BIOCHEMICAL

Vol. 82, No. 1, 1978

AND BIOPHYSKAL

RESEARCH COMMUNICATIONS Pages

May 15,1978

NADH-DEPENDENT

CINERULOSE

Tadazumi

Komiyama,

Tomio

REDUCTASE

IN RAT LIVER

Toshikazu

Oki,

Takeuchi*

and

Hamao

Taiji

March

MICROSOMES

Inui,

Umezawa*

Central Research Labolatories, Sanraku-Ocean Johnan, Fujisawa 251 and 4-9-1, *Institute of Microbial Chemistry, 3-14-23, Shinagawa-ku, Tokyo 141, Japan Received

188-195

Co.,

Ltd.,

Kamiosaki,

14,1978

Summary: In the course of studies on the metabolism of a new antitumor anthracycline antibiotic, aclacinomycin A, the new keto reductase which catalyzes the reduction of keto group of L-cinerulose of aclacinomycin A to L-rhodinose was found in rat liver microsomal membrane. The enzyme requires NADH for the reduction and showed optimum pH at 7.0. Km value for aclacinomycin A, 2.1 x low5 M and the concentration of NADH need to half maximal activity, 6.2 x 10V5 M were obtained. The activity was potently inhibited by detergents, such as Triton X-100, sodium deoxycholate and sodium dodecyl sulfate.

A new by

anthracycline

Streptomyces

galilaeus

aklavinone

aglycone, 2-deoxy-L-fucose a potent some

solid

we

found at

identical

found

of

the

the

hepatic

C-7

position

with

Through we

of

that keto

the group

shows the

low

reductase of

compound of

MA144

Nl

L-cinerulose

0 I978

by Academic Press, Inc. in any form reserved.

of reproduction

metabolism

of

0006-291X(78/0821-0188$01.00/0 Copyright All rights

and

188

was

produced

aclacinomycin

A shows leukemia

of for

NADPH-cytochrome the

of

and

(1,3).

metabolism

responsible

on

P388

toxicity

anthracyclines

studies

consists

L-cinerulose,

and

cardiac of

and

produced

Aclacinomycin

L1210

studies

microsomal

further

(1,2).

against

A is

31133),

moiety,

L-rhodosamine

and

course

(ATCC

trisaccaride

activity

tumors, the

cleavage be

and

aclacinomycin

MA144-Ml and

antitumor

In A,

antibiotic,

aclacinomycin reductive

anthracyclinones P-450 of

reductase aclacinomycin

by

the

reduction

A to L-rhodinose

to (4). A,

BIOCHEMICAL

Vol. 82, No. 1, 1978

b,y rat

tase

liver In

this

in

rat

homogenate paper, liver

we

in

the

describe

microsomal

AND BIOPHYSICAL

presence the

of

presence

membrane

and

RESEARCH COMMUNICATIONS

(Fig.

NADH of some

1).

