Effect of amytal and diethylstilbesterol on the oxidation of DPNH by mouse tumor and liver mitochondria extracts

BIOCHEMICAL

Vol. 4, No. 4,196l

EFFBCT

OF AMYTAL

OF DPNH

AND BIOPHYSKAL

AND DIBTHYLSTILBESTEROL

BY MOUSE

TUMOR

AND LIVER

L. Department School

Zeceived

February

Various genie

steroid

(1959, electron

chain

posed

that

that

by

of

vation

the

resistant

DPNH to

supporting

locus

this

oxidase

from

--et liver

tions

that

besterol,

al

(1961),

have

and

tumor

mitochondria

retain but

Experiment from in

a marked which

al.

mitochondria C57

‘Supported United

the

the

mice

by States

submitted in

this

the

of

the

procedure

(CY-4868) Health

it

of

mice

and

and Service.

of

is

299

also

toward

pyridine

nu-

administration

possible

sodium

oxidizing the

to

by

755

Baltscheffsky

a Senior

are

evidence

C reductase

to

capable C5,

of

inhibition

insensitive

livers

as directed

cytochrome to

obser-

6-aminonicotinamide,

that -

proto

amytal

simultaneous

antagonist

been

the

part,

antagonism

by

has

al.

the

identical

to in

--et in

a1.(1960)1;

laboratory

synergistic

inhibit

point

is

insensitive

susceptibility

systems

--et

to

, it

compounds

eatro-

Yielding

some

Recently

being

DPNH

Enzyme

a grant Public

at

demonstrated

apparently

utilizing

activity

[Yielding

niacin

are

of

and

are

systems

and

(1959)

which

Studies

enzymatic

Jensen

these

amytal

of

shown

C.

for

possessing

been

cytochrone

inhibition,

estrogens

Dietrich

Miami,

compounds

DPN,

systems

elucidation

synthetic

of Florida.

have

inhibition sodium

cleotide-dependent of

non-steroid

and

postulate.

a biochemical

University Miami 36,

enzymatic

DPNH of

steroid

EXTRACTS1

Dietrich

reduced

antagonizing

barbituate,

that

and

of

between

the

MITOUiONDRIA

diethylatilbeaterol,

oxidation

1960),

ON THE OXIDATION

1.961

e.g.,

enzymatic

S.

Biochemistry, Medicine,

of

hormones,

activity,

the

of

23,

RESEARCH COMMUNICATIONS

Fellowship

extract prepara-

diethylstil-

amytal. DPNH

were

isolated

adenocarcinona (1957)

grown

modified

(SF-186)

by

from

the

BIOCHEMICAL

Vol. 4, No. 4,196l

employing,

as

fugation

the

enzyme source,

at 23,000

in 95% ethanol

x G for

No differences

were used

as the

were

solvent.

procedure,

The data

Results. ground

with

fine

chrome

C is

employed

tems the

and 1.7 x 10e4M,

at a concentration

tracts

sarcosomes

electron

acceptor

oxygen

is sensitive

systems

are sensitive

synthetic

estrogen

is

stilbesterol

studies

either

antagonize

were

glycol

at 2S°C in a

obtained

Extracts

employing

the

In the

latter is

cytochrome

C as the

of llaltscheffsky

(19571,

to diethylstilbestcrol. is 50 times

C is used as the to the of mnytal that

this

electron

cytochrome oxidation I both

acceptor. by Yielding

against

cytochrore

the

system has retained that

points

300

amytal

ac-

and sodium

exthat

C as the of DPNH using

DPNH oxidation is

employed

x 10e7M vs. 2.0

one studied

one to surmise at different

(4.0

electron

mitochondria

When oxygen

as sensitive

4.0 x 10e7M

who demonstrated

the

in Table

sys-

activity.

of DPNH employing

shown

around

2.0 x lo-‘M

snd tumor

while

is

enzymatic

and amytal

liver

to amytal,

when cytoLow activity

acceptor.

of approximately

As

of mitochondria

of DTNH oxidation

electron

employing

resistant

to be similar

permits

rate

Utilizing

to amytal.

of the fact

out

of 6.7 x 10e3M is without

The ineffectiveness in spite

or propylene

carried

I.

is utilized.

the oxidation

as when cytochrome tem appears

a high terminal

the findings

in heart

in Table

has a Ki

These

confirm

exhibit

respectively.

amytal

was added to control

values

diethylstilbesterol

diethylatilbesterol

Discussion.

were

and nitrogen

as the

ceptor

by centriwas dissolved

when 60% dioxane

reactions

are presented

for

of ethanol

observed

when oxygen

found

Ki

sedimented

(1957).

A1203

however,

not

Diethylstilbesterol

amount

All

Layne

material

20 minutes.

Beckman DU spectrophotometer

observed,

all

and an equivalent

vessels.

Biuret

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The former

the

x lo-gH1 sys-

(1960). C reductase

sensitivity

system

to diethyl-

and diethylstilbesteroi

in the multi-enzyme

complex

between

Vol. 4, No. 4,196l

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TABLE I Amytal

and Diethylstilbesterol

of Mouse Liver

and Tumor

Inhibition Hitochondria

as the Terminal

of DPNH Oxidation Employing

Electron

in Extracts

Oxygen and Cytochrome

C

Acceptor* DPNH Oxidation

Inhibitor ----

Tissue

micro

moles -.

of DPNH removed/S ---. ---_---___

bwn

Liver

755 Tumor

min/mg

protein

Cytochrome

C

None

38

642

Amyt al+

25

644

Diethylstilbesterol$

18

311

None

9

62

Amyt al+

5

62

DicthylstilbesterolS

4

x lo3

36 -.

---

* Each reaction vessel contained 0.067 M Phosphate, pH 7.4, DPNH, 1.0 x 10-4M, Mitochondrial extracts and inhibitor as indicated, in a total volume of Cytochrome C was added to a final concentration of 1.1 x 10V4M. Re3 ml. actions were carried out in duplicate at 25’C. + Amytal added to a final concentration of 1.7 x 10-4M and 6.7 x 10m3M in oxygen and Cytochrome C systems, respectively. + 4 Diethylstilbesterol added to a final concentration of 4.0 x lo-‘M and 2.0 x 10m5M in the oxygen and cytochrome C systems, respectively. The change in absorbancy’ observed tion was discarded in calculating during the time periods reported.

DPNH and cytochrome one place the electron permit sensitive

on the chain

the detection

C or that electron that

diethylotilbesterol

chain. is

highly

of other

to the synthetic

during the first minute activity. The reaction

points

In the

in the

estrogen.

301

antagonizes

latter

sensitive

after rates

case,

10s~

enzyme addiwere linear

at more than of a portion

to diethylstilbeeterol electron

chain

of

could which

are

less

Vol. 4, No. 4,196l

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

--References Jensen, P. K., Nature 184: 451 (1959). Yielding, K.L. and Tomas, G. M., Proc. Natl. Acad.Sci. U.S. 45: 1730 (1959 1 Yielding, K. L., Tomhins, G. M., Munday, J. S. and Cowley, I.J., J. Biol. . Chem., 235: 3413 (1960). DietriccL.S. and Martin, D. M. Cancer Res. in press (1961). Baltscheffsky, H., Expt. Cell Res. 13: 630 (1957). Layne, E. in “Methods in Enzymology~Bds. S. P. Colowick, and N. 0. Kaplan, (New York,%ademic Press) Vol. 111 p.447 (1957).

302