Mechanism of Reductive Activation of Potato Tuber ADP-glucose Pyrophosphorylase

Abstract

The potato tuber (Solanum tuberosumL.) ADP-glucose pyrophosphorylase activity is activated by a incubation with ADP-glucose and dithiothreitol or by ATP, glucose- 1-phosphate, Ca2+, and dithiothreitol. The activation was accompanied by the appearance of new sulfhydryl groups as determined with 5,5′-dithiobis(2-nitrobenzoic acid). By analyzing the activated and nonactivated enzymes on SDS-polyacrylamide gel electrophoresis under nonreducing conditions, it was found that an intermolecular disulfide bridge between the small subunits of the potato tuber enzyme was reduced during the activation. Further experiments showed that the activation was mediated via a slow reduction and subsequent rapid conformational change induced by ADP-glucose. The activation process could be reversed by oxidation with 5,5′-dithiobis(2-nitrobenzoic acid). Incubation with ADP-glucose and dithiothreitol could reactivate the oxidized enzyme. Chemical modification experiments with [14C]iodoacetic acid and 4-vinylpyridine determined that the intermolecular disulfide bridge was located between Cys12 of the small subunits of the potato tuber enzyme. Mutation of Cys12 in the small subunit into either Ala or Ser eliminated the requirement of DTT on the activation and prevented the formation of the intermolecular disulfide of the potato tuber enzyme. The mutants had instantaneous activation rates as the wild-type in the reduced state. A two-step activation model is proposed. The potato tuber (Solanum tuberosumL.) ADP-glucose pyrophosphorylase activity is activated by a incubation with ADP-glucose and dithiothreitol or by ATP, glucose- 1-phosphate, Ca2+, and dithiothreitol. The activation was accompanied by the appearance of new sulfhydryl groups as determined with 5,5′-dithiobis(2-nitrobenzoic acid). By analyzing the activated and nonactivated enzymes on SDS-polyacrylamide gel electrophoresis under nonreducing conditions, it was found that an intermolecular disulfide bridge between the small subunits of the potato tuber enzyme was reduced during the activation. Further experiments showed that the activation was mediated via a slow reduction and subsequent rapid conformational change induced by ADP-glucose. The activation process could be reversed by oxidation with 5,5′-dithiobis(2-nitrobenzoic acid). Incubation with ADP-glucose and dithiothreitol could reactivate the oxidized enzyme. Chemical modification experiments with [14C]iodoacetic acid and 4-vinylpyridine determined that the intermolecular disulfide bridge was located between Cys12 of the small subunits of the potato tuber enzyme. Mutation of Cys12 in the small subunit into either Ala or Ser eliminated the requirement of DTT on the activation and prevented the formation of the intermolecular disulfide of the potato tuber enzyme. The mutants had instantaneous activation rates as the wild-type in the reduced state. A two-step activation model is proposed. ADP-glucose pyrophosphorylase glucose 1-phosphate 5,5′-dithiobis(2-nitrobenzoic acid) dithiothreitol polyacrylamide gel electrophoresis high performance liquid chromatography 3-phosphoglycerate bovine serum albumin. ADP-glucose pyrophosphorylase (ADPGlc PPase)1(ATP:α-d-glucose-1-phosphate adenylyltransferase, EC2.7.7.27) catalyzes the synthesis of ADP-glucose as shown below and is the first committed step toward starch synthesis.α­Glucose­1­P+ATP ↔Mg2+ ADPGlc+PPiREACTION 1This enzyme plays a major regulatory role in the biosynthesis of glycogen in bacteria and starch in plants (1Preiss J. Romeo T. Adv. Microb. Physiol. 1989; 30: 183-238Crossref PubMed Scopus (145) Google Scholar, 2Preiss J. Oxf. Surv. Plant Mol. Cell. Biol. 1991; 7: 59-114Google Scholar, 3Preiss J. Romeo T. Prog. Nucleic Acids Res. Mol. Biol. 1994; 47: 299-329Crossref PubMed Scopus (117) Google Scholar, 4Preiss J. Sivak M. Zamski E. Schaffer A. Photoassimilate Distribution in Plants and Crops. Marcel Dekker, Inc., New York1996: 63-96Google Scholar). The major allosteric activator of the plant ADPGlc PPase is 3-phosphoglycerate (3PGA), and the allosteric inhibitor is orthophosphate (Pi) (2Preiss J. Oxf. Surv. Plant Mol. Cell. Biol. 1991; 7: 59-114Google Scholar,5Preiss J. Preiss J. The Biochemistry of Plants. 14. Academic Press, San Diego1988: 181-254Crossref Scopus (189) Google Scholar, 6Ghosh H.P. Preiss J. J. Biol. Chem. 1966; 241: 4491-4504Abstract Full Text PDF PubMed Google Scholar, 7Sowokinos J.R. Preiss J. Plant Physiol. 