Purification of cerebroside galactosidase from rat brain

Abstract

A method is described for the preparation of a [beta]-galactosidase which acts on the brain lipid, ceramide galactoside. The enzyme is obtained from particles that sediment between 1000-13000 x g from rat brains and is extracted with a 6% sodium cholate solution. The enzyme is solubilized and inactive proteins removed by digestion with pancreatic enzymes and the cholate is removed by dialysis and gel filtration. Preparative electrophoresis yields a major and a minor enzyme fraction. Chromatography of the major fraction on DEAE-Sephadex yields three fractions. The major fraction from this column can be purified further by precipitation of impurities at pH 5. An overall apparent increase in specific activity of over 300-fold could be obtained, with apparent yield of 27%.While the crude enzyme was rather stable to storage, exposure to ion exchange made it unstable. It was found that 50% glycerol at -20[deg] preserved the cerebrosidase fairly well. The purified enzyme still exhibited hydrolytic activity toward ceramide and nitrocatechol sulfate. Cerebrosidase activity was found in very young as well as in adult rats.