The intracellular site of synthesis of fructose 1,6-bisphosphatase in rat liver : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University

Copyright is owned by the Author of the thesis. Permission is given for

a copy to be downloaded by an individual for the purpose of research and

private study only. The thesis may not be reproduced elsewhere without

the permission of the Author.

THE I NTRACELLULAR S ITE OF SYNTHES IS OF FRUCTOSE 1 , 6 - B I SPHOSPHATASE I N RAT L IVER

A thes i s presented in parti al ful fi l ment of the req u i rements

for the degree o f Doctor of Ph i l osophy in B i ochemi stry at MASS EY UNIVERS ITY

JOHN W I LL I AM McLEAN 1979

ii

ABSTRACT

· Fructose- 1 , 6 - b i sphospha tase ( FBPas e , E . C . 3 . 1 . 3 . 11 ) has been puri fi ed from rat l i ve r cytopl asm by a new puri fi cati on procedure . Mono s peci fi c an ti bodi es were rai sed to FBPase i n rabbi ts to enabl e the i mmunochemi cal i so l ati on and quanti tati on of FBPase i n p rotei n homogenates .

Pul se l abel l i ng of rat l i ver i n vi v o showed that the synthes i s of FBPase amounted to 0 . 89% of total sol ub l e prote i n synthe s i s . Newly synthe s i zed FBPase was found a l mos t enti rely i n l i ver cytopl asm i n con tras t to serum al bumi n whi ch was associ a ted only wi th mi crosomes , and amounted to 16 . 7%

of total mi crosomal prote i n synthe s i s .

I sol ated free and bound polysomes from l i ver synthesi zed a l most equal amounts of FBPase when i ncubated i n vi tro , whereas albumi n synthes i s wa s confi ned to bound polysomes . Premature termi na ti on o f tran s l ati on l ed to the rel ease o f parti a l prote i n trans cri pts whi ch were s peci fi cal ly i mmunopreci p i tated .

Affi ni ty- puri fi ed 125r-l abel l ed anti bodi es to FBPase and a l b umi n were used to quanti tate nas cent p rotei n chai ns on free and bound polysomes . FBPase an ti body bound to both cl asses o f po l ysome wi th al mo s t equal a ffi ni ty but al bumi n anti body was only associ ated wi th bound po l ysomes .

Poly(A) + RNA i sol ated from free and bound po lysomes by po l y ( U

)

-Sepharo s e chromatography was trans l ated i n a cel l -free protei n-synth es i zi ng sys tem deri ved from wheat germ . RNA from both cl ass es o f po l ysome synthesi zed FBPase , b u t al bumi n was onl y synthes i zed i n response to bound polysomal RN

A

. Some ful l l engths trans cri pts correspondi ng to b oth protei ns were produced i n wheat germ e xtracts , but a l arge amount o f smal l er mol ecul ar wei ght materi al was s peci fi cal l y i mmunopreci pi tated. Total trans l ation products from both cl asses of po l ysome a l so produced con s i derab l e

abnormal l y s hort mol ecu l ar wei ght mate ri a l , but only ful l l ength

trans cri pts were produced when the sys tem was programmed wi th TMV RNA.

S i nce l i ver cel l s conta i n twi ce as many bound polysomes a s free , i t has been concl uded that about 70% o f i ntracel l u l a r FB Pase synthes i s takes p l ace on bound pol ysomes . I n contras t , a l bumi n synthes i s was a lmost total ly confi ned ( >97%) to bound po lysomes and newly synthesi zed

iii

al bumi n was s egreg ated i nto m i crosomes . The parti ti oni ng o f syntheti c acti v i ty for al bumi n i s therefore a res ul t of the di s tri buti on o f mRNA codi ng for th i s protei n .

