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 requ i rements for the degree o f Doctor of Ph i l osophy in B i ochemi stry at MASSEY UN IVERS ITY JOHN W I LL I AM McLEAN 1979 ii ABSTRACT · Fructose- 1 , 6 - b i s phospha tase ( FBPas e , E . C . 3 . 1 . 3 . 11 ) has been puri fi ed from rat l i ve r cytop l asm by a new puri fi cati on procedure . Mono s pec i fi c anti bodi es were rai sed to FBPase i n rabb i ts to enabl e the i mmunochemi cal i s o l ati on and quanti tati on of FBPase i n p rotei n homogenates . P u l 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 . New ly synthe s i zed FBPase was found a l mos t enti re ly i n l i ver cytopl asm i n con tras t to serum a l bumi n wh i ch was assoc i a ted only wi th mi crosomes , and amounted to 16 . 7% of total mi crosomal prote i n synthe s i s . I so l ated free and bound polysomes from l i ver synthesi zed a l most equal amounts of FBPase when i ncubated i n v i 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 re l ease o f parti a l prote i n trans cri pts wh i ch were s pec i fi cal ly immunopreci p i tated . Affi ni ty- pur i fi ed 1 25r- l abel l ed anti bod i 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 a l mo st equal a ffi n i ty but al bumi n anti body was only associ ated wi th bound po l ysomes . Po ly (A) + RNA i sol ated from free and bound po lysomes by po l y ( U ) -Sepharo se chromatography was trans l ated i n a ce l l -free protei n-synthes i z i ng sys tem deri ved from wheat germ . RNA from both cl as s es o f po l ysome synthes i zed FBPase , b u t a l bumi n was onl y synthes i zed i n response to bound polysoma l RNA . Some fu l l l engths trans cri pts correspondi ng to b oth prote i ns were produced i n wheat germ extracts , b ut a l arge amount o f smal l er mol ecu l ar wei ght mater i a l was s pec i fi cal l y i mmunopreci p i tated . Tota l trans l at ion products from both c l a s ses 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 tota l ly confi ned (>97%) to bound po lysomes and new ly synthes i 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 a l bumi n i s therefore a res u l t of the d i 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 th i s s tudy . I a l s o wi sh to acknow ledge the iv advi ce and ass i s tance gi ven by Or Tony Cashmore of the App l i ed B i o chemi s try D i vi s i on , DS I R , P a lme 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 HK(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 Tr i s L IST O F ABBREV IATI ONS Adenos i ne 5 ' -monopho s phate Adenos i ne 5'- tr i phos phate Bromopheno l b l ue Carboxymethyl ­ D i ethyl ami noethyl ­ Deoxyr i bonucl e i c aci d Copy DNA ' D i thi othre i to 1 Ethyl ened i ami netetra-acet i c aci d Endopl asmi c reti cul um D-Fructose 1 , 6-b i s phos phate Fructose- 1 ,6-b i sphosphatase D-Fructo se 6- pho s phate Guano s i ne 5'-tri phos pha te Immunogl obu l 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 aden i ne d i nucl eot ide 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 g lyco l Phosphofructok i nase Phenyl methyl su l pho nyl fl uori de RNA hav i ng a pol yadenyl ate tract at i ts 3 ' termi nus RNA l ack i ng a po lyadenyl ate tract at i ts 3 ' termi nus Ri bonucl e i c aci d Messenger RNA Ri bosoma l RNA Transfer RNA Ri bonucl ease Sod i um dodecyl s ul phate Tri chl oroaceti c aci d N , N , N" , N"-Tetramethyl ethyl ened i am i ne Tobacco Mo sa i c V i rus tri s ( Hydroxymethyl ) ami nome thane L IST OF F IGURES FI GURE PAGE 3 . 