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Lactic dehydrogenase isozymes in lens and cornea
EaTM Eye. Res. (1966) 5, 309-:.~.t,1
Lactic D e h y d r o g e n a s e l s o z y m e s in Lens and C o r n e a * [,AllIIY ]3NlcNNq'I¢IN, ~II('ltAI,~I, .[~I,lll|LI{IAN ANI) ll:%ltl~.V MAlS~Cl.
De'l,artme~tt q[ dmt&m'~ff, l Va~/ae ,State l/~,ice~,:~itt/. ,School of 3Iedicim', Detroit, Michigan,: U.N.A. (Received. S April 19B6) t a c t . i t deh.vdrogcnetse isuzymt.s o[" b o v i n e n,ui r ~ b h i t I(.,ns Itl|(~l c'ortlogt ~X'Z~Z'OI~,It,~t]YZt'(t I~,• s t a r c h gel eh, ctroph(n'e~ s. A l t h o u g h t h e r e w a s ~ p r o g r e s s i v e loss o f e n z y m e r~etivii.v in il'm Ien.ses ~)f boll1 Sl)eeies w i t h iuercr~siug ~lg~.'. I]1(: h3s8 o f isr~zymes xv~s rnon~'elearly c~'i(i,mt ill 1 he- b o v i n e lens. I I= t.he adult, b o v i t m tons. tact.it d c ' h y d r o g c u t ~ s e i s o z 3 ' m e I w~m p r e d o m i n a u t . while in l.hc a d u l t r a b b i t Iens. i s o z y m e s 3 - 5 w e r e n m i n l y I)Vesent.. *J~he m o b i l i t y ~,f Ions i s o z y m e ~ w a s ident.ieM t o l.ilaL o f i s o z y m e s in tn.hm. Ii.~.sues..[~ni'llmrmore, t h e isoz3'n-m:~ wet~e lU)t IocMized to ~u~y m a j o r specific lens eryMn.llin. T h e eorn,,a i ~ o z y m e pat.t,ern in eatclt stu:eies <'elmira:iLl,t| m a r k e t l l y wit.ix Lhr~L +~f {.it(: c o r r e s p o m l i , ~ g lens. {['hus. in I;l'm a d u l t b o v i n e t't)l'llet*, iS~,';Z,~'II|CS : 1 - 5 ~A'ert: pre.do[l~i~tttnt, a n d in t h e rabt~i~ c o r n e a , i s n z v m e s 1-4. T h i s ditti:ron(-e bel, ween lens a n d e~,rmu~ a p p e a r s t o |m a f u n c t i o n o f t h e e n z y m e e o m p n s i t i o n o f t h e lens fiber eel[ r a t h e r t h a n o f tim lens e p i t hc.liM cell.
I. Introduction
8cvcr~*l i n v e s t i g a t o r s h a v e o b s e r v e d lactic d c h y d r o g e n a s e ( L D ] t ) activity" i,t t k e b o v i n e hms. t,) v i t r o s t u d i e s h a v e showt~ a. decrease in t h i s a.etivicy per lens wit, h progressive age afte.r birt.lt (]rioekwin, ]965). Stewar~, a n d P a p a c m l s t a x t t i n o u (1966) f m m d oJ,ly L D H [ ~md 2 i1~ t h e a d u l t I~ns fiber ceil, i~1 eon t~rasb to the epit, hetial eetl, w h i c h cont.aincd a.ll five is,,zymes. T h e s e ~ u t k o r s also showed ~t decrease in. t,he m t m b e r of (~ath.odni isozymes w i t h an increase in t h e age o f Vim lens fron~ calf t o a d u l t life. ]i'owlks (I905) isoh~tcd a. pro~uilt w i t h L D H a c t i v i t y f r o m t h e b o v i n e lens, whicl3 p r o t e i n was boCh similar to amd differm~g from he,~rl~ L I ) H . Green a n d S o l o m o n (19571 r e p o r t e d o n - t h e prodtmt, ion of" laogie acid iJt the. rabbig lens. We h a v e r e p o r t e d p r e v i o u s l y o n t h e c h a n g e ill L D l t isoz2one~ d u r i n g tim d(;velopment. of t h e c h i c k lens mid ghe s i g n i f i c a n c e of s u c h c h a n g e in relation, go t h e m e t a b o l i c a c t i v i t y o£ t h e lens (Maisel, K e r r i g a n a n d S y n e r , 1965). T h e s t u d y r e p o r t e d here i n v e s $ i g a t e d t~,e isozyane p a t t ~ r n i n ghc b o v i n e a n d r a b b i t lens anti cornea d u r i n g development. 2. M a t e r i a l s and M e l h o d s
5l'issue ezt.racts Bovine eyes (obtained from a sla.ughtcrtmtmc) were removed from the atdnmI imme(liateiy aft~er aactifice, transported to the l a b o r a t o r y in crushed ice, and thet~ dissected. ]~ach lens, freed of all surrounding Lismle bill xvit~h cnl)~ul~, inL:tet:, was ]mmogenized aL variable concentrations of 100-250mg wet. weight, of tissue]mi 0-9°, o saline solution. The homogenate was centrifuged "tt. 12,000 g {'or 20 rain at 4°C and the supernal, a n t used for furtho.r tESts. Extraet~ were. also prepared ~f lenses after the capsule and epitheli':Ll cells ha(l been removed, of whole comma, heart, skeletal muscle and liver, l)retmratiuns * "|'his invest-igatlnn was suppm-Lcd by ~ l?IqIS resenreh g r a n t {N [~ 04763} frf,m the .N'uti(mal fnst, it, tJte~ of Health, U . S . P u b l i c H e a l t h Service. 309
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I'~,,~,r~: 7. 'Lt)Ii[ i~ozyme pntLertas Of rabbit lens and eori~e~ analyzed, by ~'er~,icat ,~t~rch ge} ele¢:tr~:J•:~ht~resiswithTris tm~'at.e-lu" 1)'~ J. A buffer. ' " a, 1-year-old;-Icns (0-51 I g); b, 4-month.old lens (0.3OGg); ~'., 10.day-old lens [0.156 g); d, 0-day.old :ci~s (0"08(Ig); e, ]-year.o]~leorhe~; f, 4-mox~th-old cornea; g, 10-d~y-o.ld cornea; !a, 2-dayiold cornea. Leas c~t-ra~ts w~re prepared wit,h 250 mg ti~-.~uc/ml 0 ' 9 % saline sob, tion andC~l'l~ea[ ext,rac~ Witli ~00 mg tissuo/ml 0-97,~ saline solution. Nt, mber~ represent-the val:ious L[)I:[ isozymes/
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[~ is clearly eviclent from Plates 1 a n d 2 t h a t the isozyme p~;te, rn of the bovine, Ioa~s changes significantly wlgh age. Table I, de, rived f r o m the d a t a of Fr'meois altd -I/a.ba(;y (1957) and o f ]:[oekwin, S e l m m t t e r and Mtiller (1963), relates the lens ~-eigh~, f,) the ~.ge of t,he animal. The decrease in total L D H per gram len~ wit, h increasing a,~e is associated with a progressive, loss of .LI)I-I activity, first affecting L1)H 5 and then .LD]{ 4~ 3, ,~nd 2, in t h a t order. T.}ms, in fetM lenses, all five LDI-I isozyme.s are clearly evident, w i t h predonfinan, a.otivi~y ~t L D H 2 and 3. la the young cnlf lens, I,I)I[ 1-4 a.re el{parent, with L ] ) ] t l and 2 predominant,, h~. the m a t u r e adult lens, however, only ] , D H 1 and 2 axe clearly appm'ent, with predo,ninant)activity. ,,.t L D ] t t. Identical isozyme ].>at;terns were obtained from bovine lenses fron~ which the .capsule and cpit.helial ceils had been removed. t AnL~-;
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R e h g i o n s h i p o.f boci~te lens weight, to ~ge * Lens weight
Ago.
