Abstract
1. 1. A method for the isolation of sn-glycerol-3-phosphate oxidase from T. brucei is described. After pre-treatment with saponin, bloodstream trypanosomes can be easily disrupted by gentle homogenisation. The particulate glycerophosphate oxidase can be purified 10-fold by differential centrifugation, sedimentation occurring at 24,000 g. Further purification to 24-fold has been achieved by isopycnic sucrose gradient centrifugation. The oxidase forms a symmetrical peak with a modal density of 1.17 g/ml. sn-Glycerol-3-phosphate: (acceptor) oxidoreductase (E.G. 1.1.99.5) distributes identically with the glycerophosphate oxidase in subcellular fractions and in sucrose gradients and is therefore considered to be the dehydrogenase component of the holo-enzyme. 2. 2. FAD has been identified as a coenzyme for the glycerophosphate oxidase. Flavin, copper and iron are present in partially purified preparations in a ratio of 1:4:8. Iron liganded to acid-labile sulphide is not present. EPR studies revealed an iron signal at g = 4.1 together with a cuproprotein with a signal at g = 2.05. The latter is released from the oxidase by freezing and thawing and can not be considered a component of the oxidase. The copper remaining in the preparations after freezing and thawing can not be detected by EPR.
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