Effects of sterol manipulation on microsomal desaturase activities in Tetrahymena : With regard to thermal acclimation

Vol. 113, No. 1, 1983 May 31, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages

96-101

EFFECTSOF STEROLMANIPULATIONONMICROSOMAL DESATURASE ACTIVITIES IN TETRAHYMENA: WITH REGARD TO THERMALACCLIMATION Shigenobu Umeki and Yoshinori Nozawa Department of Biochemistry, Gifu University School of Medicine, Tsukasamachi-40, Gifu, Japan Received March 4, 1983 SUMMARY : There was a great increase in microsomal palmitoyl-CoA desaturase activity of ergosterol-replaced Tetrahvmena (ergosterol-cells), which exhibited a pronounced elevation of palmitoleate (16:lA') in fatty acid composition. At 2 hr after the growth temperature-shift from 34 to 15°C (shift-down), palmitoyl-CoA desaturase activity in ergosterol-cells increased 6-fold compared to that in native cells containing tetrahymanol before the shift-down. These results suggest that, unlike drastic increases of palmitoyl-CoA, stearoyl-CoA and oleoyl-CoA desaturase activities by the shift-down in native cells, ergosterol-cells accomplish an adaptive modification of fatty acid composition by a preferential increase in palmitoyl-CoA desaturase activity, being which is principally due to the increased content of the terminal component (cyanide sensitive factor; CSF) of the desaturase system.

INTRODUCTION: One useful

approach for

involvement

membrane functions

of

lipids

in

composition of membrane by earlier

a better

supplementation

studies demonstrated that alterations

numerous supplements influence

the physical

of membranesin many organisms (l-3). 5) 9 a unicellular

eucaryote,

manipulation

membrane lipids

addition,

of

is

with

understanding

of

the

to manipulate the lipid various

of lipid properties

compounds.

The

composition induced by and enzyme activities

As reported in our previous papers (4,

Tetrahymena pyriformis, in

can undergo Q

response to exogenous ergosterol.

we have also employed this cell

as a useful system to gain

into the molecular mechanism(s) of temperature acclimation

w In

insight

of membranelipids

(6- 8) .

In

the

present

replacement transport

in

the

study

activities

enzymes, and also their

to temperature shift-down 0006-291X/83

we investigated of

microsomal desaturation

activities

from 34 to 15°C.

$1.50

Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.

changes induced by the ergosterol

96

and electron

in sterol modified cells exposed

Vol. 113, No. 1, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

MATERIALS AND METHODS: Cell growth and isolation of microsomes: Ergosterol was added to the culture medium pre-warmed to 60°C as a sterile ethanolic solution prior to incubation to a final concentration of 2 mg/ZOO ml of culture (4). A thermotorelant strain NT-1 of 1. gvriformis was grown at 34'C for 24 hr in an enriched proteose peptone medium with or without ergosterol (9). As a native culture containing tetrahymanol, 0.2 ml of absolute ethanol was added to the medium. Both types of culture at the mid-exponential phase were cooled to 15°C over 30 min and cells were harvested after the indicated incubation periods at 15°C. Mlcrosomes were isolated from homogenates prepared in phosphate buffer (0.2 M K HP04/0.2 M KH2P04: 3 mM EDTA and 0.1 M NaCl, pH 7.4) as previously described Collected microsomes were washed once by f 10). suspending in 0.1 M potassium phosphate buffer Protein was (pH 7.4). determined by the method of Lowry & al. (11). @id composition: Lipids were extracted from whole cells by the method of Bligh -2). Fatty acid methyl esters prepared with BF3 according to the method of Morrison and Smith (13), were analyzed by gas-chromatography as previously described (14). myme assay2: The activities of pa;;i;oy,llCoA14 stearoyf-CoA and oyl-CoA desaturases w?ge assayed using 20 [ C] palmitoyl-CoA, "G [ C] stearoyl-CoA and [ C] oleoyl-CoA, respectively, 50 nmol of NADH, 0.1 M potassium phosphate (pH 7.2), 100-400 ng of microsomes in a final volume of 0.5 ml at 34°C as described before (8). The NAD(P)H-cytochrome c and NADH-ferricyanide reductase activities were determined using 20 nmol of cytochrome c or 500 nmol of potassium phosphate, 100 nmol of NADH or NADPH, 20-100 pg of microsomes, and 0.1 M potassium phosphate buffer (pH 7.4) in a total volume of 1.0 ml at 34°C (8). The act'vit'es were calculatedlusi g respective extinction coefficients of 19.6 mM-!cm-' (15) and 1.02 mM cm -P (161. activity assayed by measuring CSF (terminal component) was spectrophotometrically the palmitoyl-CoA-stimulated reoxidation rate of NADH-reduced cytochrome b by monitoring the change in absorbance difference contained microsomes with 0.2 between 425 and 410 nm. d e sample cuvette 200 nmol of fresh Na S to inhibit mitochondrial nmol of cytochrome b5' cytochrome oxidase, and 0.1 M Tris-HCl buffer (pH 3.2) in a final volume of 3.0 ml. The rate of reoxidation of cytochrome b5 reduced by 2 nmol of NADH at 34°C was calculated according to the procedure of Oshino and Sato (17) with a slight modification (18). Chemicalsi The following c&micals were obtained fsom commercial sources: [lC] palmitoyl-CoA, [lC] stearoyl-CoA and [lC] oleoyl-CoA (New England Nuclear Corp., Boston, MA); palmitoyl-CoA, stearoyl-CoA and oleoyl-CoA (P-L Biochemicals, Inc., Milwaukee, WI); NADH, NADPH, horse heart cytochrome c and ergosterol (Sigma Chemical Co., St. Louis, MO); potassium ferricyanide (Kishida Chemical Co., Ltd., Osaka). Other chemicals were of the highest purity available from commercial sources.