0-55ti
G-nmntb-old fetus ( ; - 7 - m e n t h . o l d t'etu.~ 7-8- m o n t h - old t'~t,u~ 8 - 9 - m o n t b - o l d fiet,u.~ newborn catt l - 2 - m ~ , u 0 ~ - o l d <.elf 4 - m t ) n b h - o l d call" 8- m o a t h - o l d c ~ l f l-2-ycar-ol4 cow 2-ye~).r.old 0ow 5-yeast-old cow 6-year-old cow
0.600 0- 7"18 0"834 0.926
~.062 !'21 6 1-5-21 1.8,12 2" i 53 2,317 :?.400 m
* 'L'he b~t})rm.~tion in Lhis l.ablo is b a s e d o~ t h u dmt.a c o m p i l e d by Hot~kwin, S c h , n u t e e r ~ d (19611), I " v a n ~ o "~ ~tnd R a b a e y (L957), a n d t h ~ aut.t~ors el tlsis palYer.
31(il/er
The relationship of LDI-I isozymes to bo~-ine lens crys~a}lins is shown in Plates 3-5. The p a p e r e|ectro]?horetie s e p a r a t i o n of the fetal lens e x t r a c t s~aiJled /or protein and L D ] t is t)resetlted in P l a t e 3. This ~na.lysis il~dicated ~h a,t, ]~DH 1 m i g r a t e s fllrther to ~be ~mode l~han'q.-c~wstalLin, LD_IK 2 has t h e s~m~e mobility as ~-crystallin, L D H 3 migrates to a point between ~.- aud /~-crysgallin, a n d .EDH 4 and 5 have a. mobi[it.y similar m th'J;°c of ,8-erystallin. No LDI-I isozyme corresponded t,o the 7-crys~allin b~md. The relationship of ]61)I-I isozymes and lefts erysgailins a.nMyzed by p a p e r clect,'ophoresis and followed by sta.reh gel electrophoresis is ~een in. Plates 4 and 5. Plate 4 shows t h e p a p e r s~,ri]) on w]aieh lens proteins had been eleetrophoresed in t h e diree~iort of the vertical arrow. After this inimical elee~0rophoresis, the st-rip was inserted in a starch gel, and electrol?ltoresis Was performed at right angles to tim ori~i. sepa.ration. The get was biseated and one-half stained for protein a n d t h e oth,>~::~::': Lt)It. Five isoz.yraes were detected, their relationship to the leas pro|;eins beizlg slmwn in l?lage 5, which represents the :LDH gel of Plaf, e 4, which w a s couat, ersvMne~l tbr p r o t e i n . . T h e positim~ of tshe L D I I isozymes relative ix) ~-, fl- a n d y-crystallins eor,'esponds to t,h a t seen in P l a t e 4. However, the gel aJlalysis clearly shows t h a t t h e
31~
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]~o.~jthnt.,~ of Lhe i~)zyme.~ (e.xce]~t fitr 1.1)1{ 5] do n~t c o n e | d , , w i t h ttw. i,osilit?n ~f a sl>(,>('.iii(',h:n.,l c r y s t a l | i n . ']'hu~, i~lthottgh LI.}H '2 hlt~ t h'e same. m o b i l i t y lis ~.-crystallm oll paper, in I:.ht~ g,A IAiis j.~izynit; iias ~-)]igril.i.t~d f u r t h e r t o w a r d the anode, than ~. {'.ry.sbMlin, ORIv L I ) I I or is b l l r i c d iil one o f Lho ,qlower r n i T r t i i i l i g jq.-t.:I-ysl.Mliil,~, >rhe i r i o l t i l i t y i j f no I A ) I i i,,~l~.)zyrlli) t.~orrt;sfil)lid~:4[ IO l h l i t iJf t, Jl¢: l n a j o r )~-t:ryst.allhi prot.eitL~.. I~,alibii, lent4 l . , l ) I [ i.~oZylllt.%t4 }llif.| Llie l:ililliC, i'elt%t.ion.~hill to t.lle, h~fi~4 c r y s t a l t i l l ~ it,s t ho.~. of |,hi; bovino, tentf, and If the iSfiZy IIlI.'-B{Jr rM2hit ske, lcA,al itl!ls{;ifJ. 'Fh,; eOinlJarut.ive i,,~.)zyln,: pnl0tt;ritti o f b o v i n e lens filial f'.oriieit ;ll,'e s h o w l l ill. Phi tc ~L JlOt]i ill Lh0 ~-]ttollt,}l.-old fel,tls litld tile. O-inoilt.ii-old clllf, t i m C(ilPll+.:li blho%l,-ed ttl.t~d ( . l i l i i l i l l ¢ i l , ac~ivit,v n.J. L D t { 3--D, lind the ~lilli(; [!lit.tfJl'li lt'li,q 8¢*(#!1 irl l, he 6-)'ear-<.iM l;tOVillA~, c o r l l l ? l i (~0.1).ltiate 8). T h i s t3OLitrll~t~ wit.l| the ncl,llt [e.il~ f.llitl.erll, w h i c l l f4il(tWlyd o n l y I'.,l.)l.l 1 aiid '2. '~[hi..' i.~>zviiic })~ttt~rtl,~ ()[" ill.Hi.lit wllot~~ lens and c o r l i e ~ l ~,11'(~ 8f't3it ill [>liif,o 7. 