Presence of red cell type NADH-methemoglobin reductase (NADH-diaphorase) in human non erythroid cells

Vol.49,No.4,

BIOCHEMICAL

1972

PRESENCE

OF RED CELL

TYPE

(NADH-DIAPHORASE) Alena

AND BIOPHYSICALRESEARCH

NADH-METHEMOGLOBIN

IN

Leroux::

Jean-Claude

Centre Universitaire Institut de Pathologie Groupe INSERM, Laboratoire Paris 14e,

Received

September

REDUCTASE

HUMAN NON ERYTHROID

and

COMMUNICATIONS

CELLS

Kaplan

Cochin Moleculaire, associe France

au CNRS

25,1972

SUMMARY - On the basis of immunologic and electrophoretic studiesidence is given that the NADH diaphorase which promotes methemoglobin reduction in the red cell is also present in cells which do not contain hemoglobin : leukocytes, platelets, This enzyme is not found brain, placenta, muscle and liver. in human cultured fibroblasts.

ION - In the

INTRODUCT tained

in

reductase

(MetHb-R),

electrons

(1).

artificial (2)

phorase

enzyme

plays

since

its

By use

of

jects

and

this

method

phoretic cells

(6,

binemia been

*

as

is

in

compared role

characterized

work

and

between

to

be markedly

cells is

(NBC) part

Since completion observation in mental retardation.

one

severe

of

the

RBC from

of the a second

Copyright @ 19 72 by Academic Press, Inc. All rights of reproduction in any form reserved.

congenital

945

sub-

other

blood

methemogloMetHb-R

in the to

of

an electro-

the

retardation only

been

Extension

shown

of

Thesis

(3). has

normal

5).

RBC and

not

present case of

4,

in

Therefore

a Doctoral

sub-

NADH-dia-

enzyme

has

case

reduced

diapho-

a poor

this

this

(1,

mental

(8)*.

several

(DCIP) of

tube

(4)

platelets

MetHb-R

in

to

reduce

of

methemoglobin-reductase

patients

leukocytes

a source

group test

main-

methemoglobinemia

in

methemoglobinemic

Moreover

to

DCIP (2),

of

method

blood

the

to

congenital

7).

able

is

methemoglobin

NADH as

ill-defined

induces

found

" This

the

staining

associated

white

to

a specific

similarity

is

of

indophenol

deficiency

to

uses

enzyme

a physiological

electrophoretically

hemoglobin

action

2.4.dichlorophenol

methemoglobin

the

(RBC)

by the which

this

as

belongs

Whereas for

cell

mainly

an enzyme

such

therefore

rases. strate

blood

state

In vitro

dyes

and

red

a reduced

RBC but

also

possible be

work we made methemoglobinemia

has

submitted. the

in

existence

same with

Vol.49,

No.4,

1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS l

of

the

enzyme

hemoglobin

in

was

In

the

phoretic

different

present

study for

lets,

brain,

placenta,

found

in

RBC.

cultured

EXPERIMENTAL

was

in

and this

weekly

injections,

this

not

and

human

contain

electro-

leukocytes,

liver

of

the

same

enzyme

was

not

found

tory

plateMetHb-R in

at

amounts

4'C

of

g for

as

human

gel (4).

Preliminary

37'C

increasing at

both

and

12 000

estimated

and

was

performed

of

as

had

NAOH-diaphorase

previously

shown and

to

us

the

MetHb-R

in

the

(11).

curve

obtained

found

with

crude

reacting

antigen

obtained

with

red

the

WBC,

last the

lesser

different

Figure

enzyme

of four

indicating

completely

on

cell

homogenates

with two

was

the In

observed

broblasts

- As shown

with

placenta. also

at

with

inhibi-

different

min.

centrifugation was

by

intra-venous,

from

30

enzyme

experiments

identity

AND DISCUSSION

brain,

After activity

electrophoresis

electrophoretic

RESULTS

antiserum. residual

mate-

The

MetHb-R

of

units::::/mg

chicken

and

for

amount

A

antigenic in

injection).

against

red

(9).

electrophoresis.

