title: Characterization of two membrane-associated beta-glucosidases from maize (Zea mays L.) coleoptiles.
abstract: We isolated membrane vesicles from maize (Zea mays L.) coleoptiles and identified in these vesicles a 58 kDa (pm58) and a 60 kDa (pm60) protein by photoaffinity labelling with 5-azido-[7-3H]indole-3-acetic acid ([3H]N3IAA). Photoaffinity labelling was effectively competed for by auxins as well as by flavonoids. The labelled proteins were solubilized by Triton X-114 from the vesicles and partially purified. Microsequence analysis revealed that pm60 is a beta-glucosidase. This was confirmed by biochemical and immunological analysis. We show that pm60 has a beta-D-glucoside glucohydrolase (EC 3.2.1.21) activity. It uses p-nitro-phenyl beta-D-glucopyranoside (PNPG) as a substrate, with a pH optimum of 5.0. The Km for PNPG is 0.652 mM and the Vmax. 6.24 mumol.min-1.mg-1. The beta-glucosidase activity of pm60 was competitively inhibited by IAA and 1-naphthylacetic acid as well as by gluconolactam and glucose. N-terminal amino-acid-sequence analysis of pm58 revealed similarity to pm60, suggesting that both proteins are encoded by different members of a gene family.
authors.affiliation: Max-Planck Institut für Züchtungsforschung, Köln, Federal Republic of Germany.
authors.forename: J
authors.initials: J
authors.lastname: Feldwisch
authors.forename: A
authors.initials: A
authors.lastname: Vente
authors.forename: R
authors.initials: R
authors.lastname: Zettl
authors.forename: L
authors.initials: L
authors.lastname: Bako
authors.forename: N
authors.initials: N
authors.lastname: Campos
authors.forename: K
authors.initials: K
authors.lastname: Palme
mesh_terms: D000345:Affinity Labels; D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000201:enzymology; D005960:Glucosides / Q000378:metabolism; D007210:Indoleacetic Acids / Q000378:metabolism; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D003313:Zea mays / Q000201:enzymology*; D001617:beta-Glucosidase / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D000345:Affinity Labels; D005960:Glucosides; D007210:Indoleacetic Acids; C018616:2-nitrophenyl-beta-D-glucopyranoside; D001617:beta-Glucosidase