type II 15-hydroxyprostaglandin dehydrogenase is not a prostaglandin specific enzyme

BtOCHEMICAL

Vol. 101, No. 3,198l August

AND

BtOPHYSlCAL

RESEARCH COMMUNICATIONS Pages 898-904

14, 1981

II 15-HYDROXYPROSTAGLANDIN

PROSTAGLANDIN 9-KETOREDUCTASE/TYPE

DEHYDROGENASE IS NOT A PROSTAGLANDIN SPECIFIC ENZYME David Departments

Received

Guey-Bin

of Chemistry and Texas

July

Chang'

and Hsin-Hsiung

Tai'

and Biochemistry, North Texas College of Osteopathic Medicine Denton, Texas 76203

State

University

1, 1981

SUMMARY: A homogeneous preparation of prostaglandin 9-ketoreductaseltype II 15-hydroxyprostaglandin dehydrogenase from swine kidney was employed to investigate the substrate specificity of the enzyme. In addition to prostaglandins, the enzyme reduced and/or oxidized steroids, aldo sugars, quinones, and other carbonyl compounds. Among the compounds examined, quinones were found to be the best substrates. The enzyme appears to be a g eneral aldo-keto reductase. INTRODUCTION NADPH-linked type

prostaglandin

9-ketoreductase

II 15-hydroxyprostaglandin

shown to be ubiquitously activity

dehydrogenase present

(PGE) to prostaglandin

F (PGF)

sidered

in the

to be involved

obtained

two homogeneous

protein

possessed

appears sion

that

studies

(l),

enzyme

indicated

exceptionally prostaglandins

that high (4-8).

the

from

is

enzyme could which

We are

far

intrigued

capable

act

activity (2).

kidney

as well substrate on all

by these

0006-291X/81/150898-07$01.00/0 898

either

(3).

It

interconver-

inactivation

and

and kinetic

concentrations

properties

Institute

we

that

of prostaglandins

endogenous

unusual

Present Address: Department of Immunotherapy, Wadley 2Medicine, Dallas, Texas 75235 To whom correspondence should be addressed at College of Kentucky, Lexington, Kentucky 40506

the

specificity

types

the

Recently,

activities

as the

E was con-

and showed

of catalyzing

exceeded

1

Copyright 0 1981 by Academic Press, Inc. All righls of reproduction in any form reserved.

of prostaglandin

and dehydrogenase

Furthermore,

Km values

swine

have been The reductase

the dehydrogenase

prostaglandins

of prostaglandins.

(1,2).

of prostaglandins

proteins

the same enzyme protein different

tissues

whereas

reductase

activities

the conversion

inactivation

reversible

of functionally

reactivation

for

and NADP+-dependent

(15-PGDH)

in mammalian

to be responsible

was thought

(9-PGKR

with of

and have susof Molecular

of Pharmacy,

University

Vol. 101, No. 3,198l

petted

that

the

ferred

substrates.

BIOCHEMICAL

catalyzes

those It

Using

not only

of steroids

appears

that

BIOPHYSICAL

enzyme may use substances

the oxidation

than

prostaglandins

specificity

of sugars,

MATERIALS

previously

that

and other

of general

prethe

recognized.

of prostaglandins,

quinones,

to a class

as the

we found

than

and reduction

enzyme belongs

RESEARCH COMMUNICATIONS

enzyme preparation,

substrate

and reduction the

other

a homogeneous

enzyme does have much broader It

AND

but also carbonyl

aldo-keto

compounds.

