, 1 i i 1 INTAACELLULAR FOtATE OF HlI4M F18RClllAST S 1 Soudabetl A. Kutlan1 A thesh sut.1 tted to the Facu1~ of Gr.duate~ud1es and Rase.reh in parttal fulf111.,.t of t .. requ1re.nts for the degree of Master of Science. , , i j f Dlpart.nt of Physio1ogy. Mc6tll Untve"tty. Mont ....1. P.Q .• canada. @ August. 1984. -\!i\!&~1iJ.;U4M' Ji4Jl.... ~~.,. ";;;U",,*,,*"'i U.$.14 JIU _1711&4___ #OUMI'" te. Il00 .....-_ _ __,_ .... _ "El il: • 1; RtM_J._ lBSftlCT Tbe eodogenous leyels of yarious folate .oD09la~a.!te :::oapounds in :::ultare:1 baaan fibroblasts "ere deterained Il S iog big h perf oraance l iquid chroaa tograph y for the separation of folat. aoooqlQ~aaate. Endoqeoous folates "eee conyerted to aonoqlutaaate foras using the conjuqase eaz,ae present in ra t sarua and in cu bation "as carried out at pB 6.5. Tbe use of tbis procedure ainiaized folate coenz,ae lnterconyersioD 1urin9 processin g_ Osing aet banol for precipitation of protein instead of beat aioiai zed the ~e9radst1on of labile folates. RecOyerf of all folates ( eK:::ept 10-C80 84PteGlu using tbts procedure "as aore thao 'JOI. Disruptioo of cells by boiliog appeared ta cause lass post-•• traction cbanqes of cell folates tkan did treezing and tbaving or sooicatioD. oalng beat to release _dogeoOQ8 folate, coojll'jilse teeata.nt vith rilt seru at pB 6 .. 5 and precipitation of protain vitb letbaool, aore than half of tbe iotracellular folat e of nor _1 f iJu;oblasts io collfl_nt qro.th vas 5-C113 84Pt&11u, and 10-C80 H4PteGlu and a.,teGlu co.pri•• d 2i and 6 percent of ead()()enOQs folate r_pect.i•• lr. ( i ·~~~."AI~.ftlMi~jl Z$IJ!8!Ul tt;'1iI0IS"W'AAlOkai_oas*_, ..._., "' ....." _ h '3 RESUltE Les niweaux endogene8 cles diwers co. posés de la folate aooogluta.ate dans les fibroblastes de cellules bu.ailles cultiyees ont ~té deter.ioes par l' e.ploi cle la cbro.atograph ie liquide a ha ute pre s8ion (HPLC) pour la separation de la folate .onogluta.ate. Les folates endagenés étaient transtora.r sour fora. aOllog lu ta.ate duran t une incubation a pH 6.5 a Yec en zy.e conjuga se présente dans le seru. cl. rat. Cette tecbnique .ini.lsai t la CODye rs10n de la folate clue ut le procéde. L' a.ploi clu aéthanol pour la précipitation des proteines p 1l1tot que la chaleur .i Di ai sait la degrada tion :les fols tas ( 1 nstables. La récu pérat ion de tous les folates acept 1. lo-cao B16PteGlu par cett e technique était pl us de 901. La scission des cellules sui te a une ebulli tion pIOwoquait .oios cle cJlange ••nts aprés ex tract ion clea cellules folate que le qel, le cl8qel et le déae.bra.eat. L' utilisation de la cbaleur pour libérer la fol~te endogéne , le traite ..nt a pB 6.5 a 1 Gnz,.e Ayee le •• ru. de rat et la précipitation des proteiDes aYec le .éthanol oot par.is cle déter.iDer qae pllls cle la .01 tié de la folata intracellulaire des fibroDlastes nor_ a)[ en plaa .. conf la_a;::. était 1. S-CB3 a'Pte~lll, alors 'la_ 1. 