Galactocerebrosidase (GALC, EC 3.2.1.46) was purified from human urine by a 
series of hydrophobic affinity column chromatography steps. The activity was 
enriched 176,000-fold from concentrated urine by only four columns, including 
octyl Sepharose, hydroxylapatite, butyl Sepharose and ethyl-agarose. The overall 
recovery was about 20% but only low amounts were obtained due to its low 
abundance. The estimated final specific activities of several batches were 
between 1 and 2 mmol/h per mg protein. The final purified fractions were 
essentially free of other lysosomal enzyme activities. The most pure fractions 
showed a series of bands between 50 and 53 kDa on sodium 
dodecylsulfate-polyacrylamide gel electrophoresis which were determined to have 
identical N-terminal amino acid sequence. In addition, gel filtration of 
partially purified GALC after disassociation showed one peak of activity 
estimated to have a molecular mass near 50 kDa. GALC was also purified from 
human brain and human placenta using the same methods demonstrating the 
usefulness of this procedure in obtaining GALC from solid human tissues. In 
addition to the bands migrating near 50 kDa from urine, there were also bands at 
80 kDa and 30 kDa in some preparations. By N-terminal sequencing and the use of 
antipeptide antibodies, the 80 kDa band was demonstrated to have the same 
N-terminal amino acids as the 50-53 kDa bands. The 30 kDa band had a unique 
sequence. The relationship between the different molecular weight species 
remains to be determined. The purification of GALC and the securing of amino 
acid sequence information will aid in the cloning of the GALC gene. This enzyme 
is deficient in human patients with Krabbe disease and several animal species.