25-hydroxylation of vitamin D3 by a reconstituted system from rat liver microsomes

Vol. 90, No. 2, 1979

BIOCHEMICAL

RESEARCH COMMUNICATIONS

AND BIOPHYSICAL

Pages

27, 1979

September

ZFHYDROXYLATION OF VITAMIN RAT LIVER MICROSOMES Ingemar Department Karolinska Biochemistry Received

Bjiirkhem,

Ronnie

of Clinical Institutet, University August

D3 BY A RECONSTITUTED Hansson,

Inger

Holmberg

SYSTEM

and Kjell

Chemistry and Research Centre Stockholm and Department of Uppsala, Uppsala, Sweden

615-622

FROM

Wikvall

at Huddinge Hospital, of Pharmaceutical

lo,1979 SUMMARY

Using isotope dilution - mass fragmentography as assay technique, it was shown that highly purified preparations of cytochrome P-450 from rat liver microsomes catalyzed 25-hydroxylation of vitamin D when combined with NADPH-cytochrome P-450 reductase and a phospholipid. d-l e rate of conversion was approximately linear with the amount of cytochrome P-450, and was considerably higher than the rate of conversion obtained with crude liver microsomes. The possibility is discussed that the microsomal fraction contains inhibitors of 25-hydroxylase activity, which may be of regulatory importance in vitamin D metabolism. 3 The first step in the metabolism of vitamin D3 to its active form is a 25-hydroxylation.

This

reaction

is

mitochondrial

fraction

of rat

mitochondrial

activity

is inhibited

systems

have

been characterized

of mitochondrial

activity,

cytochrome

is involved

function

P-450 as electron

The there

of the

(l-3).

This

extent

of conversion

conversions 1000 fmol/mg shown the

between fact

aciivity concerning

is

that

than

l/10

that

mitochondrial

the

in part very

from

low

about

In the

that

case

a species

ferredoxin

of

reductase

are

and

properties

different

With

the

described

authors

and enzyme

been

reported up to about report

linearity

activity.

In view

be completely

responsible

by

has been

et al. had an activity

it can not have

Thus the

of a recent in which

by Sulimovici may

(3)

and

enzyme

have

protein/min

reports

protein

of this difficulties.

exception

no previous

of the mitochondria, contaminants

has not yet been established the

0.2 fmol/mg

of microsomal

system

000 of that

as the

and both enzyme

(4,5).

shown and

be due to methodological

(2,4).

(3) there

amount

oxidases

ferredoxin

and

as well

(2,4)

conclusively

that

reports

protein/min et al.

has been and

microsomal

microsomal monoxide

function

microsomal

might

varying

The

both

(6).

conflicting

system

(l-4).

by

by carbon

as mixed it

carriers

nature

are several

Suiimovici

liver

catalyzed

of less

excluded

for part

of the

activity. Using a highly accurate assay isotope dilution - mass fragmentography, activity

to be less

than

15%

of the

for

25-hydroxylase

in general mitochondrial

activity,

on

we found the microsomal activity in homogenates of

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based

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5-08$01.00/O

@ 1979 hx Acadrmc Prrs.s. Inc. of reprodwrion III uny form reserved

BIOCHEMICAL

Vol. 90, No. 2, 1979

rat liver

(7). There

protein,

and

The varying purified of

was no linearity

the extent degree

failed

to give purified

P-450

reductase

There

was

amount

of conversion

should significant

Materials.

as described Dr.

Vitamin

prepared Cutrecasas

(The

D3

Upjohn

CL-6B

hydroxyapatite

were

with

however, vitamin

enzyme

D3.

activity

sodium

cofactors

were

913 Bio-Rad

amount other

Male

rats

was

Pharmacia

from

All

Kalamazoo).

Sepharose

from

an equal

chromatography.

between

towards

(cholecalciferol),

Co.,

Emulgen

DEAE-Sepharose mixed

work,

NADPH-cytochrome and

cholate,

obtained

from

Sigma

MO.).

CNBr-activated (9).

with

reductase

26-2H3 25-Hydroxy vitamin D3 was synthesized c 3 (8). 25-Hydroxy vitamin D3 was a generous gift from

previously from

preparations

PROCEDURE

and

(St. Louis,

J. Babcock

crude

P-450.

dilauroylglycero-3-phosphocholine Co.

and a more

P-450

activity

linearity

preparations.

(7),

in the present

25-hydroxylase

EXPERIMENTAL

Chemical

studies,

together

microsomal

different

NADPH-cytochrome

P-450

an approximate

and

to inhibitors

(7). As shown

a significant

of cytochrome

with

In preliminary

cytochrome

gave

activity

widely

be due

and

activity

microsomal at least

might

P-450

enzyme

varied

be preferred.

cytochrome

highly

between

of conversion

system

microsomal

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of

Octylamine-Sepharose

4B (Pharmacia)

obtained

from

(Uppsala,

Sweden).