ACKNOWLEDGEMENTS

I wi s h to thank my s upervi sor , Or John W . Tweedi e for h i s encouragement throughout the course of thi s s tudy . I a l s o wi sh to acknowledge the

iv

advi ce and ass i s tance gi ven by Or Tony Cashmore of the Appl i ed B i o chemi s try Di vi s i on , DS I R , P a l me rs ton No rth . Than ks are a l so extended to the Soi l Sci ence and Dai ry Hus bandry Departments , Massey Uni vers i ty for the use

of s c i n ti l l ati on and gamma spectrometers .

AMP ATP BPB CM- DE- DNA cDNA OTT EDTA ER FBP FBPase F6P GTP Hab He pes H K

(

x

)

M HNa

(

x

)

M Lab NADP+

PB PBS PEG PFK PMSF pol y

{

A

)

+ po l y

{

A

)

­ RNA mRNA rRNA tRNA RNase SOS TCA TEMED TMV Tri s

LIST O F ABBREV I ATI ONS

Adenos i ne 5 ' -monopho s phate Adenos i ne 5'- tri phos phate Bromophenol bl ue

Carboxymethyl ­ D i ethyl ami noethyl ­ Deoxyr i bonucl e i c aci d Copy DNA '

D i thi othrei to 1

Ethyl enedi ami netetra-acet i c aci d Endo pl asmi c reti cul um

D-Fructose 1 ,6-bi sphos phate Fructose- 1 ,6-bi sphospha tase D-Fructo se 6- pho s phate Guano s i ne 5'-tri phos pha te

Immunogl obul i n heavy cha i n

N-2-Hydroxyethyl p i peraz i ne-N ' -2-ethanesu l phon i c a c i d 5 0 mM Hepes , x mM KCl , 5 mM MgC1 2 , pH 7 .4

50 mM Hepes , x mM NaCl , 5 mM MgC1 2 , pH 7 . 4 Immunog l o bul i n l i ght cha i n

N i coti nami de adeni ne di nucl eotide p hosphate

Phosphate buffer

{

10 mM Na2HP04 , 1 5 mM NaCl , pH 7 . 2

)

V

Phosphate buffered sal i ne

(

10 mM Na2HP04 , 150 mM NaCl , pH 7 . 4

)

Polyethyl ene glyco l

Phosphofructoki nase

Phenyl methyl sul pho nyl fl uori de

RNA hav i ng a pol yadenyl ate trac t at i ts 3 ' termi nus RNA l acki ng a po l yadenyl ate tract at i ts 3 ' termi nus Ri bonucl ei c aci d

Messenger RNA Ri bosoma l RNA Transfer RNA Ri bonucl ease

Sodi um dodecyl s ul phate Tri chl oroaceti c aci d

N , N , N" , N"-Tetramethyl ethyl ened i ami ne Tobacco Mo sa i c V i rus

tri s

(

Hydroxymethyl

)

ami nome thane

L IST OF FIGURES

FI GURE PAGE

3 . 1 El uti on o f fructose- 1 , 6 - bi spho sphata s e from phos phocel l ul os e wi th fructose 1 , 6-bi s phosphate

3 . 2 Non -denaturi ng gel ( 7%) el ectrophores i s o f fructose- 1 ,6-

31

bi s phosphatas e 32

3 .3 SOS gel ( 10%) el ectrophores i s of fructos e- 1 ,6-bi s phos phatase 33 3 .4 Esti mation of fructose- 1 ,6- bi s phosphatase subu n i t mol ecu l ar

wei ght by SOS gel ( 10%) el ectrophores i s 34

3 .5 Effect of pH on fructos e- 1 , 6- b i s phos phatase acti v i ty 35 4 . 1 SOS gel anal ys i s o f puri fi ed oval bumi n , rat al bumi n a nd

rabb i t gamma gl obul i n 38

5 . 1 Ouchterl ony i mmunodi ffus i o n ana lys i s o f anti sera aga i nst

ova l bumi n , rat a l bumi n , FBP�se and rab bi t gamma gl obul i n 46 5 . 2 Pur i fi cation o f anti bod i es to fructo s e- 1 ,6-b i sphosphatas e

