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Characterization of maize pollen flavonoid 3′-O-Methyltransferase activity and its in vivo Products

1991; Elsevier BV; Volume: 187; Issue: 3 Linguagem: Inglês

10.1016/s0015-3796(11)80104-6

0015-3796

Rowel B. Tobias, Russell L. Larson,

Phytoestrogen effects and research

An enzyme which catalyzes the methylation of quercetin at the 3′-position was isolated and partially characterized from maize pollen. The enzyme, SAM: quercetin-3'-0-methyltransferase (EC # 2.1.1.42), was purified 60-fold by a combination of salt fractionation and column chromatographic steps. The enzyme was eluted from freeze-dried pollen with NaCl, the supernatant precipitated with ammonium sulfate, subsequently desalted by Sephadex G-50 gel filtration, and partially purified by Sephadex DEAE anion exchange chromatography, ultrafiltration, and Sephadex G-200 gel filtration. The methyltransferase assay required S-adenosylmethionine as the methyl donor, dithioerythritol, and Mg+2 or Mn 12 in the reaction mixture. Optimum conditions for the reaction were pH 8.5 and 38 °C. The enzyme could be stabilized and activity maintained by the addition of 20 % glycerol prior to storage at −70°C. S-Adenosylhomocysteine, a reaction product, and mercuric chloride strongly inhibited the methylation reaction. The transferase utilized either quercetin, a flavonol, luteolin, a flavone, or eriodictyol, a flavanone, as substrates, whereas neither isoquercitrin (quercetin 3-glucoside) nor caffeic acid served as a substrate. The type of substrates methylated by the enzyme suggest that methylation occurs on the fifteen carbon skeleton prior to glucosylation which is known to occur near the end of the reaction sequence. The Km values for SAM and quercetin were 5.5 [tM and 9.6 [tM, respectively, and the Vmax was 37.3 x 10-2 pkat. The molecular weight for the transferase was estimated at 47,000. The product of the enzyme reaction, isorhamnetin, was identified in extracts of pollen stocks singly recessive for the genes C1, C2, R, A], A2, Bz] , Bz2. However, none of these genes could be shown to have any direct regulatory effect on the methyltransferase.