'Pill': n t b b i t lens wiis ¢'}ilirili~i:~liZ(,.h; ill t h e illu.s.i, rtition. The (.'¢)i'iit._'ll, {il {~lilii, riisl, Io the l~m,~, showed }[)rodoltli?llilt[. iloLiviL), ,'it ]~D.lJ" t ,..'1, ,~lll(t ~l lllttrkl21;t iilcrt.'ii~0 itl ~l.lihliil~ is xioled w i t h illcrcas(; ill ~hi ~. lltro O f tltP. r a b b i t frunt I days t(t .| ilIOILL|I~. 'F|le iSOZ)'llio ltat.ti:rnl~ • (if the le.n.~ opiiAlelium of ll (byear.ohl. cow, itl ct.liil.rltsl, l.O t>lm h.:us fiber lua,,~% . s.h o w . e d. L D l l 't--t.I~ iit iiti(tiliO'l] t() iS~l?.)'liil.;.~. | ltiltt "j ([>hit.o ;5). S i t t i i l a r l y , i - u i o l i t h - o t d rabbil, leith epil.hcliunl alit,wc~| ili<:rt.>~at::<] a.mouill,s ~)f l.,l)It | iilltl "2 w h n n coillpltrl;(t wil, h tlic Imps |flier cell pai, t(:rn, 'I'Jic i~OZylilt: lJilLtern.~
The, rcmitt,~ ot)i,ain.cd in IAlis OXl)Oriineqit arc in ii~ret4.nli~iii, wit.h t h e (~t.,serv~ition;~ of tto¢'k~vili (1~,i65), w h o found it l)ro~re,,~sivc: dei;rense o f l A ) l i act.ivii.)" per ~,~.¢171tlII l>ox'htc ions 41it(| llt)r h;tis w i t i l in~,'rc'.lishlg age t i l l e r b i r t h . T l l i s a u t h o r lll~3~l~liircd I A ) i t a ( s t i v i t y ~l)(Jtdtrolitl.OLOlltlJ~.riclitt.V ,+lil(l d M iitll. It i.stiugui.~h |)t~.12,vt)f.-'-II {-he islJz)'llit'~l. Otir clltl~l .~ho~" {hit, l,, l..hc+ ct ~..cr.lrsc. ," +," • of I A ) [ I acti~'it.y is associiited wit.h a kiss of L D I I 'I~-5 ~'il,]~ t.h0 aging of ttiC- ]C.iL$. Thli.~l, t h o fe.t.al )mis is chart~ctorized by t.]it~ ])rl~(~rice. r.)f L I ) t ] I.--5, w i t h I.A)t:[ l - a p r t ~ | o n i i n a n t , wti{lc the li(.lull, ]eiit~ lilts [;lrt.:{.|Olliiliillltl. <'let.ivitv at I.A)J.4 I. ~tew:lrL and l ? a p a e o t l ~ t a i l t i l l o u (19C6) al.~t r e p o r t e t t l;|.tc [o,.~ of oa~h(~.|u I. it'trl.)lylllt~S w i t h incre~l+ae hi age. f r n i n c a l f i,o tidult. Ve,~tl (1965), on t.ht~ oth(:r hll.tRt, .t])ulid flint; I.]1o bovi.i.~tJ .Icn.~i fiber has tlve i,~oz),ine~, c t i a r a c t e r i z e d b v a tlredoll~illaliq~,. o f L D . t l 3. T h i s l i t l b l i o r t ho~vt:vor, d i d n o t specify t.lm lilt;,~e}, }(.G~rl~!g&It t U i d ,. yl//~r, I ~)~l), ] t.l i.hl.~i Spef,$liSO, flit'. ¢~ml)r)'re(lol.iliniilic..t: o f liet.iviLy fit L D H 3.-.& whih: aft(~r tfir~h, IA)t [ t is tirt,er m ~ prtiieitt) wlts gr,)ttt.t!r ilt 3 - - 1 - w e e k - o k ! r a M , i ~ t h a n {l! 3-} (~ir-oM a n i i m i t s ,
I,A(?TI( t I ) I . l f I Y t " ) R O C E N A ~ h t IN(.)Z~'+~lt.:~ IN ),t.;N~ ,&ND (;OIt.~'F+~++
31:1
Ih+ .... r<+++ult~ m a ' ¢ l)e into+Zpretcd it~ r++,lation t.o t.he.functi,>n+d signifii++tn .e(: o f I+I)II <] +~ + lr ' ~t ~ t" isoz?,+m+;,~, It h a s br+,m l.+r,q>o,+~d t h a t I + D H ' I +rod Lt)I+ ,:~ " h a v u .~+guth<'+~nt-iy " +" f,.m<-tk+n+'+ (CMm, K++plmt, L<:vin,: n n d Z+s+illi+>g. 1962; F r i t + , "°er+~ l .., J-~t. ( h d v ti.Pmu+~,+o.,q+ahtu ,,f t<~h.,rating rel+ttliX+o+]y ttltaOl'Vttit': e o n d i t i o u ~ Ltr+ r i c h in L D I ! 5, w Idle I A ) I t I i+< p r t , ~ + t . in high e o n e t ; n t r a t i o n in tis+~at'..s r e q u i r i n g m +t+'m+.t5' s u p p l y o f om'-rgy prc+vidrd by t h e t+,-,mplete, oxMat-iot+ ++f p y r n v a t e a n d lactnt~y v i a t.h~+ c i t r i c +leid r y e h . . In t e r m ~ n f t h i s it~t,,rpretati+m, t h e met,+ti~+lie n<+.tivity o f t h e c h i c k a n d hovim,, l e n s seem.,+ to bet~nmt, Zll~:l'(~ dt,Jl+l+lldt+.ilt till+l |lt~+ro[li{.'.. ¢x.ll~l(iit+io)l,,,I w i t h tit,;, itle, rt~tish}g a~,.~ o f t|+o+ alnLtn+d. |I+++~et.l. ~~'++qnr+ Imtr+~rn +.+ft h e rab}+it ]r.tls, hmvr..v,+r. +ulz¢+.'++t++t}u~t, it. io+ mnht|}" dr~|P~,lld0nl. +m nna+.,f++bi++; ,
.
•
tn;'t a-T[>o|i~l] i.
