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Titolo:
Characterization of new fluorogenic substrates for the rapid and sensitiveassay of cathepsin E and cathepsin D
Autore:
Yasuda, Y; Kageyama, T; Akamine, A; Shibata, M; Kominami, E; Uchiyama, Y; Yamamoto, K;
Indirizzi:
Kyushu Univ, Fac Dent, Dept Conservat Dent 2, Fukuoka 8128582, Japan Kyushu Univ Fukuoka Japan 8128582 nservat Dent 2, Fukuoka 8128582, Japan Kyushu Univ, Fac Dent, Dept Pharmacol, Fukuoka 8128582, Japan Kyushu UnivFukuoka Japan 8128582 Dept Pharmacol, Fukuoka 8128582, Japan Kyoto Univ, Primate Res Inst, Dept Cellular & Mol Biol, Inuyama, Aichi 484, Kyoto Univ Inuyama Aichi Japan 484 llular & Mol Biol, Inuyama, Aichi 484, Osaka Univ, Sch Med, Dept Cell Biol & Anat, Suita, Osaka, Japan Osaka Univ Suita Osaka Japan Dept Cell Biol & Anat, Suita, Osaka, Japan Juntendo Univ, Sch Med, Dept Biochem, Tokyo 113, Japan Juntendo Univ Tokyo Japan 113 v, Sch Med, Dept Biochem, Tokyo 113, Japan
Titolo Testata:
JOURNAL OF BIOCHEMISTRY
fascicolo: 6, volume: 125, anno: 1999,
pagine: 1137 - 1143
SICI:
0021-924X(199906)125:6<1137:CONFSF>2.0.ZU;2-O
Fonte:
ISI
Lingua:
ENG
Soggetto:
HUMAN-ERYTHROCYTE-MEMBRANES; AMYLOID PRECURSOR PROTEIN; INCREASED EXPRESSION; ACID PROTEINASE; IMMUNOHISTOCHEMICAL LOCALIZATION; AFFINITY PURIFICATION; ASPARTIC PROTEINASES; ALZHEIMERS-DISEASE; PROCATHEPSIN-E; ACTIVE-SITE;
Keywords:
aspartic proteinase; cathepsin D; cathepsin E; fluorogenic substrate;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
54
Recensione:
Indirizzi per estratti:
Indirizzo: Yamamoto, K Kyushu Univ, Fac Dent, Dept Conservat Dent 2, Fukuoka 8128582,Japan Kyushu Univ Fukuoka Japan 8128582 2, Fukuoka 8128582, Japan
Citazione:
Y. Yasuda et al., "Characterization of new fluorogenic substrates for the rapid and sensitiveassay of cathepsin E and cathepsin D", J BIOCHEM, 125(6), 1999, pp. 1137-1143

Abstract

Cathepsin E and cathepsin D are two major intracellular aspartic proteinases implicated in the physiological and pathological degradation of intra- and extracellular proteins. In this study, we designed and constructed highly sensitive synthetic decapeptide substrates for assays of cathepsins E andD based on the known sequence specificities of their cleavage sites. Thesesubstrates contain a highly fluorescent (7-methoxycoumarin-4-yl)acetyl (MOCAc) moiety and a quenching a,l-dinitrophenyl (Dnp) group. When the Phe-Phebond is cleaved, the fluorescence at an excitation wavelength of 328 nm and emission wavelength of 393 increases due to diminished quenching resulting from the separation of the fluorescent and quenching moieties, The first substrate, MOCAc-Gly-Lys-Pro-IIe-Leu-Phe-Phe-Arg-Leu-Lys(Dnp) gamma-NH2,, in which the Lys-Pro combination at positions P5 and P4 was designed for specific interaction with cathepsin E, is hydrolyzed equally well by cathepsins E and D (k(cat)/K-m = 10.9 mu M(-1.)s(-1) for cathepsin E and 15.6 mu M-1. S-1 for cathepsin D), A very acidic pH optimum of 4.0 was obtained for both enzymes. The second substrate, MOCAc-Gly-Lys-Pro-Ile-Ile-Phe-Phe-Arg-Leu-Lys(Dnp)gamma-NH2,, in which the isoleucine residue at position P2 was meant to increase the specificity for cathepsin E, is also hydrolyzed equally by both enzymes (k(cat)/K-m = 12.2 mu M-1 S-.(-1) for cathepsin E and 16.3 mu M-1 .S-1 for cathepsin D), The k(cat)/K-m values for both substrates are greater than those for the best substrates for cathepsins E and D describedso far. Unfortunately, each substrate shows little discrimination between cathepsin E and cathepsin D, suggesting that amino acids at positions far from the cleavage site are important for discrimination between the two enzymes. However, in combination with aspartic proteinase inhibitors, such as pepstatin A and Ascaris pepsin inhibitor, these substrates enable a rapid and sensitive determination of the precise levels of cathepsins E and D in crude cell extracts of various tissues and cells. Thus these substrates represent a potentially valuable tool for routine assays and for mechanistic studies on cathepsins E and D.

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Documento generato il 03/04/20 alle ore 09:57:18