1 El ut i 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 ge l ( 7% ) el ectrophores i s o f fructose- 1 , 6- 31 bi s phosphatas e 32 3 . 3 SOS gel ( 10% ) e l ectrophores i s of fructos e- 1 , 6 -b i s phos phatase 33 3 .4 Esti mation of fructose- 1 ,6- bi s phosphatase subun i t mol ecu l ar we i 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 a l bumi n a nd rabb i t gamma g l obu l i n 38 5 . 1 Ouchterl ony immunodi 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 g l obu l i n 46 5 . 2 Pur i fi cation o f anti bod i es to fructose- 1 , 6-b i sphosphatas e by affi n i ty chromatography o n Sepharose-bound a nti gen 47 5 . 3 Ouchter l ony immunodi ffu s i o n a na l 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 -fructose- 1 , 6- 48 bi s p hos phatase by crude gamma g l obu l i n 49 5 . 5 Immunopreci p i tati on o f 125 I -l abel 1ed anti �fructose- 1 , 6- b i 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 bod i e s 5 1 6 . 1 SOS gel ( 10%) analys i s of FBPase immunopreci p i tated from l i ver po s t-mi crosomal s upernatant after a 24 hour i nter- per i to neal l abel l i ng w i th c 35sJmethi o n i ne 58 F IGURE PAGE 6 . 2 SOS gel ( 10%) ana l ys i s o f a l bumi n immunoprec i p i tated from s erum a fter a 24 hour i nterper i to neal l abel l i ng wi th c 35sJmeth i oni ne 6 . 3 SOS gel ( 10% ) analy 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 prote i n subun i ts i n a l bumi n , FB Pase 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 u l ar fract ions 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 m i crosome fract i o ns after a 10 mi nute pu l se 6 1 63 l abel of c35sJmeth i o n i n e vi a the portal vei n 65 6 . 7 SOS ge l ( 7% ) ana l ys i s o f FBPase immunopreci p i tated from l i ver supernatant and mi cro some fracti ons after a 10 m i nute pul se l abel of c 35sJmeth i o ni ne v i a the porta l vei n 67 6 .8 SOS gel ( 7% ) analys i s o f a l bumi n immunopreci pi tated from l i ver supernatant and mi cro some fracti ons after a 10 m i nute pul se l abel of [3 5sJmeth i o n i ne v i a the porta l 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 Sed imentation 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 ant i bod i es to free polysomes 83 8 . 2 B i nd i 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 25 1 -l abel l ed anti bod i es to bound po lysomes 8 .4 B i nd i ng o f d i fferent concentrati o ns o f 1 251 - l a bel l ed anti ­ a l bum i n and anti -fructose-1 , 6 -b i s phos phatas e to free and 85 bound po l ysomes 86 8 . 5 Rel at i ve amount o f 1 25!-l abel l ed a nti -fructose- 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 ant i-a l bumi n b i nd i ng to free and bound pol ysomes 8 . 7 B i nd i n g o f 125 r-l a be l l ed ant i -a l bum i n and anti - fructo se- 1 , 6- b i spho sphatase to d i fferent concentrati ons o f free and bound po lysomes 9 . 1 T ime course of prote i n synthes i s o n i so l ated free and bound po lysomes i ncubated i n v i tro 9 . 2 SOS gel ( 1 0% ) anal ys i 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% ) analys i s of fructo se - 1 , 6-bi spho sphatase 87 88 89 96 97 immuno prec i pi tated from free and bound po lysome i ncubati o ns 98 9 .4 SOS gel ( 10% ) ana l ys i s o f al bumi n immunopreci p i tated from free and bound po l ysome i ncubat i ons 10 . 