Purification and characterization of guinea-pig liver microsomal deacetylase involved in the deacetylation of the O-glucoside of N-hydroxyacetanilide.
نویسندگان
چکیده
A microsomal deacetylase that catalyses the deacetylation of the O-glucoside of N-hydroxyacetanilide (GHA) was purified from guinea-pig liver. The activity was located exclusively in the microsomes and not detected in the cytosol. The purified GHA deacetylase was a trimeric protein with a molecular mass of 160+/-10 (S.D.) kDa composed of subunits of 53+/-2 kDa; its pI was 4.7. The N-terminal amino acid sequence of GHA deacetylase was similar to those reported for guinea-pig and rat liver microsomal carboxylesterases. The GHA deacetylase showed a comparable hydrolytic activity towards p-nitrophenyl acetate (PNPA), although the activities towards N-hydroxyacetanilide, acetanilide and some endogenous acylated compounds were very low or not detectable. The deacetylase activity towards GHA was inhibited by organophosphates but not by p-chloromercuribenzoate, suggesting that GHA deacetylase can be classified as a B-esterase. The enzyme exhibited a positive homotropic co-operativity towards GHA. The values of the Hill coefficient, the half-saturating concentration ([S]0.5) for GHA, and Vmax were 1.59+/-0.03, 5.51+/-0.07 mM and 32.5+/-1.4 micromol/min per mg respectively, at the optimum pH of 8.5. The bell-shaped pH dependence of the Vmax/[S]0.5 profile indicated pKa values attributed to histidine and lysine residues. The study of stoichiometric inhibition by di-isopropyl fluorophosphate and kinetic analysis with the Monod-Wyman-Changeux model suggests that GHA deacetylase has six substrate binding sites and three catalytically essential serine residues per enzyme molecule.
منابع مشابه
Organic Circulation and persistence of Foot – and – Mouth disease virus type O in guinea pig
Since, the most susceptible laboratory animal to Foot–and–Mouth disease virus (FMDV) is guinea pig, and then one milliliter of FMDV type (O) concentrations of 106-106.5 TCID50was inoculated intradermally (ID) to 10 plantar surface of guinea pig (right side). Guinea pig adapted virus has been prepared after generalization phase, and then one milliliter of virus was inoculated intradermally to 30...
متن کاملEnzymatic deacetylation of N-hydroxy-2-acetylaminofluorene by liver microsomes.
Af-Hydroxy-2-acetylaminofluorene was rapidly deacetylated by guinea pig liver and, to a lesser extent, by other guinea pig tissues examined. The enzyme activity was located in the microsomal fraction of the liver. The enzyme appeared to be stimulated by trisodium pentacyanoamine ferrate, and it was inhibited by SKF-525A (|3-diethylaminoethyldiphenylpropyl acetate hydrochloride), by sodium fluor...
متن کاملInhibitory Effect of Ruta graveolens L. Extract on Guinea Pig Liver and Bovine Milk Xanthine Oxidase
Flavonoids could serve as potent inhibitors of xanthine oxidase (XO). In the present study, the effects of Ruta graveolens L. extract and its major isolated flavonoids, quercetin and rutin, on guinea pig liver XO have been investigated. The inhibitory effects of R. graveolens, quercetin and its glycoside form, rutin, were assayed spectrophotometrically. R. graveolens...
متن کاملMetabolism of /V-Hydroxy-2-acetylaminofluorene and A/-Hydroxy-2- aminofluorene by Guinea Pig Liver Microsomes1
The guinea pig is resistant to the hepatocarcinogenic effects of 2-acetylaminofluorene and 2-aminofluorene. This resist ance, however, is not due to the lack of a A/-hydroxylating enzyme in the liver which catalyzes the first and rate-limiting step to the activation of these chemicals to proximal carcino gens. It is shown that guinea pig liver microsomes can A/-hydroxylate both of these compoun...
متن کاملDevelopment of a Sensitive Spectrofluorometric-Multivariate Calibration Method for Enzyme Kinetic of Aldehyde Oxidase
Attempts to obtain experimental values for the kinetic parameters of phenanthridine oxidation by guinea pig or rabbit liver aldehyde oxidase using common spectrophotometric methods have not been successful due to a lower limit of detection. In the present study, a new spectrofluorimetric assay in combination with a multivariate calibration method for enzymatic kinetic study of aldehyde oxidase ...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید
ثبت ناماگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید
ورودعنوان ژورنال:
- The Biochemical journal
دوره 325 ( Pt 1) شماره
صفحات -
تاریخ انتشار 1997