int. J. Biochem. Vol. 18, No. 5, pp. 431-436, 1986 0020-711X/86 $3.00+0.00 Printed in Great Britain. All rights reserved Copyright © 1986 Pergamon Press Ltd URIDINE PHOSPHORYLASE FROM ESCHERICHIA COLI B. ENZYMATIC AND MOLECULAR PROPERTIES ALBERTO VITA 1, ADOLFO AMICI1, TIZIANA CACCIAMANI l, MARINA LANCIOTT11 and GIULIO MAGNI 2. ~Dipartimento di Biologia Cellulare, Universitfi di Camerino, 62032 Camerino, Italy 2Istituto di Biochimica, Facolt/t di Medicina e Chirurgia, Universit~i di Ancona, 60100 Ancona, Italy (Received 14 October 1985) Abstract--l. Uridine phosphorylase (EC 2.4.2.3) from Escherichia coli B is an oligomeric protein composed of four identical subunits of 29,000 mol. wt. 2. The enzyme has four half-cystine residues per subunit titrable only in denaturing condition. No disulphide linkages either inter- or intra-chain are present. The isolectic point is 5.25. 3. The enzyme shows strict specificity toward uridine and 5-methyluridine and is inhibited by thymine, deoxycytidine and heavy metal ions. INTRODUCTION Pyrimidine nucleosides are readily taken up and metabolized by Escherichia coli cells. Their utilization requires the concerted action of pyrimidine metab- olizing enzymes (Munch-Petersen et al., 1979). The main feature in the catabolism of nucleosides is the phosphorolytic cleavage of the N-glycosidic bond between the base and the pentose moieties of the nucleosides. In the case of cytidine and deoxy- cytidine, a deamination must occur before the phos- phorolytic cleavage takes place. The phosphorolysis is catalyzed by uridine phosphorylase which has been detected and purified from a variety of microbial and animal cells (Krenitsky, 1976; Leer et al., 1977; McIvor et al., 1983; Bose and Yamada, 1974). In E. coli the enzyme is induced if cytidine, ad- enosine or CMP are added to growth media (Magni et al., 1985). In the case of cytidine the enzyme specific activity increases up to 4 times its original value. Addition of glucose to E. coli growing cells in the presence of cytidine, causes a drop of uridine phosphorylase specific activity, which after two hours approaches the activity value obtained by growing cells on inorganic salts-glucose media. In order to assess whether this phenomenon is due to an inac- tivation mechanism, i.e. an alteration of the molecu- lar structure of the protein, or to a cessation of the enzyme synthesis, we have developed a rapid and efficient purification procedure (Vita and Magni, 1983), allowing characterization of uridine phos- phorylase obtained from cells grown in the above described conditions. Therefore to gain further in- sight into the metabolic role of uridine phosphorylase we have investigated its molecular and enzymatic properties. Preliminary data from these investigations have been reported (Vita et al., 1984). MATERIALS AND METHODS Materials Diaflo membranes were purchased from Amicon Cor- poration; bases, nucleosides, nucleotides, Tris (Trizma *Author to whom correspondence should be addressed. base), 2-mercaptoethanol, NaDodSO 4 (sodium dodecyl sul- fate), DTNB [(5,5'-dithiobis (2-nitrobenzoate)], protein markers, and other fine chemicals were from Sigma Chemical Co. Other chemicals, reagent grade, were obtained from J. T. Baker Chemicals B.V. Ampholine carriers am- pholytes from LKB. Solutions of amino acid standards (2.5pMol/ml), constant boiling HCI (6N), fluoraldehyde OPA-reagent solution and sodium acetate (pHix buffer grade) were obtained from Pierce. Cell growth and preparation of crude extract Cells of E. coli were grown aerobically at 37°C, up to 0.9 Optical Density units at 660 nm, in 1.51 of inorganic salts medium (19 mM glutamate, 1.7 mM MgSO4, 14.3 mM K2SO4, 43 mM NaCI, 100 mM phosphate buffer, pH 7.0), in the presence of 11 mM glucose. In the case of preparations of induced uridine phosphorylase, glucose was replaced by 5 mM cytidine. The cells were harvested by centrifugation, washed twice with 100 ml of 20 mM Tris-HCl (pH 7.5), resuspended with 25 ml of the same buffer containing 5 mM 2-mercaptoethanol (Buffer A), and disrupted by passing through an Aminco French pressure cell at 20,000 Ib/in2. The homogenate was centrifuged at 40,000g for 30 min, and the supernatant was dialyzed overnight against buffer A and taken as the crude extract. All operations subsequent to harvesting procedure were performed at 0-4°C. Assay Uridine phosphorylase activity was determined spec- trophotometrically as described by Magni (1978). The method is based on the differential absorption existing between uridine and uracil at 280nm (AE mM at 280 nm = 2.10). The reaction mixture consisted of 0.167 mM uridine, and 100 mM phosphate buffer (pH 7.5), in a final volume of 1.0 ml. The reaction was initiated by the addition of 0.02--0.04 enzyme units and the reaction rate was followed by using a Varian Cary 118C spectrophotometer. One enzyme unit is defined as the amount of enzyme which catalyzes the disappearance of 1 #mol of uridine per min at 30°C. Assay conditions of inhibition experiments are re- ported on figure legends. Protein determination Protein was measured by the dye-binding procedure of Bradford (1976) for routine determinations and by the method of Schacterle and Pollack (1973) for more accurate determinations. In both methods bovine serum albumin was used as the standard. 431