Plant Physiol. (1988) 86,0260-265 0032-0889/88/86/0260/06/$0 1.00/0 Characterization of D-Enzyme (4-a-Glucanotransferase) in Arabidopsis Leaf' Received for publication July 7, 1987 and in revised form September 17, 1987 TSAN-PIAO LIN2 AND JACK PREISS* Department ofBiochemistry, Michigan State University, East Lansing, Michigan 48824 ABSTRACT MATERIAILS AND METHODS Two maor forms of D-enzyme (4-a-gucaotrnferase, EC 2A.1.25) Reagents. D-[U-'4C]Glucose was purchased from Amersham. were successfully separated from most of the amylase activity using hexokinase, glucose 6-P dehydrogenase, maltotriose, and NAD FPLC-Mono Q column chromatogrphy. Transfer of a maltosyl group were obtained from Sigma Chemical Co. Amylopectin (mol wt was observed upon the incubation of D-enzyme with maltotriose and n- > 106 D) and BCA3 protein reagents were products from Pierce [U-J4 Clucose. About 4.5% of the radioactivity was transferred to mal- Chemical Co. Soluble starch was from Merck & Co. Inc. Safety- totriose in 2 hours. End product analysis showed the accumulation of Solve was from Research Products International Corp. Acarbose, glucose and maltopentaose from maltotriose within the first 10 minutes Bay e 4609 and deoxynojirimycin were gifts from Dr. D. Schmidt of the reaction. Several other maltodextrins were also observed with of Pharma-Forschungzentrum, Bayer AG D-5600, Wuppertal, longer incubation times, althongh maltose was never produced. A quan- West Germany. titative measurement of maltodextrin production from the reaction of Plant Material and Growth Conditions. The Columbia wild- I14 Cmaltotriose with D-enzyme shoWed that the quantity of maltotriose type ofArabidopsis thaliana was used. The plants were grown at decreased from 100% to 31% after 3 hours incubation, while glucose, approximately 22C, with a 12 h photoperiod under cool-white maltotetraose, maltopentaose, maltohexaose, maltoheptaose, maltooc- fluorescent illumination (about 200 ,E m 2 s-') on a per- taose, and higher maltodextrincsinreased in amount. Glucose is the lite:vermiculite:sphagnum (1:1:1) mixture irrigated with a min- major product throughout the course of the reaction of D-enzyme with eral nutrient solution (9). maltotriose. Maltotriose, in addition to glucose, are the major products Enzyme Purification: Preparation of Crude Extract. One in the reaction ofn-enzyme with maltodextrin with a chain length greater hundred g ofleaftissue from 4 to 6 week old plants (in vegetative than maltotriose. This study confirms the existence of a trwanglycosylase stage) was homogenized in a mortar and pestle in 90 ml of 50 that disproportionates maltotriose and higher maltodextrins by transfer- mM Tris-HCl buffer (pH 7.5) containing 2 mM EDTA and ring maltosyl or maltodextrinyl groups between maltodextrins resulting centrifuged at 16,000g for 20 min. The precipitate was washed in the production ofglucose and different maltodextrins, but not maltose, once with 50 ml of the above buffer. in leaf tissue with enzymic properties very similar to the previously Polyethylene Glycol Fractionation. The supernatants from the reported n-enzyme in potato. above extraction and wash were combined and PEG 8000 (50% w/v dissolved in 20 mm Bis-Tris-propane buffer [pH 7.0] con- taining 2 mm DTT [buffer A]) was added to bring the PEG concentration to 3% (w/v). The solution was stirred for 15 min at 4°C and centrifuged. Pellets were discarded and the superna- tant adjusted to 10% (w/v) with PEG (50% w/v), stirred and centrifuged as above. The pellets were resuspended in 11.5 ml of buffer A. DEAE-Cellulose Chromatography. The solution was clarified D-enzyme was first isolated from potato tuber (7) and has also by centrifugation at 16,000g for 10 min and 250 mg of protein been reported to be present in broad bean, carrot, and tomato in 11 ml was charged onto a DEAE-cellulose column (Whatman (4). The physiological role ofthis enzyme may be to catalyze the DE-52) (1.5 x 16 cm, 10 ml resin bed volume) which had been condensation of short oligosaccharides to form larger chains equilibrated with buffer A. Fifty percent of the protein washed which are more suitable substrates for starch phosphorylase (2). through the column with the starting buffer. After the absorbance An important property observed for n-enzyme from potato is at 280 nm ofthe eluate decreased to 0.1, the enzyme was eluted that it does not generate a new reducing end. Instead, it transfers with a salt gradient containing 50 ml of buffer A in the mixing a maltodextrinyl group to the nonreducing end ofmaltodextrins chamber and 50 ml ofbuffer A and 0.4 M NaCl in the reservoir. or to glucose to form a new nonreducing group (1, 12). The fractions