Purification and characterization of a xyloglucan oligosaccharide-specific xylosidase from pea seedlings

Journal Article · Tue Dec 05 04:00:00 EST 1989 · Journal of Biological Chemistry; (United States)

OSTI ID:5967042

O'Neill, R A; Albersheim, P; Darvill, A G ^[1]

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1. Univ. of Georgia, Athens (United States)

An {alpha}-xylosidase that acts on oligosaccharide fragments of xyloglucan, a plant cell wall polysaccharide, was purified from pea (Pisum sativum) epicotyls that had been treated with an auxin analog. The enzyme had an apparent molecular mass of 85,000 Da according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 79,000 Da according to gel-permeation chromatography under nondenaturing conditions. The purified xylosidase consisted of a series of closely related, enzymatically active proteins with isoelectric points ranging from about pH 7.35 to 7.7; the xylosidases were separated by chromatofocusing. The pH optimum of the mixed xylosidase was 4.9-5.1. The substrate specificity of the xylosidase mixture was determined by purification and structural characterization of the products of treating xyloglucan-oligosaccharide substrates with the enzyme. Characterization of the substrates and products included elution volume from a gel-permeation column, glycosyl residue and glycosyl linkage composition analyses, fast atom bombardment-mass spectrometry, and {sup 1}H NMR spectroscopy. The enzyme specifically cleaved only one of the {alpha}-xylosidic linkages of xyloglucan-oligosaccharide substrates, the one attached to a 6-linked glucosyl residue, not those attached to the 4,6-linked glucosyl residues. The enzyme was unable to cleave the xylosidic linkage of p-nitrophenyl-{alpha}-D-xylopyranoside or the {alpha}-xylosidic linkage to C-6 of glucose in the disaccharide isoprimeverose. The enzyme was also unable to release measurable amounts of xylose from large xyloglucan polymers.

• DOE Contract Number: FG09-87ER13810; FG09-85ER13424
• OSTI ID: 5967042
• Journal Information: Journal of Biological Chemistry; (United States), Journal Name: Journal of Biological Chemistry; (United States) Vol. 264:34; ISSN JBCHA; ISSN 0021-9258
• Country of Publication: United States
• Language: English

Related Subjects

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
400201 -- Chemical & Physicochemical Properties
550200* -- Biochemistry
59 BASIC BIOLOGICAL SCIENCES
AUXINS
CARBOHYDRATES
CHEMICAL REACTION KINETICS
CHEMICAL REACTIONS
CHROMATOGRAPHY
DATA
ELECTROPHORESIS
ENZYMES
EXPERIMENTAL DATA
FOOD
GEL PERMEATION CHROMATOGRAPHY
INFORMATION
KINETICS
LEGUMINOSAE
MAGNETIC RESONANCE
MAGNOLIOPHYTA
MAGNOLIOPSIDA
MASS SPECTROSCOPY
NUCLEAR MAGNETIC RESONANCE
NUMERICAL DATA
ORGANIC COMPOUNDS
PEAS
PH VALUE
PISUM
PLANT GROWTH REGULATORS
PLANTS
POLYSACCHARIDES
PROTEINS
PURIFICATION
REACTION KINETICS
RESONANCE
SACCHARIDES
SEPARATION PROCESSES
SPECIFICITY
SPECTROSCOPY
STRUCTURAL CHEMICAL ANALYSIS
VEGETABLES