Respiratory enzyme studies in Tetrahymena pyriformis. 4. Stabilization of electron-transport components

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Abstract
Homogenates of Tetrahymena pyriformis aged at 0[degree], or heated at 30[degree], lose overall succinoxidase, glutamic oxidase and B-hydroxybutyric oxidase activities rapidly. Addition of a crude bovine blood-albumin preparation to fresh Tetrahymena homogenates which were then aged at 0[degree], or heated under mild conditions, stabilized the complete succinoxidase and reduced diphosphopyridine nucleotide oxidase systems, as well as succinic dehydrogenase and reduced diphosphopyridine nucleotide cytochrome c reductase, for 24 hours or more. Crystalline bovine serum albumin and other proteins were incapable of stabilizing these enzymes, whereas horse, rat, human and chick serum, proteose peptone and yeast extract all protected against the loss of succinoxidase activity. The slower decline of the succinoxidase activity of rat-liver homogenates aged at 0[degree] could also be partly prevented by ageing in the presence of the crude albumin preparation. The stabilizing material of the crude albumin preparation was shown to be water-soluble, diffusible and resistant to boiling at neutral or acid pH for short periods. Dialysis of rat serum, yeast extract and proteose peptone also abolished their ability to stabilize the succinoxidase system. Addition of sodium succinate to homogenates of T. pyriformis aged at 0[degree] also prevented for at least 24 hours the rapid loss of activity of the complete succinoxidase and reduced diphosphopyridine nucleotide oxidase systems. Moreover, the activities of reduced diphosphopyridine nucleotide cytochrome c reductase, diphosphopyridine nucleotide-linked glutamic and [beta]-hydroxybutyric dehydrogenases and fumarase were also stabilized for varying periods by ageing homogenates at 0[degree] with succinate. When succinate-supplemented homogenates were centrifuged and the resultant particulate preparations resuspended in water, the stabilization effect was not apparent unless additional succinate was added to the re-suspended preparations. Heat denaturation of succinoxidase, reduced diphosphopyridine nucleotide oxidase and succinic dehydrogenase activities was overcome by heating homogenates in the presence of succinate. The structural requirements of the succinate-stabilization effect are very rigid. It is suggested that the present findings are different from previous observations on the stabilization and protection of enzymic activities by various methods, and the results are discussed.