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Regulation of rat liver acetyl CoA carboxylase Quayle, Katherine Amanda 1986

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REGULATION OF RAT LIVER ACETYL CoA CARBOXYLASE. by KATHERINE A. QUAYLE B.Sc. Heriot-Watt U n i v e r s i t y , 1984 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Biochemistry) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA J u l y 1986 @ Kat h e r i n e A. Quayle, 1986 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Stolen isTrZy The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date |5+k Ocr DE-6 (3/81) ABSTRACT Acetyl-CoA carboxylase was p u r i f i e d 7 50-fold from rat l i v e r c y t o s o l u s i n g a m o d i f i e d p u r i f i c a t i o n p r o c e d u r e i n v o l v i n g h i g h speed u l t r a c e n t r i f u g a t i o n , sucrose d e n s i t y step g r a d i e n t s , i o n -e x c h a n g e c h r o m a t o g r a p h y and a v i d i n - s e p h a r o s e a f f i n i t y chromatography. The p u r i f i e d p r e p a r a t i o n had a s p e c i f i c a c t i v i t y of about 2 U/mg p r o t e i n and a Km of 80uM f o r a c e t y l - C o A . S i l v e r s t a i n of the sample s e p a r a t e d by d e n a t u r i n g p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s i n d i c a t e d 95% of the p r o t e i n m i g r a t e s as a s i n g l e p r o t e i n band with subunit Mr approximately 230Kd. There i s approximately 2% contamination of low molecular mass m a t e r i a l and 3-5% of the p r o t e i n runs as a minor band of 240Kd. As the major p r o t e i n band c o - m i g r a t e s on S D S - p o 1 y a c r y 1 a m i d e g e l s w i t h i m m u n o p r e c i p i t a t e d a c e t y l - C o A c a r b o x y l a s e from a f r e s h r a t epididymal f a t pad t i s s u e e x t r a c t , i t appears that the 240Kd band i n the r a t l i v e r sample may be a p r e - f o r m of the enzyme which undergoes p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n to the mature form of the enzyme. Desp i t e appearing almost homogeneous a f t e r s i l v e r - s t a i n , the p u r i f i e d enzyme p r e p a r a t i o n s t i l l c o n t a i n s both c y c l i c AMP-independent and c y c l i c AMP-dependent p r o t e i n kinase a c t i v i t y , the f o r m e r c a u s i n g i n c o r p o r a t i o n o f 0.1mol P i / m o l a c e t y l - C o A carboxylase s u b u n i t / h r , and the l a t t e r causing i n c o r p o r a t i o n of 0.5mol P i / m o l s u b u n i t / h r . A d d i t i o n of s p e c i f i c i n h i b i t o r to i i c y c l i c AMP-dependent p r o t e i n kinase e f f e c t i v e l y blocks a l l c y c l i c AMP-dependent p r o t e i n kinase a c t i v i t y . The c y c l i c AMP-independent p r o t e i n kinase a c t i v i t y c o - p u r i f y i n g with acetyl-CoA carboxylase p h o s p h o r y l a t e s t h e " c o n t r o l s i t e s " as d e m o n s t r a t e d by 2 -d i m e n s i o n a l peptide a n a l y s i s of r a d i o a c t i v e l y l a b e l l e d ACC. T h i s p h o s p h o r y l a t i o n has no a p p a r e n t e f f e c t on enzyme a c t i v i t y . P h o s p h o r y l a t i o n by c y c l i c AMP-dependent p r o t e i n kinase i n c r e a s e s p h o s p h o r y l a t i o n o f t h e c o n t r o l s i t e s and a l s o r e s u l t s i n i n c o r p o r a t i o n of [32p] i n t o the two A p e p t i d e s and one of the B group of p h o s p h o p e p t i d e s . N e i t h e r of t h e s e endogenous p r o t e i n k i n a s e a c t i v i t i e s cause i n c o r p o r a t i o n of the l a b e l i n t o the i n s u l i n responsive s i t e of acetyl-CoA carboxylase. At l e a s t 90% of the c y c l i c AMP-independent p r o t e i n k i n a s e a c t i v i t y ( a s s a yed u s i n g ACC as s u b s t r a t e ) i n the r a t l i v e r c y t o s o l c o - p u r i f i e s w i t h a c e t y l - C o A c a r b o x y l a s e upon s u c r o s e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n . Under the c o n d i t i o n s d e s c r i b e d , approximately 90% of t h i s p r o t e i n kinase a c t i v i t y i s separated from ACC by DEAE-ce11ulose c h r o m a t o g r a p h y , e l u t i n g w i t h the o t h e r unbound p r o t e i n from t h i s column. 2 - d i m e n s i o n a 1 t r y p t i c peptide a n a l y s i s of acetyl-CoA carboxylase phosphorylated by t h i s p r o t e i n kinase f r a c t i o n i n c r e a s e s i n c o r p o r a t i o n of [32p] mainly i n t o the c o n t r o l s i t e s . F u r t h e r f r a c t i o n a t i o n of t h i s sample however does not r e v e a l i n c r e a s e d I s i t e p h o s p h o r y l a t i o n . P r o t e i n kinase a c t i v i t y e l u t e d together with ACC (at high s a l t ) from D E A E - c e l l u l o s e appeared to lead mainly to p h o s p h o r y l a t i o n of t h e B g r o u p of p e p t i d e s . However, i n t h i s c a s e f u r t h e r f r a c t i o n a t i o n of the DEAE-HS f r a c t i o n on c a s e i n - s e p h a r o s e i i i p r o d u c e s a p r o t e i n k i n a s e f r a c t i o n ( e l u t e d at 0.5M KC1) which b r i n g s about p h o s p h o r y l a t i o n of the i n s u l i n - d i r e c t e d s i t e of a c e t y l - C o A c a r b o x y l a s e . Thus a c y c l i c AMP-independent p r o t e i n kinase a c t i v i t y which phosphorylates acetyl-CoA carboxylase at an i n s u l i n r e s p o n s i v e s i t e , has been p a r t i a l l y p u r i f i e d from r a t l i v e r c y t o s o l . We are t h e r e f o r e i n a p o s i t i o n to e x p l o r e the e f f e c t s of i n s u l i n - d i r e c t e d p h o s p h o r y l a t i o n on the a c t i v i t y of acetyl-CoA carboxylase. iv TABLE OF CONTENTS Page ABSTRACT 1 1 LIST OF TABLES v i i LIST OF FIGURES v i i i LIST OF ABBREVIATIONS i x ACKNOWLEDGEMENTS x INTRODUCTION 1. C h a r a c t e r i z a t i o n of Acetyl-CoA Carboxylase 1 2. A l l o s t e r i c R e g u l a t i o n of ACC 4 3. Hormonal R e g u l a t i o n and Covalent M o d i f i c a t i o n of ACC 6 4. The T h e s i s I n v e s t i g a t i o n s 13 MATERIALS AND METHODS 14 1. Chemicals 14 2. Animals 15 3. Enzyme Assays 16 4. A n a l t i c a l Procedures 17 5. P r e p a r a t i v e Procedures 22 RESULTS 25 1. Development of a P u r i f i c a t i o n Procedure f o r ACC 25 1.1. P r e p a r a t i o n of Rat L i v e r C y t o s o l 27 1.2. Sucrose D e n s i t y G r a d i e n t Studies 28 1.3. Enzyme P r e c i p i t a t i o n S t u d i e s 4 32 1.4. DEAE-Cellulose Ion Exchange Chromatography 35 1.5. Avidin-Sepharose A f f i n i t y Chromatography 39 2. I n v e s t i g a t i o n s of P r o t e i n Kinase A c t i v i t y 45 v 2.1 Endogenous P r o t e i n Kinase A c t i v i t y of ACC 47 2.2 Exogenous P r o t e i n Kinase A c t i v i t y 52 2.3 F r a c t i o n a t i o n of P r o t e i n Kinases on Hydroxyl A p a t i t e 54 2.4 F r a c t i o n a t i o n of P r o t e i n Kinases on Casein-Sepharose 56 DISCUSSION 61 1. P u r i f i c a t i o n of Rat L i v e r ACC 61 2. C h a r a c t e r i z a t i o n of Rat L i v e r P r o t e i n Kinases 68 3. Future I n v e s t i g a t i o n s 72 REFERENCES 75 v i LIST OF TABLES Table Page 1. E f f e c t of a S i n g l e S u c r o s e D e n s i t y C u s h i o n on the D i s t r i b u t i o n of ACC a c t i v i t y D u r i n g High Speed C e n t r i f u g a t i o n . 29 2. E f f e c t of V a r i a t i o n of Composition of Sucrose Cushions on the D i s t r i b u t i o n of ACC A c t i v i t y during High Speed C e n t r i f u g a t i o n 31 3. P r e c i p i t a t i o n of ACC A c t i v i t y from Rat L i v e r C y t o s o l with P o l y e t h y l e n e G l y c o l 34 4. , P u r i f i c a t i o n of ACC from Rat L i v e r C y t o s o l 44 5. C h a r a c t e r i z a t i o n of Endogenous P r o t e i n Kinase A c t i v i t y i n P u r i f i e d P r e p a r a t i o n s of Rat L i v e r C y t o s o l 50 6. Summary of A n a l y s i s of T r y p t i c Phosphopeptides Obtained F o l l o w i n g P h o s p h o r y l a t i o n of P u r i f i e d ACC by P r o t e i n Kinase F r a c t i o n s from Rat L i v e r . . 60 L I S T OF F I G U R E S Figure Page 1. P r o t e o l y t i c Degradation of ACC Fo l l o w i n g P r e c i p i t a t i o n with PEG 36 2. Binding of ACC to DEAE-Cellulose 38 3. Avidin-Sepharose A f f i n i t y Chromatography of Rat L i v e r C y t o s o l i c ACC 40 4. Appearance of P u r i f i e d ACC Follo w i n g SDS-PAGE ... 42 5. P u r i f i c a t i o n Scheme f o r Rat L i v e r ACC 43 6. Schematic Diagram of Autoradiograph 46 7. C h a r a c t e r i z a t i o n of the Endogenous P r o t e i n Kinase A c t i v i t y A s s o c i a t e d with P u r i f i e d P r e p a r a t i o n s of ACC 49 8. Autoradiographs Obtained from 2-Dimensional T r y p t i c Peptide Maps of ACC 51 9. D i s t r i b u t i o n of P r o t e i n A c t i v i t y in F r a c t i o n s of Rat L i v e r 53 10. Autoradiographs Obtained from 2-Dimensional T r y p t i c Peptide Maps of Rat L i v e r ACC 55 11. Autoradiographs Obtained from 2-Dimensional T r y p r i c Peptide Maps of Rat L i v e r ACC . ... 57 12. Autoradiographs Obtained from 2-Dimensional T r y t i c Peptide Maps of Rat L i v e r ACC 59 v l i i LIST OF ABBREVIATIONS ACS aqueous counting s c i n t i l l a n t ATP adenosine t r i p h o s p h a t e C C e l s i us C y c l i c AMP adenosine 3 1 :5'-monophosphate DEAE-Cellulose d i e t h y l - a m i n o - e t h y l c e l l u l o s e EDTA ethylened i a m i n e - t e t r a a c e t a t e F i g . F i g u r e x g g r a v i t y g gram hr hour(s) HPLC high performance l i q u i d chromatography Km Michaelis-Menten constant 1 l i t e r m meter M Molar Me methyl Mr molecular mass min minute(s) mol mole rpm r e v o l u t i o n s per minute s seconds U U n i t s v o l . volume w weight Standard p r e f i x e s are: n (nano) 10 ; u (micro) 10 m ( m i l l i ) 10 ; c (centi) 10 i x ACKNOWLEDGEMENTS My most s i n c e r e thanks go to Dr. Roger W. Brownsey f o r h i s t o l e r a n c e , guidance and enthusiasm over the course of t h i s study. I am most a p p r e c i a t i v e of the s u p p o r t a d v i c e and encouragement which came from other members of t h i s department i n c l u d i n g Zemin Yao, N e i l Ridgeway, Dr. Rosemary C o r n e l l and o t h e r s i n "The Vance Lab", I a l s o w i s h to acknowledge my d r i n k i n g f r i e n d s w i t h o u t whom I would have become a t o t a l wreck; Tom R e d e l m e i r , H e l e n Loughrey, John Lew, C r a i g Newton, Lyndsey E l t i s and Bob Winz. F i n a l l y the f i n a n c i a l support of t h i s p r o j e c t by B.C. Health Care i s g r a t e f u l l y acknowledged. x INTRODUCTION 1. C h a r a c t e r i z a t i o n of Acetyl-CoA Carboxylase Acetyl-CoA Carboxylase [ Acetyl-CoA : ca r b o n - d i o x i d e l i g a s e (ADP-forming) E.C. 6.4.1.2 ] (ACC) i s g e n e r a l l y c o n s i d e r e d to c a t a l y s e the r a t e - l i m i t i n g s t e p i n l o n g c h a i n f a t t y a c i d s y n t h e s i s . The enzyme o c c u r s as an i n a c t i v e d i m e r o f two i d e n t i c a l s u b u n i t s . Each subunit c o n t a i n s one mole of b i o t i n and a c t i v e s i t e s f o r the ATP-dependent c a r b o x y l a t i o n of b i o t i n and f o r the t r a n s f e r of the c a r b o x y l group to a c e t y l - C o A , p r o d u c i n g m a l o n y l - C o A . Each s u b u n i t has a s i n g l e h i g h a f f i n i t y b i n d i n g s i t e f o r c i t r a t e and f o r p a l m i t o y l - C o A and t h e s e i n t e r m e d i a t e s can a l t e r enzyme a c t i v i t y a l l o s t e r i c a l l y r e s u l t i n g i n a c t i v a t i o n and i n h i b i t i o n r e s p e c t i v e l y . A number of reviews d i s c u s s d e t a i l s of the de v e l o p m e n t of t e c h n i q u e s which have been used to s t u d y ACC and a s p e c t s of the s t r u c t u r e and p r o t e i n c h e m i s t r y o f the enzyme (1-5). The r e a c t i o n occurs i n two steps; ATP + HCO3- + BIOTIN-E ^ E C 0 2 - B I 0 T I N - E + A D P + P i C 02-BI0TIN-E + CH3CO-C0A • -OOCCHCO-SCoA + BIOTIN-E 1 Me2+ BIOTIN-E Sum: ATP + HCO3- + CH3CO-SC0A ^ * -QOCCHCO-SCoA + ADP + P i The. w i d e l y accepted r e a c t i o n mechanism i s d e f i n e d as b i - b i -u n i - u n i p i n g - p o n g , and t h e r e f o r e e x h i b i t s d o u b l e d i s p l a c e m e n t k i n e t i c s (6,7). More r e c e n t i n v e s t i g a t i o n s however, i n v o l v i n g steady s t a t e k i n e t i c a n a l y s i s , have provided data which suggests the f o r m a t i o n of a qua r t e r n a r y complex, with ordered a d d i t i o n of ATP, H C O 3 - f o l l o w e d by acetyl-CoA to the c i t r a t e - a c t i v a t e d form of the c a r b o x y l a s e (8). The c a r b o x y l a t i o n of a c e t y l - C o A to malonyl-CoA commits these carbon atoms to the fo r m a t i o n of long c h a i n f a t t y a c i d s c a t a l y s e d by the complex enzyme f a t t y a c i d synthetase. S e v e r a l o b s e r v a t i o n s lend support f o r the r o l e of ACC as the p r i n c i p l e enzyme i n v o l v e d i n the s h o r t term r e g u l a t i o n of f a t t y - a c i d s y n t h e s i s : 1) M a l o n y l - C o A has no apparent m e t a b o l i c f a t e other than as a s u b s t r a t e f o r f a t t y - a c i d synthetase (9). Tn v i v o c o n c e n t r a t i o n s of h e p a t i c malonyl-CoA are observed to c o r r e l a t e with i n c r e a s e s and decreases i n f a t t y - a c i d s y n t h e s i s and, f o r example, a r e seen to d e c r e a s e i n r e s p o n s e to glucagon (10). 2) ACC i s i n a c t i v a t e d on i n c u b a t i o n of l i v e r c e l l s w i t h g l u c a g o n (11) and d i b u t y r y l c y c l i c - A M P (12) and i s s t i m u l a t e d i n r e s p o n s e to i n s u l i n (13). 3) The r e a c t i o n i s d i s p l a c e d f a r from e q u i l i b r i u m i m p l i c a t i n g t h i s s t e p as r a t e l i m i t i n g . 4) Enzyme a c t i v i t y i s re g u l a t e d not only i n the lon g -t e r m by c h a n g e s i n t h e r a t e s o f p r o t e i n s y n t h e s i s and degradation, but a l s o i n the short term by a l l o s t e r i c r e g u l a t o r s and r e v e r s i b l e c o v a l e n t p h o s p h o r y l a t i o n . 2 ACC was f i r s t d e s c r i b e d by W a k i l i n 1958 when i t was demonstrated that p a l m i t a t e could be s y n t h e s i z e d from acetyl-CoA i n a v i a n l i v e r e x t r a c t s . I t was shown t h a t s y n t h e s i s was dependent upon the a d d i t i o n of Mn^ + ,ATP, NADPH and bic a r b o n a t e and t h a t t h e f i r s t s t e p i n t h i s p r o c e s s i n v o l v e s t h e c a r b o x y l a t i o n of acetyl-CoA to form a malonyl-CoA i n t e r m e d i a t e . ACC was subsequently p u r i f i e d from avian l i v e r (14) and a l s o from a v a r i e t y of o t h e r s o u r c e s i n c l u d i n g r a t e p i d i d y m a l a d i p o s e t i s s u e (15), r a t l i v e r (16,17) and r a t and r a b b i t mammary g l a n d (18,19). The t e c h n i q u e s used i n t h e s e s t u d i e s i n c l u d e ammonium s u l p h a t e p r e c i p i t a t i o n f o l l o w e d by e x t e n s i v e d i a l y s i s and i o n -exchange, a b s o r p t i o n and s i z e e x c l u s i o n c h r o m a t o g r a p h y . The i s o l a t i o n t y p i c a l l y r e q u i r e d up to a week to a c h i e v e the f i n a l p u r i f i e d enzyme p r e p a r a t i o n . P u r i f i c a t i o n by these c o n v e n t i o n a l procedures r e s u l t e d i n a "homogeneous" p r e p a r a t i o n of ACC with a s p e c i f i c a c t i v i t y of 10-15 U/mg p r o t e i n . The o b s e r v a t i o n by Swanson e t a l (20) t h a t p a r t i a l l y p u r i f i e d ACC from r a t l i v e r was a c t i v a t e d by i n c u b a t i o n w i t h t r y p s i n , p r o m p t e d f u r t h e r i n v e s t i g a t i o n s with h i g h l y p u r i f i e d enzyme p r e p a r a t i o n s . I r i t a n i e t a l (21) r e p o r t e d t h a t ACC i s h i g h l y a c t i v e a f t e r t r e a t m e n t w i t h t r y p s i n both w i t h or w i t h o u t the a l l o s t e r i c a c t i v a t o r c i t r a t e present i n the assay medium. The enzyme was a l s o shown to l o s e s e n s i t i v i t y to i n h i b i t i o n by palmitoyl-CoA. Guy and Hardie (22) have demonstrated that i n c r e a s e s i n s p e c i f i c a c t i v i t y from 1-2 U/mg p r o t e i n to 10-15 U/mg p r o t e i n are a s s o c i a t e d w i t h l i m i t e d p r o t e o l y s i s . T h i s suggests that the enzyme p r e p a r a t i o n s which e x h i b i t a high s p e c i f i c a c t i v i t y (10-15 U/mg protein) have been s u b j e c t e d to l i m i t e d p r o t e o l y t i c d e g r a d a t i o n by c e l l u l a r 3 proteases during the i s o l a t i o n procedure. A novel procedure f o r i s o l a t i n g ACC by a v i d i n a f f i n i t y c h r o m a t o g r a p h y a l l o w s r a p i d p u r i f i c a t i o n of a homogeneous enzyme p r e p a r a t i o n (23). ACC p r e p a r e d i n t h i s manner has a s p e c i f i c a c t i v i t y of about 2 U/mg p r o t e i n and a Km f o r acetyl-CoA of 80uM as opposed to a s p e c i f i c a c t i v i t y of 10- 15 U/mg p r o t e i n and a Km of 8-12uM f o r enzyme prepared by more lengthy c l a s s i c a l procedures. D e s p i t e the i n t r o d u c t i o n of the a f f i n i t y c h r o m a t o g r a p h y t e c h n i q u e f o r the p u r i f i c a t i o n of ACC, the q u a l i t y of the p u r i f i e d enzyme p r e p a r a t i o n p r o d u c e d i n d i f f e r e n t l a b o r a t o r i e s remains a matter of c o n t e n t i o n among the v a r i o u s groups working i n t h i s f i e l d . D i f f e r e n t v a l u e s have been r e p o r t e d f o r s e v e r a l p r o p e r t i e s i n c l u d i n g the molecular mass of the enzyme subunits as c a l c u l a t e d a f t e r d enaturing p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s , the s p e c i f i c a c t i v i t y , the Km f o r acetyl-CoA and the phosphate content of the enzyme on i s o l a t i o n . 