Folate-dependent enzymes in cultured Chinese hamster ovary cells: impaired mitochondrial serine hydroxymethyltransferase activity in two additional glycine--auxotroph complementation classes.

摘要: Abstract Two glycine-requiring Chinese hamster ovary (CHO) auxotrophs (GLYB and AUXB2) representative of the Gly − mutant classes B C are shown to have defects in folate metabolism. These result 10-fold lower rates whole cell l -[U- 14 C]serine-to-[ C]glycine conversion relative parental CHO lines (2 vs 20 nmol/h/10 6 cells). This restriction serine hydroxymethyltransferase (SHMT) activity is localized mitochondria. Intact mitochondria from GLYB AUXB2 convert labeled glycine at 1–4% rate with only 1–3% total capacity Yet, contain amounts cytosolic mitochondrial SHMT, latter displaying normal substrate K m values. The impairments formation corrected (but not by a prior growth 100 μ dl -folinate. They also partially restored spontaneous or chemically induced + revertants AUXB2. Subcellular fractionation experiments suggest that low content (one-fifth parental) folylpolyglutamates contributes auxotrophy GLYB. Solubilized SHMT both cleaves alternate substrates allothreonine β-phenylserine rates, without tetrahydrofolate. Consequently, exhibit presence plus 1 -allothreonine (without glycine). studies demonstrate potentially functional (GLYB) (AUXB2). impaired vivo isolated may deficiency recycling 5,10-methylenetetrahydrofolate back multiple adenosine thymidine auxotroph AUXB1 closely mimics respect synthesis, but primary cause different. It due lack folylpolyglutamate synthetase activity.