°fhtt.'+. th(+' bovin,.+ <+'+ortlrJ+ |i:(.~ +~r+.,~i¢+tl+il'tnrkt.|+- l+l.)l! 3+-5. mt~<.,s~,in~ a ln'+~10nfinanr,.', ¢2f atla,,r++f)ie ItV.'+t~tbolis}~l+ ]'+tl+-t,¢.rZl+.+t;1 ;trtt] Kin(tobit:+ {I++)5<,)) nlmo t \ m n d thr+< + IA.)I[ i+,,zvme.-+ in (;+11,1li+ e,o r n e a l e.pi t.h{;li m n, ])rt+lmbly <:orre++pon,limz to M ) I l 3...5..I n c o n l v~,m. t,, +'}tr. k+n..'+, t.tm rahlfit. (:~>+;l+:a hn~ tnai~dy I , D I I l.-:+, mi,,.'g+li~+t+' a ]we,+10min+tJ+(:<. o f
l-+,+t:,l+o|ism, it. wa.'~ t+f h+l.ere.~.t to c<):n|nxrt ~. lhe+ir im+~.ym,--, p+'dteru.~. 'l'h,.'+ Imtt,.+.rn for ra|Jl~it +tn¢l bo+¢i+l+; Ct+rtl+'atl rpith+:li]+m wa++ e+m.~etxtially idm~tir++l tt~ t.l+at ,.ff l:|~t++ w l m l e t'q+.lrlll%~(."|'|HI+ iS()Z3"l'l'l+', lml.t:w/t.s fro" k, ns mpitho.ti~.lm. | m w o v r r , d i f f e r e d f r o n t t.|t+).~e o f the ~h<,l,: ,+r~an. 'l'}m rLdd~i~ lt:n~ rl,iLhelitm~ h~M .~i!..,~dfimmt a r n ~ u m ~ o f L I ) I [ t ,~nd 2 i~ a d , i i t i o n to I A ) J l 3 - 5 , ~<> t l t : t l : f,l:t, i~<~z)'+l~- ' I.mtt,:rn nl>plx)ximJ~l+~n mot+e t:}oa+..ly th<,t. ~)f !.]i+~ c(+rl~¢a f.h'~tt <]oe~ t,h(.~ h ' n s fil.~,.r eo.l[ ]~ut,tern, S i m i h t r t y , t h e i~<.,zyn~t; |+att.,:rn <,f htJvin(P h.'.ns +>pit l m l i u n t r,.~<,mbles more. c[om:ly t,hat- ,)1' t h e {u:~n+,.'.+; t.httn d o e s t, hr. i+s,-,zyn~r. Imt-t.c-n+ ¢>f +.he fi Iw.r +:ell. St.ew,trt a n d P+q+,eo~++t:mt.i/u)+~ (J 9611) hn +,t; r e p o r + e d their adttl~ b~,~-it+e, I~+n.~ el~ithe.ti~tt e<:.ll.~ e(mt.Mn 1A.)/-[ 1+.-5 a n d tl-.] h.,m+/ib,:r~ < m | y L D / [ I ;in<| 2. l+t. i~ a p p a r e n t , therrf~n'e, t.tmt, Mgnifie+mt e . h a n g e s in i.~ozytn,+ et+mt~,~i~i+m tii~.,r e++lls :te(-ount. f(w t h e grr.at contra++t. +mtwe,+nzj'm<+ l++llA..t;,l'l+~. + St+t+~++trt. a n d ]+~tlmcun,~t+.tmimm (1966) hnv:- p¢+it~ted ~.,t+t. t h a t the, lirn+ ep+lheli+tl <'~,tl e x h i b i t + a gr,:++t.r.r d e g r e r o f ++crobie, mr.t+tboli:m+ Llmn l.|lr fi!.,e~ +-,:1t, hr, vi~>~ ldgh,0r krV('lS ~'+f cy+.ochromt+ c, gr~at.er 8ttc+inie deh)-dnL~/:nnm; a c t i v i t y , +md retie~ativ0~, a c t i v i t y t.h~m ~ , d l l t t (i(?.lLq [ l a v l ) l-HOl.t..' t~tht~.|:d i ~ z y ' m e s . (.<_lia w i t h de~rt.,a,,.:d mitoti<" ,,~:tlvi+v, +~a the +re+Ifor :~c|utt. til.,er o'.-.ll. | m y + mo.~l, act.ire ++n(mla+ i..~+zy:ne~. "['hi.~ ,,at.gumt:nt + hov~'(Px++:r, ~'ontd n o t ~ e e m v a l i d for t h e n~bbit., _~incc,. i~ thi~i Sl~.'cirm it i+~ th,: fib,~r e etl t.h,t: t:,'mt.;~i~,+ m ~ d n l y Llm e a t h o d a l i.~ozymr-~., w h i l e /.he (,pit.h~rliM e+.'.iL~, h a v e mort: ~ff [h(~ .~m+~|.~} t.~)i~,,'tn(:,'s.'. ~.tmdarts', t h e epit}te]ilxl c elt~ o f rabMt, n n d b(,virl,:, (-ov~te~ }i+~','<..~ nutrL:edly con.t.na.sting i ~ z y n ~ e , paLte.r+~.,s. 'v'e~.t:t~ (~965} bin+ .~;.tgg<:++t~+¢] : h a t tho+'e ~.~ a ..+t.,,.+e~fir. ae~'.+_×.i~ti+)n b+.+tw,~-.n 1,I).tt 5 ~ru] t.he., nuc+t0~.ts o f ,~ .ex:II a n d t h a t th,; a ~ > e h ~ t i . o n t)et.w .e+'.+n i~zy~v..+ j,ttt.o+rn~ +~,-!d tht+ :~++~ur~.'+of. r+:)etab+~lic ,~-Gvity i.~ n ~ t v M i d . lle+ ~t~',~.e
t ~ "
°
.
31~
LARRY BIgRNSTEIN, M[CI-IAEL I,:.ERIlt£:AN AND IJ£AR.gY 5[A[SI-~L
anuclear cell, isozymes composed of ibm: idcatical A subunits are constructed with greater difficulty or arc catabolized more r a p i d l y t h a n isoz}m~cs composed of one t3 and three A suburtits. This a r g a m e n t does not aj?pe~r v,~lid wheL1 comparing the isozy-mc p a t t e r n s of r a b b i t and bovine a.nterior corneal epit, t,elium. Since the rM~bit epithelial coils also contain mmlei, one would expect a high conemttra, t~ion of I,Dt-I 51 which t h e y lack. Fur.then~mro, t h e r a b b i t lens epithelial patter.l~ shows mo~e L D H 1 m~d 2 r a t h e r ~han L D H 5 when cornp~red to ~he fiber cell. pattern. I t is clear t h a t t h e r e are ma.rked species differences in t h e r a b b i t a n d bovi~e LD]:[ isozyme patterns. Furtl.mrmorc, in these species the lens and comte~ show markedly contrasting isoz)n~e p a t t e r n s . The ftmetional significance of the differing isozyme pa.t~terns, however, is yet to be clucidated. F o w l k s (1965) isola.ted a homogenem~s protein from t.lm bovine lens with low LDI:I a c t i v i t y in comparison to ~'ecrystallized h e a r t L D H . Tim protein, w i t h ,/-erysta.llin mobilit, y was idmtt.ieal immunologie;~lly to t h a t fotmd in Ow, rabbit, lens. This L D H bad ha.lf the molecular weight of t h a t fi'om bovi,~e heart. F u r t h e r m o r e , various chemical tests revealed it to be both similar to a n d diflhrent from bovine h e a r t LDIiI. W h e t h e r the isolated L])IY corresponded to a n y specific isozyme was not determined. Our d a t a indic~tte t h a t bovi~m a~d rabbit lens LD.H exist in. t h e tbrm of isozymes, ~lm n u m b e r and conce~ttra~ion of wbich depend on the age of the animal, and 1;hat the eleetrophoretic behavior of these isozymes is sinfilar to ttmt of isozymes i~ ot.her tissues. F u r t h e r m o r e , the m o s t prominent isozyme in the a d a l t lens m i g r a t e s further t~ the anode oa paper and starch gel media a t pl-[ 8.6 t h a n c/.-cryst.allin. This finding is in contr~st, wi~h Fowlks' observ,~ti(m (1965) t h a t lens L D H ha.s a 7-crystullin mobility. I t is in.deed possible t h a t if F o w l k s has isolated ;m intermediate.isozymc, it would not, have t;h~ properties of pure h e a r t or muscle LD~[, provided the latter prepa trations represented pure LDt-I 1 and .LDH 5, respectively. A C I~2N O W L E 1> G ~ [ E N "['
The aubh¢,rs are grateful to Mrs. S. Mehta for her skilled technical assistance.
