Enkephalin analogs as substrates for the assay of brain cysteine proteinase (Cathepsin L) and its endogenous inhibitors.
A series of enkephalin-like peptides (X-Tyr-Gly-Gly-R-Pro) were synthesized for assay of cathepsin L and papain. Enzymes acted only at the Gly-Gly bond to release N-terminal dipeptides. When X = dansyl and R = Phe(NO2) the substrate was suited for continuous fluorimetric assay of rat brain cathepsin L (Km 45 microM, kcat/Km 1333 mM-1 sec-1). The substituted pentapeptides provided information on the influence of P2, P2' residues on rates of Gly-Gly cleavage. The synthetic substrate provided rapid and sensitive assays for the brain cathepsin L and its interaction with 13-14 kDa (cerebrocystatin) and 70 kDa (T-kininogen) rat brain inhibitors. The suppression of cathepsin L- or papain-mediated hydrolysis of substrates by inhibitors may be the result of competition between their binding domains at the enzyme catalytic center.
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Nathan Kline Institute, Center for Neurochemistry, Wards Island, New York, NY 10035.
Berg
M J
MJ
Danho
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W
D000818:Animals; D001921:Brain / Q000201:enzymology*; D056668:Cathepsin L; D002403:Cathepsins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors; D010450:Endopeptidases; D004745:Enkephalins / Q000378:metabolism*; D004792:Enzyme Precursors / Q000378:metabolism*; D007700:Kinetics; D011480:Protease Inhibitors / Q000032:analysis*; D051381:Rats; D013379:Substrate Specificity
D015853:Cysteine Proteinase Inhibitors; D004745:Enkephalins; D004792:Enzyme Precursors; D011480:Protease Inhibitors; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D056668:Cathepsin L; C534280:Ctsl protein, rat