3-Glucuronidase VOLUME

Skorupa, A. F. et al. (1999). Sustained production of beta-glucuronidase from localized sites after AAY vector gene transfer results in widespread distribution of enzyme and reversal of lysosomal storage lesions in a large volume of brain in mucopolysaccharidosis VII mice. Exp. Neurol. 160(1), 17-27. 

For abbreviation of analyte names see Sect. Abbreviations . ACN acetonitrile, f -glucu solution of (3-glucuronidase (E. coli), EE ethyl acetate, hep-SA heptane-sulfonic acid,, soprop iso-propanol, IS internal standard, MeOH methanol, metab. metabolites, Me-O- Bu, methyl- butyl ether n.s. not specified, OAc acetate, PB phosphate buffer Numbers give volume ratio v/v... 

Chemotaxis experiments (see Note 2) Use blind well Boyden chambers, that contain a volume of 100 pL in the lower compartment (Costar, Bodenheim, Germany). As filters use selfpunched (7 pm punch) polyvinylpyrrolidone containing polycarbonate filters (pore size 3 mm, Costar), which prior to use need to be washed with 1 M NaOH in 50 % (v/v) aqueous ethanol for 7 min followed by three washes in water (see Note 3). Use p-nitrophenyl-p-D-glucuronide (Sigma) at 10 mM in 0.1 M aqueous sodium acetate, pH 4.0, for p-glucuronidase detection (see Note 4). 

Adjust the urine sample to pH 5 by the addition of dilute hydrochloric acid. For each 9 volumes of urine add 1 volume of acetate buffer pH 5) containing 5000 Fishman units/ml of mixed glucuronidase/ sulphatase (from Helixpomatia), and incubate the mixture at 31 for 24 hours. Centrifuge, pour the supernatant liquid on to a column of Amberlite XAD-2 resin, wash the column with 50 ml of water, and then elute the steroids with 100 ml of ethanol. Evaporate the ethanol using a rotary film evaporator, dissolve the residue in 0.5 ml of ethanol, and add 2 ml of cyclohexane. 

Sample preparation 1 mL Urine + 10 mg p-glucuronidase/arylsulfatase (Helix pomatia. Sigma), heat at 37° overnight, add an equal volume of buffer, centrifuge at 2000 g for 5 min, inject an aliquot of the supernatant onto column A with mobile phase A and elute to waste. After 2.5 min backflush the contents of column A onto column B with mobile phase B, monitor the effluent from column B. Re-equilibrate both columns for 12.5 min before the next injection. (Buffer was 200 mM boric acid a< justed to pH 9.5 with 5 M NaOH.)... 

Sample preparation 100 pL Plasma or urine + 100 pL 20000 U/mL p-glucuronidase in 200 mM pH 4.9 sodium acetate buffer, heat at 37° for 20 h, add 20 pL 100 p mL me-phenytoin, add 2 volumes of ethyl acetate, extract, centrifuge at 1000 g for 5 min, repeat extraction four more times. Combine the orgtmic layers and evaporate them to dryness under a stream of nitrogen, reconstitute the residue in 20 pL MeOH, irgect a 5 pL aliquot. 

For assays of relatively small amounts of beta-glucuronidase, use 1 mM MUG in Extraction Buffer (see above) with a final reaction volume of 0.5 ml. The reaction is carried out at 37 C. At time=0 and subsequent times, remove samples (e.g., 100 1) and stop the reaction by adding 0.9 ml 0.2 M Na2C03. We have observed that GUS activity remains linear (even in crude extracts) for a very long time, sometimes days. Hence the "time=0" point does not need to be at the moment of addition of extract to substrate. In fact, it is often better to allow the reaction to proceed at 37 C for several minutes to equilibrate and to achieve before the initial time point is taken. Thus, we actually take 5, 15, 25, and 35 minute time points, and always find true linearity is maintained. The resulting slope can therefore be measured independent... 

Fig. 10. Polyacrylamide gels stained for glucuronidase activity after electrophoresis (pH 9.5) of liver extracts from DBA and C3H mice. 20% (w/v) liver homogenates were prepared in cold 0.25 M sucrose. Equal volumes of homogenate and 10% Triton X-100 were mixed and incubated at room temperature for 15 minutes, after which the insoluble material was removed by centrifugation. The supernatant was then diluted fourfold with a solution containing equal parts of 10% Triton X-100 and 0.25 M sucrose. The DBA-C3H mixture (MIX) consisted of equal parts DBA and C3H extract. These extracts were then subjected to electrophoresis according to the method of Davis (1964) at 4 mamps/gel for 90 minutes. After electrophoresis, the gels were stained for glucuronidase activity with naphtol-AS-BI-gluouronide (Ganschow and Bunker, 1970).

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