Starch in food

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Starch in food

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Starch in food Structure, function and applications

Edited by Ann-Charlotte Eliasson

Published by Woodhead Publishing Limited

Abington Hall, Abington

First published 2004, Woodhead Publishing Limited and CRC Press LLC

ß 2004, Woodhead Publishing Limited

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Part I Analysing and modifying starch

1 Plant starch synthesis

J Preiss, Michigan State University, USA

1.1 Introduction: localization and function of starch in plants

1.2 Starch synthesis: enzyme reactions in plants and algaeand glycogen synthesis in cyanobacteria

1.3 Properties of plant glucan synthesizing enzymes:

1.6 Initiation of starch synthesis using a glucosyl-protein

1.7 Locating starch synthesis in plants: the plastid

1.8 In vivo synthesis of amylopectin

1.9 Regulating starch synthesis in plants

1.10 References

2 Analysing starch structure

E Bertoft, AÊbo Akademi University, Finland

2.1 Introduction: characterising structures of starch components

2.2 Fractionation of starch

2.3 Analysis of amylose

Contents

2.4 Analysis of amylopectin structure

2.5 Analysis of intermediate materials

2.6 Analysis of chemically modified starches

3.1 Introduction: the importance of starch

3.2 Technologies for genetic modification and starch profiling

3.3 Improving starch yield and structure

3.4 Physical and chemical properties of modified starches

3.5 Functionality and uses of modified starches in

4.2 Using enzymes to modify starch

4.3 Developing starch-modifying enzymes for food

processing applications

4.4 Future trends

4.5 References

5 Understanding starch structure and functionality

A M Donald, University of Cambridge, UK

5.1 Introduction: overview of packing at different lengthscales

5.2 The effect of amylopectin chain architecture on packing

5.3 Improving packing within starch granules

5.4 The gelatinisation process

5.5 Food processing: implications of starch granule structure

5.6 Conclusions and future trends

5.7 Sources of further information and advice

5.8 References

6 Measuring starch in food

M Peris-Tortajada, Polytechnic University of Valencia, Spain6.1 Introduction

6.2 Sample preparation

6.3 Methods of analysing starch in food

6.4 Determining starch in food: recent technological

developments

6.5 Future trends

6.6 Sources of further information and advice

6.7 References

Part II Sources of starch

7 The functionality of wheat starch

H Cornell, RMIT University, Australia

7.1 Introduction: manufacture of wheat starch for the

food industry

7.2 Granular and molecular structure of wheat starch

7.3 Functionality of wheat starch: granules, films and pastes

7.4 Rheological properties of starch pastes and gels

7.5 Improving and chemically modifying wheat starch for use

in the food industry

7.6 Wheat starch syrups

7.7 Analysing starch-based products

7.8 Future trends

7.9 Sources of further information and advice

7.10 References

8 Developments in potato starches

W Bergthaller, Federal Centre for Nutrition and Food, Germany8.1 Introduction

8.2 Components and rheological properties of potato starch

8.3 Techniques for producing potato starch

8.4 Improving the functionality of potato starch for use inthe food industry

8.5 Future trends

8.6 References

9 The functionality of rice starch

J Bao and C J Bergman, Texas A&M University, USA

9.1 Introduction

9.2 Rice flour and starch as food ingredient

9.3 Constituents of rice starch

9.4 Structure and functionality of rice starch

9.5 Gelatinization and the structure of rice starch

9.6 Retrogradation and other properties of rice starch

9.7 Improving rice starch functionality for food

processing applications

9.8 Future trends

9.9 Sources of further information and advice

9.10 References

10 New corn starches

P J White and A Tziotis, Iowa State University, USA

10.1 Introduction: the use of corn starch in food processing

10.2 Improving the functionality of corn starch for foodprocessing applications: natural corn endosperm mutants

10.3 Chemically modifying corn starches for use in thefood industry

10.4 Genetically modifying corn starches for use in thefood industry

10.5 Future trends

10.6 Sources of further information and advice

10.7 References

11 Tropical sources of starch

S N Moorthy, Central Tuber Crops Research Institute, India11.1 Introduction: tropical sources of starch

11.2 Characteristics and properties of cassava starch

11.3 Characteristics and properties of sweet potato starch

11.4 Characteristics and properties of yam and aroid starches

11.5 Characteristics and properties of other minor root starches

11.6 Modifying `tropical' starches for use in the food industry

11.7 Future trends

11.8 References

Part III Applications

12 Starch as an ingredient: manufacture and applications

P Taggart, National Starch and Chemical, UK

12.6 Uses and applications

12.7 Regulatory status: European label declarations

12.8 Acknowledgements

12.9 Bibliography

13 Utilizing starches in product development

T Luallen, Cargill Inc., USA

13.1 Introduction

13.2 Components of starch

13.3 Food applications for natural and modified starches

13.4 Methods of starch selection

13.5 Factors affecting starch in food products

13.6 Using the functional properties of starch to enhancefood products

13.7 References

14 Modified starches and the stability of frozen foods

H D Goff, University of Guelph, Canada

14.1 Introduction

14.2 The structure and stability of frozen foods

14.3 The role of modified starch in stabilizing frozen foods

14.4 Future trends

14.5 Sources of further information and advice

14.6 References

15 Starch-lipid interactions and their relevance in food products

A-C Eliasson and M Wahlgren, Lund University, Sweden

15.1 Introduction

15.2 The structure and properties of the starch-lipid complex

15.3 Analysis of starch: lipids and emulsifiers

15.4 The effect of lipids on starch behaviour

15.5 Enzymatic degradation of amylose-lipid complexes

15.6 Future trends

15.7 References

16 Starch-based microencapsulation

P Forssell, VTT Biotechnology, Finland

16.1 Introduction: using microencapsulation in food processing

16.2 Using starch in microencapsulation: starch hydrolysates,derivatives, polymers and granules

16.3 Starch-based shell matrices for food ingredients

16.4 Future trends

16.5 References

Part IV Starch and health

17 Development of a range of industrialised cereal-based

foodstuffs high in slowly digestible starch

V Lang, Danone Vitapole, France

17.1 Introduction

17.2 Characteristics and properties of starch and starchy foods

17.3 Low G I diets and their associated health benefits

17.4 Case study: low glycaemic index, high slowly digestiblestarch plain biscuits, the EDPÕ(`Long-lasting energy')range developed by Danone, Vitapole

17.5 Future trends

17.6 Sources of further information and advice

17.7 Acknowledgements

17.8 References

18 Starch: physical and mental performance

F Brouns, Cerestar Vilvoorde R & D Centre, Belgium and

University of Maastricht, Netherlands and L Dye, University of Leeds,England

18.1 Introduction

18.2 Physical performance: energy requirements, delivery

and availability

18.3 Mental performance: the effects of glucose

18.4 Mental performance: the effects of CHO and glucose

during the day

18.5 Future trends

18.6 References

19 Detecting nutritional starch fractions

K Englyst and H Englyst, Englyst Carbohydrates, UK

19.1 Introduction

19.2 Methods of determining RAG, SAG and RS fractions

19.3 Quality control and troubleshooting

19.4 Carbohydrate bioavailability data for selected foods

19.5 Conclusion and future trends

20.2 Effects of resistant starch on the digestive system

20.3 Improving the functional effects of resistant starch

20.4 Future trends

20.5 Sources of further information and advice

20.6 References

21 Analysing starch digestion

R E Wachters-Hagedoorn, M G Priebe and R J Vonk,

University Hospital Groningen, The Netherlands

21.1 Introduction

21.2 Starch and the prevention of hypo- and hyperglycemia

21.3 The determinants of the rate of absorption of

starch-derived glucose

21.4 Techniques for monitoring starch digestion

21.5 Current applications of slowly available starch and theprevention of hyper- and hypoglycemia

21.6 Future trends

21.7 Sources of further information and advice

21.8 References

Chapter 3

Dr A BlennowThe Royal Agricultural andVeterinary UniversityDenmark

GroningenThe NetherlandsE-mail: dbutler38@hotmail.commaarel@voeding.tno.nl

Contributor contact details

Location Detmoed and MuensterInstitute for Cereal, Potato and StarchTechnology

PO Box 1354Detmold 32756GermanyTel: + 49 5231 741320Fax: + 49 5231 741300E-mail: w.bergthaller@bagkf.de;wolfgang.bergthaller@t-online.de

