Characterization of D-Enzyme (4-Α-Glucanotransferase) In

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] containing 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 exchange 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 following 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. Bis-Tris-propane buffer (pH other maltodextrins 0.92 x I05 cpm (0.2%). The identity of[14 C] 6.5) containing 2 mMi DTT and 10% glycerol (buffer B). The maltotriose was confirmed by rechromatographing the eluted dialyzing medium was renewed once. sample against standard maltotriose. The specific radioactivity FPLC-Mono Q Column Chromatography. The dialyzed solu- of glucose, maltotriose, maltotetraose, and maltopentaose was tion was centrifuged at 16,O00g for 10 min and the clear solution checked, and all ofthem had similar values varying between 2.24 was divided into aliquots each containing 10 mg protein. The X 105 to 2.36 x 105 cpm/4mol. This indicates the presence of

Table I. Purification ofD-Enzyme Total Specific Fraction Vol Protein Activity Activity Yield mlmlmg/ml iimol/min jAmol/min-mg~~~~~~~~~~protein % Homogenate 145 5.9 33.3 0.039 100 PEG 3-10% 11.2 23.9 17.3 0.064 52 lst DE-52 11.2 4.8 21.5 0.40 64 2nd DE-52 7.1 4.2 16.9 0.89 51 FPLC-Mono Q DI 2.1 0.32 3.3 4.87 10 D2 1.9 1.7 3.7 1.13 11 262 LIN AND PREISS Plant Physiol. Vol. 86, 1988 No. 1 paper (70,000 cpm per lane) and the maltodextrins separated by paper chromatography as previously indicated. The individual substances were located by autoradiography by a 6 d exposure to the chromatogram to Kodak X-Omat AR film. The zones containing the sugars were cut from the paper chromatogram, eluted with H20, and the radioactivity was determined as described above. Protein Assay. Protein concentration was determined by the method ofSmith et al. (8) usingthe Pierce Chemical Co. prepared E lo. BCA reagent and BSA as the standard. Absorbance at 280 nm also was used to estimate the protein concentration for the eluate U Ix~~~~~~ eluted from the column during enzyme purification. K x ++,,^_ ww/ 0 5 10 15 20 2 5 30 35 RESULTS Distance from origin, cm. Purification of D-Enzyme. More than 85% ofthe recovered D- FiG. 3. The reaction of D-enzyme (DI) with D_[U'4 C]glucose and enzyme activity was located in 3 to 10% PEG fraction (data not maltotriose. The final concentration of solutes in a 250 Al digest were shown). n-Enzyme activity in the 15 to 35% PEG fraction was 283 mm D-[U-'4C]glucose (4,500 cpm/gmol glucose), 12.5 mM malto- insignificant. D-Enzyme activity was eluted from the DE-52 triose, 40 mM Na acetate (pH 6.0) and 0.75 unit (umol/min) of D- column at a salt concentration of 160 mm NaCl (Fig. 1) and enzyme. The reaction was incubated at 37°C, and the labeled products heavily contaminated with amylase activity. Chromatography of were separated by paper chromatography and were counted in a liquid n-enzyme activity on the Mono Q column yielded two major scintillation counter. The reaction mixture of 0 time contained heat peaks ofactivity, and these fractions were designated Dl and D2 denatured D-enzyme. The positions ofmaltodextrin standards are shown in order of elution (Fig. 2). The n-enzyme purification scheme and were detected using AgNO3 (10). is presented in Table I. The total activity of n-enzyme in leaf homogenate may be overestimated since the high activity of the same mole concentration of [14 C]glucose in each mole ofthe amylases (about 0.1 ,umol/min mg) may significantly contribute different maltodextrins. The concentration of the maltodextrins to the glucose produced in the reaction mixtures. was determined by digesting the oligosaccharide with glucoa- n-enzyme preparations (Dl and D2) were slightly contami- mylase and measuring the glucose formed with hexokinase, ATP, nated with amylase activity. Dl is relatively low in amylase as and glucose 6-P dehydrogenase (3). indicated by the activity ratio of n-enzyme to amylase being 44 Action ofn-Enzyme on 1X4 ClMaltotriose. The reaction mixture while the ratio for D2 is 16. n-Enzyme is stable at 4C in 20 mM contained 2.48 ,mol maltotriose (1.21 x 105 cpm/,umol), 10 Bis-Tris-propane buffer (pH 6.5) containing 10% glycerol. No Mmol sodium acetate buffer (pH 6.0), 0.25 ,umol NEM, 0.07 unit activity losses were detected over 6 months. of n-enzyme (DI) in 250 1A. Samples of 60 ,l were removed Characterization of D-Enzyme. pH Optimum. n-Enzymes Dl after incubation at 37°C for 0 (containing heat denatured en- and D2 have a pH optimum around pH 6.5 with no activity zyme), 10, 30, 90, and 180 min. They were spotted on Whatman detectable at pH 4.0. The activity in sodium succinate or sodium