RESULTS AND DISCUSSION: applied an

to many types

earlier

tetrahymanol In fluid,

addition,

study, with

of cells we

it

was with

of membrane

(19-22),

showed

ergosterol

when compared

polarization

Manipulation

either

that

that

the

native

the

can

sterol

in

ergosterol-replaced membranes,

(5). 97

composition

procaryotic

Tetrahymena

by adding

found

lipid

has been

or eucaryotic. replace

the culture membranes

as examined

by

the

In native

medium became

(41. less

fluorescence

Vol. 113, No. 1, 1983 Table

I

BIOCHEMICAL

Changes in fatty

acid composition during temperature

34OC (n=6)

RESEARCH COMMUNICATIONS

of total lipids of native and ergosterol-cells shift-down from 34 to 15’C

cells

Native

Fatty acids

AND BIOPHYSICAL

34

Ergosterol-cells

to

15Oc 6 hr

2 hr (n=3)

34°C (n=6)

(n=3)

34 to

15°C 6 hr

2 hr (n=3)

(n=3)

12:o

2.620.4

1.OfO.l

o.a+o.1

2.5iO.2

1.4t0.1

1.8tO.l

14:o

9.6t0.7

8.1?0.4**

7.2?0.7*

14.8?0.1*

8.8f0.7#

8.5?0.2#

16:0 16:l

A9

18:0 18:l 18~2 18:2

A9 A6911

A9,12 A6,9,=

18:3 16:l

A’

/16:0

U/S

ratio

12.1-10.4

7.5+0.6*

7.1?0.2*

6.650.6

13.610.8

18.1+0.9*

15.7?0.8*

7.5+0.9*

2O.OiO.5*

22.2?1.1#

5.920.7#

3.3to.4

4.5f0.8

4.4’0.5

4.6F0.2*

4.4io.3

5.6tO.6

5.8iO.3

7.OtO.6

4.5iO.2*

4.820.5

4.470.5

3.4fO.4

3.920.4

4.9t0.3*

4.1+0.2*

4.8t0.6

5.3+0.4!+

20.920.6 4.320.2

14.4tO.8

12.7i2.1

12.2t1.7

9.4?0.3*

ll.OtO.6

10.7iO.8

23.921.2

26.5t1.6

27.9r0.8*

20.2?1.4*

24.6t2.6

25.0?1.6#

1.1

2.4

2.1

2.8

3.4

3.5

2.2

3.2

3.4

2.0

3.1

3.2

Details for the quantitative analysis of fatty acid composition are described in the MATERIALS AND METHODS. Values are averages +_ S.D. of 3-6 independent experiments. U/S ratio, the ratio of major unsaturated to major saturated fatty acids. n, the number of experiments performed. *, PcO.01; **, Pt0.02 compared to native cells. #, PcO.01 compared to ergosterolcells before a shift. In the present significant (14:0)

changes

ergosterol-cells

in

fatty

and palmitoleate

long-chain

the

increased

palmitoleate

activities

decreased stearate

pathway

would

also

acid

composition

from

by

activity.

