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

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.

Molecular Cloning of a β-Galactosidase from Radish That Specifically Hydrolyzes β-(1→3)- and β-(1→6)-Galactosyl Residues of Arabinogalactan Protein

“…These enzymes can be classified into the second class of b-galactosidases, the b-galactosidase/exo-b-(1/3)(1/6)-galactanases. However, we cannot rule out the possibility that this second group of b-galactosidases participates in the degradation of both pectic b-(1/4)-galactan and the carbohydrate moieties of AGPs, because a b-galactosidase specimen (b-Gal II) purified from spinach leaves shows a broad substrate specificity, acting not only on b-(1/3)-and b-(1/6)-galactooligosaccharides but also weakly on b-(1/4)-galactooligosaccharides (Hirano et al, 1994). Further studies are required to clarify the functional differences of the second class of b-galactosidases from b-galactosidase/exo-b-(1/4)-galactanases.…”

“…The reason for this is that the structure of pectic b-(1/4)-galactan is regulated spatially during the development of plant tissues, and pectin thus plays an important role in the architecture of the cell wall and intercellular attachment (McCartney et al, 2000;Sørensen et al, 2000). On the other hand, we have previously shown that b-galactosidase specimens isolated from radish seeds and spinach leaves (spinach b-Gal I) hydrolyze specifically b-(1/3)-and b-(1/6)-galactooligosaccharides besides PNP-b-Gal, and are thereby able to degrade the b-(1/3)(1/6)-galactan backbones of AGPs, but not pectic b-(1/4)-galactan (Sekimata et al, 1989;Hirano et al, 1994). These enzymes can be classified into the second class of b-galactosidases, the b-galactosidase/exo-b-(1/3)(1/6)-galactanases.…”

“…We have previously found b-galactosidases in radish (Raphanus sativus) seeds and spinach (Spinacia oleracea) leaves, which were capable of hydrolyzing b-(1/3)-and b-(1/6)-galactosyl sequences of AGPs but not pectic b-(1/4)-galactan (Sekimata et al, 1989;Hirano et al, 1994). These enzymes belong to the second class of b-galactosidase.…”

Isolation, Characterization, and Cloning of α-l-Arabinofuranosidase Expressed during Fruit Ripening of Japanese Pear

“…This indicates that the enzyme possesses bifunctional activity as a-L-arafase/b-xylosidase as an enzymatic property. Bifunctional activity has been reported in plant a-L-arafases purified from radish (Raphanus sativus) seeds (Hata et al, 1992) and spinach (Spinacia oleracea) leaf (Hirano et al, 1994) besides barley (Lee , 2003). It is not clear whether these a-L-arafases belong to GH family 3 or not; however, it may be reasonable that several b-xylosidases grouped family 3 were called a-L-arafase rather than b-xylosidase.…”

Comprehensive analysis of beta-galactosidase protein in plants based on Arabidopsis thaliana

“…βgal is widely distributed and assessed in many creatures, such as insects (Seddigh and Bandani, 2012), and different plant tissues, like leaves (Hirano et al, 1994), seedlings (Li et al, 2001), hypocotyls (Kotake et al, 2005), and meristem zones of roots, cotyledons, vascular tissues, trichomes, and pollens (Hruba et al, 2005;Wu and Liu, 2006). The enzyme has been implicated in a number of biological processes, including plant growth and fruit ripening.…”