, which assumes 1:1 binding, gave a Kassoc of 2.2 x 10(7) M-1. Fluorescein-labeled MeCT (FMeCT) binds somewhat more weakly to alpha PT (Kassoc = 1.2 x 10(6) M-1; 0.87 binding site). Similar results were obtained by using the proflavin displacement method to determine the binding constant for MeCT with alpha 1 PI (Kassoc = 1.0 x 10(6) M-1). With alpha 1 PI (ZZ type) in which the serum level is reduced and there is a strong tendency to develop chronic obstructive pulmonary disease, the Kassoc found by the fluorescence polarization method was similar to that for alpha 1 PI (MM type) for both CT derivatives. Alpha 1 PI (MM type), modified by oxidation with N-chlorosuccinimide, shows a reduced binding affinity for FAnhCT (Kassoc = 6.5 x 10(5) M-1) and no measurable binding with FMeCT (Kassoc less than 1 x 10(4) M-1). Previous studies have demonstrated that bovine CT forms very stable complexes with alpha 1 PI. In contrast, complexes formed with both active site modified CT derivatives undergo rapid dissociation as shown by the drop in the polarization value on dilution or on the addition of excess unlabeled chymotrypsin derivative. This weakened association suggests that, for reaction with alpha 1 PI, the enzyme active site serine is important in stabilizing the enzyme-inhibitor complex.
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PMID:Fluorescence polarization studies on the interaction of active site modified chymotrypsins with alpha1-protease inhibitor. 697 49

Serine proteases are typically synthesized as proteolytically inactive zymogens that often become activated in a limited and highly localized manner. Consequently, determination of the spatial and temporal activation pattern of these molecules is of great importance to understanding the biological processes that they mediate. Until only recently, the tools to conveniently address the question of where and when serine proteases are active within complex tissues have been lacking. In order to detect spatially restricted serine protease activities in Drosophila embryos and ovaries we introduce a technique using fluorescent synthetic and protein-based inhibitors. With this approach we have detected a novel serine protease activity with a relative mobility of 37 kDa, localized to the surface of pole cells, the germ-line precursors, in embryos between nuclear cycles 11 and 14 in development. A second novel cell-specific protease activity was localized to the tissues of early gastrulating embryos. Microinjection of inhibitors into the perivitelline space of stage 2 embryos perturbed normal embryonic development. Fluorescein-conjugated chymotrypsin inhibitor and Bowman-Birk inhibitor labeled protease activity localized to the oocyte-somatic follicle cell interface of the developing egg chamber. Our results suggest that this technique holds promise to identify new spatially restricted activities in adult Drosophila tissues and developing embryos.
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PMID:Fluorescently labeled inhibitors detect localized serine protease activities in Drosophila melanogaster pole cells, embryos, and ovarian egg chambers. 1560 41

Because impaired cellular protease activities are linked to many diseases, such as cancer, inflammation, neurodegeneration, and infection, internally quenched fluorescent peptides have recently been developed as tools for analyzing the specificities of these enzymes. Here we report convenient and cost-effective approaches for the selective "in synthesis" assembly of such substrate peptides for protease assays. Fluorescein and Dabcyl groups were covalently and selectively attached during synthesis to epsilon-amino groups of internal lysines. Functionality was then tested by digestion with leucine aminopeptidase, chymotrypsin, and microsomal vesicles. All peptides proved to be appropriate substrates of the enzymes tested and of the endogenous peptidases in the microsomal vesicles. In summary, we describe an innovative and cheap method to develop completely functional quenched fluorescent peptides that are usable in specific detection of individual proteases, in particular aminopeptidases, in both in vitro and in vivo systems.
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PMID:Assembly and selective "in synthesis" labeling of quenched fluorogenic protease substrates. 1693 May 25