Chapter 9

Dr C BergmanUniversity of NevadaLas Vegas

Nevada 89154USA

Dr J BaoZhejiang UniversityHuajiachi

Hangzhou 310029China

E-mail: bergman5@univ.nevada.edujsbao@zju.edu.cn

Chapter 10

Professor P J White and Ms A

Tziotis

Department of Food Science and

Human Nutrition and Centre for

Crops Utilization Research

2312 Food Sciences Building

Iowa State University

B U

PO Box 1467Cedar Rapids

IA 52406USATel: 319 399 6187Fax: 319 399 6123E-mail: tom_luallen@cargill.com

Chapter 14

Professor H D GoffDepartment of Food ScienceUniversity of GuelphGuelph

Ontario N1G 2W1Canada

Tel: 519 824 4120Fax: 519 824 6631E-mail: dgoff@uoguelph.ca

Chapter 15

Professor A-C Eliasson and

M WahlgrenFood Technology DivisionLund University

Box 124

22100 LundSwedenTel: +46 222 9674Fax: +46 222 9517E-mail: ann-charlotte.eliasson@livsteki.lth.se

R&D Center of the Groupe Danone

Nutrition Research Department

Professor F Brouns and Dr L Dye

Cerestar Vilvoorde R&D Centre

SO16 7NPUKTel: +44 (0) 23 80 769650Fax: +44 (0) 23 80 769654Email: klaus@englyst.co.uk

Chapter 20

Dr M ChampINRA-UFDNH/CRNHRue de la GeÂraudieÁre

BP 71627

44316 Nantes Cedex 03France

Email: champ@nantes.inra.fr

Chapter 21

Professor R J VonkLaboratory of Nutrition andMetabolism

Laboratory Centre CMC V, Y2147University Hospital GroningenHanzeplein 1

P.O Box 30.001

9700 RB GroningenThe NetherlandsTel: +31-50-3632675Fax: +31-50-3611746Email: r.j.vonk@med.rug.nl

Part I

Analysing and modifying starch

1.1 Introduction: localization and function of starch in plants

This chapter reviews starch synthesis in higher plants and algae Since thereactions leading to glycogen synthesis in the cyanobacteria are similar to thoseobserved in the higher plants there will be some referral to studies in thoseorganisms particularly in regulation of cyanobacterial 1,4-glucan synthesis Theenzymology and biochemistry of the various enzymes in the plant, algal andcyanobacterial systems will also be described In view of the existing informationavailable on the properties of the starch biosynthetic enzymes and the effects ofcertain mutants on starch structure a pathway of starch synthesis is described whichpostulates specific functions for the starch synthases and branching enzymes.Finally regulation of starch synthesis at the enzymatic level will be discussed and

in relation to this regulation, recent results indicating how starch content has beenincreased in certain plants will be descibed A previous chapter1 in the secondedition of Starch Chemistry and Technology which reviewed starch biosynthesisdiscussed the various maize endosperm mutants or mutant combinations, 26 ofthem, that showed an effect on the quantity or the nature of the starch formed Thisinformation remains of interest and the reader is referred to that review However,for many of those mutants the biochemical basis for mutation effects on starchquantity or quality was unknown This review will deal with only the mutantswhere the biochemical process affected by the mutation has been elucidated to atleast some extent There are some recent reviews on starch biosynthesis2±11thatdiscuss many of the areas presented in this chapter

1.1.1 Leaf starch

Starch is deposited in granules in almost all green plants and in various types of

1

Plant starch synthesis

J Preiss, Michigan State University, USA

plant tissues and organs, e.g., leaves, roots, shoots, fruits, grains, and stems

Illumination of the leaf in bright light causes the formation of starch granules inthe chloroplast organelle and was demonstrated in the nineteenth century.12

Disappearance of the starch occurs either by exposure of the leaf to low light or

by extended exposure in the dark (24±48 hours) This is readily observed byiodine staining of the tissue13 or by light or electron microscopy.14 Starchaccumulates due to carbon fixation during photosynthesis and the starch formed

in the light is degraded in the dark to products that are in most cases utilized forsucrose synthesis Mutants of Arabidopsis thaliana unable to synthesize starch,grow at the same rate as the wild type in a continuous light regime because theyare able to synthesize sucrose,15but their growth rate is drastically reduced ifgrown in a day-night regime The reason for this is that the accumulated starch isrequired for sucrose synthesis at night; the sucrose is transported from the leaf tothe sink tissues Biosynthesis and degradation of starch in the leaf is therefore adynamic process having diurnal fluctuations in its stored levels

Starch also plays an important role in the operation of stomatal guard cells,where it is degraded during the day In the late afternoon or evening while thestomata are open, the starch is resynthesized Leaf starch is lower in amylosecontent than what is observed in storage tissues.16The amylose structure is also

of a smaller molecular size

1.1.2 Starch in storage tissues

In storage organs, fruit or seed, during the development and maturation of thetissue, synthesis of starch occurs At the time of sprouting or germination of theseed or tuber, or ripening of the fruit, starch degradation in these tissues thenoccurs and the derived metabolites are used as a source both for carbon andenergy The degradative and biosynthetic processes in the storage tissues maytherefore be temporally separated However, there is some possibility that duringeach phase of starch metabolism some turnover of the starch molecule occurs.The main site of starch synthesis and accumulation in the cereals is theendosperm, with starch granules that are located within the amyloplasts Starchcontent in potato tuber, maize endosperm, and in roots of yam, cassava andsweet potato ranges between 65 and 90% of the total dry matter Patterns ofstarch accumulation during development of the tissue are specific to the speciesand are related to the unique pattern of differentiation of the organ

Starch granules in storage tissues can vary in shape, size and composition.The shape and size of the granules depends on the source, but in each tissuethere is a range of sizes and shapes The diameter of the starch granule changesduring the development of the reserve tissue There are also some fine features,characteristic of each species, e.g., the `growth rings', spaced 4±7 m apart, andthe fibrillar organization seen in potato starch, which allows one to identify thebotanical source of the starch by microscopic examination

Two polymers are distinguished in the starch granule Amylose, which isessentially linear, and amylopectin, highly branched Amylose is mainly found aslinear chains of about 840 to 22,000 units of -D-glucopyranosyl residues linked

by -(1->4) bonds (molecular weight around 136,000 to 3.5  106) The number

of anhydroglucose units, however, varies quite widely with plant species and stage

of development Some of the amylose molecules are branched to a small extent(-1->6-D glucopyranose; one per 170 to 500 glucosyl units) Amylopectin, incontrast, which usually comprises about 70% of the starch granule, is morehighly branched with about 4 to 5% of the glucosidic linkages being -1->6.Amylopectin molecules are large flattened disks consisting of -(1,4)-glucanchains joined by frequent -(1,6)-branch points Many models of amylopectinstructure have been proposed but from these the most satisfactory models, i.e.,those that best fit the experimental data available, are those proposed by Robin

et al.17, Manners and Matheson18and by Hizukuri.19These are known as clustermodels The chemical and physical aspects of the starch granule and itscomponents amylose and amylopectin have been discussed in some recentexcellent reviews by Morrison and Karkalis20and Hizukuri.21

1.2 Starch synthesis: enzyme reactions in plants and algae and glycogen synthesis in cyanobacteria

1.2.1 Enzyme reactions of starch synthesis

The sugar nucleotide utilized for synthesis of the -1,4 glucosidic linkages inamylose and amylopectin is ADP-glucose and not UDP-glucose ADP-glucosesynthesis is catalyzed by ADP-glucose (synthetase) pyrophosphorylase (reaction