by affi n i ty chromatography o n Sepharose-bound a nti gen 47 5 .3 Ouchterlony i mmunodi ffu s i o n a nal ys i s o f affi ni ty-pur i fi ed

anti bodi es

5 .4 Immunopreci p i tati on o f 125 I -l abel l ed a nti -fructo s e - 1 , 6-

48

bi s p hos phatase by crude gamma gl obul i n 49 5 . 5 Immunopreci pi tati on o f 125I -l abel 1ed anti �fructose- 1 ,6-

bi s phos phatas e by aff i n i ty-puri fi ed a n ti body 50 5 . 6 SOS gel ( 10% ) analys i s of aff i ni ty-pur i f i ed anti bodi e s 5 1 6 . 1 SOS gel ( 10%) analys i s of FBPase i mmu nopreci pi tated from

l i ver po s t-mi crosomal s upernatant after a 24 hou r i nter-

peri to neal l abel l i ng w i th c35sJmethi o n i ne 58

FIGURE PAGE 6 . 2 SOS gel ( 10%) ana l ys i s o f a l bumi n immunopreci pi ta ted from

s erum a fter a 24 hour i nterperi to neal l abel l i ng wi th c 35sJmethi oni ne

6 . 3 SOS gel ( 10% ) anal y s i s o f oval bumi n added to and immune­

prec i p i tated from l i ver pos t-mi crosomal supernatant after

59

a 24 hour i nterperi toneal l abel l i ng wi th [35SJmeth i o n i n e 6 0 6 .4 Semi l o g pl ot of mol ecu l ar wei ght versus rel ati ve mi grati o n

for protei n subun i ts i n a l bumi n , FBPase and oval bumi n immunoprec i pi tates o n 10% SOS gel s

6 . 5 Fractio nation scheme for the i so l ati on of s ubcel l ul ar fractions from rat l i ver

6 . 6 SOS gel ( 7 % ) analys i s o f TCA-preci p i tabl e pro tei n i n l i ver s upernatan t and mi crosome fract i o ns after a 10 mi nute pul se

6 1

63

l abel of c35sJmeth i o n i n e vi a the portal vei n 65 6 . 7 SOS gel ( 7% ) ana l ysi s o f FBPase i mmuno preci p i ta ted from

l i ver supernatant and mi cro some fracti ons after a 10 mi nute

pul se l abel of c 35sJmeth i o ni ne v i a the portal vei n 67 6 .8 SOS gel ( 7% ) analys i s o f al bumi n immuno preci pi ta ted from

l i ver supernatant and mi cro some fracti ons after a 10 mi nute

pul se l abel of [3 5sJmeth i o n i ne v i a the portal vei n 68 7 . 1 I so l a t i o n scheme for free and bound po lysomes from rat l i ver 74 7 . 2 Sedimentation pro fi l es of free and bound pol ysomes o n

i so ki net i c sucro s e g radi ents 75

8 . 1 B i nd i n g o f 125! - l a bel l ed anti bodi es to free polysomes 83 8 . 2 B i ndi ng o f 125! - l abel l ed anti bod i es to bound polysomes 84

/

FIGURE PAGE 8 . 3 Effect o f RNase o n t h e b i nd i ng o f 1 251 -l abel l ed anti bod i es

to bound pol ysomes

8 .4 B i nd i ng o f di fferent concentrati o ns o f 1 251 -l a bel l ed anti ­ a l bum i n and anti -fructose-1 , 6 -bi s phos phatas e to free and

85

bound pol ysomes 86

8 . 5 Rel a t i ve amount o f 1 25!-l abel l ed a nti -fructo s e - 1 ,6- b i spho s phatase bi nd i ng to free and bound po l ysomes

8 . 6 Rel ati ve amount o f 1 251-l abel l ed anti-al bumi n b i ndi ng to free and bound pol ysomes

8 .7 B i nd i n g o f 125 r-l a bel l ed ant i -al bum i n and anti - fructo se- 1 , 6- b i spho sphatase to d i fferent concentrati ons o f free and bound pol ysomes

