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:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The synthesis of two biotinylated affinity labels for chymotrypsin and trypsin-like serine proteinases is described, along with their kinetic characterization and application to the detection of these proteinases after PAGE and Western blotting. Thus the chloromethane analogues biotinylphenylalanylchloromethane (Bio-Phe-CH2Cl; reagent 1) and biotinylarginylchloromethane (Bio-Arg-CH2Cl, reagent 2), have been shown to be potent active-site-directed inactivators of chymotrypsin and trypsin respectively. The apparent overall second-order rate constants (kobs./[I]) for the inactivation of chymotrypsin and trypsin by reagent 1 (approximately 4.9 x 10(3) M-1.min-1) and reagent 2 (approximately 1.0 x 10(5) M-1.min-1) respectively are comparable with those obtained by other workers with simple urethane-protected analogues and demonstrates that the presence of the bulky biotinyl moiety is compatible with inhibitor effectiveness. Samples of chymotrypsin and trypsin that have been inactivated by reagents 1 and 2 respectively and which have been subjected to SDS/PAGE and Western blotting can be revealed with a streptavidin/alkaline phosphatase label. We can presently detect down to 20 ng of inactivated proteinase by using this system. The utility of the arginine derivative for the detection of the plasma trypsin-like proteinases plasmin and thrombin has also been demonstrated, thus holding out the possibility that this reagent may find general application as an active-site-directed label for this class of proteinase.
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PMID:The synthesis, kinetic characterization and application of biotinylated aminoacylchloromethanes for the detection of chymotrypsin and trypsin-like serine proteinases. 157 91

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

The phenomenon of regulation of the catalytic activity of enzymes via changing their oligomeric composition in the system of reversed micelles of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in octane was studied using alpha-chymotrypsin (CT) from bovine brain and alkaline phosphatase (AP) from calf intestinal mucosa. The dependences of the enzyme catalytic activity on the AOT hydration degree (Wo = [H2O]/[AOT]), the parameter determining the radius (rc) of the inner cavity of micelles, usually represent the bell-shaped curves. The maximal catalytic activity is observed at such Wo when rc is equal to the size of the enzyme molecule. The position of this maximum strictly correlates with the enzyme oligomeric composition. Thus, in the case of CT this is observed at Wo = 12 when rc is equal to the radius (rp) of the CT globule. In the case of artificially produced conjugate containing six cross-linked CT molecules, this is observed at Wo = 43 when rc is equal to the radius of the sphere surrounding the absolute octahedron composed of six CT globules. The dependence of the catalytic activity of AP on Wo represents a curve with two maxima that are observed when rc is equal to rp of either AP monomer (Wo = 17) or AP dimer (Wo = 25). Ultracentrifugation experiments revealed that variation of Wo causes a change in the oligomeric composition of AP - its transition from monomeric (Wo less than 20) to dimeric form (Wo greater than 20). Hence, the observed maxima correspond to functioning of different oligomeric forms of AP.
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PMID:Regulation of the catalytic activity and oligomeric composition of enzymes in reversed micelles of surfactants in organic solvents. 199 3

