Purification and properties of neutral β-galactosidases from human liver

Yoav Ben-Yoseph*, Emmanuel Shapira, David Edelman, Barbara K. Burton, Henry L. Nadler

*Corresponding author for this work

Research output: Contribution to journalArticle

23 Scopus citations

Abstract

Two neutral β-galactosidase isozymes were purified from human liver. The initial step of purification was removal of the acidic β-galactosidases by adsorption on concanavalin A-Sepharose 4B conjugate. Subsequent purification steps included ammonium sulfate precipitation, diethylaminoethyl cellulose column chromatography, Sephadex G-100 gel filtration, and preparative polyacrylamide-gel isoelectric focusing. The final step of purification was affinity chromatography of the separated isoelectric forms on ε{lunate}-aminocaproyl-β-d-galactosylamine-Sepharose 4B conjugate. The purified β-galactosidase isozymes had activity toward both β-d-galactoside and β-d-glucoside derivatives of 4-methylumbelliferone and p-nitrophenol with a pH optimum around 6.2. These enzyme forms were also found to possess lactosylceramidase II activity with a pH optimum in the range of 5.4 to 5.6, but not lactosylceramidase I activity and no activity toward galactosylceramide or GM1-ganglioside. The molecular weight was found to be in the range of 37,500-39,500 for the two neutral isozymes and they had similar Km and V values; the more acidic form (designated β-galactosidase N1) was more heat stable than the other form (designated β-galactosidase N2). Antibodies evoked against the N1 and N2 β-galactosidases gave identical precipitin lines retaining enzymatic activity. No cross-reactivity was observed between the neutral and the acidic isozymes when examined with the respective antisera.

Original languageEnglish (US)
Pages (from-to)373-380
Number of pages8
JournalArchives of biochemistry and biophysics
Volume184
Issue number1
DOIs
StatePublished - Nov 1977

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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