Báo cáo Y học: Comparison of starch branching enzyme I and II from potato ppt

Comparison of starch branching enzyme I and II from potato Ulrika Rydberg 1 , Lena Andersson 2 , Roger Andersson 2 , Per A ˚ man 2 and Ha ˚ kan Larsson 1 1 Department of Plant Biology, and 2 Department of Food Science, Swedish University of Agricultural Sciences, Uppsala, Sweden The in vitro activities of purified potato starch branching enzyme (SBE) I and II expressed in Escherichia coli were compared using several assay methods. With the starch– iodine method, it was found that SBE I was more active than SBE II on an amylose substrate, whereas SBE II was more active than SBE I on an amylopectin substrate. Both enzymes were stimulated by the presence of phosphate. On a substrate consisting of linear dextrins (chain length 8–200 glucose residues), no significant net increase in molecular mass was seen on gel-permeation chromatography after incubation with the enzymes. This indicates intrachain branching of the substrate. After debranching of the products, the majority of dextrins with a degree of polymerization (dp) greater than 60 were absent for SBE I and those with a dp greater than 70 for SBE II. To study the shorter chains, the debranched samples were also analysed by high-performance anion-exchange chromatography. The products of SBE I showed distinct populations at dp 11–12 and dp 29–30, whereas SBE II products had one, broader, population with a peak at dp 13–14. An accumulation of dp 6–7 chains was seen with both isoforms. Keywords: gel-permeation chromatography (GPC); high- performance anion-exchange chromatography (HPAEC); Solanum tuberosum; starch branching enzyme; starch. Starch is composed of linear and branched chains of a- D-glucose residues. The starch branching enzymes (EC 2.4.1.18), which are responsible for forming a-1,6-linkages in the glucan, can be divided into two classes, class A (e.g. potato and maize SBE II, pea SBE I) and class B (e.g. potato and maize SBE I, pea SBE II). The A and B isoforms have highly similar amino-acid sequences but usually differ by an N-terminal extension of the B form and a C-terminal extension of the A form [1,2]. In vitro studies of the maize isoforms have shown that SBE I preferentially branches amylose, whereas SBE II preferen- tially branches amylopectin [3]. Furthermore, SBE I transfers longer chains than SBE II in vitro, and it has been suggested that SBE I takes part in the synthesis of long and intermediate chains during amylopectin biosynthesis [4]. This model is supported by the observation of an increased average chain length in amylopectin of amylose- extender maize mutants that lack SBE II [5]. There is no known mutant with reduced SBE I; however, the chain length distribution in amylopectin was not significantly affected in transgenic potato plants with a reduced level of SBE I [6,7]. Interestingly, the physical properties of the starch from transgenic potato with reduced SBE I levels are clearly changed [6–8]. SBE I from potato was first characterized as having a relative mass of 80/85 kDa [9,10]. In 1991 it was shown that intact potato SBE I had a relative molecular mass of 103 kDa [11]. The active 80/85-kDa form present in potato tubers was isolated and shown to have an almost intact N-terminus and thus thought to result from proteolytic cleavage in the C-terminal part [12]. Both intact SBE I and the 85-kDa form have been shown to transfer chains from a donor chain to an acceptor chain (interchain branching) [13,14]. The occurrence of intrachain branching, i.e. transfer within one and the same chain, could not be excluded in those experiments. Thorough studies of the activity of maize SBE I and II isolated from endosperm [3] or expressed in Escherichia coli [15,16] have been performed on various substrates. Potato SBE II was first observed to be present as a granule- bound protein in tuber starch [17]. SBE II seems to be less abundant in potato tubers than SBE I and has not been isolated from potato in amounts required for activity analysis. Recently, however, both isoforms of potato SBE have been expressed in E. coli [18–20], and the present paper reports the activity of potato SBE I and SBE II with amylose, amylopectin and linear dextrins as substrates. MATERIALS AND METHODS Branching enzyme isoforms Potato SBE I and II were expressed in E. coli and purified by ammonium sulfate precipitation, starch affinity chroma- tography, and anion-exchange chromatography, as described by Khoshnoodi (SBE I) [18] and Larsson (SBE II) [19]. The preparations of potato SBE I and SBE II expressed