During the past year we have continued to examine the role of polyglutamate forms of methotrexate (MTX) in the cytotoxic action of this drug. We have completed studies describing the formation, retention, and binding of polyglutamates in human breast cancer cells. These findings were summarized in last year's report. In new projects, we have undertaken a detailed analysis of the inhibitory effect of MTX polyglutamates on a number of enzymes involved in the synthesis of DNA precursors. We have found that MTX ia a weak, uncompetitive inhibitor of thymidylate synthetase (TS), while the polyglutamates are much more potent noncompetitive inhibitors of the same enzyme, with Kis 2 to 3 logs lower than the parent compound. In addition we have found that MTX polyglutamates inhibit the rate of formation of complex between FdUMP folate and TS, but have no effect on the dissociation rate of this complex. The competition for complex formation appears to be noncompetitive in nature. The analysis of binding studies with TS indicates that the MTX polyglutamates could have potent direct inhibitory effect on TS independent of the depletion of folate pools caused by DHFR inhibition. We have pursued the concept of additional sites of action of polyglutamates, examining enzymes involved in purine synthesis and folate interconversions. This work has established that the MTX polyglutamates potently inhibit AICAR and GAR transformylases, in contrast to the weak or nonexistent inhibition by the parent compound. We have developed a method for highly purifying AICAR transformylase by affinity chromatography, and intend to characterize the catalytic mechanism of this enzyme more fully. Enhanced inhibition by polyglutamates at this site is consistent with the notion that the competitive nature of leucovorin rescue may be the result of competition of reduced folates with the MTX polyglutamates for inhibited enzymes such as AICAR transformylase.