Tetrahydrofolate formylase

In a similar way the purine antimetabolite 6-mercapto-purine (6-MP)/ after conversion to the monophosphoribo-tide, acts as an inhibitor of this enzyme (9.10). There have been described other sites of action of 6-MP (4-7) which are marked by arrows in figure 1. We have previously demonstrated the inhibition of the enzymic formate activation (tetrahydrofolate formylase) in leukemic cells by 6-MP (12). inspite of a rather high inhibitory concentration of 6-MP between 10 and 10 M, this inhibition has some practical clinical implications for the treatment of acute leukemia/ as it is detected in sensitive leukemic cells only. In accordance with reports of several authors (8,9/10) we have postulated/ that 6-MP has to be converted into 6-thioinosinic acid for exerting its inhibitory effect on the de novo synthesis of purine-nucleotides. On the other hand DAVIDSON and WINTER have shown that both 6-MP sensitive and resistant cells con-... 

The details of cell preparations and the assay of the tetrahydrofolate formylase are described elsewhere (12). 

For determination of the tetrahydrofolate formylase activity sodium formate and tetrahydrofolate ere incubated with ATP, ATP-regenerating system and Mg ions in TRIS-puffer pH 7.4 60 minutes at 37 C. By stopping the reaction with perchloric acid all NlO-formyItetrahydrofolic acid is converted to anhydrocitrovorum factor (ACF) which was determined by measuring the absorption at 355 m/u in a Zeiss Spectrophotometer (e=22.000). 

For comparising the activity of the tetrahydrofolate formylase with the netto de novo synthesis of purine nucleotides incubations were performed with 14C-formate and the components necessary for the enzymic formate activation only and in a parallel experiment with additional KHCO3, glutamine,glycine and ribose-5-phosphate (12). Determinations of the tetrahydrofolate formylase activity and of... 

The tetrahydrofolate formylase was found in normal and leukemic leukocytes, the highest activities in immature blast cells of acute Leukemia (table 1). By determination of the 14C-formate incorporation a measurable netto de novo synthesis of purine nucleotides was only detected in immature leukemic cells. 

In figure 6 the influence of 6-MP and allopurinol on the tetrahydrofolate formylase in leukemic blast cells of 5 patients with acute leukemia is illustrated. 6-MP in a rather high concentration of 1.5 x 10 M has only a small inhibitory effect on this enzyme.By addition of allopurinol the inhibition of the enzyme by 6-MP was markedly increased, whereas allopurinol alone had no effect. This is explained by the fact, that allopurinol, by inhibition of the xanthine oxidase, reduces the inactivation of 6-MP to 6-thiouric acid. 

---