Characterization of human folylpolyglutamate synthetase expressed in Chinese hamster ovary cells.
Chinese hamster AUX B1 cells lack the enzyme folylpolyglutamate synthetase (FPGS) responsible for adding polyglutamates to folic acid. The human gene for FPGS was introduced into AUX B1 cells by cotransfection with human genomic DNA and either pSV2neo or pSV2gpt plasmid DNA. Cells were coselected for FPGS expression by growth in the absence of glycine, adenosine, and thymidine, and for drug resistance by growth in geneticin or mycophenolic acid. The presence of human enzyme in transfected cells was identified by relative enzyme activity determinations with ATP or dATP as substrates. The human HeLa cell enzyme displayed a 50% higher activity with dATP, and the hamster enzyme showed a 50% higher activity with ATP. Hamster and human enzymes also differed in the chain length of polyglutamates on cellular folate. Only monoglutamates were detected in AUX B1 cells, while wild-type hamster cells had predominantly hexa- and heptaglutamates, and human HeLa cells had predominantly hepta- and octaglutamates. Transformants with FPGS activity that showed a human enzyme preference for dATP also had folate polyglutamate chain lengths characteristic of the human enzyme.
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D000818:Animals; D002268:Carboxypeptidases / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D009693:Nucleic Acid Hybridization; D010053:Ovary; D010641:Phenotype; D010957:Plasmids; D011623:gamma-Glutamyl Hydrolase / Q000235:genetics* / Q000378:metabolism
D002268:Carboxypeptidases; D011623:gamma-Glutamyl Hydrolase