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Titolo:
A highly sensitive fluorescent microplate method for the determination of UDP-glucuronosyl transferase activity in tissues and placental cell lines
Autore:
Collier, AC; Tingle, MD; Keelan, JA; Paxton, JW; Mitchell, MD;
Indirizzi:
Univ Auckland, Sch Med, Dept Pharmacol & Clin Pharmacol, Auckland, New Zealand Univ Auckland Auckland New Zealand lin Pharmacol, Auckland, New Zealand
Titolo Testata:
DRUG METABOLISM AND DISPOSITION
fascicolo: 10, volume: 28, anno: 2000,
pagine: 1184 - 1186
SICI:
0090-9556(200010)28:10<1184:AHSFMM>2.0.ZU;2-P
Fonte:
ISI
Lingua:
ENG
Soggetto:
GLUCURONIDATION; ACID; EXPRESSION;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
16
Recensione:
Indirizzi per estratti:
Indirizzo: Collier, AC Univ Auckland, Sch Med, Dept Pharmacol & Clin Pharmacol, 85 PkRd, Auckland, New Zealand Univ Auckland 85 Pk Rd Auckland New Zealand and, New Zealand
Citazione:
A.C. Collier et al., "A highly sensitive fluorescent microplate method for the determination of UDP-glucuronosyl transferase activity in tissues and placental cell lines", DRUG META D, 28(10), 2000, pp. 1184-1186

Abstract

The fluorescent compound 4-methylumbelliferone (4MU) can be used to detecturidine diphosphate glucuronosyl transferase activity by observing the fall in fluorescence as the compound is converted to 4-methylumbelliferone glucuronide. A microplate assay has been developed that has improved sensitivity and is faster and cheaper than the historical extraction method. Activity is detectable with approximately 10% of the protein required in the extraction method. Absence of extraction and cleanup procedures and the ability to observe reaction rate directly are also of great advantage to the researcher. Michaelis-Menten kinetic data from one healthy female human liver is presented. The extraction method yielded a mean V-max of 19.9 nmol/min/mg of protein and a mean K-m of 652.5 mu M on 1 day [n = 6, coefficients of variation (CV) 15 and 24%, respectively]. For the microplate method on 1 day, the mean V-max was 36.21 +/- 1.3 nmol/min/mg of protein (CV = 3.7%), significantly (P < .0001) higher than for the extraction method. The mean K-m, 175.4 +/- 24.2 mu M (CV = 14.5%), was significantly lower (P < .0001) than observed in the extraction method. The assay was performed in replicates of six over 6 days; average intra- and interassay coefficients of variation were 9 and 22% for V-max and 8 and 35% for K-m, respectively, for the microplate method. The microplate method has also detected activity in the placental trophoblast-derived cell lines JEG-3, JAr, and BeWo (5.5, 4.1, and 2.6 nmol/min/mg of protein, respectively, at 200 mu M 4MU concentration), indicating that placental cells may be capable of glucuronidating 4MU.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 11/08/20 alle ore 15:23:08