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OTHER LONG-CHAIN FATTY AC IDS IN RAT AND SHEEP LIVER
MITOCHONDRIA
A thes i s presen ted in par t i a l fulf i lment of the requ i r ement for the degree of
Doctor of Phi losophy in Biochemi stry
at
Mas s ey Univers i ty New Z ea land
John Campbell W i l l iam Reid 1 9 8 6
LIBPJU{Y CL�TCE
1. (a) I give my permission for my thesis, entitled
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"i • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • o •
Abs tract
S he ep l iver mitochondr i a oxi d i s ed pa lmi tate , oleate and l inoleate at s lower rates than did rat l iver mi tochondr i a . Rat l iver mitochond r i a ox i d i s ed l inoleate at 1 . 2 to
1 . 7 t imes the rate observed with pa lmitate as the subs trate . However , sheep l i ver mi tochond r i a ox i d i s ed l i noleate at 0 . 7 4 to 0 . 8 4 the rate observed when palmitate was the substrate . The b iochemi cal bas i s of th i s d i f f er ence i s not unders tood .
The reaction catalysed by the en z yme carn i t i n e
acyltran s f erase I i s bel i eved t o be a n important regulatory step in the oxidat i on of long-chain fatty acids and is known to be competit ively inhib i ted by ma lonyl-CoA . Both rat and s heep l iver mitochondr i a wer e able to form acyl carni t in e when palmitoyl-CoA and l i noleate , coupled with a n acyl-CoA generat i ng system , wer e the acyl substrate s .
Malonyl-CoA was very effective in i n h i b i t i ng the CAT I react ion in sheep l iver mi tochondr i a . When l i noleate ,
coupled with an acyl-CoA generating sys tem , was the
substrate for CAT I , 1 �M ma lonyl-CoA was found to inhi b i t t h e reac t i on b y 9 0 % . However , when the same s ubstrate was a s s ayed in rat l i ver mi tochond r i a the i n h i b i t i on was muc h less , 2 2 � M ma lonyl-CoA leading t o only 5 0 % inhibit ion of the CAT I en z yme . When palmi toyl-CoA was used as
a substrate for the en z yme CAT I , l i tt l e di f ference was seen between rat and s heep l iver mi tochondr i a in the extent of i n h i b i t ion obs er ved over the concentrat ion range of 1 to 5 � M malonyl-CoA .
These exper iments ind icate that sheep l i ver
mi tochondr i a cou ld oxi d i s e palmitate rather than l inoleate at low levels of malonyl-CoA , as one might expect in v i vo .
I n contra s t , in rat l i ver mi tochondr i a , linoleate would be oxid i s ed faster than p a lmi tate at a l l concentra t i ons of malonyl-CoA inve s t igated .
I t is suggested that thi s system may be an important means whereby s heep are ab le to conserve l i noleate by p r event ing i t s oxidat i on .
I n add i t ion the mi tochondr i a l glycerol 3-phosphate acyltran s f erase react i on was inves t i gated with both sheep and rat l iver mi tochondr i a . With linoleate and an acyl-CoA generat ing sys tem , rat l iver preparations ester i f i ed
1 . 5 nmoles min/mg protei n whereas sheep l i ver mi tochondr i a e s t er i f i ed less than one tenth o f th i s . It was concluded ester i f i cation of l i noleate to glycerol 3 -phosphate i s not an important mechan i sm of cons erving linoleate in she ep l iver mi tochondr i a .
Ester i f i ca t i on of pa lmi tate to glycerol 3 -phosphate was stud i ed us ing palmitoyl-CoA as the acyl donor . At max imal rates of ester i f i cat i on i t was obs erved that rat l iver mitochondr i a e s ter i f ied palmi toyl-CoA at 2 nmo les/min/mg whereas sheep mi tochondr i a e ster i f i ed 0 . 8 nmoles/mi n/mg .
i i i
Acknowledgement
I wis h to thank my supervisor Dr D . R . Hus bands for his advice , encouragement and s ense of humour throughout this work .
I wou ld also like to thank Dr J . C . Hawke for his advice throughout this work especia l ly in the writing of this
thesis .
I am also grateful to Ear l Stevens for his help wit h statistic a l analysis and f o r his genera l support .
An extended thanks is given to members of the
Chem/Biochem department and to many other friends for t heir support throughout this work .