1982; 69: 1459-1466Crossref PubMed Google Scholar, 8Plaxton W.C. Preiss J. Plant Physiol. 1987; 83: 105-112Crossref PubMed Google Scholar). ADPGlc PPase from all sources is found to be a tetrameric protein. However, the enzyme from the enteric bacteria is homotetrameric (1Preiss J. Romeo T. Adv. Microb. Physiol. 1989; 30: 183-238Crossref PubMed Scopus (145) Google Scholar, 2Preiss J. Oxf. Surv. Plant Mol. Cell. Biol. 1991; 7: 59-114Google Scholar, 3Preiss J. Romeo T. Prog. Nucleic Acids Res. Mol. Biol. 1994; 47: 299-329Crossref PubMed Scopus (117) Google Scholar), whereas the enzyme from higher plants is a heterotetramer composed of two different subunits (4Preiss J. Sivak M. Zamski E. Schaffer A. Photoassimilate Distribution in Plants and Crops. Marcel Dekker, Inc., New York1996: 63-96Google Scholar, 9Smith-White B. Preiss J. J. Mol. Evol. 1992; 34: 449-464Crossref PubMed Scopus (177) Google Scholar). The amino acid sequence of the small subunit of plant ADPGlc PPase is highly conserved (80–95%), but the amino acid sequence of the large subunit shows less conservation (55–65%; Ref. 9Smith-White B. Preiss J. J. Mol. Evol. 1992; 34: 449-464Crossref PubMed Scopus (177) Google Scholar). The molecular masses of the small and large subunit of the enzyme from potato tuber were found to be 50 and 51 kDa, respectively (10Okita T.W. Nakata P.A. Anderson J.M. Sowokinos J. Morell M. Preiss J Plant Physiol. 1990; 93: 785-790Crossref PubMed Scopus (149) Google Scholar). Two cDNAs encoding the large subunit and small subunit of the potato tuber ADPGlc PPase have been expressed in Escherichia coli(11Iglesias A.A. Barry G.F. Meyer C. Bloksberg L. Nakata P.A. Laughlin M.J. Okita T.W. Kishore G.M. Preiss J. J. Biol. Chem. 1993; 268: 1081-1086Abstract Full Text PDF PubMed Google Scholar, 12Ballicora M.A. Laughlin M.J. Fu Y. Okita T.W. Barry G.F. Preiss J. Plant Physiol. 1995; 109: 245-251Crossref PubMed Scopus (122) Google Scholar). The properties of the recombinant enzyme are very similar to those of the enzyme purified from potato tuber (12Ballicora M.A. Laughlin M.J. Fu Y. Okita T.W. Barry G.F. Preiss J. Plant Physiol. 1995; 109: 245-251Crossref PubMed Scopus (122) Google Scholar). The activity of several chloroplast enzymes is regulated by reversible thiol/disulfide interchange (13Buchanan B.B. Annu. Rev. Plant Physiol. 1980; 31: 341-374Crossref Google Scholar, 14Wolosiuk R.A. Ballicora M.A. Hagelin K. FASEB J. 1993; 7: 622-637Crossref PubMed Scopus (59) Google Scholar). During photosynthetic electron transport in the light, covalent redox modification is mediated by a redox chain, the ferredoxin-thioredoxin system, leading to reductive activation of several stromal target enzymes, e.g.fructose-1,6-bisphosphatase, NADP-malate dehydrogenase, phosphoribulokinase, etc. (15Scheibe R. Plant Physiol. 1991; 96: 1-3Crossref PubMed Scopus (162) Google Scholar). However, little information is known on the possible activation of ADPGlc PPases via reduction. The activity of potato tuber ADPGlc PPase was found to be stimulated by dithiothreitol (DTT) (7Sowokinos J.R. Preiss J. Plant Physiol. 1982; 69: 1459-1466Crossref PubMed Google Scholar, 16Sowokinos J.R. Plant Physiol. 1981; 68: 924-929Crossref PubMed Google Scholar). The authors suggest the presence of key sulfhydryl (-SH) groups at the catalytic and/or allosteric site. The mechanism of DTT stimulation, however, is not known. In the current study, we present evidence that the activation is due to synergism involving the enzyme interacting with both DTT and its substrates. A reduction of the intermolecular disulfide bridge between Cys12 of the two small subunits of potato tuber ADPGlc PPase is involved in the activation process. ATP, ADPGlc, Glc-1-P, 3PGA, inorganic pyrophosphate, and 4-vinylpyridine were purchased from Sigma. [32P]PPi were purchased from NEN Life Science Products. [14C]Glc-1-P and [1-14C]iodoacetic acid were from ICN Pharmaceuticals, Inc. All other reagents were purchased at the highest available commercial grade. The wild-type and mutant ADPGlc PPase cDNAs were expressed as described previously (17Fu Y. Ballicora M.A. Preiss J. Plant Physiol. 1998; 117: 989-996Crossref PubMed Scopus (47) Google Scholar). The wild-type enzyme was purified to apparent homogeneity as estimated from about 4 μg of protein on SDS-PAGE. The mutants were purified as the wild-type enzyme except that the heat treatment and the hydrophobic chromatography steps were eliminated, and a second Mono Q chromatography step was added. In the second Mono Q step, protein elution was done with 50 mmHepes, pH 8.0, 5 and with a from The mutant enzymes were purified to about homogeneity as estimated by with about μg of protein. In the enzyme activity was to the of Morell M. Preiss J. Plant Physiol. 