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)