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Biosynthesis of starch; identification of potato starch enzymes
Food Hydrocolloids vol.I no.5/6 pp.387 - 391, 1987
Biosynthesis of starch; identification of potato starch enzymes G.H.Vos-Scheperkeuter, A.S.Ponstein, J.G.de Wit, W.J.Feenstra', G.T.OostergeteI 2 , E.F.J.van Bruggerr' and B.Witholt
Groningen Biotechnology Center, Departments of Biochemistry, 'Geneucs and 2Electron Microscopy, Nijenborgh 16, 9747 AG Groningen, The Netherlands Abstract: Two important starch enzymes, granule-bound starch synthase and branching enzyme, were purified from potato tubers and characterized by immunological comparison with the corresponding enzymes of other plants. Granule-bound starch synthase was identified as a 6O-kd protein homologous to the corresponding enzymes of maize and amaranth; the enzyme was missing in amylose-free potato starch granuies. Branching enzyme of potato tubers was purified as a single protein species of 79 kd which appeared to be homologous to maize branching enzyme I, but much less to branching enzymes IIa and lIb.
Higher plants store their excess of energy in the form of water-insoluble starch granules. The granules usually consist of20-25 % amylose and 75 -80% amylopectin and have a highly ordered structure. We are studying the complex processes involved in starch granule formation, in particular in potato tubers, thereby focussing on the key enzymes in the biosynthetic pathways of either amylose or amylopectin. These key enzymes are: starch synthase which produces linear a-I ,4-glucan chains (i.e. amylose) and branching enzyme which introduces a-I,6-branch points into linear glucan chains and thus produces amylopectin. Both types of enzyme occur in multiple forms, at least in maize (1), and it is our primary aim to characterize the equivalent (iso)enzymes in potato tubers. The protein compositions of various starches were compared by SDS - gel eIec-
Fig. 1. SDS-gel analysis (A) and immunoblot (B) of proteins from starch of normal maize kernels (I), waxy maize kernels (2), potato tubers (3), normal amaranth seeds (4) or waxy amaranth seeds (5). Panel B shows the reactions of antibodies directed against the major 60-kd protein of potato starch with proteins from other starches. Lane R contains reference proteins (mol. wts indicated on the left) (reproduced from ref. 2).
© IRL Press Limited, Oxford, England
387
Biosynthesis of starch
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Fig. 2. Chromatography of potato branching enzyme on DEAE-cellulose. A cleared potato tuber homogenate was concentrated by 40% ammonium sulphate precipitation and applied to the column in 10 mM Tris-HCl, pH 7.5, containing 1 mM EDTA, 1 mM DTT and 75 mM NaCI. Elution was performed with a linear gradient of 75-375 mM NaCI. BE activity was measured indirectly via stimulation of phosphorylase activity (5,7).
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Fig. 3. Chromatography of maize branching enzymes on DEAE-cellulose. A cleared maize kernel homogenate was subjected to 40% ammonium sulphate precipitation and chromatographed in 50 mM Tris-acetate, pH 7.5, containing 10 mM EDTA, 2.5 mM DTT and 10% sucrose (5)_ Elution was measured as a Figure 1. Four peaks of BE activity were detected which were pooled as indicated.
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Fig. 4. SDS-gel analysis (A) and immunoblots (B and C) of maize branching enzyme pools 1,2,3 and 4. The antibodies used in immunoblotting were raised against (B) highly purified, native potato BE (sp. act. 189 U/mg) and (C) pure, denatured potato BE isolated by preparative SDS -gel electrophoresis. Marker proteins are indicated on the left.
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,AJg IgG / assay Fig. 5. Neutralization of maize branching enzyme 1 by anti-native potato BE IgG (closed symbols) or non-immune IgG (open symbols). Maize BE I (sp. act. 2.4 U/mg) was incubated with increasing amounts of IgG during 30 min at room temperature and then assayed, either via stimulation of phosphorylase activity (panel A) or via branching of amylose (panel B) (5-1). Per assay either 3.2 )kg protein (triangles and squares in A) or 8.0 )kg protein (circles in A and B) of the enzyme preparation was used. The activity is expressed as a percentage of the activity measured in the absence of IgG.
