Purification and Partial Characterization of Potato

Invertase plays an important role in the hydrolysis of sucrose in higher plants, especially in the storage organs. In potato (Solanum tuberosum) tubers, and in some other plant tissues, the enzyme seems to be controlled by interaction with an endogenous proteinaceous inhibitor. An acid invertase from potato tubers (variety russet) was purified 1560-fold to electrophoretic homogeneity by consecutive use of concanvalin A-Sepharose 4B affinity chromatography, DEAE-Sephadex A-50-120 chromatography, Sephadex G-150 chromatography, and DEAE-Sephadex A-50-120 chromatography. The enzyme contained 10.9% carbohydrate, had an apparent molecular weight of 60,000 by gel filtration, and was composed of two identical molecular weight subunits (M, 30,000). The enzyme had a Km for sucrose of 16 millimolar at pH 4.70 and was most stable and had maximum activity around pH 5. The endogenous inhibitor was purified 610-fold to homogeneity by consecutive treatment at pH 1 to 1.5 at 370C for 1 hour, (NH4)2SO4 fractionation, Sephadex G-100 chromatography, DEAESephadex G-50-120 chromatography, and hydroxylapatite chromatography. The inhibitor appears to be a single polypeptide (M, 17,000) without glyco groups. The purified inhibitor was stable over the pH range of 2 to 7 when incubated at 370C for 1 hour. Sucrose is the most abundant transportable free carbohydrate in the plant kingdom. Sucrose serves as an important reserve carbohydrate in plants, especially in such storage organs as tuber, root, and seed. During germination, sucrose is a readily degradable source of energy. In storage organs, invertase (fl-fructofuranosidase; EC 3.2.1.26) functions in the hydrolytic degradation of sucrose to glucose and fructose. Invertase was one of the first enzymes discovered; it was first isolated from yeast more than a century ago (3). The enzyme occurs widely in microbial, plant, and animal sources. The presence of invertase in potatoes was first reported over 80 years ago (10). The first evidence for an endogenous proteinaceous invertase inhibitor in potatoes was obtained from kinetic studies on invertase in crude extract (22). Pressey (18, 19) showed an inhibitor was present by its purification to homogeneity and partial characterization. Invertase inhibitors have been found also in red beet, sugar beet, and sweet potato (13, 20) and in maize endosperm (8). These higher plant invertase inhibitors are all soluble proteins and their inhibitor activity seems to be limited to acid invertases. The present paper describes the purification of potato invertase and its endogenous inhibitor to homogeneity, with partial characterization of the two proteins. MATERIALS AND METHODS BSA (98-99% pure, catalog No. A-7030), sweet potato 3amylase (type 1-B, crystalline suspension, catalog No. A7005), bovine pancreatic trypsin (type IX, crystallized, catalog No. T-0 134), bovine thyroglobulin (type I, catalog No. T100 1), chicken lysozyme (grade I, 3X crystallized, catalog No. L-6876), methyl mannopyranoside (grade III, catalog No. M6882), DEAE-Sephadex (stock A-50-120), and Con A-Sepharose 4B (catalog No. C-9017) were obtained from Sigma Chemical Co. Human transferrin (electrophoretically pure, catalog No. 616397) and hydroxylapatite (fast flow, catalog No. 391947) were obtained from Calbiochem. Chicken ovalbumin was a gift from Dr. Robert Feeney's laboratory. A standard proteins kit for mol wt determination using SDS-PAGE was obtained from Bethesda Research Laboratories. Sephadex G-100 and Sephadex G-150, both column chromatography fine grade, were obtained from Pharmacia. All other chemicals used were of reagent grade. All water used was double deionized. Potatoes (Solanum tuberosum, var russet) were purchased in local supermarkets and screened for invertase and inhibitor activities. Potatoes with high inhibitor activity were peeled, sliced, soaked in a 0.1 M NaHSO3 solution, frozen rapidly, and stored at -20°C in polyethylene bags, each containing about 1 kg of sliced potatoes. Potatoes with high invertase activity were stored at 4°C in polyethylene bags for at least 3 weeks. Screening of Potatoes for Invertase and Inhibitor In a typical experiment, about 100 g of peeled, sliced potatoes were homogenized with 1O mL of 0.1 M NaHSO3 for 2 min in a Waring blender at 4°C. The extract was filtered through eight layers of cheesecloth, and the filtrate was centrifuged at 15,000g for 30 min at 4C. A portion of the supematant was dialyzed against three changes of water (at least 50-fold dialysate/sample ratio was always used) at 4°C for 3 to 4 h each time. The remainder of the supernatant was blended for 30 min in a Waring blender at 4C. Blending was carried out with 5 min blending periods, with 10 min cooling periods in between. The blended extract was centrifuged at 1 5,000g for 30 min at 4°C and dialyzed as above. The precipitate that formed during dialysis was removed by centrifuga386 www.plantphysiol.org on January 22, 2018 Published by Downloaded from Copyright © 1990 American Society of Plant Biologists. All rights reserved. POTATO INVERTASE AND ENDOGENOUS PROTEINACEOUS INHIBITOR tion, and the supernatants were assayed for invertase and inhibitor activities as described below. Assay for Invertase Activity Invertase activity was determined by measuring reducing sugars formed from sucrose hydrolysis by the Somogyi method as modified by Nelson (15). The incubation mixture (0.5 mL) contained a suitable amount of enzyme, 80 mM acetate buffer (pH 4.70), and 143 mm sucrose. The assay was performed at 37°C for 1 