In vitro biosynthesis of homogalacturonan by a membrane-bound galacturonosyltransferase from epicotyls of azuki bean

A membrane preparation of 7-d-old seedlings from azuki bean (Vigna angulari s) contained galacturonosyltransferase (GaIAT) capable of transferring gala cturonic acid (GaIA) from UDP-GaIA into polygalacturonic acid (PGA) as an e xogenous acceptor. The enzyme was maximally active at pH 6.8-7.8 and 25-35 degreesC in the presence of 5 mm Mn2+ and 0.5% (w/v) Triton X-100. Acid-sol uble low-M-r (average M-r 10,000) PGA was a more efficient acceptor substra te than acid-insoluble polymer (M-r 70,000). The apparent Michaelis constan ts for UDP-GaIA and low-M-r PGA were 0.14 mm and 0.02 mg/ml, respectively. Various pectins with different degrees of methyl-esterification (DE) were p oor acceptors, and the enzyme activity tended to decrease with decreasing D E of the pectins. The transfer products from incubation of the enzyme with UDP-C-14-GaIA and the low-M-r PGA yielded C-14-GaIA(2). as the major produc t upon digestion with an endopolygalacturonase (EPGase), confirming the inc orporation of GaIA into PGA through contiguous alpha -1,4-linkages.