1987; PubMed Google Scholar). The pH 8.0, 5 bovine serum and enzyme in a of was activity during enzyme In the ADP-glucose synthesis enzyme activity was at to the of J. L. E. 1966; Scholar). activity was in in the The pH 8.0, or [14C]Glc-1-P ATP, 5 bovine serum of inorganic and enzyme in a of The was The enzyme was activated with A pH 8.0, ADPGlc, and at was to as the activation In the the enzyme was with pH 8.0, and was to as the the available sulfhydryl groups of the activated and nonactivated potato tuber ADPGlc enzyme was either under the activation or were into pH 8.0, 5 and with a was to the and was with a model of was the reduction PubMed Scopus Google Scholar). A molecular of (12Ballicora M.A. Laughlin M.J. Fu Y. Okita T.W. Barry G.F. Preiss J. Plant Physiol. 1995; 109: 245-251Crossref PubMed Scopus (122) Google was to the of enzyme the The the DTT from the as by the that in the of enzyme both A and the of the the groups of the activated enzyme were as determined by under nonreducing the activity of the activated and nonactivated enzyme was as determined by a of the activity of the enzyme the was determined by the of PubMed Scopus Google Scholar). was with A at in a of of were activity in the synthesis In of 4 were and with 4 of to the reduction to as described The protein of the subunit and from were by the with a The reduction was by the of the protein of the with was done as described by PubMed Scopus Google on polyacrylamide was not to the protein under nonreducing The nonactivated potato tuber ADPGlc PPase was in under nonreducing and to a with the subunit and were as described previously A to and Academic Press, San Scholar). were to and with ADPGlc PPase The was by treatment with by with tuber ADPGlc PPase was with either A or was and the were at 50 and at were to and was to all the with the were and with in DTT was and the reduction was at 50 and at [14C]iodoacetic acid was and the were in the at The was by of 50 The were and as the were with in pH of liquid was and the were in a liquid tuber ADPGlc PPase was either under activation or as described [14C]iodoacetic acid was and in the at was by with tuber ADPGlc PPase was either under activation or as described 4-vinylpyridine was to the to the reduction of protein and to was and the were at 50 and at were to and with the were and with of in 5 of DTT was and were at 50 and to of [14C]iodoacetic acid was and the were in the was by of of tuber ADPGlc PPase was either under activation or in a of of was and the were at The were at to sequence was to a described A to and Academic Press, San on the potato tuber ADPGlc PPase with [14C]iodoacetic the of the potato tuber enzyme was to the of to and was at a to ADPGlc PPase of The was to at The was by of the at The were to a were with a by A acid) and acid). A 5 to was at a of The were and the was with a to of the from and a and a was at a of were on a from and protein were to a model amino acid sequence The mutant enzymes with Ser and Ala at of the small subunit of potato tuber ADPGlc PPase were as and The mutant was by the Ref. C. J. the mutant was by steps R. K. in Inc., New Scholar). the Ala was into the The sequence of the was by The are shown below with the to the encoding Cys12 in the small in the of 3PGA, the catalytic activity of potato tuber ADPGlc PPase was found to with of were to the enzyme during a at in both ADPGlc and DTT were to about activation of the potato tuber enzyme. In the of ADPGlc could the enzyme to However, DTT was in the in the of ADPGlc, about activity was a the of the of ADPGlc synthesis was in the presence or of DTT was eliminated from the the enzyme was in a activity The to a high activity DTT was present not suggest that reduction of a disulfide is involved in the activation process. of ATP, Glc-1-P, Ca2+, and DTT were on the activation of potato tuber ADPGlc DTT was in all the it was the activation. the Glc-1-P, and were present was as a to the activation process from showed that could as a the potato tuber ADPGlc PPase at about of the with and the apparent of the enzyme Fu and J. is similar to that of suggest that to the as but with catalytic and were to the a very high and a very in the was and be the the was in the of activation