The mechanism of the diurnal variation of hepatic HMG-CoA reductase activity in the rat

BIOCHEMICAL

Vol. 45, No. 1, 1971

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

THF, MECHANISM OF THE DIURNAL VARIATION REDUCTASE ACTIVITY Malcolm Department

Received

Higgins,

Takashi

OF HEPATIC HMG-CoA

IN THE RAT

Kawachi*

and Harry

Rudney

of Biological Chemistry, University of Cincinnati College of Medicine, Cincinnati, Ohio 45219

July 26, 1971

SUMMARY. Direct measurements of the incorporation of 3H-leucine into highly purified 8-hydroxy-pmethylglutaryl CoA reductase (E.C. 1.1.1.34) have shown that the diurnal variation in activity observed in crude fractions is due to synthesis of new enzyme protein for approximately six hours, followed by complete cessation of synthesis for a duration of approximately 15 hours. The rate of degradation does not appear to be significantly altered. The molecular

mechanisms

mammals have been a recent

review

regulated

the

CoA reductase

drastically

reduced

by various

treatments

biliary

drainage

Lynen co,

(7),

and others

of the it

synthesis

of investigation

by several

laboratories

activity

as measured

after

cholesterol

feeding

such as Triton

WR 1339

Hamprecht

and Saucier

that

and/or

degradation

* Present

address

: National

MVA, mevalonic

large

Cancer acid;

(3),

2),

is

(6),

Research

HMG-CoA,

138

Institute,

or

N&isler

and

Dugan -et al -*

in the activity liver

due to changes inhibition

is

increased (4),

(8),

variations

enzyme while Center

and is

Hamprecht,

in mouse and rat is

fractions,

noradrenaline

and Rodwell

diurnal

phenomenon

of the

(for

of HMG-CoA to

in microsomal (e.g.,

Shapiro

synthesis this

reduction

and Lynen

(3),

in

of 8-hydroxy-P-methylglutaryl

enzyme,

Back,

of cholesterol

the NADPH dependent

have demonstrated

suggested

synthesis

of this

synthesis

Abbreviations: CoA.

of isoprenoid

by the

enzyme and cholesterol

has been

control

The hepatic

catalyzes

(5).

Kandutsch

1).

extent

which

The activity

MVA.

subject

see Ref.

to a major

of the

preparations; in the rate

of

by cholesterol Tokyo,

&hydroxy-P-methylglutaryl-

Japan.

BIOCHEMICAL

Vol. 45, No. 1, 1971

feeding

may be due to the action

AND BIOPHYSICAL

of an as yet

RESEARCH COMMUNICATIONS

undiscovered

inhibitor

(e.g.,

10). In the aforementioned redu.ctase

activity

to studying

the

synthesis

work

in crude

on the

preparation

was obtained

microsomal

regulation

is

of the isolated

incorporation

measurement

to determine

into

isolated

of HMG-CoA

A more direct the

enzyme protein.

of 3H-leucine

of HMG-CoA reductase

via

preparations.

of enzyme levels

and degradation

studies

data

the

rate

of

We report

highly

by Kawachi

approach

here

purified

and Rudney

(11).

Methods Assay

of HMG-CoA Reductase.

by Kawachi

and Rudney

of the reaction lactone,

with

dried

fraction,

after

applied

MVA-lactone (pH 7.0)

markers

was scraped The amount

hydroxamate

of Hinse Animals

obtained free

from

access

radioactive intraperitoneally.

layer

which

were

chloride

and Lupten

and Isolation

amino

acid

directly in the

and Grass1

Inc.,

other (12),

One

of nitrogen,

plate

benzene:acetone

gel (1:l

with

corresponding

v/v).

2 M NH2OH to MVA-

a scintillation

fraction

was

of silica

successively

into

Female Madison,

chow and water.

dissolved

After

two aliquots.

The ether

counter

was determined

by the

or by the gas chromatographic

(13).

Sprague-Dawley, rat

into

The area

solution.

of Enzyme.

to Rockland

(5 x 1 vol).

with

of MVAwith

ether

chromatographic

termination

quantity

solution

by a stream

by spraying

the plate

of Lynen

volume

was developed

visualized

from

diethyl

as described After

known of the

was divided

to a small

of MVA-lactone

method

of an accurately

Na2SO4,

thickness),

were

1 ml or 2 ml volumes.

with

to a thin

and a ferric

lactone

method

was extracted

evaporation

mm wet

using

conditions

H2SO4 and saturation

by anhydrous

quantitatively

vial.

with

MYA-lactone

extract,

but

NaOH, addition

acidification

Na2SO4,

G (0.3

(11)

Incubation

in

rats,

Wisconsin, At

the

0.1 ml isotonic

an appropriate

139

time

approx

interval

200 g, were

and were specified saline

allowed

times

the

was injected

the rats

were

killed

BIOCHEMICAL

Vol. 45, No. 1, 1971

by decapitation, isotonic

or injected

AND BIOPHYSICAL

with

RESEARCH COMMUNICATIONS

25 mg unlabelled

leucine

dissolved

in

saline.