1, E.C 2.7.7.27; ATP:-D-glucose-1-phosphate adenylyltransferase)

ATP ‡ -glucose-1-P () ADP-glucose ‡ PPi 1:1ADP-glucose ‡ -1,4 glucan ˆ)-1,4-glucosyl--1,4 glucan ‡ ADP 1:2Elongated -1,4-oligosaccharide chain ˆ)-1,4--1,6 branched-glucan 1:3

(pro-amylopectin) (phytoglycogen)

Reaction 2 is catalyzed by starch synthase (E.C 2.4.1.21; -D-glucan 4--glucosyltransferase) A similar reaction is noted for glycogensynthesis in cyanobacteria and other bacteria (see references 22 and 23 but thereaction is referred to as glycogen synthase (also E.C 2.4.1.21) Reaction 3 iscatalyzed by branching enzyme (E.C 2.4.1.18; 1,4--D-glucan 6--(1,4--glucano)-transferase) The branch chains in amylopectin are longer (about 20 to

ADP-glucose;1,4-24 glucose units long) and there is less branching in amylopectin (~5% of theglucosidic linkages are -1,6 as seen in glycogen (10±13 glucose units long and10% of linkages are -1,6) Thus, the starch branching enzymes may havedifferent properties with respect to size of chain transferred, or placement ofbranch point, than enzyme that branches glycogen Alternatively, the interaction

of the starch branching enzymes with the starch synthases may be different fromthe interaction of the bacterial branching enzymes with their respective glycogensynthases The chain elongating properties of the starch synthases could bedifferent from those observed for the bacterial glycogen synthases and mayaccount for some of the differences observed in the amylopectin structure The

differences in the catalytic properties of the starch synthases and branchingenzymes isolated from different plant sources may also account for thedifferences observed in the various plant starch structures.

Isozymic forms of plant starch synthases (cited in references, 3, 4, 24±28) andbranching enzymes (cited in references 3, 4, 24, and in recent literature, 29±34)have been reported They seem to play different roles in the synthesis of the twopolymers of starch, amylose and amylopectin and are products from differentgenes In many different plants35±40as well as in Chlamydomonas reinhardtii41

a granule-bound starch synthase involved in the catalysis of reaction (5) hasbeen shown to be involved in the synthesis of amylose Mutants of manydifferent plants defective in this enzyme, are known as `waxy' mutants and giverise to starch granules having only amylopectin

Another enzyme, a debranching enzyme, most probably is involved insynthesis of the starch granule and its polysaccharide components amylose andamylopectin.42±45Soluble -glucan formed by reactions 1±3, is debranched toform the amylopectin present in the starch granule9, 46and possibly provide aprimer in the starch granule for amylose synthesis by the granule-bound starchsynthase The data strongly suggesting the role of a debranching enzyme insynthesis of amylopectin and the starch granule is discussed in a later section.Reaction 2 was first described by Leloir et al.47 with UDP-glucose as theglycosyl donor, but it was later shown that ADP-glucose was more efficient interms of maximal velocity and km value.48Leaf starch synthases and the solublestarch synthases of reserve tissues are specific for ADP-glucose In contrast, thestarch synthases bound to the starch granule in reserve tissues do have some lowactivity with UDP-glucose as compared to activity seen with ADP-glucose

1.3 Properties of plant glucan synthesizing enzymes: glucose pyrophosphorylase

ADP-1.3.1 Structure-function relationships

The ADP-glucose pyrophosphorylases (ADPGlc Ppase) of higher plants andgreen algae, as well as the cyanobacteria, are proteins under allosteric controland an important enzyme site for regulation of starch synthesis The enzymes arehighly activated by 3-phosphoglycerate and inhibited by inorganic phosphate.The regulation of starch and cyanobacterial glycogen synthesis via regulation ofthe photosynthetic ADPGlc Ppase will be discussed in a later section Thestructural properties of the ADPGlc Ppase will be described in this section.The kinetic and regulatory properties of the ADPGlc Ppases from the leafextracts of spinach, barley, butter lettuce, kidney bean, maize, peanut, rice,sorghum, sugar beet, tobacco, and tomato have been studied in detail and aresimilar.49±52

The spinach leaf ADPGlc Ppase has been purified by either preparative disc gelelectrophoresis,53by hydrophobic chromatography51and the use of FPLC.54Theenzyme has a molecular mass of 206,000 and is composed of two differentsubunits, of molecular masses of 51 and 54 kDa.54±57 These subunits are also

distinguished with respect to amino acid composition, amino-terminal sequences,peptide patterns of the tryptic digests on high-performance liquid chromatography(HPLC), and antigenic properties The two subunits are therefore quite distinct andare the products of two different genes The enzyme may be considered as having

an 2 2 structure Bacterial ADPGlc Ppases including the cyanobacterialenzymes, in contrast, are homotetrameric, i.e., composed of only one subunit,with a molecular mass of 50 to 55 kDa depending on the species.58

Other plant ADPGlc Ppases have been shown to be composed of twodissimilar subunits The maize endosperm ADPGlc Ppase, which has amolecular mass of 230 kDa, is composed of subunits of 55 and 60 kDa,corresponding to the spinach leaf 51 and 54 kDa.59

The maize endosperm mutants shrunken 2 (sh 2) and brittle 2 (bt2) areADPGlc Ppase activity deficient (reviewed references 3 and 4) Inimmunoblotting experiments using antibodies raised against the native spinachleaf enzyme and the individual subunits, it was found that the mutant bt2endosperm lacks the 55 kDa subunit and the mutant sh 2 endosperm lacks the 60kDa subunit These results59 strongly suggested that the maize endospermADPGlc Ppase is composed of two immunologically distinctive subunits andthat the sh 2 and bt2 mutations cause reduction in ADPGlc Ppase activitythrough the lack of one of the subunits; the sh 2 gene would be the structuralgene for the 60 kilodalton protein while the bt2 gene would be the structuralgene for the 55 kDa protein Consistent with this hypothesis is the isolation of anADPGlc Ppase cDNA clone from a maize endosperm library60which hybridizedwith the small subunit cDNA clone from rice.61 This maize ADPGlc PpasecDNA clone was found to hybridize to a transcript present in maize endospermbut absent in bt2 endosperm Thus, the bt2 mutant appears to be the structuralgene of the 55 kDa subunit of the ADPGlc Ppase

Hylton and Smith62proposed the existence of not two, but four polypeptides

of MW around 50 kDa for the ADPglucose PPase of pea embryo, and amolecular mass for the holoenzyme of about 110 kDa The relationship of thefour subunits to the constitution of the native enzyme was not explained.However, the available information of most systems indicates that both the seedand leaf ADPglucose pyrophosphorylase are heterotetramers composed of twodifferent subunits, and that, on the basis of immunoreactivity and sequencedata,63there is close homology between the subunits in the leaf enzyme and withthe subunits of reserve tissue enzyme Another point brought out by comparison

of the amino acid sequences of the the two different plant subunits with eachother and with the bacterial ADPGlc Ppase subunit amino acid sequences is thatthe plant subunits may have evolved from the bacterial subunit.63

1.3.2 Chemical modification of ligand binding sites of substrates andeffectors

Substrate sites

Because the plant native ADPGlc Ppases are tetrameric and composed of twodifferent subunits it was of interest to understand why two subunits are required

for optimal catalytic activity in contrast to the bacterial ADPGlc Ppase Theenzyme must contain ligand-binding sites for the activator, 3PGA, and inhibitor,