I~EFERENCES
Calm. ]I.. D.. I{~plan, N, 0., Levine. L. ~nd Zwilling, E. (1962). ,Science 136, 962. ~'owlks, W. L. (1965). invest. Ophthalmol. 4, 611. ?Francois, J. ~nd ].~al)~ey, hi. (]957). Arch. OphthabnoL (Chicago} 57, 672. ]?ritz, P. J. (1965). S~ience 150, 364. .F~tterman, S. vmd :K_inoshita., J. :t]~. (1959). J. Biol. Ohem. 234, 3174. Green, }I. and Solomon, S. A. (1957). Arch. Ophthal.mol. (Chic~go) 58, 23. H0ckwin~ O. (1965). l~vv~t. Ophl~dmol. 4, 496. ~tockwin, O., Schmuttcr: J. ~nd Miiller, K. 1,2. ([963). Arch. Ophtt~dmol. 166, 136. Lowry, O. H., ]'¢osebrough, N. J., Farr, A- L. and :Randall, 1~. J. (1951). J. Biol. Chem. 193, 265. ~IMsel, it., l{errigau, M. ~n¢:t S)~er, 1~. (t955). invest. Ophthalmol. 4, 362. l>onlik, )i. D. (1957.). Nature 180, ]477. Sippcl, T. O. (1965), tnvesL Ophthalmol. 4, 502, Smithies. O. (t959). Biochem. J. 71,585. Stewart, J. A. ttnd Pap~const~atino~:. J. (19(;6). Biochim. Biophys. Acta 12I, 69. Vcsell, E. S. (1965). Science 150, 1735.
Lactic D e h y d r o g e n a s e l s o z y m e s in Lens and C o r n e a * [,AllIIY ]3NlcNNq'I¢IN, ~II('ltAI,~I, .[~I,lll|LI{IAN ANI) ll:%ltl~.V MAlS~Cl.
De'l,artme~tt q[ dmt&m'~ff, l Va~/ae ,State l/~,ice~,:~itt/. ,School of 3Iedicim', Detroit, Michigan,: U.N.A. (Received. S April 19B6) t a c t . i t deh.vdrogcnetse isuzymt.s o[" b o v i n e n,ui r ~ b h i t I(.,ns Itl|(~l c'ortlogt ~X'Z~Z'OI~,It,~t]YZt'(t I~,• s t a r c h gel eh, ctroph(n'e~ s. A l t h o u g h t h e r e w a s ~ p r o g r e s s i v e loss o f e n z y m e r~etivii.v in il'm Ien.ses ~)f boll1 Sl)eeies w i t h iuercr~siug ~lg~.'. I]1(: h3s8 o f isr~zymes xv~s rnon~'elearly c~'i(i,mt ill 1 he- b o v i n e lens. I I= t.he adult, b o v i t m tons. tact.it d c ' h y d r o g c u t ~ s e i s o z 3 ' m e I w~m p r e d o m i n a u t . while in l.hc a d u l t r a b b i t Iens. i s o z y m e s 3 - 5 w e r e n m i n l y I)Vesent.. *J~he m o b i l i t y ~,f Ions i s o z y m e ~ w a s ident.ieM t o l.ilaL o f i s o z y m e s in tn.hm. Ii.~.sues..[~ni'llmrmore, t h e isoz3'n-m:~ wet~e lU)t IocMized to ~u~y m a j o r specific lens eryMn.llin. T h e eorn,,a i ~ o z y m e pat.t,ern in eatclt stu:eies <'elmira:iLl,t| m a r k e t l l y wit.ix Lhr~L +~f {.it(: c o r r e s p o m l i , ~ g lens. {['hus. in I;l'm a d u l t b o v i n e t't)l'llet*, iS~,';Z,~'II|CS : 1 - 5 ~A'ert: pre.do[l~i~tttnt, a n d in t h e rabt~i~ c o r n e a , i s n z v m e s 1-4. T h i s ditti:ron(-e bel, ween lens a n d e~,rmu~ a p p e a r s t o |m a f u n c t i o n o f t h e e n z y m e e o m p n s i t i o n o f t h e lens fiber eel[ r a t h e r t h a n o f tim lens e p i t hc.liM cell.
I. Introduction
8cvcr~*l i n v e s t i g a t o r s h a v e o b s e r v e d lactic d c h y d r o g e n a s e ( L D ] t ) activity" i,t t k e b o v i n e hms. t,) v i t r o s t u d i e s h a v e showt~ a. decrease in t h i s a.etivicy per lens wit, h progressive age afte.r birt.lt (]rioekwin, ]965). Stewar~, a n d P a p a c m l s t a x t t i n o u (1966) f m m d oJ,ly L D H [ ~md 2 i1~ t h e a d u l t I~ns fiber ceil, i~1 eon t~rasb to the epit, hetial eetl, w h i c h cont.aincd a.ll five is,,zymes. T h e s e ~ u t k o r s also showed ~t decrease in. t,he m t m b e r of (~ath.odni isozymes w i t h an increase in t h e age o f Vim lens fron~ calf t o a d u l t life. ]i'owlks (I905) isoh~tcd a. pro~uilt w i t h L D H a c t i v i t y f r o m t h e b o v i n e lens, whicl3 p r o t e i n was boCh similar to amd differm~g from he,~rl~ L I ) H . Green a n d S o l o m o n (19571 r e p o r t e d o n - t h e prodtmt, ion of" laogie acid iJt the. rabbig lens. We h a v e r e p o r t e d p r e v i o u s l y o n t h e c h a n g e ill L D l t isoz2one~ d u r i n g tim d(;velopment. of t h e c h i c k lens mid ghe s i g n i f i c a n c e of s u c h c h a n g e in relation, go t h e m e t a b o l i c a c t i v i t y o£ t h e lens (Maisel, K e r r i g a n a n d S y n e r , 1965). T h e s t u d y r e p o r t e d here i n v e s $ i g a t e d t~,e isozyane p a t t ~ r n i n ghc b o v i n e a n d r a b b i t lens anti cornea d u r i n g development. 2. M a t e r i a l s and M e l h o d s
