NAD-Independent Lactate and Butyryl-CoA Dehydrogenases of Clostridium acetobutylicum P262 |
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Authors: | Francisco Diez-Gonzalez James B Russell Jean B Hunter |
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Institution: | (1) Section of Microbiology, Wing Hall, Cornell University, Ithaca, NY 14853, USA , US;(2) Agricultural Research Service, USDA, Cornell University, Ithaca, NY 14853, USA , US;(3) Department of Agricultural and Biological Engineering, Cornell University, Ithaca, NY 14853, USA , US |
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Abstract: | Clostridium acetobutylicum P262 cells that were
growing on lactate and acetate had an NAD-independent lactate dehydrogenase
(iLDH) activity of 200 nmol mg protein−1 min−1.
Ammonium sulfate precipitation and DEAE cellulose caused a 35-fold
purification. Gel filtration indicated that the iLDH had a molecular weight
of approximately 55 kDa, but two bands were always observed. Phenyl sepharose
could not separate the two proteins, and hydroxyapatite caused a complete
loss of activity. The semi-purified iLDH had a Vmax of 13,000 nmol
mg protein−1 min−1 and a K
m
value of 3.5 mM for D-lactate. The Vmax and K
m
values for L-lactate were 300 nmol mg protein−1
min−1 and 0.7 mM. The iLDH had a pH optimum of 7.5, was not
activated by fructose-1,6-bisphosphate (FDP), and could be coupled to either
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) or
dichlorophenol-indophenol (DCPIP), but not methyl viologen (MV) or benzyl
viologen (BV). The iLDH did not have strong absorbance between 500 and 300
nm, and trichloroacetic acid or acid ammonium sulfate extracts had virtually
no fluorescence at 450 nm. The crude extracts also had MTT-linked butyryl-CoA
dehydrogenase activity (60 nmol mg protein−1
min−1). The NAD-independent butyryl-CoA dehydrogenase eluted
from DEAE-cellulose as two fractions. The yellow fraction was extremely
unstable, but the green fraction could be stored for short periods of time at
5°C. The green-colored butyryl-CoA dehydrogenase had strong absorption at
450 nm, and gel filtration indicated that it had a molecular weight of 90
kDa. The NAD-independent butyryl-CoA dehydrogenase could be coupled to MTT,
DCPIP, or MV, but not BV. Because the NAD-independent lactate and butyryl-CoA
dehydrogenase could both be linked to low potential carriers, these two
enzymes may function as oxidation-reduction system in vivo.
Received: 24 July 1996 / Accepted: 10 September 1996 |
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Keywords: | |
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