of chloroplast R.A. Ballicora M.A. Hagelin K. FASEB J. 1993; 7: 622-637Crossref PubMed Scopus (59) Google Scholar, R.A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). shown in the enzyme was activated all were present at the DTT present in of the other two were not enzyme activity from the in the of ADPGlc was all were present in the was to the activation from ADPGlc and that from ATP, and in the that ADPGlc was in the the enzyme was that ATP, Glc-1-P, and potato tuber ADPGlc PPase formation of a reduction in the activation was to the available sulfhydryl groups of the potato tuber enzyme under activated or nonactivated shown in the activation of potato tuber ADPGlc PPase was accompanied by an of about new sulfhydryl groups tetrameric enzyme the nonactivated with the reduction of a disulfide bridge in the activation. with disulfide on under nonreducing conditions, both the activated and nonactivated potato tuber ADPGlc PPases were to under nonreducing a and The activated protein as a with molecular about 50 was in with on and ADPGlc PPase (10Okita T.W. Nakata P.A. Anderson J.M. Sowokinos J. Morell M. Preiss J Plant Physiol. 1990; 93: 785-790Crossref PubMed Scopus (149) Google Scholar, 12Ballicora M.A. Laughlin M.J. Fu Y. Okita T.W. Barry G.F. Preiss J. Plant Physiol. 1995; 109: 245-251Crossref PubMed Scopus (122) Google that showed the molecular masses of the small and large subunit were 50 and 51 kDa, the small and large subunits in electrophoresis were to be The nonactivated enzyme as two to molecular masses of 50 and were to a and were the it was the The was the as the sequence of the small subunit (12Ballicora M.A. Laughlin M.J. Fu Y. Okita T.W. Barry G.F. Preiss J. Plant Physiol. 1995; 109: 245-251Crossref PubMed Scopus (122) Google Scholar), and the was the as that of the large subunit from sequence P.A. T.W. Anderson J.M. Okita T.W. Preiss J. Mol. Biol. 1991; PubMed Scopus Google except that the first was in both the was the of the small the of an intermolecular disulfide bridge between the small subunits of the potato tuber was reduced during activation. conditions, both the activated and nonactivated enzyme as a of 50 and a reduction step was involved in the activation of potato tuber ADPGlc it was of to the of reduction with the of activation. the reduction in a of the of the small subunit to its it was possible to the of the reduction by the of the protein of the with The are shown in The of enzyme activation the of reduction. shows a between the reduction and the activation that the reduction of the intermolecular disulfide is the step in the activation of potato tuber enzyme. A below the was due to protein from the small subunit during both ADPGlc and DTT were the activation of potato tuber ADPGlc an was done to the of two shown in the potato tuber enzyme was first with DTT at DTT was by a rapid of ADPGlc in instantaneous activation. The enzyme of the activity is in with the in that the reduction is the step of the activation process. During the first incubation with the enzyme activity as in and the enzyme was first with ADPGlc, subsequent of DTT could not the enzyme in the of ADPGlc both ADPGlc and DTT were to the enzyme. The suggest that reduction is a the activation. In both ADPGlc and DTT were from the incubation the activity of the activated enzyme to below that of the nonactivated enzyme A second incubation either with ADPGlc or with ADPGlc DTT the enzyme. of DTT showed in the that the intermolecular disulfide bridge was was by nonreducing that the enzyme reduced the first The that the conformational change of the reduced enzyme is of potato tuber ADPGlc incubation incubation (ADPGlc the first enzyme was with ADPGlc and DTT at the reagents were by the enzyme into a pH 8.0, 5 and In the second the enzyme was either with ADPGlc or ADPGlc DTT at pH 8.0, was in all the of μg of enzyme were treatment to the in a new In the first enzyme was with ADPGlc and DTT at the reagents were by the enzyme into a pH 8.0, 5 and In the second the enzyme was either with ADPGlc or ADPGlc DTT at pH 8.0, was in all the of μg of enzyme were treatment to the the activated of enzyme was with a of the to the could be on under nonreducing by was accompanied by a of the enzyme activity the of the intermolecular disulfide The enzyme could be by a second with ADPGlc and the intermolecular disulfide bridge was reduced are in potato tuber ADPGlc PPase in the small subunit and in the large to the intermolecular disulfide bridge between the small the