Expression of the potato tuber ADP-glucose pyrophosphorylase in Escherichia coli.
cDNA clones encoding the putative mature forms of the large and small subunits of the potato tuber ADP-glucose pyrophosphorylase have been expressed separately and together in an Escherichia coli B mutant deficient in ADP-glucose pyrophosphorylase activity. Expression of both subunits from compatible vectors resulted in restoration of ADP-glucose pyrophosphorylase activity. Maximal enzyme activity required both subunits. The expressed ADP-glucose pyrophosphorylase was purified and characterized. The recombinant enzyme exhibited catalytic and allosteric kinetic properties very similar to the enzyme purified from potato tuber. The expressed enzyme activity was neutralized by incubation with antibodies raised against potato tuber and spinach leaf ADP-glucose pyrophosphorylases but not with anti-Escherichia coli enzyme serum. 3-Phosphoglycerate was the most efficient activator and its effect was increased by dithiothreitol. In the ADP-glucose synthesis direction, 3-phosphoglycerate activated the recombinant enzyme nearly 100-fold in the presence of dithiothreitol, with an A0.5 value of 57 microM. The recombinant ADP-glucose pyrophosphorylase was less sensitive to P(i) inhibition and more sensitive to heat denaturation than the potato tuber enzyme. Results suggest that bacterial expression of potato tuber cDNAs could be used to study the role and interaction of the subunits of the native ADP-glucose pyrophosphorylase.
Iglesias
A A
AA
Department of Biochemistry, Michigan State University, East Lansing 48824.
Barry
G F
GF
Meyer
C
C
Bloksberg
L
L
Nakata
P A
PA
Greene
T
T
Laughlin
M J
MJ
Okita
T W
TW
Kishore
G M
GM
Preiss
J
J
D000494:Allosteric Regulation; D000906:Antibodies; D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D051599:Glucose-1-Phosphate Adenylyltransferase; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009500:Neutralization Tests; D009713:Nucleotidyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D011198:Solanum tuberosum / Q000201:enzymology* / Q000235:genetics
D000906:Antibodies; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D009713:Nucleotidyltransferases; D051599:Glucose-1-Phosphate Adenylyltransferase; D008274:Magnesium