Purification of cytochrome P-450 catalyzing 25-hydroxylation of vitamin D3 from rat liver microsomes

Vol. 121, No. 3, 1984 June 29, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 994-1000

PURIFICATION OF CYTOCHROME P-450 CATALYZING 25-HYDROXYLATION OF VITAMIN D3 FROM RAT LIVER MICROSOMES Shin-ichi

Hayashi,

Department School Received

April

Mitsuhide

Noshiro,

and Kyuichiro

Okuda

of Biochemistry, Hiroshima University, of Dentistry, Hiroshima 734, Japan

7, 1984

SUMMARY: Cytochrome P-450 catalyzing 25-hydroxylation of cholecalciferol (cytochrome P-450c 25) was purified from rat liver microsomes based on its catalytic activity at each purification step. The specific cytochrome P-450 content of the final preparation was 15.1 nmol/mg of protein. Reconstituted activity of 25-hydroxylation of cholecalciferol with the purified enzyme was 2.3 nmol/min/mg of protein, which was 4,300 times as high as that in microsomes. The minimum molecular weight of the enzyme was 50,000 based on SDS-polyacrylamide gel electrophoretogram. Amino terminal sequence of the P-450 c\qa,""",,",2"-;;;;;y; Pro-Val-Leu-Val-. Immunochemical study showed P-450 was homogeneous and the cytochrome was immunochemically diffe% 5 from either cytochrome P-450(PB-1) or cytochrome P-448(MC-1).

Hydroxylation essential form

at

for

C25

the

of

subsequent

mitochondrial the

conversion

of

active The

oxidase

based for

(2,

on its

also

immunochemical

which

enabled

pure

In this

us to

already

Abbreviations: ZO-methylcholanthrene,

study,

from and

compare

with

kidney

amino

other

active

by microsomal

and/or

the the

P-450 microsomal

P-450 cc25'

electrophoretogram

terminal of

as

enzyme highly

cytochrome

only

is

(1).

we purified

species

its

cytochrome

named not

into

liver

mitochondria

and obtained

tentatively judging

in

vitamin

containing

activity,

reactions

sequence cytochromes

but analysis, P-450

in

reported.

MATERIALS AND METHODS Male Wistar rats weighing sacrifice. Liver microsomes

0006491X/84

3).

the

catalyzed

system

catalytic

was

is

transport

25-hydroxylation,

cytochrome

microsomes

in

cholecalciferol

electron

terminal

enzyme

of

(cholecalciferol)

D3

(1,25-dihydroxycholecalciferol)

25-Hydroxylation

Copyright All rights

vitamin

cc:

about 200 g were starved were prepared as described

cholecalciferol, SDS: sodium dodecyl

$1.50

0 1984 by Academic Press, Inc. of reproduction in any form reserved.

PB: sulfate.

for 20 h before previously (4).

phenobarbital,

MC:

