Uricase and Allantoinase in Glyoxysomes ' Received for publication

In fat-degrading tissues of seedlings of seven different plant species examined, uricase activity (urate:02 oxidoreductase, EC 1.7.33) was associated with particulate fractions. After equilibrium density centrifugation on sucrose density gradients the enzyme activity was recovered in the glyoxysomal band (density: 1.25 grams per cubic centimeter). Allantoinase is also present in glyoxysomes but, equally, in the proplastid region (density: 1.22 grams per cubic centimeter). Xanthine oxidase, xanthine dehydrogenase, allantoicase, and urease were not detected in glyoxysomes from castor bean endosperm. Uricase in these particles shows its maximal activity at pH 8.9. The apparent Km is 7.4 AM. Urate concentrations greater than 120 MM as well as certain other purine compounds inhibit the enzyme. Cyanide at a concentration of 10 AM is a potent inhibitor. 2,6-Dichlorophenolindophenol did not substitute for oxygen as electron acceptor. The over-all reaction catalyzed by uricase (urate:02 oxidoreductase, EC 1.7.33) was elucidated by investigations with preparations from hog liver (27), fungi (15, 21), and bacteria (3). As shown in Figure 1, decarboxylation occurs at C-6, and the enzyme functions as an aerobic dehydrogenase. Highly purified preparations have been obtained from hog liver (28). Early cell fractionation studies with rat liver showed that urate oxidase sedimented with the crude mitochondrial fraction (38). Subsequently, by differential and equilibrium density centrifugation, the enzyme was shown to be associated with other oxidases that produce H202 (L-a-hydroxy acid oxidase, D-amino acid oxidase) and catalase in the class of microbody known as peroxisomes (4, 11). Urate oxidase is in fact used as a marker enzyme for these organelles and has been shown to be present in peroxisomes from rat liver (11) and from liver and kidney of birds and amphibians (41) as well as in peroxisomes from certain protozoa (30-32). The first report of urate oxidase activity in higher plants dates back to 1920 when is was shown that soybean meal was able to degrade uric acid (33). Fosse and his collaborators (13, 14) demonstrated urate oxidase activity in numerous seedlings, and Franke et al. (15) and Green and Mitchell (21) studied the enzyme from fungal cells. In addition to its role in the catabolism of purines, as in the extensive digestive process in storage tissue of germinating seedlings, urate oxidase seems to be important in nitrogen mobilization in those vascular plants where allantoin and allantoic acid are major transport forms (36, 37). Schlee and Reinbothe (39) invoked urate oxidase to account 'Supported by National Science Foundation Grant GB 13228. for the demonstration that in tissues from such plants uric acid is an intermediate in the synthesis of allantoin from glycine. However, despite the known and widespread occurrence of urate oxidase in plant tissues, little appears to have been established concerning its properties and intracellular distribution (44). In the present paper we describe properties of the enzyme from the endosperm of germinating castor bean seedlings and its subcellular localization in glyoxysomes from this and other fat-degrading tissues. MATERIALS AND METHODS Plant Material. Leaves from tobacco (Nicotiana tabacum) and corn (Zea mays) were cut from young plants grown in the greenhouse. Sunflower (Helianthus annuus), safflower (Carthamus tinctorius), and corn seeds were sterilized with 0.5% sodium hypochlorite (Clorox) solution for 20 min, soaked in distilled water for approximately 5 hr, and germinated on moist paper towels at 30 C in the dark for 5 to 6 days. Seeds of the one-leaved pinyon (Pinus monophylla) were collected in the Panamint Mountains (SW Death Valley) and of the Joshua tree at the Walker Pass (California). After sterilization with 10% Clorox solution for 20 min, the seeds were soaked for 8 to 10 hr in distilled water and germinated in moist vermiculite at 30 C and 90% relative humidity in the dark for 5 days (pinyon) or 9 days (Joshua tree). Seeds of castor bean (Ricinus communis) were germinated as described previously (20). Preparation of Cellular Organelies. One general grinding procedure was employed with slight modifications for the different plant materials. Usually 2 to 3 ml of grinding medium (described by Gerhardt and Beevers [19, 20], but with the EDTA concentration reduced to 0.001 M), was used for each gram of tissue. All preparations were carried out at 0 to 5 C. The plant part or tissue of interest was either minced with a razor blade in grinding medium (tobacco and corn leaves, roots, safflower and sunflower cotyledons, yucca perisperm) or chopped in a household onion chopper (corn scutella, pine megagametophytes, castor bean endosperm). The slurry was then ground in a mortar, and the homogenate was passed through four layers of cheesecloth. After centrifuging at 270g for 10 min the supernatant solution (supernatant I) was decanted and centrifuged for 30 min at 11,000g. This produced supernatant II and a pellet, the crude particulate fraction, which was gently resuspended in grinding medium. Two millilitrs of this suspension, corresponding to 5 to 10 g fresh weight of tissue, were layered on stepped or continuous sucrose gradients (20) and centrifuged for 4 to 5 hr at 64,300g in a Spinco rotor SW25.2. Discontinuous gradients were fractionated by puncturing the bottom of the tube and collecting separately the visible glyoxysomal, proplastid, and mitochondrial bands. An ISCO density gradient fractionator model D was used to collect successive 1-ml samples from continuous gradients. Biochemical Assays. Spectrophotometric assays were con-