2. A l l o s t e r i c R e g u l a t i o n of ACC Having e s t a b l i s h e d that ACC a c t i v i t y c o n t r o l s the rate of f a t t y - a c i d s y n t h e s i s , many workers have attempted to e l u c i d a t e the mechanism by which the enzyme i s r e g u l a t e d . Long-term r e g u l a t i o n i n v o l v e s changes i n the r a t e s of enzyme s y n t h e s i s and d e g r a d a t i o n , which o c c u r over a p e r i o d of hours (24). The mechanism by which the more r a p i d s h o r t - t e r m changes i n a c t i v i t y o c c u r as o b s e r v e d f o r example i n r e s p o n s e to hormonal s t i m u l i , remains e l u s i v e . In 1952 Brady and Gurin (25) demonstrated that 4 f a t t y - a c i d s y n t h e s i s i n a v i a n l i v e r e x t r a c t s i s s t i m u l a t e d by c i t r a t e . Subsequent in v i t r o s t u d i e s have shown that c i t r a t e and c e r t a i n other t r i c a r b o x y l i c i n t e r m e d i a t e s such as i s o c i t r a t e and to a l e s s e r extent, malonate can s t i m u l a t e f a t t y - a c i d s y n t h e s i s , and t h a t t h e s e i n t e r m e d i a t e s cause a p p r o x i m a t e l y a 5 - f o l d s t i m u l a t i o n of ACC a c t i v i t y (26-28). The a c t i v a t i o n by c i t r a t e o c c u r s i n p a r a l l e l w i t h p o l y m e r i z a t i o n of the i n a c t i v e d i m e r i c form of the enzyme i n t o an a c t i v e f i l a m e n t o u s form c o n t a i n i n g 20 or more d i m e r s (29). The in v i t r o e f f e c t s o f c i t r a t e have been s t u d i e d e x t e n s i v e l y by a v a r i e t y of d i f f e r e n t t e c h n i q u e s , i n c l u d i n g s e d i m e n t a t i o n c e n t r i f u g a t i o n which d e m o n s t r a t e s the f o r m a t i o n of a p o l y m e r i c form by a change i n the s e d i m e n t a t i o n c o e f f i c i e n t from 10-15S f o r the protomeric form to 50-55S f o r the c i t r a t e a c t i v a t e d form (30). V i s c o s i t y s t u d i e s (31), e l e c t r o n microscopy (3) and more r e c e n t l y l i g h t s c a t t e r i n g and s t o p - f l o w k i n e t i c s which have d e m o n s t r a t e d t h a t i n c r e a s e s i n the r a t e of c a r b o x y l a t i o n of acetyl-CoA probably preceeds i n i t i a t i o n of the p o l y m e r i z a t i o n p r o c e s s (32). The m a j o r i t y of work i n t h i s a r e a has been concerned with in v i t r o s t u d i e s , however there i s some e v i d e n c e t h a t the- p o l y m e r i z a t i o n of ACC does o c c u r w i t h i n the i n t a c t c e l l (33). In c o n t r a s t to c i t r a t e , l o n g - c h a i n f a t t y a c y l - C o A t h i o e s t e r s , the most p o t e n t b e i n g p a l m i t o y 1 - C o A and a l s o a v a r i e t y o f e x p e r i m e n t a l c o n d i t i o n s (e.g. low t e m p e r a t u r e , h i g h pH, h i g h s a l t ) can l e a d to i n h i b i t i o n of ACC by c a u s i n g d i s a g g r e g a t i o n of the polymer to i t s i n a c t i v e protomeric s t a t e (30, 34). The obvious i n t e r p r e t a t i o n of these o b s e r v a t i o n s i s to presume the enzyme i s r e g u l a t e d by f e e d - f o r w a r d a c t i v a t i o n by 5 c i t r a t e and f e e d - b a c k i n h i b i t i o n by p a l m i t o y 1 - C o A (35). I t has however prov e d e x t r e m e l y d i f f i c u l t to c o n f i r m a c o r r e l a t i o n between f l u c t u a t i o n s i n i n t r a c e l l u l a r c o n c e n t r a t i o n s o f f r e e c i t r a t e or p a l m i t o y l - C o A and c o r r e s p o n d i n g r a t e s of f a t t y - a c i d s y n t h e s i s (36-38). Other p o t e n t i a l r e g u l a t o r s have been d e s c r i b e d i n the l i t e r a t u r e , i n c l u d i n g r e g u l a t o r y p r o t e i n s (39), coenzyme A (40), guanine n u c l e o t i d e s (41) and p o l y p h o s p h o i n o s i t i d e s (42), however there i s no evidence to date that any of these f a c t o r s can a l t e r r a t e s of f a t t y - a c i d s y n t h e s i s by d i r e c t l y r e g u l a t i n g ACC a c t i v i t y w i t h i n i n t a c t c e l l s . 3. Hormonal R e g u l a t i o n and Covalent M o d i f i c a t i o n of ACC I t i s w e l l e s t a b l i s h e d t h a t the storage and m o b i l i z a t i o n of f a t t y - a c i d s i n a n i m a l c e l l s i s c o n t r o l l e d by hormones i_n v i v o (43) and that these e f f e c t s i n v o l v e the c o n t r o l of ACC (44). The p r i n c i p a l s i t e s of f a t t y - a c i d s y n t h e s i s i n mammals are the l i v e r , brown and w h i t e a d i p o s e t i s s u e and l a c t a t i n g mammary g l a n d . I n s u l i n a c t i v a t e s ACC, s t i m u l a t i n g f a t t y - a c i d s y n t h e s i s w h i l s t glucagon and hormones which i n c r e a s e c e l l u l a r c y c l i c AMP l e v e l s i n h i b i t ACC. The i n h i b i t o r y e f f e c t o f c y c l i c AMP on ACC a c t i v i t y was f i r s t demonstrated by A l l r e d and Roehrig (12) who showed that the a d d i t i o n o f c y c l i c AMP to the a s s a y r e a c t i o n m i x t u r e i n h i b i t e d ACC a c t i v i t y 50%. I n i t i a l i n d i c a t i o n s t h a t p h o s p h o r y l a t i o n may a l s o be in v o l v e d i n the r e g u l a t i o n of c a t a l y t i c a c t i v i t y was the 6 o b s e r v a t i o n t h a t p u r i f i e d r a t l i v e r ACC c o n t a i n e d c o v a l e n t l y bound a l k a l i n e - l a b i l e phosphate (45) and a l s o that i n c u b a t i o n of r a t l i v e r e x t r a c t s w i t h Mg-ATP r e s u l t e d i n p h o s p h o r y l a t i o n and i n a c t i v a t i o n of the enzyme (46). I t i s now w e l l e s t a b l i s h e d that a c o v a l e n t p h o s p h o r y l a t i o n / d e p h o s p h o r y l a t i o n mechanism i s a s s o c i a t e d with the s h o r t term r e g u l a t i o n of ACC, however there i s s t i l l u n c e r t a i n t y about the nature of the p r o t e i n kinases and p r o t e i n phosphatases r e s p o n s i b l e f o r c a t a l y s i n g these r e a c t i o n s . A d d i t i o n of c y c l i c AMP to a crude t i s s u e e x t r a c t i n a c t i v a t e s ACC (12) and t h i s o b s e r v a t i o n prompted i n v e s t i g a t i o n s i n t o the e f f e c t s o f c y c l i c A M P - d e p e n d e n t p r o t e i n k i n a s e on t h e p h o s p h o r y l a t i o n of p u r i f i e d ACC and the corresponding e f f e c t s on c a t a l y t i c a c t i v i t y . The r a t i o n a l e of these s t u d i e s has been that r e g u l a t i o n of ACC i s a n a l o g o u s to the c y c l i c AMP m e d i a t e d r e g u l a t i o n d e s c r i b e d f o r o t h e r p r o t e i n s , i n c l u d i n g g l y c o g e n p h o s p h o r y l a s e , p h o s p h o r y l a s e k i n a s e and g l y c o g e n s y n t h e t a s e . Experiments with ACC p u r i f i e d from a v a r i e t y of sources i n c l u d i n g r a t and r a b b i t mammary g l a n d and r a t l i v e r d e m o n s t r a t e t h a t c y c l i c AMP-dependent p r o t e i n k i n a s e can p h o s p h o r y l a t e and i n a c t i v a t e the enzyme (47-49). D e s p i t e the e v i d e n c e s u p p o r t i n g the r o l e of c y c l i c AMP-dependent p r o t e i n kinase a c t i n g d i r e c t l y on ACC i n response to a d r e n a l i n or glucagon, some i n v e s t i g a t o r s are not convinced that ACC i s a d i r e c t s u b s t r a t e f o r c y c l i c AMP-dependent p r o t e i n kinase. A c y c l i c AMP-independent p r o t e i n kinase that i n a c t i v a t e s ACC by p h o s p h o r y l a t i o n , has been i s o l a t e d from r a t l i v e r c y t o s o l (50,51). T h i s p r o t e i n kinase a c t i v i t y has been d e m o n s t r a t e d to i n a c t i v a t e p u r i f i e d ACC o n l y i f coenzyme A i s p r e s e n t (52). F u r t h e r m o r e t h e r e i s e v i d e n c e t h a t the c a t a l y t i c 7 s u b u n i t of c y c l i c AMP-dependent p r o t e i n k i n a s e a c t i v a t e s the c y c l i c AMP-independent p r o t e i n k i n a s e by p h o s p h o r y l a t i o n . The c y c l i c AMP-independent p r o t e i n k i n a s e i s r e p o r t e d to then phosphorylate ACC causing i n h i b i t i o n of c a t a l y t i c a c t i v i t y (53). The f i r s t demonstration that p h o s p h o r y l a t i o n of ACC occurs i n i n t a c t t i s s u e was a c h i e v e d by i n c u b a t i n g r a t e p i d i d y m a l f a t c e l l s w i t h r a d i o a c t i v e p h osphate (54). R a p i d i n c o r p o r a t i o n of [32p] from the medium phosphate i n t o ACC was o b s e r v e d . When t i s s u e was incubated i n the presence of epi n e p h r i n e , the i n i t i a l a c t i v i t y o f ACC was r e d u c e d by 40% and t h i s e f f e c t was a s s o c i a t e d with a 40% i n c r e a s e i n p h o s p h o r y l a t i o n of the enzyme. The i n h i b i t i o n of ACC i n r e s p o n s e to e p i n e h r i n e was r e l i e v e d subsequently i n t i s s u e e x t r a c t s under c o n d i t i o n s which favoured d e p h o s p h o r y l a t i o n . P h o s o p h o r y l a t i o n o f ACC was l a t e r d e m o n s t r a t e d to o c c u r i n v i v o by i n t r a p e r i t o n e a l i n j e c t i o n o f i n o r g a n i c [ 3 ^ p ] i n t o r a t s . A d m i n i s t r a t i o n o f e p i n e p h r i n e s t i m u l a t e d [32p] i n c o r p o r a t i o n and caused i n a c t i v a t i o n o f ACC (55). S t u d i e s with h i g h l y p u r i f i e d ACC from l a c t a t i n g r a t mammary gland revealed that m u l t i p l e s i t e p h o s p h o r y l a t i o n occurs (47) and f u r t h e r c h a r a c t e r i z a t i o n o f t h e h o r m o n e s t i m u l a t e d p h o s p h o r y l a t i o n i n d i c a t e d t h i s was a l s o the case i n i n t a c t t i s s u e . Incubation of r a t e p i d i y m a l adipose t i s s u e with medium c o n t a i n i n g [ 3 2 p ] - p h o s p h a t e , but i n the absence of hormones r e s u l t s i n i n c o r p o r a t i o n of the l a b e l i n t o ACC, however t h i s p h o s p h o r y l a t i o n has no a p p a r e n t e f f e c t on enzyme a c t i v i t y . A d d i t i o n o f e p i n e p h r i n e to the i n c u b a t i o n medium s t i m u l a t e d f u r t h e r i n c o r p o r a t i o n of [32p] a n d i n a c t i v a t i o n of ACC a c t i v i t y . 8 A d d i t i o n of p r o p r a n o l o l to the medium, to block the ^ - a d r e n e r g i c r e c e p t o r s y s t e m , p r e v e n t e d both the e p i n e p h r i n e s t i m u l a t e d i n c o r p o r a t i o n of [ 3 2 P ] and the i n h i b i t i o n of enzyme a c t i v i t y . The d r u g had no e f f e c t h o w e v e r on i n c o r p o r a t i o n o f " c o n t r o l " p hosphate u n r e l a t e d to enzyme a c t i v i t y . T h i s i n d i c a t e s t h a t p h o s p h o r y l a t i o n i s o c u r r i n g a t more than one s i t e , a t l e a s t one of which i s i n v o l v e d i n ACC i n a c t i v a t i o n i n response to hormonal s t i m u l i (55). An e a r l y study by Brownsey and Hardie (56) revealed t h a t i n an i s o e l e c t r i c f o c u s s i n g s y s t e m , the major t r y p t i c p h o s p h o p e p t i d e of ACC from mammary t i s s u e t r e a t e d w i t h c y c l i c AMP-dependent p r o t e i n kinase co-migrated with the major t r p t i c phosphopeptide i s o l a t e d f o l l o w i n g i m m u n o p r e c i p i t a t i o n of ACC from e x t r a c t s of f a t c e l l s t r e a t e d w i t h e p i n e p h r i n e . W i t t e r s e t a l (41) and Holland and Hardie (48) have s i n c e shown that mapping of [ 3 2 p ] - p h o s p h o p e p t i d e s of ACC by r e v e r s e phase h i g h p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y , r e v e a l s t h a t the major [ 3 2 P ] - p e p t i d e p h o s p h o r y l a t e d i n r e s p o n s e to i n c u b a t i o n of i n t a c t t i s s u e w i t h e p i n e p h r i n e or g l u c a g o n p r e s e n t i n the medium, c o m i g r a t e d w i t h the major [ 3 2 p ] - p e p t i d e p h o s p h o r y l a t e d in v i t r o by the c y c l i c AMP-dependent p r o t e i n kinase. A l t e r n a t i v e mechanisms f o r the r e g u l a t i o n of ACC by c y c l i c AMP have appeared in the l i t e r a t u r e and evidence that c y c l i c AMP may i n f l u e n c e c y t o p l a s m i c c i t r a t e c o n c e n t r a t i o n s has been d i s c u s s e d (57). More r e c e n t l y an i n v e s t i g a t i o n i n t o the mechanism of glucagon a c t i o n i n hepatocytes l e d to the p o s t u l a t i o n that the e f f e c t s of p h o s p h o r y l a t i o n are i n t e r - r e l a t e d to the p h y s i c a l s t a t e of the enzyme, causing polymer-protomer t r a n s i t i o n s in v i v o (58) . 9 Despite the u n c e r t a i n t y as to the s p e c i f i c p r o t e i n kinases r e s p o n s i b l e f o r the f l u c t u a t i o n s i n degree of p h o s p h o r y l a t i o n of the enzyme, a l l the i n v e s t i g a t i o n s o u t l i n e d above tend to support the f a c t t h a t ACC i s i n h i b t e d by p h o s p h o r y l a t i o n of "A" group p e p t i d e s and that t h i s i n h i b i t i o n can be r e l i e v e d by subsequent d e p h o s p h o r y l a t i o n . T h i s i m p l i c a t e s a s i m p l e p h o s p h o r y l a t i o n / d e p h o s p h o t y l a t i o n mechanism which c o r r e l a t e s with enzyme i n h i b i t i o n and a c t i v a t i o n . I n v e s t i g a t i o n s of the e f f e c t s of i n s u l i n upon the l e v e l of p h o s p h o r y l a t i o n of ACC however have r e v e a l e d t h a t i t too c a u s e s an i n c r e a s e i n the i n c o r p o r a t i o n of phosphate i n t o the enzyme (59). I n s u l i n s t i m u l a t i o n of the p h o s p h o r y l a t i o n of ACC a p p e a r s to be m e d i a t e d t h r o u g h a c y c l i c AMP-independent p r o t e i n kinase a c t i v i t y as i n t r a c e l l u l a r c y c l i c AMP l e v e l s do not change i n the presence of i n s u l i n and there i s no d e t e c t a b l e e f f e c t on c y c l i c AMP-dependent p r o t e i n k i n a s e a c t i v i t y (60). The extent of m u l t i s i t e p h o s p h o r y l a t i o n of ACC i n response to i n s u l i n has a l s o been s t u d i e d f o l l o w i n g p r o t e o l y t i c d i g e s t i o n and s e p a r a t i o n of the p h o s p h o p e p t i d e s by r e v e r s e phase HPLC techniques or by a 2-dimensional peptide mapping technique. The l a t t e r method has been a p p l i e d f o l l o w i n g i m m u n o p r e c i p i t a t i o n of the enzyme and s u b s e q u e n t d i g e s t i o n w i t h t r y p s i n . The [ 3 2 P ] -p e p t i d e s may be s e p a r a t e d on TLC p l a t e s by h i g h v o l t a g e e l e c t r o p h o r e s i s i n the f i r s t dimension and chromatography based on s o l u b i l i t y i n an o r g a n i c s o l v e n t system i n the second dimension. S e v e r a l groups of posphopeptides are separated using these techniques and are v i s u a l i z e d by autoradiography. Brownsey 10 e t a l (59,61) have d e m o n s t r a t e d t h a t the i n s u l i n s t i m u l a t e d p h o s p h o r y l a t i o n of ACC i n r a t e p i d i d y m a l f a t pads r e s u l t s i n a 5-f o l d i n c r e a s e i n the i n c o r p o r a t i o n of [32p] i n t o a peptide which i s d i s t i n c t from those phosphorylated under " c o n t r o l " (no hormone t r e a t m e n t ) or a f t e r e x p o s u re of the t i s s u e to a d r e n a l i n e . T h i s s i t e has been d e s i g n a t e d the I - s i t e . D i f f e r e n t p h o s p h o r y l a t i o n p a t t e r n s a r e o b s e r v e d i n r e s p o n s e to d i f f e r e n t hormones and appear to c o r r e l a t e w i t h the a n t a g o n i s t i c e f f e c t s of i n s u l i n (which l e a d s to a c t i v a t i o n ) and a d r e n a l i n e (which l e a d s to i n h i b i t i o n ) of ACC a c t i v i t y . S i m i l a r r e s u l t s have been reported by W i t t e r s e t a l (49) u s i n g a r e v e r s e phase HPLC sys t e m to s e p e r a t e the t r y p t i c p h o s p h o p e p t i d e s . E l u t i o n of the p e p t i d e s w i t h o r g a n i c s o l v e n t s d i s c r i m i n a t e s peaks which appear to be a s s o c i a t e d with s t i m u l a t i o n by d i f f e r e n t hormones. Having e s t a b l i s h e d that s i t e s p e c i f i c p h o s p h o r y l a t i o n occurs i n r e s p o n s e to a d r e n a l i n e and i n s u l i n i n i n t a c t f a t pads, attempts to i s o l a t e the p r o t e i n kinase a c t i v i t y r e s p o n s i b l e has r e s u l t e d i n e v i d e n c e f o r a t l e a s t two c y c l i c AMP-independent p r o t e i n kinases which can phosphorylate ACC causing i n h i b i t i o n of c a t a l y t i c a c t i v i t y (51, 62) however a s t i m u l a t o r y p r o t e i n kinase has yet to be i s o l a t e d . ACC can a l s o be phosphorylated by c a s e i n kinase I and I I , i s o l a t e d from r a t l i v e r c y t o s o l (63) but n e i t h e r of t h e s e k i n a s e s appear to a f f e c t ACC a c t i v i t y , d e s p i t e the i n c o r p o r a t i o n of s t o i c h i o m e t r i c amounts of phosphate i n t o the i n s u l i n r e s p o n s