Pi, as well as catalytic sites for the two substrates, ATP and glucose-1-P, and it ispossible that these sites may be located on different subunits Chemicalmodification was used to obtain information on the catalytic mechanism and onthe catalytic site of the ADPGlc Ppase Chemical modification studies on theADPGlc Ppase have involved the use of the following affinity labels: pyridoxal-5-phosphate (PLP), an analogue of the activator 3-PGA as well as an analog ofthe substrate, glucose-1-P; 8-azido-ATP and 8-azido-ADPGlc, photoaffinityanalogs of the substrates ATP and ADPGlc, respectively64, 3and phenylglyoxal,for the identification of arginine residues that may be involved in substrate oreffector binding These types of studies have provided information on thecatalytic and regulatory sites of the spinach ADPGlc Ppase and on the role of thelarge and small subunits

In studies with the E coli ADPGlc Ppase, Lys residue 195 has been identified

as the binding site for the phosphate of glucose-1-P65and tyrosine residue 114has been identified as involved in the binding of the adenosine portion of thesubstrate, ATP.66 The overall amino acid sequence identity of the E colienzyme when aligned with the plant and cyanobacterial ADPGlc Ppases rangesfrom 30 to 40%.63, 67, 68, 69However, there is greater sequence identity when the

E coli ATP and glucose-1-P binding sites (Tables 1.1 and1.2)are comparedwith the corresponding sequences of the plant and cyanobacterial enzymessuggesting that those sequences are still important in the plant enzyme, probablyhaving the same function Indeed, in a recent preliminary experiment with thepotato tuber ADPGlc Ppase expressed in E coli,70site-directed mutagenesis onthe lysine residue K198 of the 50kDa subunit (equivalent to the E coli ADPGlcPpase K195 to a glutamate residue, increased the S0.5 value (concentrationrequired for 50% of maximal activity) for glucose-1-P from 57 M to about

31 mM without any perceptible change on the km or Ka for the other substrates,Mg+2, ATP or for activator, 3PGA (Y Fu and J Preiss, unpublished results,

Table 1.3) The apparent affinity of glucose-1-P was lowered over 500-fold.Even a conservative mutation such as arginine replacing lysine at residue

198, caused a 135-fold decrease in the glucose-1-P apparent affinity Theseresults indicate an involvement of Lys residue 198 of the plant ADPGlc Ppase inthe binding of glucose-1-P In the case of the putative ATP binding site instead

of tyrosine there is a phenylalanine residue in the corresponding sequences ofthe plant and cyanobacterial enzymes (Table 1.2) It would be of interest todetermine in future site-directed mutagenesis and chemical modification studieswhether the WFQGTADAV region of the plant enzyme is indeed a portion ofthe ATP binding region or whether the conservative change of two amino acids

in the sequence has affected the function of that portion of the protein Theamino acids completely conserved are the tryptophan, glycine, threonine,alanine and aspartate residues and possibly mutation of those residues wouldindicate the relevancy of this region as an ATP binding site

is binding at the activator site Ball and Preiss72 showed also that three lysineresidues of the spinach leaf large subunit are also involved or close to the bindingsite of pyridoxal-P and, presumably, of the activator, 3PGA The chemicalmodification of these Lys residues by pyridoxal-P was prevented by the presence

of 3PGA during the reductive phospho-pyridoxylation process and in the case ofthe Lys residue of site 1 of the small subunit and site 2 of the large subunit (Table

Table 1.1 Conservation in plant ADPGlc Ppases of the Glucose-1-Phosphate (65) sitespresent in E coli ADPGlc PPase References to these sequences for the plant ADPGlcPpases are in Smith-White and Preiss.63The sequences for the Anabaena enzyme is inCharng et al.,67 for the Synechocystis enzyme in Kakefuda et al.68and for the wheatendosperm small subunit, in Ainsworth et al.69The number 195 corresponds to Lys195 ofthe E coli enzyme and | signifies the same amino acid as found in the E coli enzyme

PLANT SMALL SUBUNIT

PLANT LARGE SUBUNIT

1.4), Pi also prevented them from being modified by reductive pyridoxylation

Similar results were otained via reductive phosphopyridoxylation of theAnabaena ADPGlc Ppase.73The modification was also prevented by 3PGA and Pi.Lys419 was the modified residue and the adjacent sequences about that residue arevery similar to those observed for site 1 sequences of the higher plants(Table 1.4).Site-directed mutagenesis of Lys 419 to either Arg, Ala, Gln, or Glu producedmutant enzymes having 25- to 150-fold lower apparent affinities, A0.5(concentration of activator needed for 50% of maximal activation), than that ofwild-type enzyme (Table 1.5) No other kinetic constants such as affinity (km) forsubstrates and the inhibitor, Pi, were affected The heat stability or the catalyticefficiency of the enzyme were also not affected These mutant enzymes, however,were still activated to a great extent at higher concentrations of 3PGA suggestingthat an additional site was involved in the binding of the activator The Lys419Argmutant was chemically modified with the activator analog, PLP Modification ofLys382 in the Arg mutant was observed and caused a dramatic alteration in theallosteric properties of the enzyme which could be prevented by the presence of3PGA or Pi during the chemical modification process Lys382 was thus identified

as another site involved in the binding of the activator and as seen in Table 1.4, thesequence about Lys382 in the Anabaena enzyme is very similar to that seen for the

Table 1.2 Conservation in plant ADPGlc Ppases of the ATP binding sites present in E.coli ADPGlc Ppase.22References to these sequences for the plant ADPGlc Ppases are inSmith-White and Preiss.63The sequences for the Anabaena enzyme is in Charng et al.,67for the Synechocystis enzyme in Kakefuda et al.68and for the wheat endosperm smallsubunit, in Ainsworth et al.69 The number 114 corresponds to tyrosine of the E colienzyme and | signifies the same amino acid as found in the E coli enzyme

higher plants site 2 which is situated on the large subunit Thus the site directed

mutagenesis along with the reductive phosphopyridoxylation experiments stronglyindicate that in higher plants as well as in the cyanobacteria, lysine residues nearthe carboxyl terminii of the ADPGlc Ppase subunits are part of the binding domain

of the allosteric activator

Site 1 of the Anabaena enzyme corresponds to the lysyl residue near the Cterminus, Lys440, that is phosphopyridoxylated in the spinach leaf smallsubunit,71 and corresponds to Lys468 in the rice seed small subunit and toLys441 in the potato tuber ADPGlc Ppase small subunit Lys404 of the potatotuber large subunit corresponds to Site 2 of the Anabaena enzyme, Lys382

Table 1.3 The effect of site-directed mutagenesis of Lys residue 198 of the smallsubunit of potato tuber ADPGlc Ppase on the Km of glucose-1-P S refers to the 50 kDa(small) subunit and L refers to the 51 kDa (large) subunit of the potato tuber enzyme Theletters, K, R, A and E, are of the one alphabet code corresponding to the amino acids,lysine, arginine, alanine and glutamate

by pyridoxal-P are in outline The potato tuber enzyme Lys residue was identified viasite-directed mutagenesis The numbers 419 and 382 correspond to the Lys residues in theAnabaena ADPGlc Ppase subunit Site 1 is present in the small subunit of the plantADPGlc Ppase while Site 2 is present in the large subunit

Activator Site 1 Activator Site 2

Table 1.4 also shows that the amino acid sequence of the spinach leaf smallsubunit peptide containing the modified lysyl residue of Site 1 is highlyconserved in the barley,76potato tuber, maize,77rice seed and wheat endospermsmall subunits69 and the Anabaena67, 78 and Synechocystis68 ADPGlc Ppasesubunits The amino acid sequence of Site 2 of the spinach leaf large subunit ishighly conserved in the barley endosperm,79 maize80potato tuber, wheat seedand wheat leaf81 large subunit ADPGlc Ppases.