This

palmitate

(16:0)

desaturase

a

tetrahymanol activities

+ 2.8)

I).

18:2

18:3

98

) This

an

and fatty in

exogenous desaturase

of palmitoleate

compared

to

stearoyl-CoA (Fig.

the

adaptive

against

ratio

the

fluidity

palmitoyl-CoA

in the

1),

altered

membrane

of

due

in

(Fig.

,

the

desaturases A6 ,g,12

palmitoleate

In contrast,

be

and to

1,

accomplish

increase

in ergosterol-cells

to

12

’

involving

A6,11

elongation.

in ergosterol-cells

(Table decreased

-f

in

18;2

expected

oleoyl-CoA

enhancement as the

, linoleate;

desaturase

decreased

of

decreases A9

are

+

of chain

formation

was reflected (1.1

changes

18:lA

and Ag,12

in

preferential

A9

and

in myristate

significant

18:l

ergosterol-cells

increasing

via

+

decrease

Therefore,

modification

containing

16:l A9

result

may

increases

11

stearoyl-CoA

the

by

palmitoyl-CoA

-+ 18:l A9 + 18:2

(18:0

result

These

to show reproducible

great

(oleate;

I). of

+

of

ergosterol-cells.

ergosterol

(Table

(16:0

composition;

acids

activity

pathway

found

accompanied

fatty

18:3A67g’12)

were

acid

(16:lAg)

unsaturated

y-linolenate; to

study,

1).

to cells

native

and oleoyl-CoA Blank

&

a.

BIOCHEMICAL

Vol. 113, No. 1, 1983

I

f0 34oc

AND BIOPHYSICAL

I

I

1

2

4

6 34oc

TIME Fig. 1 desaturases shift 34 MATERIALS experiments, a> cells, B

(20)

have

activity

AFTER

SHIFT

( h )

Changes in activities of palmitoyl-CoA, stearoyl-CoA and oleoyl-CoA in microsomes from native and ergosterol-cells following the to 15’C. Details for the enzyme assays are described in the AND METHODS. Values are averages i S.D. of 3-6 independent each performed in duplicate. -, palmitoyl-CoA desaturase; stearoyl-CoA desaturase, -, oleoyl-CoA desaturase. A, native , ergosterol-cells.

demonstrated

in microsomes

that

was

N-isopropylethanolamine, would

there

and

result

was a decrease

of L-M cells

composition

headgroup

activity

RESEARCH COMMUNICATIONS

from

of

which

modified have

altered

desaturase

membrane

phospholipid

polar

the

treatment

with

by

suggested membrane

in stearoyl-CoA

that fluidity

the in

reduced the

desaturase

lipid

modified

L-M cells. When

native

the proportion and

and ergosterol-cells of palmitoleate

y-linolenate

reached

gradually

represented

by the

increased

acids

Within

2 hr after

(S).

desaturase

acyl-CoA

34"C-native 6-fold

cells

(Fig.

1).

the

to that

(data

on the

shift-down

was,

3-4

with

all times

a progressive

at 2 hr

of 34"C-native 99

cells,

increased

activity

shown),

after

cells,

three

being fatty fatty

compared increase

a

hand,

linoleate

(U) to saturated

of native

however,

other

whereas

not

of unsaturated

rapidly There

to 15'C,

the maximum level,

U/S ratio

desaturase

compared

shifted

increased

activities

in palmitoyl-CoA

ergosterol-cells

were

shift-down

and 2.5-fold

to

up to of to that

113, No. 1 , 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

-

oL

0' ' 34oc ZO

3E

1 4

2

I 6

4 c -0.5z h e -0.42 z 2

I I

c

-0.2

5 Q E! x @

-0.1

p” Y

I 2

1 4

r0 34OC AFTER SHIFT (h)

TIME

-0.3

1 6’:

8

n Jo

g f: c-l

Fig. 2 Changes in activities of CSF (terminal component) of the desaturase system reductases in microsomes from native and ergosterol-cells and three following the shift 34 to 15°C. Details for the enzyme assays are described in the MATERIALS AND METHODS. Values are averages I S.D. of 3-6 independent experiments. o---q , NADH-cytochrome c reductase; -, NADPH-cytochrome c reductase; -, NADH-ferricyanide reductase. u, CSF activity. A, native cells; B, ergosterol-cells.

