Hirofumi Fukushima

Studies on thermal adaptation in Tetrahymena membrane lipids Positional distribution of fatty acid in diacyl- and alkyl-acyl-phosphatidylcholinesand -(2-aminoethyl)phosphonolipids from cells grown at different temperatures

Phosphatidylcholine and 2-aminoethylphosphonolipid, major membrane phospholipids in Tetrahymena, comprise 1,2-diacyl and 1-alkyl-2-acyl moieties, whereas phosphatidylethanolamine is solely in diacyl form. The overall fatty acid composition of phosphatidylcholine and 2-aminoethylphosphonolipid fractions from 15 degrees C-grown cells mainly consisted of linoleic (C18 : 2 delta 9,12) and gamma-linolenic (C18 : 3 delta 6,9,12) acids, except for the large amount of an usual fatty acid, cilienic acid (C18 : 2 delta 6,11) in 2-aminoethylphosphonolipids. When compared with 39.5 degrees C-grown cells, the 1-position of phosphatidylcholine and 2-aminoethylphosphonolipid from 15 degrees C-grown cells undergoes a large increase in palmitoleic (C16 : 1 delta 9) and gamma-linolenic acids with a corresponding decrease of myristic (C14 : 0) and palmitic (C16 : 0) acids. At the 2-position of 15 degrees C-grown cells, linoleic and gamma-linolenic acids in phosphatidylcholine, and cilienic and linoleic acids in 2-aminoethylphosphonolipid increase with a large decrease of palmitoleic acid in both phospholipids and of gamma-linolenic acid in 2-aminoethylphosphonolipid. There was quite a similarity in fatty acid composition between 1,2-diacylphosphatidylcholine and 1,2-diacyl-(2-aminoethyl)phosphonolipid in 39.5 degrees C- and 15 degrees C-grown cells. However, a marked increase of gamma-linolenic acid at the 1-position of 1,2-diacyl-phosphatidylcholine, and of linoleic acid at the 2-position of diacyl-aminoethylphosphonolipid was observed in 15 degrees C-grown cells. The 2-position of 1-alkyl-2-acyl-phosphatidylcholine and 1-alkyl-2-acyl-(2-aminoethyl)phosphonolipid was occupied mainly by unsaturated fatty acids. In 15 degrees C-grown cells, the sum of linoleic and gamma-linolenic acids accounted for 79.9% in 1-alkyl-2-acyl-phosphatidylcholine and 87.1% in 1-alkyl-2-acyl-(2-aminoethyl)phosphonolipid. These data support the hypothesis that 2-aminoethylphosphonolipid and phosphatidylcholine would play an important role as acceptors of polyunsaturated fatty acids (C18 : 2 delta 6,11, C18 : 2 delta 9,12 and C18: 3 delta 6,9,12) for temperature acclimation. Furthermore, changes in the ratio of diacyl- to alkyl-acylphosphatidylcholine may also be involved in thermal adaptation by regulating the number of fatty acid acceptor.

Studies on lipid metabolism in Tetrahymena pyriformis: Properties of acyltransferase systems

Abstract Membrane preparations from Tetrahymena pyriformis catalyzed the acylations of glycerophosphate, isomeric monoacylglycerophosphate, and 1-acylglycerylphosphoryl-choline. Under the optimal conditions, glycerophosphate acyltransferase and 1-acylgly-cerophosphate acyltransferase used saturated and unsaturated acyl-CoA at comparable rates. The specificities of these acyltransferase systems for various acyl-CoAs as compared with the respective maximal velocities do not directly explain the fatty acid distribution in glycerophospholipids. However, the acylation of 2-acylglycerophosphate was highly selective for palmitate when the incubations were carried out in the presence of palmitoyl-CoA, oleoyl-CoA, 1-acylglycerophosphate, and 2-acylglycerophosphate. The 1-acylglycerylphosphorylcholine acyltransferase system showed relatively higher specificity for unsaturated acyl-CoA, which is consistent with the fatty acid pattern of phospholipids. Significant amounts of diglyceride and triglyceride were formed together with phosphatidic acid from acyl-CoA and glycerophosphate, indicating that the enzymes involved in triglyceride synthesis are closely associated with acyltransferase systems involved in phosphatidate synthesis in microsomes. These acyltransferase activities were found mainly in microsomes, and to a lesser extent, in pellicles, too. No significant difference was observed in the properties of acyltransferase systems in microsomes and pellicles.