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FIG. 4. The pattern ofaction ofn-enzyme (D1) on maltodextrins. For reaction conditions see the legend to Table II. The substrate and incubation times (min) are listed under each paper chromatogram. MS, maltodextrin standards. ARABIDOPSIS LEAF D ENZYME 263

'C:x U 'a 0 ~00 -6. E 0.0 0 1-e E c

60 120 180 G4 Incubation time (min) FIG. 6. Changes of the concentration of individual sugars during 3 h incubation of [14C]maltotriose with D-enzyme (Dl). Details of this ex- G5 periment are given in "Materials and Methods." At each time point, there was 67,000 cpm of maltodextrins (557 nmol of maltotriose at 0 G6 t- time). Since each sugar has same specific radioactivity (see "Preparation G7 of [14C]Maltotriose"), the cpm of each sugar can be converted to nmol G8 by dividing each cpm by 1.21 x l10 cpm/umol. At the right hand side, each sugar was calculated as % of the total radioactivity, 67,000 cpm, which is considered as 100%. (X), glucose; (0), maltotriose; (+), maltotetraose; (0), maltopentaose; (A), maltohexaose; (0), maltoheptaose; (A), maltodextrins with chain length greater than maltoheptaose. 180 90 30 10 0 min FIG. 5. Autoradiogram of maltodextrins synthesized by iD-enzyme (Dl) with ['4C]maltotriose at different incubation times. The incubation Table II. Production ofGlucosefrom Maltodextrin by D-Enzyme (DI) times are indicated. D-Enzyme (0.12 unit/ml) was incubated with 5 mg/ml substrate, 40 mm sodium acetate (pH 6.5). At indicated intervals, samples 250 Ml, were acetate buffer is the same. The pH optimum of 1)-enzyme in withdrawn and glucose was measured by following the reduction of Arabidopsis leaves is similar to that found for potato D-enzyme NADP in the presence ofhexokinase and glucose 6-P dehydrogenase. which is pH 6.7 (7), but different from that of carrot and tomato Reaction Incubation Ratio of which is pH 5.5 (4). Substrate Reactio TncubatooncGiGlc Glucose to Action pattern of D-Enzyme. D-Enzyme, a disproportionating Mixture Time Produced SusrtSubstrate transglycosylase, acts on maltodextrins by transferring a-1,4- linkages (7). Transfer of the maltosyl-group from maltodextrin 'Umol min ;smol Mmolh/mol also was observed for Arabidopsis 1-enzyme. Figure 3 shows the 0 0 0 distribution oflabel between products ofthe incubation ofD-[U- 10 0.43 0.17 14 C]glucose and maltotriose, as separated by paper chromatog- 30 0.67 0.27 raphy. Only 4.5% of the radioactivity from [14 C]glucose was Maltotriose 2.49 120 1.02 0.41 transferred to maltotriose after 2 h incubation. The same per- 0 0 0 centage ofincorporation was observed even after 24 h incubation 10 0.23 0.12 indicating that the reaction equilibrium had been reached. 30 0.37 0.20 The action pattern of maltodextrin formation as revealed by Maltotetraose 1.88 120 0.46 0.24 paper chromatography is shown in Figure 4. When maltotriose is the substrate, glucose and maltopentaose are detectable after 0 0 0 10 min incubation (Fig. 4a and 5). The intensity of glucose 10 0.13 0.08 staining increases continuously with the length of incubation. Maltopen- 30 0.25 0.17 Maltotetraose becomes perceptible at 2 h incubation (Fig. 4a) taose 1.51 120 0.34 0.22 and other oligosaccharides larger than maltotriose are also visible 0 0 0 (Fig. 5). Production of maltose was not observed but it was 30 0.13 0.12 detected after 2 h incubation when the activity ratio of D-enzyme Maltohep- 120 0.22 0.20 to contaminating amylase is lower than 10. taose 1.08 180 0.28 0.26 When maltotetraose was used as the substrate, glucose and maltotriose were detected in the early incubation (Fig. 4b). 0 0 0 Maltopentaose and higher maltodextrins become visible follow- Amylopectin 5 mg/ml 240 0 0 264 LIN AND PREISS Plant Physiol. Vol. 86, 1988 several isoforms. D-Enzyme would not have been identified by paper chromatography if the high amylase activity had not been largely removed. Glucose is the major product of the D-enzyme reaction. Jones and Whelan (1) observed that 37% of the original mole concen-

