Characterization of spinach leaf α‐l‐arabinofuranosidases and β‐galactosidases and their synergistic action on an endogenous arabinogalactan‐protein

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Abstract
Two β‐galaclosidases (β‐Galase‐I and ‐II, EC 3.2.1.23) and two α‐l‐arabinofuranosidases (α‐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 (Mr) of β‐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 β‐Galase‐I and ‐II were 6.9 and 5.2, respectively. Both enzymes hydrolyzed maximally p‐nitrophenyl (PNP) β‐galactoside at pH 4.3, and were activated about 2‐fold in the presence of BSA (100 μg ml−1). The activity of both enzymes was inhibited strongly by heavy metal ions and p‐chloromercuriberszoate (p‐CMB). d‐Galactono‐(1→4)‐lactone and d‐galactal served as potent competitive inhibitors for the enzymes. β‐Galase‐I and ‐II could be distinguished from each other in their relative rates and kinetic properties in the hydrolysis of aryl β‐galactosides as well as of lactose and galacto‐oligosaccharides. In particular. β‐Galase‐I exhibited a preferential exowise cleavage of β‐1,6‐galactotriose and β‐1.3‐galactan. α‐l‐Arafase‐l (Mr 118000) and ‐II (M, 68 000) were optimally active on PNP α‐l‐arabinofuranoside at pH 4.8 and gave Km values of 1.2 and 2.2 mM. respectively. l‐Arabino‐(1 → 4)‐lactone. Ag+, and SDS acted as inhibitors for the isozymes. α‐lArafase‐I was characterized by its activity to hydrolyze PNP β‐d‐xylopyranoside besides PNP α‐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 α‐lArafase‐II at one‐half the rate for PNP α‐larabinofuranoside, while the polysaccharide was less susceptible to α‐lArafase‐I. A spinach leaf arabinogalactan‐protein was practically resistant to the action of β‐Galases, but its susceptibility to the enzymes increased remarkably after prior hydrolysis with α‐lArafase‐Il.