Oxidation of glycerol to formaldehyde by rat liver microsomes

Vo1.153, No. 2,1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 612-617

June 16,1988

OXIDATION OF GLYCEROL TO FORMALDEHYDE B Y RAT LIVER MICROSOMES

Debra K. Winters, Liviu A. Clejan and Arthur I. Cederbaum Department of Biochemistry The Mount Sinai School of Medicine (CIINY) New York, N.Y. 10029

Received April 26, 1988

Rat liver microsomes catalyzed the oxidation of glycerol to a Nashreactive material in a time- and protein-dependent manner. Omission of the glycerol or the microsomes or any of the components of the NADPH-generating system resulted in almost a complete loss of product formation. Apparent K m and Vma x values for glycerol oxidation were about 18 mM and 2.5 nmol formaldehyde per min per mg microsomal protein. Carbon monoxide inhibited glycerol oxidation indicating a requirement for cytochrome P-450. That the Nashreactive material was formaldehyde was validated by a glutathione-dependent formaldehyde dehydrogenase positive reaction. These studies indicate that glycerol is not inert when utilized with microsomes or reconstituted mixed function oxidase systems, and that the production of formaldehyde from glycerol may interfere with assays of other substrates which generate formaldehyde as product. ® 1988AcademicPress,Inc.

Stabilization

of

proteins

against

denaturation

and

conformational

changes that may lead to inactivation can be accomplished by mimicking the natural

environment

of

the

protein.

Including

glycerol

solutions is a widely used method for doing this (i).

in

the

buffer

It has been suggested

that protein stabilization is due to the preferential binding of the protein to the water. against

Presumably,

the more

the structure so formed is less able to unfold

structured glycerol

solvent than against the water

alone

(2). Glycerol

is a product

of the metabolism of

triglycerides by

adipose

tissue and only tissues that possess the enzyme that activates the glycerol, glycerol kinase, can utilize it (3). amounts

in

liver,

mammary glands ( 4 ) .

kidney,

Glycerol kinase is found in significant

intestine,

brown

adipose

tissue,

and

lactating

This enzyme, which requires ATP, catalyzes the conver-

sion of glycerol to glycerol-3-phosphate.

Through a series

of reactions,

glycerol-3-phosphate can be converted to glyceraldehyde-3-phosphate, which is an intermediate of both the glycolytic and the gluconeogenic pathways. Most protocols for the purification of cytochrome P-450 involve the use of glycerol (added to concentrations of up to 30% v/v) as a stabilizer (5,6). In routine assays of cytochrome P-450-containing fractions, we have observed

0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

612

Vol. 153, No. 2, 1988

the production no added carried

of Nash-reactive

substrate, out

metabolize

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

to

but which

evaluate

glycerol

material contain

and

in reaction

glycerol.

characterize

to a Nash-reactive

the

product

mixtures

which

contain

Experiments

were

ability

microsomes

of

and to demonstrate

therefore

that

to this

product was indeed formaldehyde.

MATERIALS

AND METHODS

Liver microsomes were prepared by differential centrifugation from male Sprague-Dawley rats which weighed about 120 g. The mierosomes were washed twice with 125 mM KCI and stored in the same solution at a protein concentration of approximately i0 mg/ml at -70°C. Oxidation of glycerol was assayed at 37°C in a reaction mixture containing i00 mM potassium phosphate, pH 7.4, i0 mM MgCI2, 0.4 mM NADP +, i0 mM glucose-6-phosphate, 2.3 units of glucose-6-phosphate dehydrogenase, i00 mM glycerol and about 0.75 mg of microsomal protein in a total volume of 1 ml, unless stated otherwise. Reactions were initiated by the addition of glucose-6-phosphate plus glucose-6-phosphate dehydrogenase and terminated by the addition of triehloroacetic acid (final concentration of 6~ w/v). The reactions were routinely carried out for i0 min. Protein was removed by centrifugation in a table top centrifuge and an aliquot of the supernatant was used for determination of formaldehyde by the Nash reaction (7). All values were corrected for zero-time controls in which acid was added before initiation of the reaction with the NADPH-generating system. Protein was estimated by the method of Lowry et al. using bovine serum albumin as standard (8). Cytochrome P-450 content was determined by the method of Omura and Sato (9). Values for product formed are based on a standard curve carried out with known amounts of formaldehyde. For experiments involving the addition of nitrogen or carbon monoxide, the following procedure was used. The complete reaction mixture except for microsomes was placed in a test tube which contained approximately 15 ml air space, when empty. The mixture was gently sparged with a steady stream of either nitrogen or carbon monoxide for 60 sec and the tubes were sealed with rubber serum stoppers. Either 5 or I0 ml of the headspace gas was immediately removed with a syringe while the same amount of air was added with a second syringe. The reaction was initiated by the addition of microsomes via a syringe and was terminated with trichloroacetie acid, also added with a syringe. Glycerol, spectrophotometric grade, was purchased from Aldrich Chemical Co. (Milwaukee, WI). All buffers were passed through a Chelex-100 column (Bio-Rad Laboratories, Richmond, CA) to remove contaminating iron.