5l'issue ezt.racts Bovine eyes (obtained from a sla.ughtcrtmtmc) were removed from the atdnmI imme(liateiy aft~er aactifice, transported to the l a b o r a t o r y in crushed ice, and thet~ dissected. ]~ach lens, freed of all surrounding Lismle bill xvit~h cnl)~ul~, inL:tet:, was ]mmogenized aL variable concentrations of 100-250mg wet. weight, of tissue]mi 0-9°, o saline solution. The homogenate was centrifuged "tt. 12,000 g {'or 20 rain at 4°C and the supernal, a n t used for furtho.r tESts. Extraet~ were. also prepared ~f lenses after the capsule and epitheli':Ll cells ha(l been removed, of whole comma, heart, skeletal muscle and liver, l)retmratiuns * "|'his invest-igatlnn was suppm-Lcd by ~ l?IqIS resenreh g r a n t {N [~ 04763} frf,m the .N'uti(mal fnst, it, tJte~ of Health, U . S . P u b l i c H e a l t h Service. 309
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I'~,,~,r~: 7. 'Lt)Ii[ i~ozyme pntLertas Of rabbit lens and eori~e~ analyzed, by ~'er~,icat ,~t~rch ge} ele¢:tr~:J•:~ht~resiswithTris tm~'at.e-lu" 1)'~ J. A buffer. ' " a, 1-year-old;-Icns (0-51 I g); b, 4-month.old lens (0.3OGg); ~'., 10.day-old lens [0.156 g); d, 0-day.old :ci~s (0"08(Ig); e, ]-year.o]~leorhe~; f, 4-mox~th-old cornea; g, 10-d~y-o.ld cornea; !a, 2-dayiold cornea. Leas c~t-ra~ts w~re prepared wit,h 250 mg ti~-.~uc/ml 0 ' 9 % saline sob, tion andC~l'l~ea[ ext,rac~ Witli ~00 mg tissuo/ml 0-97,~ saline solution. Nt, mber~ represent-the val:ious L[)I:[ isozymes/
•(luJ/u!~1(~ad ~u~ ~'S) a~'-~:> I'lo 'a~a.~= 9.1o ,~{l~a[~!pq:l!da .m!.~a~u,,:",~:qm/~!~>~,o.~d ,~u~ ~.~) .~rm lqo-.n,a.,/:-=g .io ~;au.ma ,~loqa~ "~I'{|m/t~]a~,,.~d ~'u~ I-:,'} o~u>:) p[~-a~o.{'~-~ fi Jo ~ll~a l e ! I a l l l ! d o ~su,~I 'a :(1 tu:'u!a.load ~,tt[ g'.~) a~oa l:,lo-a ~0.C, ~ 9 .t ° ~m:~.u .I~qt t ~ua l "p i ) . . . :(]t~t/u!O~l(,td .Sttl ~t , | ) ~ /. ,q q u , t l'~[:.,-~['l~mtu :--! j o ~llOa l~riattardo a m a. o } t ' e . leam,~)/> "a . :{l~U/ma~o.ld ~ [ I ] 0 " ~ ' ) 1 1 q ' / 1 1 ! ' l p l o q ~ ~'.'m --=-1 JO s'IDa t ~ ! l a g t ! d a s u a I 'g ~{]m/u!o.~oad ~ttt <).g,c:) :~!qq ea plO-~lltlpul = l j o ss~tti Jaq !j .~uu" I "u • .,aLum' ~ y , I , ( l ~ a ~ u o q .~!a'; ~11!-~ :!~.,->,mtldo.~!,)t~la io~ ~4a:t~;1-~ pa,>!.:l.~aa Xq l~aZSB, m,' u~ua~a, p u t : .-:u,~I j o .~u,~a~,~;d atu,~'zos'! H ( I : [ "S '--1.I-v':d
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[~ is clearly eviclent from Plates 1 a n d 2 t h a t the isozyme p~;te, rn of the bovine, Ioa~s changes significantly wlgh age. Table I, de, rived f r o m the d a t a of Fr'meois altd -I/a.ba(;y (1957) and o f ]:[oekwin, S e l m m t t e r and Mtiller (1963), relates the lens ~-eigh~, f,) the ~.ge of t,he animal. The decrease in total L D H per gram len~ wit, h increasing a,~e is associated with a progressive, loss of .LI)I-I activity, first affecting L1)H 5 and then .LD]{ 4~ 3, ,~nd 2, in t h a t order. T.}ms, in fetM lenses, all five LDI-I isozyme.s are clearly evident, w i t h predonfinan, a.otivi~y ~t L D H 2 and 3. la the young cnlf lens, I,I)I[ 1-4 a.re el{parent, with L ] ) ] t l and 2 predominant,, h~. the m a t u r e adult lens, however, only ] , D H 1 and 2 axe clearly appm'ent, with predo,ninant)activity. ,,.t L D ] t t. Identical isozyme ].>at;terns were obtained from bovine lenses fron~ which the .capsule and cpit.helial ceils had been removed. t AnL~-;
I
R e h g i o n s h i p o.f boci~te lens weight, to ~ge * Lens weight
Ago.
0-55ti
G-nmntb-old fetus ( ; - 7 - m e n t h . o l d t'etu.~ 7-8- m o n t h - old t'~t,u~ 8 - 9 - m o n t b - o l d fiet,u.~ newborn catt l - 2 - m ~ , u 0 ~ - o l d <.elf 4 - m t ) n b h - o l d call" 8- m o a t h - o l d c ~ l f l-2-ycar-ol4 cow 2-ye~).r.old 0ow 5-yeast-old cow 6-year-old cow
0.600 0- 7"18 0"834 0.926
~.062 !'21 6 1-5-21 1.8,12 2" i 53 2,317 :?.400 m
* 'L'he b~t})rm.~tion in Lhis l.ablo is b a s e d o~ t h u dmt.a c o m p i l e d by Hot~kwin, S c h , n u t e e r ~ d (19611), I " v a n ~ o "~ ~tnd R a b a e y (L957), a n d t h ~ aut.t~ors el tlsis palYer.