cinerulose of

its

reduccatalytic

properties. Materials

and

Methods

Chemicals: Aclacinomycin A and MA144 Nl were obtained from the culture of St. galilaeus MA144-Ml in our labolatory (1,2). NADH was purchased from BSehringer Mannheim GmbH and Oriental Yeast Co.; LTD. Pre-coated TLC-silica gel 60F254 was obtained from Merck Co. Enzyme preparation: Adult male rats of Wistar strain were killed by decaptation, and the liver was wuickly exised, rinsed with ice-cold 0.15 M KCl, and used immediately or stored frozen at -25°C. Subcellular fractionation was carried out by the modification of De Duve et al. (5) described by Sedgwick and Hflbscher (6). The minced livers were suspended in 9 volumes of 0.3 M sucrose solution containing 2.0 mM EDTA, pH 7.0 and homogenized by a Potter homogenizer with 6 times up and down movements. The homogenate was centrifuged at 1,000 x g for 10 minutes to remove nuclei and debris. The supernatant was subsequently spun at 7,300 x g for 10 minutes to sediment the mitochondrial fraction. The supernatant was further centrifuged at 11,700 x g for 20 minutes to remove the lysosomal fraction. The resulting supernatant was spun at 100,000 x g for 60 minutes to obtain the microsomal fraction. Each fraction was suspended in the same sucrose solution as described above and 100,000 x g supernatant was refered to as the soluble fraction. Protein was determined by the method of Lowry et al. (7) using bovine serum albumin as the standard. Enzyme activity: The stock solution of aclacinomycin A was prepared by dissolving in 5.0 mM acetic acid at a concentration of 2.0 mM. The reaction mixture, in a total volume of 1.0 consisted of NADH 0.2 mM, aclacinomycin A 0.1 mM, potassium ml, phosphate buffer 0.1 M, pH 7.0 and suitable amount of enzyme. The reaction was started by the addition of NADH solution, carried out with standing at 37°C for 30 minutes, and then sto ped by the addition of 2.5 ml of chloroform-methanol (1:l v/v P . The chloroform layer obtained by centrifugation at 3,000 rpm for 5 minutes was evaporated to dryness. The residue was dissolved in 100 ~1 of chloroform-methanol (1:l v/v) and 50 ~1 of sample was spotted on silica gel TLC, then developed with a solvent system of chloroform-methanol (lo:1 v/v). The product was identified by the direct comparison with MA144 Nl obtained from microbial culture (2). The amount of the product was measured with a Shimadzu dual-wave length TLC scanner, Model CS900 at 430 nm. The unit of the activity was defined as pmoles of the product, formed under above condition, and specific activity Km value was calculated was defined as units per mg of protein. from Lineweaver-Burk's plot (8). Results The

result

of

subcellular

and

Discussion

fractionation

189

of

NADH-dependent

BIOCHEMICAL

Vol. 82, No. 1, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

OH Aclacinomycin

Fig. 1. homogenate.

Reduction

cinerulose

of

reductase in

is

shown

found

was

observed

the

microsomal

fraction

KC1

containing

10 mM Tris-HCl

0.1

M potassium

reduced

no

protein

pH 7.0

in

obtained The

These

of

Km value The

saturation

reciprocal

of

high The

activity

activity with

in

0.15

pH 7.4

on IiADH, addition

results

is

present

or

M

with

at

activity. cinerulose

buffer

and

to

that

liver

to

NADPH

NADPH

and NADH

NADH-

microsomes.

proceeded

Heating

reductase

of

in

microsomes

2).

and

indicate

proportionally

phosphate

activity

relatively

mM EDTA,

and

NADH-dependent

Tris-HCl

effects

4B.

M.

the

potassium with

4A and

(Fig.

pH for

0.25

liver

highest

by washing

dependent

by and

The

b,y rat

pH 7.4.

reductase catalyzed

Nl

fractions,

released and

2).

60 minutes

optimum

not

inactive,

(Table

abolished

and

Nl

MA144

I.

debris

strictly

concentration

Table

buffer,

were

reaction

completely

The

phosphate

for

in

and was

was

A to

fraction

nuclei

cinerulose

least

lO-5

the

effect

The

the

in

reaction

dependent

at

microsomal

glutathione

showed

MA144

aclacinomycin

was

The

the

A

linearly

the

microsomal

00°C

for

5 minutes

As

shown

in

reductase a s milar

Fig.

was result

3,

around was

buffer. the for

substrate aclacinomycin curve

the

concentration

velocity

of

are

A was the plotted

190

enzyme

shown

calculated with

against

as

NADH is the

in

Fig. 2.1

sigmoidal.

reciprocal

x

BIOCHEMICAL

Vol. 82, No. 1, 1978

AND BIOPHYSICAL

Table Subcellular

Distribution

Fraction

RESEARCH COMMUNICATIONS

1

of NADH-Dependent

Protein

Total

Cinerulose

Activity

Specific

Reductase

Activity

(mg)