389
Biosynthesis of starch
trophoresis (Figure IA). Since granule-bound starch synthase is known to be absent in waxy starch (I), this enzyme could be identified as a 61-kd protein in maize (Figure l A, lane 1) and a 65-kd protein in amaranth (Figure IA, lane 4) . The major 60-kd protein of potato starch (Figure IA, Jane 3) could be identified as granule-bound starch synthase by showing that (i) it cross-reacted with the corresponding enzymes of maize and amaranth in immunoblotting (Figure IB), and (ii) anti-60-kd antibodies were able to neutralize the activity of granule-bound starch synthase (2). Recently a waxy (or better: amylose-free) potato was isolated using X-ray mutagenesis and cellular techniques (3). As expected , this waxy potato (i) does not contain the major 60-kd protein present in normal starch granules and (ii) has a strongly decreased activity of granule-bound starch synthase (4). When branching enzyme (BE) of potato tubers or maize kernels was chromatographed on DEAE -cellulose quite different results were obtained. The potato enzyme completely bound to the column and eluted as a single peak (Figure 2), whereas the maize extract contained multiple peaks of BE activity one of which was not bound to the column (Figure 3). Comparison of the latter profile with previously published results by others allows the conclusion that our peak 1 is equivalent to maize BE I, peak 3 to maize BE Ilb and peak 4 to maize BE IIa (1,5). The nature of our peak 2 is not clear at present. Our results are consistent with those of Borovsky et ai. (6) who purified potato BE using a different isolation procedure and a different assay [i.e. they monitored branching of amylose via decrease in iodine stain (5 -7) J. These authors obtained a single protein entity running at a mol. wt of 85 kd in their hands. Potato BE appears to be analogous to maize BE I since antibodies directed against the native potato enzyme reacted with maize BE I both in immunoblotting (Figure 4, panel B) and neutralization experiments (Figure 5). The maize protein involved in these cross-reactions is a minor protein of - 81 kd which can clearly be distinguished from the major 82-kd protein previously identified as maize BE I (8). Therefore, identification of the cross-reacting 81-kd protein as maize BE I must await further evidence. Anti-native potato BE could neutralize the activity of maize BE I, but not of BIlla/b. This result confirms the view that, although there exists homology between all three branching enzymes from maize, BE I and BE IIa/b are clearly different types of BE (8). Multiple cross-reactions were observed when maize BE pools 1-4 were reacted with anti-denatured potato BE (Figure 4, panel C) . This result suggests that maize kernels and potato tubers contain additional proteins (or even branching enzymes) which are homologous. The roles and interactions of these and other starch enzymes are presently studied by performing starch biosynthesis in permeabilized potato tuber slices under conditions when one or more starch enzymes are specifically inhibited (9) . In addition, we try to elucidate the roles of various enzymes in starch granule formation by studying the molecular structure of the starch granule (J 0) and, in the near future, by localizing the starch enzymes by (immuno )electron microscopy.
Acknowledgements We thank N.Panman and K.Gilissen for drawing and photography respectively. Part of the results presented here were reproduced from ref. (2) with permission of the publisher. 390
G.H.Vos-Scheperkeuter et al.
References 1. Preiss,J. and Levi,C. (1980) Starch biosynthesis and degradation. In Preiss,J. (ed.), The Biochemistry of Plants. Vol. 3. Carbohydrates, Structure and Function. Academic Press, New York, pp. 371-423. 2. Vos-Scheperkeuter,G.H., de Boer,W., Visser,R.G.F., Feenstra,W.J. and Witholt,B. (1986) Piant Physiol., 82,411-416. 3. Feenstra et ai. (1987) Food Hydrocolloids, 1, 393-394. 4. Visser,R.G.F., Hovenkamp-Hermelink,J.H.M., Ponstein,A.S., Vos-Scheperkeuter,G.H., Jacobsen,E., Feenstra,W.J. and Witholt,B. (1987) Proc. 4th Eur. Congress on Biotechnol., 2, 432-435. 5. Boyer,C.D. and Preiss,J. (1978) Carbohydr. Res., 61, 321-334. 6. Borovsky,D., Smith,E.E. and Whe1an,W.J. (1975) Eur. J. Biochem., 59, 615-625. 7. Hawker,J .S., Ozbun,J.L., Ozaki,J., Greenberg,E. and Preiss,J. (1974) Arch. Biochem. Biophys., 160, 530-551. 8. Singh,B.K. and Preiss,J. (1985) Piant Physiol., 79, 34-40. 9. Ponstein et ai. (1987) Food Hydrocolloids, 1, 497 -498. 10.Oostergetel and van Bruggen,E.F.J. (1987) Food Hydrocolloids, 1, 527-528.
