3‐Mercaptopicolinic acid, a preferential inhibitor of the cytosolic phosphoenolpyruvate carboxykinase

in a similar fashion to quinolinic acid [2,3] an inhibitor which is thought to block gluconeogenesis at the level of phosphoenolpyruvate carboxykinase (GTP: oxaloacetate carboxylase (trans-phosphorylating) EC 4.1.1.32). Quinolinic acid appears to be a more potent inhibitor of gluconeogenesis in the rat than in the guinea-pig [ 3,4] since phosphoenolpyruvate carboxykinase (PEPCK) in the latter animal has a bimodal distribution the mitochondrial activity being inaccessible to inhibition by the mitochondrial non-penetrant quinolinic acid. In this communication we attempt to define the site of inhibition of mercaptopicolinic acid and to assess its inhibitory potency relative to quinolinate. 2. Materials and methods 2.1. Preparation of liver fractions Samples of liver taken at sacrifice were blotted, weighed and homogenized in 10 vol of ice-cold buffer containing 0.25 M sucrose, 5 mM Tris-HCl and 0.1 mM EGTA, pH 7.4. The homogenate was cen- trifuged at 600 g for 10 min to remove cell debris and the supernatant then centrifuged at 12 000 g for 10 min to give fractions containing the mitochondrial pellet, aid microsomes plus the soluble components of the cells. These fractions were used for the assay of phosphoenolpyruvate carboxykinase activity by the 12 method of Roobol and Alleyne [5], mitochondria being solubilized by the addition of Triton Xl00 (0.2,;6) prior to assay of enzyme activity. 2.2. Glrtconeogenesis in kidney cortex Kidney cortex obtained from 24-h starved rats or guinea-pigs were rinsed in cold 0.956 (w/v) NaCl and slices were cut with a Stadie-Riggs microtome. The slices were rinsed for 5 min in Krebs-Henseleit buffer, pH 7.4 [6] , and incubated for one hour under O2 + CO2 (95:5) at 37°C with 10 mM lactate, pyruvate, or L-malate. Following incubation the slices were blotted and dried for dry weight deter- mination and the medium deproteinised by addition of perchloric acid. Glucose in the medium was deter- mined enzymically by the method of Slein [7]. III separate experiments we showed that the kidney cortex glycogen content of between 0.2 and 0.5 mg/g wet weight in both rats and guinea-pigs did not decrease significantly during the course of the incuba- tion and therefore did not contribute glucose to the medium via glycogenolysis. We thank Dr H. L. Saunders of Smith Kline and French Laboratories, Philadelphia, U.S.A., for a generous gift of 3-mercaptopicolinic acid. 3. Results 3.1. Irzhibition of glrtcorzeogenesis Since it has been shown [