BIOCHEMICAL

Vol. 121, No. 3, 1984

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Microsomes were solubilized with sodium cholate and fractionated with polyethylene glycol. The precipitate obtained by 8-12% polyethylene glycol was subjected to column chromatography of aminooctyl-Sepharose DEAE-Sepharose CL-6B, and CM-Sepharose CL-6B, 4B, hydroxylapatite, subsequently. The detailed procedure will be published elsewhere. Detergent-solubilized NADPH-cytochrome P-450 reductase was purified from rat liver microsomes by the method of Yasukochi and Masters (5). Cytochrome P-450 (PB-1) and cytochrome P-448 (MC-l) were purified from PB-treated and MC-treated rat liver microsomes, respectively, as described by Kuwahara et al. Antibodies against the purified (6). proteins were prepared as described previously (4). Assay mixture of cholecalciferol 25-hydroxylation contained 0.1-0.5 nmol of cytochrome P-450, 0.5-2.5 unit of NADPH-cytochrome P-450 reductase, 20 pg of dilauroylglyceryl-3-phosphorylcholine, 100 umol of phosphate buffer (pH 7.0), 100 nmol of cholecalciferol dissolved in 10 ul of ethanol, and NADPH-generating system consisting of 0.5 limo1 of NADP , 5 umol of isocitrate, 5 umol of MgC12, and 0.44 U of isocitrate dehydrogenase in a total volume of 1 ml. Incubation was carried out for lo-30 min at 37°C. The reactions were terminated by adding 1 ml of ethanol, and the reaction products were extracted with 5 ml of petroleum ether. The solvent was evaporated to dryness, the residue was dissolved in a small amount of isopropanol, and an aliquot was subjected to high performance liquid chromatography to determine the amount of the product (25-hydroxycholecalciferol) using authentic standard. the external 25-Hydroxycholecalciferol was a generous gift from Chugai Pharmaceutical LTD. Co. (Tokyo). Content of cytochrome P-450 was dete?lminfd according to Omura and Sato using extinction coefficient of 91 mM cm (7). SDS polyacrylamide gel electrophoresis was carried out according to Laemmli (8) using 7.5% acrylamide gel. terminal portions of the Manual sequence analysis of the amino purified enzyme was performed according to the method described by Black and Coon (9). and phenylthiohydantoin derivatives of amino acids were identified by the method of Lottspeich (10). Immuno-dfffusion analysis was carried out as described previously (4). Protein was determined by the method of Lowry et al. (11) using bovine serum albumin as the standard.

RESULTS Purification

of

specific

content

nmol/mg

of protein.

activity

of

times the

as high enzyme

purification.

of cytochrome

P-450

The purified pmol/min/mg

which

microsomes,

polyethylene

P-450cc25

2,300

pmol/min/nmol, from

cytochrome

as that activity

in

to However,

final

and

4,300the

should

of this

ensued

activation

Table

and total

was 15.1

showed

a specific rate

of

purification

activity

recovered

a marked

will

152

250-fold

chromatography

at

The

1.

preparation

a turnover

suggesting

have

in

P-450cc25

in aminooctyl-Sepharose microsomes,

The reason

summarized

the

protein

corresponded

or that

in

cytochrome of

respectively.

glycol

is

an

in was 5

activation

early

be discussed

stage later.

of of

BIOCHEMICAL

Vol. 121, No. 3, 1984 Table

I

Purification

Purification

of P-450 cc25 from liver

P-450 Total Specific amount content

Protein

steps

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(mg)

(nmol)

microsomes of untreated

25-Hydroxylase Specific activity

Total activity

(nmol/mg

(nmol/min)

of protein)

rat

activity TUrnOVer rate

(pmol/minlmg of protein)

(pmol/min/nmol of enzyme)

0.53

0.6

Microsomes

4860

4278

0.88

PEG 8-12% fraction

1242

2550

2.05

13.5

663

6.22

16.9

159

25.5

29.5

266

9.03

13.3

464

51.4

17.2

187

10.9

12.9

750

68.8

10.6

117

11.1

8.8

833

75.0

Aminooctyl

Sepharose

Hydroxylapatite

107

I

DEAE-Sepharose Hydroxylapatite

II

2.51

10.9

5.3

CN-Sepharose

I

2.0

28.4

14.2

3.6

1818

128

CM-Sepharose

II

1.2

17.4

15.1

2.7

2303

152

The

SDS-polyacrylamide

preparation

showed

weight

of 50,000

the purified low

spin

peak

to

1).

cross

a single

reactivity in

Fig.

precipitin

cytochrome,

whereas

combination

of

to a minimum

molecular

the

they

antigens.

the

at 416 nm, indicating

with

sodium

other

forms

only not

These

show

the any

observations 996

indicate

and the cytochrome

and

corresponding precipitin

the

P-450cc25

protein

purified

anti-PB-1,

with

Soret

immuno-diffusion

purified of

its

showed

cytochrome for

form of

the

spectrum

used

of the

anti-P-450cc25,

line

dithionite,

purified

and was purity

toward

did

band

the

rabbit,

2,

of the oxidized

CO-difference

against

the

spectrum

Soret

and

nm,

to examine

As shown

corresponding

414

white

possible

purified

was reduced

male

order

the

When it

in in

of

band

showed

Antibody

elicited

electrophoretogram

The absolute

450 nm.

analysis

formed

(Fig.

