--B. A. Bailey and R. L. Larson
Recent attempts at purifying UDPG:2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) glucosyltransferase from maize extracts identified two peaks of activity by Q-Sepharose gel chromatography. Enzyme eluting in the initial peak of activity also had a low level of activity on the flavonoid quercetin with the first thought being that UDPG:Flavonol 3-O-glucosyltransferase (UFGT) was eluting concurrently with peak 1 of the DIMBOA glucosyltransferase. It was also considered possible that UFGT and the initial DIMBOA-glucosylating peak were the same enzyme.
To try to distinguish between UFGT and the DIMBOA glucosyltransferase, enzyme was extracted from aleurone (23 dap) of genetic stocks dominant and recessive for Bz1, the structural gene producing UFGT. Fifty aleurones were ground in a mortar with the addition of 30ml of Hepes buffer (50mM, 5mM DTE, pH 7.5). The debris was removed by filtering through Pellon followed by centrifugation (26,000g, 10min). A 30 to 60% ammonium sulfate precipitate was applied to a Sephadex G-200 superfine column and the protein eluted with 20mM Hepes, pH 7.5. Active fractions were pooled and applied to a Q-Sepharose ion exchange column and eluted with a 0.1 to 0.3M KCl gradient in Hepes buffer (20mM, pH 7.5) with fractions being assayed as described below.
The assay mixture included approximately 2µg of protein, 1mM UDPG and Hepes buffer (50mM, 5mM DTE, pH 8.2) in a volume of 200µl. Samples were incubated 10min at 37 C and included 1mM DIMBOA or 83M quercetin. The reactions were terminated by the addition of 0.8ml of a 2:1 chloroform:methanol solution (1% HCl). DIMBOA glucoside and isoquercetin were identified by HPLC methods and quantitated using standard curves based on peak height.
Assay of the fractions in the Q-Sepharose elution profile for the fully dominant Bz1 aleurone preparation identified 3 peaks of glucosyltransferase activity (Fig. 1). The initial peak of activity on DIMBOA reaches a maximum one fraction prior to the maximum for glucosylation of quercetin. The elution profiles for peak 1 of DIMBOA glucosyltransferase and UFGT overlap partially indicating UFGT is carried through as a contaminant when attempting to purify DIMBOA glucosyltransferase. Both DIMBOA glucosyltransferase peaks are observed in the recessive bz1 aleurone (Fig. 2) although activity on quercetin is completely absent further distinguishing DIMBOA glucosyltransferase from UFGT. These results clearly demonstrate that the activity observed on quercetin when attempting to purify DIMBOA glucosyltransferase is due to contamination by UFGT, and DIMBOA glucosyltransferase peak 1 does not glucosylate quercetin.
Fig. 1. Q-Sepharose elution profile for protein isolated from dominant Bz1 aleurone. DIMBOA glucosyltransferase activity (o___o); UFGT activity (•___•).
2. Q-Sepharose elution profile for protein isolated from recessive
bz1 aleurone. DIMBOA glucosyltransferase activity (o___o); UFGT
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