Analysis of TCA cycle pathways and disposition of cycle intermediates in isolated rat heart mitochondria by carbon-13-NMR

Rat heart mitochondria were incubated in an artificial cytosol for long time periods (∼90 min) to obtain kinetic data on the appearance of 13C label in the cytosolic glutamate pool as measured by 13 C nuclear magnetic resonance (NMR). The medium contained 3- 13C pyruvate as the oxidative substrate along with a pool of initially unenriched TCA cycle intermediates. The non specific transaminase inhibitor, aminooxyacetate (AOA) was used to inhibit transaminase activity almost completely in the cytosolic compartment and partially in the mitochondrial compartment. Interestingly, 13C appeared in the cytosolic glutamate pool with a similar time course in both the presence and absence of inhibitor, showing that cytosolic glutamate was not derived from cytosolic (alpha-KG. This indicates that a transporter other than the unidirectional glutamate-aspartate transporter was active and responsible for transporting labeled glutamate from the mitochondria to the cytosol. Examination of data indicates that the exchange flux directly reflects mitochondrial GOT flux. A novel atmospheric ionization source was also built for trace gas analysis. The corona discharge in this source occurred through a hollow needle thus enhancing the sensitivity of the analysis by eliminating or minimising the use for a blank gas. Poly(3-alkyl)thiophene membranes were prepared and analyzed by atomic force microscopy and scanning electron microscopy. The ultimate use for these being in studying the premeability of these membranes to various gases.