Metabolic Characteristics of Preparations of Isolated Sheep Thyroid Gland Cells. I. Activity Levels of Enzymes Concerned with Glycolysis1 and the Tricarboxylic Acid Cycle1

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Abstract
1. The activities of enzymes involved in glucose utilization via the Embden-Meyerhof and pentose phosphate pathways and the Krebs cycle were studied in fractions prepared from homogenates of intact sheep thyroid glands and isolated cells liberated from follicles by a continuous-flow trypsinization procedure. 2. The specific activities (m∧moles of substrate converted/min/mg protein) of all soluble (nonparticulate) enzymes studied were about 3 times higher in homogenate fractions obtained from cells than in those obtained from intact follicles. This attests to the usefulness of the isolated cell preparation for the study of metabolism in the thyroid gland. 3. Glucokinase activity was found both in mitochondria and in the high-speed supernatant fraction (100,000 ×g). About 70% of the glucokinase activity was associated with mitochondria isolated from both the intact sheep thyroid tissue and the liberated parenchymal cells. 4. No evidence was found for the existence in either mitochondria or cell sap of a glucokinase with a low Km value for glucose. This suggests that the gland does not contain a glucose phosphorylating enzyme that adapts to dietary treatment. 5. The presence of malic dehydrogenase (DPN+-dependent), the malic enzyme (TPN+), aconitase, isocitric dehydrogenase (TPN+-dependent) and the citrate-cleavage enzyme in the high-speed supernatant fraction suggests that there are extramitochondrial sites for the disposition of di- and tricarboxylic acids in the thyroid gland. 6. Apparently the rate-limiting step in the conversion of citrate to α-ketoglutarate in the high-speed supernatant fraction is the aconitase reaction. (Endocrinology76:178,1965)