Evidence for two separate β-ketoacyl CoA reductase components of the hepatic microsomal fatty acid chain elongation system in the rat

Vol. 165, No. 3, 1989 December

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

29, 1989

Evidence

Pages

1428-1434

for Two Separate O-Ketoacyl CoA Reductase Components of the Microsomal Fatty Acid Chain Elongation System in the Rat*

Mahmoud N. Nagi,

Lynda Cook, Sanoj K. Suneja, Peter Osei and Dominick

Juan

Paul S. Peluso, L. Cintit

Hepatic

C. Laguna,

Department of Pharmacology University of Connecticut Health Center Farmington, Connecticut 06032

Received

November

20,

1989

The hepatic microsomal fatty acid chain elongation system can utilize either NADPH or NADH. Elongation activity, measured as the rate of malonyl CoA incorporation into palmitoyl CoA, was enhanced by a fat-free diet and by bovine serum albumin (BSA) when either cofactor was employed. When the intermediate products were determined, it was observed that in the presence of BSA and NADPH, the predominant product was the saturated elongated fatty acid, whereas in the presence of BSA and NADH, the major intermediate was the O-ketoacyl derivative. Employing O-ketostearoyl CoA as substrate, BSA markedly inhibited NADH-supported O-ketoacyl CoA reductase activity and stimulated NADPH-supported activity. Furthermore, the sum of the NADH-dependent and NADPH-dependent O-ketoreductase activities approximated the activity obtained when both cofactors were present in the incubation medium, suggesting the existence of two O-ketoacyl CoA reductases, one using NADH and a 1989 .kadennc Press. Inc. the other NADPH.

The endoplasmic catalyze

the

sequential

elongation

activities

CoA reductase, (l-3).

(FACES)

include

thioester,

*This

work

the

Copyright All rights

equivalents

of malonyl

was supported

liver

possesses

through

the

of the

fatty

fatty

acid

provided

the

ability

involvement

1) condensing

CoA dehydrase,

a) an activated

form

acids

enzymes:

Requirements

~To whom correspondence 0006-291x/89

of fatty of four

b> reducing in

of mammalian

3) O-hydroxyacyl

reductase

moiety

reticulum

of

enzyme,

to

the

2) O-ketoacyl

and 4) trans-2-enoyl acid

in

chain

the

form

elongation of

coenzyme

CoA system A

by NADH or NADPH, and c> 2-carbon

CoA (l-3).

by U.S. should

Public

Health

be addressed.

$1.50

0 1989 by Academic Press, Inc. of reproduction in any form reserved.

1428

Service

Grant

AM21633.

BIOCHEMICAL

Vol. 165, No. 3, 1989 Two of the first

generates

forms

the

the

four

a secondary

saturated

conversion

in detail

final

the

8-ketoacyl

CoA to the

adequately

using

existence the

second

the

first

product.

utilizing

Although CoA to the

reduction

two pyridine

two separate

step,

8-ketoacyl

reductions

whereas the

second

which

catalyzes

evidence

the

step,

i.e.,

studied

conversion

been

of

studied

NADH and NADPH. which

strongly

CoA reductases, reduce

the

reduction

has been the

has not

which

second

reduction

CoA moiety,

nucleotides,

in

the

acyl

CoA derivative,

we provide

NADPH, which

are

intermediate

B-hydroxyacyl

communication, of

reactions

alcohol

of trans-2-enoyl

(l-71,

In this

enzyme-catalyzed

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

suggests

one utilizing

8-ketoacyl

the

NADH and

CoA to the

secondary

alcohol. Methods C3-14Cl B-ketostearyl-CoA (8-keto-18:O) was synthesized according to the procedures of Stoffel and Pruss (8) and Al-Arif and Blecher (9). The concentration of 8-keto 18:0-CoA was measured both by the method of Ellman (101, after the cleavage of the thioester bond with hydroxylamine and by the spectrophotometric measurement of the BSA-O-keto 18:0 CoA complex at 303nm using an extinction coefficient of 29mWlcm-1 (11). The purity of the B-ketostearoyl CoA as determined by radiogas chromatography and thin layer chromatography was greater than 90%. were

Liver microsomes from obtained as described

male Sprague-Dawley previously (11).