Studies on thermal adaptation in tetrahymena membrane lipids changes in positional distribution of fatty acids in diacyl-phospholipids and alkyl-acyl-phospholipids during temperature acclimation

The positioning of acyl chains in both 1-O-alkyl-2-acyl- and 1,2-diacyl-phospholipids was analyzed at various time intervals for a thermotolerant strain (NT-1) of Tetrahymena pyriformis cells during cold acclimation. During the 10 h period of adaptation, cells were not able to grow but maintained the ability to divide. The content of palmitate (16 : 0) in phosphatidylcholine and phosphatidylethanolamine was decreased after temperature-shift, with a concurrent increase of palmitoleate (16 : 1 delta 9) and gamma-linolenate (18 : 3 delta 6,9,12). An increase in gamma-linolenate at the 1-position and linoleate at the 2-position was observed in diacyl-phospholipids (phosphatidylethanolamine, phosphatidylcholine and 2-aminoethylphosphonolipid). The 2-position of 1-O-alkyl-2-acyl-phosphatidylcholine and 1-O-alkyl-2-acyl-(2-aminoethyl)phosphonolipid was occupied mainly by gamma-linolenate together with cilienate (18 : 2 delta 6,11) and linoleate (18 : 2 delta 9,12). Cilienate and gamma-linolenate at the 2-position of 1-O-alkyl-2-acyl-phosphatidylcholine were increased after temperature shift, with a small decrease of linoleate. There are little significant changes in alkyl ether lipid content of phosphatidylcholine and 2-aminoethylphosphonolipid after temperature shift. The results indicate that phosphatidylethanolamine, which is most abundant and present only in the diacyl form, would play a crucial role in thermal adaptation of membrane lipids, by replacing palmitate with gamma-linolenate at its 1-position, and also that hexadecyl/gamma-linolenoyl phosphatidylcholine would be an important molecular species in the acclimation.

Isolation and lipid composition of nuclear membranes from macronuclei of Tetrahymena pyriformis

Abstract A procedure was developed for isolation of macronuclei and nuclear membranes from the ciliated protozoan Tetrahymena pyriformis E, and the lipid composition of the isolated nuclear membranes was determined. This method involves cell lysis with octanol, separation of the nuclear membrane with 0.2 M phosphate–1M NaCl and purification on a discontinuous sucrose gradient. By phase-contrast and electron microscopic examinaton, our preparations were pure and preserved the typical nuclear membrane morphology: inner and outer nuclear membranes, and nuclear pore complexes. As for lipid distribution, the three major phospholipids in the membranes were phosphatidylcholine (31.0%), phosphatidylethanolamine (26.1%) and 2-aminoethylphosphonolipids (23.3%) and the molar ratio of a sterol-like lipid, tetrahymanol to phospholipid phosphorus was 0.036. These results were compared to other membrane fractions of Tetrahymena.

Variations in the activity of fatty ACYL-CoA desaturases and electron-transport components in Tetrahymena microsomes with temperature and age of culture

Abstract 1. 1.|Alterations in the fatty acid composition of microsomes were most marked in the exponential phase of both 39.5- or 15°C- grown Tetrahymena pyriformis NT-1. 2. 2.|Activities of palmitoyl-CoA and stearoyl-CoA desaturases were lower in 15°C cells than in 39.5°C cells, while the activity of oleoyl-CoA desaturase was higher in 15°C cells. 3. 3.|Activities of the terminal component of the desaturation system as well as all three desaturases (palmitoyl-CoA, stearoyl-CoA, oleoyl-CoA) were higher in the exponential phase than in the stationary phase for cells grown at both temperatures. 4. 4.|NAD(P)H-cytochrome c reductase activity and cytochrome b 5 content were reduced whereas NADH-ferricyanide reductase activity was increased in the stationary phase at both 39.5 and 15°C.

Immunochemical evidence for participation of NADPH-cytochrome c reductase in microsomal fatty acyl-CoA desaturation of Tetrahymena cells lacking in cytochrome P-450

Rabbit antibody was prepared against NADPH-cytochrome c reductase of Tetrahymena microsomes. When examined by the Ouchterlony double diffusion test, anti-NADPH-cytochrome c reductase immunoglobulin formed a single precipitation line with Tetrahymena reductase but not rat liver one. The antibody inhibited the NADPH-cytochrome c reductase activity of Tetrahymena microsomes, but it did not affect either NADH-ferricyanide or NADH-cytochrome c reductase activity of Tetrahymena microsomes. The NADPH-dependent desaturation of stearoyl-CoA in Tetrahymena microsomes was inhibited by anti-reductase immunoglobuline, while the NADH-dependent desaturation was affected by neither anti-reductase nor control immunoglobuline. It was suggested that the temperature associated-alteration of NADPH-cytochrome c reductase activities would be important for regulation of microsomal NADPH-dependent desaturase activities in Tetrahymena which contains no cytochrome P-450.

