Isolation of uridine diphosphate-galacturonic acid from seedlings of Phaseolus aureus

Biochimica et Biophysica Acta
1961.0

Abstract

In the present communication, we report on the isolation of UDP-D-galacturonic acid from etiolated, 4-day-old mung-bean seedlings. The isolation involved using a Dowex-I formate resin column for elution with ammonium formate, pooling ultraviolet-absorbing fractions, adsorption on Norit A charcoal, elution with 50% ethanol-0.15 N NH4OH, followed by paper chromatography on oxalic acid-washed paper and paper electrophoresis. The identification of the isolated compound (compound I) as UDP-D-galacturonic acid was based on: its typical ultraviolet spectrum of uridine 5'-nucleotides at pH 7 and 12; a 1:1 mole ratio of uronic acid to uridine; treatment with potato nucleotide pyrophosphatase yielding a compound with electrophoretic mobility of UMP at pH 3.6 (indicating an uridine 5'-pyrophosphate structure); and identity with 14C-labeled UDP-D-galacturonic acid (prepared by treating radioactive UDP-D-glucuronic acid with mung-bean particles) via co-electrophoresis in 0.2 M ammonium formate (pH 3.6) and 0.2 M ammonium acetate (pH 5.8), as well as co-chromatography in ethanol-ammonium acetate systems. The yield of this nucleotide was approximately 0.5 μmole. Although the configuration (D or L) of the uronic acid moiety was not determined, it is likely of the D form, consistent with enzymically prepared UDP-D-galacturonic acid and natural polymers like pectin.

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