rfr/rr TRANSPORT AÀID ÂDENOS,I.NE* TRIPHOSPHATASE ACTIVITIES IN TURNIP AND FRACTIONS A thesís submitted to the univetsitg of Adelaide as a teqtJitement for the degtee of DOCTOR OF PHILOSOPHY by ,toHN MTCHAEL RUNcrE, B.sc" (Hons) Botang Depattment universitg of Adelaìde November 7971 CONTENTS Page SUMMARY DECLARATION ACKNOVüLEDGEMENTS ABBREVTATIONS CHAPTER I . GENERALINTRODUCTION I-38 A" TNTRODUCTTON I B. DEFTNTTTON OF POST-MTTOCHONDRTAL FRACTTONS 2 C " ANTMAL MTCROSOT'IAT.' ANÐ SOLUBLE ACTTVÏTTES 4 7. AnimaT microsomal electlon transpott 4 (a) NADE-specific eJ-ectton transport 5 (b) NADPH-specific eJectron transport 8 (c) rnteraction between the t¡o chains 11 2" Animal soluble eJectron transport, L2 3" AnimaL micrgsomal and soTuble phosphatases L4 (a) Acid and aJ-kaline phosphatases L4 (b ) G Tucose-6- phosphatase I5 (c) Nucleoside phosphatases L6 4" Induced and deveTopmentaJ- ehanges ín ëhe anímaL L7 microsomâ.I and solubTe sgstems D" PLANT MTCROSOMAL AND SOLUBLE ACTTVTTTES 18 7" PLant mictosomaL electton ttans¡ott 18 (a) NADV-specific eTectron transport I9 (b) NADPV-specific eJ-ectron transport 20 2" Plant mictosomaT'and soLuble peroxidases 22 3. PJ-ant sofuble eþctton trans¡nrt 24 4" Plant microsomaL and soluble phosphatases 25 (a) ecid and aL:,kaline phosphatases 25 (b) Glucose-6- phosphatase 26 (c) Nucleoside phosphatases 26 5" Induced and deveTopmentaT changes in the plant 2A mictosomal and solubTe sgstems (a) lnduced phgsioTogical changes 29 (b) Induced RIVÀ and protein sgnthesis changes 32 (c) Induced changes in enzgme activities 34 (d) Induaed uLtrasttuctutal changes 36 EO THE PRESENT STUDY 37 7" General charactetization 37 2. Compatison with cawesponding animal f ractìons 38 3. Rol,e of the fractions in the overaTT energetics ot 38 èhe ce77 4 " Genetal membrane co.ncept 38 CHAPTER II - METHODS 39-5I A. PLANT MATERTAL 39 B" SLTETNG AIID AGTNG ROOT'STORAGE TTSSUE 39 C. TTSSUE PHYSTOLOGY 40 f" Manometrg 40 2" Conductivitg 40 D. PREPARATTON OF MTCROSOMES 4L E. SUBFRACTTONATTON OF MTCROSOMES 42 7. Method for Chapters rII , rv and vI 42 2. MethoQ for Chapter VII 43 3. Variations 43 F " .PREPENATTON OF SOLUBLE FRACTTON 43 G" SOLUBLE ENZYME PURTFTCATTON 44 (NHì 44 J. Z SO¿ fractÍonatìon 2 " Sephadex elution 44 H. ENZYME ASSAYS 45 7 " oxidation-teductíon activities 45 2. Mixed function oxidases 47 3" Peroxidase 48 4" AbsorpÈion spectra 48 5 " Phospltatases 49 T. CHEMTCA¿ .ASSAYS 50 J, ETJECTRON MICROSCOPY 50 K" CHEMTCALS 5I C H A P T E R I 1 I . MICROSOMAI, ELECTRON TRANSPORT 52-7L INTRODUCTION 52 RESULTS 52 A. GENERAL CHARACTERTZATTON 52 B, PROPERTTES OF REDUCTASE ACTTVTTTES 54 7. pH optima 54 2 " Inhibitor sensitivities 55 3. Concurrent reduction of two aaceptots 56 ' (a) FeCN and DCPIP 57 (b) FeCN and egtochrome c 57 (c) Cgtochrome c and DCPIP 58 C. MTCROSOMAL DTSRUPTTON 59 7" PhgsícaL 59 2 " Enzgmic 6L Ð " MTCROSOMAL SUBFRACTTONATTON 62 f, , DifferentiaT. centrifuging 62 2. Discontinuous sucrose gtadients 62 (a) Appeatance and ulttasttuctute of subftactions 63 (b) EnzVmic distribution 64 DISCUSSION 65 A. NADH ELECTRON TRANSPORT CHATN 65 B. NADPH ELECTRON TRANSPORT CHATN 68 C, MTCROSOI4AL SUBFRACTTONS 69 7" Enzgmic distribution 69 2. Origìn of ftaction 2 (niddTe) 70 D" PROPOSED ELECTRON TRANSPORT SCHEME 7T CH AP T E R I'V . CHANGES IN MICROSOMATELECTRON