Characterization and localization of three soluble invertase forms from Lilium longiflorum flower buds

Three invertase forms (EC 3.2.1.26) were identified in soluble extracts from developing flower buds of Lilium longiflorum Thunb. cv. Nellie White. The enzymes were separable on a diethylaminoethyl (DEAE)-Sephacel column and designated invertase I. II or III according to the order of elution from Sephacel. To determine tissue specificity of these floral invertases, anthers were separated from tepal. pistil and filament tissue, and analyzed for invertase activity. Invertase I was localized primarily in anthers, with invertases II and III being present in much smaller amounts (less than 5% of the invertase I activity). Much higher levels of invertases II and III were found in the nonanther organs of the flower, where essentially no invertase 1 was detectable. Further purification of each form (using gel filtration. Con-A-Sepharose affinity chromatog-raphy and hydrophobic interaction chromatography on phenyl-agarose) resulted in 135- 189- and 202-fold purification of pooled fractions from DEAE-Sephacel. respectively, and established that each invertase form is a glycoprotein. Each was an acid invertase. with pH optima between 4.0 and 5.0 and an apparent molecular mass of 77 500 Da (as determined by Sephadex gel filtration). The invertases had sucrose K_m values of 1.0. 6.4 and 6.6 m M . and temperature optima of 40. 50 and 45°C. respectively. A temperature stability study revealed that invertase III was the most thermostable, followed by II and I. Invertases II and III had lower affinity to raffinose and stachyose than invertase I. All three enzymes were completely inhibited by Hg²⁺ or Ag⁺ ions at 1.7 m M . At this concentration. Cu^2- showed differential partial inhibition . Although fructan was shown to be present in both anther and nonanther tissues of Lilium flower buds, these invertases showed no sucrose:sucrose fructosyltransferase (EC 2.4.1.99) activity.