THE EFFECT OF COSUBSTRATES ON TRICARBOXYLIC ACID CYCLE DYNAMICS DURING PYRUVATE OXIDATION*: THE FORMATION OF α‐KETOGLUTARATE AND UTILIZATION OF GLUTAMATE BY MITOCHONDRIA FROM RABBIT BRAIN

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Abstract
—Data comparing tricarboxylic acid cycle dynamics in mitochondria from rabbit brain using [2‐ or 3‐14C]pyruvate with and without cosubstrates (malate, α‐ketoglutarate, glutamate) are reported. With a physiological concentration of an unlabelled cosubstrate, from 90‐99% of the isotope remained in cycle intermediates. However, the liberation of 14CO2 and the presence of 14C in the C‐1 position of α‐ketoglutarate indicated that multiple turns of the cycle occurred. Entry of pyruvate into the cycle was greater with malate than with either α‐ketoglutarate or glutamate as cosubstrate. With malate as cosubstrate for [14C]pyruvate the amount of [14C]citrate which accumulated averaged 30nmol/ml or 23% of the pyruvate utilized while α‐ketoglutarate averaged 45 nmol/ml or 35% of the pyruvate utilized. With α‐ketoglutarate as cosubstrate for [14C]pyruvate, the average amount of [14C]citrate which accumulated decreased to 8 nmol/ml or 10% of the pyruvate utilized while [14C]α‐ketoglutarate increased slightly to 52 nmol/ml or an increase to 62%, largely due to a decrease in pyruvate utilization. The percentage of 14C found in α‐ketoglutarate was always greater than that found in malate, irrespective of whether α‐ketoglutarate or malate was the cosubstrate for either [2‐ or 3‐14C]pyruvate. The fraction of 14CO2 produced was slightly greater with α‐ketoglutarate as cosubstrate than with malate. This observation and the fact that malate had a higher specific activity than did α‐ketoglutarate when α‐ketoglutarate was the cosubstrate, indicated a preferential utilization of α‐ketoglutarate formed within the mitochondria.When l‐glutamate was a cosubstrate for [14C]pyruvate the principal radioactive product was glutamate, formed by isotopic exchange of glutamate with [14C] α‐ketoglutarate. If malate was also added, [14C]citrate accumulated although pyruvate entry did not increase. Due to retention of isotope in glutamate, little [14C]succinate, malate or aspartate accumulated. When [U‐14C]l‐glutamate was used in conjunction with unlabelled pyruvate more 14C entered the cycle than when unlabelled glutamate was used with [14C]pyruvate and led to α‐ketoglutarate, succinate and aspartate as the major isotopic products. When in addition, unlabelled malate was added, total and isotopic α‐ketoglutarate increased while [14C]aspartate decreased. The increase in [14C]succinate when [14C] glutamate was used indicated an increase in the flux through α‐ketoglutarate dehydrogenase and was accompanied by a decrease of pyruvate utilization as compared to experiments when either α‐ketoglutarate or glutamate were present at low concentration.It is concluded that the tricarboxylic acid cycle in brain mitochondria operates in at least three open segments, (1) pyruvate plus malate (oxaloacetate) to citrate; (2) citrate to α‐ketoglutarate and; (3) α‐ketoglutarate to malate, and that at any given time, the relative rates of these segments depend upon the substrate composition of the environment of the mitochondria. These data suggest an approach to a steady state consistent with the kinetic properties of the tricarboxylic acid cycle within the mitochondria.
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