Enzyme-gold cytochemistry of seed xyloglucans using two xyloglucan-specific hydrolases. Importance of prior heat-deactivation of the enzymes.
Two pure, homogeneous xyloglucan-hydrolyzing enzymes from germinated nasturtium seeds have been used to localize xyloglucans specifically in seed cell walls. The enzymes, a novel endo (1----4)-beta-D-glucanase which shows absolute specificity towards xyloglucans and a beta-D-galactosidase which is capable of removing galactosyl residues from polymeric xyloglucans, were used to stabilize gold sols. The complexes were applied to ultrathin sections of nasturtium (Tropaeolum majus L) and tamarind (Tamarindus indica L) seeds. The gold complexes prepared from the active enzyme proteins retained enzyme activity, and such complexes gave extremely weak section-labelling or no labelling at all. When the enzymes were subjected to heat-deactivation before being used to stabilize the gold sols, gold complexes were obtained which lacked enzyme activity, but which gave strong, specific labelling of xyloglucans in ultrathin sections. The specificity of the labelling was checked by substrate-competition, by pretreatment of sections with the active and heat-denaturated enzymes and by comparing the labelling of xyloglucan-containing storage cells with other cell types in the same section. The labelling was maximal at the pH which was optimal for the active enzyme. We conclude that the enzyme-gold complexes which retain high activity against the substrate to be localized are likely to be unsuitable as cytochemical probes because they may cause in situ substrate modification. In the case of the enzyme complexes described here the specific localization obtained with the gold complexes prepared from heat deactivated enzymes may be attributable to the retention by the heat-treated enzymatically-inactive proteins of substrate recognition.(ABSTRACT TRUNCATED AT 250 WORDS)
Vian
B
B
Ecole Normale Supérieure, Laboratoire des Biomembranes et Surfaces Cellulaires Végétales, Paris.
Nairn
J
J
Reid
J S
JS
D001665:Binding Sites; D005936:Glucans; D006026:Glycoside Hydrolases / Q000737:chemistry*; D006046:Gold; D006651:Histocytochemistry / Q000379:methods*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008854:Microscopy, Electron; D011134:Polysaccharides / Q000032:analysis*; D011489:Protein Denaturation; D012639:Seeds; D012680:Sensitivity and Specificity; D014990:Xylans; D001616:beta-Galactosidase / Q000737:chemistry*
D005936:Glucans; D011134:Polysaccharides; D014990:Xylans; C029353:xyloglucan; D006046:Gold; D006026:Glycoside Hydrolases; C072818:xyloglucan endo(1-4)-beta-D-glucanase; D001616:beta-Galactosidase