Partial purification and characterization of a luteolin-triglucuronide-specific β-glucuronidase from rye primary leaves ( Secale cereale)

Abstract

Primary leaves of rye contain a β-glucuronidase with high specificity for luteolin 7- O-[β- D-glucuronosyl (1 → 2)β- D-glucuronide]-4′- O-β- D-glucuronide, the major flavonoid of the leaf. The enzyme hydrolyses the glucuronic acid moiety in position 4′ (K m = 7 μM; V max = 1093 μkat/kg protein). A 330-fold purification was obtained by protein fractionation with ammonium sulphate, Sephadex G 150, hydroxylapatite and CM-Sepharose CL-6B. The enzyme has a pH optimum of 4.3 in 0.01 M citrate buffer. The molecular weight was determined to be 280 kD with active subunits of 67 kD. Isoelectric focusing indicates subunits of different isoelectric points at pH 5.5 and 6.3. The β-glucuronidase shows a temperature optimum at 55° and is inhibited by heavy metal ions such as Cu (0.85 mM) and Ag (0.25 mM), but is activated by ethyleneglycol monomethylether up to 15%. The enzyme is stable in 50% glycerol at −20° for at least 2 months. The results suggest that this β-glucuronidase is involved in the turnover of luteolin triglucuronide in vivo.