Functional studies of chloroplast glyceraldehyde-3-phosphate dehydrogenase subunits A and B expressed in Escherichia coli: formation of highly active A4 and B4 homotetramers and evidence that aggregation of the B4 complex is mediated by the B subunit carboxy terminus |
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Authors: | Elisabeth Baalmann Renate Scheibe Rüdiger Cerff William Martin |
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Institution: | (1) Pflanzenphysiologie, FB 5 Biologie/Chemie, Universität Osnabrück, D-49069 Osnabrück, Germany;(2) Institut für Genetik, Technische Universität Braunschweig, Spielmannstrasse 7, D-38023 Braunschweig, Germany |
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Abstract: | Chloroplast glyceraldehyde-3-phosphate dehydrogenase (phosphorylating, E.C. 1.2.1.13) (GAPDH) of higher plants exists as an A2B2 heterotetramer that catalyses the reductive step of the Calvin cycle. In dark chloroplasts the enzyme exhibits a molecular mass of 600 kDa, whereas in illuminated chloroplasts the molecular mass is altered in favor of the more active 150 kDa form. We have expressed in Escherichia coli proteins corresponding to the mature A and B subunits of spinach chloroplast GAPDH (GapA and GapB, respectively) in addition to a derivative of the B subunit lacking the GapB-specific C-terminal extension (CTE). One mg of each of the three proteins so expressed was purified to electrophoretic homogeneity with conventional methods. Spinach GapA purified from E. coli is shown to be a highly active homotetramer (50–70 U/mg) which does not associate under aggregating conditions in vitro to high-molecular-mass (HMM) forms of ca. 600 kDa. Since B4 forms of the enzyme have not been described from any source, we were surprised to find that spinach GapB purified from E. coli was active (15–35 U/mg). Spinach GapB lacking the CTE purified from E. coli is more highly active (130 U/mg) than GapB with the CTE. Under aggregating conditions, GapB lacking the CTE is a tetramer that does not associate to HMM forms whereas GapB with the CTE occurs exclusively as an aggregated HMM form. The data indicate that intertetramer association of chloroplast GAPDH in vitro occurs through GapB-mediated protein-protein interaction.Abbreviations GAPDH
glyceraldehyde-3-phosphate dehydrogenase
- CTE
carboxy-terminal extension
- HMM
high molecular mass
- ATP
adenosine triphosphate
- 3PGA
3-phosphoglycerate
- 1,3bisPGA
1,3-bisphosphoglycerate
- HMM
high-molecular mass |
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Keywords: | glyceraldehyde-3-phosphate dehydrogenase enzyme structure light regulation redox enzyme activation |
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