Takehito Watanabe

Studies on Tetrahymena membranes in vivo manipulation of membrane lipids by 1-O-hexadecyl glycerol-feeding in Tetrahymena pyriformis

1. Tetrahymena pyriformis NT-I cells were grown in the medium supplemented with 1-O-hexadecyl glycerol which is the precursor for alkyl ether-containing phospholipids; choline phosphoglyceride and 2-aminoethylphosphonolipid, and alterations in the plasma membrane and microsome lipid composition were examined. No incorporation of supplemented 1-O-hexadecyl glycerol was seen in ethanolamine phosphoglyceride. 2. The hexadecyl glycerol fed membranes contain more polyunsaturated fatty acids than do the native membranes. However, the level of oleic acid (C 18:1) drops strikingly in the phospholipids of plasma and microsome membranes. 3. The hexadecyl glycerol-feeding induced a remarkable alteration in the polar headgroup composition of plasma membrane, especially a large increase in 2-amino-ethylphosphonolipid with a compensatory decrease in ethanolamine phosphoglyceride of plasma membranes. 4. The fatty acyl chain composition of phospholipids, especially ethanolamine phosphoglyceride, of the hexadecyl glycerol-fed plasma membranes and microsomes was found to be significantly different from that of the native membranes. 5. These results may indicate that marked alterations in polar headgroup as well as fatty acyl chain composition of membranes induced by glyceryl ether-feeding would be required for maintaining proper membrane fluidity.

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 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.

Studies on temperature adaptation in tetrahymena positional distribution of fatty acids and species analysis of phosphatidylethanolamine from terahymena pyriformis grown at different temperatures

Phosphatidylethanolamine of 15 degrees C-grown Tetrahymena pyriformis (NT-I) cells contains more polyunsaturated fatty acids than 39.5 degrees C-grown cells. This increase in unsaturation is due to an increase in linoleic (C18 : 2) and linolenic (C18 : 3) acids, and a decrease in myristic (C14 : 0), palmitic (C16 : 0), palmitoleic (C16 : 1) and heptadecanoic (C17 : 0) acids. Compared with 39.5 degrees C-grown cells, the proportion of palmitic acid (C16 : 0) decreased in the 1-position as does at the 2-position in 15 degrees C-grown cells. On the contrary, there is a significant increase in linoleic (C18 : 2 delta 9, 12) and gamma-linolenic (gamma-C18 : 3) acids in the 1- and 2-positions, respectively. Phosphatidylethanolamine has been subfractionated into seven different diglyceride species. In 15 degrees C cells, the amounts of fractions 2 (1-linolenoyl-2-linoleoyl) and 3 (1-linolenoyl-2-palmitoleoyl, 1-linolenoyl-2-oleoyl) increased while there was a great decrease in subfraction 7 (1-myristoyl-2-palmitoleoyl, 1-palmitoyl-2-palmitoleoyl). Since subfractions 1 and 2 contain over 70% linoleic (C18 : 2) and linolenic (C18 : 3) acids, these fractions might be composed mainly of 1-linolenoyl-2-linolenoyl and 1-linolenoyl-2-linoleoyl molecular species at 15 degrees C. These data support evidence that phosphatidylethanolamine would play a principal role as an acceptor of acyl chains for temperature acclimation.

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.

Effects of various inhibitors on desaturase and reductase enzyme activities in Tetrahymena microsomal electron transport system

Abstract 1. 1. The effects of several inhibitors on the Tetrahymena microsomal desaturase and cytochrome reductase activity were studied. 2. 2. Cyanide strongly inhibited Tetrahymena microsomal desaturation supported by either NADH or NADPH. Moreover, NADH- and NADPH-dependent desaturase activity was inhibited by cytochrome c and ferricyanide which are artificial electron acceptors. 2′-AMP, a competitive inhibitor versus NADPH, inhibited NADPH-dependent desaturation, while NADH-desaturase activity was not affected by this nucleotide. 3. 3. NADH-ferricyanide and NADPH-cytochrome c reductase activities as well as microsomal desaturase activity were sensitive to p -chloromercuribenzoate, mercuric chloride and N -ethylmaleimide respectively. This indicated that the sulfhydryl groups of NADH-ferricyanide reductase and NADPH-cytochrome c reductase are essential for the interaction of nucleotides with these flavoproteins in Tetrahymena microsomes.