TRANSPORT 72-A9 TNDUCED BY SLICING AND AGING OF TISSUE INTRODUCTION 72 RESULTS 72 .72 A. EFFECT ON MTCROSOTTAL ACTTVTTTES TN VTVO 7" Changes in mícrosomaJ- h.ctivities induced bg sTicing and 72 agíng of tissue 2. ReTation between induced microsomaT changes and RNA and 73 protein sgnÈhesis in tutnip disks 3. Effect of disk size on inQuced microsomal changes 74 B" CHARACTERTZATTON OF LOSSES OF MTCROSOMAL ACTTVTTTES 75 7, rncubation of microsomes with sofuble fraction in vitro 75 2 " Effect of IAA on microsomal activities in vivo 78 3. Effect of sJicing and aging in isotonic sucrose 79 C, EFFECT ON DTSTRTBUTTON OF ACTTVTTIES WTTHTN MTCROSOMAL 80 SUBFRACTTONS DI SCUSS ION 83 A. RELATTON TO REPORTED BTOCHEMTCAL CHANGES TN MTCROSOMES IíTTH 83 AGTNG B. RELATTON TO REPORTED ULTRASTRUCTURAL CHANGES -r¡V ER WTTH AGTNG 84 C. NATURE OF TNDUCED T'TTCROSOMAL ACTTVTTY LOSSES 86 1,. iVature of sLicing-induced stimuTus 86 2 " Nature of the inactivation 8l 3. Nature of the developed insensitivittl 89 CHAP TE R V . SOLUBLEELECTRONTRANSPORT ACTIVITIES 90-106 TN FRESH AND AGED TISSUE INTRODUCTION 90 RESULTS 90 A" REDUCTASE ACTTVTTTES TN THE CRUDE SOLUBLE FNECTTON FROM FNNSH 90 ANÐ AGED TTSSUE B" SOLUBLE REDUCTASE PURTFTCATTON 92 (NH/ 7. Z So4 fractionation 92 2. Sephadex eLution 93 3 " Ptoperties 95 C. CHARACTERTZATTON OF THE DEVET,APMENT OF THE SOLUBLE REDUCTASE 96 ACTTVTTTES D. SOLUBLE REDUCTASE ACTTVTTTES TN OTHER TTSSUES 99 DISCUSSTON 101 A" SUMMARY OF THE SOLUBLE ACTTVTTTES 101 B" COMPARTSON WTTH REPORTED SOT'UBLE ACTTVTTTES FROM PLANTS LO2 C" COMPARTSAN WTTH REPORTED SOLUBLE ACTTVTTTES FROM ANTMALS LO2 D. COMPARTSON WTTH SOLUBTLTZEÐ MEMBRANE-BOUND ACTTVTTTES LO2 E. FUNCTTONS OF THE SOLUBLE ACTTVTTTES r04 F" DEVELOPMENT OE THE ACTTVITTES WTTH AGTNG r05 C H A P T E R V T - MICROSOMAL AND SOLUBLE OXYGENASES LO7-L20 IN FRESH A}ÍD AGED TISSUE INTRODUCTION IO7 RESULTS 108 A" MTCROSOMAL NAD(P)E OXTDATTON, REQUTRTNG O AND ORGANTC 108 2 SUBSTRATE 7. Characterization of the reaction 108 2 " Sensitivitg to irùtibitors and actÍvators 109 (a) EJectron transpnzt effectors 109 (b) Cgtochrome P-450 effectots 110 (c) Peroxidase effectots 111 (d) othets lrl 3. Products lL2 (a) DinethgTaniTìne TL2 (b) AniTine LL2 (c) PhenoL 113 B" SOLT]BLE NAD(P)H OXTDATTON, REQUTRTNG O AND ORGANTC 113 2 SUBSTRATE C, MTCROSOMAL AND SOLUBT,E PEROXTDASE 113 l. Characterìzation 113 2. Specificitg of membrane binding 115 D" AGTNG TNÐUCED CHANGES TN MTCROSOMAL AND SOLUBLE NAD(P)H. 116 ANTLTNE OXTDASE AND PEROXTDASE DISCUSSTON 118 CHAPTER V]I MICROSOMAL AI{D SOLUBLE PHOSPHATASES 121-132 IN FRESH AND AGED TISSUE ]NTRODUCTION L2L RESULTS L2I " A. ACTD ÞHOSPHATASE L2L B " GLI]COSE -6- PHOSPHATASE L22 C. MTCROSOMAL ATPASE L23 l. Elinination of acid phosphatase activitg 123 2. Subftactionation of mictosomal membranes l-23 3. Conditions for optimum activítg L23 4. Speclficitg of salt stimulation L24 5" Effect of membtane distuptìon 125 6" Relatíon to other ATPases L25 7. Changes utith tjssue aging L26 DISCUSSION I26 A. RELATTON OT TURN|P MTCROSOMAL ATPASE TO OTHER PLAI{T 126 T'TTCROSOMAL ATPASES ,'MTTCHELI',, B" ,RET'ATTON TO ATPASE L28 C" ORTGTN OF THE ATPASE r31 C H A P T E R V I I I - SLICING AND AGING INDUCED 13 3-140 PHYSIOLOGICAL CHANGES TNTRODUCTION 133 RESULTS 133 DISCUSSTON 136 A. TNDUCED RESPTRATTON r36 