Comp. Biochem. Physiol. Vol. 94B, No. 3, pp. 469~.74, 1989 0305-0491/89 $3.00 + 0.00 Printed in Great Britain © 1989 Pergamon Press plc LIVER URICASE IN CAMELUS DROMEDARIUS: PURIFICATION AND PROPERTIES AHMED MOHAMUD OSMAN,* ANTONELLA DEL CORSO,t PIER LUIGI IPATAt and UMBERTOMURAt~ *Department of Physiology, Faculty of Veterinary Medicine, National University of Somalia, P.O. Box 1738, Mogadishu, Somalia; and tDipartimento di Fisiologia e Biochimica, Laboratori di Biochimica, Universit~ di Pisa, via S. Maria, 55, 56100 Pisa, Italy (Tel: 050 500292) (Recewed 13 March 1989) Abstract--1. Uricase (urate:oxygen oxidoreductase, EC 1.7.3.3) was purified 750-fold from the liver of Camelus dromedarius. 2. The enzyme is a tetramer with a Mr of 100,000, displays high specificity for uric acid with a Krn of 12/zM and is inhibited by a selected number of purine derivatives carrying oxygen at the C 2 position. 3. The effect of pH and the inhibition by thiol compounds and chelating agents on the enzyme activity is reported. 4. Some lines of evidence suggesting the possibility of interaction of camel liver uricase with oligonucleotides are presented. INTRODUCTION Uricase (urate:oxygen oxidoreductase, EC 1.7.3.3) is the enzyme responsible for the breakdown of the purine skeleton to allantoin in a variety of living systems. Probably due to its potential use in clinical chemistry and in therapy, uricase has been isolated and studied from various sources (Mahler, 1970; Venter et al., 1975; Wang and Marzluf, 1980; Kinsella et al., 1985; Nakamura et al., 1986). The enzyme appears to be located in peroxisomes, as shown by several studies on rat, mouse and fish liver and it is actually used as a marker enzyme for this organelle (Tsukada et al., 1968; Leighton et al., 1969; Yokota, 1973; Noguchi et al., 1979; Angermuller et al., 1986). Among mammalian enzymes, the most character- ized uricases are from pig liver, which is reported to be a copper protein (Mahler et al., 1955; Baum et al., 1956a, b; Pitts et al., 1974) and from ox kidney (Truscoe et al., 1964; Truscoe and Williams, 1965). We already showed that camel liver possesses a rather singular purine metabolism with a limiting step in the catabolic flow at the level of xanthine oxidase and guanase and with a high capability of purine recovery (Mura et al., 1986; Mura et al., 1987). With the aim of a better definition of the purine catabolic pathway, we describe here the purification and the characteri- zation of uricase from camel liver. MATERIALS AND METHODS Materials Liver of camel (Camelus dromedarius) was removed from freshly slaughtered animals, in the Public Slaughterhouse of Mogadishu and immediately frozen. Mol. wt markers for polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS), Pentyl agarose, Concanavalin ~Author to whom correspondence should be addressed. A-Sepharose 4B, uric acid, xanthine and other purine and pyrimidine derivatives were from Sigma. Blue Sepharose CL-6B, Sephacryl S-200 and Mono Q HR5/5 were from Pharmacia. Hydroxylapatite (Biogel HTP) was from Biorad. DEAE-Cellulose 23-SS was obtained from Serva. YM 30 ultrafiltration membranes and Centricon 30 centrifu- gal microconcentrator were from Amicon. 8-Aminoxan- thine-agarose (Mazzoni et al., 1982) was a gift from Prof. A. Lucacchini, Faculty of Pharmacy, University of Pisa. All other chemicals were of reagent grade and were used without further purification. Assay of uricase The enzyme activity was measured at 37°C essentially according to Kalckar (Kalckar, 1947) by following the decrease in absorbance at 293 nm which accompanies the breakdown of the purine ring of uric acid. Unless otherwise stated, the assay mixture contained in a final volume of 0.7 ml, 0.1 M Tris-HCl, pH 9, 0.1 mM uric acid, and vary- ing amounts of enzyme preparation. The molar extinction coefficient used to calculate the enzyme units was 11,400. One unit of uricase is the amount of the enzyme which catalyzes the transformation of 1 #mol of uric acid/min. Purification of liver uricase All procedures were carried out at 4°C, unless specified otherwise. Twenty-three g of frozen camel liver were thawed, added to 4.5 vol of 80 mM sodium phosphate pH 8 containing 80/~M dithiothreitol (S-buffer) and homoge- nized with Polytron for 5 rain. The homogenate was cen- trifuged at 35,000 g for 20 min, and the supernatant, filtered through cheesecloth, is referred to as crude extract (Table 1). The crude extract was subjected to acetone precipitation by a slow addition under magnetic stirring in a ice/salt bath of 2 vol of acetone precooled at -20°C. The suspension was then centrifuged for 20 rain at 35,000 g. The pellet was kept under nitrogen flow for a few minutes, resuspended in S-buffer and stirred for 30 min before centrifugation at 35,000 g. Protamine sulphate was added to the supernatant to 0.4% final concentration. The resulting suspension was centrifuged and the supernatant subjected to ammonium sulphate fractionation; ammonium sulphate to a final con- centration of 25% of saturation was added and the solution was stirred overnight. After centrifugation, the supernatant 469