Purification and partial characterization of cytochrome b5 from Tetrahymena pyriformis

Abstract With the use of detergents and successive column chromatographies, Tetrahymena b-type cytochrome was purified from microsomes to a specific content of 36.0 nmol per mg of protein. The purified form showed a single band on SDS-polyacrylamide gel with molecular weight of 22,000. The spectral properties of the reduced b-type cytochrome, the α-peak of which is situated at 560 nm and asymmetric with a shoulder at 556 nm, was different from that of rat liver microsomal cytochrome b 5 . However, it was reducible by NADH in the presence of NADH-cytochrome b 5 reductase purified from rat liver microsomes. The results indicated that the microsomal b-type cytochrome should be designated as cytochrome b 5 of a ciliated protozoan, Tetrahymena pyriformis .

Changes in microsomal hemoprotein content and glucose 6-phosphatase activity during low-temperature acclimation of Tetrahymena pyriformis

To examine the temperture‐associated alterations in microsomal enzymes in an eukaryote, Tetrahymena cells were transferred from 39.5–15°C. The content of cytochrome b‐560ms, a cytochrome similar to but not identical with liver microsomal cytochrome b 5, was slightly increased within 2 h after temperature shift‐down, then gradually decreased, and reached the cytochrome b‐560ms level of the 15°C‐isothermally grown cells within 10 h following the temperature‐shift. Cytochrome P‐450 was not detected in Tetrahymena microsomes. The presence of a co‐binding hemoprotein with a main peak at 420 nm was, however, observed. The amount of this hemoprotein was unchanged for 2 h after cold‐shift. Later, it increased gradually and reached the maximum level at 10 h. Glucose 6‐phosphatase activity increased slightly at 10 h, when cell division had already been regained. We conclude that cytochrome b‐560ms, which is linked to the desaturase, may be essential for short‐term adaptation (2 h after shift), while the amount of co‐binding hemoprotein and the activity of glucose 6‐phosphatase would be associated with long‐term adaptation (10 h after temperature shift) in Tetrahymena cells.

Studies on Tetrahymena microsomal electron transport systems: Solubilization of microsomal electron transport enzymes involved in fatty acid desaturation

Tetrahymena microsomes were solubilized with five different detergents and the effect on electron transport enzymes involved in fatty acid desaturation was studied. Cytochrome b560ms and NADPH-cytochrome c reductase were solubilized with a low concentration detergent (0.25%), in the order of sodium deoxycholate greater than Renex 690 greater than Triton X-100 greater than octylglucoside greater than sodium cholate, whereas all of these detergents at the high concentration (1%) could solubilize preferentially both enzymes (70-100%). Increasing the concentration of various detergents from 0.5 to 1.0% did not produce an incremental change in NADH-ferricyanide reductase solubilization. NADH-cytochrome c reductase system, which would be catalyzed by the cooperation action of NADH-ferricyanide and cytochrome b560ms, was relatively inactivated by all detergents. Compared to the other four detergents, octylglucoside has a much higher recovery of stearoyl-CoA desaturase activities in the supernatant. Our study suggests that octylglucoside may be more useful for the isolation in active form of cyanide-sensitive factor (CSF) from Tetrahymena microsomes.

Modification of microsomal lipid composition and electron transport enzyme activities in isovalerate-supplemented cells of novelTetrahymena ISO

Tetrahymena ISO cells, which have an unusually high level ofiso odd-numbered fatty acids, were grown medium supplemented with various concentrations of isovalerate. There was a marked increase in the total proportion ofiso odd-numbered fatty acids in supplemented whole cells (28.9→70.3%) and microsomes (37.7→84%), with a corresponding decrease in normal fatty acids, although no significant alteration of phospholipid composition was observe during 11 hr isovalerate-supplementation. Microsomal palmitoyl-CoA and stearoyl-CoA desaturase activities in isovalerate-supplemented cells decreased by 45.7% and 30.6% during 11 hr, respectively. NADH-cytochrome c reductase and NADH-ferricyanide reductase activities as well as the content of cytochrome b560ms, which is similar to mammalian microsomal cytochrome b5, were reduced in microsomes from 11 hr-supplemented cells, whereas NADPH-cytochrome c reductase activity was constant. It is suggested that the alteration of the cross-sectional area of lipid molecules in the bilayer, which results from the replacement of normal fatty acids withiso-15∶0 andiso-17∶1, would result in the decline of palmitoyl- and stearoyl-CoA desaturation in the isovalerate-supplemented cells, in order to maintain membrane fluidity at a functional level.