CHARACTERIZATION OF SPINACH LEAF ALPHA-L-ARABINOFURANOSIDASES AND BETA-GALACTOSIDASES AND THEIR SYNERGISTIC ACTION ON AN ENDOGENOUS ARABINOGALACTAN-PROTEIN

Two beta-galactosidases (beta-Galase-I and -II, EC 3.2.1.23) and two alpha-L-arabinofuranosidases (alpha-L-Arafase-I and -II, EC 3.2.1.55), were purified from mesophyll tissues of spinach (Spinacia oleracea L.), using chromatography on DEAE-cellulose, lactose-conjugated Sepharose CL-4B, and Sephadex G-100, or on hydroxylapatite and Sephadex G-150. The apparent molecular mass (M(r)) of beta-Galase-I and -II, respectively, were estimated to be 38 000 and 58 000 on SDS-PAGE and 64 000 and 60 000 on gel-permeation chromatography, indicating that the former was a dimeric protein. The isoelectric points of beta-Galase-I and -II were 6.9 and 5.2, respectively. Both enzymes hydrolyzed maximally p-nitrophenyl (PNP) beta-galactoside at pH 4.3, and were activated about 2-fold in the presence of BSA (100 mu g ml(-1)). The activity of both enzymes was inhibited strongly by heavy metal ions and p-chloromercuribenzoate (p-CMB). D-Galactono-(1-->4)-lactone and D-galactal served as potent competitive inhibitors for the enzymes. beta-Galase-I and -II could be distinguished from each other in their relative rates and kinetic properties in the hydrolysis of aryl beta-galactosides as well as of lactose and galacto-oligosaccharides. In particular, beta-Galase-I exhibited a preferential exo-wise cleavage of beta-1,6-galactotriose and beta-1,3-galactan. alpha-L-Arafase-I (M(r) 118 000) and -II (M(r) 68 000) were optimally active on PNP alpha-L-arabinofuranoside at pH 4.8 and gave K-m values of 1.2 and 2.2 mM, respectively. L-Arabino-(1-->4)-lactone, Ag+, and SDS acted as inhibitors for the isozymes. alpha-L-Arafase-I was characterized by its activity to hydrolyze PNP beta-D-xylopyranoside besides PNP alpha-L-arabinofuranoside, inhibition by D-xylose and D-glucono-(1-->5)-lactone, and less sensitivity to Hg2+, Cu2+, and p-CMB. Sugar beet arabinan was hydrolyzed rapidly by alpha-L-Arafase-II at one-half the rate for PNP alpha-L-arabinofuranoside, while the polysaccharide was less susceptible to alpha-L-Arafase-I. A spinach leaf arabinogalactan-protein was practically resistant to the action of beta-Galases, but its susceptibility to the enzymes increased remarkably after prior hydrolysis with alpha-L-Arafase-II.