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ACETYL-CoA CARBOXYLASE IN PHOTOSYNTHETIC T I S SUE

A the s i s pre sented in parti al ful f i lment of the

requirements for the degree of Doctor of Philo sophy

in B iochemi stry at

MASSEY UNIVERS ITY

BASIL JOHN N IKOLAU

1 9 8 1

i i

ABSTRACT

Acetyl -CoA carboxylase catalyses the rate-limit ing reaction

in de novo f atty acid biosynthesis in a wide var iety of organisms. I n plants however , the signifi cance of this

enzyme in regulating de novo fatty ac id biosynthesis is

unknown.

In th is invest igat ion acetyl-CoA carboxylase of ma i z e , barley

and spinach leaves has been studied in order to compare some

features of this enzyme in the three pl ants . In mai ze ,

acetyl-CoA c arboxylase is located in chloroplasts , where i t occurs as a soluble enzyme in the stromal fraction . Both

mesophyll and bundle sheath ce l ls also exhibit acetyl -CoA

carboxylase activity . Activities of acetyl-CoA carboxylase

in isol ated c hloroplasts have been compared to rates of l ipid

synthesis from acetate in orde r to investigate the ro le o f acetyl-CoA carboxyl ase i n regul ating de novo fatty ac id bio­

synthesis from acetate . Although acetyl -CoA carboxylase

activity was higher than that expected from the rate of

acetate incorporat ion into l ip ids of isolated chloroplasts o f mai ze and bar ley , the opposite was found for chloroplasts

isolated from spinach . In chloroplasts from the emerg ing

leaves of ma i ze seedl ings , l ipid synthesis was max imal with chloroplasts isolated from the leaf segment corresponding to

leaf greening . However , a relationship between leaf deve lop­

ment and acetyl -CoA carboxylase activity was less apparent .

Consequently a regul atory role for acetyl-CoA carboxylase in de � fatty acid biosynthesis could not be d irectly estab­

l ished from a compar ison of the rates of l ipid synthesis and

acetyl-CoA c arboxyl ase act iv i t ies .

An alternative approach to the establishment of a regulatory

role for acetyl -CoA carboxylase was to purify the enzyme

from mai ze and barley leaves , and study its response to var i ­

able concentrations o f substrates , products and certain

cellular metabolites . Pur i ficat ion of both enzymes was per-

lll

formed by identi c a l procedure s , includ ing polyethylene glycol

fract i onation , hydrophobic chromatography and ge l fi ltration .

Acetyl -CoA carboxylase from b?th mai z e and barley leave s

appeared to be an integral enzyme , a s no evidence for its

d i s sociation was found , contrary to the find i ngs of the

bacteri a l enzyme . The kinet ic properties o f the partially

puri fied enzyme from mai ze and bar ley were very similar . The

apparent Michael i s constants for the substrat e s , acetyl-CoA and HCO� , were about O . lmM and 2mM , respective ly for both

enzyme s . The enzymically active form of the substrate , ATP , was found to be Mg . ATP . Furthermore , free ATP inhibited

enzymic activity , while f ree Mg 2 + act ivated the enzyme from

both p l ant source s . Monovalent cations , particular ly K+,

were pos itive e f fectors o f acetyl-CoA carboxyl ase , on the

other hand , the products o f the acetyl -CoA c arboxylase re-

action , malonyl -CoA and ADP were inhibitor s . ADP inhibit ion

was compet itive with respect to ATP , but uncompetitive with

respect to acetyl-CoA . I nh ibition of acety l -CoA carboxylase

activ i ty by CoA was noncompet it ive with re spect to acetyl­

CoA , while palmitoyl-CoA inhibition wa s uncompetit ive with

re spect to acetyl-CoA .

From the view point of regulation o f acetyl -CoA carboxylase

act iv i ty , the observed response o f activity to change s in pH ,

and the concentrat ions o f Mg2+, K

+ , ATP and ADP in vitro , may be o f s ign i f i c ance . Changes in the chloroplastic leve l s of

the se e f fecto r s of acety l-CoA carboxylase act ivity have been reported to occur during l ight-dark tran s i tion of chloro­

plasts , and would be cons i stent in regulating acetyl -CoA carboxylase a c tivity in the light and dark .

iv

ACKNOWLEDGEMENTS

I wi sh to thank my superv i sor , Dr J C Hawke , for his invalu­able advice , encouragement and a s s i s tance throughout the

cour se o f thi s study . I am a l so indebted to Dr C R S lack of

the Plant Physiology Divis ion , DSIR , for hi s advice and

a s s i stance , particularly dur ing the absence of Dr Hawke .

