MATHEMATICAL-MODEL FOR EVALUATING THE KREBS CYCLE FLUX WITH NONCONSTANT GLUTAMATE-POOL SIZE BY C-13-NMR SPECTROSCOPY - EVIDENCE FOR THE EXISTENCE OF 2 TYPES OF KREBS CYCLES IN CELLS

A practical method using matrix operations is proposed for studying th e isotopic transformation of glutamate, or any other metabolite isotop omers, in the Krebs cycle. Two mathematical models were constructed fo r evaluating the Krebs cycle flux where the enrichment of [2-C-13]acet yl-CoA is not 100% and the total glutamate concentration remains const ant or varies during incubation. A comparative study of [1-C-13]glucos e metabolism was subsequently carried out using Saccharomyces cerevisi ae cells from two different strains (ATCC-9763 and NCYC-239) by C-13-N MR spectroscopy and biochemical techniques. The results show that ther e are two types of Krebs cycles in cells. The first is represented by the ATCC cells which contain a small amount of 2-oxoglutarate dehydrog enase and hence the flux in the Krebs cycle is negligible. With [1-C-1 3]glucose as a carbon source, the C-13-NMR spectra of glutamate exhibi t the C2 and C4 resonances that are almost equivalent and much greater than that of the C3. Labeled metabolites derived from [1-C-13]glucose enter the Krebs cycle at two points: oxaloacetate and citrate. The se cond cell type is represented by NCYC-239. The C2 and C3 areas are equ ivalent and smaller than the C4 resonance. The results suggest that la beled metabolites enter the Krebs cycle only at the citrate level via acetyl-CoA, 2-oxoglutarate dehydrogenase is present but pyruvate carbo xylase is virtually absent or inactivated. When both are incubated wit h glucose, the total concentration of glutamate was found to decrease with the incubation time. The fraction of glutamate in isotopic exchan ge with the Krebs cycle in NCYC-239 cells is about 2.6% and the reduct ion in glutamate concentration is about 0.5 %/min. Using our model, wi th a variable glutamate pool size, good agreement between the theoreti cal and experimental data is obtained.