Activation properties of the redox-modulated chloroplast enzymes glyceraldehyde 3-phosphate dehydrogenase and fructose-1,6-bisphosphatase

Rapid changes of enzyme activity are obtained by post-translational modification of cysteine residues of some chloroplast enzymes. Individual fine-tuning is achieved by specific factors acting upon the redox cycle. In order to study the regulatory properties of these enzymes, they are purified from leaves or in a recombinant form from Escherichia coli . The various factors acting upon the enzyme in vivo can be simulated in vitro. However, in these studies, some subtle technical problems can be encountered. Two cases are presented in this article, and an attempt is made to explain some previous, seemingly contradictory results. The Calvin-cycle enzyme glyceraldehyde 3-phosphate dehydrogenase in its less active A8B8 form can be dissociated and thereby activated in vitro simply by diluting out the protein. On the other hand, activation requires the presence of reduced thioredoxin (Td) and an increase in ionic strength when performed at a high protein concentration, as present in vivo. Chloroplast fructose-1,6-bisphosphatase (FBPase) is purified from E. coli as an enzyme similar to that purified from leaves. However, using the standard protocol for lysis of the bacteria leads to a form with some unusual properties as changed isoelectric point, lack of Ca2+/fructose-1,6-bisphosphate (FBP) dependency of reductive activation, and lack of activity at high pH and high Mg2+ concentration. These observations are used in order to better understand the characteristics of the activation/inactivation process.