nature.

shifted

P-450.

the major

cytochrome

maximum at was

gel

anti-NC-1 isozyme

lines

in

the

purity

of

other of

Vol. 121, No. 3, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

AB

01

02 Fig. 1. SDS-plolyacrylamide gel electrophoresis of the purified cytochrome P-450 Electrophoresis was carried out according to the method of LaemH5;8) using 7.5% gel. Lane A contained standard proteins with molecular weight of 94,000, 67,000, 43,000, 30,000, 20,100, and 14,400, lane B 0.25 ug of the purified P-450cc25, respectively. Fig. 2. Immune-diffusion analysis of P-450 1.2% agar containing 50 mM phosphate bufferccfps F?;y ?~%m~~~l~pOw~:k sodium azide and 0.5% cholate. Wells designated A-CC, A-PB, and A-XC contained antibodies against P-45O(PB-1) and P-4480X-l), respectively. Wells designated CC, and MC contained the purified P-450(PB-1) and P-448(MC-l), respectively. p-450cc25'

cytochrome

from

p-450cc25 The

and

p-450cc25

amino

either

sequence

published

sequences

methionine

as many of

However, P-450a

or

6th

the

determined

of

but

residue

so far

cytochrome

was quite

was similar

3). 997

first

studied,

different to PB-2

our

compared

The

P-450RLM3

(7),

to where

and

P-450.

cytochromes

sequence

P-450(PB-1)

was

cytochromes

P-450(PB-2)

following

RLM5 up to the (Fig.

of

(7), the

difference

PB-1 or MC-l.

terminal

P-450(PB-1)

immunochemical

sequence

was

.P-450a(12),

P-450RLM5

from

that

(13).

of either

and was identical analysis

other

residue e.g.

and

to

with

was performed

BIOCHEMICAL

Vol. 121, No. 3, 1984

P-450c&

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1 Met-Asp-Pro-Val-Leu-Val-

:

5

Fig. 3. Amino terminal sequence of cytochrome degradation was carried out as described bv Black and Phenylthiohydantoin derivatives of amino-acid were method of Lottspeich (10).

P-450 *5’ Coon t!+?. identified

Edman by

the

DISCUSSION A number and

cytochrome

P-450

liver

microsomes

(6,

preparation

of

rat

final various

of

exogenous

investigate important

substrate,

based

on

the

Only

a

few

it

reports

have

e.g.

so

(20).

cholecalciferol

25-hydroxylase

toward

vitamin

The

specific

vitamin

In

this

activity more

However,

a marked

observed

in

activity

in microsomes

the

of than

P-450 step. met

this (18)s

and the

specific

to

physiologically

which

(19),

the

purified

testosterone

purification

catalytic

in

the

could

not

reductase

of activity

in

significantly

factor(s) as

in in

suggested

microsomes

microsomes by

Bjb’rkhem

998

might al.

activity

limited

which

provides

to

was

enzymatic amount

did Thus, to

removed

(21).

of

reducing

externally

be due be

toward

microsomes.

total

shown).

seemed

et

lower

reductase

not

which

of

total

since

P-450 (data

that

to the

microsomes reaction,

P-450 cc25

the

The

be attributed

hydroxylation

activity

of

steps.

the

25-hydroxylase

than

recovery

NADPH-cytochrome

25-hydroxylation

cytochrome higher

purification

P-450

for

the

detergent-solubilized

purification

order

7a-hydroxylase

on its

4,300-fold

increase initial

NADPH-cytochrome

inhibitory

published

the

using

a particular

we described

based

studied

at any purification

been

study,

of

in

the

to purify

activity

rabbit

D 3'

D3 was

electrons

of

12a-hydroxylase

15a-hydroxylase

been

However,

taurodeoxycholate

7a-hydroxy-4-cholesten-3-one

from

properties

have

reaction

far

purified

catalytic

substrates.

may be necessary hydroxylation

been

P-450

hydroxylation

specific

requirement,

The

cytochrome

endogenous

a specific

have

14-17).