Starch

cells

as

elicited

incubation

human

West

= 2 500

used

was per

antibody a given

the

of

intra-muscular

after

chicken to

described

the of

15 min.,

submitted

subsequently MetHb-R

mg protein

measured

from

technique activity

alternately of

was

and

(5-7

purified

the

(specific

against

capacity hours

partially of

enzyme

material

tissues

was

obtained

Antiserum

cell

muscle

a modification

rial.

was

do

immunologic

presence

However

purified

protein)

red

we bring

the

- MetHb-R

using

700-fold

15

which

fibroblasts.

cells

of

tissues

considered.

evidence the

human

was

to

liver,

that

muscle,

additional

slope

of at least another contrast the slope

lines from

inactivation

identical an

In

affinity.

different

the

platelets,

tissues presence

cultured

1,

of that

human given

embryo by the

fired

enzyme. These

studies.

results Two

were

further

confirmed

electrophoretically

:::: One unit is the amount of methemoglobin-ferrocyanide

distinct of

enzyme which complex 946

by

electrophoretic

bands

(A and

B,

reduces 1 mumole per minute (10).

x

Vol.49,No.4,

BIOCHEMICAL

1972

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

-ENZYME *.-.a

100;

-

ENZYME

-

HEMOLYSATE

,-----,

PLATELETS

c-a.4

LEUKOCYTE,

1

LIVER

,----.ERAlN

-ENZYME

-

ENZYME

t---m,

RAXNYA

)c----dt

FloRoBLAs1s

*.-..A

NUSCLE

~----*FiBRoeL*sTs YRISOMIC

25

Fig.

1 -

fig.

Inactivation curves of MetHb-R from various human tissues by chicken antiserum prepared against human red cell methemoglobin-reductase. The 30 000 x g supernatant of homogenates prepared in 1 volume of cold 0.06 M Tris buffer pH 7.6 containing 1 mM EDTA was incubated with antiserum (see text) and subsequently assayed for residual MetHb-R activity according to Hegesh (10). The results are expressed as percentage of the activity measured in control experiments where antiserum was replaced by normal chicken serum. "Enzyme" refers to the standard curve obtained with the antigenic material used to produce the antiserum.

2)

of

platelets

NADH-diaphorase and

incubation resis nit

of (fig.

anodic tical

of It

that

D,

fig.

This

band

A and

an 2),

fresh in

additional only

bands

prior

indicates

band our

B in of

erythrocyte

suppressed

to

electropho-

and

blood

assumption leukocytes

B were

band sensitive

is

antige-

that (6,

by

cells

might

extracts

slow-moving A and

leukocytes, were

a common

brain

previous

947

brain,

bands

antiserum

extracts

aged

in

Both clearly

confirms in

seen

where

found

with

3).

also seen

to

placenta,

extract

2 and

band

were

RBC extracts.

the

reactivity

extracts.

(band

aged

the be

7).

idenIn

observed to

anti-

Vol.49,No.4,

1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

+

Origin-

-

_

RBC

Fig.

-

-

$$ ‘ENZY&,,‘BC extract

Starch gel electrophoresis tase in various human ned as NADH diaphorase

2 -

-

-

-

PLAT.

BRAIN

PLAC.

of

methemoglobin The enzyme

tissues. (4).

Bands which are completely by incubation of extract prior to electrophoresis.

0

Bands

not

inhibited

reducwas stai-

inhibited with antiserum

by

antiserum.

