2764-95-6Relevant articles and documents
A novel histochemical method for the visualization of thrombin activity in the nervous system
Bushi,Gera,Kostenich,Shavit-Stein,Weiss,Chapman,Tanne
, p. 93 - 104 (2016)
Although thrombin has an important role in both central and peripheral nerve diseases, characterization of the anatomical distribution of its proteolytic activity has been limited by available methods. This study presents the development, challenges, validation and implementation of a novel histochemical method for visualization of thrombin activity in the nervous system. The method is based on the cleavage of the substrate, Boc-Asp(OBzl)-Pro-Arg-4MβNA by thrombin to liberate free 4-methoxy-2-naphthylamine (4MβNA). In the presence of 5-nitrosalicylaldehyde, free 4MβNA is captured, yielding an insoluble yellow fluorescent precipitate which marks the site of thrombin activity. The sensitivity of the method was determined in vitro using known concentrations of thrombin while the specificity was verified using a highly specific thrombin inhibitor. Using this method we determined the spatial distribution of thrombin activity in mouse brain following transient middle cerebral artery occlusion (tMCAo) and in mouse sciatic nerve following crush injury. Fluorescence microscopy revealed well-defined thrombin activity localized to the right ischemic hemisphere in cortical areas and in the striatum compared to negligible thrombin activity contralaterally. The histochemical localization of thrombin activity following tMCAo was in good correlation with the infarct areas per triphenyltetrazolium chloride staining and to thrombin activity measured biochemically in tissue punches (85 ± 35 and 20 ± 3 mU/ml, in the cortical and striatum areas respectively, compared to 7 ± 2 and 13 ± 2 mU/ml, in the corresponding contralateral areas; mean ± SEM; p 0.05). In addition, 24 h following crush injury, focal areas of highly elevated thrombin activity were detected in teased sciatic fibers. This observation was supported by the biochemical assay and western blot technique. The histochemical method developed in this study can serve as an important tool for studying the role of thrombin in physiological and pathological conditions.
Polymerase and hydrolytic activities of purified goat brain cysteine proteinase dipeptidyl peptidase I.
Pal, Suresh,Raghav, Neera,Kamboj, Ramesh C.,Singh, Hari
, p. 97 - 101 (2007/10/02)
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TRIPEPTIDE DERIVATIVES AND THEIR APPLICATION IN ASSAYING ENZYMES
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, (2008/06/13)
Tripeptide derivatives having the formula STR1 wherein R. sup.1 is alkanoyl or ω-aminoalkyanoyl, phenylalkanoyl or p-aminophenylalkanoyl, cyclohexylcarbonyl or 4-aminomethyl-cylcohexylcarbonyl, benzoyl optionally substituted with methyl, amino, or halogen in the o-or p-position, alkoxy carbonyl, benzyloxy carbonyl optionally substituted with methoxy, methyl, or chlorine in the p-position, alkylsulfonyl, phenylsulfonyl or naphthylsulfonyl, R 2 is straight-chained or branched alkyl, hydroxyalkyl, alkoxyalkyl, benzoxyalkyl, ω-carboxy-alkyl, ω-alkoxy carbonylalkyl, ω-benzyloxycarbonylalkyl, cyclohexyl, cyclohexylmethyl, 4-hydroxycyclohexylmethyl, phenyl, benzyl, 4-hydroxybenzyl or imidazol-4-yl-methyl, R 3 is hydrogen or alkyl, R 4 is hydrogen, methyl or ethyl, and R 5 is an amino group substituted with aromatic or heterocyclic radicals, R 5 being capable of being split off by enzymatic hydrolysis to form a colored or fluorescent product H-R 5. The tripeptide derivatives of formula I are used for assaying certain enzymes, and in particular, factor Xa. Enzyme-bearing materials are reacted with the said tripeptide derivatives. The quantity of split product H-R 5 released by the enzymatic action on the tripeptide derivative is determined photometrically, spectrophotometrically, fluorescence-spectrophotometrically, or electrochemically. The quantity of released split product H-R 5 per time unit is proportional to the quantity of enzyme present in the starting material.