Lab.

(Richmond,

CF-1

were

as described

Kao-Atlas

and

SM-2

and

Hydroxyapatite

powder

standard

by

(Tokyo)

Bio-Beads

Calif.).

cellulose

chemicals

48 was

was

(Whatman)

before

commercial

high

purity

materials. Methods. about

200 g, were

used.

for

were

and treated

der

Hoeven

prepared with

and

essentially

sodium 80 mM

with (IO).

and

Sprague-Dawley

1 mg per

Liver

ml of

homogenates,

potassium

P-450

livers

(11,12)

potassium

P-450 phosphate

column

equilibrated

with

potassium

buffer

5 mM phosphate

and buffer.

applied The

P-450

fraction

called

cytochrome

P-450

fraction

8. The

cytochrome

treated

with

and calcium

phosphate

gel and

pH 7.4,

containing

The

the

solubilization column

was

eluted

5 mM phosphate

35 mM

phosphate P-450

dialyzed

20% (v/v) glycerol

616

was

to a DEAE-Sepharose column

cytochrome

buffer

buffer.

rats)

the hydroxyapatite

and

A and

by van

and DEAE-Sepharose

5 mM

Bio-Beads

phosphate

from

of Wistar

involving

called

Tris-acetate

35 mM

was eluted

was

and microsomes

as described

octylamine-Sepharose-hydroxyapatite

Cytochrome

weighing

water,

33% (w/v), (from

by Guengerich

strain,

drinking

pyrophosphate

Cytochrome

as described

cholate,

chromatography. with

1 to 2 weeks. Coon

Wistar

Phenobarbital,

administered prepared

of the

with

eiuate eluate

fractions

was was were

against

50 mM

and 0.1 mM

EDTA.

Vol.

90,

No.

The final

2, 1979

BIOCHEMICAL

cytochrome

cytochrome

P-450

P-450

per

as

to Lowry

(15,16)

fractions

by Omura et al.

cytochrome

P-450

apparent

molecular

weights

one band,

corresponding

NADPH-Cytochrome Sprague-Dawley

rats (18)

Reductase

activity

0.3 of

had

preparations

fraction

of untreated

were

vitamin

D3 dissolved

P-450,

3 units

The

1.5 ml

of 0.1 reductase

min,

vitamin

at

37 ‘C

NADPH.

at 37’C

incubations described

previously

focused at m/e

26-2H3

buffer

that were

extracted

ion

the

spectrum amount

to

between

of the

reaction

first

the

the

was added

reductase x g

using

livers

of deuterium m/e

586

the

and

the

peak

at

20°C

at

addition

as

detector

ion

of the

spectrum

ion

was

589. The

25-hydroxy

vitamin in the

vitamin

using

The

derivatized

ion

calcuIated 589

of and

standard.

and

mass

3

was preincubated by

25-hydroxy m/e

for

of 3 ml of toluene

corresponding

D3 was

of

NADPH-cytochrome

multiple

in the

of

volume

on the ion at m/e

labeled

vitamin

25 yg

in a total

purified

ion

50 pg of

of cytochrome

started

of unlabeled

to

with

as internal

of the

molecular

30 min

and the mixture

product

channel

25-hydroxy at

of 1 umol

pH 7.4,

preincubated

by addition

D3 was

corresponds

derivative peak

the

derivative

589

of unlabeled

using

100,000

P-450,

were

at m/e 586 and the second

at m/e

at 30°C

reductase,

of NADPH

added

and

The

corresponds

ion

(19)

1.5 nmol

P-450

terminated

t-butyldimethylsilyl/trimethylsilyl and

0.75 or

then

vitamin

(7).

of protein.

P-450

20 or

pH 7.4. Cytochrome

were

25-hydroxy

were

on the 586

1

with

dilauroyl-3-phosphocholine

incubations

[

of

73 ng

of

as described

to reduction

buffer,

for

and 1.5 ymol

3 and buffer 1 min. After

for

0.1 M Tris-Cl

in

B

livers

mg

electrophoresis.

