209

Biochimica

et Biophysics

0 Elsevier

Scientific

Acta,

Publishing

398

(1975)

Company,

209-216 Amsterdam

- Printed

in The Netherlands

BBA 56640

THE EFFECTS OF ISOVALERATE SUPPLEMENTATION ON GROWTH AND FATTY ACID COMPOSITION OF TETRAHYMENA PYRIFORMIS W

ROBERT

L. CONNER

of Biology,

Department

(Received

January

and ANN E. REILLY Bryn

Mawr College,

Bryn

Mawr, Pa. 19010

(U.S.A.)

6th, 1975)

Summary Cultures of Tetrahymena pyriformis W respond to isovalerate supplementation by an increase in odd-numbered saturated, unsaturated and cr-hydroxy iso-fatty acids and by a decrease in even-numbered normal fatty acids in the glycerolipids and sphingolipids. Supplementation, however, did not alter the relative amount of unsaturated fatty acids found in the polar lipids. The unsaturated acids 17 : l(i) and 19 : l(i) were isolated from cells grown with [l-l 4 C] isovalerate and found to have a higher specific activity than the monoenes of the normal series. Isotopic and gas-chromatographic analyses also indicated the presence of dienoic and trienoic acids of the iso-acid series. The iso-fatty acid content was elevated with isovalerate levels up to 5.0 mM and an inhibition of growth was noted. At higher concentrations of the short chain precursor, no further increase in total cellular iso-acids was detected although growth inhibition was more pronounced. The a-hydroxy iso-fatty acids of the sphingolipids, however, were elevated in a fashion that paralleled the external concentration of isovalerate; thus, the amount of cw-hydroxy isoacids and the degree of growth inhibition show a direct relationship. The increase in cY-hydroxy iso-acid content of the sphingolipids was at the expense of the saturated normal and iso-acid components. The impact on the physiology of the cells can be envisaged as the result of changes in membrane fluidity due to the presence of high levels of iso-fatty acids without an accompanying reduction in unsaturated acids.

Introduction The fatty acid composition of the polar lipids of many types of cells can be altered by a number of procedures which include temperature shifts, addition of specified long-chain acids, or supplementation with short-chain fatty

210

acids that are, in turn, primers for elongation. All these techniques have been found to be effective in the ciliate, Tetrahymena pyriformis [l-9 J . The use of short chain precursors has shown that sodium propionate, isobutyrate, oc-methyl-n-butyrate [8], and Tris isovalerate [9] result in an increase in normal odd-numbered, iso-even-numbered, anteiso-odd-numbered [ 81 and iso-odd-numbered fatty acids [9], respectively. Isovalerate supplementation also leads to growth inhibition that is a function of the external concentration of the short-chain acid [9]. It was postulated that the growth inhibition may be the result of abnormal membrane function due to an alteration in the fatty acid composition of the polar lipids. To determine if such a relationship exists between the observed growth inhibition and membrane fatty acid content, the ciliates were subjected to a range of isovalerate concentrations and the fatty acid composition of the glycerophospholipids and sphingolipids was determined; The findings of these experiments are detailed in this report. Further, it was anticipated that these data would allow a determination of the degree of modification possible in the polar lipid fatty acid composition and, hence, presumably on membrane fatty acid content, as well as to ascertain the acyltransferase specificity in this organism. Materials and Methods Growth studies were carried out in an enriched peptone medium supplemented with a series of concentrations of Tris isovalerate as previously described [9]. Mass cultures of Tetrahymena pyriformis W were grown at 28.5 + 0.5”C. Due to the growth inhibition by isovalerate, the following schedule was employed to obtain population densities sufficient for analysis: 10.0 mM Tris chloride, 21 1~; 2.5 mM isovalerate, 21 11; 5.0 mM isovalerate, 24 h; 7.5 mM isovalerate, 28 h; 10.0 mM isovalerate, 30 h. A separate incubation was carried out in which 8.4 PCi sodium [l-’ 4 C] isovalerate (spec. act. 2 Ci/mol, International Chemical and Nuclear Corp., Irvine, Calif.) was provided in 500 ml culture fluid. The ciliates from all incubations were harvested, lyophilized, and extracted with chloroform/methanol. 2 : 1 (v/v), as previously described [lo]. The lipids were separated from non-lipid contaminants by Sephadex column filtration [ 111. The purified lipids were divided into neutral and polar lipids by absorption chromatography on a column packed with methanolwashed, 100-200 mesh Unisil (Clarkson Chemical Co., Williamsport, Pa.) [12]. The neutral lipids were eluted with chloroform, while the polar lipids were removed with chloroform/methanol 2 : 1 (v/v). The polar lipids collected from the Unisil column were subjected to differential methanolysis [13]. The fatty acid methyl esters from the glycerophospholipids and the sphingolipids were obtained as described by Ferguson et al. [14]. The fatty acid methyl esters of the glycerophospholipids were resolved further into saturates and unsaturates by argentation chromatography [ 151. The unsaturated fatty acid fractions were applied to a Unisil column and eluted with benzene to remove traces of silver before hydrogenation with PtO,! as a catalyst. The unsaturated fatty acid methyl esters from the [l-l 4 C] - and 5.0 mM isovalerate supplemented incubations were combined and separated into