391
Biosynthesis of starch; identification of potato starch enzymes G.H.Vos-Scheperkeuter, A.S.Ponstein, J.G.de Wit, W.J.Feenstra', G.T.OostergeteI 2 , E.F.J.van Bruggerr' and B.Witholt
Groningen Biotechnology Center, Departments of Biochemistry, 'Geneucs and 2Electron Microscopy, Nijenborgh 16, 9747 AG Groningen, The Netherlands Abstract: Two important starch enzymes, granule-bound starch synthase and branching enzyme, were purified from potato tubers and characterized by immunological comparison with the corresponding enzymes of other plants. Granule-bound starch synthase was identified as a 6O-kd protein homologous to the corresponding enzymes of maize and amaranth; the enzyme was missing in amylose-free potato starch granuies. Branching enzyme of potato tubers was purified as a single protein species of 79 kd which appeared to be homologous to maize branching enzyme I, but much less to branching enzymes IIa and lIb.
Higher plants store their excess of energy in the form of water-insoluble starch granules. The granules usually consist of20-25 % amylose and 75 -80% amylopectin and have a highly ordered structure. We are studying the complex processes involved in starch granule formation, in particular in potato tubers, thereby focussing on the key enzymes in the biosynthetic pathways of either amylose or amylopectin. These key enzymes are: starch synthase which produces linear a-I ,4-glucan chains (i.e. amylose) and branching enzyme which introduces a-I,6-branch points into linear glucan chains and thus produces amylopectin. Both types of enzyme occur in multiple forms, at least in maize (1), and it is our primary aim to characterize the equivalent (iso)enzymes in potato tubers. The protein compositions of various starches were compared by SDS - gel eIec-
Fig. 1. SDS-gel analysis (A) and immunoblot (B) of proteins from starch of normal maize kernels (I), waxy maize kernels (2), potato tubers (3), normal amaranth seeds (4) or waxy amaranth seeds (5). Panel B shows the reactions of antibodies directed against the major 60-kd protein of potato starch with proteins from other starches. Lane R contains reference proteins (mol. wts indicated on the left) (reproduced from ref. 2).
© IRL Press Limited, Oxford, England
387
Biosynthesis of starch
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400
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30
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20
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100
120
140
160 Fraction number
Fig. 2. Chromatography of potato branching enzyme on DEAE-cellulose. A cleared potato tuber homogenate was concentrated by 40% ammonium sulphate precipitation and applied to the column in 10 mM Tris-HCl, pH 7.5, containing 1 mM EDTA, 1 mM DTT and 75 mM NaCI. Elution was performed with a linear gradient of 75-375 mM NaCI. BE activity was measured indirectly via stimulation of phosphorylase activity (5,7).
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Fig. 3. Chromatography of maize branching enzymes on DEAE-cellulose. A cleared maize kernel homogenate was subjected to 40% ammonium sulphate precipitation and chromatographed in 50 mM Tris-acetate, pH 7.5, containing 10 mM EDTA, 2.5 mM DTT and 10% sucrose (5)_ Elution was measured as a Figure 1. Four peaks of BE activity were detected which were pooled as indicated.
388
G.H.Vos-Scheperkeuter et
c
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Fig. 4. SDS-gel analysis (A) and immunoblots (B and C) of maize branching enzyme pools 1,2,3 and 4. The antibodies used in immunoblotting were raised against (B) highly purified, native potato BE (sp. act. 189 U/mg) and (C) pure, denatured potato BE isolated by preparative SDS -gel electrophoresis. Marker proteins are indicated on the left.
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,AJg IgG / assay Fig. 5. Neutralization of maize branching enzyme 1 by anti-native potato BE IgG (closed symbols) or non-immune IgG (open symbols). Maize BE I (sp. act. 2.4 U/mg) was incubated with increasing amounts of IgG during 30 min at room temperature and then assayed, either via stimulation of phosphorylase activity (panel A) or via branching of amylose (panel B) (5-1). Per assay either 3.2 )kg protein (triangles and squares in A) or 8.0 )kg protein (circles in A and B) of the enzyme preparation was used. The activity is expressed as a percentage of the activity measured in the absence of IgG.