in E. coli were judged to be highly pure as SDS/PAGE followed by Coomassie blue staining revealed only one additional, faint band for SBE II and none for SBE I [19]. The protein concentration was measured by the Bradford method with BSA as standard. Aliquots of 1 m M in 50 mM Correspondence to H. Larsson, Department of Plant Biology, SLU, PO Box 7080, SE-750 07, Uppsala, Sweden. Fax: 1 46 18 673279, Tel.: 1 46 18 673396, E-mail: Hakan.Larsson@vbiol.slu.se Enzymes: starch branching enzyme (EC 2.4.1.18); isoamylase (EC 3.2.1.68). Note: a wep page is available at http://www.vbiol.slu.se/ (Received 4 July 2001, revised 27 September 2001, accepted 28 September 2001) Abbreviations: dp, degree of polymerization; GPC, gel-permeation chromatography; HPAEC-PAD, high-performance anion-exchange chromatography with pulsed amperometric detection; SBE, starch branching enzyme. Eur. J. Biochem. 268, 6140–6145 (2001) q FEBS 2001 Tris/HCl, pH 7.5, containing 1 mM dithiothreitol and 10% glycerol buffer were stored at 270 8C until use. Determination of branching enzyme activity on amylose and amylopectin with the starch–iodine assay Amylose (type III, Sigma) and amylopectin (Sigma) from potato were typically dissolved at 10 mg : mL 21 in 0.5 M NaOH. The solutions were buffered with 1 M KH 2 PO 4 and pH adjusted to 7.5 with NaOH. The reaction mixtures contained 0.6 mg : mL 21 substrate, 90 mM KH 2 PO 4 , and 0.01 m M branching enzyme. Incubations were performed at room temperature (22 8C), and aliquots were withdrawn at several intervals between 5 and 180 min after the addition of the SBE and terminated by heating at 95 8C for 5 min. A 100-mL sample of each aliquot was mixed with 900 mL iodine solution (0.0125% I 2 and 0.04% KI, freshly made from a 100 Â stock solution), and the absorbance between 400 and 800 nm was measured immediately on a Beckman PU-70 spectrophotometer. The control did not contain branching enzyme, but was otherwise treated as the other samples. The experiments were repeated at least twice with essentially the same results. When phosphate stimulation was investigated, Tris/HCl, pH 7.5 (final concentration 50 m M) was used to buffer the reaction mixtures and KH 2 PO 4 , pH 7.5, was added to obtain increasing concen- trations of phosphate. Incubations were terminated at 120 min and analysed by the starch –iodine assay. Incubation of linear dextrins with branching enzyme for gel-permeation chromatography (GPC) and high-performance anion-exchange chromatography (HPAEC) Linear dextrins with a relatively narrow weight range were produced by enzymatic degradation of retrograded starch by the method of Andersson et al. [21]. The linear dextrins were dissolved in a small volume of 2 M KOH and diluted with Tris buffer to a final concentration of 4mg : mL 21 dextrins and 50 mM Tris/HCl, pH 7.6. To 900 mL of this solution was added 100 mL1m M branching enzyme or water (control sample). The samples were incubated at room temperature for 16 h, and the reactions terminated by heating at 100 8C for 5 min. After addition of 150 mL1 M acetate buffer, pH 3.6, the samples (1 mL) were debranched with 295 U isoamylase (Hayashibara Biochemical Laboratories Inc., Okayama, Japan) for 5 h at 38 8C. Before injection on to a column, the reaction was terminated by heating to 100 8C for 5 min, and the pH adjusted to . 10 with NaOH as described by Andersson et al. [21]. Chromatographic methods GPC was conducted as previously described [22] using a Sepharose CL-6B column eluted with 0.25 M KOH. The relative amounts of carbohydrate in the collected fractions were measured by the phenol/sulfuric acid method [23]. HPAEC-PAD and a CarboPac PA-100 column was used as described by Koch et al. [24]. In this method, correction for detector response is performed. All experiments were run in duplicate with only small differences between the samples. RESULTS AND DISCUSSION Comparison of SBE I and SBE II on the amylose and amylopectin substrates To compare the activity properties of potato SBE I and SBE II, commercially available amylose and amylopectin were used as substrates in a kinetic study using the starch – iodine method (Fig. 1). SBE I was more active than SBE II on the amylose substrate, whereas SBE II was more active than SBE I on the amylopectin substrate. For both enzymes, the greatest effect was observed on the amylose substrate where the A 655 with SBE I decreased to 27% of that of the control, and with SBE II it decreased to 46% (Fig. 1A). On the amylopectin substrate, the A 520 with SBE I decreased to 74% of the control and with SBE II to 64% (Fig. 