9 . 1 Time course of prote i n synthes i s o n i sol ated free and bound pol ysomes i ncubated i n v i tro

9 . 2 SOS gel

(

1 0%

)

anal ysi s of TCA-preci pi tabl e pro te i n produced i n free and bound pol ysome i ncubati ons

9 .3 SOS gel

(

10%

)

analysi s of fructo se - 1 , 6-bi spho sphatase

87

88

89

96

97

immuno preci pi tated from free and bound polysome i ncubati o ns 98 9 .4 SOS gel

(

10%

)

anal ys i s o f al bumi n i mmunopreci pi tated from

free and bound po l ysome i ncubat i ons

10 . 1 Radi ochemi ca l pur i ty anal ys i s o f L-c35sJmeth i oni ne by TLC

99

on cel l ul ose 1 15

1 0 . 2 Formamide gel ana l ys i s o f TMV RNA 1 16 10 . 3 Time course o f protei n synthes i s a nd effect o f RNA

concentration on the transl ation o f TMV RNA i n wheat germ 1 1 7 extracts

FIGURE

10 . 4 Effect o f K+ a nd Mg2+ concentration o n the transl ation o f

PAGE

TMV RNA i n wheat germ extracts 118

10 . 5 SOS gel ( 7 % ) analysis o f transl ation products produced i n wheat germ extracts programmed wi th TMV RNA

10 . 6 I so l ation o f pol y(A)+ RNA by chromatography o f rat l i ver pol ysomal RNA on pol y ( U ) -Sepharo se

10 . 7 Non-denaturi ng gel (2.4%) anal ysi s of pol y(A) + a nd pol y (A)­

RNA from free pol ysomes

10 .8 Non-denatur i ng gel (2 .4%) anal ys i s of poly(A) + and pol y( A ) -

119

120

121

RNA from bou nd pol ysomes 122

10 . 9 T i me course of pro tei n synthes i s and effect of s perm i ne concentration on the transl ation o f poly(A)+ RNA i n whea t

germ extracts 123

10 . 10 The effect o f K+ and Mg2+ concentrati ons on the transl ation

o f pol y(A) + RNA from total pol ysomes i n wheat germ extracts 124

10 . 11 Effect of RNA concentration on the translation o f pol y(A) + and po l y(A) - RNA from free and bound pol ysomes i n wheat germ

extracts 125

10 . 12 SOS gel (7%) anal ysi s of rel eased products produced i n wheat germ extracts programmed wi th pol y (A) RNA from free and +

bou nd pol ysomes 126

10 . 13 SOS gel (7%) analys i s o f r i bosome-bound trans l at i on products produced i n wheat germ extracts pro grammed wi th pol y(A) + RNA from free a nd bound pol ysomes

10 . 14 SOS gel ( 10%) anal ysi s of transl ation products produced i n wheat germ extracts pro grammed wi th pol y(A)+ RNA from free a nd bound pol ysomes which were i mmunopreci pi tated wi th ant i ­ fructose-1 , 6-b i sphosphatase

127

129

FIGURE PAGE 10 . 15 SDS gel ( 10%) analysi s o f tra ns l atio n products produced i n

wheat germ extracts programmed wi th pol y (A) + RNA from free and bound pol ysomes wh ich were immuno preci p i ta ted wi th anti­

a l bumi n 130

LIST O F TABLES

TABLE PAGE

3 . 1 Puri fi cati on o f fructose- 1 , 6-bi s phosphatase from rat l i ver 30 5 . 1 Anti body puri fi cati on by affi ni ty chromatography 45

5 . 2 Anti body quanti tati on 48

6.1 Inmunoprec i p i tat i on of a l b umi n from serum, FBPase and carri er oval bumi n from l i ver pos t-mi crosomal s upernatant a fter a 24 hou r i nterperi toneal l abel l i ng wi th