Abbrev iations CAT
CPT GPAT FFA DTT PPO PO POP NbS 2 EGTA
.9:
g 1 1 6 : 0 1 8 : I
1 8 : 2
carn i t ine acyltran s f erase
carn i t ine palmitoyltran s f erase
g lycerol 3 -phosphate acyltransferase free fatty acid
d i t h i othre itol
2 , 5 -d iphenyloxa z ole
1 , 4 - b i s [ 2 - ( 5 -phenyloxa z olyl } ] ben z ene 5 , 5 ' -dith i ob i s - ( 2 -n i troben z o ic ac id } ethy leneg lycol-b i s - < S -amino-ethyl ether } N , N ' -tetra-acet i c ac id
gravitational force grams
l i tr e
palmi t i c acid oleic acid l i no l e i c a c i d
V
L i s t of Contents
ABSTRACT ii
ACKNOWLEDGEMENT iv
ABBREVIATIONS V
LIST OF TABLES X
LIST OF FIGURES xi
CHAPTER 1 - INTRODUCT I ON
1 . 1 T he requ i r ement of an imal s for l i no l e i c ac i d 1 1 . 2 S ynthes i s of long-cha in f atty acids 1
by an imal ce l ls
1 . 3 Hydrog enation of po lyuns aturated fatty ac ids 3 i n the rumen
1 . 4 Function of long-cha i n polyuns aturated 4 f atty acids in cell membranes
1 . 5 P lasma entry rates of fatty acids in sheep 6
1 . 6 Metabo l i sm of l i no l e i c ac id 6
i n the an imal cell
1 . 6 . 1 8-oxidat i on 7
1 . 6 . 2 Ester i f ication 7
1 . 6 . 3 E longation/Desaturat i on 9
1 . 7 Free fatty ac ids as mitochondr i a l substrates 1 0 1 . 7 . 1 Act ivat ion o f long-cha i n fatty ac ids 1 0 1 . 8 Oxidat i on and es ter i f i cation
of acyl-CoA in mi tochondr i a
1 . 8 . 1 T h e es ter i f i ca t i on o f acyl-CoA to g lycerol 3 -phosphate in l i ver mi tochondr i a
1 . 8 . 2 The ester i f i ca t i on o f acyl-CoA to carn i t ine in l i ver mi tochondr ia 1 . 9 E f f ect of malonyl-CoA on CAT I activity
1 2 1 3
1 5 1 9
1 . 1 0 Oxidat ion of polyuns aturated fatty ac ids 1 . 1 1 P er ox i s omal ox idat i on
AIMS OF THE STUDY
CHAPTER 2 - MATERIALS AND METHODS 2 . 1 Chemical r eagents
2 . 2 Preparat i on of reagents
2 . 2 . 1 [1 4c ] g lycerol 3 -phosphate 2 . 2 . 2 [1 4c ] carn i t i n e
2 . 3 P r epara t i on of intact l i ver mi tochond r i a 2 . 3 . 1 Treatment of an ima l s and l iver
t i s sue pr ior to the preparat ion of rat and s heep mitochondr i a
2 . 3 . 2 Preparation of mitochondr i a 2 . 4 Preparat i on of peroxi s omes from
l i ver t i s sue
2 0 2 7 2 9
3 1 3 1 3 2 3 2 3 2 3 4
3 4 3 6 2 . 4 . 1 P r eparat i on o f a perox i s ome-enr i ched 3 7
fraction from l i ver
2 . 5 Preparation of long-cha in fatty ac id- f r ee albumi n
2 . 6 Ass ay cond i t i on s
2 . 6 . 1 Rate of S -ox i dat ion
2 . 6 . 2 As s ay of fatty a c id oxidat i on by l iver perox i s omes
2 . 6 . 3 As s ay for car n i t ine acyltransferase ( CAT ) I in l iver mi tochondr i a
2 . 6 . 4 Ass ay f o r glycerol 3 -phosphate acyltransferase ( GPAT ) in l i ver mi tochondr i a
2 . 7 P r eparat i on of electron micrographs 2 . 8 Determinat i on of protein
37 37 3 7 4 0 4 1 4 3
4 3 4 4
v i i
CHAPTER 3 - RESULT S
3 . 1 Integr i ty o f mi tochondr i a
3 . 2 Oxidat ion of long-chain f r e e fatty ac ids 3 . 3 The oxidat i on of long-chai n acyl-CoA es ter s
by r a t and s he ep l iver peroxi somes 3 . 4 Fatty acyl substrates for CAT I
and GPAT a s s ays
3 . 5 Glycerol 3 -phosphate acyltran s f erase activity in l iver mi tochondr i a
3 . 6 Car n i t i n e acyltransferase activity i n l iver mitochond r i a
3 . 7 Compet i t i on for acyl-CoA between glycerol 3 -phosphate acyltran s f erase ( GPAT ) and carnit ine acyltran s f er a s e ( CAT ) I in rat l iver mi tochondr i a
3 . 8 Ef f ect of malonyl-CoA o n car n i t i n e acyl
