OXIDATION OF GLUTAMINE IN HELA-CELLS - ROLE AND CONTROL OF TRUNCATED TCA CYCLES IN TUMOR MITOCHONDRIA

The oxidative metabolism of glutamine in HeLa cells was investigated u sing intact cells and isolated mitochondria. The concentrations of the cytoplasmic amino acids were found to be aspartate, 8.0 mM; glutamate , 22.2 mM; glutamine, 11.3 mM; glycine, 9.8 mM; taurine, 2.3 mM; and a lanine, <1 mM. Incubation of the cells with [C-14]glutamine gave stead y-state recoveries of C-14-label (estimated as exogenous glutamine) in the glutamine, glutamate, and aspartate pools, of 103%, 80%, and 25%, respectively, indicating that glutamine synthetase activity was absen t and that a significant proportion of glutamate oxidation proceeded t hrough aspartate aminotransferase. No label was detected in the alanin e pool, suggesting that alanine aminotransferase activity was low in t hese cells. The clearance rate of [C-14]glutamine through the cellular compartment was 65 nmol/min per mg protein. There was a 28 s delay af ter [C-14]glutamine was added to the cell before C-14-label was incorp orated into the cytoplasm, while the formation of glutamate commenced 10 s later. Aspartate was the major metabolite formed when the mitocho ndria were incubated in a medium containing either glutamine, glutamat e, or glutamate plus malate. The transaminase-inhibitor AOA inhibited both aspartate efflux from the mitochondria and respiration. The addit ion of 2-oxoglutarate failed to relieve glutamate plus malate respirat ion, indicating that 2-oxoglutarate is part of a well-coupled truncate d cycle, of which aspartate aminotransferase has been shown to be a co mponent [Parlo and Coleman (1984): J Biol Chem 259:9997-10003]. This w as confirmed by the observation that, although it inhibited respiratio n, AOA did not affect the efflux of citrate from the mitochondria. Thu s citrate does not appear to be a cycle component and is directly tran sported to the medium. Therefore, it was concluded that the truncated TCA cycle in HeLa cells is the result of both a low rate of citrate sy nthesis and an active citrate transporter. DNP (10 mu M) induced a sta le III-like respiration only in the presence of succinate which suppor ts the evidence that NAD-linked dehydrogenases were riot coupled to re spiration, and suggests that these mitochondria may have a defect in c omplex I of the electron transport chain. Arising from the present res ults with HeLa cells and results extant in the literature, it has been proposed that a major regulating mechanism for the flux of glutamate carbon in tumour cells is the competitive inhibition exerted by 2-oxog lutarate on aspartate and alanine aminotransferases. This has been dis cussed and applied to the data. (C) 1998 Wiley-Liss, Inc.