Purification and properties of sphingolipid β-galactosidases from human placenta

J. Lo, K. Mukerji, Y. C. Awasthi, E. Hanada, K. Suzuki, S. K. Srivastava

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Abstract

β-Galactosidases have been purified to homogeneity from human placentae. The purification steps involved ammonium sulfate precipitation, concanavalin A-Sepharose affinity chromatography, Sepharose 4B-ε-aminocaproyl p-aminophenyl β-D-N-thiogalactopyranoside affinity chromatography, and Sephadex G-200 gel filtration. The enzyme was purified about 54,000-fold with a yield of about 37%. Throughout the purification, no detergent was used and the enzyme was stabilized with 30 mM sodium chloride. In the last step, i.e. Sephadex G-200 gel filtration, the β-galactosidases separated into two enzyme activity peaks designated Peaks I and II. Both Peaks I and II were found to be devoid of galactosylceramide β-galactosidase activity and had no or negligible lactosylceramide β-galactosidase I activity. However, both peaks exhibited enzyme activity toward lactosylceramide β-galactosidase II and G(A1). β-D-Galactosylamine, 0.5 mM, inhibited the enzyme activity of Peaks I and II towards 4-methylumbelliferyl β-D-galactoside, whereas substrate analogs, galactose and galactone-γ-lactone inhibited the enzyme by about 50%. The approximate molecular weight of Peaks I and II determined by gel filtration was found to be 420,000 to 480,000 and 220,000, respectively. During guanidine hydrochloride-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in the presence or absence of β-mercaptoethanol, Peak II dissociated into a single protein band corresponding to a molecular weight of 77,000 and Peak I into three bands corresponding to molecular weights of 77,000, 31,000, and 22,000. The presence of a common subunit between Peaks I and II enzymes and unique subunits in Peak I enzyme was subsequently confirmed by immunological techniques.

Original languageEnglish (US)
Pages (from-to)6710-6715
Number of pages6
JournalJournal of Biological Chemistry
Volume254
Issue number14
StatePublished - Dec 1 1979

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Lo, J., Mukerji, K., Awasthi, Y. C., Hanada, E., Suzuki, K., & Srivastava, S. K. (1979). Purification and properties of sphingolipid β-galactosidases from human placenta. Journal of Biological Chemistry, 254(14), 6710-6715.