Wheat Invertases

Abstract

Wheat coleoptiles have two distinct invertases, a soluble and a cell wall-bound form as indicated by results from cytochemical and biochemical studies. These enzyme activities differ in their pH optima, chromatographic behavior on diethyaminoethyl cellulose, kinetic properties, thermal stability, and response to light treatment. The soluble invertase was purified to near homogeneity by diethylaminoethyl-cellulose, concanavalin-A Sepharose, and Sephacryl S-300 chromatography. MATERIALS AND METHODS Buffer Solutions. The buffer solutions used were as follows: A, 20 mm sodium phosphate (pH 6.5), 1 mm EDTA, 1 mM DTT, 0.1 mm phenylmethylsulfonyl fluoride, and 1 mM benzamidine; B, 10 mm sodium phosphate (pH 6.5), 1 mM EDTA, and 0.1 mM DTT; C, 100 mm sodium phosphate (pH 6.5), 1 mM MnCI2, 1 mM CaCl2, 1 mM MgCl2, 1 mM DTT, and 0.5 M NaCl; D, 50 mm sodium phosphate (pH 6.5), and 1 mM DTT.Plant Material. Seeds of Triticum aestivum L. cv Yecovo Rojo were sown on two layers of filter paper moistened with distilled H20 on Petri plates. One set was grown under continuous light and the other placed in total darkness at room temperature. The coleoptiles and roots were harvested after 4 d and analyzed for invertase activity as discussed below.Invertase Extraction. Five g of tissue were homogenized with a mortar and pestle containing 30 ml of buffer A and then centrifuged at 3000g for 20 min. The resulting pellet was washed by repeated cycles of suspension in 30 ml of buffer A and centrifugation as described above. All supernatant fluids were combined and centrifuged at 31,000g for 20 min, and the clear supernatant fluid was designated as the soluble invertase fraction. The final pellet was resuspended in 30 ml of buffer A containing 1 M NaCl and shaken overnight at 4C. The resuspended pellet was then centrifuged at 31,000g for 20 min, and the resulting supernatant fluid was designated as the cell wall enzyme fraction. Both the soluble and cell wall enzyme fractions were dialyzed separately against buffer B to remove endogenous reducing sugars.Invertase Assay. Appropriate amounts of an enzyme preparation were added to a test tube containing 2 ml of20 mm sucrose in 50 mm sodium acetate buffer, pH 5.5, and incubated at 37C for 30 min. The reaction was terminated by adding 2 ml of Somagi reagent (17)

Cell Wall Invertase in Tobacco Crown Gall Cells

“…A strong inhibition of the cell wall invertases from U. dioica (3) and Triticum aestivum (15) was demonstrated for micromolar concentrations of p-chloromercuribenzoyl sulfonate, while millimolar concentrations of DTT (3) were needed to activate the enzyme. The absence of any inhibitory effect of NEM, as previously reported for the T. aestivum enzyme (15), suggests that the sensitive SH-group(s) may be either exposed only at the enzyme's pH optimum (here NEM is not reactive) or during the catalytic cycle. The binding of cell wall invertase to a organo-mercurial derivative of Bio-gel (13) in the absence of substrate argues against the second interpretation, but different SH-groups may be involved.…”

“…Recently, a number of higher plant cell wall invertases (EC 3.2.1.26) have been purified and characterized (3,5,6,9,13,15,17,(22)(23)(24)37). The enzyme, which has an acidic pH optimum and is positively charged (pI 9-10) at the pH of the cell wall, hydrolyzes sucrose in the apoplast in rapidly growing tissues and has been proposed to be involved in establishing metabolic sinks (25,33, and literature cited therein).…”

Acid and Neutral Invertases in the Mesocarp of Developing Muskmelon (Cucumis melo L. cv Prince) Fruit

“…The neutral invertase from muskmelon mesocarp has a higher Km value (10 mM) for sucrose than that of the acid invertase (2.2 mM) as in the case for other plant tissues (7,13) and cultured cells (14). Inhibition of activity by Hg2" ions seems to be common to most plant invertases (6,8,13) and the partial inhibition of the neutral invertase by Cu2> and Zn 2 ions resembles the effects of these ions on the alkaline invertase from sugar beet (13).…”

“…Some of the wallbound invertases can be released by treatment with a highmolarity solution of NaCl (6,8,11,12,14) and with chelating agents such as EDTA (11,14). Schaffer et al (18) found considerable invertase activity in cell walls of muskmelon mesocarp tissue which was not an artifact of extraction.…”

Sucrolytic Enzyme Activities in Cotyledons of the Faba Bean (Developmental Changes and Purification of Alkaline Invertase)

“…Plant invertases are usually categorized according to their pH and their spatial location. Acid invertases are generally considered to be localized within the vacuole and/or the cell wall (Pressey, 1966;Krishnan et al, 1985;Salzer and Hager, 1993). Alkaline invertase is thought to be a cytosolic enzyme (Ricardo and ap Rees, 1970), although no direct evidence is available as yet.…”