Discussion

y-BBH was purified to homogeneity by a four-step column chromatographic method. First, the influence of various compounds on y-BBH activity at different storage temperatures was determined. Storing the homogenate at 4°C in the presence of 200g/1 glycerol and l0mM DTT was found optimal for storage during the purification. For long term storage, -20 °C with the same additives is recommended An important observation was that addition of EDTA results in total loss of y-BBH activity, which is probably caused by the chelation of y-BBH bound Fe2+. Addition of Fe2+in the presence or absence of ascorbate had no effect on the stability of the enzyme.

y-BBH was subsequently purified 280-fold from rat liver cytosol and identified as a 44 kDa protein by SDS-PAGE analysis, which agrees with the previously purified rat liver y-BBH.13'14 The purified protein will be used to raise an antiserum and the N-terminal amino acid sequence will be determined with the ultimate goal to identify the cDNA encoding rat y-BBH.

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