i v e p e p t i d e of the c a r b o x y l a s e s u b u n i t . T h i s l e a v e s u n c e r t a i n the p o t e n t i a l i n v o l v e m e n t of t h e s e p r o t e i n k i n a s e s i n r e g u l a t i n g ACC a c t i v i t y . In a d d i t i o n the d i r e c t r e l a t i o n s h i p between ACC a c t i v i t y and p h o s p h o r y l a t i o n of the I-11 p e p t i d e i s a l s o questioned and may perhaps suggest the p o t e n t i a l i m p o r t a n c e of s p e c i f i c p r o t e i n or o t h e r l i g a n d i n t e r a c t i o n s i n t h e a c t i v a t i o n and i n h i b i t i o n o f ACC. F a t c e l l membrane p r e p a r a t i o n s c o n t a i n a c y c l i c AMP-independent p r o t e i n k i n a s e a c t i v i t y w h ich p h o s p h o r y l a t e s and i n c r e a s e s the a c t i v i t y of p a r t i a l l y p u r i f i e d ACC (64). The p r o t e i n k i n a s e a c t i v i t y was found to r e q u i r e Mg-ATP, but was i n s e n s i t i v e to c y c l i c AMP, a s p e c i f i c i n h i b i t o r of c y c l i c AMP-dependent k i n a s e i n h i b i t o r p r o t e i n and c a l c i u m . Changes i n c a t a l y t i c a c t i v i t y of ACC i n the p r e s e n c e of t h i s p r o t e i n k i n a s e appeared s i m i l a r to a c t i v i t y changes induced by exposure of the t i s s u e to i n s u l i n . Subsequent s t u d i e s i n d i c a t e d t h at the a c t i v i t y of a s i m i l a r p r o t e i n kinase i n high speed supernatant f r a c t i o n s i n c r e a s e d a f t e r i n c u b a t i o n of the i n t a c t f a t c e l l p r e p a r a t i o n s w i t h i n s u l i n (65). T h i s d a t a supports the hypothesis that b i n d i n g of i n s u l i n to i t s receptor a c t i v a t e s a p r o t e i n k i n a s e c a p a b l e of p h o s p h o r y l a t i n g ACC and t h i s i n t u r n r e s u l t s i n an i n c r e a s e i n ACC c a t a l y t i c a c t i v i t y . R ecent i n v e s t i g a t i o n s w i t h t r i t o n e x t r a c t s of human p l a c e n t a membranes a l s o supports the idea that a p r o t e i n s e r i n e kinase i s i n v o l v e d with i n s u l i n s i g n a l l i n g (66). I t i s , s t i l l unclear wether inc r e a s e d p h o s p h o r y l a t i o n of ACC i s the d i r e c t cause of enzyme a c t i v a t i o n , as the e f f e c t of i n s u l i n on c a t a l y t i c a c t i v i t y does not s u r v i v e p r o t e i n p u r i f i c a t i o n on a v i d i n sepharose d e s p i t e the f a c t t h a t the enzyme remains phosphorylated on s p e c i f i c ( i n s u l i n -d i r e c t e d ) r e s i d u e s (49,67). The requirement of p r o t e i n p h o s p h o r y l a t i o n f o r r e g u l a t i o n of ACC a c t i v i t y , need not n e c e s s a r i l y i n v o l v e r e g u l a t i o n of p r o t e i n 12 k i n a s e a c t i v i t y , as the degree of p h o s p h o r y l a t i o n c o u l d be c o n t r o l l e d by p r o t e i n p h o s p h a t a s e a c t i v i t y . S e v e r a l p r o t e i n p h o s p h a t a s e p r e p a r a t i o n s have been d e s c r i b e d w h i c h c a n dephosphorylate ACC which has been phosphorylated by c y c l i c AMP-dependent p r o t e i n k i n a s e and t h e r e b y r e l i e v e the i n h i b i t o r y e f f e c t on c a t a l y t i c a c t i v i t y (68-70). These phosphatases however tend to have broad s p e c i f i c i t y and i t i s d i f f i c u l t to contemplate t h a t s p e c i f i c e f f e c t s would be i n i t i a t e d by a r a t h e r n o n - s p e c i f i c s i g n a l . 4. The T h e s i s I n v e s t i g a t i o n s C l a r i f i c a t i o n of the d e t a i l s of the r e g u l a t i o n of ACC by a p r o t e i n p h o s p h o r y l a t i o n / d e p h o s p h o r y l a t i o n mechanism r e q u i r e s f u r t h e r i n v e s t i g a t i o n . . My i n i t i a l work i n v o l v e d c h a r a c t e r i z a t i o n o f a m o d i f i e d p u r i f i c a t i o n p r o c e d u r e to p r o d u c e a s t a b l e p r e p a r a t i o n of ACC w i t h the p r o p e r t i e s considered a p p r o p r i a t e f o r the i n t a c t enzyme. T h i s p r e p a r a t i o n was s u b s e q u e n t l y used as a s u b s t r a t e f o r d e t e c t i n g c y c l i c AMP-dependent and c y c l i c AMP-independent p r o t e i n kinase a c t i v i t y o c c u r i n g i n f r a c t i o n s of r a t l i v e r c y t o s o l . The c y c l i c AMP-independent p r o t e i n kinase a c t i v i t y has been c h a r a c t e r i z e d i n terms of i t s p o t e n t i a l to phosphorylate the i n s u l i n r e s p o n s i v e p e p t i d e of ACC by 2 - d i m e n s i o n a l t r y p t i c p eptide mapping a n a l y s i s . The development of techniques f o r the p u r i f i c a t i o n of t h i s p r o t e i n k i n a s e a c t i v i t y from r a t l i v e r c y t o s o l i s d e s c r i b e d . 13 MATERIALS AND METHODS 1. Chemicals The c h e m i c a l s and o t h e r m a t e r i a l s used i n t h i s s t u d y are l i s t e d below under t h e i r supply company. A l l other chemicals used were reagent grade. Sigma Chemical Company, St. L o u i s , MO EDTA, T r i s , G l y c i n e , MOPS, p-Glycerophosphate, A v i d i n , B i o t i n , p-Amino Benzamidine d i - H y d r o c h l o r i d e , B a c i t r a c i n , P r o t e i n K i n a s e I n h i b i t o r P r o t e i n , P e p s t a t i n , Coenzyme A, L e u p e p t i n , A d e n o s i n e 3':5' C y c l i c Monophosphate, Coenzyme A (Lithium s a l t ) , P a l m i t o y l Coenzyme A, P r o t e i n Kinase C a t a l y t i c Sub-unit, T r y p s i n , Spermine T e t r a h y d r o c h l o r i d e , P r o t e i n A, B o v i n e Serum A l b u m i n , P h e n y l m e t h y l - S u l p h o n y l F l u o r i d e (PMSF) , N-2 ,4-DNP-L-Lysine , P o l y e t h y l e n e G l y c o l , Sodium A z i d e , C o o m a s s i e B r i l l i a n t B l u e R, C i t r i c A c i d BDH Chemicals Canada Ltd., Vancouver B.C. Potassium C h l o r i d e , H y d r o c h l o r i c A c i d , A c e t i c A c i d ( g l a c i a l ) , D-G l u c o s e , P o t a s s i u m Hydrogen C a r b o n a t e , S i l v e r N i t r a t e , Sodium Hydroxide, G l y c e r o l , P y r i d i n e F i s h e r S c i e n t i f i c Ltd., Vancouver, B.C. T r i c h l o r o a c e t i c a c i d , P h o s p h o r i c A c i d , Ammonium B i c a r b o n a t e , Ammonium Sulphate, Formaldehyde Bio-Rad L a b o r a t o r i e s , Richmond, C a l i f o r n i a N,N'-Methylene-bis-Acrylamide, Acrylamide, Ammonium Persulphate, 14 Bromophenol B l u e , TEMED, 2 - M e r c a p t o e t h a n o l , Sodium D o d e c y l Sulphate, Coomassie B r i l l i a n t Blue G-250, Sucrose E. Merck L a b o r a t o r i e s , c/o BDH Chemicals Ltd. (see above) Magnesium C h l o r i d e Calbiochem, La J o l l a , CA Aquacide I I I ( f l a k e p o l y e t h y l e n e g l y c o l ) , Hydroxyl A p a t i t e Eastman Kodak Company, Rochester, NY Adenosine 5'-Tri Phosphate Disodium S a l t , X-Omat AR f i l m XAR-5, Chromagram Sheet (for t h i n l a y e r chromatography) 13255 C e l l u l o s e , Kodak GBX D e v e l o p e r and R e p l e n i s h e r , Kodak R a p i d F i x e r and Hardner (H SO s o l u t i o n UN 1830) Amersham, O a k v i l l e , Ont. Sodium [ 1 4 C ] - B i c a r b o n a t e , Adenosine 5'- [ $ - 3 2 p ] Triphosphate , Aqueous Counting S c i n t i l l a n t (ACS) Terochem L a b o r a t o r i e s Ltd., Edmonton, A l b e r t a D i e t h y l Amino E t h y l C e l l u l o s e (DE 52) Pharmacia Fine Chemicals AB, Uppsala, Sweden Sepharose CL-4B 2. Animals Male Wistar r a t s weighing 200-240g and fed ad l i b i t u m with a s t a n d a r d l a b o r a t o r y d i e t , used t h r o u g h o u t t h i s s t u d y were s u p p l i e d by The U n i v e r s i t y of B r i t i s h Columbia Animal U n i t , and housed i n the Department of B i o c h e m i s t r y f o r a p p r o x i m a t e l y 1-2 weeks, p r i o r to use. 15 3. Enzyme assays A c e t y l - C o A C a r b o x y l a s e - ACC was a s s a y e d e s s e n t i a l l y as d e s c r i b e d by H a l e s t r a p and Denton (71). The a s s a y i n v o l v e s the measurement of i n c o r p o r a t i o n of [ 1 4 C ] - b i c a r b o n a t e i n t o malonyl-CoA. A s s a y s were i n i t i a t e d by a d d i n g an a l i q u o t (50ul) of the sample, preincubated with 10mM c i t r a t e and 5mg/ml albumin unless otherwise s p e c i f i e d to 0.45ml of assay medium (100mM T r i s - H c l ; pH 7.4, 10mM MgS0 4 , 0.5mM EDTA, 5mM ATP, 5mM 2 - m e r c a p t o e t h a n o l , 15mM K H 2 C O 3 ( s p e c i f i c r a d i o a c t i v i t y ; 500-600 dpm/nmol), 10mg/ml albumin and 150uM acetyl-CoA) i n a sealed m i c r o - c e n t r i f u g e tube. The assay was terminated a f t e r 2 min by the a d d i t i o n of 200ul of 5M HC1 . A sample (0.6ml) was t r a n s f e r r e d to a s c i n t i l l a t i o n v i a l and e v a p o r a t e d to d r y n e s s a t 85 C. The r e s i d u e was d i s s o l v e d i n water (0.5ml) and a s s a y e d f o r r a d i o a c t i v i t y i n an Isocap/300 l i q u i d s c i n t i l l a t i o n c o u n t e r , a f t e r the a d d i t i o n of 7.5ml of A.C.S. l i q u i d s c i n t i l l a t i o n f l u i d . A l l a s s a y s were p e r f o r m e d i n d u p l i c a t e and c o r r e c t e d f o r [ 1 4 C ] counts o c c u r i n g i n the absence of added ACC f r a c t i o n . (This c o n t r o l g i v e s values very s i m i l a r to those obtained by o m i s s i o n of acetyl-CoA). P r o t e i n Kinase Assay- P u r i f i e d ACC p r e p a r a t i o n (5ug) was p r e i n c u b a t e d w i t h MgCl2 (8mM) f o r 2 min at 30 °C i n a s e a l e d m i c r o c e n t r i f u g e tube i n a f i n a l volume of 5 0 u l . A d d i t i o n of a p r o t e i n kinase f r a c t i o n or other f a c t o r s being i n v e s t i g a t e d were a l s o added at the p r e i n c u b a t i o n stage. The r e a c t i o n was i n i t i a t e d by the a d d i t i o n of r a d i o a c t i v e ATP (100uM, s p e c i f i c r a d i o a c t i v i t y 16 500-1000 dpm/pmol ). The a s s a y was t e r m i n a t e d a f t e r 30 rain, u n l e s s o t h e r w i s e s p e c i f i e d by the a d d i t i o n of 1 volume of SDS-d i g e s t i o n b u f f e r f o l l o w e d by immediately heating the sample to 95 °C i n a h e a t i n g b l o c k f o r 5 min. A l l of the sample was a p p l i e d to a s i n g l e g e l t r a c k and then s u b j e c t e d to 5% SDS p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s . The g e l was s u b s e q u e n t l y f i x e d , s t a i n e d with Coomassie Blue, d e s t a i n e d and d r i e d between cellophane. The d r i e d g e l was exposed to X - r a y f i l m f o r 2-5 days, and the i n c o r p o r a t i o n o f [ 3 2 P ] i n t o the ACC 230Kd s u b u n i t e s t i m a t e d by s c a n n i n g d e n s i t o m e t r y or d i g e s t i o n of the e x c i s e d p r o t e i n band f o l l o w e d by l i q u i d s c i n t i l l a t i o n counting. 4. A n a l y t i c a l procedures P o l y a c r y l a m i d e G e l E l e c t r o p h o r e s i s - S e p a r a t i o n of p r o t e i n s was c a r r i e d out by SDS-polyacrylamide g e l e l e c t r o p h o r e s i s i n 0.6 cm t r a c k s on 5% (w/v) s l a b g e l s (18 cm x l 6 cm) run a c c o r d i n g to t h e p r o c e d u r e o f L a e m m l i ( 7 2 ) . P r o t e i n s a m p l e s f o r e l e c t r o p h o r e s i s were prepared by p r e c i p i t a t i o n with i c e - c o l d 10% (w/v) t i c h 1 o r o a c e t i c a c i d and s u b s e q u e n t l y d i s s o l v e d i n s t a c k i n g - g e l b u f f e r c o n t a i n i n g 100mg SDS/ml, 200mg sucrose/ml, 0.2mg Bromophenol Blue/ml and 100mM 2-mercaptoethanol by heat i n g at 95 °C f o r 5 min. A f t e r e l e c t r o p h o r e s i s , the p r o t e i n bands were v i s u a l i z e d by s t a i n i n g w i t h Coomassie B l u e or S i l v e r s t a i n and the g e l s s a n d w i c h e d between c e l l o p h a n e and l e f t to d r y i n the a i r stream of a fume hood ove r n i g h t . 17 C o o m a s s i e B l u e P r o t e i n S t a i n - P o l y a c r y l a m i d e g e l s were f i x e d f o r 20 min i n TCA (20% w/v) MeOH (40% w/v) i n H 2 0 ' then t r a n s f e r r e d to Coomassie Blue s t a i n i n g s o l u t i o n HOAc : MeOH: H 2 O (7:45:52, by vol.) c o n t a i n i n g 2.5g Coomassie B l u e R - 2 5 0 / l i t r e ) f o r at l e a s t 1 h r . The background was d e s t a i n e d w i t h d e s t a i n i n g s o l u t i o n HOAc : MeOH : H 2 0 (7:45: 52 by vol.) u n t i l c l e a r . The g e l was soaked i n 2% (w/v) g l y c e r o l f o r 15 min to remove t r a c e s of a c e t i c a c i d which cause the g e l to c r a c k w h i l s t d r y i n g , t h e n l a i d between c e l l o p h a n e and l e f t to d r y i n the a i r s t r e a m of a fumehood overnight. S i l v e r S t a i n - S i l v e r s t a i n i n g of p o l y a c r y l a m i d e g e l s was achieved e s s e n t i a l l y by the method d e s c r i b e d by Wray et a l (73). The g e l was washed with 50% methanol a f t e r e l e c t r o p h o r e s i s , f o r at l e a s t 2hr u s i n g a sha k e r and r e p l a c i n g the methanol a t l e a s t twice. The methanol was then poured o f f the g e l and repla c e d with s t a i n i n g s o l u t i o n f o r 15min. S t a i n i n g s o l u t i o n was p r e p a r e d by t h e f o l l o w i n g p r o c e d u r e : - D i s s o l v e 0.8g o f AgNO 3 i n approximately 5ml of water ( S o l u t i o n A). Add 190ul of 10N NaOH to 21ml H 20 and then add 1.4ml of 7.4M N H 4 O H ( S o l u t i o n B). W i t h r a p i d s t i r r i n g , add s o l u t i o n A s l o w l y to the b a s i c s o l u t i o n B and then add an a d d i t i o n a l 70ml of water. Once the g e l had been s t a i n e d , the s t a i n i n g s o l u t i o n was decanted i n t o s a t u r a t e d N a c l , as p r e c i p i t a t i o n of the s i l v e r p r e v e n t s a p o t e n t i a l e x p l o s i o n . The g e l was then washed with d i s t i l l e d water f o r 6 min r e p l a c i n g the water once. The water was then r e p l a c e d w i t h d e v e l o p i n g s o l u t i o n (1ml of 1% c i t r i c a c i d and 100ul of 37% formaldehyde i n 150ml H 2 O ) . When t h e bands had d e v e l o p e d to t h e d e s i r e d 18 i n t e n s i t y , or when a l i g h t brown background began to appear, the d e v e l o p i n g s o l u t i o n was r e p l a c e d w i t h 5% methanol. A f t e r 5min w i t h s h a k i n g t h i s was r e p l a c e d w i t h MeOH:HOAc:H2o (4:1:5, by vol.). The developed g e l s were sandwiched between cellophane and l e f t to dry i n the a i r - s t r e a m of a fume-hood o v e r n i g h t . P r o t e i n A s s a y - P r o t e i n was a s s a y e d by the method of B r a d f o r d (74). P r o t e i n samples c o n t a i n i n g 10-70ug p r o t e i n i n 0.1ml were p i p e t t e d i n t o a t e s t - t u b e , 5ml of B r a d f o r d r e a g e n t was added to the t e s t tube and the c o n t e n t s mixed by v o r t e x i n g . The absorbance at 595nm was measured 10-60 min a f t e r a d d i t i o n of c o l o u r r e a g e n t , i n 3ml c u v e t t e s a g a i n s t a r e a g e n t b l a n k c o n t a i n i n g 0.1ml of the a p p r o p r i a t e b u f f e r and 5ml of B r a d f o r d r e a g e n t . S t a n d a r d c u r v e s were p r e p a r e d u s i n g b o v i n e serum albumin, on each o c c a s i o n . Autoradiography- R a d i o l a b e l l e d p o l y a c r y l a m i d e g e l s and TLC p l a t e s were autoradiographed by exposing to f i l m (X-Omat XAR-5) i n a Permacon c a s s e t t e with an i n t e n s i f y i n g screen, f o r 2-7 days a t -70°C. The f i l m was d e v e l o p e d f o r 5 min i n Kodak d e v e l o p e r , washed under r u n n i n g tap water f o r 5 min , then f i x e d f o r 5 min in Kodak f i x i n g s o l u t i o n . A f t e r washing with water f o r a f u r t h e r 5 min the f i l m was a i r d r i e d . S c a n n i n g D e n s i t o m e t r y _ -phosphate i n t o p r o t e i n bands e l e c t r o p h o r e s i s was estimated I n c o r p o r a t i o n o f r a d i o a c t i v e s e p a r a t e d by p o l y a c r y l a m i d e g e l by scanning densitometry, using a 19 Bio-Rad video densitometer model 620 at the f o l l o w i n g s e t t i n g s : F i l t e r frequency 4.0 Enhancement frequency 1.0 Boost f a c t o r 3 E s t i m a t i o n of I n c o r p o r a t i o n of X — p l i n t o ACC by L i q u i d  S c i n t i l l a t i o n Counting- The r a d i o l a b e l l e d 230Kd subunit of ACC, l o c a t e d by a u t o r a d i o g r a p h y or C o o m a s s i e Bl u e s t a i n , was e x c i s e d from the d e n a t u r i n g p o l y a c r y l a m i d e g e l and p l a c e d i n a g l a s s s c i n t i l l a t i o n v i a l . 0.2ml of H 202 was added and the v i a l was capped s e c u r e l y and h e a t e d i n an oven a t 80°C f o r 1-2 hr or u n t i l the g e l s l i c e had t o t a l l y d i s s o l v e d . The cap was then removed and the sample l e f t a t 80 °C u n t i l a l l the H 2 O 2 had evaporated. The residue was d i s s o l v e d i n 0.5ml of 1M Hel and then 7.5ml of ACS f l u i d was added to the v i a l . The samples were assayed f o r r a d i o a c t i v i t y i n an Isocap/300 l i q u i d s c i n t i l l a t i o n c o u n t e r . B l a n k s were p r e p a r e d by c u t t i n g g e l s l i c e s from the background of the g e l and t r e a t i n g i t i n the same manner as the p r o t e i n bands. 2-Dimensional Peptide Mapping of Rat L i v e r ACC- A n a l y s i s of p h o s p h o p e p t i d e s was p e r f o r m e d e s s e n t i a l l y as d e s c r i b e d by Brownsey and Denton (59). 