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PLANT PHYSIOLOGY , Vol 101, Issue 3 1073-1080, Copyright © 1993 by American Society of Plant Biologists
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METABOLISM AND ENZYMOLOGY |
Pyrophosphorylases in Solanum tuberosum (IV. Purification, Tissue Localization, and Physicochemical Properties of UDP-Glucose Pyrophosphorylase)
J. R. Sowokinos, J. P. Spychalla and S. L. Desborough
Department of Horticultural Science, University of Minnesota, St. Paul, Minnesota 55108
The enzyme UDP-glucose pyrophosphorylase (UGPase) from potato (Solanum
tuberosum L. cv Norchip) tubers was purified 177-fold to near homogeneity
and to a specific activity of 1099 international units/mg of protein. The
molecular mass of the purified enzyme was 53 kD as determined by SDS-PAGE
and gel filtration. Immunological and activity assays detected UGPase at
similar levels in potato stems, stolons, and tubers. Leaves and roots
contained lower levels of UGPase activity and protein. Lineweaver-Burk
plots for substrates inorganic pyrophosphate and UDP-glucose were linear in
the pyrophosphorolytic direction, yielding Km values of 0.13 and 0.14 mM,
respectively. However, Lineweaver-Burk plots for the substrates glucose-1-P
and UTP were biphasic in nature when UGPase was assayed in the direction of
UDP-glucose synthesis. At physiological substrate concentrations (i.e. from
0.05-0.20 mM), Km values of 0.08 mM (glucose-1-P) and 0.12mM (UTP) were
obtained. When substrate concentrations increased above 0.20 mM, Km values
increased to 0.68 mM (glucose-1-P) and 0.53 mM (UTP). These kinetic
patterns of potato UGPase suggest a "negative cooperative effect" (A.
Conway, D.E. Koshland, Jr. [1968] Biochemistry 7: 4011-4022) with respect
to the substrates glucose-1-P and UTP. The biphasic substrate saturation
curves were similar to the kinetics of the dimeric form of UGPase purified
from Salmonella typhimurium (T. Nakae [1971] J Biol Chem 246: 4404-4411).
The in vivo significance of the enzyme's "negative cooperativity" in the
direction of UDP-glucose synthesis and potato sweetening is discussed.
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