Myelin oxidase, oxidation

Treatment with galactose oxidase. Oxidation of myelin with galactose oxidase was performed as described previously for similar oxidation of rat spinal cord preparations (4). Typically, myelin containing 0.2-1.1 mg protein is incubated with 100-500 units of galactose oxidase in 1-3 ml of phosphate buffer (10-100 mM, pH 7.2-7.4) with or without catalase. After the incubation at room temperature to 30°C for the duration of 30 min to overnight, myelin is recovered by centrifugation, washed, and lyophilized. Total lipids were extracted from the dried residue and the oxidized cerebroside as well as unaltered cerebrosides were analyzed as described above. Alternatively, the incubation was stopped by the addition of 5 volumes of chloroform/methanol (2/1, v/v) and mixed. The lower layer after centrifugation of the mixture is washed and then evaporated to dryness, and the total lipids obtained were analyzed as described above. 

We obtained unexpected findings using this method to study myelin the oxidation by galactose oxidase of myelin-bound cerebrosides could not be detected. The oxidation did not occur either with the intact spinal cord preparation, with isolated myelin, or even with lyophilized myelin. In one experiment, lyophilized myelin containing 5 mg each of dry weight was incubated with 100, 200, and 500 units of galactose oxidase for 60 min at room temperature, and no cerebroside oxidation occurred. 

Oxidation of myelin surface cerebrosides by galactose oxidase. Fig. 4 shows silica HPLC of a mixture containing benzoylated-non-hydroxy and hydroxycerebroside and benzoylated derivatives of 2,4-dinitrophenylhydrazone of oxidation products from nonhydroxy- and hydroxycerebroside. Standard curves of two 6-dehydro-derivatives were shown in Fig. 5. These standard curves demonstrate that the response of the benzoylated dinitrophenylhydrazones are linear between 0.025 nmol and 0.6 nmol. Since cerebrosides containing 5 nmol can be determined without tailing to these peaks, this method should allow the determination of as little as 0.5% of the oxidation product. The fact that each curve intersects 0 point in both the abscissa and ordinate indicates that even smaller amounts of these compounds can be detected by this technique. 

To further examine the inability of galactose oxidase in oxidizing myelin-bound cerebrosides, one mg each of lyophilized... 

One possible explanation for the lack of oxidation by galactose oxidase was thought to be steric hindrance. To investigate this possibility, lyophilized myelin containing 5.45 mg protein in 5 ml 0.1 M phosphate buffer, pH 7.4, was mixed with 0.5 ml of the same buffer solution containing 1400 units of trypsin and incubated for 1 h at 37°C. 5 mg Of trypsin inhibitor in 1 ml of... 

Copper and Zinc in Aerobic Metabolism. Cytochrome oxidase, the terminal oxidase in the electron transport chain contains an atom of copper. On this enzyme the protons and electrons generated during oxidative metabolism combine with elemental oxygen to form water. During copper deficiency the tissue concentration of cytochrome oxidase is reduced. While the effects of lower cytochrome oxidase activity on exercise has not been described, it is likely that aerobic energy metabolism will be diminished. This effect of copper deficiency was first described in animals with myelin aplasls — the degeneration myelin (86). The oxidative process of phospholipid synthesis, a primary component of myelin, was depressed. Liver mitochondria had impaired respiratory activity (87). Cytochrome oxidase activity was also depressed in brain, heart and liver. 

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