Isocitrate dehydrogenase of Tetrahymena pyriformis

Summary We have studied the isocitrate dehydrogenase ofTetrahymena pyriformis. This enzyme is able to utilize both NAD and NADP, but kinetic studies suggest that the enzymatic activity with NAD is not of physiological significance.Some of the factors that might regulate the NADP-dependent isocitrate dehydrogenase were also studied. This enzyme has an absolute requirement for divalent cations; Mg²⁺ and Mn²⁺ will serve as cofactors but the latter is more effective than the former.It is known that this enzyme is subject to a concerted inhibition by oxaloacetate and glyoxylate. Either glyoxylate or oxaloacetate alone also are capable of inhibiting the enzyme although higher concentrations are required. We have found concerted inhibition also for the NAD-dependent isocitrate dehydrogenase from rat liver and yeast. The activity of theTetrahymena pyriformis enzyme is inhibited by NADPH. This inhibition is competitive with NADP. The Ki and Km values are, respectively, 23µm and 18µm.     

The transsulfuration pathway in Tetrahymena pyriformis.

Four enzymes necessary for the metabolism of methionine by the trans-sulfuration pathway, methionine adenosyltransferase (EC 2.5.1.6), adenosylhomocysteinase (EC 3.3.1.1), cystathionine beta-synthase (EC 4.2.1.22) and cystathionine gamma-lyase (EC 4.4.1.1) were identified in Tetrahymean pyriformis. The ability of these cells to transfer 35S from E135S]methionine to form [35S] cysteine was also observed and taken as direct evidence for the functional existence of this pathway in Tetrahymena. An intermediate in the pathway and an active methyl donor, S-adenosylmethionine, was qualitatively identified in Tetrahymena and its concentration was found to be greater in late stationary phase cells than in early stationary phase cells.     

The metabolism of pyrimidine compounds by Tetrahymena pyriformis

The pyrimidine requirements for growth of T. pyriformis and for reversal of the growth inhibition caused by folate deprivation have been studied. The effects of thymidine and 5-fluorodeoxyuridine have been shown to be quantitatively different from the effects of these compounds on growth and the rate of DNA synthesis in mammalian cells. Labelled nucleosides added to the medium have been found to be converted to the corresponding bases with the exception of deoxycytidine, which is first deaminated to deoxyuridine. As a result no deoxynucleosides other than thymidine specifically label DNA. The results allow deductions to be made concerning the enzymes involved in pyrimidine utilization by this organism. It is suggested that pyrimidine utilization is always channeled through uracil in the case of those compounds that can supply the pyrimidine requirement for growth.     

The Induction of Endocytosis in Starved Tetrahymena pyriformis

SYNOPSIS. The formation of digestive vacuoles by starved Tetrahymena pyriformis could be induced by mixtures of latex particles and a variety of potentially digestible solutes. Latex particles themselves had little effect in inducing vacuole formation. Protein, polypeptide, and RNA were highly effective inducers, while glutamate, amino acid mixtures, polysacharides, and glucose were moderately effective. Sodium-β-glycerophosphate had a slight effect and sodium acetate was ineffective. The possible stimulus to endocytosis is discussed. The endocytic response to inducers does not appear to be an all-or-none phenomenon and varies with the concentration of inducer. The stimulatory effect for protein-related inducers seems to be produced by a large number of stimulatory molecules acting upon a single cell and the magnitude of the response appears to be related to molecular size.     

Intracellular phospholipase activities of Tetrahymena pyriformis

Phospholipase activity was studied in the protozoan Tetrahymena pyriformis NT-1 by using exogenous phosphatidylethanolamine and phosphatidylcholine. Several phospholipase activities were found in Tetrahymena homogenates. They were distinguished with respect to pH optimum, activity dependence on Ca2+, substrate specificity and positional specificity. Ca2+-Dependent phospholipase activity had an optimal pH around 9 and gave rise to free fatty acid and lysophospholipid. This enzyme hydrolyzes phosphatidylethanolamine but not phosphatidylcholine. The alkaline phospholipase with A1 activity was located mainly in the surface membrane (pellicle fraction). The enzyme activity had a pH optimum ranging from 8 to 9, and required 2 mM CaCl2 for the maximal activity. All detergents tested inhibited the enzyme activity. Ca2+-Independent phospholipase activity had an optimal pH from 4 to 5 and gave rise to free fatty acid, lysophospholipid, diacylglycerol, and monoacylglycerol. We concluded that there are at least three phospholipase in Tetrahymena homogenates, i.e., alkaline phospholipase A and acidic phospholipases A and C.