RESUMEN
Human liver carboxylesterases catalyze the hydrolysis of apolar drug or xenobiotic esters into more soluble acid and alcohol products for elimination. Two carboxylesterases, hCE-1 and hCE-2, have been purified and characterized with respect to their role in cocaine and heroin hydrolysis. The binding of meperidine (Demerol) and propoxyphene (Darvon) was examined in a competitive binding, spectrophotometric assay. The hCE-1 and hCE-2 bound both drugs, with Ki values in the 0.4- to 1.3-mM range. Meperidine was hydrolyzed to meperidinic acid and ethanol by hCE-1 but not hCE-2. The Km of hCE-1 for meperidine was 1.9 mM and the kcat (catalytic rate constant) was 0.67 min-1. Hydrolysis of meperidine by hCE-1 was consistent with its specificity for hydrolysis of esters containing simple aliphatic alcohol substituents. Hence, hCE-1 in human liver microsomes may play an important role in meperidine elimination. Propoxyphene was not hydrolyzed by hCE-1 or hCE-2. This observation is consistent with the absence of a major hydrolytic pathway for propoxyphene metabolism in humans.
Asunto(s)
Analgésicos Opioides/metabolismo , Hidrolasas de Éster Carboxílico/metabolismo , Hígado/metabolismo , Meperidina/metabolismo , Biotransformación , Cromatografía Líquida de Alta Presión , Dextropropoxifeno/metabolismo , Inhibidores Enzimáticos/farmacología , Cromatografía de Gases y Espectrometría de Masas , Semivida , Humanos , Hidrólisis , Técnicas In Vitro , Isoenzimas/metabolismo , Cinética , Hígado/enzimologíaRESUMEN
Human liver alcohol dehydrogenase catalyzes the NAD+-dependent oxidation of alcohols. Isoenzymes are produced in liver by five different genes, two of which are polymorphic. We have studied the three beta beta isoenzymes produced at ADH2 because they exhibit very different kinetic properties and they appear with different frequencies in different racial populations. The beta 3 beta 3 isoenzyme which appears in 25% of black Americans was purified to homogeneity, and conditions were found to stabilize this labile isoenzyme. The comparison of substrate specificity among beta beta isoenzymes for primary straight-chain alcohols indicates that there is a positive correlation between Vmax/KM and the log octanol/water partition coefficient for alcohols with beta 2 beta 2 and beta 3 beta 3 but not with beta 1 beta 1. Methyl substitutions at C1 or C2 of these alcohols reduce the catalytic efficiency with all three isoenzymes. The KM and Ki values of beta 3 beta 3 for NAD+ and NADH are substantially higher than values for beta 1 beta 1 or beta 2 beta 2. The Vmax of beta 3 beta 3 ethanol oxidation is 90 times that of beta 1 beta 1. Sequencing of the beta 3 subunit and gene indicates that the polymorphism results from a single amino acid exchange of Cys-369 in beta 3 for Arg-369 in beta 1 and beta 2 [Burnell et al. (1987) Biochem. Biophys. Res. Commun. 146, 1227-1233]. In horse alcohol dehydrogenase and beta 1 beta 1, the guanidino group of Arg-369 is thought to stabilize the NAD(H)-enzyme complex by bonding to one of the pyrophosphate oxygens.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Alcohol Deshidrogenasa/aislamiento & purificación , Isoenzimas/aislamiento & purificación , Hígado/enzimología , Alcohol Deshidrogenasa/metabolismo , Cromatografía de Afinidad , Cromatografía DEAE-Celulosa , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Electroforesis en Gel de Almidón , Humanos , Focalización Isoeléctrica , Isoenzimas/metabolismo , Cinética , Peso Molecular , Polimorfismo Genético , Análisis de Regresión , Especificidad por SustratoRESUMEN
The beta 3 beta 3 (formerly called beta Indianapolis) and beta 1 beta 1 isoenzymes of human alcohol dehydrogenase differ substantially in their catalytic properties. Specifically, the KM value for NAD+ of beta 3 beta 3 is 70 times greater than that of beta 1 beta 1, and the Ki value for NADH is 35 times greater than that of beta 1 beta 1. To identify the structural basis of these catalytic differences, we sequenced regions of the beta 3 subunit and the beta 3 gene. beta 3 differs from beta 1 by the substitution of Cys for Arg-369. Based on x-ray crystallography of horse ADH, Arg-369 should interact with the nicotinamide phosphate moiety of NAD(H). The Cys for Arg-369 substitution would decrease the enzyme's affinity for coenzyme and, thus, account for the observed kinetic differences between beta 3 beta 3 and beta 1 beta 1.
Asunto(s)
Alcohol Deshidrogenasa/genética , Arginina , Cisteína , Isoenzimas/genética , Hígado/enzimología , NAD/metabolismo , Alcohol Deshidrogenasa/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Humanos , Isoenzimas/metabolismo , Sustancias Macromoleculares , Oxidación-Reducción , Fragmentos de Péptidos/análisis , Unión ProteicaRESUMEN
The use of extradural (epidural) steroids in the management of selected cases of lumbosciatic syndrome is well known. This paper reports the appearance of systemic side-effects in four cases after each had received an injection of 6-10 ml of methylprednisolone and 0.25% bupivacaine into the extradural space at the site nearest to the affected nerve roots.