211 mono-, di- and tri-unsaturates by argentation chromatography. The monoenes were eluted from a column packed with 25% AgN03 /Unisil (w/w) with 30% benzene in petroleum ether (boiling point 40-6O”C), the dienes with 40-55s benzene, and the trienes with 70-100% benzene. All fractions were monitored by gas-liquid chromatography. Appropriate fractions were pooled and an aliquot removed from each for a determination of radioactivity employing a Packard scintillation spectrometer, Model 3320 and a cocktail composed of 2,5-diphenyloxazole and 1,4-bis-( 2-( 5-phenyloxazole))-benzene. The monoene fraction was resolved further on hydrophobic Sephadex (Lipidex TM-5000, Packard Instrument Co., Downers Grove, Ill.) as previously described [9]. The remaining fractions were hydrogenated. The sphingolipids were subjected to acid methanolysis at 75°C for 16 h. The fatty acid methyl esters were separated from the long-chain bases (sphingosines) on a Unisil column as previously described [ 141. All fatty acid methyl ester mixtures were analyzed isothermally on the model 402 F & M biochemical gas chromatography (Hewlett-Packard) using a 6 ft glass column packed with 80-100 mesh Gas Chrom P coated with 12% diethylene glycol succinate at 160” C for the fatty acid esters from glycerophospholipids and 180°C for those from sphingolipids. N2 flow was 35 ml/min. Each component was identified by a comparison of the retention time relative to methyl stearate and by mass spectrometry (Perkin-Elmer Hitachi RMS 4 mass spectrometer interfaced with a Perkin-Elmer model 990 gas chromatograph equipped with a 6 ft stainless steel column packed with 12% diethylene glycol succinate on SO-100 mesh Gas Chrom P and operated isothermally at 140°C). The lipid phosphorus content was determined by the method of Ames

[161. Results Cell growth of T. pyriformis W was inhibited by Tris isovalerate which confirms a prior investigation [9]. At a concentration of 12.5 mM isovalerate, the cells had undergone only one fission after 21 h; however, when these cells were transferred to an unsupplemented medium, growth occurred that was comparable to controls. This experiment indicated the inhibitory effects of isovalerate were readily reversible. Isovalerate supplementation at any level did not appear to influence the amount of lipid per g dry weight or the percentage of neutral and polar lipids in a consistent fashion. The saturated fatty acids of unsupplemented cultures of T.pyriformis W contain a relatively high 21.7% of odd-number iso-acids. Growth of the ciliates with 2.5 mM isovalerate elevated this value to 46.4% (Table I). The amount of iso-saturated acids remained constant and independent of the external concentration of the short-chain acid above 5 mM. There was a corresponding decrease in even-numbered straight chain fatty acids. The saturated fatty acids in unsupplemented cells consist predominantly of normal C, 4 with smaller amounts of normal C, 6, while in isovalerate supplemented cells, iso C, 7 is present in greatest abundance. The proportion of unsaturated fatty acids (74 + 3%) did not change significantly at any level of supplementation. The unsaturates were hydro-

212

genated and the distribution by carbon skeleton ascertained (Table 1). The fatty acids from unsupplemented cells contained less than 1% of odd-numbered iso-unsatura~s. This value was increased to approxima~ly 10% with an isovalerate concentration of 5.0 mM (Table I); no further change was observed with higher levels of the short chain precursor. The unsaturated acids of T. pyriformis consist primarily of normal C, x with smaller amounts of normal cells although the C, 6. This pattern was unaltered in isovalerate supplemented relative amount of normal even acids was depressed. The increase in odd-numTABLE

I

FATTY

ACID

Jf \‘!-bJENA

COMPOSITION

/‘\~KJ~ORJJJ,S

OF

CONTROL

AND

ISOVALEKATE:

TETRA-

SUPPLEMENTED

W

-.. .I ..-0.0 ~__ Saturated

2.5

m&f

mM

5.0 -__

__-_,

7.5

mM

mM

10.0

acids*

: I) (*I)* * 13: O(i)

t

14

: 0 (n)

1.1

3.0

3.1

1.0

2.3

2.8

1.4

32.0

24.1

20.5

23.1

19.5

12

2. 6 .*c

1.6

15:

O(1)

6.1

13.9

17.7

19.6

16.5

15

: 0 (11)

2.2

1.8

2.7

3.0

2.0

16

: 0 (i J

0.9

1.1

0.5

0.8

16

: I) (II)

30.7

16.9

17.3

15.5

14.6

11.8

24.5

26.9

27.3

30.5

17:0(i) 17 18

: 0 (11) : 0 (II)

19:0(i)

Odd

nc~rmal

(r

The effects of isovalerate supplementation on growth and fatty acid composition of Tetrahymena pyriformis W.

209 Biochimica et Biophysics 0 Elsevier Scientific Acta, Publishing 398 (1975) Company, 209-216 Amsterdam - Printed in The Netherlands BB...
578KB Sizes 0 Downloads 0 Views