1B). Thus, the potato isoforms differed in that SBE I was more active than SBE II on amylose and SBE II was more active than SBE I on amylopectin, which is in accordance with the results obtained with maize SBE I and II [3,15,16]. The l max of the amylose substrate shifted from 616 nm to 543 nm after incubation for 180 min with SBE I and to 574 nm after incubation with SBE II for the same time Fig. 1. Activity of SBE I and SBE II over time. Absorbance of the starch–iodine complex after incubation of amylose substrate (A), measured at 655 nm, or amylopectin substrate (B), measured at 520 nm, for different periods of time with SBE I (O) or SBE II (Â)in90m M phosphate buffer. The absorbance of the control samples without enzyme (set to 100%) was 1.09 for the amylose substrate and 0.53 for the amylopectin substrate. q FEBS 2001 Comparison of SBE I and II from potato (Eur. J. Biochem. 268) 6141 (Fig. 2A). Incubation overnight did not notably further change the l max (data not shown). The difference in final l max values and a comparison of the shapes of the two spectra indicate that SBE I reduced more efficiently than SBE II the long linear chains that mainly give rise to absorbance above 600 nm. Similar differences between the final l max values with amylose as a substrate were previously observed with maize SBE I and II [3,15,16]. The l max values after incubation with the amylopectin substrate shifted from 551 nm to 522 nm with SBE I, and to 538 nm with SBE II, after 180 min of incubation (Fig. 2B). Similar values were obtained after incubation overnight (not shown). These results differed from those with the maize isoforms, which both reduced the l max from 530 nm to about 490 nm [15,16]. Although this suggests that there may be a difference between the enzymes from maize and potato, the divergent results could also be due to a difference between the substrates, with relatively long and linear chains in potato amylopectin as indicated by the relatively high l max of 551 nm as compared with a l max of 530 nm with the maize amylopectin [15,16]. Effect of phosphate on the activity of potato SBE I and II Phosphate has been reported to increase the branching activity of SBE I and II from wheat [25] and SBE I from potato [26]. The activity assay shown in Fig. 1 was performed in 90 m M phosphate. To investigate the effect of phosphate, increasing concentrations of phosphate from 0 to 135 or 180 m M were included in the iodine-activity assay with commercially available amylose and amylopectin as substrates. The delta absorbance at 655 nm for the amylose substrate and 520 nm for the amylopectin substrate, after 120 min of incubation, as a function of phosphate concentration is shown in Fig. 3. Phosphate concentration did not affect the absorbance of the starch–iodine complex in samples without enzyme (data not shown). Close to maximal activation of both SBE I and II was obtained at 90 m M phosphate with the amylose substrate as well as with the amylopectin substrate. With the amylopectin substrate, the stimulatory effect was 130% and 40% for SBE I and II, respectively. Half-maximal Fig. 2. Activity of SBE I and SBE II after 180 minutes. Absorbance spectra of the starch–iodine complex of the amylose substrate (A) and the amylopectin substrate (B) incubated for 180 minutes with SBE I (dashed lines), SBE II (dotted lines), or control sample without enzyme (solid lines) in 90 m M phosphate buffer. The vertical lines denote the l max of the spectra. Fig. 3. Effect of phosphate on the activity of SBE I and SBE II. The delta absorbance of the amylose substrate (A), measured at 655 nm, and the amylopectin substrate (B), mesured at 520 nm, after 120 min of incubation with SBE I (K) or SBE II (Â) in increasing concentrations of phosphate. Delta absorbance is defined as the difference between the absorbance of the starch–iodine complex of the control and the samples. 6142 U. Rydberg et al.(Eur. J. Biochem. 