of 34”C-ergosterol-cells. the

lowered

In addition,

activities

of stearoyl-CoA

ergosterol-replacement thermal

adaptation

indicate

that,

to a lower to the

the

(Fig.

reductases, cells,

indicating

ergosterol-cells terminal

hand,

other

CSF

21,

there

to

component

make

activity

with

that

activities

the

(cyanide

in

over

sensitive

fluid

during

These

membranes

results

were

exposed

of palmitoleate

due

NAD(P)H-cytochrome

increased

by

at

after

of overall to

factor;

100

by

34’C-ergosterol-cells

2-fold

increases

may be due principally

fluid

of

slightly

increased

caused

activity.

unchangeable were

of

more

lipids.

accumulation

desaturase the

membranes

less

enhancement

desaturases

membrane

was a further

palmitoyl-CoA

34”C-native

Nevertheless,

required

of ergosterol-Tetrahymena

temperature,

NADH-ferricyanide to

is

and oleoyl-CoA

when ergosterol-cells

increased

On

the shift-down-induced

the CSF)

2 hr

desaturase increased of the

c

and

compared the

shift-down. a

shift-down

activities content

desaturase

in of

the

system.

Vol. 113, No. 1, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

REFERENCES: 1. Schroeder, F., Holland, J.F. and Vagelos, P.R. (1976) J. Biol. Chem. 251, 674716756: 2. Esko, J.D., Gilmore, J.R. and Glaser, M. (1977) Biochemistry 16, 1881-1890. 3. Ferguson, K.A., Davis, F.M., Conner, R.L. and Landrey, J.R. (1975) J. Biol. Chem. 250, 6998-7005. 4. Nozawa, Y., Fukushima, H. and Iida, H. (1975) Biochim. Biophys. Acta 406, 248-263. 5. Shimonaka, H., Fukushima, H., Kawai, K., Nagao, S., Okano, Y. and Nozawa, Y. (1978) Experientia 34, 586-587. 6. Nozawa, Y., Iida, H., Fukushima, H., Ohki, K. and Ohnishi, S. (1974) Biochim. Biophys. Acta 367, 134-147. 7. Nozawa, Y. and Thompson, G.A. (1979) Biochemistry and Physiology of Protozoa, pp. 275-338, Academic Press, New York. 8. Umeki, S., Fukushima, H., Watanabe, T. and Nozawa, Y. (1982) Biochem. Intern. 4, 101-107. 9. Nozawa, Y. and Kasai, R. (1978) Biochim. Biophys. Acta 529, 53-66. 10. Nozawa, Y. (1975) Methods in Cell Biology, Vol. X, pp. 105-133, Academic Press, New York. 11. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 12. Bligh, E.G. and Dyer, W.J. (1959) Can. J. Biol. Physiol. 31, 911-917. 13. Morrison, W.R. and Smith, L.M. (1964) J. Lipid Res. 5, 600-608. L. and 14. Martin, C.E., Hiramitsu, Y., Kitajima, Y., Nozawa, Y., Skriver, Thompson, G.A. (1976) Biochemistry 1.5, 5218-5227. 15. Yonetani, T. (1965) J. Biol. Chem. 240, 4509-4514. 16. Shallenberg, K.A. and Hellerman, L. (1958) J. Biol. Chem. 231, 547-556. 17. Oshino, N. and Sato, R. (1971) J. Biochem. 69, 169-180. 18. Umeki, S., Maruyama, H. and Nozawa, Y. (1983) Biochim. Biophys. Acta in press. 19. Nunn, W.D. (1977) Biochemistry 16, 1077-1081. 20. Blank, M.L., Lee, T-C., Piantadosi, C., Ishaq, K.S. and Snyder, F. (1976) Arch. Biochem. Biophys. 177, 317-322. 21. Glaser, M., Ferguson, K.A. and Vagelos, P.R. (1974) Proc. Natl. Acad. Sci. USA 71, 4072-4076. 22. Goto, M., Banno, Y., Umeki, S., kameyama, Y. and Nozawa Y. (1983) Biochim. Biophys. Acta in press.

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