0-- tration of maltotriose in the reaction with potato 1-enzyme was converted to glucose after 2 h reaction. Within 2 h, D-enzyme from Arabidopsis was able to produce 1.02,umol ofglucose from an initial 2.48 umol of maltotriose (Table II). The production of glucose is also dominant in the reaction of D-enzyme with and with maltopentaose (TableII). This observa- 0 10 50 75 100 150 maltotetraose tion may substantiate the scheme for degradation of leaf starch Inhibitor, reaction mixture Atg/250O,1 which was proposed by Okita et al. (6). They suggested that

FIG. 7. Pseudo-oligosaccharides, acarbose (x) glucose is one of the major products of starch degradation. However, the longer the chain length of maltodextrin, the less inhibition of D-enzyme (DI). 1-Enzyme was first the production of glucose (Table II). This may be because the and then other components used in 1-enzyme transfer of a maltosyl group from maltodextrins to the nonre- control assay showed that different quantities of ducing end ofan acceptor is much favored than transfer of larger same quantity of 1)-enzyme did not cause any reading at absorbance 340 nm. maltodextrinyl group. The products generated from glucose and amylopectin included maltotriose in an early incubation time ing 30 min incubation. Again maltose was not (Fig. 4c). Maltotriosyl group transfer also occurs but at decreased maltopentaose or maltoheptaose was used as level (Fig. 4c). oligosaccharides were detected; however, glucose maltotriose It is an interesting observation that maltotriose quickly be- were the major products after 2 h incubation comes dominant in the reaction of D-enzyme with maltotetraose Figure 6 shows the distribution of different maltodextrins (Fig. 4b), maltopentaose, or with maltoheptaose (data not the reaction of 1-enzyme with C]maltotriose. (GI) shown). These results might be explained as follows. Maltohep- formed quickly; maltodextrins with a chain length taose and glucose are the first products in the reaction of D- maltooctaose (G8) also can be detected, this indicates enzyme withmaltotetraose. The following reaction involves three todextrins with a chain length greater than maltotetraose substrates, i.e. maltotetraose, glucose, and maltoheptaose. Trans- also be used as an acceptor. It seems glucose and fer of maltosyl group from maltoheptaose to glucose (or malto- accumulating gradually, while other higher maltodextrins tetraose) generates maltopentaose and maltotriose (or malto- equilibrium in the exchanges as an acceptor and hexaose). Another transfer ofmaltosyl group from maltopentaose Glucose production from incubating different to glucose or other maltodextrins will generate maltotriose. with E-enzyme (D l) has been measured at different Transferring of maltotriosyl group from maltohexaose to other and maltotriose is the best substrate (Table II). Glucose, maltodextrins also gives rise to maltotriose. However, transfer- was not detected after incubating amylopectin D-enzyme ring of a maltosyl group from maltodextrins is more favorable for 4 h. than a maltotriosyl group (Fig. 4c). Walker and Whelan (12) showed that in the presence Maltotriose as the major product also has been observed in and glucose, 1-enzyme transferred maltosyl moieties the reaction of D-enzyme with C-glucose and Lintner soluble to glucose. The paper chromatogram in Figure starch (see "Materials and Methods"). Of the total radioactivity, maltotriose is the only detectable product after mmi 8.5% was incorporated in maltotriose, which is far more abun- of glucose and amylopectin. After 1.5 h, in addition dant in quantity than the sum of the other maltodextrins. One triose, maltotetraose appears as a minor product. hypothesis comes up from these experiments. During the starch maltodextrins can be detected even after 5 h degradation in the chloroplast, maltotriose probably is the pref- Two major D-enzyme activity peaks were obtained erential product from the reaction of D-enzyme with maltodex- leaf tissue in this study. Even though D2 may D- trins with long chain length. Removing the glucose by shunting enzyme, in terms of action pattern, Dl and it to other metabolic pathways, and the activity of the starch difference in using maltotriose as substrate. It hydrolytic enzymes will change the concentration ofthe different the physiological role of the different forms of D-enzyme maltodextrins. Maltotriose and other short maltodextrins even- in the leaf tissue. tually will lead to more glucose production as seen in Figure 4, Other Properties of The heat stability 1-enzyme D-Enzymes. a and b. The various maltodextrins which formed in the D- has been (Dl and D2) studied. D-enzymes enzyme reaction, can also be further depolymerized by D-enzyme when the enzyme preparations were initially 60°C itself or by starch phosphorylase. for 10 mmn either in the presence or absence of mm CaCl2. The absence of maltose formed from the incubation of D- Several pseudo-oligosaccharides known as inhibitors enzyme with maltotriose, or with maltotetraose (Fig. 4), or with and (1 1) were tested for their lase glucosidase maltopentaose indicate the penultimate linkage is not available Bay e 4609 was an effective inhibitor. The concentration for 1-enzyme. The products formed from the incubation of D- e 4609 and acarbose needed for 50% inhibition,ug/ml enzyme with glucose and maltotriose (Fig. 3), and D-enzyme 220 pg/mI, respectively (Fig. 7). Deoxynojirimycin, a-amylase with glucose and amylopectin (Fig. 4c) indicate the nonreducing inhibitor (extracts of wheat germ, Sigma Chemical a-1and ,4-inkage ofthe maltodextrin is not available for cleavage NEM, however, were not inhibitors. either. These are the two forbidden a-l1,4linkages that were first observed by Peat et al. (7). It would be interesting to see what is DISCUSSION the mechanism of 1-enzyme imposing these constraints in the