Data from differential scanning calorimetry (DSC) may be used to estimate very large binding constants that cannot be conveniently measured by more conventional equilibrium techniques. Thermodynamic models have been formulated to describe interacting systems that involve either one thermal transition (protein-ligand) or two thermal transitions (protein-protein) and either 1:1 or higher binding stoichiometry. Methods are described for obtaining binding constants and heats of binding by two different methods: calculation or simulation fitting of data. Extensive DSC data on 2'CMP binding to RNase are presented and analyzed by the two methods. It is found that the methods agree when binding sites are completely saturated, but substantial errors arise in the calculation method when site saturation is incomplete and the transition of liganded molecules overlaps that of unliganded molecules. This arises primarily from an inability to determine TM (i.e., the temperature where concentrations of folded and unfolded protein are equal) under weak-binding conditions. Results from simulation show that the binding constants and heats of binding from the DSC method agree quantitatively with corresponding estimates obtained from equilibrium methods when extrapolated to the same temperature. It was also found from the DSC data that the binding constant decreases with increasing concentration of ligand, which might arise from nonideality effects associated with dimerization of 2'CMP. Simulations show that the DSC method is capable of estimating binding constants for ultratight interactions up to perhaps 10(40) M-1 or higher, while most equilibrium methods fail well below 10(10) M-1. DSC data from the literature on a number of interacting systems (trypsin-soybean trypsin inhibitor, trypsin-ovomucoid, trypsin-pancreatic trypsin inhibitor, chymotrypsin-subtilisin inhibitor, subtilisin BPN-subtilisin inhibitor, RNase S protein-RNase S peptide, avidin-biotin, ovotransferrin-Fe3+, superoxide dismutase-Zn2+, alkaline phosphatase-Zn2+, and assembly of regulatory and catalytic subunits of aspartate transcarbamoylase) were analyzed by simulation fitting or by calculation. Apparent single-site binding constants ranged from ca. 10(5) to 10(20) M-1, while the interaction constant for assembly of aspartate transcarbamoylase was estimated as 10(37) in molarity units. For most of these systems, the DSC interaction constants compared favorably with other literature estimates, for some it did not for reasons unknown, while for still others this represented the first estimate. Simulations show that for proteins having two binding sites for the same ligand within a single cooperative unit, ligand rearrangement will occur spontaneously during a DSC scan as the transition temperature of the unliganded protein is approached.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Study of strong to ultratight protein interactions using differential scanning calorimetry. 220 24

Male Wistar rats were fed for four weeks on defined diets containing no fiber additions, 10% levels of insoluble fiber derivatives (cellulose or alfalfa), or 5% levels of viscous fiber derivatives (pectin, guar gum, or metamucil). After an overnight fast, the pancreas was assayed for protein, amylase, lipase, trypsin, and chymotrypsin. Homogenates of small intestinal mucosa were analyzed for protein, alkaline phosphatase, invertase and thymidine kinase. There were, with few exceptions, no dietary effects on the exocrine pancreatic enzymes. The specific activities of the villus marker enzymes (invertase and alkaline phosphatase) tended to be higher in the proximal (but not middle or distal) intestines of the fiber-fed groups, while total activities were the same in all groups. In contrast, the activity of the crypt marker, thymidine kinase, was highest in the distal intestinal segments, and even higher in animals given the alfalfa, guar gum or metamucil-supplemented diets.
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PMID:Dietary fiber and intestinal adaptation: effects on intestinal and pancreatic digestive enzyme activities. 240 60

Two monoclonal antibodies that recognize Alzheimer's neurofibrillary tangles (ANTs), AD10 and AB18, have been characterized by immunoblotting against human and calf spinal cord neurofilament (NF) and calf brain microtubule preparations. Both antibodies bind to the 200-kilodalton (kd) (NF-H) and 160-kd (NF-M) but not to the 68-kd (NF-L) NF triplet proteins. They also bind to high-molecular-weight microtubule-associated proteins (MAPs) and tau. AD10 immunostains MAP2 and MAP1 families, whereas AB18 stains mainly MAP1 bands. Preincubation of intact filament preparation or nitrocellulose strips containing electroblotted NF proteins with Escherichia coli alkaline phosphatase completely blocks AD10 binding and partially blocks binding of AB18. These results suggest that the determinants recognized by these antibodies are phosphorylated. Immunoblotting of peptide fragments generated by limited proteolysis of NF proteins with alpha-chymotrypsin and Staphylococcus aureus V8 protease shows that the localization of the antigenic determinants to AD10 and AB18 in NF-H is approximately 100 and 60 kd, respectively, away from the carboxy terminal, a region previously shown to form the NF projection side arm. In NF-M, the antigenic determinants to both antibodies are located also in the projection side arm, in a 60-kd polypeptide adjacent to the alpha-helical filament core. The results show that ANTs contain at least two phosphorylated antigenic sites that are present in NF and MAPs, a finding suggesting that ANTs may be composed of proteins or their fragments with epitopes shared by cytoskeletal proteins.
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PMID:Two monoclonal antibodies recognize Alzheimer's neurofibrillary tangles, neurofilament, and microtubule-associated proteins. 243 80