h. One-half mL of copper reagent was then added to terminate the reaction, and the tubes were immediately heated for 30 min in a boiling water bath. After cooling, 0.5 mL of arsenomolybdate reagent followed by 3.5 mL of water were added. Solutions were mixed, and 1.0 mL ofeach was centrifuged for 2 min using a high speed microfuge and the absorbance was read at 660 nm. Boiled enzyme blanks included as controls did not vary significantly in absorbance from reagent blanks. Centrifugation was required only with impure preparations of invertase or inhibitor. A unit of invertase is defined as the amount of enzyme that catalyzed the breakdown of 1 ,tmol of sucrose/min at 37°C and pH 4.70. The specific activity is expressed as units of invertase per mg of protein. Assay for Inhibitor Activity Inhibitor activity was determined by preincubating at 37°C a constant amount of invertase with varying amounts of inhibitor in 0.4 mL incubation mixture containing 80 mM acetate buffer, pH 4.70. At the end of 1 h of incubation, 100 ,gL of 720 mm sucrose (final concentration of 144 mM) was added to each tube and the remaining invertase activity left was measured as described above. Enzyme without inhibitor added was used as control. A plot of residual activity versus amount of inhibitor used was linear over the range of 0 to 80% inhibition. The amount of inhibitor in each sample was estimated from the corresponding plot. A unit of inhibitor was defined as that amount which inhibits one unit of invertase 50% at pH 4.70 and 37°C. The specific activity is expressed as units of inhibitor per mg of protein. Protein Determination Protein was determined by the method of Lowry et al. (12) with BSA as a standard protein. For column fractions, the protein concentrations were estimated by absorbance at 280 nm. Purification of Invertase Storage of potatoes at 4°C for 3 to 5 weeks usually increased the invertase content twoto threefold. However, with some batches of potatoes, the effect of storage at low temperatures was not as pronounced. Purification of invertase was always performed on low temperature-stored potatoes. All the purification procedures were performed at 4°C and with centrifugation at 15,000g for 30 min, unless otherwise noted. Potatoes (1 kg), peeled and sliced, were homogenized with 100 mL of 100 mm NaHSO3 for 2 min in a Waring blender at 4°C. The extract was filtered through eight layers of cheesecloth and the filtrate was centrifuged. The supernatant, normally about 600 mL, was dialyzed against three changes of water. After dialysis, the precipitate formed was removed by centrifugation, and the supernatant was freeze-dried. The lyophilized powder was dissolved in 190 mL of 20 mM TrisHCI/500 mm NaCl buffer (pH 7.40), containing 1 mm each CaCl2, MgCl2, and MnCl2, and the solution was applied to a Con A-Sepharose 4B affinity column (2.5 x 10 cm) equilibrated with the same buffer. Elution (using the same buffer) proceeded until the 280 nm absorbance of the fractions was 0.040 or less. The enzyme was then eluted with 30 mm methyl a-D-mannopyranoside in the above buffer. The fractions with invertase activity were combined, concentrated 10-fold by ultrafiltration using an Amicon PM-10 membrane, and dialyzed three times against 20 mM sodium phosphate buffer (pH 6.0). After the Con A-Sepharose 4B step, the dilute solutions of invertase were very unstable, even at low temperature. Immediate and rapid concentration by ultrafiltration stopped loss of activity. The enzyme solution from the Con A-Sepharose 4B affinity column was applied to the top of a DEAE-Sephadex A-50120 column (2.5 x 18 cm) equilibrated with 20 mm phosphate buffer, pH 6.0. The column was washed with the same buffer until the 280 nm absorbance was zero. The enzyme then was eluted with a linear gradient produced by using 250 mL of the phosphate buffer in one chamber and 250 mL ofthe same buffer containing 500 mm NaCl in an identical mixing chamber. The fractions with invertase activity were combined and concentrated 10-fold by ultrafiltration as above. The enzyme solution from the DEAE-Sephadex A-50-120 column was applied to a Sephadex G150 column (1.5 x 90 cm) equilibrated with 50 mm sodium phosphate/100 mM NaCl buffer (pH 7.2). The column was eluted with the same buffer. The main fractions with invertase activity were combined and concentrated as above. The leading and trailing side fractions were combined, concentrated, and rechromatographed on the same Sephadex column. The main fractions were combined, concentrated, and combined with the first main pool. The enzyme solution from the Sephadex G-50 column was dialyzed three times against 20 mm sodium phosphate buffer, pH 6.0, and rechromatographed on a DEAE-Sephadex A-50120 column (1.5 x 18 cm) equilibrated with the same buffer. After the column was washed with the starting buffer, the enzyme was eluted with 100 mM NaCl/20 mm sodium phosphate buffer (pH 6.0). The main fractions were combined and concentrated as above. The enzyme was stored frozen in the same buffer in 25 ,uL aliquots at -20°C until used. Aliquots of invertase were diluted with appropriate buffers containing a final concentration of 0.1 mg/mL of BSA to stabilize the enzyme. Purification of Inhibitor The inhibitor content of potatoes did not vary significantly during frozen storage. All purification procedures were carried out at 4°C and with centrifugation at 15,000g for 30 min, unless otherwise stated. 387 www.plantphysiol.org on January 22, 2018 Published by Downloaded from Copyright © 1990 American Society of Plant Biologists. All rights reserved.