[35s Jmeth i on i ne 62

6 . 2 Di s tr i buti on o f TCA-preci p i ta b l e protei n i n l i ver subcel l u l ar fracti ons after a 10 mi nu te pul se l abel o f

[35s Jmethi oni ne v i a the portal vei n 64

6,3 Di s tri buti on o f radi oacti vi ty i n a l b umi n and FBPase i mmu nopreci pi tates from l i ver superna ta nt and mi crosome fracti ons after a 10 mi nute pul s e l abel of [ 35s Jmeth i oni ne

v i a the porta l vei n 66

7.1 Sed i mentati on di s tri bution o f polysome s i ze cl as ses 9 . 1 Di s tri buti on o f i mmunopreci p i tabl e al bumi n and fructose-

1,6 -b i s phosphatase pepti des synthes i zed by i sol ated free and bound po lysomes i n v i tro

76

100 10 . 1 Di s tr i bution o f i mmunopreci p i tabl e al bumin and fructos e- 1 , 6-

bi sphos phatase pepti des produced in wheat germ extracts

programmed wi th poly

(

A

)

+ RNA from free and bound pol ysomes 128

ABSTRACT

ACKNOWLEDG EMENTS LIST OF ABBREV IATIONS LIST OF F I GURES

LIST OF TABLES

TABL E OF CONTENTS

CHAPTER 1 1 . 1

I NTRODUCTION AND H I STOR I CAL REV I EW

The i ntracel l u l ar s i tes o f protei n synthesi s i n animal cel l s

1 . 2 The segregation o f secreto ry pro tei ns duri ng pro tei n

PAGE

ii iv

V

vi xi

1

synthes i s 5

1 . 3 Obj ectives of th i s i nvesti gation 10

1 .4 The di scovery and i so l ati on of fructose-1 , 6-

bi s pho s phatase 1 1

1 .5 Physi cal and enzymi c properti es of fructo se-1 , 6 -

b i sphosphatase 1 2

1 . 6 Meta bol i c regul ation by fructo s e- 1 ,6-bi sphos phatase

in

v i vo

CHAPTER 2 GENERAL METHODS

14

2 . 1 I ntroductio n 18

2 . 2 Experimental animal s 18

2 . 3 Centri fuga t i o n 18

2 .4 Ul trafi l tration 18

2 . 5 Spectrophotometry 19

2 . 6 Protei n and RNA determ i nati on 19

2 .7 Po l yacryl ami de gel el ectrophore s i s of protei n s

2 . 7 . 1 Di s conti nuou s non-denatu r i ng gel s 19 2 . 7 . 2 Sod i um dodecyl sul phate conti nuous denatur i ng

gel s 20

2 .8 Es tima tion of protei n subun i t mo l ecul ar wei ghts 2 1 2 . 9 Polyacryl amide gel el ectro phores i s o f RNA

2 . 9 . 1 Conti nuous non -denaturi ng gel s 22

2 . 9 . 2 Formamide denatu r i ng gel s 23

2 . 10 Determ i nation of radioact i v i ty

2 . 10.1 Gel sl i ces 24

2 . 10 . 2 Tri chl oroaceti c aci d- preci pi tabl e protei n 24

CHAPTER 3 PURI F I CATION O F FRUCTOSE- 1 , 6-BI SPHOSPHATASE FROM RAT L IVER

3 . 1 I ntroduction 3 . 2 Mater ial s 3 . 3 Methods

3 . 3 . 1 Assay o f fructose- 1 ,6-bi s phospha tase

3 . 3 . 2 Pur i f i cation o f fructose- 1 , 6- b i s pho sphata s e 3 . 3 . 3 Treatment of anima l s wi th tri amci nol one

al l oxan

3 . 3 .4 Quanti ta t i o n o f pur i f i ed enzyme protei n 3.4 Resu l ts and D i scussion