Phenylglyoxal, a modifier of arginine residues causes inactivation of theenzyme that can be prevented by 3PGA or by Pi This is evidence for one ormore arginine residues being present at the allosteric sites of the spinach leafenzyme.82 Both subunits were labelled when [14C]phenylglyoxal was used.Where the Arg residues are located in the sequence is presently unknown butthere is a possibility it may be close to the Lys residue at activator Site 2.cDNA clones encoding the putative mature forms of the large and smallsubunits of the potato tuber ADPGlc Ppase have been expressed together, usingcompatible vectors, in an E coli mutant deficient in ADPGlc Ppase activity.70, 83

The ADPGlc Ppase activity expressed was high and the enzyme displayed catalyticand allosteric kinetic properties very similar to the ADPGlc Ppase purified frompotato tuber.83 The enzyme activity was also neutralized by antibody preparedagainst potato tuber and not by antibody prepared against the E coli ADPGlcPpase.83 This expression system is a very useful tool to perform site directedmutagenesis to further characterize the allosteric function of the lysyl residuesidentified via chemical modification with pyridoxal-P of the spinach enzyme.Indeed in preliminary results, shown in Table 1.5, site directed mutagenesis ofLys441 of the potato ADPGlc Ppase small subunit to Glu and Ala results in mutant

Table 1.5 Effect of mutagenesis of the allosteric activator binding site Lys residues ofcyanobacterial and plant ADPGlc Ppases The amino acid residues are indicated by a one-letter code The Anabaena ADPGlc Ppase data was obtained from Charng et al.73andfrom Sheng et al.74The potato tuber is from Preiss et al.75

conservative mutation to arginine resulted in only a twofold increase in A0.5, thusindicating that the positive charge of the cationic amino acid was important for thebinding of the activator.

1.3.3 Possible functions of the small and large subunits of the higher plantADPGlc Ppase

The ability to express cDNA clones representing the potato tuber small and largesubunits together in E coli70 to obtain a highly active enzyme enables one toexpress the subunits separately to determine their specific functions It wasfound that the potato tuber small subunit when expressed alone had highcatalytic activity provided that the 3PGA concentration was increased to 20 mM.The 3PGA saturating concentration for the expressed transgenic or (normal)potato tuber heterotetrameric enzyme is 3 mM It was found that the Ka of thetransgenic enzyme in ADPGlc synthesis is 0.10±0.16 mM while for the smallsubunit alone it is 2.4 mM Thus, the small subunit by itself has about 15- to 24-fold lower affinity for the activator The small subunit expressed alone is moresensitive to Pi inhibition than the transgenic heterotetrameric enzyme with an 8-fold lower Ki The kinetics of 3PGA activation and the Pi inhibition were themain kinetic differences between the homotetrameric small subunit and therecombinant heterotetrameric ADPGlc Ppase These results are consistent withthose obtained for the Arabidopsis thaliana mutant ADPGlc Ppase lacking thelarge subunit84where the enzyme had lower affinity for the activator and highersensitivity towards Pi inhibition than the heterotetrameric normal enzyme.85Thepotato tuber large subunit expressed by itself has negligible activity Thedominant function of the small subunit therefore is catalysis while the majorfunction of the large subunit is to affect the sensitivity of the small subunit toallosteric activation and inhibition

N-terminal sequence analysis of the purified catalytic (50 kDa) subunitexpressed in E coli and of the purified expressed heterotetrameric enzyme havingboth subunits (50 and 51 kDa) are shown in Table 1.6 The expected initialmethionine residue has been cleaved for the most part from both subunits On thebasis that there are equal amounts of tyrosine and valine at residues two for thesubunits and roughly equal amounts of valine and aspartate at residues 4, it may beconcluded that the expressed heteromeric enzyme has an 2 2 structure

1.3.4 Comparison of ADPGlc Ppase sequences

A high degree of amino acid sequence identity is observed when comparing thesequences of corresponding ADPGlc Ppase subunits from different species, aresult that could be expected from the spinach leaf catalytic (lower molecularweight) subunit antibody effectively reacting with the equivalent subunits of theenzymes from maize endosperm,59, 86 rice seed,61, 87 A thaliana88 and potatotuber.89The antibody for the lower MW spinach leaf subunit reacts weakly withthe regulatory (higher molecular mass) subunit of the other species ADPGlcPpases Not much homology, therefore, was expected between sequences of the

small and large subunits The degree of identity between the large and smallsubunits (obtained by Edman degradation or deduced from nucleotide sequences

of cDNAs or genomic DNA) is around 40 to 60%.63Sequence analyses indicate

a greater identity between the 54 kDa subunit of the spinach leaf enzyme, thesubunit coded by the Sh-2 gene from maize, and the subunit encoded by thecDNA insert, we7, from wheat endosperm81 suggesting that the latter twocorrespond to the large molecular weight subunit of the ADPGlc Ppase.Because of the relatively low but certain homology between the two subunits ofthe ADPGlc Ppase it is speculated that they have arisen originally from the samegene The bacterial ADPGlc Ppase has been shown to be a homotetramercomposed of only one subunit.58The cyanobacterial ADPGlc Ppase has 3PGA as

an allosteric activator and Pi as an inhibitor, similar to the enzyme from higherplants,90and unlike the bacterial enzymes Both bacterial22, 58and cyanobacterial91

ADPglucose pyrophosphorylases are homotetrameric, unlike the higher plantenzymes, indicating that regulation by 3PGA and Pi is not related to theheterotetrameric nature of the higher plant enzyme It is quite possible that duringevolution there was duplication of the ADPGlc Ppase gene and then divergence ofthe genes produced two different genes coding for the two peptides, both required

Table 1.6 Amino terminal sequence analysis of the potato tuber ADPGlc Ppaseexpressed in E coli either as the homotetrameric 50 kDa subunit or as theheterotetrameric enzyme having both the 50 and 51 kDa subunits Only those PTHamino acid residues found in substantial number (> 4 pmol) are shown Other amino acids

in the HPLC analysis were of lower amounts Because the PTH-Ser derivative is lessstable it is found in smaller amounts than the other PTH derivatives The analysisindicates that all of the initial methionine is cleaved from the 50 kDa subunit while about14% of the 51 kDa subunit retains the initial methionine residue

Concluded sequence for the 51 kDa subunit: Ala-Tyr-Ser-Val-Ile

for optimal activity of the native higher plant enzyme

As previously indicated above, one can tentatively assign catalytic function to thesmall subunit of the ADPGlc Ppase and this is consistent with the identity andsimilarity in sequence between the small subunits isolated from different plants andtissues In the case of the large subunit in which amino acid sequences have lowersimilarity to what is observed for the small subunits, it could be postulated that thedifferent large subunits promote or affect different regulatory properties to theheterotetrameric ADPGlc Ppases of different species and in different tissues As theneeds and amounts of starch required for each type of tissue and plant are different, adifferent sensitivity in regulation of the ADPGlc Ppase may be required and thatdifference in regulation may be affected by the different large subunits Becausesequences of the large subunits reflect their occurrences in different plant tissues,e.g., leaf, stem, guard cells, tuber, endosperm, root,63it is possible these sequencedifferences evoke different allosteric properties for the enzyme from each tissue.

1.3.5 Prediction of the two-dimensional structure of the ADPGlc Ppasesubunits

Unfortunately, no satisfactory crystals of the ADPGlc Ppase from any source areavailable for determination of the three-dimensional structure of the enzyme.However, the secondary structures can be analyzed on the basis of their aminoacid sequences and by using partial proteolysis to see if the peptide fragmentsobtained are consistent with the analysis

Hydrophobic cluster analysis (HCA)92was applied to ADPGlc Ppases fromdifferent sources representing enzymes having different allosteric effectors, andfrom different tissues, such as E coli, Anabaena, potaoto tuber small subunitand the large subunits from Chlamydomonas, maize embryo, maize endosperm,potato tuber and Arabidopsis thaliana The HCA shows that the ADPGlc areidentical in the position of many clusters, and in others the differences are small

(Fig 1.1) Even though the similarities in amino acid sequences are lowerbetween the bacterial and plant enzymes, all the clusters present in the E.colienzyme are also present in both subunits of the potato tuber ADPGlc Ppase.There are some insertions and deletions between the sequences but the generalpattern of the clusters is not altered due to the insertions not having any buriedamino acids The important point of this analysis is that the small insertions ordifferences seen among the ADPGlc Ppases are not part of the `core' of theprotein and this indicates that the ADPGlc Ppases from different sources share acommon folding pattern, despite having different quatenary structure, the plantheterotetramers and the bacterial homotetramers, and different activatorspecificities (E coli, fructose-1,6-bis-P and Anabaena and higher plants, 3PGA)

If ADPGlc Ppases from different sources have similar three-dimensionalstructures, the structure of one should help predict the secondary structure ofanother The sequences of the above seven ADPGlc Ppase subunits were alsoanalyzed using the PHD program.93Fig 1.2shows the predicted general structurethat fits all of these proteins The ADPGlc Ppase is an alpha/beta protein with someparts being mainly beta as the C-terminal region and what is noted in Fig 1.2, asDomain 3 The model is consistent with results obtained through controlled

Fig 1.1 Hydrophobic cluster analysis plots of the E coli and Anabaena ADPGlc Ppases b stands for `buried' amino acids, usually hydrophobic.

