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Metabolism and Enzymology

-L-Arabinofuranosidase from Radish (Raphanus sativus L.) Seeds

Department of Biochemistry, Faculty of Science, Saitama University, 255 Shimo-okubo, Urawa 338, Japan

An -L-arabinofuranosidase has been purified 1043-fold from radish (Raphanus sativus L.) seeds. The purified enzyme was a homogeneous glycoprotein consisting of a single polypeptide with an apparent molecular weight of 64,000 and an isoelectric point value of 4.7, as evidenced by denaturing gel electrophoresis and reversed-phase or size-exclusion high-performance liquid chromatography and isoelectric focusing. The enzyme characteristically catalyzes the hydrolysis of p-nitrophenyl -L-arabinofuranoside and p-nitrophenyl -D-xylopyranoside in a constant ratio (3:1) of the initial velocities at pH 4.5, whereas the corresponding -L-arabinopyranoside and -D-xylofuranoside are unsusceptible. The following evidence was provided to support that a single enzyme with one catalytic site was responsible for the specificity: (a) high purity of the enzyme preparation, (b) an invariable ratio of the activities toward the two substrates throughout the purification steps, (c) a parallelism of the activities in activation with bovine serum albumin and in heat inactivation of the enzyme as well as in the inhibition with heavy metal ions and sugars such as Hg²⁺, Ag⁺, L-arabino-(14)-lactone, and D-xylose, and (d) results of the mixed substrate kinetic analysis using the two substrates. The enzyme was shown to split off -L-arabinofuranosyl residues in sugar beet arabinan, soybean arabinan-4-galactan, and radish seed and leaf arabinogalactan proteins. Arabinose and xylose were released by the action of the enzyme on oat-spelt xylan. Synergistic action of -L-arabinofuranosidase and -D-galactosidase on radish seed arabinogalactan protein resulted in the extensive degradation of the carbohydrate moiety.

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