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

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

Comparison of starch branching enzyme I and II from potato

Ulrika Rydberg1, Lena Andersson2, Roger Andersson2, Per A˚man2and Ha˚kan Larsson1

1 Department of Plant Biology, and2Department 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

M A T E R I A L S A N D M E T H O D S 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 chromachroma-tography, 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 in 50 m

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.

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:mL21 in 0.5M

NaOH The solutions were buffered with 1MKH2PO4and

pH adjusted to 7.5 with NaOH The reaction mixtures

contained 0.6 mg:mL21 substrate, 90 mM KH2PO4, and

0.01 mMbranching 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% I2 and 0.04% KI, freshly made

from a 100  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 mM) was used to buffer the reaction mixtures and

KH2PO4, 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 2MKOH and

diluted with Tris buffer to a final concentration of

4 mg:mL21 dextrins and 50 mM Tris/HCl, pH 7.6 To

900 mL of this solution was added 100 mL 1 mMbranching

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 mL 1M 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 0.25M 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

R E S U L T S A N D D I S C U S S I O N

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 A655with 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 A520with 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 lmaxof 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 () in 90 m 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.

(Fig 2A) Incubation overnight did not notably further

change the lmax(data not shown) The difference in final

lmax 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 lmax values with amylose as a substrate were

previously observed with maize SBE I and II [3,15,16]

The lmax 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 lmax 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

lmaxof 551 nm as compared with a lmaxof 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 mMphosphate To investigate the effect of phosphate, increasing concentrations of phosphate from 0 to

135 or 180 mM 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 mMphosphate 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 () 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.

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 mM 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 Inter-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 mMphosphate and 100 mMcitrate, 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 mM 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 () 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.

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

A C K N O W L E D G E M E N T S

We are grateful to Dr E Johansson who expressed and purified the starch branching enzymes used in our experiments This work was funded by the Swedish Foundation for Strategic Research and the Swedish Farmer’s Foundation for Agricultural Research.

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