10-CIO a.'teGlu et gu. le I14Pteala co.posaient 29 et 6 poGJ:Ca.t r ..pectiy __ t. 11 Tl BEL OF COlT BIT 5 CHAPrER 1 rlTBODOCTIOI 1.1 Folate Structure and Blocbe.istry •••••••• 1 1.2 Polate !azy.a•••••••••••••••••••••••••••• 8 1.3 Polypolygluta .. te Sjntbesis ••••••••••••• 27 1." fI••• all. an Polate Transport •••••••••••••• J 1 1. 5 Eni OCJiI DOUS Pola te in fI ....1ian Cell. •••• ]5 CHAPTES 2 fliCJb Perforaan ce Liq uid C broaatogra pb, TacbniqIl8 •••••••••••••••••••••.•••••••••••• 38 ':1I1PTER J Tbe Parpose of Preaent Stady ••••••••••••••• 'l fil" CHAPTB. Il flaterials aad ..t.ods •••••••••••••••••••••• 115 CHAPTal 5 a ..alts ...•...•••••••••..•••••.••••....•.•• 55 CHAPTD 6 Diac.. sioa •••••••••••••••••••••••••••••••• 18 8I8LIOGI1PBI •••••••••••••••••••••••••••••••••••••••• 85 111 1 ".. Dedicated to: .J bQsbaDd, "ho ù"aJs iDspired .e to achie.e ., goals. ( ( ( 1 ICKIOVLB DGBIIDTS ',lrst l woœld like to eKpress.y siocere appreciati~n and tbanks, to ., saper.isar Prof. B.A. Cooper for Ilis ,JQ.l.d~nce, encoara'J.ent and patience duriog tbis res.arcb. l IIoold like~to tbank PIs. sally Loe-Shing for tea=blQ~ .e the tech.n ical 5k1.115, an d for ber ad. ice and belp IIi tb tbe .1crobiological assay. l "ould like to tbanJt Dr. Oayid 80senblatt liDO proY1.ded the cell lioa œs.d in tbis stœ«ty and Dr. Rob .. rt ftaclenzie for Bis belpfull suggestions tbroU')hoat tois ":t res_arch. r IIould also lite to tbank "s. Dorli Itoifel for ( rl83istlog .e "itb t,pioC] and PIs. Deoise Besuliel1 far praparation of tbe Praodl abstract for the th.sis, and !la. catby Grauer for ber assistance IIith tbe celi c1llture. 1 a. 'lrat.fal to the "edical aes.arch Council of ) ~4nad3 for grants proYided to De. cooper tbrou'Jàout tàis st",dy. Word caD ~ardlJ azpre~s., siocere appr.ciatioo to .J 1 .otber for her loy., e.courage.. nt, support and patia.ce tàrOUgAout ., lifa. ( ( w~_... ;:." J.qll';ICU\\tl1JlUU'~ '_4. "_._~"I,~ .. III :M4tI.1'''''.''''" ..........._ .. __ ......... (~"" ........ ".... ,............ ..,MMlM ... J .... 4)i!... 41... Ii4A..."r#lhŒ.1tj!.1:1l.-lblfi,..~r '~--- C81PT~ 1 1.1 P~late structore and biocb.. istry: At tbe beCJinniD9 of tais c8lltury a series Qf pigaeJlts If er e l.Solated froa tbe v iDCJs of the botte rf l" dll of vrli=A wera foand to baYe tba sue basic strucrt-are. lIienland (1) Ilsed the tera' Pteridine' for the bicyclie nitrogen ring s,ste. of tbese piqaeuts. 1 Du.ber Qf studies on nutrition&! factors in Mn !ed t:> tbe diseoyer, e>f a substance containiDq the Pteriiiae str~tore, vbicb has b ••n found to pla, a ka, role 1D intr~eellQlar aetabolisa. 11115(2) reportad tbat eKtra::ts ( of lltolfsed ,east or li Yer vere effectiYe in tbe tr_taent :> tropical aacroc,tic an.aia. It vas lat~r shovn t8at 3Dkeys aaintained 00 di8ts siailar to tho. associated vita huaan tropical aa::rocytic aneaia &lso de.eloped an aneaia vhich responded to the adainiatration of ,east or liyer fi Ktracts (3,4). A li yer factor vas f ollnd to he an eS:Jantial Blltrient for laetic aeld Daet.ria (5, 6' a.