In all cases, control experiments showed that a serum from a normal chicken had no effect on the enzyme mobility. "Enzyme" refers to the semi-purified red cell MetHb-R used as antigen. Differences in cellular environment probably account for the discrepancy between red cells, where the A-band not seen in fresh hemolysates only appears upon aging or purification (4, 6), and the other cells where the A-band is already present in fresh homogenates.

serum

inactivation.

a different

mobility

hibited

by the

reactivity is

the

by

(band

C,

is

easily

2)

a single

was

It

seen.

confirming

the

band with was not in-

different

immuno-

enzyme.

that

electrophoresis

extracts fig.

thus

fibroblast

noteworthy

which

phorase,

fibroblasts

antiserum,

of

It rase

In

the

red

cell

NADPH-dependent

distinguishable was

(4),

from

totally

the

dia-

NADH-diapho-

insensitive

to

the

antiserum. An antiserum

for

liver, slope firm

identical

affinity the

muscle, of this

the

enzyme

brain

and

inactivation

finding.

Our

of

the

present

anti-human in

placenta

is

curve.

The

results

RBC,

suggest

948

red

cell

leukocytes,

indicated

by

MetHb-R platelets,

the

electrophoretic that

the

identical data

enzyme

con-

asso-

BIOCHEMICAL

Vol.49,No.4,1972

Fig.

3 -

AND BIOPHYSICALRESEARCH

Photograph of starch gel electrophoresis moglobin-reductase from WBC homogenate supernatant) preincubated with : 1 = 1 vol. normal chicken serum chicken antiserum ; 3 = 2 vol. serum ; 4 = 1 vol. saline.

ciated

with

in

different

the

a common

methemoglobin-reductase tissues

antigenic

The

fact

reductase

in

placenta,

brain

question not

of

contain

might

is

MetHb-R The

thesis with

that mental

the

as the in

the

enzyme

RBC is and

which

also

might

be

at

least

might

as

a methemoglobin

is

identical share

identity

RBC which

severe

that In

has strongly

form is

of

the so-called methemoglobin-reductase ticularly brain (8, 13).

cells

agreement

of

cytochrome

been

recently

the

which

do

heme

proteins

with

this

hy-

b5 reductase postulated

support

our

(12).

starting

hypo-

methemoglobinemia

due

to in

949

raises

other

congenital

actually

pla.telets,

liver, in

likely

acceptors.

findings

retardation

leukocytes,

and

significance

possible the

in

muscle

It

electron

acts

present

presumably

physiological

present the

methe000 g

determinant. that

its

or

of (30

; 2 = 1 vol. chicken anti-

activity

investigated,

hemoglobin.

serve

pothesis and

COMMUNICATIONS

a major

many

tissues

defect and

of par-

Vol. 49, No.4,

1972

BIOCHEMICAL

AND BIOPHYSICALRESEARCH

COMMUNICATIONS

ACKNOWLEDGMENTS We thank This

study

National

Dr

was de

J.C.

DREYFUS

supported

la

Sante

in et

de

for

part la

fruitful

discussions.

by a grant

Recherche

the

from

Institut

Medicale.

REFERENCES

1.

JaffC,

E.R.

and

2.

Scott,

E.M.

and MC Graw,

Hsieh,

H.S.,

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417(1971).

Chem., 237,

249

(1962). 3. 4.

Scott, E.M. and Griffith, -34, 584 (1959). Kaplan, J.C. and Beutler, 29, 605 (1967).

Biochem.

Hopkinson, and Harris,

6.

Kaplan, J.C. and 52, 1243 (1970).

7.

Kaplan, J.C., in : "Clinical Aspects of Red ture and Metabolism" B. RAMOT ed., Academic York, 1 vol., 125 (1971).

8.

Kaplan, J.C., Hanzlickova-Leroux, C.R. Acad. Sci., Paris, 270, West,

Leroux,

A.,

Thesis,

Leroux,

12.

Hultquist, 229, 252

13.

Kaplan,

A.

and D.E. (1971).

J.C.,

Kaplan, and Biochimie,

Bull.

serie

Univ.

Hegesh, E., Calmanovici, Med., 72, 339 (1968).

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