Urate oxidase activity and copper content in the liver of macular mutant mouse, a model animal for human congenital copper deficiency, Menkes' kinky hair disease.

The macular mouse is an X-linked recessive inherited mutant and is considered to be a model for human congenital copper deficiency, Menkes' kinky hair disease. The activity of urate oxidase, which has been believed to be a copper enzyme, and copper content in the liver of the mutant mouse were determined. The oxidase activity was maintained at normal level even though there was very low level of copper present in the liver through days 7 to 14. Copper administration increased the copper content in the liver to the normal level, but did not affect the oxidase activity.

Urate oxidase [urate: oxygen oxidoreductase, EC 1.7.3.3] is a peroxisomal enzyme that catalyzes the oxidation of uric acid to allantoin in most mammals , and has been believed to be a copper enzyme, since the purified enzyme from porcine liver was reported to contain copper (14). In humans and certain other primates , h owever, the enzyme has been lost by a sudden mutational event (15) . Although many studies on copper-containing enzymes have been carried out , no reports have been available on urate oxidase in the mutant mice.
This paper reports that the urate oxidase activity in the liver of macular mice is almost the same as that of the normal mice, in spite of copper deficiency in the liver of the mutant mice. MATERIALS AND METHODS

RESULTS
Body weight, liver weight and liver copper content Changes in body weight, liver weight and copper content in the liver of 7-to 14-day-old mice are shown in Table 1. No significant difference in body weight was observed through days 7 to 14 in macular males (Ml/y). Although the body weight of normal littermates (+/y) at 7 days of age was almost the same as that of Ml/y, the former gained more weight than the latter at 10 days of age, and no significant increase was observed after 10 days.
The copper contents per wet weight of the liver in M1/y was significantly low compared to those in +/y. The most significant difference in the copper content was observed at 7 days of age and +/y contained 6-fold higher than that of M1/y. The copper content in Ml/y did not change significantly through days 7 to 14; however, that in +/y showed significant decrease. These findings are consistent with the previous report (9). The analyses of variance revealed that significant differences in body weight and copper content were observed throughout the described age, and between the two genotypes and in their interaction.
Liver weights of both Ml/y and +/y at 7 days of age were almost the same. After 7 days, liver weights in +/y increased gradually, whereas those in Ml/y decreased gradually and were about half of those in +/y at 14 days of age. Likewise, significant difference between the two genotypes was observed by the analyses of variance.
Urate oxidase activity Changes in urate oxidase activities per protein and per liver weight shown in Table 2 revealed no significant difference between macular males and normal littermates. The total activities in Ml/y were significantly lower than those in +/y throughout days 7 to 14, but this may probably be due to a decrease in the liver weight of the mutant ( Table 1).

Effect of copper treatment
The effect of copper treatment on macular males and normal littermates is shown in Tables 3 and 4. Since significant difference in copper contents of the treated groups was observed by Bartlett test due to their large deviation, the logarithms of the values were used for the statistical analyses.
Copper treatment did not significantly affect the body weight and liver weight at 10 days of both Ml/y and +/y. However, the copper contents in the liver markedly increased in both Ml/y and +/y. The copper contents in the treated Ml/ y reached a level comparable to that in untreated +/y at 10 days of age (Table 3). Significant difference was observed in the interaction between copper treatment and genotypes, suggesting that the effect of the treatment was different in the two genotypes.
In spite of the increase in copper content, no significant change in urate oxidase activities in both Ml/y and +/y was observed by the copper treatment (Table 4).  Table 3. Effect of copper treatment on body weight, liver weight and liver copper content at 10 days old macular males (Ml/y) and normal littermates (+/y).  Experimental conditions were the same as in Table  3. No significant difference between the two genotypes was found by the analyses of variance.  (7,9), and by decrease in activities of copper-containing enzymes, including lysyl oxidase in skin and aorta (10,19,20), cytochrome c oxidase in brain and heart (11,12,21), superoxide dismutase in brain (11,21) and tyrosinase in melanocytes (13 Urate oxidase is present in the liver but not in the brain (23). Like the other enzymes in the liver, no significant difference in the activity was observed between macular males and normal littermates. The oxidase activity was not affected by the copper treatment either. Possible explanations for these results are as follows: 1) The concentration of copper is low compared to the normal, but this enzyme activity is strictly regulated and preferentially maintained in the liver. 2) The decreased copper is mainly due to the stored one but not to the functional one as proposed previously (11,12). 3) The copper is not a functional metal for the enzyme.
Since Mahler et al. reported that copper is the essential cofactor of urate oxidase from porcine liver (14), the mammalian enzyme has been believed to be a copper-requiring enzyme. However, the enzyme from ox kidney was reported not to contain any heavy metals (24), and the ones from microbials contain iron as the cofactor (25,26). Urate oxidase has been purified from camel liver recently , but the role of copper in the purified enzyme has not been clarified (27). On the basis of these reports and the data presented in this paper, the possibility that copper is not functioning in the enzyme catalysis cannot be ruled out. To confirm the possibility, we tried to purify urate oxidase from the livers of the present normal mice according to the method of Conley and Priest (28 sodium dodecyl sulfate. The specific activity and the copper content were obtained to be approximately 3.7 U/mg and 0.01mol/mol of the subunit, respectively. Characterization of the enzyme is now in progress in our laboratory. In spite of similarities in biochemical and histological features between human disease and model mice, there is a critical difference between the two. In human disease, many trials to improve the symptoms and save the life have been unsuc cessful (29,30), whereas the symptoms of the model mice can be significantly improved and the life span can be prolonged by a single administration of copper at around 7 days after birth (31,32). However, the different distribution of urate oxidase in mouse and human seems not to be correlated to the difference in response to the copper treatment, since the activity in the liver of the mutant mice remained the same as the normal one and was not affected by the copper treatment. Disorders.