purified

and

isozymes

added

not

enhance

the

lower

an unknown during

Immunochemical

the

Vol. 121, No. 3, 1984

study

BIOCHEMICAL

indicated

anti-P-450

that and

cc25

anti-P-450(PB-1) It

may,

which

is

be

to

The amino

was

no

and

estimate

possible the

cytochrome

perform

quantitative

of

the

PB-1,

sequence

of cytochrome

that

of

the

amino

terminal

sequence

of PB-2

The amino

terminal

sequence

of RLM5 purified

PB-2,

as a constitutive

analysis

hydroxylations

recently

least

performed.

be

reported

cholecalciferol electrophoresis. present

cytochrome

cytochrome,

cytochrome

up

to

the

described

On

neither P-450ccz5. immuno-

in

microsomes,

data

their

is

though

the

an

preparation at

et

from

al.,

to our

identical

position the

to

available

similar

different

though

preparation.

by Cheng and Schenkman

P-450

to

towhere cytochrome activity

hand,

cytochrome

25-hydroxylation Although

very

other

is

by Kuwahara

that

the

a constitutive

is

6th

testosterone,

studied.

P-450cc25

described

of

They

of to

MC-1 or MC-2

form

at

remained

furthermore

recognized to

amount

between

investigation.

terminal

preparation

cross-reactivity

P-448(MC-l),

anti-P-450(MC-1)

therefore,

under

there

P-450(PB-1)

nor

precipitation

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

P-450 apparent

that

for

Andersson highly

of

our was

active

al.

active

homogeneity

are

not

for

vitamin et

our

sequence

D3 (22)

for

the

based

on

to

the

seems to be similar present

(13)

sufficient

for

conclusion.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

DeLuca, H.F. and Schnoes, H.K. (1976) Ann. Rev. Biochem. 45, 631-666 J. 184, 491-499 Madhok, T.C. and DeLuca, H.F. (1979) Biochem. Bjorkhem, I. and Holmberg, I. (1978) J. Biol. Chem. 253, 842-849 83, 61-77 Noshiro, M. and Omura, T. (1978) J. Biochem. Yasukochi, Y. and Masters, B.S.S (1976) J. Biol. Chem. 251, 5377-7344 Kuwahara, S., and Harada, N., Yoshioka, H., Miyata, T. and Omura, T. (1984) J. Biochem. 95, 703-714 Omura, T. and Sato, R. (1964) J. Biol. Chem. 239, 2370-2378 Laemmli, U.K. (1970) Nature 227, 680-685 Black, S.D. and Coon, M.J. (1982) J. Biol. Chem. 257, 5929-5938 Lottspeich, F. (1980) 2. Physiol. Chem. 361, 1829-1834 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 Botelho, L.H., Ryan, E.E. and Levin, W. (1979) J. Biol. Chem. 254, 5635-5640 Cheng, K.C. and Schenkman, J.B. (1983) J. Biol. Chem. 258, 11738-11744

Vol. 121, No. 3, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

14. Haugen, D.A. and Coon, M.J. (1976) 251, 7929-7939 15. Aoyama, T., Imai, Y. and Sato, R. (1982) in Microsomes, Drug Oxidations and Drug Toxicity, Japan Scientific Sot. Press, Tokyo, pp. 83-84 16. Ryan, D.E., Thomas, P.E. Korzeniowski, D. and Levin, W. (1979) J. Biol. Chem. 254, 1365-1374 17. Guengerich, F.P. Dannan, G.A. Wright, S.T., Martin, M., and Kaminsky, L.S. (1982) Biochemistry 21, 6019-6030 18. Murakami, K., Wakeshima, C. and Okuda, K. (1980) Biochem. Biophys. Res. Commun. 94, 1098-1105 19. Murakami, K. and Okuda, K. (1981) Biochem. Biophys. Res. Commun. 100, 91-99 20. Harada, N. and Negishi, Pi. (1984) J. Biol. Chem. 259, 1265-1271 21. Bjb'rkhem, I., Hansson, R., Holmberg, I. and Wikvall, K. (1979) Biochem. Biophys. Res. Commun. 90, 615-622 22. Andersson, S., Holmberg, I. and Wikvall, K. (1983) J. Biol. Chem. 258, 6777-6781

1000