RESULTS A N D DISCUSSION

As

shown

glycerol with somal

the

length

protein

the

the

about

range

1 to

conditions.

presence

of

system and the microsomes.

effectively

material.

of incubation over

were

reaction

required

i, microsomes

to a Nash-reaetive

production these

in Fig.

The consumption

up to at least of

0.5

2 nmol The

catalyze

to

the metabolism

of glycerol was linear

15 min and linear with micro-

1.5

mg.

Rates

of

per min per mg microsomal production

glycerol,

the

of

formaldehyde

components

of

the

formaldehyde protein from

under

glycerol

NADPH-generating

Omission of any of these agents resulted

613

of

in about

Vol. 153, No. 2, 1988

• BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS I .50

A

25

B

/

E20 o E

E

1.00 c Q

815

i., o

~,10

0.50

Q 0

o.

0

O nn

5

i

i

|

i

15

10 TIME(rain)

5

20

i

0.50

0

i

1.00

1.50

MICROSOMAL PROTEIN (rag) FIGURE LEGENDS

Fig. I.

a

95%

Effect of incubation time and microsomal protein concentration on glycerol oxidation. The oxidation of I00 mM glycerol was assayed at 37°C as described under Materials and Methods with increasing incubation time (panel A) and with increasing concentration of microsomal protein for I0 min (panel B). Product formed is based on a Nash-reactive material and calculated with formaldehyde as standard. Results are from 3 experiments and are corrected for appropriate zero-time controls (A) and controls which contain no microsomal protein (B).

depression

of

c o u l d n o t be r e p l a c e d Increasing resulted

in

an

kinetics

could

the

formaldehyde

concentration

increase be

production

b y an e q u i v a l e n t

in

observed

amount

of

the

I).

of b o v i n e

glycerol

formaldehyde for

(Table

added

production,

reaction

(Fig.

Microsomal serum to

and

2).

An

the

microsomes

typical

saturation

apparent

TABLE i Requirements

for the Oxidation of Glycerol by Microsomes

Reaction condition

Glycerol oxidation

(nmol/min/mg) Complete system Minus glycerol Minus NADP + Minus glucose 6 P and glucose 6 P dehydrogenase Minus microsomes Minus microsomes, plus BSA

Effect

(%)

2.31 0.13 0.09 0.Ii

-94 -96 -95

0.i0 0.06

-96 -97

The oxidation of glycerol was assayed as described under Materials and Methods. Results are from two experiments and are corrected for corresponding zero-time controls.

614

protein

albumin.

K m value

Vol. 153, No. 2 , 1 9 8 8

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

2.5

2.0 E E "~ 1.5 o E

I-cO

./

1.0

60 40

/

O 0 nn

20

y

0.5 30 I

0

I

I

I

90

120

150

I

30

60

60 90 120 150 GLYCEROL (raM)

G L Y C E R O L (mM) Fig. 2.

for

Effect of the concentration of glycerol on the rate of metabolism of glycerol by microsomes. Incubations were for I0 min at 37°C with microsomes corresponding to 0.75 mg protein per sample. Results are the average of three experiments with different preparations of mierosomes and are corrected for rates found in the absence of added glycerol. The insert shows a Hanes-Wolf plot of the substrate concentration curve.

glycerol

data

with

an

microsomal about

of

3.5

about

apparent

protein nmol

18 mM was Vma x

(Fig.

product

of

2,

calculated

2.55

nmol

insert).

formed

apparent K m value for glycerol

per

from

product

This w o u l d

min

per

nmol

to formaldehyde, compared

a role for cytochrome

the ability

to the effect

33~ N 2 did not

change

P-450

of

formed per min per mg correspond of

to

cytochrome

a value P-450.

the of of The

(I0,Ii). in the oxidation of glycerol

of carbon monoxide

of an appropriate the

plot

is similar to that found for the oxidation of

alcohols such as ethanol, propanol and 2-butanol To demonstrate

a Hanes-Wolf

rate

over

to inhibit

nitrogen control.

that of i00~ air

the reaction was The presence

(Table

2).