NADPH-cytochrome

D

The

upon

in 10 ~1 of acetone

M Tris-Cl and

(17)

NADPH-cytochrome

performed

of

corresponds

fraction of 50,000.

from per

to

rats.

dilauroylglycero-3-phosphocholine P-450

One unit

determined

P-450

et al.

was

corresponding

Masters

to Masters

P-450

weight

55 units

of

electrophoreses

prepared

of

18 nmol

was gel

Cytochrome

and

activity

according

was prepared

Incubations

protein bands

was

homogenous

Sprague-Dawley

11 and

molecular

to Yasukochi

buffer.

were

and

protein

reductase

minute.

used

two

COMMUNICATIONS

Cytochrome

and 50,000.

a specific

phosphate c per

(13)

to an apparent

was assayed

M potassium cytochrome

Supernatant

of 46,000

according

and

Sato

A gave

P-450

previously

B contained

On SDS- po 1yacrylamide

fraction

gave only

RESEARCH

respectively.

and

(14).

BIOPHYSICAL

A and

mg of protein

described

determined

according

AND

from

D3 mass

D3. the

a standard

The ratio curve

(6,7). RESULTS of

Incubation

cytochrome synthetic

P-450, phospholipid

vitamin

D3 with

a reconstituted

NADPH-cytochrome resulted

into

P-450 a significant

617

system

reductase, conversion

consisting NADPH into

of and

25-hydroxy

a

Vol. 90, No. 2, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A

0

m/e=586 kc

m/e

=586

m/e

= 589

m/e=589

6

4 Retention

Fig. 1.

1 10

8 time

4

D 9. Thus mass fragmentographic of such an incubation, to which deuterium

been

added,

(Fig.

1A). The two

derivative

gave of

ion

at

of

The different height curve

analyzed

significant

peaks

obtained

589

analysis labeled

P-450 at m/e

exact

quantity

at m/e

586

of

and

tracing

of

the

added,

isopyro

586

form

of

in the tracing

of

internal

incubation

standard. to

which

gave no significant

25-hydroxy

unlabeled

calculated

peak

(cf. Experimental Procedure In order to ascertain the by

labeled

m/e

no peaks

1B).

be

the

had been

of the extract (A) and P-450 vitamin prior to

of an extract vitamin D3 had

and

peaks obtained

a corresponding

reductase

could

two

deuterium

10

(min)

of the ion at

to the pyro-

D3. The

of

of derivative 25-hydroxy

in the tracing

correspond to the

586 (Fig.

incubations

peaks

vitamin

corespond

extract

an

NADPH-cytochrome in the tracing

two

25-hydroxy

m/e

Analysis

a time

MID recordings at m/e 586 and m/e 589 trimethylsilyl/t-butyldimethylsilyl derivative of incubation of vitamin b with the complete reconstituted system the reconstitdted system minus $JADPH-cytochrome reductase (B). A fixed amount of H3-labeled 25-hydroxy D3 (73 ng) had been added to the incubation mixture extraction (cf. Experimental Procedure). -

vitamin

the

6

Retention

(min)

height and

identity

ions at m/e

vitamin

from

the

at m/e

ref.

ratio

589,

D3 formed between

with

the

in the peak

use of a standard

6). of

131

618

the and

product,

m/e

some

134.

The

incubations results

were

obtained

BIOCHEMICAL

Vol. 90, No. 2, 1979

AND BIOPHYSICAL

Table Reconstitution

of

(fraction 25 (2.

rat

contained:

system

B),

vitamin

1.5

1.

D3

P-450

NADPH-cytochrome

NADPH

w

liver

cytochrome

2%hydroxylase

0.75

P-450

pmol.

RESEARCH COMMUNICATIONS

nmol

activity. (fraction

reductase

Standard

3 units,

incubation

The A)

or

complete 1.5

nmol

phosphatidylcholine

conditions

were

used

Methods).

System

Cytochrome

Complete NADPH-cytochrome

P-450

reductase

Minus

cytochrome

Minus Minus

+The

A

pmol

formed

108

Minus

P-450

fraction

Cytochrome

per

73

13

18

1

NADPH

10

8

phosphatidylcholine

93

20

figure

within

parentheses

B

incubation

(205)+

3 (lo)+ P-450

P-450

fraction

represents

another

experiment

using

the

same

conditions.

were m/e

almost

identical

those

Table

1

different

summarizes

A and

B.

significant

The

formation

of

P-450

reductase

effect

when

a stimulatory cytochrome Addition

untreated

with

tracing

the

P-450 of

ions

rat reduced

than

the

50% (results 2 shows

that

the

amount

of added

cytochrome

P-450

or NADPH

at

m/e

586

and

higher

cytochrome with

A

P-450

than

seen

when

cytochrome

was

omitted.

The

P-450

fraction

with

B.