The asterisk represents glycine residues and black squares represent proline residues

proteolysis of the Anabaena and E coli enzymes with trypsin (Charng, Y Y andPreiss, J., unpublished results) Exposed loops would be more sensitive toproteolysis and the protease studies, do show the protease cleaving at sitespredicted as loops in the model Proteolysis is seen in the alpha helix predicted nearthe C-terminal of the Anabaena enzyme and may be regarded as contrary to theprediction However this may be regarded as an insertion of about 20 amino acids,absent in the E coli enzyme and it it is not predicted as being buried in Anabaena

in the HCA study Thus, this helix may be regarded as not part of the core but part

of a loop and sensitive to partial proteolysis

It is also important to point out that most of the amino acids shown to have roles

in the binding of the substrates, ATP and glucose-1-P, the binding of the activators,are situated in loops or are very close to loops Also a common super secondarystructure, the glycine loop motif, seen in nucleotide binding proteins,94 is alsopresent in the model in what is labelled in Fig 1.2 as Domain 1 This domainwould bind the phosphates of the ATP, and with the Domains 2 beta and alphahelices comprise a Rossman fold where the purine portion of the ATP is bound It

is likely that Domains 1, 2 and 3 form a catalytic domain, having a typical alpha/beta structure where the substrates bind in the loop parts exposed to the aqueousmedia The secondary structure predicted for the ADPGlc Ppase in Domains 1 and

2 is identical to the accepted structure of the oncogenic protein H-Ras21which is

Fig 1.2 Profile neural network (PHD) prediction of the secondary structure of theADPGlc Ppase Domain 1 contains the fructose-1,6-bis-P activator site, KRAKPAV of E.coli in a loop Domain 2 has the putative ATP binding site, Tyr114, also in a loop regionbetween a -strand and an -helix starting at GTAD The glucose-1-P binding site is alsoseen in a loop series of predicted -strands The secondary structure between Domains 1

and 2 cannot be determined and is drawn as a dotted line

used as one of the folding models for binding of GTP.95

In Domain 2, the loops on the N- side of the beta sheets (the C-end of thealpha helices) have no amino acids conserved in all the sequences of the knownADPGlc Ppases This is in agreement with the idea that the ATP binding site islocated on the other side of the alpha/beta structure For topological reasonsthese loops would not be accesible to the substrate and as a consequence,evolutionary pressure to conserve the amino acids in these loops would be lowerthan in the loops located at the C-end of the beta sheets.

1.4 Properties of plant glucan synthesizing enzymes: starch synthase

1.4.1 Enzyme properties

Starch synthase activity is measured as the transfer of [14C]glucose fromADPglucose into a primer such as rabbit glycogen or amylopectin.96, 97Thereare many unknowns regarding starch synthases that remain to be answered Thegaps in our understanding of starch biosynthesis arise partly from the difficultiesinherent to the starch granule itself, which is insoluble in water, has a verycomplicated structure and is still the object of much research and speculation.20

In vivo, starch synthesis occurs by deposition on the granule surface by theconcerted action of starch synthases and branching enzyme Starch synthaseactivity is associated with the starch granules or in the supernatant of crudeextracts Thus we can have granule-bound forms and soluble forms Theelucidation of the roles of the multiple forms of starch synthase and branchingenzyme in the biosynthesis of starch, and the determination of its fine structurewill require purification and characterization of each isoform

In maize endosperm there are at least four starch synthases, two soluble98and

at least two granule-bound.97The number of isoforms may vary with the plantspecies and the developmental stage, but those that have been studied morecarefully seem to have a similar number of isoforms Indeed as in the case of peaembryo an isozyme of starch synthase, starch synthase II, can exist as a solubleand starch-granule bound.99The question remains whether the activities solubleand granule bound are both functional Indeed, Mu Forster et al.,100 havereported that in maize endosperm, more than 85% of the starch synthase Iprotein may be associated with the starch granule This was determined by usingantibody prepared against the starch synthase However no evidence waspresented to indicate that the starch synthase I was active in the particulatestage.The cDNA clones that encode the two isozymes of granule bound starchsynthase of pea embryo are optimally expressed at different times duringdevelopment;40 while isozyme II is expressed in every organ, isozyme I is notexpressed in roots, stipules or flowers.40

Purification of the starch synthase and branching enzymes in large amounts and

to a large specific activity has proven to be difficult, and partly for this reason ithas not been possible so far to find out how the enzymes interact to produce thetwo carbohydrates, amylose and amylopectin, that form the starch granule At

present, knowledge of the elongating properties of the starch synthase isozymes areunknown and whether there are differences between the isozymes is also unknown.Also whether the isozymes have a preference in elongating A,B or even C chains isnot known Purification of the isoforms to high specific activity and lack ofinterfering activities will facilitate the characterization of the isoforms with respect

to primer specificity and interaction with isoforms of branching enzyme, supplyinginformation about their role in vivo

1.4.2 Identification of the waxy locus as the structural gene for the granulebound starch synthase

Genetic studies implicate one granule-bound starch synthase (GBSS) isoform inthe synthesis of amylose In waxy (wx) mutants there is virtually no amylose,GBSS activity is deficient35, 36, 101 and the wx protein is missing The finalproduct of the wx locus is a protein of molecular weight 58 kDa associated withthe starch granule

Shure et al.36 prepared cDNA clones homologous to wx mRNA and, insubsequent experiments102restriction endonuclease fragments containing part ofthe wx locus were cloned from strains carrying the different wx alleles to furthercharacterize the controlling insertion elements activator (ac) and dissociation(ds) Excision of the ds element from certain wx alleles produces two new allelesencoding for wx proteins with altered starch synthase activities.103

The DNA sequence of the wx locus of Zea mays was determined by analysis

of both a genomic and an almost full length cDNA clone103and the wx locusfrom barley has been cloned and its DNA sequenced.104Amino-acid sequencesare also available for rice,105potato,38cassava,106wheat107and pea isozymes.40Table 1.7compares three regions of the deduced amino acid sequences frombarley, cassava, maize, potato, rice, isozyme I of pea embryo and wheat wxclones with the amino acid sequence for the E coli glycogen synthase,108andthe rice soluble starch synthase.109Region 1 starts with the first 27 amino acids

of the N terminal of the E.coli glycogen synthase Thirteen are identical to theamino acid sequences deduced for the plant wx proteins Of particularsignificance is the sequence starting at residue Lys 15 of the bacterial enzyme KTGGL The lysine in the bacterial glycogen synthase has been implicated

in the binding of the substrate, ADPglucose110 on the basis of the chemicalmodification of that site by the substrate analogue ADP-pyridoxal Thesimilarity of sequences between the bacterial glycogen synthase, the solublestarch synthase and the wx protein provides further evidence that the wx gene isindeed the structural gene for the granule bound starch synthase