When

of the

concentration of N 2 was

increased to 67~,

of that under i00~ air.

Under an atmosphere containing either 33~ or 67~ CO,

the production

of formaldehyde

comparison

the

to

N2

the rate of product formed was 85~

from glycerol was

controls

(Table

2).

Thus,

inhibited by about 75~, the

inhibition

by

CO

in of

glycerol m e t a b o l i s m is not due to an anaerobic effect but rather to an effect on the catalytic activity of cytochrome P-450. Formaldehyde specific An

dehydrogenase

is

a glutathione-dependent

for the oxidation of formaldehyde

aliquot

of

the

supernatant,

obtained

in the presence after

the

enzyme of NAD +

addition

n e u t r a l i z e d and incubated with 1.0 m M NAD +, 2.0 m M glutathione

615

of

which

is

(12-14). TCA,

was

and i unit of

Vol. 153, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 2

Inhibition of Glycerol Oxidation by Carbon Monoxide

Reaction condition

Air 33% 33% 67% 67%

Glycerol oxidation

Effect of CO

(nmol product/min/mg )

(~)

2.39 2.35 0.59 2.04 0.48

-75

N2 CO N2 CO

-77

The oxidation of glycerol was assayed as described under Materials and Methods for experiments with nitrogen or carbon monoxide. Results are from two experiments and are corrected for corresponding zero-time controls. The % effect for CO refers to the appropriate nitrogen control.

formaldehyde

dehydrogenase

little or no increase

from an experimental was

the reaction

in absorbance

controls were utilized.

glycerol

until

at 340 nm when aliquots

An increase

in absorbance

sample which originally

assayed.

glutathione-dependent,

The thus

ran to completion.

did occur when an aliquot

increase

the

was

of the zero-time

contained microsomes,

absorbance

indicating

There

presence

was of

both

NADPH

and

NAD +o

formaldehyde

and

in the

sample. In

summary,

metabolized material

in is

the rat

liver

oxidized

glutathione-dependent inhibition by

microsomes

high,

mierosomes

here

to

a

formaldehyde

manner,

P-450.

or

reconstituted

experiments. an important

is usually

that

Moreover,

in high

the production

consideration

of formaldehyde

it

is

is

not

that

inert

NAD +-

and

concentrations

The

it is mediated when

added

NADPH-cytochrome

of formaldehyde

from substrates

an

is This

formaldehyde.

the K m for glycerol

during assays which

glycerol

material. in

suggests

containing

Although

present

that

Nash-reactive

glycerol

systems

P-450.

indicate

dehydrogenase

indicating

Therefore,

and cytochrome

glycerol

production

by

presented

of this reaction by carbon monoxide

cytochrome

reductase

results

to

P-450

is relatively

in reconstitution

from glycerol

involve demethylations,

such as aminopyrine,

may be e.g.,

benzphetamine

or dimethylnitrosamine.

ACKNOWLED(~IENTS

These studies were supported by USPHS Institute on Alcohol Abuse and Alcoholism. typing the manuscript.

616

Grant AA-06610 from the National We thank Ms. Roslyn C. King for

Vol. 153, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES

i. 2. 3. 4. 5. 6. 7. 8. 9. i0. Ii. 12. 13. 14.

Scopes, R. (1982) Protein Purification: Principles & Practice. Springer-Verlag. New York. Ackko, K. and S.N. Timasheff (1981) Biochemistry 20, 4677-4686. Stryer, L. (1981) Biochemistry. W.H. Freeman & Company. San Francisco, CA. Martin, D.W., P.A. Mayes and V.W. Rodwell (1981) Harper's Review of Biochemistry. Lange Medical Publications. Los Altos, CA. Sato, R. and T. Omura (1978) Cytoehrome P-450, Academic Press, New York. Ortiz de Montellano, P.R. (1986) Cytochrome P-450 Structure, Mechanism and Biochemistry, Plenum Press, New York. Nash, T. (1953) Biochem.J. 55, 416-421. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall (1951) J.Biol. Chem. 193, 265-275. Omura, T. and R. Sato (1964) J.Biol.Chem. 239, 2370-2378. Teschke, R., Y. Hasumura and C.S. Lieber (1975) J.Biol. Chem. 250, 73977404. Krikun, G. and A.I. Cederbaum (1984) Biochemistry 23, 5489-5494. Strittmatter, P. and E.S. Ball (1955) J.Biol. Chem. 513, 445-461. Goodman, J.L. and T.R. Tephly (1971) Biochim. Biophys.Acta 252, 489-505. Uotila, L. and M. Koivusalo (1974) J.Biol. Chem. 249, 7652-7663.

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