1 Rad i ochemi ca l pur i ty anal ys i s o f L-c35sJmeth i on i ne by TLC 99 on cel l u l o se 1 15 1 0 . 2 Formamide gel ana lys i s o f TMV RNA 1 16 10 . 3 T ime course o f protei n synthes i s a nd effect o f RNA concentration on the transl at i on o f TMV RNA i n wheat germ 1 1 7 extracts FIGURE 10 . 4 Effect of K+ a nd Mg2+ concentrat ion o n the trans l at ion o f PAGE TMV RNA i n wheat germ extracts 118 10 . 5 SOS gel ( 7 % ) analys i s o f transl at ion products produced i n wheat germ extracts programmed wi th TMV RNA 10 . 6 I so l at ion o f po l y (A)+ RNA by chromatography o f rat l i ver pol ysomal RNA on pol y ( U ) -Sepharo se 10 . 7 Non-denatur i ng gel (2 . 4% ) anal ys i s of po l y(A ) + a nd po l y (A )­ RNA from free pol ysomes 10 .8 Non-denatur i ng gel ( 2 . 4% ) anal ys i s of po ly (A ) + and po l y (A ) - 1 19 1 20 12 1 RNA from bou nd pol ysomes 122 10 . 9 T ime course of pro tei n synthes i s and effect of s perm i ne concentration on the trans l at ion o f po ly (A )+ RNA i n wheat germ extracts 123 10 . 10 The effect of K+ and Mg2+ concentrati ons on the transl at ion o f pol y (A ) + RNA from tota l pol ysomes i n wheat germ extracts 1 24 10 . 11 Effect of RNA concentrat ion on the trans lat ion 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 ys i s of rel eased products produced i n wheat + germ extracts programmed wi th po l y (A ) RNA from free and bou nd pol ysomes 126 10 . 13 SOS gel ( 7% ) ana lys i s o f r i bosome-bound trans l at i on products produced i n wheat germ extracts pro grammed wi th po l y (A ) + RNA from free a nd bound pol ysomes 10 . 14 SOS gel ( 10% ) anal ysi s of trans l at ion products produced i n wheat germ extracts pro grammed wi th po l y (A)+ RNA from free a nd bound po l ysomes which were immunopreci pi tated wi th ant i ­ fructose-1 , 6-b i sphosphatase 127 1 29 FIGURE PAGE 10 . 15 SDS gel ( 10% ) analys i 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 immunopreci p i ta ted wi th anti­ a l bum i n 130 LIST O F TABLES TABLE PAGE 3 . 1 Puri f i cati on o f fructose- 1 , 6 -b i s phosphatase from rat l i ver 30 5 . 1 Anti body puri fi cati on by affi n i ty chromatography 45 5 . 2 Ant i body quanti tati on 48 6.1 Inmunoprec i p i tat i on of a l b umi n from serum, FBPase and carr i 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 D i 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 pu l se l abel o f [35s Jmethi oni ne v i a the portal vei n 64 6,3 Di s tr i buti on o f radi oacti v i ty i n a l b umi n and FBPase immunopreci p i tates from l i ver s uperna 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 tr i but ion o f polysome s i ze cl as ses 9 . 1 Di s tr i buti on o f immunopreci p i tab l e al bumi n and fructose- 1,6 -b i s phosphatase pepti des synthes i zed by i so l ated free and bound po lysomes i n v i tro 76 100 10 . 1 D i s tr i bution o f i mmunopreci p i tabl e al bum in and fructos e- 1 , 6 - b i sphos phatase pepti des produced i n wheat germ extracts programmed wi th po ly (A ) + RNA from free and bound pol ysomes 1 28 ABSTRACT ACKNOWLEDGEMENTS L IST OF ABBREV IATIONS LIST OF F I GURES L IST OF TABLES TABL E OF CONTENTS CHAPTER 1 1 . 