reductases

AND METHODS

Materials: Prostaglandins were a generous gift of Dr. John Pike of the Upjohn Company. Steroids, D-glucuronate, D-glucuronolactone, D-galacturonate, 9,10phenanthrengquinone, menadione, hydrindantin, 4-nitrobenzaldehyde, gluthathinone (GsH), NADP , and rutin were obtained from Sigma Chemical Co. Ampholine was purchased from Pharmacia Fine Chemical Co. Prostaglandin 9-ketoreductase/l5hydroxyprostaglandin dehydrogenase was purified from swine kidney according to Chang and Tai (3). Enzyme Assays: Enzyme activities were assayed as described previously (3). Briefly, prostaglandin 9-ketoreductase activity was assayed by following the decrease in absorbance at 340 nm at 25°C. The reaction mixture contained: NADPH, 0.1 mM; varied concentrations of PGE or other carbonyl compounds and enzyme in 0.5 ml of 0.1 M Tris-HCl buffer, $H 7.5. The reaction was initiated by the addition of enzyme. 15-hydroxyprostaglandin dehydrogenase activity was assayed by following the increase in absorbance at 340 nm or the increase in fluorescence at 468 pm with excitation at 340 nm at 25°C. The reaction mixture contained: NADP , 0.8 mM; varied concentrations of PGE2 or other hydroxyl compounds and enzyme in 0.5 ml of 0.1 M Tris-HCl buffer, pH 7.5. The reaction was initiated by the addition of enzyme. In some cases, oxidation or reduction of prostaglandins at a specific position (C-9 or C-15) was determined by a specific radioimmunoassay as described earlier (3). RESULTS AND DISCUSSION Previously, tionation, isoelectric

we reported

Sephadex focusing

hydroxyprostaglandin step

yielded

re'duction for

G-100

renal

homogeneous

homogeneous

well

as the oxidation ability

in two different proteins

enzyme to catalyze

899

with

prostaglandin

chromatography

(Figure

the reduction

The major

and

focusing

1).

oxido-

was assayed

as PGBl oxidation,

peaks

can catalyze

of prostaglandin. of the

The isoelectric

of this

frac-

9-ketoreductase/l5-

enzyme proteins

as well

acetone

chromatrography

prostaglandin

to homogeneity.

reduction

copurified

either

the

swine

involving

TEAE-cellulose

When each fraction

activities.

that

investigate

procedure

filtration,

dehydrogenase

two different

were

gel

to purify

9,10-phenanthrenequinone

activities

a purification

the two This

indicates

of a quinone

form was used

to further

the oxidation

or the

as

(9).

BIOCHEMICAL

Vol. 101, No. 3,198l

AND

BIOPHYSICAL

FRACTION

Fig.

1:

reduction

bony1 compounds.

values

as shown

previously

activity in k cat/Km'

to serve

as substrates

for

(5),

for

reduction

the avian

than

steroids, substrate

are

for

the

(6),

for enzyme.

swine

Cagan and Pisano three

900

decrease

in the ability

enzyme

of GSH-PGA1 was at least

adrenal

is

orders

showed

high

kidney cortex

Km

(4),

(7).

substrates in Km values

GSH for and the

of GSH conjugates

not as dramatic (10)

car-

some apparent

unusually

rabbit

to be better

by the

increase

renal

from

and other

with

but with

and rat

found

PGAs as evidenced the

specificity

enzyme reported

A (PGA) were

However,

quinones,

substrates

human placenta

of prostaglandin

increase

ported

I shows the

Prostaglandins

human erythrocytes conjugates

of prostaglandins,

Table

constants.

reductase

NO.

Isoelectric focusing of swine renal prostaglandin 9-ketoreductase/l5hydroxyprostaglandin dehydrogenase. The TEAE-cellulose fraction was isoelectrically focused in the pH range 4 to 6.5 as described preThe activity was assayed by using 200 PM 9,10-phenanviously (3). threnequinone or 60 JJM prostaglandin B1 as substrate. 9,10-phenanthrenequinone, tt; prostaglandin Bl, -o-o-; pH, ---A---A---.

of a variety

kinetic

RESEARCH COMMUNICATIONS

that

of magnitude

as that

re-

the activity greater

than

Vol. 101, No. 3,19Bl

TABLE

1:

BIOCHEMICAL

Substrate Specificity Dehydrogenase

of

k cat

x lo3

(set

kcat/Km

-5

(M -'

125

3.3

PGA2

670

0.7

1,o

15-keto-PGFpa

200

3.3

16.5

set-'

)

26.4

PGA1-GSH

41

12.2

297.6

PGA2-GSH

43

10.3

238.4

556

7.0

12.5

90

2.3

25.6

18.2

14.5 9.9

Dehydroisoandrosterone

1,250

Estrone

500

5.0

Progestrone

333

3.3

9.9

D-Glucuronate

1,000

28.4

28.4

D-Glucuronolactone

2,500

39.6

15.9

D-Galacturonate

1,000

5.5

9,10-phenanthrenequinone

49.5

5

10.6

2,110.o

20

180.2

9,OlO.O

Hydrindantin

434

18.2

641.8

4-Nitrobenzaldehyde

250

22.1

88.4

Menadione

Dehydrogenase PGE2

140

0.08

PGEl

153

1.6

10.5

PGA2

80

1.1

13.2

PGAl

180

3.1

17.2

PGB2

200

4.4

22.0

PGB,

500

38.8

69.3

Estradiol

220

0.06

0.3

Testosterone

400

0.11

0.3

*Km and Vmax values were determined kcat by dividing by Et. Et was assumed M.W. of 30,000 (8).