I would l ike to thank Mr I J Warr ington of the P l ant

Phys iology Divi s ion , DS I R , for helpful advice in the use o f

the c l imate control rooms for the growing o f mai ze and

barley plants .

In add it i on , particular thank s i s extended to Ms Joy Watson

for typi ng this manuscript .

ACP

ADP

AMP

ATP

BCCP

BSA

chl

P-CMB

CoA

DTT

EDTA

Hepe s

Mes

NADH

NADPH

NEM

PEG

PEP

2 -PGA

3 -PGA

LIST OF ABBREV IATIONS

acyl-carrier prote i n

adenos ine 5'-diphosphate

adenosine 5'-monophosphate

adenos ine 51-tr iphosphate

biotin carboxyl-carrier protein

bovine serum albumi n

chlorophy l l

P-chloromercuriben z oate

coenzyme A

d ithiothre itol

ethylenedi amine tetraace tic ac id

N-2-hydroxyethylpiperaz ine-N'- 2�ethane sulphonic

ac id

2[ N-Morphol i no] ethane sulphonic ac id

V

8-nicotinamide adenine dinucleotide , reduced form

n icotinamide adenine dinuc leotide pho sphate , re­

duced form

N -ethylmale imide

pol�t hylene glycol

phosphoeno lpyruvate

2 -phosphoglyceric ac id

3 -phosphoglyceric ac id

PO POP

ppGpp

PPO

pppGpp

RNA

RuDP

TEMED

TLC

Tr i c in e

Tr i s

1 , 4 -bi s[2 ( 5 -phenylox a z o l y l )]Benzene

guano s ine 5 ' -d ipho sphate- 3 ' -d ipho sphate

2 , 5 -diphenyloxazole

guanos ine 5 ' -triphosphat e- 3 ' -d ipho sphate

r ibonucleic ac id

ribu lose 1 , 5 -diphosphat e

N , N , N ' , N'-Tetramethylethy lened iamine

thin-layer chromatography

N -tris[Hydroxymethyl]-methyl glyc ine

t r i s ( hydroxymethyl ) aminomethane

vi

vii

TABLE OF CONTENTS

AB STRACT i i

ACKNOWLEDGEMENTS iv

L I ST OF ABBREVIAT IONS

TABLE OF CONTENTS

L I ST OF F I GURES

LIST OF TABLES

CHAPTER 1 INTRODUCT ION

1 . 1 General Background

V

Vll

x i i i

xvi i i

1

1 . 2 Acety l -CoA Carboxylase from An imal Sources

1 . 2 . 1 Mo lecu lar prope rties 2

1 . 2 . 2 Regu lat ion o f cata lytic activity

1 . 2 . 2 . 1 Long-term control o f

acetyl-CoA carboxylase 2

1 . 2 . 2 . 2 Short-term control of

acetyl -CoA carboxy lase 4

1 . 3 Acetyl -CoA Carboxylase from Escherichia col i

1 . 3 . 1 Molecular propert ies 8

1 . 3 . 2 Regulat i on o f cata lytic act iv ity 9

1 . 4 Acetyl -CoA Carboxylase from Yea st 10

1 . 5 Acet y l-CoA Carboxyl ase f rom Plant s 1 1

1 . 6 Acetyl -CoA Carboxylase from Other S ources 1 2 .

