Factors influencing the activity of xanthine oxidase in aged homogenates of rat liver
McLennan, Barry Dean
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A study has been made of the factors influencing increases in the xanthine oxidase activity of ageing homogenates of rat liver. The previous finding that liver xanthine oxidase activity depends upon the dietary protein content was confirmed. It was found that the xanthine oxidase activity of homogenates of livers of rats fed normal or low protein diets increased with the age of the homogenate. A study was made of the kinetics of the enzyme assay with both fresh and aged, normal or low protein homogenates. There was no significant change in the kinetics of enzyme action during the ageing process. The increase in enzyme activity was dependent upon the ageing temperature. The enzyme activity was found to increase with ageing at 37°C, 4°C and -20°C. Dialysis of an ageing homogenate at 4°C rapidly decreased the enzyme activity. When the concentrated dialysate was added to a normal ageing homogenate, it initially had no effect, then transiently interfered with and later stimulated enzyme activity. Methylene blue is reduced by the homogenate and stimulates the xanthine oxidase activity of both fresh and aged homogenates. However, the stimulus decreased with the age of the homogenate. It is suggested that methylene blue acted as an additional hydrogen acceptor and permitted unrestricted enzyme activity. The increase in enzyme activity in the ageing homogenate seems to depend on the availability of hydrogen acceptors, most likely flavin adenine dinucleotide. It has been shown that uricase is not rate limiting in the assay of xanthine oxidase in ageing rat liver homogenates. The isolation of uric acid from the dialysate of an ageing normal homogenate suggests that at 4°C uricase activity is rate limiting in the xanthine oxidase system. The dialysate from a normal ageing homogenate was analyzed to determine which of its components stimulated the enzyme activity of normal and low protein ageing homogenates. A chemical analysis indicated the absence of glycogen, protein, reducing sugars, calcium and magnesium. Chloride, phosphorus, ferrous iron, sulphur and non-protein nitrogen were detected. The dialysate contained a considerable amount of 260 mμ-absorbing material. Column and paper chromatographic analysis suggested that the dialysate contained uric acid, AMP-5', adenine, guanine, cytosine, uracil, and thymine. The presence of these substances was confirmed by a spectral analysis of the isolated components. It was found that uric acid enhanced the enzyme activity of xanthine oxidase in an ageing rat liver homogenate. AMP-5' caused a delayed stimulation of enzyme activity. However, the stimulatory effects of these substances was not as great as the initial stimulation of xanthine oxidase activity by methylene blue.