The most effective step in the separation and transferring maltodextrinyl group between maltodextrin.

D-enzyme from Arabidopsis leaf tissue in this study D-Enzyme are probably the only starch degradativeenzymt of FPLC-Mono Q column chromatography. This that cannot change the total mole concentration of original lowed the resolution of 1-enzyme and amylase -su-bstrate. The observation that no maltose is produced in the ARABIDOPSIS LEAF D ENZYME 265 incubation of maltodextrins with D-enzyme ofArabidopsis, and 6. OKITA TW, E GREENBERG, DN KUHN, J PREISS 1979 Subcellular localization of starch degradative and biosynthetic enzymes of spinach leaves. Plant the changes of the pattern and the relative quantity of products Physiol 64: 187-192 produced from the reaction of D-enzyme with maltotriose is very 7. PEAT S, WJ WHELAN, WR REES 1956 The enzymic synthesis and degradation similar to the potato D-enzyme. of starch, part 20, the disproportionating enzyme of potato. J Chem Soc p 44 LITERATURE CITED 8. SMITH PK, RI KROHN, GT HERMANSON, AK MALLIA, FH GARTNER, MD PROVENZANO, EK FuJIMoTO, NM GOEKE, BJ OLSON, DC KLENK 1985 1. JONES G, WJ WHELAN 1969 The action pattern of D-enzyme, a transmaltodex- Measurement of protein using bicinchoninic acid. Anal Biochem 150: 76- trinylase from potato. Carbohydr Res 9: 483-490 85 2. LEE EYC, WJ WHELAN 1971 Glycogen and starch debranching enzyme. In 9. SOMERVILLE CR, WL OGREN 1981 Isolation of photorespiration mutants in A. PD Boyer, ed, The Enzymes, Ed 3, Vol 5. Academic PRess, New York, pp Thaliana. In M Edelmam, R Hallick, NH Chua, eds, Methods in Chloroplast 191-234 Molecular Biology. Elsevier, New York, pp 129-138 3. LEVI C, J PREISS 1978 Amylopectin degradation in pea chloroplast extracts. 10. TREVELYAN WE, DP PROCTER, JS HARRISON 1950 Detection of sugars on Plant Physiol 61: 218-220 paper chromatograms. Nature 166: 444 445 4. MANNERS DJ, KL ROWE 1969 Studies on carbohydrate-metabolizing enzymes, 11. TRUSCHEIT E, W FROMMER, B JUNGE, L MULLER, DD SCHMIDT, W WINGEN- part 21, the a-glucosidase and D-enzyme activity of extracts of carrots and DER 1981 Chemistry and biochemistry of microbial a-glucosidase inhibitors. tomatoes. Carbohydr Res 9: 441-450 Angew Chem Int Ed Engl 20(9): 744-761 5. NELsON N 1944 A photomeric adaptation of the Somogyi method for the 12. WALKER GL, WJ WHELAN 1957 The mechanism of carbohydrase action. For determination of glucose. J Biol Chem 153: 357-380 the mechanism of D-enzyme action. Biochem J 67: 548-551