389
Biosynthesis of starch
trophoresis (Figure IA). Since granule-bound starch synthase is known to be absent in waxy starch (I), this enzyme could be identified as a 61-kd protein in maize (Figure l A, lane 1) and a 65-kd protein in amaranth (Figure IA, lane 4) . The major 60-kd protein of potato starch (Figure IA, Jane 3) could be identified as granule-bound starch synthase by showing that (i) it cross-reacted with the corresponding enzymes of maize and amaranth in immunoblotting (Figure IB), and (ii) anti-60-kd antibodies were able to neutralize the activity of granule-bound starch synthase (2). Recently a waxy (or better: amylose-free) potato was isolated using X-ray mutagenesis and cellular techniques (3). As expected , this waxy potato (i) does not contain the major 60-kd protein present in normal starch granules and (ii) has a strongly decreased activity of granule-bound starch synthase (4). When branching enzyme (BE) of potato tubers or maize kernels was chromatographed on DEAE -cellulose quite different results were obtained. The potato enzyme completely bound to the column and eluted as a single peak (Figure 2), whereas the maize extract contained multiple peaks of BE activity one of which was not bound to the column (Figure 3). Comparison of the latter profile with previously published results by others allows the conclusion that our peak 1 is equivalent to maize BE I, peak 3 to maize BE Ilb and peak 4 to maize BE IIa (1,5). The nature of our peak 2 is not clear at present. Our results are consistent with those of Borovsky et ai. (6) who purified potato BE using a different isolation procedure and a different assay [i.e. they monitored branching of amylose via decrease in iodine stain (5 -7) J. These authors obtained a single protein entity running at a mol. wt of 85 kd in their hands. Potato BE appears to be analogous to maize BE I since antibodies directed against the native potato enzyme reacted with maize BE I both in immunoblotting (Figure 4, panel B) and neutralization experiments (Figure 5). The maize protein involved in these cross-reactions is a minor protein of - 81 kd which can clearly be distinguished from the major 82-kd protein previously identified as maize BE I (8). Therefore, identification of the cross-reacting 81-kd protein as maize BE I must await further evidence. Anti-native potato BE could neutralize the activity of maize BE I, but not of BIlla/b. This result confirms the view that, although there exists homology between all three branching enzymes from maize, BE I and BE IIa/b are clearly different types of BE (8). Multiple cross-reactions were observed when maize BE pools 1-4 were reacted with anti-denatured potato BE (Figure 4, panel C) . This result suggests that maize kernels and potato tubers contain additional proteins (or even branching enzymes) which are homologous. The roles and interactions of these and other starch enzymes are presently studied by performing starch biosynthesis in permeabilized potato tuber slices under conditions when one or more starch enzymes are specifically inhibited (9) . In addition, we try to elucidate the roles of various enzymes in starch granule formation by studying the molecular structure of the starch granule (J 0) and, in the near future, by localizing the starch enzymes by (immuno )electron microscopy.
Acknowledgements We thank N.Panman and K.Gilissen for drawing and photography respectively. Part of the results presented here were reproduced from ref. (2) with permission of the publisher. 390
G.H.Vos-Scheperkeuter et al.
References 1. Preiss,J. and Levi,C. (1980) Starch biosynthesis and degradation. In Preiss,J. (ed.), The Biochemistry of Plants. Vol. 3. Carbohydrates, Structure and Function. Academic Press, New York, pp. 371-423. 2. Vos-Scheperkeuter,G.H., de Boer,W., Visser,R.G.F., Feenstra,W.J. and Witholt,B. (1986) Piant Physiol., 82,411-416. 3. Feenstra et ai. (1987) Food Hydrocolloids, 1, 393-394. 4. Visser,R.G.F., Hovenkamp-Hermelink,J.H.M., Ponstein,A.S., Vos-Scheperkeuter,G.H., Jacobsen,E., Feenstra,W.J. and Witholt,B. (1987) Proc. 4th Eur. Congress on Biotechnol., 2, 432-435. 5. Boyer,C.D. and Preiss,J. (1978) Carbohydr. Res., 61, 321-334. 6. Borovsky,D., Smith,E.E. and Whe1an,W.J. (1975) Eur. J. Biochem., 59, 615-625. 7. Hawker,J .S., Ozbun,J.L., Ozaki,J., Greenberg,E. and Preiss,J. (1974) Arch. Biochem. Biophys., 160, 530-551. 8. Singh,B.K. and Preiss,J. (1985) Piant Physiol., 79, 34-40. 9. Ponstein et ai. (1987) Food Hydrocolloids, 1, 497 -498. 10.Oostergetel and van Bruggen,E.F.J. (1987) Food Hydrocolloids, 1, 527-528.
391