CHAPTER 4 PUR I FICATIO N O F OVALBUM I N , RAT ALBUM I N AND SHEEP GAMMA GLOBUL I N

4 . 1 Introducti on 4 . 2 Material s

4 . 3 Methods and Res u l ts 4 . 3 . 1 Ovalbumi n 4 . 3 . 2 Rat a l bum i n

4 . 3 . 3 Rabb i t gamma g l o bu l i n

CHAPTER 5 ANTI BODY PRODUCTI ON AND PUR I FI CATION 5 . 1 I ntroduct i o n

5 . 2 Mater i al s 5 . 3 Methods

5 . 3 . 1 Immu n i zation pro tocol s

5 . 3 . 2 Sepa ra t i o n o f gamma gl obul i n from serum 5 . 3 . 3 Anti body speci fici ty

5 . 3 .4 Preparation of Sepharo se-bound antigen aff i ni ty col umns

5 . 3 . 5 Aff i n i ty chroma tography 5 . 3 . 6 Anti body ti tre

5 .4 Res u l ts and Di scuss i o n

C HAPTER 6 LABELL I NG OF L I V ER PROTE I NS I N V IVO 6 . 1 I ntroductio n

6 . 2 Materia l s 6 . 3 Methods

and

PAGE

25 25 26 26 27 28 28

36 36 36 36 37

39 39 39 40 40 41 41 42 42

52 53

6 .4 CHAPTER 7 7 . 1 7 . 2 7 .3

7 .4 CHAPTER 8 8 . 1 8 . 2 8 . 3

8 .4

6 . 3 . 1 Interperi toneal

(

24 hour

)

l abel l i ng 6 .3 . 2 Intraportal

(

10 mi nute

)

l abel l i ng 6 . 3 .3 Anal ys i s o f l abel l ed products 6 . 3 .4 Immunoprec i p i tati o n assay Resul ts and Di scus s ion

POLYSOME ISOLATION Introducti o n

Materia

i

s Methods

7 . 3 . 1 Preparation o f free and bound pol ysomes 7 . 3 . 2 Preparati o n of total pol ysomes

7 . 3 . 3 Pol ysome sedimentation analys i s Resul ts and

D

i scus s ion

125I-LABELLED ANTIBODY BINDING TO POLYSOMES Introducti on

Mater i a l s Methods

8 . 3 . 1 Pol ysome preparati o n 8 . 3 . 2 Ant i body iodi nati o n

8 . 3 . 3 Pol ysome b i nd i ng experiments Res u l ts and Di scuss ion

CHA PTER 9 PROTEIN SYNTHESIS BY ISOLATED POLYSOME$ IN VITRO

PAGE 53 54 54 55 55

69 70 70 72 72 72

77 78 79 79 79 80

9 . 1 Introduction 90

9 . 2 Mater i a l s 91

9 . 3 Methods

9 . 3 . 1 Cel l sap preparati o n 9 . 3 . 2 Transl a t i o n assays

9 . 3 .3 Analysi s o f trans l ation products 9 . 3 .4 Immunoprec i pi tati on assay

9 .4 Resul ts and Di scu s s ion

CHAPTER 10 PROTEIN SYNTHESIS IN WHEAT GERM EXTRACTS IN VITRO

91 92 92 92 93

10 . 1 Introduction 101

10 . 2 Materia l s 10 . 3 Methods

102

10 . 3 . 1 10 . 3 . 2 10.3 . 3 10 . 3 .4 10 . 3 . 5 10 . 3 . 6 10 . 3 . 7 10 . 4 Res u l ts

PAGE Radi ochemical pur i ty o f L-c35sJmeth i o n i ne 103 Preparati o n of Tobacco Mo saic V i ru s RNA 103 Extracti o n o f pol y( A ) + RNA from pol ysomes 104 Preparati on of wheat germ extracts 106

Transl ati o n assays 106

Anal ysi s o f transl ati on products 107

Immunoprec i p i tation a ssay 107

a nd Di scu s s i on 108

CHAPTER 11 DISCUSSION 131

BIBLIOGRAPHY 136