Purification and properties of a folate-catabolizing enzyme.
We have identified and purified to homogeneity an enzyme from rat liver that catalyzes the oxidative catabolism of 5-formyltetrahydrofolate to p-aminobenzoylglutamate and a pterin derivative. Purification of the enzyme utilized six column matrices, including a pterin-6-carboxylic acid affinity column. Treatment of crude rat liver extracts with EDTA or heat decreased the specific activity of the enzyme by up to 85%. Peptides generated from the purified protein were sequenced and found to be identical to primary sequences present within rat light chain or heavy chain ferritin. Commercial rat ferritin did not display catabolic activity, but activity could be acquired with iron loading. The purified enzyme contained 2000 atoms of iron/ferritin 24-mer and displayed similar electrophoretic properties as commercial rat liver ferritin. The ferritin-catalyzed reaction displayed burst kinetics, and the enzyme catalyzed only a single turnover in vitro. Expression of rat heavy chain ferritin cDNA resulted in increased rates of folate turnover in cultured Chinese hamster ovary cells and human mammary carcinoma cells and reduced intracellular folate concentrations in Chinese hamster ovary cells. These results indicate that ferritin catalyzes folate turnover in vitro and in vivo and may be an important factor in regulating intracellular folate concentrations.
Suh
J R
JR
Division of Nutritional Sciences, Cornell University, Ithaca, New York 14853, USA.
Oppenheim
E W
EW
Girgis
S
S
Stover
P J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002460:Cell Line; D002846:Chromatography, Affinity; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005293:Ferritins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification*; D005492:Folic Acid / Q000378:metabolism*; D005971:Glutamates / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D007501:Iron / Q000378:metabolism; D007525:Isoelectric Focusing; D007700:Kinetics; D002955:Leucovorin / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008956:Models, Chemical; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011622:Pterins / Q000378:metabolism; D051381:Rats; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D005971:Glutamates; D010455:Peptides; D011622:Pterins; D005293:Ferritins; D005492:Folic Acid; C011398:4-aminobenzoylglutamic acid; D004492:Edetic Acid; D007501:Iron; D002955:Leucovorin