1 . 7 Acety l -CoA Carboxylase a s a B iot in Enzyme 1 4

1 . 8 Fatty Ac id Biosynthe s i s in Plants 1 5

CHAPTER 2 MATERIALS

2 . 1 P lant Mater i a l s 2 0

CHAPTER

viii

2.2 Reage nts

3 METHODS

3.1 Preparation o f Acy l -CoA Esters

3.2 En z yme Assays

3. 3 Preparation o f C e l l - free Extracts

3. 4 Preparation o f Chloropl asts

3.5 Preparation o f Me sophyl l Protop lasts and

Bund le Sheath Strand s

3. 6 Preparation o f E . co l i Acety l-CoA Carbox­

lase Component s

3.7 I ncorporation o f [ l - 1 �C ] Acetate into Lipids

3. 7. 1 [ l - 1 4C ] acetate incorporat ion into

2 0

2 2

2 3

2 5

2 6

2 6

2 8

lipids by i solated chloroplasts 2 9

3. 7. 2 [ l- 1 4C] acetate incorporat i on into

l ipids by leaf s l i ces

3. 8 Preparation of Ma i z e Lea f Section s

3. 9 Preparation o f S epharo se -N-Propane

3.1 0 Polyacry lamide Ge l Electrophere s i s

3.1 1 Centr ifugat ion

3.1 2 Ana l yti cal Methods

3.1 2. 1 Determinat i on o f prote in

3.1 2. 2 Determinat i on of chlorophy l l

3.1 3 Determination o f Radi oact ivity

3.1 4 Chromatographic P rocedure s

3.1 4. 1 Gel f i ltrat ion chromatography

3.1 4. 2 Thin�layer chromatography

3.1 4. 3 P aper chromatography

3.1 5 Pur i f i cation o f Acetyl-CoA Carboxylase

from Leave s

2 9

3 0

3 0

3 0

3 1

3 2

3 2

3 3

3 3

3 3

3 3

3 4

CHAPTER 4 RESULTS

GENERAL FEATURE S AND STAB I LITY OF LEAF

ACETYL-CoA CARBOXYLASE

4.1 General Feature s of Acetyl -CoA Carboxy­

lase Act iv ity

4.2 Stab i l i zat ion o f Acetyl -CoA Carboxylase

in Ce l l -Free Extract s by Sul fhydryl Pro­

tect ing Agent s

LOCAT I ON OF ACETYL-CoA CARBOXYLASE ACTIVITY

IN MAI Z E LEAVES

4 . 3 Locat ion of Acety l -CoA Carboxy lase in

Chlorop last s o f Ma i z e Leave s

4. 4 Solub i l ity o f Ma i z e Chloropl astic Acetyl­

CoA Carboxylase

4 . 5 Location o f the B iot in Prosthetic Group

ix

3 6

4 2

4 6

4 8

o f Chloroplast i c Acetyl -CoA Carboxylase 5 1

4. 6 Distr ibut ion o f Ac ety l -CoA Carboxyl a s e

Between Mesophy l l and Bund le She at h C e l l s

o f Mai z e Leave s

LEVELS OF ACETYL-CoA CARBOXYLASE ACTIVITY IN

LEAVES

4 . 7 I nve st igat ion o f the Re lat ionship B etween

Leaf Deve lopment , Acetyl -CoA Carboxylase

Activity and [ l - 1 4C] acetate Incorpor ation

5 5

into Lipids 5 5

4. 7.1 I ncorporat ion o f [ l- 1 ljC] acetate

by ma i z e l e a f s l ic e s 6 0

4. 7. 2 I ncorporat i on o f [ l - 14 C J acetate

into l ip i d s by i solated chloro­

p l a sts from deve loping ma ize

l eave s

4.7.3 Acetyl -CoA carboxylase activity

in c e l l - free extract s of deve lop­

ing mai ze l eaves

6 0

6 2

4 . 7 . 4 Acety l -CoA carboxylase act ivity in

isolated chloropla st s from deve lop­

ing ma i z e leave s

4 . 8 Acetyl-CoA Carboxyla se Activity and Lipid

Synthe s i s in Leave s o f Ma i z e , Barley and

X

6 5

Spinach 6 5

4 . 9 E f fect o f Light on Ac etyl-CoA Carboxylase

Act ivity in Ma i z e Leave s

PURIFICATI ON OF LEAF ACETYL-CoA CARBOXYLASE

4 . 1 0 Pur i f ic at ion o f Acety l -CoA Carboxy l a s e

from Ma i z e Leave s

4 . 1 1 Pur i f i c at ion o f Ac etyl -CoA Carboxy lase

from Barley Leave s

K I NET IC PROPERT IES OF ACETYL-CoA CARBOXYLASE

4 . 1 2 E f fect of Acety l -CoA and HC03 Concentra-

7 0

7 0

8 3

t ions on Acetyl -CoA Carboxylase Act ivity 8 4

4 . 1 3 E f f ect o f ATP and MgC12 Concentrat ions