B. DEVELOPMENT OF NOONO- At:lD ANTIMYCIN A-INSENSITM r37 RESPTRATTON 7. DeveTopment of a reductase 137 ..NveToprtent tesistance 138 2 " of a 3. Discussion of two hgPotheses 139 C. TNDUCED TON ACCUMUT'ATTON I40 CH APTE R I X - GENERÃL DISCUSSION ANDFUTURET,IORK LAL-L44 BIBLIOGRAPHY 145-161 SUMMARY l. Microsomes htere isolated from turnip (Btassica rapa L') rqot sÇorage,tissue. subfractíonation (using discontinuous sucrose gradients ín the presence of ions) resulted in (a) the separation of (b) the majority of the ribosomes from the membranous vesicles and the further separation of at Ieast two types of smoo€h vesicles - one conçidered to be deríved from the endoplasmic reticulum and the other from the plasmamembranes" 2. specific NADH and NADPH dehydrogenases (Ín the presence of a variety of acceptors), and cytochrome b555, but not cytoohrQme P-450, were detected in the microsome fraction. The NADH dehydrogenase aêtivities, NADPH-cytochrome o reductase and cytoehrome bUUU were concentrated in one,of the smooth membrane fractíons¡ while the other throughout NADPH dehydrogenase actiVities were distributed more evenly the subfractions. The structure of the electron transport system was determined. The resuLts indicated that, as ín animal microsomes¡ there were t\¡ro electron transport chainsi one specific for each red.uced pyridinç nucleotid,e. Attempts to detect mixed function oxi- dase activity. involving the NADPH electron transport chain were unsuccessful. oxygen consumptíon \^'as detected in the presence of NAD(P)Ha4dorganicsubstrate¡butwasshowntobeduetoperoxidase.' 3. The microsome,fraction also contained a salt-stimulated ++ Mg---activated ATPase which \^/as .concentrated in the smooth membrane fractions. Activity was assayed "a-t high pH {7'"8} to eliminate inter- fe.rence f,rom aci.d. phosphatase whic.h,wag .also present in the microsomes" There was a specif,icity for the anion but not oation in the salt- stimulation of, the Mg++-ATPase. This aetùui"ty was further stimulated by carbonyl cyanide m+eh.lorophenylhydrazone (CCCP) , 2,A-dinitrophenol , valinomycin, nigericin and NH4CI" with.. a sy.nerg.isÈic effect between cccP and valinomyc-in. Activåty was i¡rsensitive to oligomycin, phlorízin a¡d ouabain. Based ,on .simùJ"ar;iÈy 'to-the chloroplast ATPase, it'was proposed .that this ACPase was s.iÆuated on the outside of the vesicle and moved,H* to the insdde-, J.eanr.íng the outstde alkaline. IÈ -is sugges.ted tha"t .tlrís resuLted ;in movemÊrrt of- the anLon to maintain electrieal neutrality¡ and exch.ange o-f, tlre proton for the cation to maintain pH neutr.alitY. 4. The. soluble su¡rernatant fracùion con-ta-ined- two reductase acLÍví.ties. pa:rtial. purificatíon,showed.tl¡'atrone-.was- speeific for both 'NADH and FeCN but that the other showed little specificity for either donor or acceptor. It was suggested that the former may be solubil- ized from the microsomal membranes during homogenizing of the tissue and that the latter may be the equivalent of the a¡rimal DT diaphorase" 5. Slicing turnip tissue índuced 20-100% loss of microsomal- NADH dehydrogenase activities within IO minutes. partial recovery of some activities occurred on subsequent aging of the disks, and these recoveries l^lere sensitive to cycloheximide andr'in part¡ to 6'methyl- purine.