22 IIAfBaiALS Atm HBTBODS 21lt•&oal!a Httby1 &lyo:xal ( ,.. ID wat•r), Glutatllloae (reduced), Tbeopby111n1 Catfe1ae 1 Cyolio AHP (Be ealt), Cbloroqtd.ae, ColobiclDe ver• ol»talaed lroa ·st.a• (USA). P.D1tropheQF1 pboepbate Ba •alt aDd folio aDd Clooal teu' a pbenol reoaeat ( IR) ••• purcllaautd troa CSlft Centre tor Dtoobeatoale 1 V.P. Cbeat laatitute, Delhi. Trlcllloroacetto act4 waa obtained froa Herek, lacU.a. 'a tbyalcU.ae wae obtained fro• leotope D1Y1eloD1 BARC, Bombay. Dt.~~athyl POPOP wa• obtduod trn Paoleard loetro• aeate, USA, PPO froa W.w BaalancJ Nuolear1 VSA. Htlltpore tlltera ('5 •) were obtained troa Haxflov Filters, BoabQF. Baoterlologloal peptoae from Glaxo• Bombay al1d Yeaat extract fro• CentroD Beeearcb Prodaote1 Bo•bq. All otber chemical a were ot •Gauanteed aeoaeat • grade troa Sarabllat .,. Cbemioole, India or • ADalar• arade tro• BDB 1 Judith All solution• were prepared ta class double distilled water. 23 Glyonolaoo X ~tlvity ootvloally accordlQl to ~ao nso~o~ Zoiso opootropllo'Comotor-Pt1Qll. upon 'tho forroattoa oil tbiol ub!ob eboorbo oanaoollV ee coooyod (!9S!) at opoc~vophoto as~c eolna Csvl 'lbo oooay t1ao dopoodone ootono~ s Lnotoylalutnth!ooo e~ono. ~o otandard aoaar olnturo oooto!110d !m n gs.ual voluoo of ! o!.t Sodium pboop~a~o boffo~ ?.o, o.e n o.$ o.a o.a o.a ol BDnyDO o.os ol lietorr Oo09 ol !)!! notbylglyonnl• !7.5 crJ Glutathloao ~oducoa; naanoo!uo oulphoto; 'alo otntu-o !7.0 o~ O.!G ~ tJCO 1\0t ~ '' ~ ol o1 ' · ' ctl '·' ~ o1 tncubatod forr go ct.o. boforo tbo a.dd! t!oa of tho onayoo to lot tllo noa-oonyotre!o ronctioa to~art bot~eeo C3tbylglyone1 aed alutnthlomo ao oqu!!ltbrluo. L}llfZfP Onl !L Ono unlt of onnyco is dof!aod ao tho OCDtmt of eDDyoo oatolyst.oa tbo foroatioe og s. Laotoylalutctb!ooo por otnuto !n oystots. ap Cl)lo o1l ot~c~ aooav 24 Aold pb.oopbotooo "co aooovod colozrlootvt.oo.lly accor4log to BorlJt1070tr (!97t.l) ot 25°Co i'b.o asoay t1QO dopendoBe upoa tbo roloooo of p.m. tnpl!ouol fB"'o tbo subotvoto £l.D1 'ropbonylpboopbato 0 tdtlcb cally at 1;3:05 aboo~rbo oant- ib.o otal242er6 oOOQf olnttaro coota1ao4 DOe 111 o .tt.uol voluco ot loOS ol: Cosoont~ot!oa !a talqga .•AAomx oanwo C!thrato buB~Ol"l o.n fl 0 98 5.6 1.0 oA P.ottnpbo1101pboopbato 0 Oa oalt (o.Gra) o.O:i ol Bnnyo eoletlon o.oa socnun b.ydron14oo o.s ibo rroaottoa 0' ra olntoro Sol t100 95°C after thO etddl t!OD ot a ol o~ o.s D so4Aum o1 !aoubatoci l1orr Olla)'r.lO brd~ron14o oo!utiono n of. to. Tboa uoo c44o4 to atop tbo reaction. Oao au~ oi' onayoo ls 4o1?lDOd ao tbo oanyce Dooded to tlywolyeo l p f~oc p.aAtBeghouy!pboopba~o o.osoy o!Bturo. ~lo ot QC)UD.t of p.ei~Jropbcal por ctoato !a otoa6agQ 25 Coaweraloo ot Cbanae la absorbance to enzy110 uD1 ta file coaveraton waa done by tollowlJli fontnllaa Iu a ,.a IIOlea/minute/ml wllere A 1 Cbange 1n absorbanoe Tva total voluae lVI BDayme Yo lu,me b a Ltgh t Patb e a Molor absorgttoa ol the prodUct It is expressed aa unita per .. ol protein. Protein wae esUmntecl by the aethod of Lowery et al (1951) w1tb bovtae eeru11 albulllft aa standard. 