Localization and Substrate Specificity of Glycosidases in Vacuoles of Nicotiana rustica

Vacuoles isolated from Nicotiana rustica var brasilia have been shown to contain significant levels of glycosidase activity when assayed using p-nitrophenyl-glycosides as substrates. The substrate specificity for the glycosidases in the vacuolar fraction closely paralleled that found in the protoplasts, and the leaf tissue from which the vacuoles were isolated. The substrate specificity of the vacuolar enzyme(s) was different from glycosidic activity found in the commercial digestive enzyme preparations used to isolate the protoplasts from leaf tissue. It was demonstrated that 70 to 90% of the glycosidases that were found in the protoplasts appeared to be localized within the vacuole, when the p-nitrophenyl substrates α- and β-;d-galactose, β-d-glucose, and α-d-mannose were used. Neither the vacuolar nor the protoplast enzymes were active towards the naturally occurring phenolic glycoside, rutin. α-Mannosidase appears to be a valuable marker enzyme for vacuoles isolated from mesophyll leaf cells of tobacco.