on Acety l -CoA Ca rboxylase Act iv ity

4 . 1 4 Spec i f i c ity of the Divalent Metal Ion

Requi red for Acety l-CoA Carboxylase

Act ivity

4 . 1 5 E f fect of pH on Acetyl -CoA Carboxylase

Act ivity

4 . 1 6 E f fect of Temperature on Acetyl -CoA

Carboxyl a se Act ivity

4 . 1 7 Act ivation of Acetyl -CoA Carboxylase

Activity by Monova l ent Cat ions

4 . 1 8 S ubstrate Spec i f i c ity of Acetyl-CoA

Carboxy l a s e

4 . 1 9 I nhibition o f Ma i z e L e a f Acetyl - CoA

Carboxylase by Ma lonyl -CoA

9 2

1 0 5

1 0 5

1 0 7

1 0 7

1 1 1

1 1 1

4 . 2 0 Effect o f ADP on Acety l -CoA Carboxy lase

Act ivity

4 . 2 1 E f fect of CoA on Acetyl -CoA Carboxyl a s e

Act ivity

4 . 2 2 E f f ect of Palrnitoyl -CoA on Acetyl -CoA

Carboxy l a s e Act ivity

4 . 2 3 E ffect of AMP , Pho sphoenolpyruvate ,

Oxaloacet ate and Citrate on Acetyl -CoA

Carboxyla s e Act ivity

4 . 2 4 E f f ect o f Sul fhydryl Reacting Reagent s

on Acetyl -CoA Carboxylase Act ivity

CHAPTER 5 DI SCUS S I ON

5 . 1 Acetyl-CoA Carboxylase - it s General

Feature s a nd Stabi l ity

5 . 2 Cellular Location o f Acetyl-CoA Carboxy­

lase in Ma i ze Le ave s

5 . 3 Re l ationship Between Acetyl-CoA Carboxy­

l ase Act iv ity and Lipid Synthe s i s in

I so l ated Ch loropl a st s

5 . 4 Re lation sh i p Between Acetyl-CoA Carboxy­

lase Act ivity , Lipid Synthe s i s and Lea f

Deve lopment

x i

1 1 6

1 1 6

1 2 5

1 2 5

1 2 8

1 3 0

1 3 2

1 3 5

1 3 7

5 . 5 Pur i f icat ion o f Acety l -CoA Carboxylase 1 3 9

5 . 6 K inetic Properties o f Acetyl-CoA Carboxy-

lase

5 . 6 . 1 E f f e ct of sub strate s on acety l -CoA

carboxylase 1 4 2

5� 6 . 2 Sub strate spec i f icity o f acetyl -

CoA c arboxy lase

5 . 6 . 3 E f fectors o f acety l-CoA c arboxy­

l a s e a ctivity

5 . 7 L i ght-Dark Modulation o f Acetyl - CoA

Carboxy l a s e Act ivity

1 4 3

1 4 4

1 4 6

5 . 8 Summary and Sugge st ions for Further

Study

BIBLIOGRAPHY

x i i

1 4 8

1 5 1

x i i i

LI ST O F FIGURE S

F igure Page

1 Structura l c l a s s i f ication of biot in c ontaining

e n zyme s

2 I dent i f icat i on o f the product of acetyl -CoA

c arboxy l a s e as say by TLC

3 Identificat ion o f the product of the acety l -

1 6

3 7

CoA carbox y l ase a s say by paper chromatography 3 8

4 Dependence o f acetyl -CoA c arboxylase activity

on protein l eve l s of cell- free extracts o f

mai ze leave s

5 E f fect o f t ime on acetyl-CoA carboxyl a s e

4 0

act ivity i n c e l l - free extracts of ma i z e leaves 4 1

6 Stab i l i z at ion o f acetyl -CoA carboxylase act iv­

ity in ce l l - free extracts of ma ize by 8 -mer-

c aptoethano l 4 3

7 Stabi l i zat i on o f acetyl -CoA carboxylase act iv­

ity in cel l - free extracts of barley by 8 -mer­

c aptoethano l

8 D i stribut ion o f enzymes , chlorophy l l and pro­

te in fol lowing the centrifugat ion of pur i f ied

ma i ze chloropl ast s in a suc ro s e density

gradient

9 Repre sentat ive f i e ld o f a preparation o f i so­

l ated mai z e chloroplasts

4 4

4 7

4 9

x iv

Figure Page

1 0 D i str ibut ion o f enzyme s , chlorophy l l and

prote in f o l lowing the centri fugation of d i s ­

rupted ma i z e chlorop l asts i n a di scont i nuou s

sucro se den s ity gradient 5 0

1 1 Representat ive f i e ld o f a preparation o f iso­

l ated mai z e me sophyl l protopl ast s

1 2 Representat ive f i e ld o f a preparation o f mai z e

