Xenopus Allantoicase: Molecular Cloning, Enzymatic Activity and Developmental Expression Davide Vigetti, Claudio Monetti, Loredano Pollegioni, Roberto Taramelli, and Giovanni Bernardini 1 Dipartimento di Biologia Strutturale e Funzionale, Universita ` degli Studi dell’Insubria, Via J. H. Dunant 3, I-21100 Varese, Italy Received February 1, 2000, and in revised form April 6, 2000 Allantoicase is one of the enzymes of the purine deg- radation pathway and, interestingly, it appears to be lost, together with uricase and allantoinase, during mammalian evolution. Only allantoicases from the as- comycetes S. pombe, S. cerevisiae, and N. crassa have already been cloned, although the activity has been reported also in fishes and amphibians. By screening a cDNA expression library of Xenopus liver, we have cloned a 1491-bp-length cDNA coding for a 389 amino acid protein that shows an high similarity with the enzyme allantoicase. We have found that allantoicase mRNA is abundantly expressed in kidney and liver, but at much lower level is also present in brain, testis, intestine, and lung. We have detected enzymatic activ- ity in crude extract from kidney, liver, and lung; we have also determined kinetic parameters (K m  8.44 mM, V max  6.94 mol min 1 per mg protein) in kidney. During embryo development, we have detected allan- toicase transcript and activity starting from 1 and 5 days after fertilization, respectively. © 2000 Academic Press Key Words: allantoicase; allantoinase; amphibia; pu- rine degradation; Xenopus embryo. The catabolic pathway which degrades purines to uric acid is common to all vertebrates. Uric acid is then degraded to allantoin by urate oxidase (uricase, EC 1.7.3.3). This does not occur in humans where uricase is absent, but it occurs in most mammals that excrete allantoin in the urine as the end product of purine metabolism. Purine catabolism proceeds further in lower vertebrates, since allantoin is degraded by allan- toinase (EC 3.5.2.5) to allantoic acid, which in turn is degraded to urea and ureidoglycollate by allantoicase (EC 3.5.3.4); ureidoglycollate is degraded by ureidogly- collate lyase to urea and glyoxylate. Urea is then de- graded to CO 2 and NH 3 by urease (1–3). The causes of the progressive extinction of these enzymatic activities during the phylogenetic evolution are still to be elucidated. The loss of uricase activity in humans is due to nonsense mutations in the gene cod- ing for the enzyme (4, 5). Information on the other uric acid degrading enzymes in mammals or higher verte- brates is absent. Allantoinase and allantoicase activity could be loss in mammals because of gene delection or deleterious mutation. In fishes, allantoicase seems to have a peroxisomal localization and two isoformes with identical weight are present: a membrane-bound enzyme and a matrix enzyme (6). In Rana catesbiana, allantoicase and al- lantoinase activities were copurified from liver extract and this suggested that both reactions could be carried out by a single protein consisting in a 48-kDa subunit (allantoicase) and a 54-kDa subunit (allantoinase) (3). More recently, R. catesbiana allantoinase cDNA se- quence has been published; the protein consists of 483 amino acids, it has two potential transmembrane do- mains and a mitochondrial localization (7). In contrast, only activity data are available on amphibian allanto- icase, that seems to have a peroxisomal localization (8). This allantoicase subcellular localization would be in contrast with the mitochondrial localization of the com- panion enzyme allantoinase. In this paper, we provide the first charaterization of a vertebrate allantoicase cDNA together with the pre- dicted aminoacid sequence, its tissue distribution, and developmental regulation. We have also determined the kinetic properties of the amphibian enzyme and compared them with those of allantoicases from differ- ent organisms. 1 To whom correspondence and reprint requests should be ad- dressed. Fax ++39 0332 421300. E-mail: Giovanni.Bernardini@ unimi.it. 90 0003-9861/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. Archives of Biochemistry and Biophysics Vol. 379, No. 1, July 1, pp. 90 –96, 2000 doi:10.1006/abbi.2000.1863, available online at http://www.idealibrary.com on