The Effect of Temperature on the Fatty Acid Composition of Tetrahymena pyriformis WH-14

SYNOPSIS. A reduction in the growth temperature of Tetrahymena pyriformis strain WH-14 from 35 C to 15 C resulted in distinct alterations in the fatty acid composition of the glycerophospholipids. The proportion of normal saturated acids declined from 26 to 19%; palmitoleic acid increased by 6%, and the composition of the polyunsaturated fatty acids increased in 18:2 Δ^6,11(n) and decreased in 18:2 Δ^9,12(n) and 18:3 Δ^6,9,12(n). The unsaturation index (the average number of double bonds/100 molecules) did not change with a shift in temperature.
Two biosynthetic pathways exist in Tetrahymena for the formation of unsaturated fatty acids. The observed changes in fatty acid composition that accompany a lowering of the environmental temperature can be accounted for by a reduction in the accumulation of products of the fatty acid pathway leading to the formation of γ-linolenic acid [16:0(n) → 18:0(n) → 18:1 Δ⁹(n) → 18:2 Δ^9,12(n) → 18:3 Δ^6,9,12(n)] and an increase in the components of the pathway leading to the formation of 18:2 Δ^6,11(n) [16:0(n) → 16:1 Δ⁹(n) → 18:1 Δ¹¹(n) → 18:2 Δ^6,11(n)]. The data suggest that the regulatory mechanism in Tetrahymena differs from that found in some bacteria where a simple substitution of unsaturated fatty acids for saturated fatty acids occurs at low culture temperatures.     

The presence of acyl-CoA: cholesterol acyltransferase in Tetrahymena pyriformis W

1. The esterification of cholesterol was studied in Tetrahymena pyriformis an organism which does not synthesize sterols nor are sterols required for growth. 2. Microsomes catalyzed the esterification of cholesterol in the presence of oleoyl-CoA but not oleic acid or lecithin. 3. The enzyme has a similar sterol substrate specificity to that of mammalian acyl-CoA: cholesterol acyltransferase (ACAT) and was inhibited by the specific ACAT inhibitor 58-035. 4. The enzyme is constitutive since activity was observed in cells grown in sterol-free medium when cholesterol was added to the in vitro assay.     

The Methylated Purines of Tetrahymena pyriformis Transfer Ribonucleic Acid*

SYNOPSIS. By phenol extraction and DEAE cellulose column chromatography, tRNA was isolated from Tetrahymena pyriformis strain GL. Following acid hydrolysis of the tRNA, the methylated purine content was determined by Dowex 50 column chromatography and paper chromatography. The most abundant methylated guanine derivative was found to be N²-DMG. Also present were 1-MG, N²-MG, and 7-MG. The most abundant methylated adenine was found to be 1-MA; no 2-MA was detected. Small amounts of the N⁶-methyladenines were detected.     

Characterization of the extracellular ribonuclease of Tetrahymena pyriformis W

Several investigations have indicated that Tetrahymena pyriformis secretes ribonuclease activity into culture media. The extracellular ribonuclease from strain W has been purified and partially characterized. The molecular weight was determined by gel filtration to be 26,500. The amino acid composition of the enzyme was compared with those of the three intracellular ribonucleases characterized by Trangas, and substantial differences were demonstrated. The extracellular enzyme hydrolyzed both polyadenylic and polyuridylic acids, indicating lack of absolute base specificity. The hydrolysis of polyadenylic acid followed normal Michaelis-Menten kinetics, but substrate inhibition occurred at high concentrations of polyuridylic acid. The hydrolysis of polyuridylic acid was competitively inhibited by 2'- and 3'-cytidine, guanine, and uridine nucleotides, and by 2'AMP. No inhibition of the hydrolysis of Torula yeast RNA was detected. The kinetic properties of the extracellular ribonuclease are compared with those of the intracellular enzymes.     