reduction

occurrence In oids

9-Ketoreductase/l5-Hydroxyprostaglandin

PGE2

Corticosterone

for

RESEARCH COMMUNICATIONS

Reductase

Androstendione

B.

BIOPHYSICAL

Prostaglandin

Km (uM) -~

Compounds

A.

AND

were

of of

addition tested

PGEl

either

(11). PGA or

to

prostaglandins

as

possible

reductase/l5-hydroxyprostaglandin

by Lineweaver-Burk determined by dividing

However,

no

GSH-PGA

in and

plots the

convincing

mammalian their

alternative

0.6

evidence systems

GSH

901

for has

conjugates,

substrates

dehydrogenase.

and Vmax values converted protein concentration by

for The

been

the

prostaglandin was

natural

produced.

a number

enzyme

to the

of

ster-

9-ketofound

to

reduce

BIOCHEMICAL

Vol. 101, No. 3,19Bl

and oxidize these

steroids

steroids

for

It

also

is

the enzyme.

explored.

as readily

were

centrations.

glucose

and D-galactose

involved

as well

in L-ascorbic

--in vivo.

Other

strates.

To our

substrates of menadione

was 350 times

and high

k cat/Km

vitamine

K (menaquinones)

quinones

are

little

is

Recent

evidence

cribed

herein

quinones.

(12).

In addition

in the

that

the

cytosolic

be involved to quinones,

of PGE2.

such as 4-nitrobenzaldehyde

acid

cycle,

In fact, of the

--in vivo.

Km and

However,

these

like

and

oxidoreductases. reduces the

active

aromatic

albeit

vitamin

enzyme des-

of biologically

and hydrindantin

low

of membranes

soluble

reduced

k,,,/K,,,

Q (ubiquinones)

environment

reductases

sub-

to be excellent

In view

for

or both,

alternative

found

substrates

enzyme also

D-

the enzyme may be

coenzyme

the metabolism

the

toward

NADPH dehydrogenase

quinone in

substrates

enzyme were

kcat/Km.

as substrates

cytosolic

that

were

hydrophobic

con-

of the enzyme to reduce

as possible

endogenous

availability

Other

may also

and ketones

derivatives,

of

D-glucuronolactone

and high

may be the preferred

indicates

liver

examined

that

this

glucuronic

derivatives

than

natural

for

suggests

Km values

greater

embedded

known on their

K in rat

low

of quinone

normally

were

were

Km values

was displayed

or in the

some quinone

as shown by their

endogenous

The ability

as D-glucoronate

suprise,

to their

D-glucoronate,

shown),

compounds

the

steroids

of reactivitiy

synthesis

However,

substrates

reduced

not

acid

carbonyl

tested

endogenous

level

(data

RESEARCH COMMUNICATIONS

as compared

the

readily

and a lower

D-glucuronolactone

high

that

possible

The reductase

BIOPHYSICAL

as prostaglandins.

unusually

unlikely

Other

and galacturonate

AND

aldehydes

at a lesser

degree. It

is

carbonyl

interesting

that

compounds

of a broad

of carbonyl

compounds

unresolvable

separate

to be homogeneous enzyme protein derived

from

of very

spectrum. different

enzyme proteins

by electrophoretic

catlayzing heat

the enzyme

inactivation

the

is

capable One logical

structures in the

(Fig.

concern could

preparation

criteria.

reduction

of catalyzing

different inhibition

is

that

that

of

reduction

be catalyzed

although

Evidences

of two very 2) and rutin

well

reduction

it

appeared

support

the same

compounds studies

by

were

(Fig.

3).

Vol. 101, No. 3,1981

BIOCHEMICAL

0

AND

5

10

BIOPHYSICAL

15

20

( min

)

TIME

Fig.