HMG-CoA reductase Kawachi

and Rudney

material

was isolated

(11)

through

was contaminated

accordkg

to the method

chromatography

by a minor

on Sephadex

component

(as shown

described G-200.

by This

by disc

gel

electrophoresis). Results Table from

the

specific tion

livers

the data

of rats

from

injected

radioactivity

stepis

of the Table

1 displays

and Discussion

with

and specific

evidence

that

an HMG-CoA reductase 3H-leucine. enzyme

purified

The correspondence

activity

the radioactivity

preparation

observed

in the is

final

indeed

in

purificaconstitutive

enzyme molecule. 1.

Purification

of 31NiMG-CoA-Reductase

Enzyme Activity

from Rat Liver

mg Protein

% Recovery of Activity

3H dpm

dpm 3H/mg Protein

Microsomal fraction

1.9

890

100

1.35

x 106

1,521

Deoxycholate supernatant

5.7

425

140

6.05

x

1,400

Ammonium sulfate fraction

10.1

169

109

2.48

x lo5

1,470

DEAR cellulose

78.0

18.1

82

3.5 x 104

1,950

Hydroxyapatite

277.0

3.1

51

7.65

x lo3

2,470

G-200

301

2.7

49

7.0 x 103

2,630

-

0.5

w

1.35

2,700

Sephadex

Disc -gel electrophoresis

*

lo5

x 103

Twenty rats were injected Fntraperitoneally with 50 p.Cf each of 3H-leucine (390 uCi/nmtole) in 0.1 ml isotonic saline. Protein in column fractions was precipitated with trichloroacetic acid after addition of 1 mg bovine albumen to each tube and filtered on Whatman 3GA discs. After digestion with Beckman Soluene-100 radioactivity was determined directly using a toluene based scintillant. Protein was determined by the Lowry-Folin methot. Enzyme activities expressed as nmoles MVA/mg protein/hr. 0.5 mg protein was subjected to polyacrylamide disc gel electrophoresis in 12 tubes. Gels were sliced, dissolved in piperidine and counted (15).

140

Vol. 45, No. 1, 1971

BIOCHEMICAL

Figure

obtained

1 shows data

the injection at each the

with

time

3H-leucine.

poFnt

same rats,

and the

AND BIOPHYSICAL

by isolation

of the

The microsomal

enzymic

total

radioactivity

was subsequently

RESEARCH COMMUNICATIONS

enzyme 35 min after activity

was measured

of the purified

enzyme,

from

determined.

. 280 240 200 160 120

0800

I200

1600

TIME

2000

2400

“; k$. Q +! s zi k

0400

OF DAY

Figure 1. Variation of Microsomal HMG-CoA Reductase Activity and Incorporatjon of 3H-leucine. Groups of 8 rats were injected with 40 p,Ci of H-leucine (390 mCi/mmole) at the times shown. Livers were removed 35 min later and enzyme purification commenced. Assays are a8 described in the text and legend to Table 1.

The specific the diurnal reflect8

activity

variation the rise

the diurnal remains

period;

constant

for could

indicate

and fall

of the

however,

an artifact

synthesie

purified

enzymes

The variation

the It

seems to precede

may be merely increased

period.

as expected.

in incorporation This

of the

is the

of the

of protein

a more complex

control

in crude

radioactivity interesting rise

of leucine fractions

that

in microsomal

during fractions

the

enzymic

reflecting

to be reflected

during

in microsomal to note

system,

where

change

in incorporation

enzyme activity total

does not

increase activity.

the time

required

a8 enzyme activity,

the enzyme

is

inhibited

or it at that

time. .A88uming

that

the

incorporation

of amino

141

acid

reflects

enzymic

syn-

BIOCHEMICAL

Vol. 45, No. 1, 1971

thesis

(and

since

not

decrease

begin

to feed

diurnal

variation

the animals

suggest

that

change

in rate

the

degradation then

of synthesis.

of the

35 min later

and specific time

tion In the

before

former

rapidly,

case,

while

Conversely,

in Figure

total

specific

of rats

time

is

were

diluted

into

the role with

were

and the

of these after by newly

played

determined

and the

essentially

is

results approximately synthesized

labeled is

at

fnjec-

striking. declines

constant,

of isolatable

“pulse”

by

3H-leucine,

and protein

amount

of the

due to a

the results

to increase

remains

to

radioactivity

between

radioactivity

radioactivity

not

partly

of maximum incorporation

isolatable

stops

least

The total

starts

here

was reasonable

injected

The contrast

radioactivity

essentially

is at

- unlikely

?lMG-CoA reductase

time

activity

it

leucine.