30ug of p u r i f i e d ACC p r e p a r a t i o n was p r e i n c u b a t e d w i t h MgCl2 a n d a p r o t e i n k i n a s e f r a c t i o n where a p p r o p r i a t e f o r 2 min at 30 °C i n sealed m i c r o - c e n t r i f u g e tubes. The p h o s p h o r y l a t i o n r e a c t i o n was i n i t i a t e d by the a d d i t i o n of [ -32p] l a b e l l e d ATP (100uM, s p e c i f i c r a d i o a c t i v i t y 1000dpm/pmol, ) and i n c u b a t e d f o r a f u r t h e r 30 min at 30°C. The r e a c t i o n was 20 t e r m i n a t e d by the a d d i t i o n of 0.5ml of quench b u f f e r ( 50mM glycerophosphate; pH 7.2, 10mM EDTA, 50mM potassium f l u o r i d e , 5mM 2-mercaptoethanol, 2ug/ml p e p s t a t i n , 2ug/ml l e u p e p t i n ) . 15ul of p o l y c l o n a l ACC anti-serum was added to the samples and vortexed thoroughly. The assays were incubated f o r a f u r t h e r 30 min at 30 °C to a l l o w the a n t i b o d y to b i n d to the a n t i g e n . P r o t e i n A (2mg/assay of crude aureus Type 11 powder) was then added, and the samples were l e f t a t room t e m p e r a t u r e w i t h o c c a s i o n a l v o r t e x i n g f o r 20 m i n . T h i s i n d u c e s p r e c i p i t a t i o n o f t h e a n t i b o d y / a n t i g e n complex. The samples were then spun i n an ep p e n d o r f c e n t r i f u g e f o r 2 min to p e l l e t the complex and the supernatant was removed and d i s c a r d e d . The p r o t e i n A/antibody/ACC p e l l e t was washed three times with 1ml of quench b u f f e r and two times with 1ml of 100mM ammonium bicarb o n a t e pH 8.2. The p e l l e t was then resuspended i n 200ul of the ammonium bicarb o n a t e b u f f e r and 1 0 u l of a lmg/ml s o l u t i o n of t r y p s i n ( d i s s o l v e d i n the same bu f f e r ) was added to each sample. The samples were incubated f o r 3hr a t 30 °C, when t h e p e l l e t was sp u n down as d e s c r i b e d p r e v i o u s l y and the s u p e r n a t a n t removed. The p e l l e t was a g a i n r e s u s p e n d e d i n 200ul of the ammonium b i c a r b o n a t e b u f f e r and a s i m i l a r a d d i t i o n of t r y p s i n made to both the p e l l e t and the supernatant f r a c t i o n s . A f t e r a f u r t h e r i n c u b a t i o n f o r 3hr at 30 °C, b o t h s u p e r n a t a n t s were p o o l e d t o g e t h e r and taken to d r y n e s s under an a i r stream. A l i q u o t s of the r e a c t i o n mixture were saved a f t e r the i n i t i a l p h o s p h o r y l a t i o n r e a c t i o n and the a d d i t i o n o f a n t i s e r a and p r o t e i n A. These samples were run on d e n a t u r i n g p o l y a c r y l a m i d e g e l s and subsequently subjected to autoradiography 21 as d e s c r i b e d above. T h i s a l l o w s assessment of recovery of [32p]_ l a b e l l e d carboxylase. The t r y p s i n - d i g e s t e d residue was washed and r e d r i e d t w i c e w i t h 1ml of d i s t i l l e d water to f u l l y s u b l i m e any r e s i d u a l ammonium b i c a r b o n a t e , then the t r y p t i c p e p t i d e s were re s u s p e n d e d i n 10ul of d i s t i l l e d water. The e n t i r e d i g e s t was a p p l i e d to t h i n - l a y e r c e l l u l o s e p l a t e s . T w o - d i m e n s i o n a l s e p a r a t i o n of the pep t i d e s i n v o l v e d high v o l t a g e e l e c t r o p h o r e s i s i n the f i r s t d i m e n s i o n at pH 3.6 (3hr, 400V), u s i n g a p y r i d i n e : HOAc : H2O (1:9:189, by vol.) s o l v e n t s y s t e m and a s c e n d i n g c h r o m a t o g r a p h y i n the second d i m e n s i o n u s i n g a b u t a n - l - o l : p y r i d i n e : HOAc : H 20 (15:10:3:12. by vol.) s o l v e n t system. D i n i t r o p h e n y l l y s i n e was s p o t t e d w i t h the sample onto the t h i n l a y e r p l a t e s as a m a r k e r . [ 3 2 p ] _ 1 at,e 11 ed p e p t i d e s were v i z u a l i s e d by autoradiography f o r 3-7 days. 5. P r e p a r a t i v e Procedures P r e p a r a t i o n of DE A E - C e l l u l o s e - D E A E - c e l l u l o s e was prepared f o r use by b a t c h washing w i t h 5 v o l s , of 0.5M Hel f o l l o w e d by 0.5M NaOH, f o l l o w e d by 0.5M HC1. The r e s i n was then washed e x t e n s i v e l y with 10mM sodium phosphate b u f f e r , pH 7.0 to r e s t o r e the pH to n e u t r a l i t y . The r e s i n was e q u i l i b r i a t e d w i t h the a p p r o p r i a t e sample b u f f e r and used f o r batch/column s e p a r a t i o n of r a t l i v e r f r a c t i o n s . The r e s i n was r e g e n e r a t e d by r e p e a t i n g the a c i d - a l k a l i n e - a c i d wash and s t o r e d i n 10mM sodium phosphate b u f f e r c o n t a i n i n g 0.02% sodium azi d e at 4°C. P r e p a r a t i o n of Avidin-Sepharose- A v i d i n - s e p h a r o s e was p r e p a r e d 22 by a c t i v a t i n g Sepharose 4B with CNBr (72). Approximately 15ml of r e s i n was made pH 11 with NaOH and a c t i v a t e d by the a d d i t i o n of l g of CNBr. The pH and temperature were c a r e f u l l y monitored, and m a i n t a i n e d between pH 10.8-11.5 and 15-20 °C. r e s p e c t i v e l y . The r e s i n was suspended i n 10mM sodium phosphate b u f f e r , pH 7.0 and s t i r r e d w i t h 10mg of a v i d i n o v e r n i g h t a t 4°C. E t h a n o l a m i n e / H c l (0.5M, pH 7.0) was used as a b l o c k i n g a g ent, then the r e s i n was r e - e q u i l i b r a t e d w i t h sodium phosphate b u f f e r . The r e s i n was monomerized by the method of G r a v e l et a l (73). To e f f e c t monomerization, the r e s i n was poured i n t o the column (1.5 cmx 10 cm) and washed w i t h 6M g u a n i d i n e - H c l i n 0.2M KC1, pH 1.5. The column was clamped o f f and l e f t o v e r n i g h t at 4 °C. A f t e r removal of g u a n i d i n e - H C l w i t h washing of column w i t h sodium phosphate b u f f e r (6 v o l s . ) , the a v i d i n b i n d i n g s i t e s were s a t u r a t e d by washing the column w i t h the same b u f f e r c o n t a i n i n g 2mM b i o t i n . E x c h a n g e a b l e b i o t i n was removed by washing w i t h a t l e a s t 5 volumes of 0.1M g l y c i n e / H c l , pH 2.0. The e x c h a n g e a b l e b i o t i n b i n d i n g c a p a c i t y of the column was 4mM/ml of r e s i n as determined u s i n g [ 3 H ] - b i o t i n . The column was r e g e n e r a t e d a f t e r use by washing e x t e n s i v e l y w i t h the g l y c i n e b u f f e r and s t o r e d i n 10mM phosphate b u f f e r pH 7.0 c o n t a i n i n g 0.02% sodium azide at 4°C. Hydroxyl A p a t i t e C h r o m a t o g r a p h y - 5g of h y d r o x y l a p a t i t e was suspended i n 25ml of 10mM sodium phosphate b u f f e r , pH 7.2. The r e s i n was spun i n a bench top c e n t r i f u g e (2min, 1500 xg) and the f i n e s d e c a n t e d . The r e s i n was poured i n t o a column and e q u i l i b r a t e d w i t h t h e same b u f f e r c o n t a i n i n g 5mM 2-23 mercaptoethanol and ImM EDTA. 25ml of sample was a p p l i e d to the column at 10ml/hr. The unbound p r o t e i n was e l u t e d with s t a r t i n g b u f f e r a t 35ml/hr. A phosphate g r a d i e n t 10mM-lM phosphate i n a t o t a l volume of 100ml was used to e l u t e the bound p r o t e i n from the column and the e l u a t e was c o l l e c t e d i n 8ml f r a c t i o n s . The column was regenerated by washing e x t e n s i v e l y with 2M phosphate b u f f e r , t h e n s t o r e d a t 4 °C i n 10mM sodium phosphate b u f f e r c o n t a i n i n g 0.02% sodium a z i d e . C a s e i n - S e p h a r o s e C h r o m a t o g r a p h y - C a s e i n - s e p h a r o s e was prepared by a c t i v a t i n g sepharose 4B as p r e v i o u s l y d e s c r i b e d f o r the p r e p a r a t i o n of a v i d i n - s e p h a r o s e . A p p r o x i m a t e l y 10ml of the a c t i v a t e d r e s i n was resuspended i n 10mM sodium-phosphate b u f f e r pH 7.0 and s t i r r e d w i t h 10mg o f c a s e i n o v e r n i g h t a t 4°C. E t h a n o l a m i n e / H C l (0.5M, pH 7.0) was used as a b l o c k i n g agent, then the r e s i n was e q u i l i b r i a t e d i n an a p p r o p r i a t e b u f f e r ready f o r use. 25ml of sample was a p p l i e d to the column at 10ml/hr and the unbound p r o t e i n e l u t e d with s t a r t i n g b u f f e r at 25ml/hr. The column was then e l u t e d with 500mM KC1 and the e l u a t e c o l l e c t e d as a s i n g l e f r a c t i o n . The column was r e g e n e r a t e d by washing w i t h 5 v o l . o f 10mM so d i u m - p h o s p h a t e b u f f e r c o n t a i n i n g 1M KC1, pH7.0 and s t o r e d at 4°C i n 10mM sodium-phosphate b u f f e r c o n t a i n i n g 0.02% sodium a z i d e . 24 RESULTS 1. Development of a Procedure f o r the P u r i f i c a t i o n of a S t a b l e  Homogeneous P r e p a r a t i o n of Acetyl-CoA Carboxylase from Rat L i v e r . D e spite the f a c t that p u r i f i c a t i o n procedures f o r acetyl-CoA c a r b o x y l a s e have been documented i n the l i t e r a t u r e s i n c e the e a r l y 1960's (14), the present day techniques f o r the i s o l a t i o n of a s t a b l e homogeneous p r o d u c t s t i l l p r e s e n t s some i n t r i n s i c p r o b l e m s . The s t a n d a r d p u r i f i c a t i o n p r o c e d u r e f o r ACC used by many l a b o r a t o r i e s i n v o l v e s the p r e c i p i t a t i o n of the enzyme a c t i v i t y from a 100,000 xg c y t o s o l i c f r a c t i o n w i t h ammonium s u l p h a t e or p o l y e t h y l e n e g l y c o l or a c o m b i n a t i o n of the two techniques. The p r o t e i n p e l l e t produced i s then resuspended i n an a p p r o p r i a t e b u f f e r and a p p l i e d d i r e c t l y to an a v i d i n -sepharose a f f i n i t y column. The column i s washed e x t e n s i v e l y to remove the unbound p r o t e i n and the ACC e l u t e d by the a d d i t i o n of f r e e b i o t i n to the column b u f f e r . T h i s p r o c e d u r e a p p e a r s to be s u f f i c i e n t f o r i s o l a t i o n of the enzyme from a d i p o s e t i s s u e or mammary g l a n d , but p u r i f i c a t i o n of the l i v e r enzyme by t h e s e techniques r e s u l t s i n very poor y i e l d s of c a t a l y t i c a c t i v i t y . In my hands, a major p r o b l e m w i t h t h i s p r o c e d u r e has been a s s o c i a t e d w i t h p r e c i p i t a t i o n of the enzyme from the c y t o s o l i c f r a c t i o n by e i t h e r of the two p r e c i p i t a t i o n techniques d i s c u s s e d above. The p r o t e i n p e l l e t has p r o v e d to be e x t r e m e l y d i f f i c u l t to r e s o l u b i l i z e w i t h a s i g n i f i c a n t p r o p o r t i o n of the i n s o l u b l e p r o t e i n being ACC. The l o s s i n enzyme a c t i v i t y at t h i s step i s 25 very v a r i a b l e a c c o r d i n g to r e p o r t s from s e v e r a l l a b o r a t o r i e s , and c a n r a n g e f r o m 40%- 80% (23,77 ). In t h e s e s t u d i e s , my r e c o v e r i e s were t y p i c a l l y between 15-25%. A second problem appears to r e s u l t from prolonged exposure of the enzyme to endogenous p r o t e a s e a c t i v i t y p r e s e n t i n the i n i t i a l f r a c t i o n , s i n c e i n order to bind to the a v i d i n - s e p h a r o s e a f f i n i t y column, the enzyme i s r e q u i r e d to be i n the i n a c t i v e d i m e r i c form. T h i s form of the enzyme i s o b t a i n e d by removing c i t r a t e from the sample and under these c o n d i t i o n s , the enzyme i s r a t h e r s u s c e p t i b l e to p r o t e o l y s i s . The l o n g e r the enzyme i s e x p o s e d to p r o t e o l y t i c a c t i v i t y w h i l s t i n d e s t a b i l i z i n g c o n d i t i o n s r e s u l t i n g from the removal of c i t r a t e , the g r e a t e r i s the chance of f r a g m e n t a t i o n of the i n t a c t form of the enzyme. Assuming the i n t a c t enzyme does i n d e e d have the k i n e t i c p r o p e r t i e s d e s c r i b e d by Song and Kim (23), i . e . a s p e c i f i c a c t i v i t y of about 2 U/mg p r o t e i n and a Km f o r a c e t y l - C o A of 80uM, then d e v i a t i o n s from these c r i t e r i a p r o vide an i n d i c a t i o n of the i n t e g r i t y of the p u r i f i e d p r o t e i n . A d d i t i o n a l l y i f a l l of the p h o s p h o r y l a t i o n s i t e s are present, then presumably the p r o t e i n i s i n t a c t . P u r i f i c a t i o n of r a t l i v e r ACC by the p r o c e d u r e o u t l i n e d above y i e l d s l e s s than 10% r e c o v e r y of enzyme a c t i v i t y . T h i s i n v e s t i g a t i o n d e s c r i b e s the development of m o d i f i c a t i o n s of the s t a n d a r d p r o c e d u r e which a l l o w p r o t e c t i o n of the enzyme from p r o t e o l y t i c d egradation i n the crude f r a c t i o n and i n c r e a s e s the o v e r a l l y i e l d of c a t a l y t i c a c t i v i t y to 35-40%. The i n i t i a l approach to the problem was to f r a c t i o n a t e the 26 enzyme a c t i v i t y by s u c r o s e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n i n o r d e r to reduce the t o t a l amount of p r o t e i n as much as p o s s i b l e b e f o r e p r e c i p i t a t i o n w i t h ammonium s u l h p h a t e or p o l y e t h y l e n e g l y c o l . I t was e x p e c t e d t h a t t h i s would r e s u l t i n removal of a s i g n i f i c a n t p r o p o r t i o n of the protease a c t i v i t y which occurs i n the c y t o s o l and f a c i l i t a t e r e s o l u b i l i z a t i o n o f the p r o t e i n p e l l e t . 1.1 P r e p a r a t i o n of Rat L i v e r C y t o s o l . Male W i s t a r r a t s fed ad l i b i t u m and weighing approximately 200-240g were stunned and k i l l e d by d e c a p i t a t i o n , the l i v e r s were removed i m m e d i a t e l y and p l a c e d on i c e . The l i v e r s were then r i n s e d w i t h d i s t i l l e d water and homogenized g e n t l y i n t h r e e volumes of e x t r a c t i o n b u f f e r , (10mM T r i s - H c l , 20mM MOPS; pH 7.4, 250mM s u c r o s e , 2mM EDTA, 5mM c i t r a t e , 5mM 2 - m e r c a p t o e t h a n o l , 0.5mM PMSF, l m g / m l b a c i t r a c i n , 2ug/ml p e p s t a t i n , 2 u g / m l l e u p e p t i n , 2.5mM b e n z a m i d i n e , 0.02% sodium a z i d e ) u s i n g a Pot t e r Elvejhem f o r 30s. at 4*C. A l l subsequent o p e r a t i o n s were c a r r i e d out a t 4°C, e x c e p t where i n d i c a t e d . The h o m o g e n i z a t i o n procedure i s based on methods shown to produce mi t o c h o n d r i a with good r e s p i r a t o r y q u o t i e n t s , suggesting that o r g a n e l l e i n t e g r i t y i s r e t a i n e d . T h i s i s important s i n c e i t i n d i c a t e s that lysosomes, a m a j o r s o u r c e o f p r e o t e a s e s may a l s o r e m a i n i n t a c t . The homogenate was c e n t r i f u g e d at 3,000*g f o r 10 min. The supernatant was c o l l e c t e d and f i l t e r e d t h r o u g h 4 l a y e r s of c h e e s e c l o t h to remove the f l o a t i n g l i p i d l a y e r which appears at the su r f a c e of the tubes a f t e r c e n t r i f u g a t i o n , then c e n t r i f u g e d at 25,000xg f o r 27 25 min, to p e l l e t the " m i t o c h o n d r i a l " f r a c t i o n . The s u p e r n a t a n t was d e c a n t e d o f f and f i l t e r e d as p r e v i o u s l y d e s c r i b e d . T h i s f r a c t i o n was then c e n t r i f u g e d f o r 60 min a t 125,000/g to p e l l e t the " m i c r o s o m a l " f r a c t i o n . The s u p e r n a t a n t was c o l l e c t e d and designated the c y t o s o l i c f r a c t i o n . Samples (1ml) of both the high speed p e l l e t (resuspended i n e x t r a c t i o n b u f f e r ) and the c y t o s o l i c f r a c t i o n were r e t a i n e d f o r assay of ACC a c t i v i t y . 1.2 Sucrose D e n s i t y G r a d i e n t S t u d i e s . 1.2.1 S u c r o s e Cushions. The c y t o s o l i c f r a c t i o n was incubated f o r 30min at 30 C with 10mM c i t r a t e and b o v i n e serum a l b u m i n (BSA, 3mg/ml) to e n s u r e complete p o l y m e r i z a t i o n and to s t a b i l i z e the polymerized form of the enzyme. A f t e r the i n c u b a t i o n , 7ml samples were measured i n t o p o l y c a r b o n a t e c e n t r i f u g e tubes and 2ml of 20%, 30% or 40% s u c r o s e s o l u t i o n s of e x t r a c t i o n b u f f e r was i n j e c t e d by s y r i n g e below the sample t h r o u g h a Y a l e h y p o d e r m i c n e e d l e (17G, 3.5, r e g u l a r b e v e l ) , p r o d u c i n g a s u c r o s e c u s h i o n . The samples were c e n t r i f u g e d a t 100,000*g f o r 90 min a t 15*C. S u c r o s e c u s h i o n , i n t e r f a c e and supernatant f r a c t i o n s were obtained by removing two 2ml samples and a 5ml sample c o n s e c u t i v e l y from the bottom of the tube u s i n g the s y r i n g e . The p e l l e t was r e s u s p e n d e d i n 2ml of e x t r a c t i o n b u f f e r and a l l f r a c t i o n s were a s s a y e d f o r ACC a c t i v i t y and t h e r e s u l t s a r e shown i n t a b l e 1. T h i s d a t a demonstrates that i n c r e a s i n g the d e n s i t y of the sucrose cushion 28 TABLE 1. E f f e c t of a S i n g l e S u c r o s e D e n s i t y C u s h i o n on the D i s t r i b u t i o n of ACC A c t i v i t y During High-Speed C e n t r i f u g a t i o n ACETYL CoA CARBOXYLASE ACTIVITY (% OF TOTAL ACTIVITY) FRACTION 20% SUCROSE 30% SUCROSE 40% SUCROSE SUPERNATANT INTERFACE 6.1 14.4 17.4 SUCROSE CUSHION 22.7 23.8 24.4 PELLET 71.2 61.8 56.2 125,000 xg s u p e r n a t a n t f r e s h l y p r e p a r e d from r a t l i v e r was i n c u b a t e d a t 30°C f o r 30min to ens u r e p o l y m e r i z a t i o n of ACC. F r a c t i o n s were subsequently l a y e r e d above sucrose cushions of the i n d i c a t e d c o n c e n t r a t i o n s . F o l l o w i n g c e n t r i f u g a t i o n f o r 90min at 130,000xg (15°C), samples were removed f o r assay of ACC a c t i v i t y . R e s u l t s a r e e x p r e s s e d as a p e r c e n t a g e of the t o t a l a c t i v i t y a p p l i e d to each tube. 2 9 from 20%-40% r e d u c e s p r e c i p i t a t i o n of the enzyme a c t i v i t y as a p e l l e t by 15%, with a concurrent i n c r e a s e of a c t i v i t y appearing a t the i n t e r f a c e . In the absence of s u c r o s e a t l e a s t 80% of the ACC a c t i v i t y o c c u r s i n the p e l l e t . R e s o l u b i l i z a t i o n of t h i s a c t i v i t y however r e s u l t s i n a very low recovery of the enzyme. 