2:

In both paring

Effect of heat upon the or 4-nitrobenzaldehyde 0.1 M Tris-HCl buffer, protein) were cooled in 113 PM prostaglandin E strate and 0.1 mM NADP6

cases,

parallel

Numerous been described

aldo-keto (9).

to heat

from

recently,

Wermuth

0.02

INHIBITOR

3:

and rutin

mammalian

I I 0.01 Fig.

30

were

observed

when com-

as substrates.

reductases Very

25

enzyme activity as assayed by prostaglandin E2 as substrate. Enzyme was incubated at 50°C in At the indicated time, aliquots (20 ug pH 7.5. ice and assayed for the enzyme activity using (0) or 50 PM 4-nitrobenzaldehyde (0) as subas coenzyme.

sensitivities

PGE2 and 4-nitrobenzaldehyde

RESEARCH COMMUNICATIONS

CONC.

(13)

and submammalian reported

I

I

0.03

0.04

( mM

a carbonyl

species

reductase

)

Inhibition of the enzyme activity by rutin using prostaglandin E2 or 4-nitrobenzaldehyde as substrate. The enzyme (20 Pg protein) activity was assayed by using 113 PM prostaglandin E2 (0) or 100 PM 4-nitrobenzaldehyde (0) as substrate in the presence of 0.1 mM NADPH and rutin at the indicated concentrations.

903

have

Vol. 101, No. 3,198l

having

three

BIOCHEMICAL

different

enzyme reduces These

a number

aldo-keto

one reductase hibit

ibrium.

BIOPHYSICAL

was purified

of biologically

reductases

show similarity substrates

in tissue

distribution,

weight, In view

sulfhydryl

the nonspecific

glandins

of the enzyme should of the

nature

proposed

than

cofactor

being

and the

the other. requirement,

be taken

into

for

although They also

and reaction

equil-

prostaglandin

9-

Km values

consideration

ex-

pH optimum,

in physiological high

The

prostaglandins.

specificity

proposed

unusually

human brain.

including

noninducibility,

dehydrogenase

(14-16),

nificance

compounds

better

roles

ketoreductase/l5-hydroxyprostaglandin

from

in substrate

sensitivity,

of the increasing

RESEARCH COMMUNICATIONS

to homogeneity active

may use certain

similarities

molecular

forms

AND

systems for

in assessing

prostathe sig-

roles. ACKNOWLEDGMENTS

We thank supported

Dr.

by grants

John

Pike

from

for

the

kind

USPHS (GM-25247)

gift

of prostaglandins.

and the American

This Heart

work was

Association

(78-865). REFERENCES

:: 3. 4. 5. 6. 7. 8. 1:: 11. 12. 13. 1':: 16.

Lee, S.C. and Levine, L. (1974) J. Biol. Chem. 249:1369-1375. Lee, S.C. and Levine, L. (1975) J. Biol. Chem. 250:548-552. Chang, D.G.B., Sun, M. and Tai, H.H. (1981) Biochem. Biophys. Res. Comm. 99:745-751. xone, K.J. and Hart, M. (1975) Prostaglandins 10:273-288. Kaplan, L., Lee, S.C. and Levine, L. (1975) ArcE Biochem. Biophys. -167: 287-293. Jarabak, J. and Fried, J. (1979) Prostaglandins 18:241-246. Levasseur, S., Friedman, T. and Burke, G. (1980)xiochem. Biophys. Res. Comm./ 95:236-242. mang, D.G.B. and Tai, H.H. (1980) Fed. Proc. 39:1897. Bachur, N.R. (1976) Science 193:595-597. Cagen, L.M., Fales, H.M. andPisano, J.J. (1976) J. Biol. Chem. 251:6550-6554 Cagen, L.M. and Pisano, J.J. (1979) Biochim. Biophys. Acta 573:547-551. Wallin, R., Gebhardt, 0. and Prydz, H. (1978) Biochem. J. 169:95-101. Wermuth, B. (1981) J. Biol. Chem. 256:1206-1213. Weber, P., Larson, C. and Scherer, B. (1977) Nature 266:65-66. Limas, C.J. and Limas C. (1977) Am. J. Physiol. 2(1):H87-H92. Ziboh, V.A., Lord, J.T. and Penneys, N.S. (1977)-J. Lipid Res. --18.37-43.

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