Screening of antigenically reactive fragments of alpha S1-casein (alpha S1-CN), the major casein in bovine milk, was done by using HPLC and enzyme-linked immunosorbent assay (ELISA). BALB/c mice (6-week-old) were injected intraperitoneally with alpha S1-CN and complete Freund's adjuvant, and 14 days later, all the mice were boosted with alpha S1-CN and incomplete Freund's adjuvant. Twenty-one days after the 1st immunization, the mice were bled and antiserum was separated. Anti alpha S1-CN antibody fraction was obtained by precipitation from the antiserum with 50% saturated ammonium sulfate. alpha S1-CN was digested with trypsin and chymotrypsin, and 35 peptides were purified from the digests by reversed-phase HPLC with ODS (octadecylsilica) columns. Reactivity of peptides with the antibody were examined by ELISA. The solid phase in the wells of the polystyrene microtiter plate was coated with peptides, and the plate was successively incubated with anti alpha S1-CN antibody, conjugate of anti mouse immunoglobulin with alkaline phosphatase (ALP) and substrate of ALP. Two tryptic fragments (the residues 104-119 and 133-151) and three chymotryptic fragments (33-54, 105-121, and 174-199) were positive in an ELISA test. These five fragments would correspond to four antigenic sites. We could thus find antigenically reactive fragments of alpha S1-CN by the direct and simple detection of specific antigen-antibody interaction.
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PMID:Rapid screening of antigenically reactive fragments of alpha s1-casein using HPLC and ELISA. 244 84

The interaction of alpha-chymotrypsin, invertase, alcohol dehydrogenase and alkaline phosphatase with some ionic and non-ionic surfactants, viz. sodium dodecyl sulphate, dioctyl sodium sulphosuccinate, hexadecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide and Triton X-100, has been examined by studying the effect of varying surfactant concentrations on enzyme activities as well as by determining the time-dependent inactivation and the time-independent inhibition. The kinetic parameters, Km and Vmax, for alpha-chymotrypsin-catalysed reaction in presence of sodium dodecyl sulphate were evaluated. Anionic surfactants markedly decreased enzyme activity, whereas cationic surfactants were less effective. Nonionics showed no effect. This change in enzyme activity was also dependent on the nature of enzyme.
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PMID:Stability and kinetic behaviour of some enzymes in surfactant environment. 263 63

It has recently been shown that a monoclonal antibody SM 1-36-2 against connectin, an elastic filament of striated muscles, binds to the "elastic" domain of the molecule, and that the H subunit of neurofilament (NF-H), an intermediate filament of nerve cells, shares a homologous domain (Shimizu, T. et al. (1988) Biomed. Res. 9, 227-234 and Itoh, Y. et al. (1988) J. Biochem. 104, 504-508). In order to characterize (1) the intramolecular localization of the domain in the NF-H and (2) the effect of the phosphorylation state on the immunoreactivity, the homologous domain in the NF-H was analyzed by Western blotting after limited digestion with trypsin or alpha-chymotrypsin and dephosphorylation with E. coli alkaline phosphatase. It was found that (1) the epitope was located not in the core region but in the carboxyl-terminal peripheral (cross-bridge) region of NF-H and (2) the epitopes in connectin and NF-H were not affected by the phosphorylation state.
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PMID:The immunological homology between two filamentous cross-linker phosphoproteins, connectin and cross-bridge region of neurofilament-H, is not affected by the phosphorylation state. 272 66

Hybrid enzymes which have two different enzyme activities linked together covalently may be useful reagents for various applications, such as the determination of complex biological structures. The present paper describes the preparation and purification of two such enzyme-enzyme conjugates, namely, trypsin-chymotrypsin and trypsin-alkaline phosphatase. Whereas the former has been prepared by using the well-known bifunctional reagent glutaraldehyde, the latter exploited the Schiff base formation between the oxidized carbohydrate moiety of alkaline phosphatase and the free amino groups of trypsin.
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PMID:Preparation of heteroenzyme conjugates: trypsin-chymotrypsin and trypsin-alkaline phosphatase. 284 96


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