containing D-enzyme activity were pooled and n-Enzyme has been studied only from nonphotosynthetic concentrated to 12 ml using an Amicon PM30 membrane. The tissue in higher plants, and the presence and properties of D- solution was dialyzed twice against 500 ml ofbuffer A each time enzyme from photosynthetic tissues have still not been well for 5 h. The precipitate formed was removed by centrifugation. established. The presence of n-enzyme in leaf tissues was first The clear solution containing 54 mg protein in 11.2 ml was reported by Okita et al. for spinach (6). This communication rechromatographed on a second DE-52 column (1 x 10 cm; 6.5 reports on the action pattern and some properties of n-enzyme ml resin bed volume) which was preequilibrated with buffer A. purified from Arabidopsis leaves. D-Enzyme was eluted in the same way as described above in a total gradient volume of 70 ml. The salt concentration in the ' Supported in part by National Science Foundation Grants DMB 85- eluate was determined by conductivity. Fractions coninin% o 10088 and 86-10319. 2 McKnight Foundation Post-doctoral Fellow. 3 Abbreviations: BCA, bicinchoninic acid; NEM, N-ethylmaleimide. 260 ARABIDOPSIS LEAF D ENZYME 261 solution was chromatographed on a FPLC Mono Q anion ex- change HR 5/5 column (Pharmacia, Uppsala, Sweden) that had been equilibrated with buffer B. After the sample was loaded on to the column, the column was washed with 3 ml of buffer B I and eluted with a 30 ml linear KC1 gradient (0.10-0.35 M) in buffer B at 0.5 ml/min. D-Enzyme active fractions were pooled as above. LC and concentrated described Enzyme Assays. Amylase activity was measured in 1 ml reac- .E 0 tion mixtures containing 5 mg of amylopectin, 40 Amol sodium \ 0.5 rt acetate buffer (pH 6.0) and enzyme (6). The Nelson method (5) 0 was used to determine reducing sugar formation. D-Enzyme was z measured in 250 A reaction mixtures containing 10,umol sodium acetate or sodium succinate buffer (pH 6.5) and 2.48 ,mol maltotriose. The reaction mixtures were incubated at 37°C for 0 30 min and terminated by immersing the reaction tubes in FRACTION NO. boiling water for 30 s. Released glucose was measured by follow- ing the reduction of NADP in the presence of hexokinase and FIG. 1. Second DEAE-cellulose column chromatography of the 3 to 6-P dehydrogenase (3). 10% PEG fraction. The chromatography procedure is described in "Ma- glucose D-Enzyme Catalysis of [14 Exchange with Malto- terials and Methods." CqGlucose triose. The reaction mixture in a total volume of250 Al contained 283 mM D_[U-'4C]glucose (4,500 cpm/,mol glucose), 12.5 mM maltotriose, 40 mm sodium acetate buffer (pH 6.5) and 0.075 unit (Amol/min) of D-enzyme. The high ratio of glucose to 0.8 16 maltotriose effectively rendered glucose the only acceptor in the reaction mixture, so that no polymers larger than the initial maltotriose could be formed (1). The reaction mixture was 'tA~~~~~~~~~~ incubated at 37°C and at intervals, 120 Al of the mixture was boiled for 30 s and 110 ,l was subjected to paper chromatogra- Di D2, ~ phy. The chromatogram was cut into 2 cm strips, mixed with 500 Al distilled water, and 5 ml of scintillant cocktail. The radioactivity was quantified by liquid scintillation counting. 1220 2I03 Preparation of [14 CqMaltotriose. The reaction mixture con- A~~~~~~~ taining 50 mg Lintner soluble starch, 277 'Umol D-[U-'4 C]glucose (2.50 x I05 cpm/,mol glucose), 0.12 unit (,mol/min) D-enzyme (Dl), 4 Amol NEM and 20 Mmol sodium succinate buffer (pH 6.5) in a total volume of 4 ml. The reaction mixture was covered with toluene and incubated at 30C for 19 h. The reaction was FRACTION NO. terminated by immersing the tube in boiling water for 5 min. FIG. 2. Elution profile of Arabidopsis leaf n-enzyme in FPLC-Mono The solution was then applied to Whatman 3MM paper, and Q chromatography. The sample was prepared from the 3 to 10% PEG chromatographed in a system of butanol:pyridine:water (6:4:3) fraction and chromatographed twice by DEAE-cellulose chromatogra- (10) for 35 h. The maltodextrins were individually cut from the phy. Fractions were assayed for n-enzyme activities. Fractions 20 to 22, paper and eluted off with distilled water. The final recoveries for and 30 to 33 were pooled separately and designated as Dl and D2, each sugar were: glucose 4.76 x 107 cpm (86.4%), maltose 4.92 respectively. x I05 cpm (0.9%), maltotriose 46.9 x I05 cpm (8.5%), maltote- enzyme activity were pooled and concentrated 3-fold using a traose 15.5 x 105 cpm (2.8%), maltopentaose 5.08 x 105 cpm PM30 membrane. The nenzyme fraction then was dialyzed (0.9%), maltohexaose 2.0 x I05 cpm (0.4%), maltoheptaose and overnight against 500 ml of 20 mM.