5 8

bundle sheath strands 5 9

1 3 E f fect o f l ight on the activity o f acety l -CoA

c arboxyla s e in ma i z e leave s

1 4 E lut ion o f a c etyl -CoA carboxylase of ma i z e from

S epharo se-N -Propane

1 5 Ge l f i ltrat ion o f acety l -CoA carboxy l a s e o f

ma i z e on U lt rogel AcA2 2

1 6 Ge l f i ltrat i on o f acetyl -CoA carboxy l a s e o f

mai z e on U ltrogel AcA2 2

1 7 Polyacrylamide gel e l ectropher e s i s o f acetyl ­

CoA c arboxyl as e o f ma i z e l eave s

1 8 Rel at ionship b etween HC03 , acetyl-CoA concen­

trat i ons and the activity o f ma i ze leaf

acet y l-CoA c arboxylase

1 9 Re l at ionship between HC03 , acety l-CoA concen­

trat ions and the activity o f bar l ey leaf

acetyl -CoA carboxylase

2 0 Lineweaver -Burk analy s i s o f the e f fect o f

acetyl -CoA on the activity o f maize leaf

acet y l-CoA c arboxy lase

7 1

7 7

7 8

8 0

8 1

8 6

8 7

8 8

XV

Figure Page

2 1 Lineweaver -Burk analy s i s o f the e f fect o f

acety l -CoA o n t h e act ivity o f bar ley l e a f

acety l - CoA carboxy lase

2 2 Re l at ionshi p between HC03 , acety l -CoA concen­

trat ions and the activity o f ma i z e leaf

acety l - CoA carboxyl ase

2 3 Re lat ionship be tween HC03 , ac etyl -CoA conc en­

trat ions and the activity of bar ley l e a f

acetyl -CoA c arboxylase

2 4 Lineweaver -Burk analy s i s o f the e f fect o f

HCO� o n the activity o f ma i z e leaf acetvl-CoA �

carbox y l a s e

2 5 Lineweaver -Burk a naly s i s o f the e f fect o f

HCO� on the activity of bar ley leaf acetyl -CoA

carboxylase

2 6 Effect o f MgC 1 2 o n the act ivity o f ma i z e leaf

acety l - CoA carboxylase

2 7 Effect o f MgC l 2 o n the act ivity o f barley l e a f

acetyl -CoA carboxylase

2 8 Ef fect o f ATP o n the act ivity o f ma i z e leaf

acety l - CoA c arboxylase

2 9 E f fect o f ATP on the activity o f bar ley leaf

acety l-CoA carboxy lase

3 0 Re lationship between ATP , MgC l 2 concentration s

and t h e activity o f mai z e l e a f acety l - CoA

carbox y l a s e

8 9

9 0

91

9 3

9 4

9 5

9 6

9 7

9 8

1 0 0

xvi

Figure Page

3 1 Re l ation ship between ATP , MgC l2 concentrations

and the act ivity of barley leaf acety l -CoA

carboxy l a s e

3 2 Ana ly s i s b y Hil l -plot of the relation s h ip be­

tween ATP , MgC12 concentrat ions and the

activity of ma ize l e a f acetyl -CoA carboxylase

3 3 Ana ly s i s by Hill-plot o f the re lat ions h ip be­

tween ATP , MgC l2 concentrat ions and the activ-

1 0 1

1 0 2 , 1 0 3

i ty o f bar l ey leaf acetyl-CoA carboxyl a s e 1 0 4

3 4 E f fect o f d iva lent metal ions o n the a c t ivity

o f ma i z e l ea f acety l -CoA carboxylase

3 5 E ffect o f pH on the act ivity o f ma i z e l e a f

acety l -CoA c arboxy l a se

3 6 E f fect o f pH on the act ivity o f bar l ey l e a f

acetyl -CoA carboxyl a s e

3 7 E f fect o f temperature on the activity o f ma i z e

1 0 6

1 0 8

1 0 9

l e a f acetyl -CoA c arboxylase 1 1 0

3 8 E f fect o f KCl on the activity o f ma i z e l e a f

acety l -CoA carboxylase

3 9 Spec ific ity of carboxylation by ma i z e l e a f

a c e tyl -CoA carboxylase

4 0 E f fect o f malonyl-CoA on the activity o f mai z e

l e a f acetyl -CoA carboxylase

4 1 ADP inhibition o f the ac tivity o f ma i z e l e a f

acetyl-CoA carboxylase

1 1 2

1 1 4

1 1 5

1 1 7

xv ii

