Characterization of cyanogenic glucosides and β-glucosidases in Triglochin maritima seedlings

Nahrsted, Adolf; Hösel, Wolfgang; Walther, Axel

doi:10.1016/0031-9422(79)80121-1

Cited by 42 publications

(16 citation statements)

References 13 publications

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“…Purely competitive inhibition with galactose was verified for the 50,000-mol wt form, but was assumed for the 25,000-mol wt form because of insufficient enzyme. Purely competitive inhibitor constants (Ki values) for the two forms were 0.65 and 0.45 mm for the 25,000-and 50,000-dalton forms, respectively, as estimated using plots of-versus [I] yet catalyze the hydrolysis of f-glucosides found in the plant (7,9,12). If an a-galactosidase of this type were present in cucumber leaves, it may not have been identified in the present study.…”

Section: Kvid B 3 I I A~~7mentioning

confidence: 68%

Characterization of α-Galactosidase from Cucumber Leaves

Smart

Pharr²

1980

Plant Physiol.

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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.

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Section: Kvid B 3 I I A~~7mentioning

confidence: 68%

Characterization of α-Galactosidase from Cucumber Leaves

Smart

Pharr²

1980

Plant Physiol.

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“…In cassava [2] and Trigllochin [23] much higher purification levels are recorded showing that the tissues used contain a lower level of cyanogenic fl-glucosidase compared to white clover. Wilkinson and Millar [29] have shown that white clover linamarase is bound to CM cellulose at pH 5.0, however their studies on the enzyme are based on a 24h hydrolysis period.…”

Section: Discussionmentioning

confidence: 99%

Biochemical characterisation of the Li locus, which controls the activity of the cyanogenic ?-glucosidase in Trifolium repens L.

Hughes

Dunn

1982

Plant Mol Biol

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The cyanogenic β-glucosidase (linamarase) was purified from white clover leaf tissue. The enzyme is a homodimer with a molecular weight of 105 300-103 400 daltons estimated from molecular exclusion chromatography. The effect of buffer ions on the pH optimum and charge properties of the enzyme are presented. A combination of molecular exclusion chromatography and CM cellulose ion exchange chromatography purified linamarase 16 fold to a single 62 000 dalton polypeptide on SDS polyacrylamide gel electrophoresis. This polypeptide represented about 5% of the total soluble leaf protein and can be seen as a prominent band in SDS polyacrylamide gel electrophoresis of crude leaf extracts from Li Li plants. Screening backcross progeny showed that extracts from li li plants, which have no linamarase activity, lack this 62 000 dalton polypeptide. Linamarase is the major glycoprotein in white clover leaf extracts which binds to Concanavalin A-Sepharose.

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“…The purified enzyme I was able to bind strongly to Sepharose, Sephadex, and Ultrogel (LKB) and could be resolubilized with 0.5 it4 NaCl. The enzyme extract of Triglochin maritima (392) hypocotyls was resolved by a DEAE column into three fractions. Fraction I was eluted unadsorbed; I1 and I11 were eluted with a concentration gradient of Tris-HC1 buffer, pH 7.0.…”

Section: Prakash M Dey and Elena Del Campillomentioning

confidence: 99%

Biochemistry of the Multiple Forms of Glycosidases in Plants

1984

Advances in Enzymology - And Related Areas of Molecular Biology

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Characterization of cyanogenic glucosides and β-glucosidases in Triglochin maritima seedlings

Cited by 42 publications

References 13 publications

Characterization of α-Galactosidase from Cucumber Leaves

Characterization of α-Galactosidase from Cucumber Leaves

Biochemical characterisation of the Li locus, which controls the activity of the cyanogenic ?-glucosidase in Trifolium repens L.

Biochemistry of the Multiple Forms of Glycosidases in Plants

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