Characterization of α-Galactosidase from Cucumber Leaves

Abstract

Two forms of a-galactosidase (a-D-galactoside galactohydrolase, E.C. 3.2.1.22) which differed in molecular weight were resolved from Cucumis sativus L. leaves. The enzymes were partially purified using ammonium sulfate fractionation, Sephadex gel filtration, and diethylaminoethyl-Sephadex chromatography. The molecular weights of the two forms, by gel filtration, were 50,000 and 25,000. The 50,000-dalton form comprised approximately 84% of the total a-galactosidase activity in crude extracts from mature leaves and was purified 132-fold. The partially purified 25,000-molecular weight form rapidly lost activity unless stabilized with 0.2% albumin and accounted for 16% of the total a-galactosidase activity in the crude extract. The smaller molecular weight form was not found in older leaves.The two forms were similar in several ways including their pH optima which were 5.2 and 5.5 for the 50,000-and 25,000-dalton form, respectively, and activation energies, which were 15.4 and 18.9 kilocalories per mole for the larger and smaller forms. Both enzymes were inhibited by galactose as well as by excess concentrations of p-nitrophenyl-a-D-galactoside substrate. Km values with this substrate and with raffinose and melibiose were different for each substrate, but similar for both forms of the enzyme. With stachyose, Km values were 10 and 30 millimolar for the 50,000-and 25,000-molecular weight forms, respectively.The present study involved the resolution, partial purification, and characterization of two forms of a-galactosidase in cucumber leaves as a step in understanding the role of a-galactosidase in the breakdown of the transport sugars, stachyose and raffmose (17). Several properties of the two forms were compared, including their mol wt, substrate and inhibitor specificities, activation energies, and their occurrence as a function of leaf age.a-Galactosidase (a-D-galactoside galactohydrolase, E.C. 3.2.1.22) has also been studied in squash (15, 16), another cucurbit that transports stachyose. Thomas and Webb (15,16) Enzyme Extraction. Crude extracts were prepared by homogenizing 25 g of cucumber leaves with 80 ml of 0.1 M Na-acetate buffer (pH 5.2) for 3 min in a VirTis homogenizer. The homogenate was centrifuged at 27,000g for 15 min, and the supernatant was retained and filtered through cheesecloth.Assays for a-Galactosidase. a-Galactosidase activity was monitored routinely using I mm p-nitrophenyl-a-D-galactoside as substrate at pH 4.6 in 0.1 M Na-acetate buffer and at 30 C, as described by Pharr et al.

Cloning and functional expression of alkaline α‐galactosidase from melon fruit: similarity to plant SIP proteins uncovers a novel family of plant glycosyl hydrolases

“…Total RNA was used as a template for ®rst-strand ß Blackwell Publishing Ltd, The Plant Journal, (2003), 33, 97±106 cDNA synthesis with the aid of Superscript II reverse transcriptasesubstrates stachyose, raf®nose and melibiose (Sigma) as previously described by . The assay for hydrolysis of natural saccharide substrates was based on the measurement of released galactose by using galactose de-hydrogenase (Sigma) in an enzyme-linked assay, according to Smart and Pharr (1980). K m values were determined in the non-puri®ed E. coli extract since activity was lost during the Ni-puri®cation step.…”

A Potential Pathway for Galactose Metabolism in Cucumis sativus L., A Stachyose Transporting Species

“…The initial step in stachyose and raffmose catabolism is thought to be hydrolysis by a-galactosidase (9,18,25,28). However, the pathway by which the released gal moieties are metabolized in a stachyose utilizing tissue has only been suggested and not documented experimentally (1,5,16).…”

“…After centrifugation, the supernatant was desalted using a Sephadex G-25 column and assayed. The complete reaction mixture (100 y1) contained 25 1 h. An aliquot of this mixture was then spotted on a DEAEcellulose strip which was developed as described above. The neutral product formed during phosphatase treatment was eluted from the strip with dH20 and identified using paper chromatography.…”

“…One possibility is that it results from the enzymic hydrolysis of Gal moieties from stachyose and raffmose in vivo. It has been shown that cucumber a-galactosidases are capable of hydrolyzing the galactosidic linkages of stachyose, raffmose, and melibiose (9,25). Also, cucumber callus cells contained a pool of Gal in their free space only when cultured on media containing raffmose saccharides as a carbon source (9).…”

“…Stachyose is also a major reserve carbohydrate in many seeds (1, 4, 24). The enzymes involved in raffmose saccharide biosynthesis are known, but the pathways of degradation of these sugars have not been studied extensively (5).The initial step in stachyose and raffmose catabolism is thought to be hydrolysis by a-galactosidase (9,18,25,28). However, the pathway by which the released gal moieties are metabolized in a stachyose utilizing tissue has only been suggested and not documented experimentally (1,5,16).…”