31(il/er
The relationship of LDI-I isozymes to bo~-ine lens crys~a}lins is shown in Plates 3-5. The p a p e r e|ectro]?horetie s e p a r a t i o n of the fetal lens e x t r a c t s~aiJled /or protein and L D ] t is t)resetlted in P l a t e 3. This ~na.lysis il~dicated ~h a,t, ]~DH 1 m i g r a t e s fllrther to ~be ~mode l~han'q.-c~wstalLin, LD_IK 2 has t h e s~m~e mobility as ~-crystallin, L D H 3 migrates to a point between ~.- aud /~-crysgallin, a n d .EDH 4 and 5 have a. mobi[it.y similar m th'J;°c of ,8-erystallin. No LDI-I isozyme corresponded t,o the 7-crys~allin b~md. The relationship of ]61)I-I isozymes and lefts erysgailins a.nMyzed by p a p e r clect,'ophoresis and followed by sta.reh gel electrophoresis is ~een in. Plates 4 and 5. Plate 4 shows t h e p a p e r s~,ri]) on w]aieh lens proteins had been eleetrophoresed in t h e diree~iort of the vertical arrow. After this inimical elee~0rophoresis, the st-rip was inserted in a starch gel, and electrol?ltoresis Was performed at right angles to tim ori~i. sepa.ration. The get was biseated and one-half stained for protein a n d t h e oth,>~::~::': Lt)It. Five isoz.yraes were detected, their relationship to the leas pro|;eins beizlg slmwn in l?lage 5, which represents the :LDH gel of Plaf, e 4, which w a s couat, ersvMne~l tbr p r o t e i n . . T h e positim~ of tshe L D I I isozymes relative ix) ~-, fl- a n d y-crystallins eor,'esponds to t,h a t seen in P l a t e 4. However, the gel aJlalysis clearly shows t h a t t h e
31~
[,A|~I?,Y
HI-~I~-NS'FI~I,%', M I C H A f ~ [ o
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.-%.N'J~ H A R I ' , V
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]~o.~jthnt.,~ of Lhe i~)zyme.~ (e.xce]~t fitr 1.1)1{ 5] do n~t c o n e | d , , w i t h ttw. i,osilit?n ~f a sl>(,>('.iii(',h:n.,l c r y s t a l | i n . ']'hu~, i~lthottgh LI.}H '2 hlt~ t h'e same. m o b i l i t y lis ~.-crystallm oll paper, in I:.ht~ g,A IAiis j.~izynit; iias ~-)]igril.i.t~d f u r t h e r t o w a r d the anode, than ~. {'.ry.sbMlin, ORIv L I ) I I or is b l l r i c d iil one o f Lho ,qlower r n i T r t i i i l i g jq.-t.:I-ysl.Mliil,~, >rhe i r i o l t i l i t y i j f no I A ) I i i,,~l~.)zyrlli) t.~orrt;sfil)lid~:4[ IO l h l i t iJf t, Jl¢: l n a j o r )~-t:ryst.allhi prot.eitL~.. I~,alibii, lent4 l . , l ) I [ i.~oZylllt.%t4 }llif.| Llie l:ililliC, i'elt%t.ion.~hill to t.lle, h~fi~4 c r y s t a l t i l l ~ it,s t ho.~. of |,hi; bovino, tentf, and I
The, rcmitt,~ ot)i,ain.cd in IAlis OXl)Oriineqit arc in ii~ret4.nli~iii, wit.h t h e (~t.,serv~ition;~ of tto¢'k~vili (1~,i65), w h o found it l)ro~re,,~sivc: dei;rense o f l A ) l i act.ivii.)" per ~,~.¢171tlII l>ox'htc ions 41it(| llt)r h;tis w i t i l in~,'rc'.lishlg age t i l l e r b i r t h . T l l i s a u t h o r lll~3~l~liircd I A ) i t a ( s t i v i t y ~l)(Jtdtrolitl.OLOlltlJ~.riclitt.V ,+lil(l d M iitll. It i.stiugui.~h |)t~.12,vt)f.-'-II {-he islJz)'llit'~l. Otir clltl~l .~ho~" {hit, l,, l..hc+ ct ~..cr.lrsc. ," +," • of I A ) [ I acti~'it.y is associiited wit.h a kiss of L D I I 'I~-5 ~'il,]~ t.h0 aging of ttiC- ]C.iL$. Thli.~l, t h o fe.t.al )mis is chart~ctorized by t.]it~ ])rl~(~rice. r.)f L I ) t ] I.--5, w i t h I.A)t:[ l - a p r t ~ | o n i i n a n t , wti{lc the li(.lull, ]eiit~ lilts [;lrt.:{.|Olliiliillltl. <'let.ivitv at I.A)J.4 I. ~tew:lrL and l ? a p a e o t l ~ t a i l t i l l o u (19C6) al.~t r e p o r t e t t l;|.tc [o,.~ of oa~h(~.|u I. it'trl.)lylllt~S w i t h incre~l+ae hi age. f r n i n c a l f i,o tidult. Ve,~tl (1965), on t.ht~ oth(:r hll.tRt, .t])ulid flint; I.]1o bovi.i.~tJ .Icn.~i fiber has tlve i,~oz),ine~, c t i a r a c t e r i z e d b v a tlredoll~illaliq~,. o f L D . t l 3. T h i s l i t l b l i o r t ho~vt:vor, d i d n o t specify t.lm lilt;
I,A(?TI( t I ) I . l f I Y t " ) R O C E N A ~ h t IN(.)Z~'+~lt.:~ IN ),t.;N~ ,&ND (;OIt.~'F+~++
31:1
Ih+ .... r<+++ult~ m a ' ¢ l)e into+Zpretcd it~ r++,lation t.o t.he.functi,>n+d signifii++tn .e(: o f I+I)II <] +~ + lr ' ~t ~ t" isoz?,+m+;,~, It h a s br+,m l.+r,q>o,+~d t h a t I + D H ' I +rod Lt)I+ ,:~ " h a v u .~+guth<'+~nt-iy " +" f,.m<-tk+n+'+ (CMm, K++plmt, L<:vin,: n n d Z+s+illi+>g. 1962; F r i t + , "°er+~ l .., J-~t. ( h d v ti.Pmu+~,+o.,q+ahtu ,,f t<~h.,rating rel+ttliX+o+]y ttltaOl'Vttit': e o n d i t i o u ~ Ltr+ r i c h in L D I ! 5, w Idle I A ) I t I i+< p r t , ~ + t . in high e o n e t ; n t r a t i o n in tis+~at'..s r e q u i r i n g m +t+'m+.t5' s u p p l y o f om'-rgy prc+vidrd by t h e t+,-,mplete, oxMat-iot+ ++f p y r n v a t e a n d lactnt~y v i a t.h~+ c i t r i c +leid r y e h . . In t e r m ~ n f t h i s it~t,,rpretati+m, t h e met,+ti~+lie n<+.tivity o f t h e c h i c k a n d hovim,, l e n s seem.,+ to bet~nmt, Zll~:l'(~ dt,Jl+l+lldt+.ilt till+l |lt~+ro[li{.'.. ¢x.ll~l(iit+io)l,,,I w i t h tit,;, itle, rt~tish}g a~,.~ o f t|+o+ alnLtn+d. |I+++~et.l. ~
.