There are two other regions of high conservation of the various GBSSs withthe E.coli glycogen synthase In region II, only one or two amino acids of thethirteen amino acids are different from the E coli sequence and in region III, allthe GBSSs are completely identical with respect to the amino acid sequencewhile the bacterial enzyme differs in only two of nine amino acids, an Arg for aSer and an Ala for a Val

1.4.3 The wx protein is a starch synthase

The genetic evidence points to the wx locus as the structural gene for a starchsynthase bound to the starch granule However, direct biochemical evidence waslacking, mainly because of the difficulties involved in studying the proteinsassociated with starch Starch was solubilized using amylases, and the starch pro-teins liberated into the supernatant were fractionated by chromatography on

Table 1.7 Conserved regions of amino acid sequences of the E coli glycogen synthase,rice soluble starch synthase and the various granule-bound starch synthases The numberspreceding the sequence indicate the residue number from the N-terminus in the sequence.The underlined sequence in KTGGL, has been shown for the E coli glycogen synthase to

be involved in binding of the sugar nucleotide substrate.110 References to the othersequences may be obtained from Preiss and Sivak.9

DEAE97The GBSS I was clearly associated with the wx protein (recognized by itsmobility on SDS polyacrylamide gels and its reaction with antibodies raisedagainst the pure wx protein) throughout purification The molecular mass of theGBSSI, determined by gel filtration or by sucrose density gradients, was about

59 kDa.111

Because of the failure to demonstrate that the wx protein from pea endospermhad starch synthase activity, Smith112suggested that the waxy protein of pea isnot the major granule-bound starch synthase However, when starch wasextracted from developing embryos of pea a starch synthase activity wassolubilized and was determined to be the waxy protein on the basis of thefollowing results.111 The MW of the pea starch synthase is about 59 kDa, asdetermined by ultracentrifugation in sucrose density gradients The pea granule-bound starch synthase preparation displayed a relatively high specific activityand when this enzymatic fraction was subjected to SDS polyacrylamide gelelectrophoresis it migrated the same as the waxy protein and gave a strongimmunoblot with antibody prepared against the waxy protein either from peaembryo or maize.111 Thus the immunological data indicated that the activityassayed by Sivak et al.111was due to the granule bound starch synthase (waxyprotein) and not due to the truncated soluble starch synthase of 60 kDa assuggested by Edwards et al.99

When the gene coding for the mature waxy protein from maize kernel wasexpressed in E coli, the recombinant protein had a MW similar to the maizeprotein as determined by SDS PAGE, reacted with antibody raised against the plantprotein and had starch synthase activity (M N Sivak, H P Guan and J Preiss,unpublished data) Thus, the biochemical re-examination of starch synthase present

in starch granules from two species, maize and pea, strengthens the geneticevidence supporting the role of the wx protein as a granule-bound starch synthasewith a major role in the determination of amylose content of starch

It has been shown by many experiments involving anti-sense RNA inpotato39, 113 and in rice,114that disappearance of amylose correlates very wellwith the loss of wx gene expression It is possible that the interior of the granule

is devoid of branching enzyme or, if branching enzyme is in the granule itself, it

is not appreciably active The presence of an active chain-elongating enzyme,i.e., starch synthase, without an active branching enzyme present (in thepresence or absence of some debranching activity), may then explain whyamylose formation occurs However, this situation may be more complicatedsince more than one isozyme of the GBSS has been found for a number ofplants

It is quite possible that the GBSS may also be involved in the initialformation of amylopectin near the exterior portion of the granule along with thesoluble starch synthases In Chlamydomonas reinhardtii a wx mutant deficient

in GBSS was isolated.41In this mutant not only was the isolated starch deficient

in amylose but also one of the amylopectin fractions, amylopectin II, wassignificantly lower Amylopectin II has longer chains than the amylopectin Ifraction as judged from the increase in max of the glucan-I2 complex When

GBSS is active, it would not be rate limiting and thus amylose and amylopectinare seen as normal components of the starch granule When there is a loss of themajor GBSS activity, then the rate of formation of the amylose and initialamylopectin structures may be limiting in the wx mutant and only the higherbranched amylopectin I fraction would be present.

A normal amount of starch is made even if the GBSS activity is deficient andthis may suggest that there is sufficient activity of the minor granule-boundstarch synthases present or that the soluble starch synthases activity maysubstitute to form sufficient unbranched chains allowing normal amylopectinsynthesis to proceed at the same rate as in the wild type However these limitingamounts of GBSS isozyme activity are not sufficient to produce substantialamounts of amylose Alternatively, if the GBSS activity is deficient and isinvolved in amylopectin synthesis then it is possible that the soluble starchsynthase activities may substitute for the normal GBSS function for onlyamylopectin synthesis.The involvement of GBSS in amylopectin and amylosesynthesis synthesis is discussed in a later section

1.4.4 Characterization of the soluble starch synthases

A variety of plant systems have shown the presence of multiple forms of solublestarch synthases (SSS) Studies with barley, maize, pea, rice, sorghum andteosinte seeds, and wheat endosperms, spinach leaf and potato tuber extracts,have indicated the presence of at least two major forms of SSS (reviewed inreferences 3,4, 9, 24) designated as types I and II In most cases, SSS I usuallyelutes from an anion exchange column at lower salt concentrations than SSS II.Although starch synthase I (SSSI) has been partly purified from maizekernels,115, 116 starch synthase II (SSSII), a more unstable isoform, has beenmore difficult to purify

The properties observed for the isoforms of maize endosperm tissue reflectthe properties of the corresponding enzyme forms in other plant materials andthe properties of the starch synthase isozymes have been reviewed.9 Theapparent affinity for ADPglucose, measured by the km, is similar for the twoforms The maximal velocity of the type I enzyme is greater with rabbit liverglycogen than with amylopectin and the type II enzyme is less active withglycogen than with amylopectin Citrate stimulation of the primed reaction isgreater for type I than for type II Both forms can use the oligosaccharidesmaltose and maltotriose as primers when present at high concentrations Starchsynthase I seemed to have more activity than SSSII with these acceptors.The lower activity for SSSI with amylopectin as a primer as compared toglycogen, suggests that SSSI may prefer the short exterior chains (A-chains) thatare more prevalent in glycogen than in amylopectin The reverse may be true forSSSII where it may prefer the longer chains (B-chains) seen in amylopectin.Differences were also noted in the apparent affinities with respect to primer Forexample, the km of the type I enzyme for amylopectin is nine times lower thanthat of the type II enzyme It is worth noting that the type I enzyme is active

without added primer in the presence of 0.5 M citrate while the type II enzyme isinactive in these conditions Citrate decreases the km of amylopectin for bothtypes of enzymes; 160 fold for the type I enzyme and about 16 fold for the type

II starch synthase with 0.5 M citrate

The starch synthase isozymes in maize endosperm have different molecularmasses The GBSS isozyme I has a molecular mass of 60 kDa, that of GBSS II

95 kDa, the SSS I a molecular mass of 72 kDa, and SSS II, 95 kDa (reviewed in9) Mu et al.,117have reported the molecular mass of maize endosperm SSSI as

76 kDa which is similar to the value reported previously for SSSI Thesemolecular mass values for the starch synthases are all higher than that of the E.coli glycogen synthase with a molecular weight of 52 kDa.108It appears that themaize endosperm SSS I and II are immunologically distinct.97 Antibodyprepared against maize endosperm SSSI showed very little reaction with SSSII

in neutralization tests

In summary, the maize SSSI and II seem to be distinct forms distinguished onthe basis of their physical, kinetic and immunological properties and areprobably products of two different genes Because of their different kineticproperties and different specificities with respect to primer activities they mayhave different functions in the formation of the starch granule

In rice, three isoforms of soluble starch synthase were separated by anionexchange chromatography which, in immunoblot, reacted with antibodies raised

to the rice waxy protein.109After affinity chromatography of the active fractions,amino-terminal sequences were obtained for the protein bands of 55±57 kDa(separated by SDS PAGE) that cross-reacted weakly with serum raised againstthe rice waxy protein It is worth noting that this experimental approach does notexclude the possibility that other soluble starch isoforms were present which didnot cross-react with the antiserum, and the authors indicate that other resultssuggest that another soluble starch synthase isoform, with a mw of 66 kDa, isalso present in seed extracts