268) q FEBS 2001 activation was obtained for both isoforms at 15–20 mM phosphate with both substrates, which is similar to that reported for wheat SBE I and II [25]. A fivefold activation by 10 m M phosphate of potato and wheat SBE I has been reported previously [25,26]. The effect of phosphate is dependent on the buffer conditions [26], which could explain the divergent results for potato SBE I. From the studies performed by us and others, it cannot be excluded that the observed stimulatory effect is a consequence of the phosphate ions interacting with the substrate, and thereby changing its structure, leading to enhanced enzyme reactions. Further investigations are required to clarify this and whether the effect of phosphate is of relevance in vivo. Branching of linear dextrins To obtain a more detailed comparison of the mode of action of SBE I and SBE II, the branching products were further examined using linear dextrins, prepared from commer- cially available retrograded high-amylose maize starch [21], as substrate. The majority of the chains of this substrate were longer than 8 but shorter than 200 glucose residues and had a peak maximum at degree of polymerization (dp) < 60. These dextrins were less complex than commercially available amylose or amylopectin and therefore more suitable as substrates for the analysis of the branching properties of SBEs by chromatographic methods. The molecular mass distribution of the dextrin substrate and the products formed after the branching process were analysed by GPC. The elution profiles of the dextrins after incubation with SBE I or SBE II for 16 h revealed only small changes compared with the original substrate (Fig. 4A). The absence of an increase in molecular mass indicates that both enzymes mainly produced intrachain branches, as we have previously reported for SBE I [21]. However, interchain branching cannot be excluded. Inter- chain transfer of chains by 80/85-kDa potato SBE I has been demonstrated by Borovsky et al. [14]. In a more recent study, materials with higher molecular mass were formed, possibly by multiple chain-transfer reactions, from linear dextrins with relatively low molecular masses (dp 30 –40) when incubated with 103-kDa SBE I from potato [13]. The results show that potato SBE I has the ability to incorporate glucans into starch in an interchain catalytic reaction, although intrachain reactions could not be excluded. Thus, in contrast with these previous studies, the results in Fig. 4A suggest that potato SBE I and SBE II also produce intrachain branches. The discrepancies between the studies may be explained by differences in molecular masses and phosphorylation of the substrates [13] or by differences between the enzymes used. The experiments of Viksø- Nielsen et al. [13] and Borovsky et al. [14] were performed in 50 m M phosphate and 100 mM citrate, respectively. The results shown in Fig. 4 were obtained in the absence of phosphate, but the same elution pattern was obtained in the presence of 90 m M phosphate (data not shown). Thus it seems that SBE can produce branches by both intrachain and interchain branching, depending on external factors. After debranching with isoamylase, the GPC elution profiles were shifted to lower molecular masses compared with the original substrate, showing that extensive branching had taken place (Fig. 4B). A more pronounced effect was seen for SBE I than for SBE II. It is notable that, for both enzymes, essentially all high-molecular-mass material had disappeared. For SBE I, the majority of the dextrins with a dp greater than 60 were missing and for SBE II those greater than 70. At the same time, the proportion of short chains was slightly increased for both enzymes and some new chains shorter than those in the original substrate were detected. These results are in agreement with the results from the starch–iodine assay. Similarly, the product of maize SBE II contained a higher amount of the longest chains than the SBE I product [4]. To obtain a more detailed picture of the individual chains produced by the enzymes, quantitative analyses of the shorter unit chains (dp 6–47) were performed by HPAEC. The relative distribution of the original substrate showed a broad peak with no distinct populations with chains down to dp 6 (Fig. 5C). By debranching the substrate with Fig. 4. Activity of SBE I and SBE II on linear dextrins analysed with GPC. Elution profiles of linear dextrins after incubation with SBE I (K), SBE II (Â) or control samples without SBE added (W)in 50 m M Tris buffer. Elution profiles were obtained before (A) and after (B) debranching with isoamylase. Data for SBE I has previously been published in Andersson et al. [21]. Dp values obtained after column calibration with pullulan standards are shown on the upper axes. q FEBS 2001 Comparison of SBE I and II from potato (Eur. J. Biochem. 