rats

(150-2009)

on fat-free

diet

The assay mixture for measuring the total elongation activity contained the following components (final concentration) in a total volume of l.Oml: O.lM Tris-HCl buffer, pH 7.4, 5pM rotenone, 40PM palmitoyl-CoA, 20pM BSA, 60PM C2-14Cl+malonyl-CoA (0.037 PCi), 500PM NADPH or NADH. Following a I-min preincubation at 37°C. the reaction was started by adding 25Opg microsomal protein. After 5-min incubation, the reaction was stopped by adding l.Oml 15% KOH in methanol, followed by addition of carrier lipids (15OPg each of cold methyl-8-ketostearate, B-hydroxystearate and stearic acid) and saponification at 65°C for 45 min. After acidification with l.Oml of 5N HCl, the free fatty acids were extracted three times with 3ml hexane. The pooled hexane fractions were placed into scintillation vials, dried and the incorporated radioactivity was counted. In other experiments for identification and measurement of intermediates, the pooled hexane extracts were evaporated and the residues redissolved in 1OOpl CHCl spotted on 0.3mm silica gel G plates and developed in petroleum ether 3 diethyl ether/formic acid (75/25/l, v/v). The plates were sprayed with 0.2% dichlorofluorescein in ethanol and the separated intermediates - 8-hydroxy, B-keto, saturated and trans-2-enoic acids - were carefully scraped into separate scintillation vials and counted. The incubation mixture for measuring the C3-14Cl.B-ketoacyl-CoA reductase activity contained the following components (final concentration) a total volume of l.Oml: O.lM Tris-HCl, pH 7.4, 5HM rotenone, 55PM 8-ketostearyl-CoA, 27.5pM BSA when used, 750pM NADH or NADPH or both. The reaction was initiated by addition of 1OOpg microsomal protein and incubated at 37°C for 4-min. Termination of the reaction, acidification,

1429

in

Vol. 165, No. 3, 1989

saponification, above or were (12).

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and separation of intermediates were by radiogas chromatography as reported

extraction identified

Results In their Sprecher

investigation

(3)

was markedly incubation

that

enhanced

by both

of the

Table

this

at a ratio stimulation

II).

than

CoA:BSA)

In the

presence

elongation

the

of malonyl

activity

stimulation

of the

step

(3,

hepatic

with

assay

medium.

The addition

with

of BSA

3-fold

NADPH or NADH. chromatography a different

of NADPH and omission

in

NADH (1.70

in approximately

products

microsomal

CoA elongation

observed

radio-gas

7).

CoA incorporation)

rate

either

and

of BSA to the

limiting

of palmitoyl

the

using or

elongation

addition

the

rate

resulted

elongation

the

of

as rate

from

FACES, Bernert

and NADH-dependent

The rate

chromatography

and quantitate

and the

the overall

28% faster

(palmitoyl

When thin-layer

(Table

of BSA.

of

microsomal

was a reflection

when BSA was omitted of 2:l

step

diet

CoA (measured

of microsomal

identify

a fat-free

NADPH-dependent

and presence

1.33)

rate-limiting

enzyme,

the

NADPH was approximately versus

and Discussion

NADPH-dependent

increase

of palmitoyl

absence

the

the

condensing

I compares

elongation the

observed

assay;

activity

of

as described previously

was employed picture

of BSA, greater

emerged than

TABLE I TOTAL FATTY ACID CHAIN ELONGATION ACTIVITY IN HEPATIC MICROSOMES OBTAINED FROM RATS ON A FAT-FREE, HIGH CARBOHYDRATEDIET Elongation Activitya nmol/min/mg protein NADPH

NADH

Malonyl

CoA + Palmitoyl

CoA

1.70 * 0.44

1.33 + 0.d'

Malonyl

CoA + Palmitoyl

CoA + BSA

4.36 f 1.09

3.83 + 1.35

"Elongation activity is expressed as nmols C2-14Cl malonyl CoA incorporation; assay conditions were as described under "Methods". b These experiments were performed in duplicate with three separate microsomal preparations; each preparation contained two pooled livers from rats on a fat-free diet. The values represent the mean + standard deviation. 1430

to

90% of

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

BIOCHEMICAL

Vol. 165, No. 3, 1989

TABLE

IDENTIFICATION

II

OF INTERMEDIATE PRODUCTSOF HEPATIC

MICROSOMAL

PALMITOYL

CoA ELONGATIONa,b

8-Keto

B-Hydroxy-

18:0

18:0

x

x

trans-2-18:1

+ 18:0

&-9+,8:lc

NADPH NADPH

2.0

+ 0.6

2.9 2.3

+ 0.8 f 0.2

95.2 86.6

f 0.9 + 2.5

20.6 3.0

+ 2.8 * 0.4

65.0 11.3

+ 4.8 + 3.8

11.1 f 3.8

+ BSA

NADH NADH + BSA

14.3 85.7

f 2.0 + 4.0

x

aThe conditions are as described in Table I and under "Methods". bAs in Table I. c Analysis by radlogas chromatography showed that greater than 90% of the radioactivity was associated with Q-9-18:1.

the

elongation

unsaturated

(18:O);

more

product

than

stimulated

Replacing

90% of

the

When 8SA was included

in

f-2-18:1

+ 18:D + cis

This mixture.