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°fhtt.'+. th(+' bovin,.+ <+'+ortlrJ+ |i:(.~ +~r+.,~i¢+tl+il'tnrkt.|+- l+l.)l! 3+-5. mt~<.,s~,in~ a ln'+~10nfinanr,.', ¢2f atla,,r++f)ie ItV.'+t~tbolis}~l+ ]'+tl+-t,¢.rZl+.+t;1 ;trtt] Kin(tobit:+ {I++)5<,)) nlmo t \ m n d thr+< + IA.)I[ i+,,zvme.-+ in (;+11,1li+ e,o r n e a l e.pi t.h{;li m n, ])rt+lmbly <:orre++pon,limz to M ) I l 3...5..I n c o n l v~,m. t,, +'}tr. k+n..'+, t.tm rahlfit. (:~>+;l+:a hn~ tnai~dy I , D I I l.-:+, mi,,.'g+li~+t+' a ]we,+10min+tJ+(:<. o f
l-+,+t:,l+o|ism, it. wa.'~ t+f h+l.ere.~.t to c<):n|nxrt ~. lhe+ir im+~.ym,--, p+'dteru.~. 'l'h,.'+ Imtt,.+.rn for ra|Jl~it +tn¢l bo+¢i+l+; Ct+rtl+'atl rpith+:li]+m wa++ e+m.~etxtially idm~tir++l tt~ t.l+at ,.ff l:|~t++ w l m l e t'q+.lrlll%~(."|'|HI+ iS()Z3"l'l'l+', lml.t:w/t.s fro" k, ns mpitho.ti~.lm. | m w o v r r , d i f f e r e d f r o n t t.|t+).~e o f the ~h<,l,: ,+r~an. 'l'}m rLdd~i~ lt:n~ rl,iLhelitm~ h~M .~i!..,~dfimmt a r n ~ u m ~ o f L I ) I [ t ,~nd 2 i~ a d , i i t i o n to I A ) J l 3 - 5 , ~<> t l t : t l : f,l:t, i~<~z)'+l~- ' I.mtt,:rn nl>plx)ximJ~l+~n mot+e t:}oa+..ly th<,t. ~)f !.]i+~ c(+rl~¢a f.h'~tt <]oe~ t,h(.~ h ' n s fil.~,.r eo.l[ ]~ut,tern, S i m i h t r t y , t h e i~<.,zyn~t; |+att.,:rn <,f htJvin(P h.'.ns +>pit l m l i u n t r,.~<,mbles more. c[om:ly t,hat- ,)1' t h e {u:~n+,.'.+; t.httn d o e s t, hr. i+s,-,zyn~r. Imt-t.c-n+ ¢>f +.he fi Iw.r +:ell. St.ew,trt a n d P+q+,eo~++t:mt.i/u)+~ (J 9611) hn +,t; r e p o r + e d their adttl~ b~,~-it+e, I~+n.~ el~ithe.ti~tt e<:.ll.~ e(mt.Mn 1A.)/-[ 1+.-5 a n d tl-.] h.,m+/ib,:r~ < m | y L D / [ I ;in<| 2. l+t. i~ a p p a r e n t , therrf~n'e, t.tmt, Mgnifie+mt e . h a n g e s in i.~ozytn,+ et+mt~,~i~i+m tii~.,r e++lls :te(-ount. f(w t h e grr.at contra++t. +mtwe,+n
t ~ "
°
.
31~
LARRY BIgRNSTEIN, M[CI-IAEL I,:.ERIlt£:AN AND IJ£AR.gY 5[A[SI-~L
anuclear cell, isozymes composed of ibm: idcatical A subunits are constructed with greater difficulty or arc catabolized more r a p i d l y t h a n isoz}m~cs composed of one t3 and three A suburtits. This a r g a m e n t does not aj?pe~r v,~lid wheL1 comparing the isozy-mc p a t t e r n s of r a b b i t and bovine a.nterior corneal epit, t,elium. Since the rM~bit epithelial coils also contain mmlei, one would expect a high conemttra, t~ion of I,Dt-I 51 which t h e y lack. Fur.then~mro, t h e r a b b i t lens epithelial patter.l~ shows mo~e L D H 1 m~d 2 r a t h e r ~han L D H 5 when cornp~red to ~he fiber cell. pattern. I t is clear t h a t t h e r e are ma.rked species differences in t h e r a b b i t a n d bovi~e LD]:[ isozyme patterns. Furtl.mrmorc, in these species the lens and comte~ show markedly contrasting isoz)n~e p a t t e r n s . The ftmetional significance of the differing isozyme pa.t~terns, however, is yet to be clucidated. F o w l k s (1965) isola.ted a homogenem~s protein from t.lm bovine lens with low LDI:I a c t i v i t y in comparison to ~'ecrystallized h e a r t L D H . Tim protein, w i t h ,/-erysta.llin mobilit, y was idmtt.ieal immunologie;~lly to t h a t fotmd in Ow, rabbit, lens. This L D H bad ha.lf the molecular weight of t h a t fi'om bovi,~e heart. F u r t h e r m o r e , various chemical tests revealed it to be both similar to a n d diflhrent from bovine h e a r t LDIiI. W h e t h e r the isolated L])IY corresponded to a n y specific isozyme was not determined. Our d a t a indic~tte t h a t bovi~m a~d rabbit lens LD.H exist in. t h e tbrm of isozymes, ~lm n u m b e r and conce~ttra~ion of wbich depend on the age of the animal, and 1;hat the eleetrophoretic behavior of these isozymes is sinfilar to ttmt of isozymes i~ ot.her tissues. F u r t h e r m o r e , the m o s t prominent isozyme in the a d a l t lens m i g r a t e s further t~ the anode oa paper and starch gel media a t pl-[ 8.6 t h a n c/.-cryst.allin. This finding is in contr~st, wi~h Fowlks' observ,~ti(m (1965) t h a t lens L D H ha.s a 7-crystullin mobility. I t is in.deed possible t h a t if F o w l k s has isolated ;m intermediate.isozymc, it would not, have t;h~ properties of pure h e a r t or muscle LD~[, provided the latter prepa trations represented pure LDt-I 1 and .LDH 5, respectively. A C I~2N O W L E 1> G ~ [ E N "['
The aubh¢,rs are grateful to Mrs. S. Mehta for her skilled technical assistance.
I~EFERENCES
Calm. ]I.. D.. I{~plan, N, 0., Levine. L. ~nd Zwilling, E. (1962). ,Science 136, 962. ~'owlks, W. L. (1965). invest. Ophthalmol. 4, 611. ?Francois, J. ~nd ].~al)~ey, hi. (]957). Arch. OphthabnoL (Chicago} 57, 672. ]?ritz, P. J. (1965). S~ience 150, 364. .F~tterman, S. vmd :K_inoshita., J. :t]~. (1959). J. Biol. Ohem. 234, 3174. Green, }I. and Solomon, S. A. (1957). Arch. Ophthal.mol. (Chic~go) 58, 23. H0ckwin~ O. (1965). l~vv~t. Ophl~dmol. 4, 496. ~tockwin, O., Schmuttcr: J. ~nd Miiller, K. 1,2. ([963). Arch. Ophtt~dmol. 166, 136. Lowry, O. H., ]'¢osebrough, N. J., Farr, A- L. and :Randall, 1~. J. (1951). J. Biol. Chem. 193, 265. ~IMsel, it., l{errigau, M. ~n¢:t S)~er, 1~. (t955). invest. Ophthalmol. 4, 362. l>onlik, )i. D. (1957.). Nature 180, ]477. Sippcl, T. O. (1965), tnvesL Ophthalmol. 4, 502, Smithies. O. (t959). Biochem. J. 71,585. Stewart, J. A. ttnd Pap~const~atino~:. J. (19(;6). Biochim. Biophys. Acta 12I, 69. Vcsell, E. S. (1965). Science 150, 1735.