Evidence is presented to suggest that in chick liver, xanthine dehydrogenase and aldehyde oxidase activities are associated with only one protein species. The results of SDS electrophoresis of the purified material indicate a subunit MW of 120000.
I. INTRODUCTION
The mammalian enzymes xanthine oxidase (EC 1.2.3.2) and aldehyde oxidase (EC 1.2.3.1) are closely related molecules with broadly overlapping substrate specificities, molecular weights, and content of fiavin adenine dinucleotide, molybdenum, and iron [1]. The analogous enzymes xanthine dehydrogenase (XDH) and aldehyde oxidase (AOX) in Drosophila are particularly interesting because their activities are dependent upon the functioning of a gene locus that is not genetically linked to the known structural genes of the two enzymes [2]. One hypothesis to account for this 'regulation' of the activities of two related enzymes by a third locus is that the two enzymes may have subunits in common [3]. Assuming that a similar relationship between the two enzymes from vertebrate species may exist, we set out to test this hypothesis by analyzing the subunits of liver XDH, which had been studied extensively in previous investigations [4], and AOX from the same organism, the chick.
To our surprise, the two activities in the chick appear to be associated with only one protein. We have purified the activities to apparent homogeneity; they co-purify and co-electrophorese. These and other observations suggest that the two activities in chicken, in contrast to the rabbit, may be associated with only one protein or that they may have very similar physicochemical properties.
II. MATERIALS AND METHODS
Commercial chicken and rabbit liver were stored frozen at -70 ~ until used. Unless stated otherwise, crude extracts were prepared by homogenizing 250 g liver in 750 ml H20 in a Waring blendor. The general purification protocol for XDH is that of [4].
XDH was assayed at 25 ~ in an incubation mixture containing one ml of 0.05 M potassium phosphate buffer, pH 7.9, 0.15 mM xanthine, 0.5 mM NAD+, 0.1 mM EDTA, and 5 to 50 lal enzyme solution. The generation of NADH (340 m~t) or uric acid (290 mix) was recorded. AOX was assayed at 25 ~ in the same buffer, which contained phenazine methosulfate (PMS, 20 Ixg ml-1), 2,6-dichlorophenol indophenol (DCPIP, 20 txg ml-1), and acetaldehyde (50 mM); 5-200 ~tl of 81