Metabolism of branched-chain fatty acids in Tetrahymena pyriformis W

Cultures of Tetrahymena pyriformis W were supplemented with the branched short-chain acids, isobutyrate and α-methyl-n-butyrate. Growth inhibition occurred which was directly related to the concentration of the supplement. Growth of the cells with isobutyrate resulted in an enhancement in the levels of even iso and normal fatty acids, a decrease in odd iso fatty acids, and an elevation in fatty acids less than 18 carbons in chain length. The data indicate a reduction in overall cellular branched-chain synthesis. Polyunsaturated, long-chain iso acids were not detected.Addition of α-methyl-n-butyrate led to minimal incorporation (2%) of saturated long-chain anteiso fatty acids and to a marked increase in odd normal acids at the expense of odd iso and even normal acids in the glycerophospholipids and in neutral lipids. An elevation of odd normal α-hydroxy acids in the sphingolipids was observed. The metabolism of α-methyl-n-butyrate to propionyl-CoA which serves as a primer for odd normal acids would account for the observed changes. Unsaturated anteiso components were not detected. Enhancement of the amount of anteiso acids occurred when the cells were grown at low temperature with α-methyl-n-butyrate.Long-chain anteiso acids (C₁₅, C₁₇ and C₁₉) were incorporated extensively into the glycerophospholipids at the expense of odd iso, odd normal and even normal acids. Anteiso unsaturates were not detected. Elongation of 15 : 0(ai) and 17 : 0(ai) occurred. Retroconversion of 17 : 0(af) and 19 : 0(ai) was observed. Growth with 17 : 0(ai) and 19 : 0(ai) but not 15 : 0(ai) led to the appearance of 19 : 0(ai, α-OH) in the sphingolipid fraction. The fact that addition of these long-chain anteiso saturates led to an enhanced incorporation into the glycerophospholipids compared to supplementation with α-methyl-n-butyrate indicates that a defect in the synthesis of long-chain acids occurs and that acyltransferase activity is not limiting in anteiso acid incorporation.A proposal is made to account for the low levels of unsaturated even iso acids and the lack of unsaturated anteiso acids based on Δ⁹ desaturase specificity toward carbon chain length and the position of the methyl substituent in the fatty acid.     

Characterization of the Extracellular Ribonuclease of Tetrahymena pyriformis W

Several investigations have indicated that Tetrahymena pyriformis secretes ribonuclease activity into culture media. The extracellular ribonuclease from strain W has been purified and partially characterized. The molecular weight was determined by gel filtration to be 26,500. The amino acid composition of the enzyme was compared with those of the three intracellular ribonucleases characterized by Trangas, and substantial differences were demonstrated. The extracellular enzyme hydrolyzed both polyadenylic and polyuridylic acids, indicating lack of absolute base specificity. The hydrolysis of polyadenylic acid followed normal Michaelis-Menten kinetics, but substrate inhibition occurred at high concentrations of polyuridylic acid. The hydrolysis of polyuridylic acid was competitively inhibited by 2'- and 3'-cytidine, guanine, and uridine nucleotides, and by 2'AMP. No inhibition of the hydrolysis of Torula yeast RNA was detected. The kinetic properties of the extracellular ribonuclease are compared with those of the intracellular enzymes.     

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.     

Mirex and Aroclor® 1254: Effect on and Accumulation by Tetrahymena pyriformis Strain W*

Effects of 2 toxicants, Mirex and Aroclor 1254, on Tetrahymena pyriformis strain W in axenic cultures were investigated. Mirex is a chlorinated hydrocarbon effective against the fire ant, and Aroclor 1254 is a compound structurally related to DDT and used extensively in various industrial processes. Both toxicants reduced growth rates and population densities of T. pyriformis grown at 26 C generally in proportion to concentrations of the chemicals, their effects becoming statistically significant (P < 0.05) at 0.9 μg/liter for Mirex and 1.0 and 10.0 μg/liter for Aroclor 1254. Ciliates exposed to the toxicants for 7 days concentrated Mirex 193 × and Aroclor 60 × as compared to the initial concentrations of these compounds. It is suggested that the chief effect of the 2 toxicants on populations of T. pyriformis and of similarly responding ciliates in nature would be to reduce the availability of these protozoa as food organisms and nutrient regenerators. The ability of the ciliates to concentrate the tested compounds would permit the toxicants to enter into and to be translocated through aquatic food chains. In this manner the compounds could exert toxic effects at higher trophic levels.     

Cell cycle-dependent modulations of fenestrin expression in Tetrahymena pyriformis

In Tetrahymena, besides apparent cell polarity generated by specialized cortical structures, several proteins display a specific asymmetric distribution suggesting their involvement in the generation and the maintenance of cell polarization. One of these proteins, a membrane skeleton protein called fenestrin, forms an antero-posterior gradient, and is accepted as a marker of cell polarity during different cellular processes, such as cell division or oral replacement. In conjugating cells, fenestrin forms an intracytoplasmic net which participates in pronuclear exchange. The function of fenestrin is still unknown. To better understand the role of fenestrin we characterized this protein in an amicronuclear Tetrahymena pyriformis. We show that in this ciliate not only does fenestrin localization change in a cell division-dependent manner, but its mRNA and protein level is also cell cycle-regulated. We determine that the two available anti-fenestrin antibodies, 3A7 and 9A7, recognize different pools of fenestrin isoforms, and that 9A7 is the more general. In addition, our results indicate that fenestrin is a phosphoprotein. We also show that the level of fenestrin in the amicronuclear T. pyriformis and the amicronuclear BI3840 strain of T. thermophila is several times lower than in micronuclear T. thermophila.