F igure Page

4 2 L i neweaver-Burk analys i s o f the e f fect o f ADP

on the activity o f ma i z e leaf acety l-CoA

c arboxylase

4 3 Ana l y s i s by Dixon-plot o f the e f fect o f ADP

�n the act ivity o f ma i z e leaf acety l - CoA

c arboxylase

4 4 ADP inhibi t ion o f the a ctivity o f ma i z e l e a f

acety l -CoA c arboxyl a s e

4 5 L ineweaver-Burk ana l y s i s of the e f fect o f ADP

o n the act ivity o f ma i z e leaf acetyl-CoA

c arboxylase

4 6 CoA inhibition o f the activity o f ma i z e leaf

acetyl -CoA carboxyl ase

4 7 L ineweaver-Burk ana ly s i s of the ef fect o f CoA

o n the act ivity of ma i z e leaf a cety l - CoA car-

1 1 8

1 1 9

1 2 0

1 2 1

1 2 2

boxylase 1 2 3

4 8 Ana l y s i s by D ixon-plot o f the e f fect o f CoA

o n the activ ity o f ma i z e leaf acety l - CoA car-

boxy l ase 1 2 4

4 9 P a lmitoyl -CoA inhibition of the activity o f

ma i z e leaf acetyl-CoA c arboxylase

5 0 L i neweaver -Burk analys i s of the e f f ec t o f

pa lmitoyl-CoA on the activity o f ma i z e leaf

acetyl-CoA c arboxylase

1 2 6

1 2 7

xv i ii

Table

I

L I ST OF TABLES

Protomer-polyrner tran s i t ion of an imal

acetyl -CoA carboxylase

I I Co f actor requ irements o f acetyl-CoA c arboxy-

5

l a s e activity in cel l - free extracts o f ma i z e 3 9

I l l Ace ty l -CoA c arboxylase ac t ivity i n c e l l - free

extracts o f sp inach l eave s

IV

V

Preparation o f biot in c arboxylase from E . coli

Co f ac tor requ i rements for the carboxylation of

the biotin pro s thetic group in ma i z e chloro­

p l a s t s by biot i n c arboxylase of E. col i

VI Ma l onyl -CoA format ion from carboxyl -biotin by

mai z e chloropl a st s

VI I D i s t r ibut ion o f the biotin pro sthet ic group in

chloroplasts i so l ated from the leave s o f three

4 5

5 2

5 3

5 4

p l an t s 5 6

VI I I D i s tribution o f en zymes between me sophy l l proto-

p l a s t s and bundle sheath s trand s 5 7

IX E f fe c t of leaf deve lopment on [l - 1 4 C ] acetate

incorporation into l ip i d s by leaf s l i c e s of

ma i z e ·

X E f fect of l ea f deve lopment on [l - 1 4C ] acetate

incorporat ion i nto l ipids by i sol ated chloro­

p l a s t s of mai z e

6 1

6 3

Tabl e

XI Acety l-CoA carboxylase act ivity in c e l l - free

extracts obta ined from section s of maize

leave s

XI I Acetyl -CoA carboxylase act ivity in c hloro-

xix

6 4

pl ast s isolated from sect i ons o f ma i ze leave s 6 6

XI I I [l- 1'"'C J acetate incorporat ion into l ipids o f

chloropl asts and acetyl -CoA carboxyla se

act iv ities in c e l l - free extracts and chloro­

pl ast s from ma i z e , bar l ey and spinach

XIV Acety l-CoA carboxylase in ma i z e and spinach

leave s

XV Pur i f ication o f acetyl-CoA carboxylase of

ma i z e leave s us ing procedure I

XVI Pur i f ication o f acety l-CoA carboxy lase of ma i z e

leave s us ing proc edure I I

XVI I

XVI I I

I nte ract ion of ac etyl-CoA c arboxyl ase with

Sepharo se-N-Cx

Stabil ity of part ially pur i f ied ac etyl -CoA

carboxylase

XIX Pur i f ication o f acetyl-CoA carboxyl a s e of

barley leave s u s i ng procedure I

XX Act ivation of ma i z e lea f acetyl-CoA c arboxy­

lase by monovalent cat ions

XXI Inhibition of ma i z e leaf ac etyl -CoA c arboxy-

6 7

6 9

7 3

7 4

7 5

8 2

8 5

1 1 3

lase act ivity b y sulfhydryl reacting reagent s 1 2 9