(unit)

Nuclei+Debris

711

10.9

0.015

Mitochondria

381

0.7

0.002

Lysosomes

169

2.0

0.012

Microsomes

269

29.3

0.109

Soluble

704

2.8

0.004

The rat liver, 13.5 g, was fractionated in Materials and Methods. Table Cofactor

Requirement

and assayed

as described

2

of Microsomal

Cofactor

(unit/mg)

Cinerulose

Reductase

MA144 Nl Production

(nmoles/tube) NADH

22.8

NADPH

0.9

NADH + NADPH

21.9

Reduced glutathione

Microsomal protein added were at the of the square

0.23 mg was used in the assay and cofactors concentration of 0.2 mM.

concentration

The concentration

0.4

of NADH gave a straight

of NADH to exhibit

191

half

maximal

line activity

(Fig.

4B). was

~~IOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 82, No. 1, 1978

Protein

Fig. 2. (left) Effect cinerulose reductase. Methods were used with

( mg )

PH

of the concentration of microsomal Assay conditions described in Materials various amount of microsomal protein.

Fig. 3. (right) Effect of pH on microsomal cinerulose Microsomal protein 0.23 mg was used in the assay with values of 0.1 M potassium phosphate buffer.

0

2

4 l/NADH2

and

reductase. various pH

10 ,:06

$1

Fig. 4. Effect of the substrate concentration on microsomal cinerulose reductase. Microsomal protein 0.23 mg was used in the assay with various amount of aclacinomycin A or NADH. A: Effect of aclacinomycin A concentration. B: Effect of NADH concentration. as 6.2 x 10-5

calculated

The enzyme activity detergents, dodecyl

(Table

detergents

detergents

3),

at the

60, Tween 40 and Brij three

was highly

such as Triton

sulfate

by these

M. X-100, over

sensitive

to various

sodium deoxycholate 90 % of the

concentration

activity

of 0.05

35 showed a milder

effect

at the same concentration.

of Triton

X-100

and sodium deoxycholate,

inhibited

the reaction

There are several

and sodium was inhibited

$, while than

the above

A higher tested

Tween

concentration

up to 1.0 $,

more strongly. NADH-dependent

192

reactions

in liver

micro-

BIOCHEMICAL

Vol. 82, No. 1, 1978

AND BIOPHYSICAL

Table Effect

of

Detergent

3

on Microsomal

Detergent

RESEARCH CO~~~UNI~ATIONS

Cinerulose

Reductase

Concentration

Control

Activity

($)*

($1

----

100

Triton

X-100

0.05

9

Sodium

deoxycholate

0.05

3

Tween

60

0.05

56

Tween

40

0.05

44

0.05

26

0.05

3

Brij

35

Sodium

dodecyl

sulfate

Microsomal protein 0.23 mg was used in the assay and detergent was added to reaction mixture as the indicated concentration. *The percentage was expressed as v/v for Triton X-100, Tween and Tween 40, and w/v for sodium deoxycholate, Brij 35 and sodium dodecyl sulfate. somal

membrane;

reduction

desaturation

(lo),

reduction

benzo(a)pyrene phenetol

and and

phetamine of (20),

these

above

Although this

enzyme

(12,13),

be

vitamin

the a new

of

(21),

(19),

microsomal

cinerulose

benz-

cyclization DT diaphorase

~CZ- and

20&hydroxysteroid specificity

p-nitroof

(17), K3

substrate

IlS-hydroxydehydrogenase

and

other

reductase

properties described

enzyme.

aclacinomycin to

of

N-demethylation

of

and

hydroxylamine

hydroxylation

epoxidation

the

seems

(ll),

(14,15),

(22)

