CHARACTERIZATION OF 2 MEMBRANE-ASSOCIATED BETA-GLUCOSIDASES FROM MAIZE (ZEA-MAYS L) COLEOPTILES

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-H-3]indole-3-acetic acid ([H-3]N(3)IAA). 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 P-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-nitrophenyl beta-D-glucopyranoside (PNPG) as a substrate, with a pH optimum of 5.0. The K-m for PNPG is 0.652 mM and the V-max 6.24 mu mol 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.