Other forms of starch synthase may be present in plants Recently, Marshall

et al.118 have reported the presence of a starch synthase, 140 kDa, in potatotubers which may account for 80% of the total soluble starch synthase activity

A cDNA representing the protein gene was isolated Expression of an anti-sensemRNA caused a reduction of about 80% of the soluble starch synthase activity

in the tuber extracts Of interest was that the severe reduction in activity had noeffect on starch content or on the amylose/amylopectin ratio of the starch.However, there was a change in the morphology of the starch granulessuggesting an alteration in the starch structure The specific change in structurecausing the morphology change remains to be determined

1.4.5 Cloning of the soluble starch synthases

Baba et al.109 isolated cDNA clones coding for the putative soluble starchsynthase from maize from an inmature rice seed library in gt 11 using as probessynthetic oligonucleotides designed on the basis of the amino-terminal amino

acid sequences available The insert of about 2.5 kb, was sequenced and shown

to code for a 1878-nucleotide open reading frame Comparison with thecorresponding amino-terminal sequences led the authors to conclude that theprotein is initially synthesized as a precursor, carrying a long transit peptide atthe amino acid terminus and that the same gene would be expressed both inseeds and in leaves

1.4.6 Soluble starch synthase II mutants of Chlamydomonas reinhardtii

In order to understand the various functions of the different starch synthases Balland his associates in Lille, France set out to isolate various mutants ofChlamydomonas deficient in starch synthase activities They have beensuccessful in isolating a soluble starch synthase II deficient mutant119 anddouble mutants deficient both in GBSS and in SSS II.120 These studies haveprovided significant information on the function of both these enzymes and theirinvolvement in amylopectin biosynthesis The SSSII mutant contained only 20±40% of the starch seen in the wild-type organism and the percent amylose of thetotal starch increased from 25% to 55% This mutant also contained a modifiedamylopectin which had an increased amount of very short chains (2 to 7 DP) and

a concomitant decrease of intermediate size chains (8 to 60 DP) This suggestedthat the SSSII was involved in the synthesis or maintenance of the intermediatesize chains (B-chains) in amylopectin The higher amylose content could beexplained because if an unbranched amylose-like intermediate was a precursorfor amylopectin synthesis, in the SSSII mutant it could not be effectivelyutilized It is quite possible that this amylose fraction may be more highlybranched than the usual amylose The absorption spectra of its I2 complex haslower maximal wavelength than the wild-type amylose fraction, suggesting thatmore branching has occurred The mutant amylose fraction may therefore have agreater amount of branched amylose intermediates on the route to amylopectinbiosynthesis

The double mutants defective in SSSII and a GBSS,120had an even lowerstarch content, 2% to 16% of the wild-type and the amount of starch present wasinversely correlated with the severity of the GBSS defect of the double mutant.The authors suggest that the GBSS is required to form the basic structure of theamylopectin and these effects of the GBSS absence are exacerbated due to thediminished SSSII activity Of interest is that the SSSI may, in addition to a smallamount of starch, synthesize a small water soluble polysaccharide Analysis ofboth fractions suggests that they may be intermediate in structure betweenamylopectin and glycogen with respect to the extent of branching

These studies of the Chlamydomonas mutants by Ball and his colleagues areimportant in that they provide good evidence for involvement of the GBSS inamylopectin as well as in amylose synthesis and suggest that an importantfunction for SSSII would be in its involvment in synthesis of the intermediatesize (B) branches in amylopectin

1.5 Properties of plant glucan synthesizing enzymes:

A unit of activity is defined as decrease in absorbance of 1.0 per min at 30 ëC atthe defined wavelength

The phosphorylase-stimulation assay121-123is based on the stimulation of the'unprimed' (without added glucan) phosphorylase activity of the phosphorylase

a from rabbit muscle as the branching enzyme present in the assay mixtureincreases the number of non-reducing ends available to the phosphorylase forelongation One unit is defined as 1 mol transferred from glucose-1-P per min

at 30 ëC

In contrast to the two assays described above, the branch-linkage assay124is

an assay that measures the actual number of branch chains formed by branchingenzyme catalysis The enzyme is incubated with the substrate, a NaBH4-reducedamylose, and the reaction is then stopped by boiling The product is incubatedwith purified Pseudomonas isoamylase for debranching Finally, the reducingpower of the oligosaccharide chains transferred by the enzyme is measured by ahighly sensitive reducing sugar assay like the Park-Johnson method Reduction

of amylose with borohydride gives about 2% of the reducing power of the reduced amylose, resulting in lower blanks

non-The branching-linkage assay is the most quantitative assay for branchingenzyme but amylolytic activity would interfere with this assay Thephosphorylase stimulation assay is the most sensitive particularly if labelledglucose-1-P is used and the I2 assay although not very sensitive, does allowassaying branching enzyme specificity with various -1,4-dextrins andproviding information on the possible role of the different branching enzymeisoforms It is best to employ all three assays when studying the properties of thebranching enzymes, but, above all, before studying the branching enzymesproperties, they must be purified to the extent that all degradative enzymes areeliminated if reliable information is being sought

Enzyme characterization of the isozymes

Maize endosperm has three branching enzyme isoforms.31, 121, 125Reports onother tissues are consistent with the presence of more than one isoform, such ascastor bean.126BE I, IIa and IIb from maize kernels31, 121, 125have been purified

to the extent that they no longer contained amylolytic activity.31, 125Molecularweights were 82,000 for isoform I and 80,000 for isoforms IIa and IIb.121, 122

Table 1.8 summarizes the properties of the various maize endosperm BEisozymes from the studies of Takeda et al.124 and Guan and Preiss.31 Of thethree isoforms, BEI had the highest activity (using the iodine assay) in branchingamylose (Table 1.8) and its rate of branching amylopectin was about 3% of thatwith amylose In contrast, the BEIIa and IIb isoforms branched amylopectin attwice the rate they branched amylose, and catalyzed branching of amylopectin at2.5 to 6 times the rate observed for BEI.

Takeda et al.124have analyzed the branched products made in vitro, fromamylose by each BE isoform This was done by debranching the products ofeach reaction using isoamylase, followed by gel filtration BEIIa and BEIIbare very similar in their affinity for amylose and the size of chain transferred.When presented with amyloses of different average chain length, the threeBEs have higher activity with the longer chain amylose, but while BEI couldstill catalyze the branching of an amylose of average c.l of 197 with 89% ofthe activity shown with the c.l of 405, the activity of BEII dropped sharplywith chain length The study of the reaction products showed that in vitro, theaction of BEIIa and BEIIb results in the transfer of shorter chains than thosetransfered by BEI These results suggest that BEI catalyzes the transfer oflonger branched chains and that BEIIa and IIb catalyze transfer of shorterchains Thus, it is quite possible that BEI may produce slightly branchedpolysaccharides which serve as substrates for enzyme complexes of BE IIisoforms and starch synthases to synthesize amylopectin; BE II isoforms mayplay a major role in forming the short chains present in amylopectin Also BEImay be more involved in producing the more interior (B-chains) chains of theamylopectin while BE IIa and BEIIb would be involved in forming theexterior (A) chains

Table 1.8 Properties of the maize endosperm branching isozymes The units are forphosphorylase stimulation and branching linkage assays, mol/min and for the iodinestain assay, a decrease of one absorbance unit per min

Iodine stain assay (c)

of tyrosine... secondarystructure, the glycine loop motif, seen in nucleotide binding proteins,94 is alsopresent in the model in what is labelled in Fig 1.2 as Domain This domainwould bind the phosphates... waslacking, mainly because of the difficulties involved in studying the proteinsassociated with starch Starch was solubilized using amylases, and the starch pro-teins liberated into the supernatant