(21% to 3%). concomitant

pattern

l-2-18:1, increase

results

about

in O-keto-18 both

in Table

is

the

in

and the absence

pattern

was associated while

1431

products

the

when

amounts

formed

i.e.,

decrease

accumulate. of two separate

of 14% and 20X,

in

results

and NADH-dependent

II).

products;

8SA was included

These

was

TLC-spot

(65% to 11%) occurred,

0 (14% to 86%).

existence

with

a significant

products

elongation

of elongation

accumulated,

altered

and 18:0

was not

of 8SA (Table

significant

(13-OH-18:O)

This

desaturation

I, the

showed

oleate.

NADPH-dependent

in Table

NADPH-d e pendent different

II

assay,

a different

g-18:1

with

both

and

chromatography

conditions

produced

in Tab e II,

-cis-9-18:l

3-fold,

the

was markedly

As seen

BSA stimulates

activities

activity

and O-hydroxystearate

respectively. reaction

NADH, yielded

elongation

assay

as seen

to those

by radiogas

to contain

(a-9-18:1)

was associated

our

3-fold

presumed

and oleate

latter

under

total

TLC spot

(t-2-18:1)

radioactivity

since

NADPH with

O-keto-18:0

the

the

in magnitude

containing

while

in the

of the

approximately

similar

65% of

present

separation

was expected

inhibited.

were

were

trans.-2-octadecenoate

stearate that

products

chain

in

R-OH-18:O with

a

indicate

that

elongation

One interpretation reductases

the

- an

of

Vol. 165, No. 3, 1989

NADH-specific

BlOCHEMlCALANDBlOPHYSlCALRESEARCH

O-ketoacyl

the

accumulation

is

NADPH-specific

of O-keto

that

our

by organic

The O-ketoacyl

the

presence

O-ketoacyl

(ILOH-18:0,

t-2-18:1

presented

in Table

intermediate

and used activity

III

(R-hydroxy

only 18:O)

is

the

CoA reductase and 18:O)

by BSA resulting

O-ketoacyl

to measure the

product

equivalents

inhibited

CoA reductase

or stimulated

separately

O-hydroxy

of reducing

is

were

is

the

reduction of

immediately

dehydrated

activity,

all

product three

However,

as the

rate

of formation

for

reasons

indicated

step.

activity microsomes;

final

measured.

first

intact

saturated

which

CoA was

one enzymatic in

is

and

formed.

In

the

below.

enzyme

activity

8-Hydroxy

As shown

Activitya

18:0

NADH

14.88 * 3.64c

NADPH

6.33 = 1.11

}21.21 20.50 + 3.30

NADH + NADPH NADH + BSA"

6.36 zt 2.30

NADPH + BSA

15.12 i 6.38

NADH + NADPH + BSA

20.58 e 7.34

}21.48

a The 8-ketoacyl CoA reductase activity was measured in the presence of 55PM 8-ketostearyl CoA, 1OOpg microsomal protein, 750PM NADH and/or 750PM NADPH and 4 minute incubations as described under "Methods", and expressed as nmols formed/minute/mg protein. b When BSA was included in the assay mixture, a 2:l ratio of substrate to BSA was always used. c The values represent the mean + standard deviation of experiments performed in duplicate with 8 separate microsomal preparations. 1432

is

initial in

HEPATIC MICROSOMAL 8-KETOACYL CoA REDUCTASE ACTIVITY IN THE PRESENCE OF NADH AND/OR NADPH

Additions

in the

intermediates

of the

CoA Reductase

FACES

in other

TABLE III

8-Ketostearoyl

in

by 8SA.

C3- "CI-13-ketostearoyl

be measured

intermediate

determining

unaffected

interpretation,

synthesis

cannot

which

and a second

is

CoA reductase

ostensibly

words,

18:0,

and either

To substantiate prepared

CoA reductase

COMMUNICATIONS

Table

Vol.