of disulfide of potato tuber ADPGlc PPase was determined by [14C]iodoacetic acid The nonactivated enzyme was first with to all sulfhydryl were by subsequent of the protein was reduced with DTT with [14C]iodoacetic In the oxidized groups be was found that were tetrameric protein. was to the activated was tetrameric from that is disulfide bridge in the potato tuber ADPGlc the of the intermolecular disulfide both the activated and nonactivated enzyme were with [14C]iodoacetic acid and with The were by shown in major of was the activated enzyme. by sequence showed that its sequence to in the small subunit of potato tuber ADPGlc PPase in was at the nonactivated the was not sulfhydryl groups were in the protein. The other were not determined due to of of and ADPGlc or acid and to purified of the of and and to the activated and nonactivated and potato tuber ADPGlc and two to the small and large The sequence to subunit was from the known of the two subunits (12Ballicora M.A. Laughlin M.J. Fu Y. Okita T.W. Barry G.F. Preiss J. Plant Physiol. 1995; 109: 245-251Crossref PubMed Scopus (122) Google Scholar, P.A. T.W. Anderson J.M. Okita T.W. Preiss J. Mol. Biol. 1991; PubMed Scopus Google Scholar). were to the small The of the first amino acid of A to the sequence of the small subunit (12Ballicora M.A. Laughlin M.J. Fu Y. Okita T.W. Barry G.F. Preiss J. Plant Physiol. 1995; 109: 245-251Crossref PubMed Scopus (122) Google Scholar). The were and were by with the elution of the two as determined the major sequence shown in the was a sequence to in the small that amino acid could be in the in a new A and to purified of the of and and to the activated and nonactivated and potato tuber ADPGlc and two to the small and large The sequence to subunit was from the known of the two subunits (12Ballicora M.A. Laughlin M.J. Fu Y. Okita T.W. Barry G.F. Preiss J. Plant Physiol. 1995; 109: 245-251Crossref PubMed Scopus (122) Google Scholar, P.A. T.W. Anderson J.M. Okita T.W. Preiss J. Mol. Biol. 1991; PubMed Scopus Google Scholar). were to the small The of the first amino acid of A to the sequence of the small subunit (12Ballicora M.A. Laughlin M.J. Fu Y. Okita T.W. Barry G.F. Preiss J. Plant Physiol. 1995; 109: 245-251Crossref PubMed Scopus (122) Google Scholar). The were and were by with the elution of the two as determined the major sequence shown in the was a sequence to in the small that amino acid could be in the the that the of A was due to the of sulfhydryl groups in the activated a and the oxidized was the nonactivated enzyme. shown in major was sequence was determined The major sequence to in the small subunit in was at is in with the from A. was a sequence present in to in the small not was on the activated could be not In to sequence was on 4-vinylpyridine protein the of the small subunits were in the disulfide The shows two to both the small and large on the known of the two the sequence of subunit could be shown in Cys12 in the small subunit of the activated enzyme was by In sequence not at the in the small subunit of the nonactivated the presence of is not during The the by in and in by [14C]iodoacetic that the located at of the two small subunits are by a disulfide bridge in the nonactivated potato tuber ADPGlc The disulfide was reduced during the activation. The of mutant ADPGlc PPase cDNAs was by the on SDS-PAGE. tuber ADPGlc PPases were by with ADPGlc PPase that been shown to be with the potato tuber enzyme (10Okita T.W. Nakata P.A. Anderson J.M. Sowokinos J. Morell M. Preiss J Plant Physiol. 1990; 93: 785-790Crossref PubMed Scopus (149) Google Scholar). The two mutant enzymes, and were at similar to the wild-type enzyme on the of the apparent were the as that of the the prevented the formation of the intermolecular disulfide between the small the mutant and wild-type enzymes were to under and nonreducing and to showed that the mutant as a under nonreducing the wild-type as two to molecular masses of 50 and conditions, the mutant and wild-type enzymes as a eliminated the intermolecular disulfide in potato tuber enzyme. the from modification shown in of the small subunit by either Ser or Ala eliminated the requirement of DTT the activation of the potato tuber ADPGlc between the mutant and wild-type enzymes was the of activation. The wild-type to the two mutant enzymes were activated the mutant enzymes the activation as the reduced wild-type The present shows that the activation of the potato tuber ADPGlc PPase via a reduction of the intermolecular disulfide bridge between the small subunits and a subsequent conformational change induced by the substrates. The that all ATP, Glc-1-P, and are to be present to have an activation as ADPGlc on the enzyme is with an mechanism as previously shown ADPGlc PPases from E. Preiss J. J. Biol. Chem. Full Text PDF PubMed Google Preiss J. J. Biol. Chem. Full Text PDF Google Scholar), and Preiss J. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). and was the of ATP, but not the of ADPGlc Preiss J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). all ATP, Glc-1-P, and are to the catalytic in to In it that both the and are to be in to the conformational change the reduction of an intermolecular disulfide bridge in a of the of the small subunit to its in By analyzing protein from different of the the reduction could be be to the reduction of intermolecular disulfide in 5 and that the activation of the potato ADPGlc PPase is mediated via a slow reduction and a rapid conformational the enzyme to be reduced first in the conformational change to enzymes as chloroplast the of the reduction process is by conformational induced by M.A. R.A. J. 1994; PubMed Scopus Google Scholar). However, of ADPGlc not of the reduction of potato tuber ADPGlc PPase not that the activation of potato tuber enzyme in a with the reduction to the sulfhydryl groups in in formation of in PubMed Scopus Google Scholar). treatment could the activation of potato tuber ADPGlc PPase by the reduced intermolecular disulfide bridge of ADPGlc and DTT the enzyme. suggest that the activated and the nonactivated disulfide of the enzyme could be The intermolecular disulfide bridge to as a the activation process. treatment in formation of disulfide in ADPGlc the that of was of protein sulfhydryl is to the activation induced by the DTT from that induced by the All the on the reductive activation of potato tuber ADPGlc PPase were in the of was in the the enzyme showed and the activity was about higher in the ADPGlc synthesis and higher in the that from the reductive activation. disulfide are involved in the of its in the activation of potato tuber ADPGlc the intermolecular disulfide bridge between the small subunits is involved in the enzyme in a state. The potato tuber ADPGlc PPase DTT was present in the A and the disulfide bridge was either ADPGlc or ATP, to the enzyme from with is that reduced wild-type and mutant enzymes are at 5 the wild-type enzyme is at (12Ballicora M.A. Laughlin M.J. Fu Y. Okita T.W. Barry G.F. Preiss J. Plant Physiol. 1995; 109: 245-251Crossref PubMed Scopus (122) Google Scholar). Mutation of Cys12 in the small subunit into either Ala or Ser mutants with instantaneous activation rates as the wild-type in the reduced state. that the role of Cys12 was to its its but to its to a disulfide of all the plant ADPGlc PPases available that Cys12 and its is conserved in the small subunit of enzymes from all etc. is conserved in the small subunit of plant that from and J. However, ADPGlc PPase from could not be activated by ADPGlc and By analyzing the ADPGlc PPase on under nonreducing it was found that an intermolecular disulfide bridge between its small of disulfide bridge the enzyme heat as in the of the potato tuber enzyme. the reductive activation of the other ADPGlc PPases with the conserved is not chloroplast enzymes are regulated by reversible interchange mediated by ferredoxin-thioredoxin R.A. Ballicora M.A. Hagelin K. FASEB J. 1993; 7: 622-637Crossref PubMed Scopus (59) Google Scholar). the potato ADPGlc PPase small subunit is expressed both in and Ref. P.A. Anderson J.M. Okita T.W. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). However, the in is that in is not the potato tuber ADPGlc PPase is expressed in potato reduced from was DTT to the potato tuber ADPGlc could be is a that a different of be However, the of the reductive activation in enzyme is in the enzyme be to the activator the be that an plays a role in the of the potato tuber enzyme. A activation mechanism of potato tuber ADPGlc PPase is shown in The intermolecular disulfide bridge between the small subunits the protein in a nonactivated the and subsequent of ADPGlc a rapid conformational change of the enzyme to the activated state. of ADPGlc the enzyme to its nonactivated of the intermolecular disulfide bridge the enzyme to its nonactivated disulfide the small subunits are but it be in that the reduction of the intermolecular disulfide bridge to a of the small and large subunits during the activation. with