26 B,JAoterlmgs L. Cftli, ~olocular a voo ollttdaod irroo Prot. H.K. Dec;, Dloloay Lob, SObool of nnv!~oaoontol SCioocoo. Jls41D.L Cultaro CGdtuo Poptoao 0.5~ Glacoao 0.5~ Sodlua Cblorl6o 0.3~ tlotrloDt oodtf30 PoptOD$ ~~ Glucooo 1~ Yoao' ontrac~ 1~ o.s~ Sod!oo Cblor!Go woro onun aozroblon11y oa e rotfl£7 Cul~uroo cotton abahon ot 'ooc. ovomGbt culture t1oro uooa to ioooulato troob oodta. G~~tb ~oo Eollo~od coo.sur1au tbo optical donot ty ot 6a5 • 660 1'¥1 b.V flltou ia o ooloriootor. Vloblo call oounto umro o'bto!nod by platlma oa autr!ont ca~ codluo !a aBtpllootoo. 11oJr oaeymo otud!eo, oollo oGntrlfunatloo &~ SOOO X llt ~oc ~oD"' lltunreoeod l3y for tO eta. i'ho 27 pollot uao ~acbo4 ~tb o.oatl sodluo ,boopkaeo bufZor1 pH 7.0 en4 rosuopondod la 5 c1 of oaco bo£gov. colla at soutootod ia 11sn Sowd.cattnr fott' 3 oto. t1GI'O i'bo (1) 0 0. ibo aonnlco~od collo t1oro coatn.tuaoa ot 16000 lt g fo1r '0 oto. nllfa tho ocgoi"DG\Dt uao oto41od for ensyco actlvlQy. n.npt. tlo. a io otu4f tt'Ooponoo of Oultureo aroun tor ttoro troatod ~ltb ~ bgoo (oar!y loa pbaoo) Gjtbylglyozal to o f!nal co&eon- a to () cn. trn'e1oa of ootbyla!yonalo cultu~o ~o ~ GJr8"~ l:1QO obSQI"VOd torbl· d.t.metr!cally ond aloo by v!ablo ooll ooontAaa. nxtp~. no; a io otu4F slyonaleoo X cot1v1ty 4ur1DB aroHtb. io atudy tbe easymo oottvtty dutrtnJ otageo of (ln"Ol1tb 0 ot cul twro at ' doocrtbod !a nttpt. vadot~o cello ttoro tuurvostect gvoo B5 o1 t!uroo(Sb 8 lUro. on11 tlt'OetofJ ao ODQY~ otu41oo. no. ' i'o otudy ono~ ac tlvt ty dt!ri ll!;; atr0t1~b iE!bl· b1t.1oa. io oultureo g~oun tor ~ btt'a. weo oGQod ootbyl- alyonal to a final conceutrat1oa ot e ~ am6 ~ ~. 28 Colle t:1oro lllarvoo~oa o~ portocUonl tatorvo.lo oa1l studlod for tbo oosyoo oottv1ty. Bnpt. mo. () ~ ctu4f ounyco acttv!t,r durlaa a~ou~ rovoveal. fo oultaB'oo auotJn tor (l l'Uro. tsao add.od mtbyl- alyonal ~ a fi"Del oo®oo~retioa of ~ d'-1. alyottnl lnb!bltod cul~uoo ttoro tbon rle\'shYl• ~oobo4 Sroo of tbo dna o.ftor oDO nmt tbroo bowro by contdfua!DQ colla 8114. t1&&h!QB tt1!co 1.1ltb 0.9~ SOdluc Oblotr!do. Collo vaobGd flt'Oo of tho drua t1aro ouspel!:4io4 1\.n trooh oul twro codJ.uo. Dur1cg tbo rolnl U.atod Grtot7tll !a tbono cultures, tbo onayo::;, ootlv!ty tvao otudioct at varioue intervale. n.n:pt. mo. s io et~dy alyonalaoo 1 octtv!t.v Cul turoo Gff'Ottll ~o~e to~r ~~aa Sb!Kt up. Ia bro. !D cult.uro t:.Pdluo barveato4 and ouogoodod !a nutrlont luoubctoQ at )0°0 oa a rotar,v obu~er. ~dluo ~ Soopleo ttoro cirat1o at vonoue lntorvnla to otudy for tbo onnyco oc'C1 v1 ty. llUCQ!Dt1Q.Sgptg\}1 A!!!S!lQa 0 • 10 t1oor:to old St1l oo elbiato o!oo t1oro ~esulorl~ obtalDGd froo Dloeaoo froo QDioal bouoo, 29 Be.ryODG AQrtculturo UD!voa-o!ty, B!soorr. i'bo &ldoalo t1oro aalnta!DOd a.'e 12/ta l!abt amt dart: period nD4 fod ~ttb Blndusthan L!vor Pooa ad !lb!teo. a~ !FJ:ad1 a! lent AD!calo ia-radlatoQ !a tTOirO 0 Co60 cb.OObOI' obtaleod fnm flABC., Docboy to. e glo.otio coco. Anlaalo trore lrrodlotoci et 200R, tiG!"' at Q dOOO rrato of !361/m!amto ttbllo tbo onao Arrotaatod o-e ~OOA Qlld 600R t:JOl"' ot a dooo ~roto of itJ vciio/ooc. Tho dooo rate uao cGlculatoa oulpbato b.y otcnao~ tor~uo dos1D3t~. :R,rgatgga£ 1 Gnep X Coottrol GDlmaloa W»D-irrrodi atod noof.voG only oolluo Group II Cbeolcol control: IJ'oroal aoD-ltrvL\dlatoea CD!t:lalo fl'Gcolvod cbom!colo oo &a Gntap IV. Group Ill Badlat!oa eontrrola Aa!solo uoro 1rradlato4 aoon aad ue.rro !aJocte~ ttl th oDly ocllll<'h Gtroup IV finperiontol& ADI.oalo at eooa t~orro ODd uoro tnJoc9od cbocicals. arrrad!nto4ll ~ath tollo~taa All tbo cbeaicalo uore dimoo- lvod !a oalino ODd !oJootod tntrnporltoldoally la a tlaal voluco of o.a!) o1. 30 a woo 'i'beopbyl11c uao gtvon at ana 5 o:!0/100 lm!l aod a dl0 ao boaly of t oa tJt., Coffelae at oycl!o At1P at 5 ~ o.nd 10 t!l 0 cbloroqu!no at 1 o.:; Gild 10 os/tta body ttoiakt 1 Glatotbioao roducod at 1 anci S ~1 otUl Colob!olno o.t l oa/1!£t:J ~ of body uolnbt. J7or ott.adytaa mtA eyntboo!o 0 3n tbytlld.tao ttoa !DJoctod 1\.otsrogorl. toD!olly tt1o taoutro be!oro oaori.fy• o!ua tile oolmnle at 5 fA C!/ no QDQXO@ Studie t»dy uotallt. ru. iho aotoa!o tiore sncrlftcod by looatlon. ibo op!oooo ~o~o reoovod 0 oo~ool die- ~cobod t~1oo vitb chilled 0.02 11 SOd!uc phoopbato batforr, boco- aenlsed !a 5 m1 of pB 7 .o. o.oa ~ Sodl~o Phospbato butfor, i'bo bocoBeuisatioa uao .oatntolttsci eoaotant for all tho oploono. tuao4 ot 16000 X th tbe bo~engonnteo ~oc fo1l" SO oto. ttotro contri• Tbo oupornaat t.tao otwUoci forr a!yonolaoo I nllii ao!ti pboopbotaoo cctlvl.ty. PM SYatJ}etd.o ,S§t.!§!9JU. A part of oploou, aboat 10·15 C30 vao oat after oao~ttlolag ~olahod tbo aotoala and nocnaoniaod in lOSl 'i'rtcbloroooeUo acid. Tbo AomogoDGto ttco 31 filtered tbroalb Dillipore filters, waabe4 thrice vttb s• uitb o.s Ob111e4 trlcblo~acetlo aold QDd •1 of bundnd per oont aloobol. t.lnolly Tbe filters were dried and were oouated tor tbe couata. PPO, POPO.P 1 SUlphur acintillaUon tluid. tree toluene ••• .... a• Tile cout• were tallea Ia Paok- ar4 SC1nt111atloo CoWlter.