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PLANT PHYSIOLOGY , Vol 109, Issue 1 245-251, Copyright © 1995 by American Society of Plant Biologists
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BIOCHEMISTRY AND ENZYMOLOGY |
Adenosine 5[prime]-Diphosphate-Glucose Pyrophosphorylase from Potato Tuber (Significance of the N Terminus of the Small Subunit for Catalytic Properties and Heat Stability)
M. A. Ballicora, M. J. Laughlin, Y. Fu, T. W. Okita, G. F. Barry and J. Preiss
Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824 (M.A.B., Y.F., J.P.)
cDNAs encoding the large subunit and a possibly truncated small subunit of
the potato tuber (Solanum tuberosum L.) adenosine
5[prime]-diphosphate-glucose pyrophosphorylase have been expressed in
Escherichia coli (A.A. Iglesias, G.F. Barry, C. Meyer, L. Bloksberg, P.A.
Nakata, T. Greene, M.J. Laughlin, T.W. Okita, G.M. Kishore, J. Preiss, J
Biol Chem [1993] 268: 1081-1086). However, some properties of the
transgenic enzyme were different from those reported for the enzyme from
potato tuber. In this work, extension of the cDNA was performed to elongate
the N terminus of the truncated small subunit by 10 amino acids. This
extension is based on the almost complete conservation seen at the
N-terminal sequence for the potato tuber and the spinach leaf small
subunits. Expressing the extended cDNA in E. coli along with the large
subunit cDNA yielded a transgenic heterotetrameric enzyme with similar
properties to the purified potato tuber enzyme. It was also found that the
extended small subunit expressed by itself exhibited high enzyme activity,
with lower affinity for activator 3-phosphoglycerate and higher sensitivity
toward inorganic phosphate inhibition. It is proposed that a major function
of the large subunit of adenosine 5[prime]-diphosphate-glucose
pyrophosphorylases from higher plants is to modulate the regulatory
properties of the native heterotetrameric enzyme, and the small subunit's
major function is catalysis.
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