Brain Research, 98 (1975) 607-611
607
© ElsevierScientificPublishing Company, Amsterdam - Printed in The Netherlands
Citrate transport and oxidation by isolated rat brain mitochondrla
M. S. PATEL Departments of Medicine and Biochemistry, General Clinical Research Center and Fels Research Institute, Temple University School of Medicine, Philadelphia, Pa. 19140 (U.S.A.)
(Accepted July 16th, 1975)
Citrate plays a regulatory role in glycolysis21 and lipogenesis 8 in the cytosol. Although citrate can be formed by a carboxylation of 2-oxoglutarate by NADP-isocitrate dehydrogenase (EC 1.1.1.42) and subsequent action of aconitate hydratase (EC 4.2.1.3) in the cytosol, the major source of cytosolic citrate is of mitochondrial origin. In rat liver mitochondria citrate is transported across the inner mitochondrial membrane via a specific transporter system~,10,12. However, very little is known about such a transporter system in rat brain mitochondria. Using the osmotic behavior of mitochondria, Chappell 2 and Minn et al. 11 have observed a low activity of citrate permease in rat brain mitochondria. Clark and Nicklas 3 and Patel and Tilghman 17 presented data to suggest that the metabolism of exogenous citrate by rat brain mitochondria was stimulated by L-malate and inhibited by n-butylmalonate, a specific inhibitor of malate transport 18. Since the metabolism of exogenous citrate is dependent on its transport into mitochondria, the oxidation of [6-14C]citrate to 14CO2 was monitored for the transport and oxidation of citrate by brain mitochondria. Using this parameter some properties of the citrate transporter system in rat brain mitochondria are reported in this communication. Mitochondria from adult rat brain were isolated by the method of Clark and Nicklas 3 using a discontinuous Ficoll-sucrose gradient. Rat brain mitochondria isolated by this procedure have previously been shown to have functional integrity as judged by the P/O and respiratory control ratios 13. The composition of the reaction mixture for the oxidation of [6-14C]citrate to 14CO2 is described in the legend to Fig. 1. Incubations were carried out in 25 ml Erlenmeyer flasks which were sealed with rubber serum stoppers equipped with hanging polyethylene cups. 14CO2 was collected into hydroxide of Hyamine-10X as described previously 17. Mitochondrial protein was measured as described by Lowry et a l p using bovine serum albumin as a standard protein. In preliminary studies it was observed that the oxidation of exogenous 5 m M [6-14C]citrate to 14CO2 by rat brain mitochondria was markedly stimulated by 2 m M L-malate (also see Fig. 1A) and the rate of oxidation under such conditions was linear for at least 30 min (results not shown). Increasing the concentration of [14C]citrate in the incubation medium resulted in a marked increase in the rate of oxidation of
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607
© ElsevierScientificPublishing Company, Amsterdam - Printed in The Netherlands
Citrate transport and oxidation by isolated rat brain mitochondrla
M. S. PATEL Departments of Medicine and Biochemistry, General Clinical Research Center and Fels Research Institute, Temple University School of Medicine, Philadelphia, Pa. 19140 (U.S.A.)
(Accepted July 16th, 1975)
Citrate plays a regulatory role in glycolysis21 and lipogenesis 8 in the cytosol. Although citrate can be formed by a carboxylation of 2-oxoglutarate by NADP-isocitrate dehydrogenase (EC 1.1.1.42) and subsequent action of aconitate hydratase (EC 4.2.1.3) in the cytosol, the major source of cytosolic citrate is of mitochondrial origin. In rat liver mitochondria citrate is transported across the inner mitochondrial membrane via a specific transporter system~,10,12. However, very little is known about such a transporter system in rat brain mitochondria. Using the osmotic behavior of mitochondria, Chappell 2 and Minn et al. 11 have observed a low activity of citrate permease in rat brain mitochondria. Clark and Nicklas 3 and Patel and Tilghman 17 presented data to suggest that the metabolism of exogenous citrate by rat brain mitochondria was stimulated by L-malate and inhibited by n-butylmalonate, a specific inhibitor of malate transport 18. Since the metabolism of exogenous citrate is dependent on its transport into mitochondria, the oxidation of [6-14C]citrate to 14CO2 was monitored for the transport and oxidation of citrate by brain mitochondria. Using this parameter some properties of the citrate transporter system in rat brain mitochondria are reported in this communication. Mitochondria from adult rat brain were isolated by the method of Clark and Nicklas 3 using a discontinuous Ficoll-sucrose gradient. Rat brain mitochondria isolated by this procedure have previously been shown to have functional integrity as judged by the P/O and respiratory control ratios 13. The composition of the reaction mixture for the oxidation of [6-14C]citrate to 14CO2 is described in the legend to Fig. 1. Incubations were carried out in 25 ml Erlenmeyer flasks which were sealed with rubber serum stoppers equipped with hanging polyethylene cups. 14CO2 was collected into hydroxide of Hyamine-10X as described previously 17. Mitochondrial protein was measured as described by Lowry et a l p using bovine serum albumin as a standard protein. In preliminary studies it was observed that the oxidation of exogenous 5 m M [6-14C]citrate to 14CO2 by rat brain mitochondria was markedly stimulated by 2 m M L-malate (also see Fig. 1A) and the rate of oxidation under such conditions was linear for at least 30 min (results not shown). Increasing the concentration of [14C]citrate in the incubation medium resulted in a marked increase in the rate of oxidation of
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