1 I NTRODUCT ION AND H I STOR I CAL REV I EW The i ntrace l l u l ar s i tes o f prote i n synthes i s i n an imal cel l s 1 . 2 The segregat ion o f secretory pro tei ns duri ng pro te i n PAGE ii iv V vi xi 1 synthes i s 5 1 . 3 Obj ect ives of th i s i nve sti gation 10 1 . 4 The d i scovery and i so l ati on of fructose-1 , 6- b i s pho s phatase 1 1 1 .5 Phys i cal and enzymic properti es of fructo se-1 , 6 - b i sphosphatase 1 2 1 . 6 Metabol i c regul ation by fructose- 1 ,6-bi sphos phatase in v i vo CHAPTER 2 GENERAL METHODS 14 2 . 1 I ntroductio n 18 2 . 2 Exper imenta l an imal s 18 2 . 3 Centri fugat i o n 18 2 .4 U l 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 ectrophores 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 s u l phate conti nuous denatur i ng gel s 20 2 .8 Es timation of protei n subun i t mo l ecul ar we i ghts 2 1 2 . 9 Po lyacryl am ide gel el ectrophores i s o f RNA 2 . 9 . 1 Conti nuous non -denatur i ng gel s 22 2 . 9 . 2 Formamide denatu r i ng gel s 23 2 . 10 Determ i nat ion of rad ioact i v i ty 2 . 10.1 Gel s l i ces 24 2 . 10 . 2 Tr i ch l oroaceti c ac i d- prec i pi tabl e prote i n 24 CHAPTER 3 PURI F I CATION O F FRUCTOSE- 1 , 6-B I SPHOSPHATASE FROM RAT L IVER 3 . 1 I ntroduction 3 . 2 Mater ia l s 3 . 3 Methods 3 . 3 . 1 Assay o f fructose- 1 ,6-b i s phosphatase 3 . 3 . 2 Pur i f i cat ion o f fructose- 1 , 6- b i s pho sphata s e 3 . 3 . 3 Treatment of an ima l s wi th tri amci nol one a l l oxan 3 . 3 .4 Quant i tat i on o f pur i f i ed enzyme protei n 3 . 4 Resu l ts and D i scuss ion CHAPTER 4 PUR I F ICATIO N O F OVALBUM I N , RAT ALBUM I N AND SHEEP GAMMA GLOBUL I N 4 . 1 I ntroducti on 4 . 2 Mater ia l s 4 . 3 Methods and Resu 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 ANT I BODY PRODUCT I 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 toco l s 5 . 3 . 2 Separat i on o f gamma gl obul i n from serum 5 . 3 . 3 Anti body speci f ic i ty 5 . 3 .4 Preparat ion of Sepharo se-bound antigen aff i n i ty col umns 5 . 3 . 5 Aff i n i ty chroma tography 5 . 3 . 6 Anti body ti tre 5 .4 Resu l ts and D i 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 Mater ia 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 Interper i toneal ( 24 hour ) l abel l i ng 6 . 3 . 2 Intraportal ( 10 m i 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 Resu l t s and D i scus s ion POLYSOME ISOLATION Introducti o n Materia i s Methods 7 . 3 . 1 Preparat ion o f free and bound po l ysomes 7 . 3 . 2 Preparat i o n of tota l po l ysomes 7 . 3 . 3 Polysome sedimentat ion ana lys i s Resu l ts and Di scus s i on 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 Resu l ts and Di scus s ion CHAPTER 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 Introduct ion 90 9 . 2 Mater i a l s 91 9 . 3 Methods 9 . 3 . 1 Cel l sap preparati o n 9 . 3 . 2 Trans l at i o n assays 9 . 3 . 3 Analysi s o f trans l at ion products 9 . 3 . 4 Immunoprec i pi tati on assay 9 .4 Resu l t s and D i 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 Rad i ochemical pur i ty o f L-c35sJmeth i on i ne 103 Preparat i o n of Tobacco Mo saic V i ru s RNA 103 Extracti o n o f pol y(A ) + RNA from po l ysomes 104 Preparati on of wheat germ extracts 106 Transl ati o n assays 106 Anal ys i s o f trans l ati on products 107 Immunoprec i p i tat ion a ssay 107 a nd Di scu s s i on 108 CHAPTER 11 D ISCUSSION 13 1 B IBL IOGRAPHY 136