tran s f erase ( CAT ) I activity in both rat and s heep l i ver mi tochond r i a CHAPTER 4 - D I SCUSS I ON
4 . 1 Metabo l i sm in the rumen
4 . 1 . 1 The l imi ted avai la b i l i ty of poly
unsaturated fatty ac ids to ruminants
4 . 2 Conservat i on of l i no l e i c ac id by rumi nants 4 . 3 Oxi dat ion of long-chain fatty ac ids in
r a t and s heep l i ver mitochondr i a
4 . 4 Compar i s on o f relat ive rates o f oxidat i on of both saturated and uns aturated fatty ac ids
4 . 5 Long-cha in f r ee fatty acids as
uncouplers of oxidat ive phosphorylation 4 . 6 Long-cha in f r ee fatty ac ids as
inhib itors of en z yme activity
4 . 7 Fatty acid concentrat ions produc ing maximum rates of ox idat ion
4 5 4 5 4 5 5 8 6 1
6 2 6 8 7 5
7 8 8 3 8 3 8 3
8 4 8 5 8 6
8 8 8 9 9 0
4 . 8 Mi tochondr i a l oxidation and
es ter i f i cation of long-cha in fatty acids 4 . 9 Mitochond r i a l g lycerol 3 -p ho s phate
acyltran s f erase activity i n r at and s h eep l iver mitochondr i a
4 . 10 Carn i t i ne acyltrans ferase I activ ity i n rat and s he ep l iver mi tochondr i a
4 . 11 E f f ect of malonyl-CoA o n CAT I act i v i ty in rat and s he ep l iver mitochond r i a 4 . 1 2 Regula t i on of t h e ox idat ion
of linole i c a c i d in s heep
4 . 1 3 Inh i b i t i on of CAT I by malonyl-CoA in ruminants
4 . 1 4 Metabo l i sm of acetate i n ruminants 4 . 1 5 E f fect of methylma lonyl-CoA on
CPT I activity in s heep l i ver CONCLUS I ON
SUMMARY
FURTHER RESEARCH B IBLIOGRAPHY
APPENDI X - Pub l i ca t i on in the B iochemical Journal ( 1 9 8 5 )
Reid , J . C . W . and Husbands , D . R . ( 1 9 8 5 ) B i ochem . J . 2 2 5 , 2 3 3 -2 3 7 .
T i t le - Ox idat ive metabol ism of long-cha in fatty ac ids in mi tochondr i a f r om s heep and rat l iver
i x
9 1 9 2
9 3 9 4 9 7 9 9 lOO 1 0 1 1 0 2 1 0 2 1 0 4 1 0 5
Table No .
1 2
3
4
L i s t of Tables
T i t l e
Character i sa t i on of mi tochond r i a from r a t and s heep l i ver
A compar i s on of the rates of oxida t i on of palmi t i c , o l e i c and l i no l e i c ac ids i n rat l iver
mi tochondr i a
A compar i son o f the rates of oxidat i on of palmi t i c , ole i c and l i nole i c ac ids in sheep l i ver mi tochond r i a
The e f f e c t of incuba t i on time pr ior to the add i t ion of ADP on the rate of oxidation of 0 . 0 1 mM palmi tate in the presence of 2 . 5 mM glycerol 3 -phosphate
in rat l i ver mi tochondr i a
5 0 5 9
6 0
7 7
L i s t of Figures
F igur e No . T it l e
1 The biosynthe s i s of long-cha in polyunsaturated fatty acids from l i no l e i c ac i d by a combination of desaturat i on and elongation reac t i ons 2 Schema t i c representat i on of the f luid
mosa i c model for membrane structure proposed by S inger and N i cholson 3 Met abol i sm of linol e i c acid in the
mammal i an hepatocyte
4 Schemat i c representat i on of the
metabol i sm of long-cha i n fatty acids in l i ver mi tochondr i a
5 Metaboli sm of long-cha i n acyl-CoA formed within the inter-membrane space of l iver mi tochondr i a
6 Transport of long-cha i n fatty acids acros s the i nner-membrane of
mi tochond r i a
7 s -oxidat i on of saturated fatty acids in mi tochond r i a
8 Oxidation of linoleic ac i d in mi tochond r i a
9 Metabol i sm of acetyl-CoA in l i ver mi tochond r i a
1 0 T h e Clark-type oxygen electrode 11 Oxygen electrode trace
1 2 Oxygen electrode trac i ng us i ng 1 0 mM
succ inate as a subs trate in rat l iver mi tochond r i a
1 3 Oxygen electrode trac i ng us i ng 1 0 mM
succinate as a substrate in sheep l iver mi tochondr i a
