Resolution of rat liver 10-formyltetrahydrofolate dehydrogenase/hydrolase activities.
10-Formyltetrahydrofolate dehydrogenase (EC 1.5.1.6) catalyzes the NADP-dependent conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO2. Previous studies of 10-formyltetrahydrofolate dehydrogenase purified from rat or pig liver homogenized in phosphate buffers indicated the presence of copurifying 10-formyltetrahydrofolate hydrolase activity, which catalyzes conversion of 10-formyltetrahydrofolate to tetrahydrofolate and formate. We find that the supernatant from rat liver homogenized in mannitol/sucrose/EDTA medium contains essentially all of the total cellular 10-formyltetrahydrofolate dehydrogenase activity, but no measurable hydrolase activity. Treating mannitol/sucrose/EDTA-washed mitochondria with Triton X-100 (0.5%) releases hydrolase activity in soluble form. 10-Formyltetrahydrofolate dehydrogenase purified from the mannitol/sucrose/EDTA supernatant has no 10-formyltetrahydrofolate hydrolase activity. Results of kinetic experiments using the hydrolase-free dehydrogenase give a complex rate equation with respect to (6R,S)-10-formyltetrahydrofolate. Double-reciprocal plots fit a 2/1 hyperbolic function with apparent Km values of 3.9 and 68 microM. Our results indicate that 10-formyltetrahydrofolate hydrolase and dehydrogenase are not alternate catalytic activities of a single protein, but represent two closely related and separately compartmentalized hepatic enzymes.
Case
G L
GL
Department of Nutritional Sciences, University of Wisconsin, Madison 53706.
Kaisaki
P J
PJ
Steele
R D
RD
D000581:Amidohydrolases / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D002852:Chromatography, Ion Exchange; D007202:Indicators and Reagents; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000302:isolation & purification*; D051381:Rats; D000099295:Formyltetrahydrofolate Dehydrogenase
D000581:Amidohydrolases; D007202:Indicators and Reagents; D000587:Oxidoreductases Acting on CH-NH Group Donors; D000099295:Formyltetrahydrofolate Dehydrogenase; C056575:formyltetrahydrofolate deformylase protein, rat