Purification and properties of sphingolipid β-galactosidases from human placenta

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

Research output: Contribution to journalArticle

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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 - 1979
Externally publishedYes

Fingerprint

Galactosidases
Sphingolipids
Placenta
Purification
Enzyme activity
Enzymes
Affinity chromatography
Gels
Molecular weight
Gel Chromatography
Galactosylceramidase
Thiogalactosides
Molecular Weight
Galactosides
Affinity Chromatography
Mercaptoethanol
Guanidine
Ammonium Sulfate
Lactones
Electrophoresis

ASJC Scopus subject areas

  • Biochemistry

Cite this

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

Purification and properties of sphingolipid β-galactosidases from human placenta. / Lo, J.; Mukerji, K.; Awasthi, Y. C.; Hanada, E.; Suzuki, K.; Srivastava, Satish.

In: Journal of Biological Chemistry, Vol. 254, No. 14, 1979, p. 6710-6715.

Research output: Contribution to journalArticle

Lo, J, Mukerji, K, Awasthi, YC, Hanada, E, Suzuki, K & Srivastava, S 1979, 'Purification and properties of sphingolipid β-galactosidases from human placenta', Journal of Biological Chemistry, vol. 254, no. 14, pp. 6710-6715.
Lo J, Mukerji K, Awasthi YC, Hanada E, Suzuki K, Srivastava S. Purification and properties of sphingolipid β-galactosidases from human placenta. Journal of Biological Chemistry. 1979;254(14):6710-6715.
Lo, J. ; Mukerji, K. ; Awasthi, Y. C. ; Hanada, E. ; Suzuki, K. ; Srivastava, Satish. / Purification and properties of sphingolipid β-galactosidases from human placenta. In: Journal of Biological Chemistry. 1979 ; Vol. 254, No. 14. pp. 6710-6715.
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