In vitro methotrexate polyglutamate synthesis by rat liver folylpolyglutamate synthetase and inhibition by bromosulfophthalein.
We have investigated the properties of the rat liver folylpolyglutamate synthetase using methotrexate (MTX; 4-NH2-10-CH3-PteGlu) as a substrate. Many characteristics of the synthetase (e.g., the apparent Km values for L-glutamate and ATP, and the optimal concentrations of KC1 and 2-mercaptoethanol) are virtually identical whether MTX or tetrahydrofolate is the "folate" substrate. There are, however, several significant differences between the reactions catalyzed with these two substrates. The length of products synthesized from tetrahydrofolate are inversely related to the initial monoglutamate concentration. Low tetrahydrofolate concentrations allow synthesis of longer (n greater than or equal to 3) polyglutamates, up to pentaglutamate length, while high concentrations lead to predominantly diglutamate synthesis. However, 4-NH2-10-CH3-PteGlu2 predominates regardless of the initial MTX concentration, under otherwise identical conditions. Also, tetrahydrofolate can be readily converted to pentaglutamate lengths, the same as predominates in rat liver in vivo. In contrast, MTX forms species containing only up to a total of three glutamates, i.e., 4-NH2-10-CH3-PteGlu3. Finally, the ultimate product of synthesis from tetrahydrofolate, H4PteGlu5, is a fairly good inhibitor of synthetase activity with either MTX or tetrahydrofolate as the substrate. The ultimate product of MTX synthesis, 4-NH2-10-CH3-PteGlu3, however, is a poor inhibitor of activity with either substrate. We have also investigated the inhibitory effect of bromsulfophthalein (BSP) on the rat liver synthetase. Gewirtz et al., showed that this organic anion inhibited the uptake of MTX and 5-CH3-tetrahydrofolate into hepatocytes, and presented indirect evidence that BSP effected polyglutamate biosynthesis. We have demonstrated that BSP is a potent (Ki = 1 microM) inhibitor of the rat liver synthetase. Inhibition is noncompetitive with rspect to L-glutamate, ATP, and MTX. Pre-incubation and time course experiments demonstrated that inhibition is not stoichiometric and is not caused by slow inactivation of the synthetase. Since BSP is transported into mammalian cells, it is the first inhibitor of polyglutamate biosynthesis which has potential for use in in vivo studies.
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003352:Corynebacterium / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D009492:Neurospora crassa / Q000201:enzymology; D010453:Peptide Synthases / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D011099:Polyglutamic Acid / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D011817:Rabbits; D051381:Rats; D013045:Species Specificity; D013448:Sulfobromophthalein / Q000494:pharmacology*
D010455:Peptides; D013448:Sulfobromophthalein; D011099:Polyglutamic Acid; C014085:methotrexate polyglutamate; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase; D008727:Methotrexate