2.

Our explanation

rapidly.

size

some insight

of incorporation

the enzyme

the

at this

rate

specific

new enzyme protein labeled

groups

at the

the

the

while

rising,

decreased

the

pool

in enzyme

of purified

injections

acid

RESEARCH COMMUNICATIONS

at the same time),

25 mg unlabeled

as shown

from

just

protein, with

in amino

To gain

radioactivity

intervals

obtained

were

a large

AND BIOPHYSICAL

that six

protein protein

synthesis hours.

of Rrotein

enzyme.

*9* \P

DECREASING ENZYMIC ACTIVITI

t

2 14 :: 1 IO 2 $6 B $2 z sG

I2345678

12345678

TIME (in hours 1 Figure 2. The Variation of Specific Radioactivity after a Single Injection of 3H- leucine . Groups of 36 rats ware injected with 25 @i 3H-leucine each (440 l.UX/mmole) at 2200 hr (A) or 1630 hr (B) and with 25 mg unlabeled leucine 35 min later. Livers of 6 rats were excised at the times shown and the enzyme isolated. Rotted lines represent specific radioactivity, histogram the total radioactivity.

142

Vol. 45, No. 1, 1971

BIOCHEMICAL

The isolatable is plotted

radi.oactivity

on semilogarithmic

both while the enzyme activity while it is falling, data the half differences the

life

AND BIOPHYSICAL

at intervals

scales (Fig. 3).

after

RESEARCH COMMUNICATIONS

a single pulse label

The decay in radioactivity,

in the q icrosomal fraction

follows approximately of the protein

first

is rising

order kinetics.

is approximately

3 hr.

and From this

The slight

in slope of the two lines can be explained by reutilization

of

radioact ive amino acid.

TIME

(hours)

Associated with RMC-CoAReductase. Figure 3. Decay of Radioactivity Experimental conditions are as described for Figure 2. If

the assumption is made that the enzyme behaves identically

purification

procedures at high and low levels,

experiments (which will inhibitor equivocally

and in the light

be reported elsewhere) which failed

of the enzyme during the low part of the cycle, demonstrates that the diurnal variation

in the of mixing

to show an this data un-

in activity

is due to

an increase in the rate of synthesis of the enzyme during the rising

portion

of the cycle and an apparent complete cessation of enzyme synthesis during the

falling

phase. Present experimental conditions make it difficult

pin point the exact initiation

of these 143

to

phases. Changes in the rate of

BIOCHEMICAL

Vol. 45, No. 1, 1971

degradation

do not

AND BIOPHYSICAL

seem to be significantly

RESEARCH COMMUNICATIONS

different

during

both

phases .

of

the cycle. The conclusion rather

than

data

a marked

obtained

Randutsch using

that

it

change

by others

and Saucier

puromycin,

prevention

of the decrease

protein

also

with

a very

sis

playing

a role

the

signals

for

work

on complete

variation

* 9 * P \

is

was supported

Diseases,

Grant

Foundation

in activity

on the

by cycloheximide is

effected

repression

by a

The nature of the

of

synthesis

to be investigated.

by the National

No. AM-12402.

(14)

of enzyme synthe-

of note.

remains

work

synthesis.

any light

derepression

biosynthesis

rise

with

in the experiments

throw

worthy

and subsequent

the Wellcome

cyclic

about

of thia

This

from

brought

life.

and Metabolic grant

do not

half

Acknowledgements.

a travel

the

short

the repression

of protein

of RMG-CoA reductase

key enzyme in isoprenoid

of Arthritis

to prevent

in activity

in a diurnal

is consistent

inhibitors

Our results

(14).

of enzyme synthesis,

had the same effect

the degradation

The demonstration

of this

able

rate

of degradation,

antibiotic

cycloheximide

and Rodwell

changing

in the rate

using

of Shapiro

of course,

a rapidly

(3) were

while

unless,

is

Institute

MR acknowledges

(London).

DECREASING ENZYMlC *CT,“,Ty

t 2 14 :: 1 IO 2 $6 B $2 z G s

I2345678

12345678

TIME (in hours 1

Figure 2. The Variation of Specific Radioactivity after a Single Injection of 3H- leucine . Groups of 36 rats ware injected with 25 @i 3H-leucine each (440 uCi/mmole) at 2200 hr (A) or 1630 hr (B) and with 25 mg unlabeled leucine 35 min later. Livers of 6 rats were excised at the times shown and the enzyme isolated. Dotted lines represent specific radioactivity, histogram the total radioactivity.

142