Invertases of Lilium Pollen

Abstract

ITwo different forms of invertase are found in pollen of lily (Lilium anratum). One form is cytoplasmic (Invertase 1) and the other is bound to the pollen wall (Invertase 2). Invertase 1 has been partilly purifed and is a glycoprotein (apparent molecular weight, 450 kilodaltons) with a K, of 0.65 millimolar for sucrose. The two invertases differ in pH optimum and thermal stability. Invertases of lily pollen are ,@-fructofuranosidases which can hydrolyze sucrose but not melizitose. The mature pollen grains have enzyme activity in both cytoplasmic and wall fractions, and no increase in the activity of either occurs during germtion. The wall-bound enzyme could not be released by treatments with detergents or high salt concentrations.Invertase (EC 3.2.1.26) or ,B-fructofuranoside fructohydrolase hydrolyzes sucrose and related oligosaccharides. Sucrose represents the major soluble sugar present in the majority of angiosperm pollen grains and is also the most commonly detected sugar in natural substrates for pollen germination (25). Germinating pollen grains show an extremely high rate of growth, i.e. 400 Am/h (13) and thus have a high demand for carbon skeletons required for pollen tube wall synthesis as well as a substrate for respiration. In vivo, the pollen tubes of lily grow on the surface of stigmatic and stylar canal secretion products. By means of labeling experiments, it has been shown that carbohydrate material of the secretion product of styles is used by growing pollen tubes for tube wall synthesis (17). Although invertase was among the first enzyme activities recognized in pollen, no detailed characterization of this pollen enzyme has been attempted. In the present study, we report the purification and properties of invertase from the pollen of Lilium auratum and discuss its functional significance. 12,000g for 10 min, and the supernatant was dialyzed for 20 h at 2C against extraction buffer and the dialyzed extracts were then used as a crude extract for enzyme assays. The wall pellets were again washed with extraction buffer three times and the washings were discarded. The resulting pellet was then suspended in 50 mM citrate-phosphate buffer (pH 6.0), and the suspension was used as a source of wall-bound invertase. Aliquots of wall fraction were assayed for hexokinase activity (11) to ascertain the absence of contaminant cytoplasmic proteins. The enzymic activity of soluble invertase was assayed as follows: a 0.05-ml sample of invertase preparation was added to 0.45 ml of 0.1 M acetate buffer (pH 4.8) which contained sucrose at a final concentration of 100 mm. The mixture was incubated for 20 min at 35C. The reactions were terminated by placing the reaction tubes in a boiling water bath for 5 min. After cooling the tubes to 35C, glucose released in the reaction mixture was estimated by a colorimetric assay using glucose oxidase and peroxidase. The assay was carried out by adding 0.5 ml ofchromogen reagent (4) to 0.5 ml ofthe invertase reaction mixture. In some instances, the reducing sugars relea...

Wheat Invertases

“…This result is consistent with studies of lily pollen invertases where the involvement of a sulfhydryl group in the enzyme's catalytic site has been suggested (23). However, 100 Mm DTNB and 100 gM NEM, reagents known to react with sulfhydryl groups, failed to inhibit the wheat invertase activity to any measurable extent.…”

“…The purified enzyme is probably a glycoprotein based on its ability to bind to the lectin Con-A. This is consistent with other studies (4,18,23) where it has been shown that invertase is a glycoprotein. The precise role of the carbohydrate moiety of the enzyme is not well understood since it is not necessary for enzymic activity or stability (4).…”

“…Results obtained by gel filtration showed an apparent mol wt of 158,000, indicating that wheat invertase is a multimeric protein. There is a considerable variability in the mol wt of plant invertases, and they range from 48,500 (4) to 450,000 (23). It should be pointed out, however, that the glycosylated nature of this enzyme may lead to a lack of precision in estimating the mol wt.…”

“…Analysis of this protein peak by SDS-PAGE indicated a major polypeptide band at 53,000 D with several faint bands of larger and smaller mol wt (Fig. 4) (20,23) were tested against the purified soluble invertase from wheat coleoptiles. AgNO3 and 12 at 4 gM concentration and HgCl2 at 2 gm concentration completely abolished invertase activity.…”

Effect of time of day for harvest and postharvest treatments on the sugar metabolism of broccoli (Brassica oleracea var. italica)

“…INV activity was also 62 and 73% lower at day 2 and 4 of storage in HT samples. Last fact could be due to the sensitivity of INV to stress produced by the high temperatures (Singh andKnox 1984, Cheikh andJones 1995). In the case of samples treated with 1-MCP, INV activity was similar to that of controls (Fig.…”