levoleucovorin and 4-aminobenzoylglutamic-acid

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.

Topics: Amino Acid Sequence; Animals; Blotting, Western; Cell Line; CHO Cells; Chromatography, Affinity; Cricetinae; DNA, Complementary; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Ferritins; Folic Acid; Glutamates; Hot Temperature; Humans; Iron; Isoelectric Focusing; Kinetics; Leucovorin; Liver; Models, Chemical; Peptides; Pterins; Rats; Time Factors; Transfection; Tumor Cells, Cultured

A densitometric thin-layer chromatographic method for the analysis of process impurities in leucovorin calcium was developed and validated. Using this method, folic acid, N10-formyldihydrofolic acid, p-aminobenzoic acid, and N-(4-aminobenzoyl)-L-glutamic acid were monitored with a limit of detection of approximately 0.1% each. Both absorbance and fluorescence densitometric evaluations were utilized; fluorescence evaluation selectively detected impurities that were not chromatographically resolved from leucovorin.

Topics: 4-Aminobenzoic Acid; Chemistry, Pharmaceutical; Chromatography, Thin Layer; Densitometry; Folic Acid; Glutamates; Leucovorin; Light; Reproducibility of Results

Thymocyte growth peptide (TGP) promotes DNA synthesis of immature thymocytes. TGP has been purified from sheep, human and calf thymus and recently characterized as an N-terminally blocked nonapeptide. Evidence is presented here that the blocking moiety consists of a formylpteroyl group bound to the N-terminal glutamyl residue of the nonapeptide. The pterin part of the TGP molecule has a ribityl substituent in analogy with riboflavin, which explains the pronounced hydrophilic property of TGP in contrast to unsubstituted and unconjugated folates. The compound can be classified as a ribofolate peptide, a novel class of growth factor. Zn2+ counteracts degradation of the molecule and is required for full biological activity; mass spectrometric data confirm that native TGP contains zinc.

Topics: Amino Acid Sequence; Animals; Cells, Cultured; Chromatography, Gel; DNA; Folic Acid; Glutamates; Growth Substances; Intercellular Signaling Peptides and Proteins; Leucovorin; Mass Spectrometry; Molecular Sequence Data; Molecular Structure; Oxidation-Reduction; Peptides; Sheep; T-Lymphocytes; Thymus Gland; Zinc