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Applied and Environmental Microbiology, December 1998, p. 4958-4964, Vol. 64, No. 12
Institut Maurice-Lamontagne, Mont-Joli,
Québec, Canada G5H 3Z4
Received 7 July 1998/Accepted 11 October 1998
Variations of intracellular concentrations of isocitrate and
NADP+ were measured throughout all growth phases of the
marine bacterium Pseudomonas nautica. The intracellular
isocitrate concentration tracked the intracellular protein
concentration throughout all phases of growth. It rapidly increased in
early exponential phase to a maximum and fell to nearly zero in
parallel with pyruvate exhaustion in the culture medium. The
intracellular NADP+ and protein concentrations increased in
parallel during the exponential phase but were poorly correlated. Even
after carbon exhaustion, the intracellular NADP+
concentration stayed high, as did protein levels. The results demonstrated that the intracellular isocitrate concentration, but not
the intracellular NADP+ concentration, was affected by the
carbon availability in the culture. They also suggest that,
because of its variability, isocitrate, but not NADP+,
plays the larger role in the control of the respiratory CO2 production rate (RCO2). From
initial rate studies, bisubstrate Michaelis constants and the
dissociation constant were determined for NADP+-specific
isocitrate dehydrogenase (IDH) from P. nautica. These studies support the hypothesis that the mechanism of IDH's
activity involves the ordered addition of the substrates,
D-isocitrate and NADP+. Furthermore, the
results support the use of a bisubstrate enzyme kinetic equation to
model RCO2 in P. nautica.
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
NADP-Isocitrate Dehydrogenase from
Pseudomonas nautica: Kinetic Constant Determination and
Carbon Limitation Effects on the Pool of Intracellular
Substrates
*
Corresponding author. Present address: Carrer de
Jesus 3, 2-2, 43201 Reus (Tarragona), Spain. Phone:
34-97-734-5891. Fax: 34-93-221-7340. E-mail:
packard{at}colon.net.
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