P-450,

phospholipid

B but

not

when

A.

enzyme

the

fraction

was

x H supernatant

not shown

Fig.

with

was

using cytochrome

fraction

100,000

experiments

product

NADPH-cytochrome

more

by

of conversion

degree

had using

obtained

589.

fractions No

with

fluid

activity

of the

a liver

homogenate

reconstituted

of

system

an

with

in Tabe). of conversion

rate

from

was

P-450

cytochrome

at

least

fraction

approximately

linear

A (up to 0.5 nmol)

and

B (up to 0.5 nmol). DISCUSSION

The identical a mass

identification properties spectrum

characteristic between

of the ions

C24

of the in thin-layer

product

as 25-hydroxy

and gas chromatography

vitamin

D3 is based on

and

the

t-butyldimethylsilyl/trimethylsilyl

at m/e

586

(molecular

and C25 in a C2, -steroid

ion)

side chain

619

on

ether and

at m/e

containing

fact

that

contained 131

the

(cleavage

an TMSiO-group

at

Vol. 90, No. 2, 1979

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

-450

Cytochrome

Fig.

very

small

spectrum

amounts

unlikely There

system: The

that was

had

of the two have

microsomal

reported The crude

should define

microsomes

were

conversion.

(7),

optimal to

In the

publications

using the most

Bhattacharyya

and

of

for

cytosol

on the

present

cytosol

to the activity.

present Lack

activity

purified

was much higher l/4

that

of the maximal

it

system

of has

obtained

activity

cytochrome

was

(about

than the activity

mitochondrial

however,

NADPH.

(3,6,7).

in the

containing

when

25-hydroxylase

for assay in the present

most

of

effect

and was about

conditions

linearity

Addition

on microsomal

P-450/min)

in mind,

In contrast

(1).

vitamin

and

requirement

inhibitory

obtained

system

the optimal

approximate

an

cytosol

cytochrome

be borne

conditions

activity had

only

is

reconstituted

reductase

P-450.

mass it

2%hydroxy

of the

effect

a full

however,

else than

P-450

absolute

almost

to obtain

findings,

components

an

activity

in a reconstituted

three stimulatory

also in some other

5 pmol/nmol

be anything

of cytochrome

by the

maximal

above

preparations

system effect

for

the

a significant

25-hydroxylase

stimulatory

could

form of the derivative for identity. In view of

was not possible

NADPH-cytochrome

reported

reconstituted

it From

the product

P-450,

phospholipid

DeLuca

with

of product, material.

a requirement

cytochrome

homogenous

been

of both the pyro- and isopyro is another specific evidence

of the isolated

highly

(nmol)

Effect of amount of added cytochrome P-450 (cytochrome P-450 fraction A and B) on the rate of 25-hydroxylation of vitamin D3 by the reconstituted system.

2.

C25 (20)). The fo rmation in the gas chromatography the

P-450

6

obtained

P-450

(6). It

not

ascertained

that

work.

Experiments

designed

all to

are now in progress. previous

between evaluation

work

amount

with

crude

microsomes,

of cytochrome

of the results

620

obtained

P-450

(Fig.

there and

2, Table

was an

extent

l),

of

it must

Vol.

90,

No.

2, 1979

be pointed 25-hydroxy precision

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

out that the present method does not allow measurement of vitamin D3 in amounts less than about 5-10 pmol, and that the is rather

low in the determination

of amounts

lower

than about

20

pmol. In addition

to vitamin

D3,

the present

preparations

of cytochrome

P-450

are also able to catalyze 25-hydroxylation of 5B-cholestane-3a ,7c(, 12cl-trio1 and 5 Bcholestane-3a ,7cc-diol (Hansson and Wikvall, unpublished observation, -cf. ref. 21). Thus it is possible that the species of cytochrome P-450 involved in these hydroxylations is the same as that involved in 2%hydroxylation of vitamin D3. It

may

be

mentioned,

however,

that

25-hydroxylation

of

5 Bcholestane-3a, 7cl,l2a-trio1 by crude microsomes is not affected by rachitis (Bjorkhem and Holmberg, unpublished observation). According to DeLuca et -al. microsomal

25-hydroxylation

of vitamin

D3 is stimulated

by the rachitic

state

(1). with

Interestingly, the purified

the rate of 25-hydroxylation of vitamin D3 was much higher cytochrome P-450 fractions than with the crude microsomal

fraction

(cf. in the crude - ref. 7). It seems likely that there are inhibitor(s) microsomal fraction, which are removed in the purification procedure. It can not be excluded that these inhibitor(s) are of regulatory importance, and this possibility is at present under study. ACKNOWLEDGEMENT The skilful technical assistance of Anne Bjerkerud, Mannerberg and Catharina Nystriim is gratefully acknowledged. supported

by the Swedish

Medical

Research

Council

(Project

Britt-Marie This work was 03X-3141

and

03X-218). REFERENCES 1.

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