1.2.2 Sucrose Step G r a d i e n t s . The c y t o s o l i c f r a c t i o n was i n c u b a t e d as d e s c r i b e d above to produce the polymerized form of the enzyme. Samples (19ml) were measured i n t o polycarbonate c e n t r i f u g e tubes and 3ml each of 30% f o l l o w e d by 60% s u c r o s e s o l u t i o n s of e x t r a c t i o n b u f f e r were i n j e c t e d below the sample as p r e v i o u s l y d e s c r i b e d . In p r e l i m i n a r y experiments the samples were c e n t r i f u g e d at 115,000xg f o r 90 min at 25"C. S u c r o s e f r a c t i o n s were c o l l e c t e d by removing 5.5ml samples from the bottom of each tube. T h i s f r a c t i o n i n c l u d e s the 30%/60% i n t e r f a c e l a y e r , but the s u p e r n a t a n t / 3 0 % i n t e r f a c e i s i n c l u d e d i n the s u p e r n a t a n t f r a c t i o n . The s u p e r n a t a n t and the s u c r o s e f r a c t i o n were a s s a y e d f o r ACC a c t i v i t y . T h i s type of e x p e r i m e n t was r e p e a t e d w i t h d i f f e r e n t r a t i o s of s u c r o s e c o n c e n t r a t i o n s i n the step g r a d i e n t , and with c e n t r i f u g a t i o n at v a r i o u s speeds, d u r a t i o n s and temperatures i n order to o p t i m i z e the recovery of a c t i v i t y i n the sucrose f r a c t i o n . The r e s u l t s are shown i n T a b l e 2. The h i g h e s t r e c o v e r y of a c t i v i t y was o b t a i n e d u s i n g 2ml of 25% s u c r o s e and 3ml of 60% s u c r o s e i n the s t e p g r a d i e n t . Optimum c e n t r i f u g a t i o n c o n d i t i o n s were 130,000Xg f o r 90 min a t 25°C. Under t h e s e c o n d i t i o n s , more than 80% of the ACC 30 TABLE 2 E f f e c t of V a r i a t i o n of Composition of Sucrose Cushions on the D i s t r i b u t i o n of ACC A c t i v i t y d u r i n g High-Speed C e n t r i f u g a t i o n ULTRA- SUCROSE LAYERS (ml) VOLUME OF SUCROSE ACC ACTIVITY (%) CENT. 25% 30% 60% FRACTION (ml) SUPT. SUCROSE (xg) FRACTION 120,000 3 — 3 120,000 — 3 3 125,000 — 2 2 130,000 2 — 3 5.5 47 53 5.5 34 66 3.5 27 73 4.5 18 82 125,000 *g s u p e r n a t a n t f r e s h l y p r e p a r e d from r a t l i v e r was i n c u b a t e d a t 30°C f o r 30min to ensure p o l y m e r i z a t i o n of ACC. 18ml f r a c t i o n s were a l i q u o t e d to each c e n t r i f u g e tube and s u c r o s e step g r a d i e n t s were prepared as d e s c r i b e d i n the text. F o l l o w i n g c e n t r i f u g a t i o n at the i n d i c a t e d speed (90min, 25°C), samples were removed f o r a s s a y of ACC a c t i v i t y . R e s u l t s are e x p r e s s e d as a percentage of the t o t a l a c t i v i t y a p p l i e d to each tube. N e g l i g i b l e a m o unts o f p r o t e i n p r e c i p i t a t e d u n d e r t h e s e c o n d i t i o n s , c o n t a i n i n g l e s s than 1% of the t o t a l ACC a c t i v i t y a p p l i e d . 31 a c t i v i t y migrates i n t o the sucrose f r a c t i o n and the t o t a l amount of p r o t e i n a s s o c i a t e d w i t h ACC a c t i v i t y i n t h i s f r a c t i o n i s reduced to 36%. A s m a l l amount of p r o t e i n p e l l e t e d at the bottom of the tubes under these c o n d i t i o n s but contained l e s s than 1% of the t o t a l c y t o s o l i c ACC a c t i v i t y . Above 130,000*g the p r o p o r t i o n of ACC p r e c i p i t a t e d became s i g n i f i c a n t and was d i f f i c u l t to recover as d i s c u s s e d above. 1.3 Enzyme P r e c i p i t a t i o n S t u d i e s . 1.3.1 Ammonium Sulphate. The c y t o s o l i c f r a c t i o n was f r a c t i o n a t e d by s u c r o s e d e n s i t y g r a d i e n t c e n t r i f ugation under the optimum c o n d i t i o n s d i s c u s s e d above. The s u c r o s e f r a c t i o n was d i l u t e d 2 - f o l d and the ACC a c t i v i t y i n the sucrose f r a c t i o n was p r e c i p i t a t e d by the dropwise a d d i t i o n o f an ammonium s u l p h a t e s o l u t i o n u n t i l a f i n a l c o n c e n t r a t i o n of 35% ammonium sulphate was reached (2g/10ml). The a d d i t i o n was made over 20 min w h i l s t s t i r r i n g on i c e , and the pH m a i n t a i n e d a t 7.0. The s o l u t i o n was l e f t on i c e f o r a f u r t h e r 2 hr to e n s u r e c o m p l e t e p r e c i p i t a t i o n . I n s o l u b l e p r o t e i n was p e l l e t e d by c e n t r i f u g a t i o n ( 25,000Xg, 20 m i n , 4 °C) and r e s u s p e n d e d i n b u f f e r A (20mM MOPS; pH 7.2, ImM EDTA, 10mM c i t a t e , 5mM 2 - m e r c a p t o e t h a n o l ) . D e s p i t e the r e m o v a l of some p r o t e i n by c e n t r i f u g a t i o n of the c y t o s o l i c f r a c t i o n on s u c r o s e c u s h i o n s , the p r o t e i n p e l l e t p r o d u c e d by ammonium s u l p h a t e p r e c i p i t a t i o n was e x t r e m e l y d i f f i c u l t to r e s o l u b i 1 i z e . The p r o t e i n which remained i n s o l u b l e was removed by c e n t r i f u g a t i o n 32 (10,000xg, 10 min, 4'C) and the s u p e r n a t a n t f r a c t i o n d i a l y s e d a g a i n s t b u f f e r A f o r 6 hr p r i o r to a s s a y of ACC a c t i v i t y . The recovery of ACC a c t i v i t y using t h i s procedure represented 20% of the a c t i v i t y o r i g i n a l l y i n the c y t o s o l i c f r a c t i o n . The presence of i n d i v i d u a l p r o t e a s e i n h i b i t o r s (or a c o m b i n a t i o n t h e r e - o f , i n c l u d i n g p e p s t a t i n 2ug/ml, benzamidine 2mM and b a c i t r a c i n ImM ) d i d not s t a b i l i z e the enzyme a c t i v i t y i n the ammonium sulphate p e l l e t s . T h i s i n d i c a t e s that p r o t e o l y s i s during and a f t e r sucrose d e n s i t y g r a d i e n t f r a c t i o n a t i o n and ammonium s u l p h a t e p r e c i p i t a t i o n i s n o t t h e e x p l a n a t i o n o f p o o r r e c o v e r y . I n c i d e n t a l l y t h e s e r e c o v e r i e s a re not u n l i k e t h o s e r e p o r t e d i n other s t u d i e s of ACC p u r i f i c a t i o n from r a t l i v e r . 1.3.2 P o l y e t h y l e n e g l y c o l 6000 (PEG). The sucrose cushion f r a c t i o n prepared as d e s c r i b e d above (1.2.2), was d i l u t e d 3 - f o l d with e x t r a c t i o n b u f f e r , then made 3% (w/v) i n PEG by the dropwise a d d i t i o n of a 50% s o l u t i o n of PEG , over 20 min w h i l s t s t i r r i n g on i c e . The pH o f t h e s o l u t i o n was c o n t i n u a l l y adjusted to pH 7.2 durin g the a d d i t i o n of PEG and the s o l u t i o n was then l e f t on i c e f o r 1 hr to o p t i m i z e p r o t e i n p r e c i p i t a t i o n . The p r e c i p i t a t e d p r o t e i n was c o l l e c t e d by c e n t r i f u g a t i o n (25,000Xg 5min, 4 °C) and the s u p e r n a t a n t was removed and f u r t h e r t r e a t e d i n a s i m i l a r f a s h i o n to produce 6% and 10% PEG f r a c t i o n s . The p e l l e t s were resuspended i n a minimum volume of b u f f e r A and assayed f o r ACC a c t i v i t y ( t a b l e 3). The 3-6% PEG p r o t e i n p e l l e t c o n t a i n e d 54% of the t o t a l ACC a c t i v i t y 33 TABLE 3. P r e c i p i t a t i o n of ACC A c t i v i t y from Rat L i v e r C y t o s o l with P o l y e t h y l e n e G l y c o l FRACTION ACC ACTIVITY ACC ACTIVITY (mU) (% OF TOTAL) CYTOSOLIC FRACTION 8237 100 SUCROSE FRACTION 7968 96 3 %-SUPERNATANT 7223 87 3% PELLET 570 7 6%-SUPERNATANT ND ND 6% PELLET 4470 54 10%-SUPERNATANT 10% PELLET 93 1 ACC a c t i v i t y was p r e c i p i t a t e d from a f r e s h l y prepared sample of r a t l i v e r c y t o s o l by the drop-wise a d d i t i o n of a 50% s o l u t i o n of p o l y e t h y l e n e g l y c o l , w h i l s t s t i r r i n g on i c e . S u p e r n a t a n t and p e l l e t f r a c t i o n s were c o l l e c t e d and assayed f o r ACC a c t i v i t y . ND - Not determined 3 4 p r o t e i n appeared to have undergone l i m i t e d p r o t e o l y s i s , as the s u b u n i t Mr appeared to d e c r e a s e from 230Kd to 215Kd ( F i g . 1. ). As w i t h t h e ammonium s u l p h a t e p r e c i p i t a t i o n t e c h n i q u e , r e s o l u b i l i z a t i o n of the p r o t e i n p e l l e t s proved to be p r o b l e m a t i c . A b r i e f c e n t r i f u g a t i o n (25,000g, 10 min, 4°C ) p r e c i p i t a t e d the i n s o l u b l e p r o t e i n , b u t t h e ACC a c t i v i t y r e m a i n i n g i n t h e supernatant was reduced to 40% of the o r i g i n a l a c t i v i t y c o l l e c t e d i n the r e s p e c t i v e PEG f r a c t i o n s . 1.4. DEAE-Cellulose Ion Exchange Chromatography. The previous s t u d i e s e s t a b l i s h e d that p r e c i p i t a t i o n of the enzyme both at an e a r l y stage of the p r e p a r a t i o n by d i r e c t p r e c i p i t a t i o n of the c y t o s o l i c f r a c t i o n , or a f t e r f u r t h e r p a r t i a l p u r i f i c a t i o n on s u c r o s e d e n s i t y s t e p g r a d i e n t s r e s u l t e d i n poor r e c o v e r y of ACC a c t i v i t y . I t t h e r e f o r e seemed a p p r o p r i a t e to a v o i d p r e c i p i t a t i o n t e c h n i q u e s i n the i s o l a t i o n p r o c e d u r e i f a t a l l p o s s i b l e . The enzyme must be i n s p e c i f i c c o n d i t i o n s f o r a p p l i c a t i o n to a v i d i n - s e p h a r o s e : 1) A l l the c i t r a t e must be removed from the sample to a l l o w ACC to bind onto the column as c i t r a t e b i n d s a l l o s t e r i c a l l y to ACC c a u s i n g c o n f o r m a t i o n a l changes i n the q u a r t e r n a r y s t r u c t u r e which p r e v e n t s a c c e s s of a v i d i n to the b i o t i n b i n d i n g s i t e . 2) I t i s p r e f e r a b l e to have the sample i n as s m a l l a volume as p o s s i b l e to reduce the sample a p p l i c a t i o n time onto the column and so reduce the length o f t i m e t h e enzyme i s e x p o s e d to p r o t e a s e a c t i v i t y u n d e r d e s t a b i l i z i n g c o n d i t i o n s . 3) I t i s b e n e f i c i a l to apply the sample 35 F i g . 1. P r o t e o l y t i c D e g r a d a t i o n of ACC d u r i n g P r e c i p i t a t i o n  with PEG Approximately 20-30ug of p r o t e i n from each sample were subjected to e l e c t r o p h o r e s i s on a 5% p o l y a c r y l a m i d e g e l which was subsequently s t a i n e d with Coomassie Blue. F r a c t i o n s a p p l i e d were (a) 125,000Xg s u p e r n a t a n t f r e s h l y p r e p a r e d from r a t l i v e r (b) p r o t e i n c o l l e c t e d as s u c r o s e c u s h i o n f r a c t i o n (c) p r o t e i n from f r a c t i o n (b) p r e c i p i t a t e d i n the presence of 6% PEG 3 6 i n a h i g h s a l t b u f f e r so as to reduce n o n - s p e c i f i c b i n d i n g o f c o n t a m i n a t i n g p r o t e i n s to the a f f i n i t y column. One of s e v e r a l a p p r o p r i a t e t e c h n i q u e s I c o n s i d e r e d as a r e p l a c e m e n t f o r the p r e c i p i t a t i o n s t e p was DEAE-52 c e l l u l o s e i o n - e x c h a n g e c h r o m a t o g r a p h y , s i n c e t h i s has been a p p l i e d p r e v i o u s l y i n p u r i f i c a t i o n of ACC. The s u c r o s e f r a c t i o n was d i l u t e d 2 - f o l d w i t h b u f f e r A ( pH 7.5), to l o w e r the s u c r o s e c o n c e n t r a t i o n to a p p r o x i m a t e l y 30%. T h i s d i l u t e d f r a c t i o n was then i n c u b a t e d f o r 30 min w h i l s t s t i r r i n g g e n t l y on i c e , with D E A E - c e l l u l o s e which had been pre-e q u i l i b r i a t e d with b u f f e r A (pH 7.5). The s l u r r y was then poured i n t o a column (20 cm x 1.5 cm) and washed w i t h 5 column volumes o f b u f f e r A (pH 7.5 ) to remove a l l the unbound p r o t e i n . T h i s f r a c t i o n was designated the DEAE-low s a l t f r a c t i o n (DEAE-LS). The column was then e l u t e d w i t h b u f f e r A (pH 7.5) c o n t a i n i n g 500mM KC1; the washing and e l u t i n g b u f f e r s contained no added c i t r a t e . The h i g h s a l t e l u a t e was c o l l e c t e d as a s i n g l e f r a c t i o n and designated DEAE-HS. Both f r a c t i o n s were assayed f o r ACC a c t i v i t y ; the low s a l t f r a c t i o n c o n t a i n e d v i r t u a l l y no d e t e c t i b l e ACC a c t i v i t y , and SDS-PAGE i n d i c a t e s no d e t e c t i b l e 230Kd p r o t e i n band i n t h i s f r a c t i o n . The h i g h s a l t f r a c t i o n c o n t a i n e d a t l e a s t 60% of the t o t a l a c t i v i t y loaded onto the column. SDS-polyacrylamide g e l s o f t h e f r a c t i o n s p r o d u c e d by t h e i o n - e x c h a n g e chromatography f r a c t i o n a t i o n i n d i c a t e d that a l l of the 230Kd band i n t h e s a m p l e ( p r o b a b l y a m i x t u r e o f ACC and F a t t y - a c i d S y n t h e t a s e ) b i n d s t o D E A E - c e l l u l o s e u n d e r t h e c o n d i t i o n s d e s c r i b e d ( F i g 2.) and i s e l u t e d i n the DEAE-HS f r a c t i o n . T h i s 37 a b _250 _225 Fig.2. B i n d i n g of ACC to DEAE-Cellulose ACC p u r i f i e d f r o m r a t l i v e r t h r o u g h s u c r o s e d e n s i t y c e n t r i f u g a t i o n was a p p l i e d to DEAE-cellulose. Samples (20-30ug) of p r o t e i n e l u t e d a t low s a l t (a) or i n the p r e s e n c e of 0.5M KC1 (b) were a p p l i e d to 5% p o l y a c r y l a m i d e g e l and the g e l was f i x e d and p r o t e i n s v i z u a l i s e d w i t h s i l v e r s t a i n . V a l u e s ( X 1 0 ? ) i n d i c a t e m i g r a t i o n of the heavy c h a i n s of human e r y t h r o c y t e s p e c t r i n . 38 DEAE f r a c t i o n a t i o n step r e s u l t s i n a 3 - f o l d p u r i f i c a t i o n of ACC which e l u t e s i n approximately 20ml. Both the c i t r a t e and sucrose i n the i n i t i a l a p p l i e d sample are washed d i r e c t l y t h r o u g h the column, a p p e a r i n g w i t h the unbound p r o t e i n i n the DEAE-LS f r a c t i o n . E l u t i o n w i t h h i g h s a l t b u f f e r l e a v e s the enzyme i n i d e a l c o n d i t i o n s f o r a p p l i c a t i o n to a v i d i n - s e p h a r o s e . 1.5 Avidin-Sepharose A f f i n i t y Chromatography. The f i n a l s t e p of the p u r i f i c a t i o n p r o c e d u r e i n v o l v e s a v i d i n -s e p h a r o s e a f f i n i t y c h r o m a t o g r a p h y . The r e s i n i s e q u i 1 i b r i a t e d w i t h b u f f e r B ( T r i s - H c l 100mM; pH 7.5, g l y c e r o l 5%, EDTA ImM, 2 - m e r c a p t o e t h a n o l ;5mM, p e p s t a t i n 2ug/ml, l e u p e p t i n 2ug/ml, sodium a z i d e 0.02%). The DEAE-HS f r a c t i o n was i n c u b a t e d w i t h 15ml of a v i d i n - s e p h a r o s e ( b i o t i n b i n d i n g c a p a c i t y : 18mg ) f o r 30min w h i l s t s t i r r i n g on i c e . The s l u r r y was poured i n t o a column (10cm * 1.5cm) and washed w i t h b u f f e r B u n t i l a l l the unbound p r o t e i n had e l u t e d (as d e t e c t e d by the B r a d f o r d a s s a y ) . The ACC a c t i v i t y was e l u t e d from the column w i t h b u f f e r B c o n t a i n i n g 0.8mM b i o t i n . A t y p i c a l e l u t i o n p r o f i l e from the a v i d i n - s e h p a r o s e a f f i n i t y c o l u m n i s shown i n F i g . 3. A l l f r a c t i o n s c o n t a i n i n g p r o t e i n were pooled and 5mM c i t r a t e added to s t a b i l i z e t h e p r e p a r a t i o n . The p r o t e i n c o n c e n t r a t i o n was i n c r e a s e d to 0.5-1.0 mg/ml by r e d u c i n g the volume of the p o o l e d f r a c t i o n s . The p o o l e d samples were t r a n s f e r r e d to d i a l y s i s t u b i n g , c o v e r e d w i t h a q u a c i d e 111 a f l a k e form of p o l y e t h y l e n e g l y c o l which i s h i g h l y h y g r o s c o p i c , and the volume reduced to 39 . 3 F R A C T I O N NUMBER F i g . 3 . A v i d i n - s e p h a r o s e A f f i n i t y C hromatography of Rat L i v e r  C y t o s o l i c A c e t y l CoA Carboxylase. The DEAE-HS f r a c t i o n (30ml) was a p p l i e d to an a v i d i n - s e p h a r o s e a f f i n i t y column (1.5 cm x 10cm) The column was washed w i t h 8 volumes of b u f f e r i n t o 5ml f r a c t i o n s (1-23), to remove a l l unbound p r o t e i n . Subsequent e l u t i o n was c a r r i e d out w i t h 40ml of b u f f e r c o n t a i n i n g 0.8mM b i o t i n i n t o 2ml f r a c t i o n s (24-35). 40 0.5ml. The concentrated ACC p r e p a r a t i o n was then d i l u t e d i n t o 3-4ml of b u f f e r A, pH 7.2 to reduce the s a l t c o n c e n t r a t i o n to more p h y s i o l o g i c a l l e v e l s . The enzyme was s t o r e d a t -70°C i n 100ul a l i q u o t s . T h i s f r a c t i o n shows a 735-fold p u r i f i c a t i o n of ACC a c t i v i t y compared to the i n i t i a l c y t o s o l i c f r a c t i o n and c o n t a i n s a major p r o t e i n band of 230Kd on a s i l v e r s t a i n e d denaturing p o l y a c r y l a m i d e g e l (Fig. 4.). The s p e c i f i c a c t i v i t y of the sample i s about 2 U/mg p r o t e i n and the Km f o r a c e t y l - C o A i s 80uM. The p r o t e i n a c t i v i t y and subunit s t r u c t u r e i s s t a b l e at 70°C f o r a t l e a s t 2 months. The o v e r a l l p u r i f i c a t i o n scheme i s shown i n F i g . 5. and t y p i c a l y i e l d s f o r each step are presented i n Table 4. 41 _250 .225 F i g . 4. Appearanee of P u r i f i e d ACC F o l l o w i n g Sodium dodecy1  s u l p h a t e - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s . Approximately 10ug of the ACC p r e p a r a t i o n was s u b j e c t e d e l e c t r o p h o r e s i s on a 5% p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s . The g e l was f i x e d and s t a i n e d w i t h s i l v e r s t a i n . V a l u e s (*103 ) r e p r e s e n t m i g r a t i o n , of the molecular weight marker s p e c t r i n . 42 RAT LIVER • HOMOGENIZATION t 3,000 xg-SPIN, 25,000 Xg-SPIN, 125,000 *g-SPIN (4 °C) 130,000Xg-SPIN (25 *C, SUCROSE STEP GRADIENT, 10mM CITRATE) • DEAE-52 CELLULOSE 500mM K c l ELUANT AVIDIN-SEPHAROSE BIOTIN ELUANT F i g . 5. P u r i f i c a t i o n Scheme f o r Rat L i v e r A c e t y l - C o A Carboxylase. 43 TABLE 4. P u r i f i c a t i o n of Acetyl-CoA Carboxylase from Rat L i v e r C y t o s o l . TOTAL SPECIFIC FRACTION PROTEIN ACTIVITY ACTIVITY PURIFICATION YIELD (mg) (mU) (mU/mg) (f o l d ) (%) CYTOSOL 5525 15458 2.8 X 10 1.0 100 (125,000 * g) 60% SUCROSE 2093 12650 6.1 x 10"* 2.2 82 DEAE-52 1128 9872 8.8 % 10 _ 5 3.1 64 BIOTIN 2.67 5874 2.2 785 38 The t a b l e presents the mean values f o r 5 d i f f e r e n t p r e p a r a t i o n s , beginning with 5 or 6 r a t l i v e r s . 44 2. I n v e s t i g a t i o n s of P r o t e i n K i n a s e A c t i v i t y i n Rat L i v e r  C y t o s o l . ACC i s known to be phosphorylated both in v i t r o , i n i n t a c t t i s s u e and i^n v i v o . P h o s p h o r y l a t i o n / d e p h o s p h o r y l a t i o n m e c h a n i s m s a r e t h o u g h t to p l a y a r o l e i n t h e s h o r t t e r m r e g u l a t i o n of ACC a c t i v i t y which occurs i n response to hormonal s t i m u l i . S i n c e ACC i s p h o s p h o r y l a t e d a t a number of s i t e s , the a n a l y s i s of the p h o s p h o r y l a t i o n of ACC has i n v o l v e d 2-dimensional p e p t i d e m a p p i n g , a t e c h n i q u e f o r s e p a r a t i n g l a b e l l e d p h o s p h o p e p t i d e s of ACC a f t e r t o t a l t r y p s i n h y d r o l y s i s . I t was a n t i c i p a t e d t h a t the l a b e l l e d p h o s p h o p e p t i d e s of r a t l i v e r ACC would f a l l i n t o t h r e e main gr o u p s , d e s i g n a t e d C, A, I . "C" r e f e r s to the c o n t r o l s i t e s which appear p h o s p h o r y l a t e d to a c e r t a i n degree i n a l l circumstances, yet appear to have no e f f e c t on c a t a l y t i c a c t i v i t y e i t h e r i n crude e x t r a c t s or i n the p u r i f i e d s y s t e m . "A" r e f e r s to the a d r e n a l i n e or c y c l i c AMP-dependent p r o t e i n k i n a s e r e s p o n s i v e s i t e s and " I " r e f e r s to the i n s u l i n r e s p o n s i v e s i t e . In f a c t a f o u r t h group of p h o s p h o r y l a t e d s i t e s have a l s o been observed (designated the "B" group), and are s i t e s which are phoshporylated upon i n c u b a t i o n with c e r t a i n p a r t i a l l y p u r i f i e d p r o t e i n k i n a s e f r a c t i o n s from r a t l i v e r c y t o s o l ( F i g . 6.). The B s i t e s have not p r e v i o u s l y been i d e n t i f i e d as b e i n g p h o s p h o r y l a t e d i n r e s p o n s e to hormonal s t i m u l i w i t h i n i n t a c t c e l l s , and the e f f e c t of i n c r e a s e d phosphate i n c o r p o r a t i o n i n t o these s i t e s on c a t a l y t i c a c t i v i t y r e q u i r e s f u r t h e r i n v e s t i g a t i o n . The s t u d i e s d e s c r i b e d below w i l l show t h a t p r o t e i n k i n a s e 45 group C group A group A group I group B w j or ig in i I 0 1 E l e c t r o p h o r e s i s r e l a t i v e t o D N P - l y s i n e F i g . 6. S c h e m a t i c d i a g r a m of a u t o r a d i o g r a p h , showing [32p]_ l a b e l l e d p h o s p h o p e p t i d e s r e l e a s e d by t r y p s i n d i g e s t i o n of ACC i n d i c a t i n g the r e l a t i v e m o b i l i t y of groups A, I, C and B as d e s c r i b e d i n t h e t e x t . The p e p t i d e s a r e s e p a r a t e d by 2-dimensional t h i n - l a y e r mapping. 4 6 a c t i v i t y c a p a b l e o f p h o s p h o p e p t i d e s have c y t o s o l . p h o s p h o r y 1 a t i n g C, B and I been p a r t i a l l y p u r i f i e d from g r o u p s of r a t l i v e r 2.1 Endogenous kinase a c t i v i t y of p u r i f i e d ACC. 2.1.1. C h a r a c t e r i z a t i o n of Endogenous P r o t e i n K i n a s e A c t i v i t y . I n c u b a t i o n of p u r i f i e d ACC w i t h [J-32p]-MgATP and s p e c i f i c (Walsh) i n h i b i t o r of c y c l i c AMP-dependent p r o t e i n k i n a s e , r e s u l t s i n p h o s p h o r y l a t i o n of the 230Kd s u b u n i t . The maximum i n c o r p o r a t i o n of phosphate under these c o n d i t i o n s occurs w i t h i n a p p r o x i m a t e l y l h r at 30 °C . The i n c o r p o r a t i o n e s t i m a t e d by s c a n n i n g d e n s i t o m e t r y and g e l h y d r o l y s i s f o l l o w e d by l i q u i d s c i n t i l l a t i o n c o u n t i n g was 0.1mol [32p]/mol 230Kd ACC s u b u n i t . C y c l i c AMP-dependent p r o t e i n kinase a c t i v i t y can be s t i m u l a t e d i n t h i s a s s a y by o m i t t i n g the i n h i b i t o r p r o t e i n and a d d i n g c y c l i c AMP (5-10uM). Under t h e s e c o n d i t i o n s , the i n c o r p o r a t i o n of phosphate i n t o ACC i s increased 5 - f o l d . A d d i t i o n of cyclic-AMP causes i n c r e a s e d i n c o r p o r a t i o n i n t o the major 230Kd band of ACC and the minor 240Kd p r o t e i n band. Q u a n t i t a t i v e l y the minor s p e c i e s of the enzyme a p p e a r s to a c t as a b e t t e r s u b s t r a t e f o r the c y c l i c AMP-dependent p r o t e i n kinase a c t i v i t y than the major s p e c i e s s i n c e i n c o r p o r a t i o n of [32p] i s s i m i l a r i n t o the two bands, d e s p i t e the p r o t e i n r a t i o being 9:1 i n favour of the 230Kd s p e c i e s . A d d i t i o n of p u r i f i e d c y c l i c AMP-dependent p r o t e i n kinase c a t a l y t i c s u b - u n i t to t h e a s s a y , d o e s n o t i n c r e a s e t h e i n c o r p o r a t i o n of phosphate above that produced by the endogenous 47 c y c l i c AMP-dependent p r o t e i n k i n a s e a c t i v i t y . Other p o t e n t i a l e f f e c t o r s o f p r o t e i n k i n a s e a c t i v i t y t e s t e d i n c l u d e d p h o s p h o l i p i d , c a l c i u m , p h o s p h a t i d y l c h o l i n e , d i a c y l g l y c e r o l and c a l c i u m (requirements f o r assaying p r o t e i n kinase C a c t i v i t y ), coenzyme A, c i t r a t e , albumin, detergent and polyamines. Of these o n l y t h e p o l y a m i n e s had any s i g n i f i c a n t e f f e c t on t h e s t o i c h i o m e t r y of i n c o r p o r a t i o n , producing a 2-3 f o l d s t i m u l a t i o n of the c y c l i c AMP-independent p r o t e i n kinase a c t i v i t y ( F i g . 7., T a b l e 5.). 2.1.2. 2 - D i m e n s i o n a 1 P e p t i d e A n a l y s i s o f P r o t e i n K i n a s e A c t i v i t i e s Present i n P u r i f i e d P r e p a r a t i o n s of ACC. Further a n a l y s i s of the c h a r a c t e r i s t i c s of the endogenous p r o t e i n k i n a s e a c t i v i t y by 2 - d i m e n s i o n a l p e p t i d e m a p p i n g o f t h e p h o s p h o p e p t i d e s i n d i c a t e d t h a t c y c l i c AMP-independent p r o t e i n k i n a s e a c t i v i t y l e d to the i n c o r p o r a t i o n of the [32p] l a b e l p r i m a r i l y i n t o the c o n t r o l s i t e s of ACC. T o t a l r e c o v e r y of r a d i o a c t i v e phosphate on the TLC p l a t e s r e f l e c t e d the i n c r e a s e i n i n c o r p o r a t i o n s t i m u l a t e d by the a d d i t i o n o f c y c l i c AMP. Under these c o n d i t i o n s , i n c o r p o r a t i o n i n t o both the c o n t r o l s i t e s and the A s i t e s was i n c r e a s e d (as d e s i g n a t e d a c c o r d i n g to F i g . 6.). A u t o r a d i o g r a p h s of the 2 - d i m e n s i o n a l t r y p t i c p e p t i d e maps are p r e s e n t e d i n F i g . 8. 48 a b c d e f g h _ .250 • ».225 F i g . 7. C h a r a c t e r i z a t i o n of the Endogenous P r o t e i n K i n a s e  A c t i v i ty A s s o c i a t e d with P u r i f i e d P r e p a r a t i o n s of ACC. P r o t e i n k i n a s e a c t i v i t y was a s s a y e d b~y the s t a n d a r d p r o c e d u r e w i t h f u r t h e r a d d i t i o n s as f o l l o w s : (a) no a d d i t i o n s , (b) c i t r a t e 10mM and a l b u m i n 5mg/ml. (c) 3 ' - 5 ' - c y c l i c AMP 10uM. (d) as (c) p l u s p u r i f i e d c y c l i c AMP-dependent p r o t e i n k i n a s e (2 u n i t s ) , (e) ph o s p h a t i d y l c h o l i n e 40ug/ml. (f) spermine 5mM. (g) 2mM EGTA. (h) ca l c i u m 10uM (as balanced Calcium/EGTA b u f f e r ) . 49 TABLE 5. C h a r a c t e r i z a t i o n of Endogenous P r o t e i n Kinase A c t i v i t y i n P u r i f i e d P r e p a r a t i o n s of Rat L i v e r ACC PROTEIN KINASE ACTIVITY (U/ug ACC) PURIFIED ACC PREPARATION 13.4 + 0.5 + CYCLIC AMP ( 1 0 U M ) 52.9 + 1.2 + COENZYME A (25uM) 18.3 + 1.9 + CALCIUM (20uM) 9.8 + 0.4 + SPERMINE (ImM) 32.2 + 2.1 + DETERGENT (.02% NP-40) 8.2 + 2.9 + PS DG CALCIUM 20.0 + 0.8 + PC (50ug/ml) 20.8 + 1.8 P u r i f i e d ACC was incubated w i t h [ "tf -32p]-ATP with the a p p r o p r i a t e a d d i t i o n s and then the samples were a p p l i e d to SDS-polyacrylamide g e l s (5% w/v ac r y l a m i d e ) . The valu e s represent i n c o r p o r a t i o n of [32p] i n t o ACC a f t e r 30min i n c u b a t i o n at 30 °C. and are presented as a r b i t r a r y u n i t s measured by densitometry of autoradiographs. A b r e v i a t i o n s a r e u s e d f o r p h o s p h a t i d y 1 s e r i n e ( P S ) , p h o s p h a t i d y l c h o l i n e ( P C ) , d i a c y l g l y c e r o l (DG) and n o n i d e t NP-40 d e t e r g e n t (NP-40). 50 a P i g . 8. A u t o r a d i o g r a p h s O b t a i n e d from 2 - D i m e n s i o n a l T r y p t i c  P e p t i d e Maps of ACC. 30ug of p u r i f i e d ACC p r e p a r a t i o n was subjected to p h o s p h o r y l a t i o n f o l l o w e d by d i g e s t i o n with t r y p s i n and 2 - d i m e n s i o n a l a n a l y s i s , w i t h the f o l l o w i n g a d d i t i o n s ; (a) s p e c i f i c (Walsh) i n h i b i t o r of c y c l i c AMP-dependent p r o t e i n kinase, (b) c y c l i c AMP 10uM. 51 2.2 P h o s p h o r y l a t i o n of Acetyl-CoA Carboxylase by Exogenous  P r o t e i n Kinase P r e p a r a t i o n s . 2.2.1 D i s t r i b u t i o n of P r o t e i n Kinase A c t i v i t y i n F r a c t i o n s from Rat L i v e r . The d i s t r i b u t i o n of c y c l i c AMP-independent p r o t e i n k i n a s e a c t i v i t y i n the f r a c t i o n s p r o d u c e d d u r i n g the i s o l a t i o n of ACC, was determined using an i m m u n o p r e c i p i t a t i o n technique. ACC was phosphorylated by the standard procedure, then immunoprecipitated from the a s s a y m i x t u r e w i t h ACC a n t i s e r a and p r o t e i n A. The p e l l e t was washed thoroughly and then d i s s o l v e d i n SDS-digestion b u f f e r and h e a t e d a t 95°C f o r 5 min. P r o t e i n s were s e p a r a t e d by S D S - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s as d e s c r i b e d i n the methods s e c t i o n . The r e s u l t s of t h i s experiment are shown i n F i g . 9. T h i s i n d i c a t e s t h at the m a j o r i t y of p r o t e i n kinase a c t i v i t y i n r a t l i v e r c y t o s o l c o - p u r i f i e s with ACC i n t o the sucrose f r a c t i o n . A f t e r a p p l i c a t i o n of t h i s sample to D E A E - c e l l u l o s e however, the m a j o r i t y of the p r o t e i n kinase a c t i v i t y appears i n the low s a l t wash w i t h the r e s t of the unbound p r o t e i n . The p r o t e i n k i n a s e a c t i v i t y o c c u r i n g i n t h i s f r a c t i o n accounts f o r approximately 90% of the s o l u b l e c y t o s o l i c c y c l i c AMP-independent p r o t e i n k i n a s e a c t i v i t y i n r a t l i v e r u s i n g ACC as the phosphate a c c e p t o r . The r e m a i n i n g 10% c o - p u r i f i e s w i t h ACC t h r o u g h DEAE- c e l l u l o s e chromatography, but mostly e l u t e s with the unbound p r o t e i n a f t e r a v i d i n - s e p h a r o s e a f f i n i t y chromatography. 52 a b e d e f g • - • .250 .225 a F i g . 9. D i s t r i b u t i o n of P r o t e i n Kinase A c t i v i t y i n F r a c t i o n s of  Rat L i v e r . The f r a c t i o n s p roduced d u r i n g the i s o l a t i o n o f ACC were a s s a y e d f o r p r o t e i n k i n a s e a c t i v i t y by the a d d i t i o n o f 5ug of p u r i f i e d ACC f o l l o w e d by i n c u b a t i o n i n the p r e s e n c e o f [ -32p]-ATP. ACC was i s o l a t e d by i m m u n o p r e c i p i t a t i o n and subsequent S D S - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s . The f i g u r e shows an a u t o r a d i o g r a p h f o l l o w i n g i n c u b a t i o n o f ACC w i t h : - (a) " c y t o s o l i c " , 125,000Xg s u p e r n a t a n t f r a c t i o n (b) " m i c r o s o m a l " 125,000Xg p e l l e t (c) h i g h speed s u p e r n a t a n t (130,000*g, 25 6C) (d) s u c r o s e f r a c t i o n (e) DEAE-high s a l t f r a c t i o n (f) DEAE-low s a l t f r a c t i o n (g) avidin-sepharose v o i d f r a c t i o n 5 3 2.2.2. 2 - D i m e n s i o n a l P e p t i d e A n a l y s i s of ACC P h o s p h o r y l a t e d by P a r t i a l l y P u r i f i e d P r o t e i n Kinases from Rat L i v e r C y t o s o l . 2 - D i m e n s i o n a l a n a l y s i s of ACC p h o s p h o r y l a t i o n by f r a c t i o n s p r o d u c e d d u r i n g the i s o l a t i o n showed t h a t the p r o t e i n k i n a s e a c t i v i t y appearing i n the DEAE-LS f r a c t i o n and the p r o t e i n kinase a c t i v i t y w h ich c o - p u r i f i e d w i t h ACC t h r o u g h D E A E - c e l l u l o s e p h o s p h o r y l a t e d d i f f e r e n t s i t e s on ACC ( F i g . 10). The DEAE-LS f r a c t i o n s t i m u l a t e s i n c o r p o r a t i o n i n t o the c o n t r o l s i t e s , the A s i t e s and one of the phosphopeptides a s s o c i a t e d with the B group of p e p t i d e s . The DEAE-HS f r a c t i o n s t i m u l a t e s i n c o r p o r a t i o n p r i m a r i l y i n t o the B s i t e s . The dark s p o t s on the l e f t of the a u t o r a d i o g r a p h a r e caused by r e s i d u a l [Y-32p]-MgATP, and f r e e [32p]-phosphate. 2.3. S e p a r a t i o n of P r o t e i n K i n a s e s by H y d r o x y l A p a t i t e F r a c t i o n a t i o n Very l i t t l e i n c o r p o r a t i o n i n t o the I - s i t e could be detected upon t r e a t m e n t of ACC w i t h p r o t e i n k i n a s e a c t i v i t y of the DEAE-LS f r a c t i o n . As t h i s f r a c t i o n c o n t a i n s the m a j o r i t y of the c y t o s o l i c p r o t e i n kinase a c t i v i t y present i n l i v e r , i t was a n t i c i p a t e d t h at s u b s e q u e n t p u r i f i c a t i o n o f the p r o t e i n k i n a s e s may r e v e a l s i g n i f i c a n t I - s i t e p h o s p h o r y l a t i o n . The DEAE-LS f r a c t i o n was f r a c t i o n a t e d on a hydroxyl a p a t i t e column, using a s a l t g r a d i e n t e l u t i o n . The f r a c t i o n s p r o d u c e d by t h i s p r o c e d u r e were s u b s e q u e n t l y d i a l y s e d to reduce the s a l t c o n c e n t r a t i o n to approximately 50mM phosphate and then assayed f o r p r o t e i n kinase 54 t I F i g . 10. A u t o r a d i o g r a p h s O b t a i n e d from 2 - D i m e n s i o n a l P e p t i d e  Maps of Rat L i v e r ACC. 30ug of p u r i f i e d ACC p r e p a r a t i o n was s u b j e c t e d t o p h o s p h o r y l a t i o n i n t h e p r e s e n c e o f (a) t h e endogenous p r o t e i n k i n a s e a c t i v i t y , (b) DEAE-low s a l t f r a c t i o n , (c) DEAE-high s a l t f r a c t i o n , f o l l o w i n g i m m u n o p r e c i p i t a t i o n and su b s e q u e n t d i g e s t i o n w i t h t r y p s i n , the p h o s p h o p e p t i d e s were s e p a r a t e d by 2 - d i m e n s i o n a l e l e c t r o p h o r e s i s and t h i n - l a y e r chromatography. 55 a c t i v i t y . A l l of the f r a c t i o n s c o n t a i n e d some p r o t e i n k i n a s e a c t i v i t y . F r a c t i o n s e l u t i n g w i t h 0.5-0.6M phosphate c o n t a i n e d c y c l i c AMP-dependent p r o t e i n k i n a s e a c t i v i t y , as i n c o r p o r a t i o n i n t o c a r b o x y l a s e by t h e s e f r a c t i o n s c o u l d be s t i m u l a t e d by the a d d i t i o n of c y c l i c AMP. 2-dimensional a n a l y s i s of the c y c l i c AMP-independent p r o t e i n kinase a c t i v i t y revealed the f r a c t i o n e l u t i n g between 80-100raM phosphate appeared to s t i m u l a t e i n c o r p o r a t i o n of [32p] i n t o the I s i t e ( F i g . 11.). D e n s i t o m e t r i c s c a n n i n g of the 2 - d i m e n s i o n a l p l a t e s however i n d i c a t e d t h a t the i n c o r p o r a t i o n i n t o the C, A and I s i t e s was p r o p o r t i o n a l l y the same as the endogenous p r o t e i n k i n a s e a c t i v i t y ( T a b l e 6.), s u g g e s t i n g the p r o t e i n kinases present i n t h i s f r a c t i o n are i d e n t i c a l to those contaminating the p u r i f i e d enzyme p r e p a r a t i o n . 2.4 P r e l i m i n a r y I n v e s t i g a t i o n s I n v o l v i n g C a s e i n - S e p h a r o s e  F r a c t i o n a t i o n of the A v i d i n V o i d F r a c t i o n of Rat L i v e r C y t o s o l . The a v i d i n v o i d f r a c t i o n was a p p l i e d to c a s e i n - s e p h a r o s e , and f r a c t i o n s were e l u t e d from the column w i t h a KC1 s t e p g r a d i e n t composed of 100mM KC1 f o l l o w e d by 500mM KC1 ( i n b u f f e r B). The two r e s u l t i n g f r a c t i o n s were designated CASEIN-LS and CASEIN-HS r e s p e c t i v e l y . Both c a s e i n kinase f r a c t i o n s were d i a l y s e d to 50mM KC1 and assayed f o r p r o t e i n kinase a c t i v i t y towards exogenous ACC and s u b s e q u e n t l y s u b j e c t e d to 2 - d i m e n s i o n a l t r y p t i c p e p t i d e a n a l y s i s . A p p r o x i m a t e l y 75-85% o f the p r o t e i n k i n a s e a c t i v i t y appeared i n the CASEIN-LS f r a c t i o n , the remainder e l u t e d i n the CASEIN-HS f r a c t i o n . The 2-dimensional peptide a n a l y s i s revealed s e p a r a t i o n of at l e a s t two d i f f e r e n t p r o t e i n k i n a s e s had been 56 a F i g . 11. A u t o r a d i o g r a p h s O b t a i n e d f r o m 2 - D i m e n s i o n a l P e p t i d e Maps of Rat L i v e r ACCT 3 0ug of pur i f Ted ACC p r e p a r a t i o n was p h o s p h o r y l a t e d i n the p r e s e n c e of (a) the endogenous p r o t e i n k i n a s e a c t i v i t y and (b) 80- 100mM h y d r o x y l a p p a t i t e f r a c t i o n . The samples were subsequently analysed as d e s c r i b e d p r e v i o u s l y (see l e g e n d Fig.10. ) 57 achieved. The CASEIN-LS f r a c t i o n c o n f i n e d p r o t e i n kinase a c t i v i t y p r e d o m i n a n t l y i n c r e a s i n g i n c o r p o r a t i o n i n t o the B s i t e s of c a r b o x y l a s e , w h i l s t the CASEIN-HS f r a c t i o n c o n t a i n e d p r o t e i n kinase a c t i v i t y r e s u l t i n g i n i n c o r p o r a t i o n i n t o the c o n t r o l s i t e s and the I s i t e ( F i g . 12.). T a b l e 6. summarizes the d a t a o b t a i n e d from d e n s i t o m e t r i c s c a n n i n g of the 2 - d i m e n s i o n a l p e p t i d e maps produc e d i n r e s p o n s e to p h o s p h o r y l a t i o n of ACC by the v a r i o u s p r o t e i n kinase f r a c t i o n s d e s c r i b e d . The most promising r e s u l t as r e g a r d s the i s o l a t i o n o f a p r o t e i n k i n a s e which i n c r e a s e s p h o s p h o r y l a t i o n of the I s i t e a p p e a r s to a r i s e from c a s e i n -s e p h a r o s e f r a c t i o n a t i o n o f the DEAE-HS f r a c t i o n . F u r t h e r p u r i f i c a t i o n and c h a r a c t e r i z a t i o n of t h i s p r o t e i n kinase a c t i v i t y i s underway. 58 a b F i g u r e 12. 2-Dimensional T r y p t i c Peptide Maps of Rat L i v e r ACC 30ug of p u r i f i e d ACC p r e p a r a t i o n was s u b j e c t e d to p h o s p h o r y l a t i o n i n the p r e s e n c e of (a) CASEIN-LS f r a c t i o n and (b) CASEIN-HS f r a c t i o n . The samples were s u b s e q u e n t l y a n a l y s e d as p r e v i o u s l y d e s c r i b e d (see l e g e n d F i g . 10.). 59 TABLE 6. Summary of A n a l y s i s of T r y p t i c Phosphopeptides Obtained F o l l o w i n g P h o s p h o r y l a t i o n o f P u r i f i e d ACC by P r o t e i n K i n a s e F r a c t i o n s from Rat L i v e r FRACTION % INCOROPRATION OF [32p] INTO TRYPTIC PEPTIDES ENDOGENOUS PROTEIN KINASE CYCLIC AMP DEPENDENT KINASE DEAE-HS OH-AP-LS OH-AP-LS 61+4 (4) 17+6 (4) 38+5 (4) DEAE-LS 38+2 (2) 22+2 (3) AVIDIN VOID 15+9 (3) CASEIN-LS 23 (1) CASEIN-HS 39 (1) 38 (1) 34 (1) 67+11 (4) 46+4 (2) (3) (3) (1) 15 (1) 54 (1) 54 (1) 19+2 (4) (4) (2) (3) 25+2 (3) 16 (1) 34 (1) 18 (1) 12 (1) — (4) — (4) 18+2 (2) 73+8 (3) 67+3 (3) 61 (1) 11 (1) — (1) — (1) 60 DISCUSSION 1. P u r i f i c a t i o n of Rat L i v e r ACC ACC has been s t u d i e d e x t e n s i v e l y i n many l a b o r a t o r i e s over the l a s t decade. R e p o r t s i n the l i t e r a t u r e d e s c r i b i n g the c h a r a c t e r i s t i c s of the enzyme however, are o f t e n i n c o n s i s t e n t and even c o n t r a d i c t o r y and c l e a r l y much r e m a i n s to be e s t a b l i s h e d c o n c e r n i n g s t r u c t u r e and r e g u l a t i o n . ACC i s a p a r t i c u l a r l y i m p o r t a n t s y s t e m to s t u d y because i t i s the o n l y one of a f a m i l y of p r o t e i n s showing i n c r e a s e d p h o s p h o r y l a t i o n i n response to i n s u l i n which so f a r shows p a r a l l e l changes i n s p e c i f i c a c t i v i t y c o r r e l a t i n g with s i t e s p e c i f i c p h o s p h o r y l a t i o n . F u r t h e r i n v e s t i g a t i o n s w i t h t h i s enzyme may t h e r e f o r e b r i n g us c l o s e r to d e t e r m i n i n g the r o l e of p r o t e i n p h o s p h o r y l a t i o n i n the mechanism of i n s u l i n a c t i o n on c e l l u l a r metabolism. I f ACC i s to be u s e d as a s u b s t r a t e i n t h i s t y p e o f i n v e s t i g a t i o n , i t i s n e c e s s a r y to have some c o n f i d e n c e i n the c o n s i s t e n c y o f t h e p r e p a r a t i o n o f t h e p u r i f i e d p r o d u c t . P u r i f i c a t i o n schemes f o r ACC have evolved i n s e v e r a l l a b o r a t o r i e s o v e r the y e a r s and t h e r e now seems to be g e n e r a l agreement t h a t r a p i d i s o l a t i o n techniques i n v o l v i n g a v i d i n - s e p h a r o s e a f f i n i t y c h r o m a t o g r a p h y a r e s u p e r i o r to t h e c l a s s i c a l p r o c e d u r e s documented i n the 1970's. The c l a s s i c a l p r o c e d u r e s i n v o l v i n g a s e r i e s of ammonium sulphate p r e c i p i t a t i o n s f o l l o w e d by e x t e n s i v e d i a l y s i s combined with ion-exchange and a b s o r p t i o n chromatography t e c h n i q u e s , produce an enzyme p r e p a r a t i o n of h i g h s p e c i f i c 61 a c t i v i t y (15 U/mg p r o t e i n ) . The p r o t e i n a p p e a r s as a 215Kd s u b u n i t on d e n a t u r i n g p o l y a c r y l a m i d e g e l s . W ith more r a p i d techniques and with e x t e n s i v e p r e c a u t i o n s to prevent p r o t e o l y s i s , enzyme p r e p a r a t i o n s e x h i b i t d i f f e r e n t c h a r a c t e r i s t i c s w i t h the s p e c i f i c a c t i v i t i e s i n the range of l-3U/mg p r o t e i n and the m o l e c u l a r mass of the s u b u n i t r e p o r t e d to be 230-260Kd. The i m p l i c a t i o n s of these o b s e r v a t i o n s are very important i f p r o t e i n p h o s p h o r y l a t i o n i s i n v o l v e d i n r e g u l a t i n g ACC a c t i v i t y . I t seems l i k e l y that the 215Kd and s m a l l e r s p e c i e s of the enzyme represent p r o t e o l y t i c d e g r a d a t i o n p r o d u c t s of the n a t i v e enzyme produced duri n g the i s o l a t i o n procedure and t h i s l i m i t e d p r o t e o l y s i s has been r e p o r t e d to r e s u l t i n the l o s s of a f r a g m e n t c o n t a i n i n g p o t e n t i a l p h o s p h o r y l a t i o n s i t e s . The amino a c i d r e s i d u e s a v a i l a b l e f o r r e v e r s i b l e p h o s p h o r y l a t i o n are presumably l o c a t e d on the s u r f a c e of the p r o t e i n and consequently w i l l be extremely s u s c e p t i b l e to l i m i t e d p r o t e o l y t i c e f f e c t s . The most r e c e n t i s o l a t i o n procedure f o r ACC d e s c r i b e d i n the l i t e r a t u r e i n v o l v e s ammonium s u l p h a t e or PEG p r e c i p i t a t i o n of the enzyme a c t i v i t y from a c y t o s o l i c f r a c t i o n f o l l o w e d by avid i n - s e p h a r o s e a f f i n i t y chromatography. Attempts to p u r i f y the p r o t e i n from r a t l i v e r by t h i s p r o c e d u r e r e s u l t e d i n p r o b l e m s o c c u r i n g a t both s t e p s . The problems a s s o c i a t e d with r e s o l u b i l i z a t i o n of p r o t e i n p e l l e t s and p r o t e o l y t i c d e g r a d a t i o n have been overcome by e l i m i n a t i n g a p r e c i p i t a t i o n step e n t i r e l y and r e p l a c i n g i t with sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n and D E A E - c e 1 1 u l o s e i o n - e x c h a n g e chromatography. The molecular c h a r a c t e r i s t i c s of ACC a l l o w a c e r t a i n amount 62 o f v e r s a t i l i t y i n t h e d e s i g n o f a p r o c e d u r e f o r i t s f r a c t i o n a t i o n . P o l y m e r i z a t i o n of t h e enzyme a l t e r s i t s s e d i m e n t a t i o n v e l o c i t y i n a s u c r o s e d e n s i t y g r a d i e n t (30), a l l o w i n g f a c t i o n a t i o n of the enzyme by high speed c e n t r i f u g a t i o n i n sucrose g r a d i e n t s . P r e p a r a t i o n of the c y t o s o l i c f r a c t i o n i s c a r r i e d out a t 4 C and t h i s p r e s e r v e s the enzyme i n i t s i n a c t i v e p r o t o m e r i c form. Under t h e s e c o n d i t i o n s 85-90% of the enzyme a c t i v i t y a p p e a r s i n the s u p e r n a t a n t , w i t h about 10% r e m a i n i n g a s s o c i a t e d with the microsomal f r a c t i o n . T h i s d i s t r i b u t i o n of a c t i v i t y between the s o l u b l e f r a c t i o n and the membrane f r a c t i o n i s c o n s i s t e n t w i t h the r e s u l t s r e p o r t e d by W i t t e r s e t a l (79), who demonstrated that the a c t i v i t y a s s o c i a t e d with the microsomal f r a c t i o n ranged from 6-40% depending on the n u t r i t i o n a l s t a t e of the animal. As the microsomal p e l l e t i s not washed e x t e n s i v e l y , t h i s s u g g e s t s t h a t 10% i s the maximum p o s s i b l e l o s s i n the f r a c t i o n . Indeed t h i s p o i n t has been c o n f i r m e d w i t h washing microsomal p e l l e t s from adipose t i s s u e . (Roger Brownsey p e r s o n a l communication). P o l y m e r i z a t i o n i s s t i m u l a t e d by i n c u b a t i n g the c y t o s o l i c f r a c t i o n a t 30°C f o r 30 min. A number of e x p e r i m e n t s i n d i c a t e d t hat a d d i t i o n of albumin p r i o r to i n c u b a t i o n improved the r e c o v e r y of ACC a c t i v i t y and t h i s i s l i k e l y to be a complex e f f e c t . The e f f e c t of albumin was ( s u r p r i s i n g l y ) mainly due to a r e d u c t i o n of the p r e c i p i t a t i o n of ACC i n t o the p e l l e t f r a c t i o n d u r i n g the second h i g h speed c e n t r i f u g a t i o n but the r e a s o n f o r t h i s i s not c l e a r . Albumin i s expected to enhance the a l l o s t e r i c b i n d i n g of c i t r a t e to ACC because of i t s c a p a c i t y to bind f a t t y -a c i d s and f a t t y - a c y l C o A e s t e r s which w i l l compete with c i t r a t e . Albumin would t h e r e f o r e tend to promote aggregation of ACC and to 63 s t a b i l i z e the p o l y m e r i c form of the enzyme. The a d d i t i o n of albumin a l s o r e i n f o r c e s the e f f e c t s of the protease i n h i b i t o r s i n c l u d e d i n the e x t r a c t i o n b u f f e r , by c o n t r i b u t i n g to the t o t a l p r o t e i n p r e s e n t and so a c t i n g as a s u b s t r a t e f o r the l e s s -s p e c i f i c proteases. T h i s i s an important c o n s i d e r a t i o n as at t h i s stage of the procedure, the temperature of the e x t r a c t i s r a i s e d to 30°C, i n order to maximise ACC p o l y m e r i z a t i o n . The c o m b i n a t i o n of d i f f e r e n t volumes and d e n s i t i e s of the s u c r o s e l a y e r s used i n the g r a d i e n t p r o v e d to be c r i t c a l f o r recovery of enzyme a c t i v i t y i n the sucrose cushion. S i m i l a r l y the s p e e d , t e m p e r a t u r e and d u r a t i o n of t h e s e c o n d h i g h s p e e d c e n t r i f u g a t i o n a f f e c t e d the s e d i m e n t a t i o n of ACC t h r o u g h the s u c r o s e l a y e r s . I n c l u s i o n of two s u c r o s e l a y e r s i n the s t e p cushion served to reduce the p r e c i p i t a t i o n of the enzyme a c t i v i t y i n t o the p e l l e t f r a c t i o n s . A n a l y s i s of step cushions composed of s e v e r a l sucrose l a y e r s (20%, 30%, 40% and 60%) i n d i c a t e d t h at ACC a c t i v i t y d i s t r i b u t e d t h r o u g h o u t the e n t i r e sequence. T h i s s u g g e s t s t h a t the enzyme forms a h e t e r o g e n e o u s m i x t u r e o f polymers of d i f f e r e n t l e n g t h s , each p o p u l a t i o n having a d i f f e r e n t s e d i m e n t a t i o n v e l o c i t y i n the s u c r o s e c u s h i o n s . The c o n d i t i o n s f i n a l l y a dopted p r o v i d e f o r two i m p o r t a n t c o n s i d e r a t i o n s ; f i r s t l y , to ensure s u f f i c i e n t p o l y m e r i z a t i o n of ACC to a l l o w a h i g h p r o p o r t i o n to s e d i m e n t from s u p e r n a t a n t i n t o s u c r o s e and s e c o n d l y , to p r o v i d e s u f f i c i e n t l y h i g h s u c r o s e to p r e v e n t complete p r e c i p i t a t i o n . The l a t t e r p o i n t was s u r p r i s i n g l y aided by the p r e s e n c e of a l b u m i n s u g g e s t i n g t h a t a l b u m i n i n some way l i m i t s the maximum s i z e to which the enzyme may p o l y m e r i z e or 64 perhaps the extent to which the enzyme may a s s o c i a t e with l a r g e r molecular aggregations i n s o l u t i o n . Ammonium sulphate p r e c i p i t a t i o n i s a standard procedure used i n many l a b o r a t o r i e s f o r the p u r i f i c a t i o n of many d i f f e r e n t p r o t e i n s . P r e c i p i t a t i o n a t t h i s s t a g e of the p r e p a r a t i o n has s e v e r a l advantages; ( 1 ) The p r o t e i n p e l l e t can be resuspended i n a s m a l l volume of b u f f e r , s h o r t e n i n g the t i m e i n v o l v e d i n ap p l y i n g the sample to the av i d i n - s e p h a r o s e a f f i n i t y column, and e f f e c t i v e l y r e d u c i n g the l e n g t h of t i m e the enzyme i s i n i t s most u n s t a b l e form, ( 2 ) the c i t r a t e p r e s e n t i n the e x t r a c t i o n b u f f e r i s e f f e c t i v e l y removed by the p r e c i p i t a t i o n step, ( 3 ) the presence of s m a l l amounts of ammonium sulphate does not appear to a f f e c t the b i n d i n g of ACC to a v i d i n - s e p h a r o s e , and the co n s e q u e n t o m i s s i o n of d i a l y s i s h e l p s i n making the p r o c e d u r e extremely r a p i d . Having experienced problems with the r e s o l u b i l i z a t i o n of the p r o t e i n p e l l e t a f t e r d i r e c t p r e c i p i t a t i o n of the c y t o s o l i c f r a c t i o n with ammonium sulph a t e , i t was p o s s i b l e that reducing the t o t a l amount of p r o t e i n p r e s e n t would be s u f f i c i e n t to overcome t h e s e d i f f i c u l t i e s . U n f o r t u n a t e l y the a d d i t i o n a l p u r i f i c a t i o n a c h i e v e d by c e n t r i f u g a t i o n i n t o a s u c r o s e c u s h i o n d i d not improve the r e c o v e r y o f ACC a c t i v i t y t h r o u g h ammonium s u l p h a t e p r e c i p i t a t i o n or r e s o l u b i l i z a t i o n of the ammonium sulphate p e l l e t . An a t t r a c t i v e a l t e r n a t i v e to p r e c i p i t a t i o n w i t h ammonium sulphate, was to use p o l y e t h y l e n e g l y c o l (PEG). Hardie and Cohen (79) p u b l i s h e d a s i m p l e t h r e e s t e p p r o c e d u r e f o r p u r i f y i n g ACC from l a c t a t i n g r a b b i t mammary g l a n d . The p r o c e d u r e i n v o l v e d 65 ammonium sulphate p r e c i p i t a t i o n f o l l o w e d by two PEG p r e c i p i t a t i o n steps which produced a homogeneous p r e p a r a t i o n of ACC. Although p r e c i p i t a t i o n of the r a t l i v e r enzyme with PEG was 2-3 times more s u c c e s s f u l l i n terms of r e c o v e r y of a c t i v i t y than ammonium s u l p h a t e , t h e r e appeared to be c o n s i d e r a b l e p r o t e a s e a c t i v i t y a s s o c i a t e d w i t h t h i s f r a c t i o n . The n a t i v e 230Kd s u b u n i t was degraded, appearing as a 215Kd fragment. These r e s u l t s sugested t h a t i t may be advantageous to a v o i d the p r e c i p i t a t i o n of the enzyme i f at a l l p r a c t i c a l . D E A E - c e l 1 u l o s e i o n - e x c h a n g e c h r o m a t o g r a p h y o f f e r s the a d v a n t a g e s of a p r e c i p i t a t i o n s t e p y e t a v o i d s the a s s o c i a t e d problems. The sucrose f r a c t i o n was incubated d i r e c t l y with DEAE-c e l l u l o s e at pH-7.5 and under these c o n d i t i o n s e s s e n t i a l y a l l of the ACC a c t i v i t y b i n d s to the r e s i n . The ACC can then be e l u t e d from D E A E - c e l l u l o s e by i n c r e a s i n g the s a l t c o n c e n t r a t i o n i n the b u f f e r to approximately 0.5M. V a r i o u s c o n d i t i o n s were t e s t e d to c h a r a c t e r i s e t h i s step and a most s i g n i f i c a n t f a c t o r was pH s i n c e l o w e r i n g the pH even s l i g h t l y (to 7.0) r e s u l t e d i n d i s p l a c e m e n t of more than 60% of the ACC a c t i v i t y from the D E A