No abstract

Purified ␥-glutamyl hydrolase secreted from rat H35 hepatoma cells has been characterized as a diffuse band of 55 kDa on SDS-polyacrylamide gel electrophoresis that is converted to bands of 35 and 33 kDa after enzymatic removal of N-linked carbohydrate. Polyclonal antibodies against 55-kDa ␥-glutamyl hydrolase captured the enzyme activity and recognized the glycosylated and both deglycosylated forms of ␥-glutamyl hydrolase. A complete cDNA sequence of ␥-glutamyl hydrolase was obtained using degenerate oligonucleotides derived from peptide sequences, screening of a rat hepatoma cDNA library, and reverse transcription polymerase chain reaction. Based upon the deduced amino acid sequence the peptide component of ␥-glutamyl hydrolase had a molecular weight of 33,400. The results of amino acid analysis of the purified protein agreed with the deduced amino acid sequence in which there are seven potential asparagine-containing glycosylation sites.

A methotrexate (MTX) analog containing fluorine at the rcarbon of the glutamate moiety, -fluoromethotrexate (FMTX), has been synthesized and evaluated for its biochemical and pharmacological properties. FMTX inhibition of dihydrofolate reductase from several sources is nearly equivalent to that shown by MTX. Most important, FMTX is an exceedingly poor substrate for folylpoly (-glutamate) Utilizing the H35 hepatoma cell culture system (3, 5, 6), we are attempting to definitively establish the role of glutamylation in the activity of MTX. The approach has been to employ a MTX derivative that is accumulated by cells and inhibits DHFR as effectively as does MTX but cannot be glutamylated. The derivative we have employed, t-fluoromethotrexate (FMTX), is an analog of MTX containing fluorine in place of hydrogen on the y carbon of glutamate (Fig. 1). Recent studies with partially purified folylpoly(-glutamate) synthetase (FPGS; EC 6.3.2.17) isolated from rat liver have shown that DL-threo-4-fluoroglutamate (threo-FGlu) acts as a chain-terminating inhibitor of the FPGS reaction (15). The data support the hypothesis that the incorporation of a molecule of FGlu into growing polyglutamate chains leads to a product, R-Glu-y-FGlu, that undergoes further glutamylation at a markedly reduced rate. These results suggest that the synthesis of reduced folates or antifolates (e.g., MTX) containing FGlu in place of glutamate would lead to molecules with greatly diminished ability to form poly(-glutamate) derivatives. The topic of this report is the influence of the fluorine substitution on the toxicity of MTX in cultured hepatic cells. In particular, short-term exposure to FMTX causes growth inhibition that is several orders of magnitude weaker than that obtained with MTX. To establish that limited glutamylation is a major reason for these results, it is necessary to demonstrate (i) that both agents exert similar inhibitory effects toward the target enzyme DHFR, (ii) that FMTX and MTX are accumulated to a similar extent in H35 cells, and (iii) that FMTX is poorly glutamylated by H35 cells. These observations are evaluated with Abbreviations: MTX, methotrexate; FMTX, y-fluoromethotrexate; FGlu, DL-4-fluoroglutamate; DHFR, dihydrofolate reductase; FPGS, folylpoly(y-glutamate) synthetase. 2598The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.