1 4 Electron micrographs of rat and sheep l iver mi tochondr i a l
pr eparations ( magn i f i ca t ion =
8 , 2 0 0 t imes )
x i
2
5
8
1 1
1 4
1 7
21 25 26
3 8 3 9
4 6
4 7
48
1 5
1 6
1 7
1 8
1 9
2 0
2 1
2 2
2 3
2 4
Electron mi crographs o f rat and s he ep l iver mi tochondr i a l
preparat i ons ( magn i f i cat i on =
1 9 , 2 0 0 t imes )
The inf luence of coen z yme A
concentrat ion on the rate of oxygen upt ake by rat and s heep l iver
mi tochond r i a us i ng 0 . 0 1 mM palm itate as the acyl substrate
The inf luenc e of carn i t ine
concentrat ion on the rate of oxygen uptake by rat and s heep l i ver
mi tochondr i a us i ng 0 . 0 1 mM palmitate as the acyl subs trat e
The effect o f palmitate concentration on the uptake of oxygen by rat and s heep l iver mitochondr i a
The eff ect of s tearate c oncentrat ion on the uptake of oxygen by rat and s heep l iver mi tochond r i a
The ef fect o f t h e concentrat ion of C1a unsaturated fatty acids on the uptake of oxygen by rat and sheep l i ver mi tochondr i a
The effect of palmitoyl-CoA
concentration on the act i v i ty of glycerol 3 -phosphate acyltrans ferase
( GPAT ) i n rat and s heep l i ver mitochondr i a
The effect o f l inoleate , coupled to an acyl-CoA generat ing sys tem , on the act ivity of glycerol 3 -phosphate
acyltran s f er a s e ( GPAT ) in rat and s heep l iver mi tochond r i a
The relat ionship between glycerol 3 -phosphate acyltran s f erase ( GPAT ) and the amount of mitochondr i a l protein in r a t and s heep l i ver mi tochond r i a
The effect o f incubat ion t ime on the act ivity of glycerol 3 -phosphate acyltran s f erase ( GPAT ) in rat l iver mi tochondr i a
4 9
5 1
5 2
5 5
5 6
5 7
6 3
6 4
6 6
6 7
2 5
2 6
2 7
2 8
2 9
3 0
3 1
3 2
3 3
3 4
The ef f ect o f incubat i on time on the activity of carn i t i n e acyltransferase
{ CAT ) I in rat l i ver mi tochond r i a T h e relat ionship between carn i t in e acyltran s f erase { CAT ) I activity and the amount of mi tochondr i a l protein in rat l i ver mi tochond r i a
The r e lationship between carni t ine acyltrans f erase { CAT ) I act ivity and the amount of mi tochondr i a l protein in sheep l iver mi tochond r i a
The inf luence of palmi toyl-CoA concentrat ion on the activity of carn i t in e acyltrans ferase { CAT ) I i n rat and s heep l iver mi tochond r i a The inf luence of l i no leate , coupled to an acyl-CoA generating sys tem , on the act i v i ty o f carn i t i n e acyltran s feras e
{ CAT ) I in rat and s heep l i ver mi tochondr i a
The inf luence o f carn i t ine
concentrat ion on the activity of glycerol 3 -phosphate acyltransferase
( GPAT ) i n rat l i ver mi tochondr i a The inhib i t i on of carn i t ine acyl
tran s f erase ( CAT ) I by malonyl-CoA in mitochond r i a from rat l i ver mi tochondr i a us ing pa lm i toyl-CoA as the acyl substrate
The inhibit ion of carn i t ine acyl
tran s f erase { CAT ) I by malonyl-CoA in mi tochondr i a from rat l iver
us i ng l i noleate , coup led to an acyl
CoA genera t i ng sys tem , as the acyl substrate
The inhibit ion of carn i t ine acyl
tran s f erase ( CAT ) I by malonyl-CoA in mitochondr i a from s heep liver us ing palm itoyl-CoA a s the acyl substrate
The inhib i t ion of car n i t ine acyl
trans ferase ( CAT ) I by malonyl-CoA in mi tochondr i a from s heep l iver us ing l i noleate , coupled to an acyl
CoA generating system , as the acyl subs trate
x i i i
6 9
7 0
7 1
7 2
7 4
7 6
7 9
8 0
8 1
8 2
