Expression and characterization of pea chloroplastic glyceraldehyde-3-phosphate dehydrogenase composed of only the B-subunit.
A cDNA fragment coding for the pea (Pisum sativum L.) chloroplastic glyceraldehyde-3-P dehydrogenase (EC 1.2.1.13) B-subunit and a truncated form corresponding in length to the A-subunit have been cloned into an expression vector, expressed in the absence of the A-subunit in a gap- Escherichia coli strain, purified, and studied. Like the isolated enzyme from higher plant chloroplasts, the recombinant enzymes have dual specificity for NADPH and NADH. The recombinant glyceraldehyde-3-P dehydrogenases have the same optimal pH as the enzyme isolated from pea chloroplasts. Like the native chloroplast enzyme, the recombinant B-subunit has a marked tendency to form large aggregates, whereas the truncated B-subunit exists as the tetramer. The recombinant B-subunit glyceraldehyde 3-P dehydrogenase is more sensitive to dithiothreitol than its truncated form. It seems likely that a different pair of cysteines is responsible for the redox sensitivity of the activity of the enzyme composed of B-subunits than the cysteine residues implicated in the modulation of the activity of the enzyme composed of A-subunits by previous work in this laboratory.
Li
A D
AD
Department of Biological Sciences, University of Illinois, Chicago 60607-7060, USA.
Anderson
L E
LE
D000595:Amino Acid Sequence; D002736:Chloroplasts / Q000201:enzymology*; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D018532:Pisum sativum / Q000201:enzymology*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D011994:Recombinant Proteins; D009243:NAD; D009249:NADP; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases