Abstract
Particle-free extracts of rat brain contain an inhibitor of the Nat i-K+-ATPase* activity of rat brain microsomal fraction, and this inhibitor has been identified as Lascorbate (Boxall & Phizackerley, 1972). Some of the features of the inhibition of the enzyme by ascorbate have now been investigated and, as shown in Table 1, ascorbate had no effect under anaerobic conditions. It was found that 45 units of catalase (EC 1.11.1.6) did not protect the enzyme from ascorbate, and 0 . 1 m ~ H ~ O ~ was inert. In contrast, although 1OpM-vitamin A alcohol and 0.1 mM-cr-tocopheryl acetate had no effect, the antioxidants listed in Table 1 completely protected the enzyme from inhibition by ascorbate under aerobic conditions. Table 1 also shows that EDTA abolished the inhibition, which implies that there is a requirement for metal ions, and desferrioxamine (CIBA Laboratories Ltd., Horsham, Sussex, U.K.) had a similar effect. Since desferrioxamine has a stability constant for Fe3+ of l O 3 I (Tripod, 1964) it is possible that iron is involved in this reaction. On the other hand, the metal ion requirement is not specific because the effect of ~ O ~ M E D T A was reversed by 20p~-Fe*+, Fe3+, Mn, Zn2+ or Pb2+ but not by Ca2+, Co2+ or Ni2+, although in the absence of EDTA these metal ions were inert. Taken together these results suggest that ascorbate inhibition is not due to H202 generation (cf. Mapson, 1967) and that it is unlikely to be a consequence of the fact that ascorbate is a reducing agent. It is more probably a result of lipid peroxidation (Hunter et al., 1964), and this conclusion was tested by measuring malonaldehyde formation under various conditions by the method of Ernster & Nordenbrand (1967). Table 1 shows that there is a good correlation between the extent of enzyme inhibition by ascorbate and malonaldehyde production. Inhibition by ascorbate is critically dependent on both the concentration of ascorbate and the concentration of microsomal fraction. Fig. 1 shows that inhibition increased as the concentration of ascorbate was increased to about l O p ~ , remained fairly constant until the concentration reached about 1 mM and then decreased. At 2 . 5 m ~ ascorbate, which approximates to the concentration found in brain (Rajalakshmi & Patel, 1968), the inhibition was very slight. It is also shown in Fig. 1 that malonaldehyde production showed a similar dependence on ascorbate concentration. Further, when the ascorbate concentration was held constant at 0.1 m M and the concentration of microsoma1 fraction was increased over a ten-fold range, inhibition of enzyme activity decreased and so did the amount of malonaldehyde produced per mg of protein. By using the assay method for Na++K+-ATPase described by Hansen et al. (1971) it was shown that ascorbate inhibition developed gradually over a period of about lOmin at 37°C. Enzyme activity was not restored by washing microsomal fraction that had been preincubated with ascorbate. The supernatant fluid obtained in this way gave a strong reaction for malonaldehyde, and very little was formed by the washed particles. This result shows that the inhibition is not due to malonaldehyde or to other solubleproducts of lipid peroxidation. Further, since malonaldehyde production was little affected by incubating microsomal fraction with ascorbate at 0°C or by incubating previously boiled microsomal fraction with ascorbate at 37C, it seems likely that ascorbateinduced lipid peroxidation is non-enzymic. Enzyme-catalysed NADPH-dependent
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