165,

III

No.

when

3, 1989

8-keto-stearoyl

approximately reaction in

BIOCHEMICAL

CoA reductase

15 nmols

formation

absence

and presence

was more

15.1).

formation

a Z-fold

other

total

product

chromatography

thiolase

activity

formation

of

in

stearate

the

concentration

not

shown)

and presence

Conclusive

and separate

obtained

when both

in Table

III,

for from

electron

in

oleate,

rate

of

of

may be attributed

in preliminary

experiments

by the

to (data

trans-2-enoyl

of 40 PM O-ketostearoyl

CoA

CoA in the

was

were

included

reductase

the

presence

with of

electron

8-ketoacyl new findings, flow

chains

to these

in the

activities

with

BSA was included

of these

transport

The low

CoA to stearate

measured

two separate

of

by the presence

CoA reductase

sum of 8-OH-18:O

pathway(s)

formed

by

8-ketoacyl

the

In light

product

a NADPH-dependent

For example,

of

since

total

20% was identified

(7).

product,

only

that

activity

proposal

preparations

the

to

the

represented

60 and 70% of

latter

(6.3

surprise,

and oleate

assay,

CoA reductase

sum of the

when

to our

the

8-ketoacyl

the

obtained

formed

the

presence

of D-OH-18:O

of R-OH-1B:O

rate

the

a NADH-dependent

approximates

were

formation

the

remaining

desaturated

NADPH in

in

measured,

CoA, the

cofactors

measured

of

occurred

of BSA.

distinct

activity

opposite with

same decrease

BSA included

between

in the

evidence

the

rate

formation;

substrate,

exact

When the

and with

were

of t-2-18:1

was inhibited

the

as stearate

its

conversion

the

was measured

in

microsomal

or

was a significant

identified

of O-keto

the

reductase

the

NADH,

of BSA, there

III);

as palmitate,

with

protein.

protein,

was 8-hydroxystearoyl

COMMUNICATIONS

formed/min/mg

activity

intermediates

RESEARCH

was measured

However,

increase

peaks

radiogas

absence

(6.4).

nmols/min/mg

radiochromatographic 10% of

presence

of BSA (Table

than

When the

about

the

BIOPHYSICAL

activity

were

CoA reductase

NADPH was 6.3

there

in

of l3-OH-18:O

when O-ketostearoyl

with

8-OH-18:O

was measured

the

AND

both

assay

mixture.

obtained cofactors

As reported

with

in the

NADH and NADPH assay

NADH and NADPH was 21.2, both

cofactors

in the

assay

was 20.5. mixture,

medium.

whereas Similar

supporting

studies

two reductases

(13-20). 1433

are from

focussed the

the results

our

CoA reductases. current

is

on the

microsomal

Vol. 165, No. 3,1989

BlOCHEMlCALANDBlOPHYSlCAL

RESEARCH COMMUNICATIONS

References :: 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

Nugteren, O.H. (1965) Biochim. Biophys. Acta 106, 280-290. Seubert, W. and Podack, E.R. (1973) Mol. Cell Biochem. 1, 29-40. Bernert, J.T., Jr. and Sprecher, H. (1977) J. Biol. Chem. 252, 6736-6744. Podack, E.R. and Seubert, W. (1972) Biochem. Biophys. Acta 280, 235-247. Cinti, O.L., Nagi, M.N., Cook, L. and White, R.E. (1982) J. Biol. Chem. 257, 14333-14340. Prasad, R.M., Nagi, M.N., Cook, L. and Cinti, O.L. (1983) Biochem. Biophys. Res. Commun. 113, 659-665. Bernert, J.T., Jr. and Sprecher, H. (1978) Biochim. Biophys. Acta 531, 44-55. Stoffel, W. and Pruss, H.O. (1967) J. Lipid Res. 8, 196-201. Al-Arif, A. and Blecher, M. (1971) Biochim. Biophys. Acta 248, 416-429. Ellman. G.L. (1959) Arch. Biochem. Biophys. 82, 70-77. Nagi, M.N., Cook, L., Suneja, S.K., Osei, P. and Cinti. O.L. (1989) Anal. Biochem. 179, 251-261. Nagi, M.N., Cook, L., Ghesquier, 0. and Cinti, O.L. (1986) J. Biol Chem. 261, 13598-13605. M.R. and Cinti, O.L. (1986) Biochem. Nagi, M.N., Cook, L., Prasad, Biophys. Res. Commun. 140, 74-80. Keyes, S.R.. Alfano, J.A., Jansson, I. and Cinti, D.L. (1979) J. B 01. Chem. 254, 7778-7784. Keyes, S.R. and Cinti, D.L. (1980) J. Biol. Chem. 255, 11357-11364 Ilan, Z., Ilan, R. and Cinti, D.L. (1981) J. Biol. Chem. 256, 10066-10072. M.R. and Cinti, D.L. (1983) J. Biol. Chem. Nagi, M.N., Cook, L., Prasad, 24, 14823-14828. Nagao, M., Ishibashi, T., Okayasu, T. and Imai, Y. (1983) FEBS Lett. 155, 11-14. M. and Yubisui, T. (1982) J. Neurochem. 39. 1047-1049 Takeshita, M.. Miki, Takeshita, M., Tamura, M. and Yubisui, T. (1983) Biochem. J. 214, 751-756.

1434