Orthophosphate flux across the membrane of Tetrahymena pyriformis W

Tetrahymena pyriformis W suspended in a buffered glucose solution accumulated orthophosphate [³²P] from the external solution at a measurable rate. The uptake of orthophosphate by the organisms was linear with respect to time when corrections were made to account for a constant efflux observed during the one hour time course of the experiments. Such corrections were based on the measured lowering of the relative specific activity of the suspension medium and led to the derivation of the expressions for the influx and efflux coefficients. The derived expressions for the coefficients are based solely upon the isotopic measurements and by means of these equations it is possible to describe the observed net inward flux of orthophosphate in quantitative terms. The dependence of the uptake of orthophosphate on the external concentration of orthophosphate followed Michaelis-Menten kinetics, and the temperature coefficient (18–28°) of 1.7 for the process fell into the range normally associated with a chemical reaction. The kinetic pattern, per se, does not distinguish a membrane transport mechanism from metabolic incorporation of P_i. Deviations from the expected pattern of uptake were observed at low temperatures.     

Cholesterol Inhibition of Pentacyclic Triterpenoid Biosynthesis in Tetrahymena pyriformis*†

SYNOPSIS. Tetrahymena pyriformis synthesizes tetrahymanol and “diplopterol” from acetate, mevalonate or squalene. The formation of these pentacyclic triterpenoid alcohols is inhibited by the addition of cholesterol to the culture fluid of the ciliates. Cholesterol also inhibits the biosynthesis of squalene from acetate or mevalonic acid. The synthesis of other terpene derivatives from acetate and mevalonate continues in the presence of cholesterol, thus suggesting that a major block occurs after “isoprene” formation and before squalene formation. Further, inhibition of squalene conversion to the pentacyclic alcohols by cholesterol has been established.     

Photolysis in a culture medium for Tetrahymena pyriformis

Considerable variability has been found in the yield of cells in batch cultures of Tetrahymena pyriformis grown axenically in 1% tryptone/0.05% yeast extract. This variability has been traced to the photolysis by visible light of the flavin mononucleotide and thiamine components of yeast extract.     

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.     

Cytomembrane differentiation in a ciliate,Tetrahymena pyriformis

Summary Two special kinds of smooth surfaced differentiations of the endoplasmic reticulum (ER) of the ciliate Tetrahymena pyriformis are described. (A) A novel type of cytomembrane structure is represented by localized bifacial regions in which one side of the cisterna is studded with ribosomes, flexible in outline and of a cytomembraneous ultrastructure and the other side has a smooth, straight profile and a plasma membrane-like triple-layered appearance. Such smooth patches of predominantly rough ER-cisternae have a tendency to pair with a separation of ca. 250 Å. The micrographs suggest a participation of such patches in the formation of vesicles and/or dictyosomes. (B) Tubular structures, including those with microtubular as well as with macrotubular (300–650 Å) diameters, can be in continuity with ER profiles. Possible origins and functions of these tubular forms are discussed.The work was supported in part by the Deutsche Forschungsgemeinschaft.The authors are indebted to Miss Sigrid Krien for skilful technical assistance as well as to Drs. Ch. Bracker, D. J. Morré (both Purdue University, Lafayette, U.S.A), and H. Falk (this institute) for helpful discussions.     

Serotype Expression and Transformation in Tetrahymena pyriformis

SYNOPSIS. Of the 4 serotypes of Tetrahymena pyriformis, syngen 1, the H (expressed at 20–35 C) and T (expressed at 36–40 C) serotypes and their genetic bases have received the most attention. The present study describes the L and I serotypes, considers their transformations to and from the H serotype, and compares inter-locus repression with allelic repression. The L serotype occurs below 20 C. No strain differences have been found between L antigens. However, the rate of transformation between H and L varies in the families, which can be ranked according to their preference for the H or L serotype. A3 is strongly L; D1 is strongly H; the other families form a continuum between these extremes. Preference for the H or L serotype is not correlated with the H allele present. Serotype differences may be hereditary, by the “dilution” test for hereditary differences. Altho the environment largely determines which serotype will be expressed, sublines can maintain established H and L serotypes for more than 40 fissions in a common environment. Crosses between H and L cells suggest macronuclear control of serotype determination and a cytoplasmic influence on macronuclear differentiation. Evidence for the cytoplasmic influence comes from both conjugants and nonconjugants. The I serotype is induced from the H serotype at room temperature by treatment with specific H antibodies, which are also necessary for I maintenance. The I serotype consists of a series of unstable serotypes which transform among themselves. Clones go thru an initial I type one day after induction and then diversify to express secondary types. The initial type and the secondary types are characteristic of the family but are not related to the H genotype. Family C1 lacks 2 secondary types found in other families studied. The differences between inter-locus repression and allelic repression are marked. Altho inter-locus repression is labile, all efforts to reverse allelic differentiations have failed. H heterozygotes maintained for long periods of time in the I or L state showed no reversal of H allelic repression.     

Metabolism of extracellular phospholipids in Tetrahymena pyriformis

We studied the metabolism of phospholipids exogenously added to cultures of the protozoan, Tetrahymena pyriformis. Tetrahymena cells were found to metabolize the extracellular phospholipids and the fatty acyl chains of the latter were accumulated predominantly as a form of triacylglycerol in the cells. This metabolism was considered to be initiated via endocytosis of phospholipid vesicles, as judged from the following facts: Cytochalasin B, an inhibitor of endocytosis, suppressed the metabolism almost completely. Phospholipid vesicles were incorporated into a phagosome-like structure in Tetrahymena cells, as observed under an electron microscope. When phospholipids doubly labeled with 14C and 3H at the glycerol moiety and fatty acyl chain, respectively, were incubated with Tetrahymena cells, the glycerol moiety and fatty acyl chain at the sn-2-position of the exogenous phospholipids were incorporated into the cellular triacylglycerol fraction in a 1 to 1 ratio. Monoacylglycerol acyltransferase activity was detected in the microsomal fraction of Tetrahymena cells. From these results, together with those of our previous study on lysosomal phospholipid hydrolysis in Tetrahymena (J. Biochem. 99, 125-133 (1986)), it is suggested that the extracellular phospholipids which were taken up by the cells via endocytosis were hydrolyzed through the action of lysosomal phospholipases A1 and C, and also that one of the products, sn-2-monoacylglycerol, served as an acyl acceptor for the synthesis of triacylglycerol via the microsomal "monoacylglycerol pathway."