E - c e l l u l o s e . The r a t mammary gland enzyme has been p a r t i a l l y p u r i f i e d by n e g a t i v e a b s o r p t i o n to D E A E - c e l l u l o s e a t pH 6.5 (18). These o b s e r v a t i o n s s u g g e s t the p i o f the enzyme i s near n e u t r a l . A t t e m p t s to determine the p i of the enzyme by standard i s o e l e c t r i c f o c u s i n g t e c h n i q u e s however has p roven p r o b l e m a t i c s i n c e the enzyme appears to smear on the f i r t dimension g e l , with no apparent peak of a c t i v i t y o c c u r i n g . (Roger Brownsey p e r s o n a l c o m m u n i c a t i o n ) . Perhaps t h i s e f f e c t may be due to the p r e s e n c e of d i f f e r e n t l y 66 p h o s p h o r y l a t e d f orms of the enzyme. I f the p r o t e i n e x i s t s as a f a m i l y of p h o s p h o r y l a t e d forms, each form may have a d i f f e r e n t o v e r a l l n e t c h a r g e and t h i s w o u l d a f f e c t t h e b i n d i n g c h a r a c t e r i s t i c s of the enzyme w i t h D E A E - c e l l u l o s e and would be e x p e c t e d to produce m u l t i p l e bands upon i s o e l e c t r i c f o c u s s i n g . Having e l u t e d the enzyme a c t i v i t y from D E A E - c e l l u l o s e with high s a l t , the sample i s i n i d e a l c o n d i t i o n f o r a p p l i c a t i o n to the a v i d i n - s e p h a r o s e a f f i n i t y column. F o l l o w i n g the b i n d i n g of ACC and r e m oval of unbound p r o t e i n , the a f f i n i t y column i s e l u t e d with b u f f e r c o n t a i n i n g f r e e b i o t i n which competes f o r the a v i d i n -s e p h a r o s e b i n d i n g s i t e s d i s p l a c i n g the ACC from the column. F r a c t i o n s c o n t a i n i n g p r o t e i n were c o n c e n t r a t e d u s i n g a q u a c i d e I I I , a f l a k e form of p o l y e t h y l e n e g l y c o l which i s h i g h l y hygroscopic and absorbs moisture from the sample through d i a l y s i s t u b i n g . T h i s method p r o v e d to be p r e f e r a b l e to o t h e r methods of c o n c e n t r a t i n g w h i c h were t e s t e d , i n c l u d i n g a c e n t r i f u g a l t e c h n i q u e , an e l e c t r o p h o r e t i c t e c h n i q u e and an i m m e r s i b l e u l t r a f i l t r a t i o n u n i t . The p r o b l e m s e n c o u n t e r e d r e s u l t e d e i t h e r because of the prolonged time necessary to achieve the r e q u i r e d c o n c e n t r a t i o n , i r r e v e r s i b l e b i n d i n g of the p r o t e i n i n the sample to the membrane, or p r e c i p i t a t i o n of the p r o t e i n from s o l u t i o n . The p u r i f i e d enzyme p r e p a r a t i o n has t h e p r o p e r t i e s p r e v i o u s l y reported by others f o r the n a t i v e , i n t a c t form of the enzyme and a p p e a r s to m i g r a t e i n a s i m i l a r f a s h i o n to an i m m u n o p r e c i p i t a t e d form of the enzyme from both f r e s h r a t e p i d i d y m a l f a t pad e x t r a c t s and r a t l i v e r c y t o s o l i n d i c a t i n g p r o t e o l y s i s has been a v o i d e d . The r a t l i v e r p r e p a r a t i o n does however c o n t a i n a minor p r o t e i n band of 240Kd which a p p e a r s to 67 migrate more s l o w l y on denaturing p o l y a c r y l a m i d e g e l s than the band produced from the i mmunopr ec i p i t a te of f r e s h f a t pad e x t r a c t s . One p o s s i b l e e x p l a n a t i o n f o r the a p p e a r a n c e of t h i s band i s t h a t i n . l i v e r the enzyme i s t r a n s l a t e d as a pre-enzyme which r e q u i r e s p o s t - t r a n s l a t i o n a l p r o c e s s i n g to the mature 230Kd form of the enzyme. In c o n c l u s i o n , the procedures developed a l l o w the p u r i f i c a t i o n of l-2mg a c e t y l - C o A c a r b o x y l a s e from 6 r a t l i v e r s w i t h i n 24hr. and the enzyme p r e p a r a t i o n i s s t a b l e at -70°C f o r a t l e a s t 2 months. Beyond t h i s t i m e , t h e r e i s v a r i a b l e a p p e a r a n c e of the c l a s s i c a l " c l i p p e d " form of s u b u n i t Mr 215Kd. From these o b s e r v a t i o n s , i t seems a p p r o p r i a t e to assume that the p u r i f i e d enzyme p r e p a r a t i o n i s s u i t a b l e f o r use as a s u b s t r a t e i n i n v e s t i g a t i o n s of c y t o s o l i c p r o t e i n kinase a c t i v i t y . 2. C h a r a c t e r i z a t i o n of Rat L i v e r P r o t e i n Kinases. The p r i m a r y o b j e c t i v e of t h e s e s t u d i e s was to i s o l a t e the p r o t e i n kinase a c t i v i t y r e s p o n s i b l e f o r i n c r e a s i n g i n c o r p o r a t i o n o f p h o s p h a t e i n t o t h e I - s i t e and to d e t e r m i n e i f t h i s p h o s p h o r y l a t i o n e f f e c t c o u l d d i r e c t l y a c t i v a t e ACC. I n i t i a l e x p e r i m e n t s i n v o l v e d i n c u b a t i o n o f t h e p u r i f i e d enzyme p r e p a r a t i o n with r a d i o a c t i v e Mg-ATP. Despite the p r e p a r a t i o n of ACC appearing near homogeneous on denaturing p o l y a c r y l a m i d e g e l s , t h i s r e s u l t e d i n the i n c o r p o r a t i o n of phosphate i n t o both the major 230Kd band and the minor 240Kd band. T h i s i n d i c a t e s the p u r i f i e d enzyme p r e p a r a t i o n i s contaminated with t r a c e amounts of p r o t e i n k i n a s e a c t i v i t y w h i c h have c o - p u r i f i e d w i t h ACC 68 t h r o u g h o u t the i s o l a t i o n p r o c e d u r e . Endogenous p r o t e i n k i n a s e a c t i v i t y can be d e t e c t e d i n the p r e s e n c e of the s p e c i f i c c y c l i c AMP-dependent p r o t e i n kinase i n h i b i t o r p r o t e i n (Walsh i n h i b i t o r ) but can a l s o be s t i m u l a t e d 5 - f o l d by the a d d i t i o n of c y c l i c AMP. T h i s s u g g e s t s both c y c l i c AMP-independent and c y c l i c AMP-dependent p r o t e i n k i n a s e a c t i v i t y i s c o n t a m i n a t i n g the enzyme p r e p a r a t i o n . These r e s u l t s are comparable to those p u b l i s h e d by Hardie and Cohen (79) studying r a b b i t mammary gland ACC. A d d i t i o n of a l a r g e e x c e s s of Walsh i n h i b i t o r i n the a s s a y overcomes the problem of contaminating c y c l i c AMP-dependent p r o t e i n kinase as t h i s a c t i v i t y i s e f f e c t i v e l y b l o c k e d . Walsh i n h i b i t o r was s u b s e q u e n t l y added to a l l a s s a y s i n v o l v i n g exogenous p r o t e i n k i n a s e s . The p r e s e n c e of endogenous c y c l i c AMP-independent p r o t e i n k i n a s e a c t i v i t y i n t h e p u r i f i e d ACC p r e p a r a t i o n , must be recognised but does not p r o v i d e an insurmountable problem, s i n c e i t i s s u f f i c i e n t l y low l e v e l (0.1 mole[32p]/mole ACC subunit/hr) and the phosphorylated s i t e s are a p p a r e n t l y " s i l e n t " i.e. produce no apparent a c t i v i t y changes. I t i s a l s o p o s s i b l e to d i s c r i m i n a t e the e f f e c t s of the endogenous p r o t e i n k i n a s e a c t i v i t y from hormone s t i m u l a t e d p r o t e i n p h o s p h o r y l a t i o n , by 2 - d i m e n s i o n a l p e p t i d e a n a l y s i s . E n d o g e n o u s p r o t e i n k i n a s e l e a d s to i n c o r p o r a t i o n o f [32p] i n t o c o n t r o l (C) s i t e s and not A or I s i t e s , which are phosphorylated i n response to hormones. Further attempts to c h a r a c t e r i z e t h i s endogenous c y c l i c AMP-independent k i n a s e a c t i v i t y i n c l u d e d e x a m i n i n g the e f f e c t of a d d i t i o n of p h o s p h o l i p i d , polyamines, c a l c i u m ions and coenzyme A. From the r e s u l t s , i t appears that the endogenous p r o t e i n kinase a c t i v i t y 69 i s n e i t h e r s t i m u l a t e d by n o r d e p e n d e n t on c a l c i u m i o n s , p h o s p h o l i p i d or coenzyme A. The a d d i t i o n of p o l y a m i n e s (ImM) however can s t i m u l a t e the i n c o r p o r a t i o n of [32p] i n t o the C s i t e s 2-3 f o l d . The s i g n i f i c a n c e of t h i s o b s e r v a t i o n r e q u i r e s f u r t h e r i n v e s t i g a t i o n . The e f f e c t s o f p h o s p h o r y l a t i o n o f ACC by c y c l i c AMP-dependent p r o t e i n k i n a s e have been shown to be d i s t i n c t from those due to i n s u l i n r e s p o n s i v e p r o t e i n kinase i n r a t epididymal f a t p a d t i s s u e . 2 - d i m e n s i o n a 1 p e p t i d e a n a l y s i s o f i m m u n o p r e c i p i t a t e d ACC from t i s s u e t r e a t e d w i t h a d r e n a l i n e i n d i c a t e s that the c y c l i c AMP-dependent p h o s p h o r y l a t i o n c a u s e s i n c r e a s e d i n c o r p o r a t i o n i n t o the c o n t r o l s i t e s and a l s o i n t o the A group of p h o s p h o p e p t i d e s . I n c o r p o r a t i o n of [32p] i n t o the I s i t e i s u n a f f e c t e d under t h e s e c o n d i t i o n s . In t h e s e s t u d i e s 2-D i m e n s i o n a l p e p t i d e a n a l y s i s o f p u r i f i e d r a t l i v e r ACC phosphorylated i n the presence of p u r i f i e d c y c l i c AMP-dependent p r o t e i n k i n a s e c a t a l y t i c s u b u n i t or s i m p l y by the a d d i t i o n of c y c l i c AMP, produced very s i m i l a r r e s u l t s . P r e t r e a t m e n t of i n t a c t f a t pad t i s s u e w i t h i n s u l i n and s u b s e q u e n t 2 - d i m e n s i o n a 1 p e p t i d e a n a l y s i s o f t h e i m m u n o p r e c i p i t a t e d c a r b o x y l a s e r e v e a l s an i n c r e a s e i n the i n c o r p o r a t i o n of [32p] i n t o the I group of p h o s p h o p e p t i d e s . Peptide a n a l y s i s thus provides an important c r i t e r i o n r e q u i r e d to i d e n t i f y p o t e n t i a l i n s u l i n - d e p e n d e n t p r o t e i n k i n a s e a c t i v i t y o c c u r i n g i n f r a c t i o n s of r a t l i v e r c y t o s o l . The d i s t r i b u t i o n of p r o t e i n kinase a c t i v i t y i n the f r a c t i o n s p roduced d u r i n g the i s o l a t i o n of ACC was d e t e r m i n e d u s i n g 70 i m m u n o p r e c i p i t a t i o n t e c h n i q u e s . D i r e c t p h o s p h o r y l a t i o n o f ACC f o l l o w e d by SDS-PAGE presented d i f f i c u l t i e s i n q u a n t i t a t i o n , as t h e more c r u d e f r a c t i o n s c o n t a i n a v e r y l a r g e number o f r a d i o a c t i v e l y l a b e l l e d p h o s p h o r y l a t e d p r o t e i n bands and ACC repr e s e n t s o n l y a s m a l l percentage of the t o t a l p r o t e i n a p p l i e d to the SDS-gel. Immunoprecipitation of ACC a f t e r p h o s p h o r y l a t i o n overcomes t h i s p r o b l e m and a l l o w s g r e a t e r d i s c i m i n a t i o n by removing r e s i d u a l P i , ATP and l a b e l l e d p h o s p h o l i p i d s which may be present i n the f r a c t i o n s . The m a j o r i t y o f the c y c l i c AMP-independent p r o t e i n k i n a s e a c t i v i t y i n r a t l i v e r c y t o s o l appears to c o - p u r i f y with ACC i n t o the sucrose f r a c t i o n . A f t e r ion-exchange chromatography however, 90% of the a c t i v i t y a p p e a r s i n the DEAE-LS f r a c t i o n w i t h the unbound p r o t e i n . The remaining 10% of p r o t e i n kinase a c t i v i t y co-p u r i f i e s w i t h ACC t h r o u g h D E A E - c e l l u l o s e and i s s u b s e q u e n t l y l a r g e l y removed from ACC on the av i d i n - s e p h a r o s e a f f i n i t y column. These o b s e r v a t i o n s i m p l y t h a t a l t h o u g h the p u r i f i e d enzyme p r e p a r a t i o n i s c o n t a m i n a t e d w i t h p r o t e i n k i n a s e a c t i v i t y , the con t a m i n a t i o n represents l e s s than 1% of the t o t a l s o l u b l e c y c l i c AMP-independent p r o t e i n k i n a s e a c t i v i t y found i n the r a t l i v e r c y t o s o l . 2-Dimensional peptide a n a l y s i s of ACC phosphorylated by the v a r i o u s d i f f e r e n t f r a c t i o n s produced d u r i n g the i s o l a t i o n procedure i n d i c a t e s s e v e r a l d i f f e r e n t p r o t e i n kinase a c t i v i t i e s have been e f f e c t i v e l y s e p a r a t e d . None of t h e s e p r o t e i n k i n a s e s however appear to in c r e a s e i n c o r p o r a t i o n of phosphate i n t o the I -s i t e . F u r t h e r f r a c t i o n a t i o n of the DEAE-HS f r a c t i o n on c a s e i n -sepharose however revealed a p r o t e i n kinase f r a c t i o n e l u t i n g with 71 500mM KC1 which l e d to the i n c o r p o r a t i o n of phosphate i n t o the I s i t e o f ACC w h i c h r e p r e s e n t s 30-35% of t h e t o t a l [32p] i n c o r p o r a t e d . T h i s d e m o n s t r a t e s t h a t i t i s i m p o r t a n t to s c r e e n i n i t i a l f r a c t i o n s a f t e r f u r t h e r p u r i f i c a t i o n , as p r o t e i n kinase a c t i v i t y i n t h e more c r u d e f r a c t i o n s may be i n h i b i t e d . P u r i f i c a t i o n to homogeneity and su b s e q u e n t c h a r a c t e r i z a t i o n of the p r o t e i n kinases which phosphorylate d i f f e r e n t s i t e s on ACC i s underway. P r e l i m i n a r y experiments measuring the e f f e c t s of s i t e s p e c i f i c p h o s p h o r y l a t i o n by d i f f e r e n t p r o t e i n kinase f r a c t i o n s on the c a t a l y t i c a c t i v i t y of ACC, suggest that under the c o n d i t i o n s examined to d a t e , p h o s p h o r y l a t i o n a l o n e i s i n s u f f i c i e n t to br i n g about the a c t i v a t i o n of p u r i f i e d ACC. 3. Future I n v e s t i g a t i o n s . E v a l u a t i o n o f the r o l e o f p h o s p h o r y l a t i o n o f ACC iri v i v o w i l l r e q u i r e an understanding of the e f f e c t s of p h o s p h o r y l a t i o n o f the enzyme i n a p u r i f i e d system. In o r d e r to o b t a i n such i n f o r m a t i o n , the p u r i f i c a t i o n of the p r o t e i n kinases i n v o l v e d i s e s s e n t i a l . P r o t e i n k i n a s e f r a c t i o n s w h i c h c a t a l y s e t h e i n c o r p o r a t i o n o f a s u b s t a n t i a l amount of [32p] (60%+ of t o t a l i n c o r p o r a t i o n ) , i n t o the A, C and B s i t e s of ACC have been p a r t i a l l y p u r i f i e d f r o m r a t l i v e r . The e f f e c t o f A s i t e p h o s p h o r y l a t i o n i n r e s p o n s e to c y c l i c AMP, has been w e l l documented i n the l i t e r a t u r e . We are now i n a p o s i t i o n to i n v e s t i g a t e the e f f e c t s of both C and B s i t e p h o s p h o r y l a t i o n o f ACC i n the presence of the v a r i o u s a l l o s t e r i c ligands.The maximum 72 i n c o r p o r a t i o n of phosphate i n t o the I s i t e a c h i e v e d to d a t e represents 34% of the t o t a l i n c o r p o r a t i o n . I t i s a n t i c i p a t e d that f u r t h e r p u r i f i c a t i o n of the p r o t e i n kinase f r a c t i o n i n v o l v e d w i l l i n c r e a s e the p r o p o r t i o n of [32p] i n c o r p o r a t i o n i n t o the I s i t e . Comparable s t u d i e s of the c a t a l y t i c p r o p e r t i e s of the enzyme i n an i n s u l i n s t i m u l a t e d p h o s p h o r y l a t e d form, can then be made. A n o t h e r avenue f o r i n v e s t i g a t i o n c o u l d i n v o l v e a m i n o - a c i d sequencing of the phosphopeptides of the A, C, I, and B groups of a c e t y l - C o A . T h i s would c o n f i r m how many s e r i n e r e s i d u e s a r e i n v o l v e d i n the r e v e r s i b l e p h o s p h o r y l a t i o n and would p r o v i d e s t r u c t u r a l i n f o r m a t i o n about the i n t a c t p r o t e i n . P r e l i m i n a r y i n v e s t i g a t i o n s s u g g e s t t h a t s m a l l a l l o s t e r i c l i g a n d s or e f f e c t o r s do not n e c e s s a r i l y c o n t r i b u t e to the k i n e t i c c h a r a c t e r i s t i c s of the enzyme prod u c e d i n r e s p o n s e to i n s u l i n . The l o s s of the i n s u l i n e f f e c t d u r i n g p u r i f i c a t i o n with a v i d i n -s e p h a r o s e , may i m p l i c a t e a r o l e f o r s p e c i f i c p r o t e i n - p r o t e i n i n t e r a c t i o n i n the r e g u l a t i o n of ACC a c t i v t y . H e t e r o - b i f u n c t i o n a l c r o s s - l i n k i n g reagents such as the s u c c i n i m i d y l e s t e r maleimides, r e a c t i n i t i a l l y w ith one component on an amine group, forming a t h i o l r e a c t i v e maleimide. T h i s can then react with a t h i o l group from a second component. These compounds may prove to be u s e f u l t o o l s f o r studying the i n t e r a c t i o n s of p r o t e i n kinases and other p r o t e i n s w i t h ACC. A l t h o u g h i n v e s t i g a t i o n s o f t h e c h a r a c t e r i s t i c s of the p u r i f i e d enzyme system are e s s e n t i a l , i t i s e q u a l l y important to study, i n p a r a l l e l , the c h a r a c t e r i s t i c s of the i n s u l i n s t i m u l a t e d enzyme from i n t a c t t i s s u e . By using r a t f a t pads, we can s t u d y when d u r i n g the p u r i f i c a t i o n of ACC t h a t the i n s u l i n e f f e c t i s l o s t and attempt r e c o n s t i t u t i o n experiments 73 by a d d i n g back v a r i o u s f r a c t i o n s i n an a t t e m p t to r e c o v e r the l o s t i n s u l i n e f f e c t . Subsequently i t may be p o s s i b l e to attempt to i s o l a t e the component(s) r e s p o n s i b l e f o r the r e s t o r a t i o n of the hormonal e f f e c t . 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