~ to pre.ant aacroertie an.. 1. in eb1eks (1,8). The Daaa 'folie acid' vas first proposed b, !lite.ell (9) et al lD 19.0 for the SUbstance vb1ch tbe, had isolated fro. spinach snd vbicla vas a .etrit ional factor for Streptococe. fa.cali.. It appr3Kiaatly tla. s ••• ti.. a fact()r st1•• 1atiJlCJ growtla of Lac:toAlacUlus c ...i, ..&s 1 1!!ItlI.,IJ!II!.IiJlitillllJ*l.UII:ltn_._.IJI'IIln.!_I!II!$ .... """ .... ~". __ .,.a .... ' .......... ' ... __ • __ • _._._"_•• _"._,_.. _____ ~ . _ _ _ ~ _ • __• _. _.____ ........ _ ••,..,p_ .... , .... i4I11J1!I- isolated fro.liyer(10,11,12). ln 19_6 &Dgier e1: al.(12. pllrified factors isolated fro. liftr, yeas1:, and spins:h tb3t vere effectiye in the tr.at.ent of .acrocytic aneaia. Purification of tbe factors responsible for tbe aboye nutritional effects reyea~ed that the basic structure ;,f these factors vas identieal aDd included t he structure 1-( 11- (2-a.ino-.... hydroxyl-6-p1:eridine' .ethyl} a.ino] beDz;,yl ~llltaa1e aeid. Osually pt.ridine refers to 2-a.ino-~- bydroz, pterin ring and ~erin is Qsed to aIl co.pounds cOlitaiDinq the pterrin. 'olie acid is pteddine liDked by a .ethylene bridge to p--a.inobenzoic aeid group, vhich i5 attached throu~b a pept.1.de bond to a gluta.ie acid .oleca~e(Pigure 1). ( rolie acid 15 poorly soluble in va ter and iD organ ie solyents. The 2-a.1no and lI-hydroxy groops haye "n insolublizing affect. lpparaotly bydrogen bonding batveen pterriA._olecales ls reioforced by h1gh dipole .caents (13 •• The qre.a1:er solabilit.y of the di-sodio. salt of folie acid thsn of tA. acid apparently is due to the effect of t iODuatioa of the glota.aie acid residue (14•• f ptarins are relatiyely resistant to oaidation bat 1 heatlag vith chlorate at acid pH degrades the ring sfste. .ith 1:b. for.a1:ioD of ~uaDidlo. fro. the 2-a_ioo pyriaidi~8 1 pottioD of the aolecole. Lite .ost pteroyl coapounds, folle f acid 15 stable to ùJtall QJlder aDaarabic conditions, bot ! { ( ~lkaline llfdrolfsis aDder aerobic coacJitioJlS cl ..Y8S the 2 J " ,....', imaM MU aM tS.ilXllIaM;;:;_,""..,.__ ...""'.z: ... __ lIII'i __ "''''' __ '"" .... ...' ,,,""'w""" ...... "... LI!lIlI..... n..... s'""'~""', ......... ,_,_, __ ~._. __.... \ .'1.32 1 d!4!\tlll!lN ~ 1 si~e chain to yield p-aaino benzoylglutallic acid and ptarin-6-earboxyli= acid (15). Aerobic bydrolysis in aeid ,ields 6-.et by Ipter r in (15). Folie acid is only active as a coenzy.e wben it is in a reduced for.. F:>lic acid and .any:lf its derivitives ean easiIy be reduced by a variety of agents to the corresponding dihydro or tetrahydro folate. H2PteGlu and B4PteGIu (16) are rapidly o~idized by air. The lIain pr~duct :l f B4PteGlu o~idat ion is p- aainobenzy 1-g luta.ic acid (1 G, 17 ) and a nu.ber of pterins. Oxidative degradation of reduced f:>lates is greatly retarded by tbiol and a s::orbate. One-carbon units can substitute on the 85 or N10 post tions of El4 PteG lu or lia y for.