Effect of coenzyme A on cholesterol formation by rat liver homogenates

ltions and Prelimin

ME A ON r LIVER

CHOLESTEROL

,tes

FO]

HOMOGENATES by

B. B. M I G I C O V S K Y AND D. M. G R E EE~N B E R G

vision o/Chemistry, Science Service, Canada Department o] Ag;,riculture, £ Department o/Physiological Chemistry, School of Medicine, University, Berkeley, Cali/ornia (U.S.A.)

a) and

"he f o r m a t i o n of cholesterol f r o m acetate b y a ceil-free a d u l t r a t liver ) [emonstrated b y BUCHER 1 a n d R A B I N O W l T Z AND G U R I N 2 w wh~ ho prepared ICHER p r e p a r a t i o n . significant feature of the BUCHER s y s t e m is the fact tthai h a t w h e n gri ged or carried o u t in a t i g h t fitting homogenizer, the rresuB e s u l t a n t supel= t to cholesterol formation. I n v e s t i g a t i o n of the s u p e r n a t e from fro t h o r o u g ~d t h a t its i n a c t i v i t y lay in t h e presence of an i n h i b i t o r y factor f~ or fa I. As a result of this feature successive p r e p a r a t i o n s of ac active supei y.

?stem has t e m from liver was :five w i t h zed livers s h o w n in greatly in

TABLE I E F F E C T OF S U P E R N A T E FROM THOROUGHLY HOMOGENIZED LIVER (I.S.) ON ACETATE* INCORPORATION I N T O CHOLESTEROL CHOLESTERO

E~ach a c h flask contained I ml of s u p e r n a t e f r o m loosely h o m o genized enb liver centrifuged for 5 m i n u t e s at

2OOO o rr.p.m., . 1. 3 m g ATP, 3.8 m g D P N , o.263 ml 14C-acetate solution sc a n d buffer to equalize ttlh e

v o l u m e in all flasks to 2.46 ml. The gas p h a s e w a s 02, iincubatior n c u b a t i o n was carried o u t for three h hou ours ad to each flask p r i o r to s a p)oniot a t 37 ° C w i t h c o n s t a n t shaking. Five m g carrier cholesterol w a s added 1 0 3. fication. n. R e s u l t s are expressed as micromoles acetate i n c o r p o r a t e d into il the t o t a l cholesterol times ic c . p . m . / m g digitonide × 2o. The fa, This value is e q u i v a l e n t to factor of 2o is the cholesterol digidi~ c . p . m . / m i c r o m o l e acetate tonide e q u i v a l e n t of ,5 m g cholesterol. Exp. No.

I

2

Addition to active system

p M acetate incorporated

LS. - - o. 5 ml

16. i 5.2

none I . S . - i.o ml

1.4 o.2

none I.S. - - i.o ml

2.5 o.2

none

2.6

I.S. - - 0.5 ml boiled liver juice - - 0. 5 m l boiled liver juice - - 0. 5 ml plus I.S. - - o. 5 ml

0. 4 6.6

none

o.9

* 1.6 micromoles c o n t a i n i n g 2.I :.I 5. lO6 c.p.m, w a s e m p l o y e d t h r o u g h o u t .

t are a p r e l i m i n a r y i n v e s t i g a t i o n of tk ~ted t h e n e c e s s i t y of " A c t i v e a c e t a t e " I{LEIN4 d e m o n s t r a t e d t h e ne e d for co( m w a s e m p l o y e d t h r o u g h o u t . Choleste scribed by RABINGVITZ AND GREENBEt flow c o u n t e r . D e c o m p o s i t i o n of t h e d pecific a c t i v i t y . leral experiments. They indicate that c T high concentration. In the presence o y m e A i n h i b i t s c h o l e s t e r o l f o r m a t i o n oi s a m e a m o u n t of e o e n z y m e A i n c r e a s e s t

J

be a c e t y l y e a s t for :ed as t h e onide w a s Lp y r i d i n e e h a v e s as w acetate ~ct. W h e n holesterol

TABLE II EFFECT

OF

COENZYME

A*

ON A C E T A T E

I N C O Rt RP PO O R ARTAITOIIO N I N T O CH

s e v e r a l e xpm 7 a r i a b i l i t y of t h e d e g r e e of a c e t a t e i n c o r p o r a t i o n in t h e se~ :oncentration d l i t y in t h e q u a n t i t a t i v e r e s p o n s e to C o A a n d a c e t a t e corn n s i t i v i t y of t h e p r e p a r a t i o n . Addition to active system

Exp. No.

Amour Amount of acetate m~

o.61 none

CoA--

I.O

mg

none

1.6 16.1 o.8

6.6

11.6

21

12.o

20. 7 16-3

20.6 23 .8

23 34

3o.4 29.9 25. 4

7 °.2 85.3 83.0

76.o 84.3 113.2

64 123.5 lO8.1

CoA - - 0.06 m g none CoA - - 0.o 5 m g CoA - - o . I o m g none C o A - - o.xo m g CoA - - 0.4o m g none

CoA -- o.Io mg CoA - - 0.40 m g

s and the ly due to

12.5 i I.O

5.1 1.8 0. 5

5-9 9-5 1.9

5.2

8.6

5.3

18.2

3"°

19.3

* P r o d u c t of P a b s t I t a p p e a r s t h a t c o e n z y m e A c a n be s h o w n to be a n a c t i v a t i n g f a c t o r for c h o l e s t e r o l s y n t h e s i s u p o n t h e p r o p e r c o n c e n t r a t i o n s of a c e t a t e a n d cofactor. s y s t e m s , c o n d i t i o n) na al l u nificant A p o s s i b l e e x p l a n a t i o n of t:his h i s b e h a v i o u r lies in t h e f a c t t h a t c o e n z y m e A p l a y s a significa bility role in s e v e r a l of t h e a l t e r n a t e mneettaa b o l i c r o u t e s for a c e t a t e a n d t h u s a l t e r s t h e c o m p e t i t i v e aabili )ossible of t h e c h o l e s t e r o l s y s t e m for t hhee a v a i l a b l e s u b s t r a t e . E x p e r i m e n t s t o s u b s t a n t i a t e t h e possi] p a r a l l e l i s m b e t w e e n i n a c t i v e s u p e r n a t e a n d CoA are in progre s s . by homogenate

The valuable

assistance

[VI. of M . RABINOVITZ is g r a t e f u l l y a c k n o w l e d g e d .

REFERENCES

* N. Buci~EE, J . A m . Chem. Soc. ,75 (I953) 498. IN, Biochim. Biophys. Acta, IO (1953) 345. ' J. R. R ~ m o w I T z AND S. GURIN ormone Con[., 6 (I951) 111-129. 8 K. BLOCH, Proc. Laurentian Hormone 4 N. P. KUzlN, Federation Proc., IO (I951) 2o9. Arch. Biochem. Biophys., 4 ° (1952) 472. 5 M . R A B I N O V I T Z A N D D. M. G R E E~,ENBERG, NBERG R e c e i v e d O c t o b e r i 2 t h , ic 1953