JBC, Vol. 250, Issue 2, 585-592, Jan, 1975
Partial purification and characterization of beta-galactosidase from rat brain hydrolyzing glycosphingolipids
T. Miyatake and K. Suzuki
Adult rat brain beta-galactosidase was partially purified with the use of
lactosylceramide,
galactosyl-N-acetylgalactosaminyl-galactosyl-glucosylceramide,
galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)galactosyl-glucosylc
eramide, and 4-methylumbelliferyl theta-galactoside as substrates.
Approximately 50-fold purification was achieved by solubilization, ammonium
sulfate fractionation, Sephadex G-200 and DEAE-cellulose column
chromatography. Activities toward the above four substrates behaved
essentially identically throughout the pruification procedure. Considerable
differences were noted between the apparent properties determined with
whole homogenate and those of the purer enzyme preparations. Based on these
studies, assay procedures for the purified preparation for the three
glycosphingolipid substrates were standardized. Inhibition studies with the
use of varieties of simple sugars, oligosaccharide chains prepared from
glycosphingolipids, and intact sphingolipids suggested that the enzyme
which cleaves lactosylceramide may be different from the enzyme(s) which is
active toward the other two glycosphingolipids. The oligosaccharide chains
of the glycosphingolipids were much poorer inhibitors for the respective
glycosphingolipid beta-galactosidases than the original intact
glycosphingolipids or ceramide, and in some instances, even unrelated
sphingolipids. These findings indicated the importance of the lipophilic
groups and perhaps of the entire molecular configuration of
glycosphingolipids in determining the specificity of these
glycosphingolipid theta-galactosidases.
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