(9),

0-deethylation

17@dehydrogenase

enzymes,

might

N-oxide

hydroperoxide

(18),

Considering

acid

of

dehydrogenase

(23). of

aniline

fatty

decomposition

testosterone

steroid

of

7-ethoxycoumarin

(16),

squalene

of

60

have

A is

not

a naturally

193

a common occuring

molecule substrate

in

mammals, and

play

BIOCHEMICAL

vol. 82, No. 1, 1978

an low

important

role

Km value More

for precise

in

AND BIOPHYSICAL

because

vivo,

aclacinomycin

the

A,

properties

of

enzyme

3.1

this

RESEARCH COMMUNICATIONS

showed

x 10 -5 enzyme

a relatively

M. will

be

reported

elsewhere. Acknowledgement The Science,

authors Kyushu

are

grateful

University

to for

Dr.

T.

Omura

helpful

of

the

Faculty

of

advice.

References 1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Y., Yoshimoto, A., Numata, K., Kitamura, Oki, T., Matsuzawa, Takamatsu, A., Umezawa, H., Ishizuka, M., I Hori, S., H., Hamada, M., and Takeuchi, T. (1975) Nakanawa, H., Suds, J. Antibiotics, 78, 830-834. Oki, T., Shibamoto, N., Matsuzawa, Y., Ogasawara, T., Yoshimoto, A., Kitamura, I. Inui, T., Naganawa, H., Takeuchi T and Umezawa, H. (19773 J. Antibiotics, 30, 683-687. ;IorI: S., Shirai, M., Hirano, S., Oki, T., Inui, T., Tsuka oshi, S., Ishizuka, M., Takeuchi, T., and Umezawa, H. 68, 685-690. (l;77? Gann, T Komiyama, T., Tone, H., Inui, T., Takeuchi, T., and UmeLawa: H. (1977) J. Antibiotics, 30, 613-615. De Duve, C., Pressmann, B. C., Gianetto, R., Wattiaux, R., and Applemens, F. (1955) Biochem. J. 60, 604-617. G. (19651 Biochem. Biophys. Acta, Sedgwick, B., and Hsbscher, 106, 63-77. Lowry, 0. II., Rosenbrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem., 193, 265-275. Lineweaver, H., and Burk, D. (1934) J. Amer. Chem. Sot., 56, 658-666. Shimakata, T., Mihara, K., and Sato, R. (1972) J. Biochem., 72, 1163-1174. Kadlubar, F. F., and Ziegler, D. M. (1974) Arch. Biochem. Biophys., 162, 83-92. Sugiura, M., Iwasaki, K., and Kato, R. (1976) Mol. Pharmacol. 12, 322-334.

12. 13. 14. 15. 16. 17. 18. 19. 20.

428. Shigematsu, H., Yamano, S., and Biochem. Biophys., 173, 178-186. Ullrich, V., and Weber, P. (1972) Chem., 353, 1171-1177. Nash, T. (1953) Biochem. J., 55, Hryca , E. G. (1975y Arch. ~i~",~~~: ~io~~&s!~‘l~k Popjak, G., Gosselin, L., Gore, (1958) Biochem. J., 69, 238-248. Sadowski, J. A., Shones, H. K., Biochemistry, 16, 3856-3863. Danielson, L., Ernstcr, L., and Chem. Stand., 14, 1837-1838. 194

Yoshida,

H.

(1976)

Hoppe-Seyler's

Arch. Z.

Physiol.

416-421. yi4ti;5F”d

I. and

G.,

and

Suttie,

Ljunggren,

Levin’

Gould, J. M.

”

R. W.

(1960)

G.

(1977) Acta

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21. 22. 23.

BIOCHEMICAL

Villee, C. A., and Spencer, 3615-3619. Hurlock, B., and Talalay, 80, 468-470. Rockagel, R. 0. (1957) J.

AND BIOPHYSICAL

J. P. Biol.'

195

M. (1959)

(1960)

RESEARCH COMMUNICATIONS

J.

Arch.

Chem.,

Biol. Biochem.

277,

273-284.

Chem - , 735, Biophys.,