268) 6143 isoamylase, the presence of 1,6-linkages in the substrate could be excluded (not shown). After incubation with SBE I and debranching with isoamylase (Fig. 5A), major popu- lations were found around dp 11 –12 and 29–30, respectively, as previously reported [21]. The unit chains with a high dp were present in only small amounts. Incubation with SBE II revealed a different picture (Fig. 5B). The most abundant chains, on a weight basis, had a dp around 13 –14 and a considerable quantity of chains with dp 6 was produced. SBE II seems to be less efficient in using the longer chains as a substrate than SBE I as the longer unit chains were present in larger amounts in the SBE II product. The original substrate had a broad range of chains that to some extent interfered with the product chains, making it difficult to interpret the results quantitatively. The results from all three analyses show that SBE I was capable of branching chains that were not branched by SBE II. The mechanism of chain transfer for maize branching enzymes has previously been investigated using reduced amylose (chain length 405) as substrate. The study of maize SBE I showed populations of transferred chains with a dp of 11 –14 and 31 after debranching of the enzyme products [4]. A more detailed investigation of the shorter chains (, dp 34) produced by maize SBE I revealed an increase in chains of dp 11–12 as well as of dp 6 [27]. Maize SBE II has been shown to transfer shorter chains than maize SBE I, and the most abundant chains were reported to be around dp 9 by Takeda et al. [4], whereas Guan et al. [27] reported an increase in chains of dp 6 –7 with a smaller peak at dp 10–12. In accordance with this, incubation with potato SBE I and II generated chains of dp 6–9, in decreasing concentrations, which has been shown to be a general feature for amylopectin in potato [28]. Thus, it is possible that during biosynthesis of amylopectin the branching enzymes produce a fraction of very short chains which are normally elongated by starch synthase III, as indicated by the interesting results of Edwards et al. [29] and work by Abel, as reviewed in Kossmann & Lloyd [8], showing that the relative amount of dp 6 chains in amylopectin was significantly higher in transgenic potato lines with reduced levels of starch synthase III. The presence of phosphate interfered with the chroma- tography of the carbohydrates on the HPAEC column. Therefore the samples shown here were incubated in a Tris- buffer. However, samples incubated in a phosphate buffer gave the same elution patterns (not shown). The absence of phosphate, which has been shown to influence branching enzyme activity, did not qualitatively change the branching patterns of the isoforms in our study. This study was performed with purified potato SBE I and II that had been expressed in E. coli. The specific activity of expressed SBE I was about twofold higher than SBE I isolated from potato tubers [18], indicating that the expressed SBE I was fully active. We have failed to isolate active SBE II from potato and to our knowledge it has not been achieved. However, as the activity of expressed SBE II was higher on the amylopectin substrate compared with that of expressed SBE I, it is resonable to assume that the expressed SBE II was also fully active. The results presented here show that there are significant differences in activity characteristics between potato SBE I and II. Further studies are needed in order to fully understand the functions of the two enzymes and the detailed structure of the products obtained. In conclusion we found that: (a) potato SBE I was more active than SBE II on long linear substrates and SBE II was more active than SBE I on an amylopectin substrate; (b) the activity of both isoforms increased in the presence of phosphate; (c) GPC results indicate that both SBE I and SBE II mainly branched the linear dextrins used in this study by intrachain branching; (d) debranching of the products showed that both isoforms produced a small fraction of dp 6–7 chains and a larger fraction of chains < dp 11–14, and in addition SBE I produced a population of dp 29–30 chains. ACKNOWLEDGEMENTS We are grateful to Dr E. Johansson who expressed and purified the starch branching enzymes used in our experiments. 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Biochem. 268) 6145 . branching enzyme activity on amylose and amylopectin with the starch iodine assay Amylose (type III, Sigma) and amylopectin (Sigma) from potato were typically dissolved at 10 mg : mL 21 in 0.5 M NaOH and 2 Department of Food Science, Swedish University of Agricultural Sciences, Uppsala, Sweden The in vitro activities of purified potato starch branching enzyme (SBE) I and II expressed in Escherichia coli. activity of potato SBE I and II Phosphate has been reported to increase the branching activity of SBE I and II from wheat [25] and SBE I from potato [26]. The activity assay shown in Fig. 1 was performed