Aging of pyruvate kinase isozymes in rabbit lens

E~J. E//e RPS. (1978) 27, 42’7-434

Aging of Pyruvate Kinase Isozymes in Rabbit Lens

liirrase activity wcw Usilkg a(qlamide gel electrofocusinp. several bands of pyrrl\atr found, corresponding to “11” bands: the isoektric points of those bunds differed awording to t Iw zonox of rabbii lens: four bands \vere seen in the epithelium (where protein syntmhesis is :Il.tive). and only two hands in the core of the adalt lens (where thrre is no prot.ein biosynthesis). On the contrary. the four bands exist in the lenr core of 1%d~hy-old rabbits. liinctir results ;tnd thermostahility experiment.n ronfirmrd that the hands of the adult lens vow correspond t.o the “$1,” itnd “-21,” isozymcn. It scwns t,hat. the disappewance of certain molecular forms of pyruvutt‘ kituw nr;cy be due to their shorter life-sp;tn. Thaw modifications m;ty hnvc some pl~~siolog:icnL or p>xthologic~al

consequences.

1. Introduction Lens is an interesting material cha.nges tluring aging. Different scnexwnce in viw can he given.

for bhe sturlv of prc,t’ein Ilwtlifications ant1 ckller 01 the niolecula,r nxchanisms of interpretations The “error catast~qhe” t~tieor\- propost~l l?.v Orgel

(1963). and Holliclay and Tarrxnt (1972) on t.hc accurnulatiou (pi’ errors in prut,ein svnthesis machinery in aging. seemcclvery mttractiw. Eut,. inore recently the imj;ortance uf post, translational inodifications of proteins had lwen stressed (Gershon and Gershon. 1976; Dreyfus: Kahn and Schapirzt. in the pws) : these alterat8ions11ra,xlw very simi1a.rto those causedI)\- a disturl)ance of the hiospt~hetic pat~llway.

Post-t,raiislational nlotlificat~ionsof lens crystdlim are no\\- well known (I~loetnentlal. I%:rlw Van cler ( Meraa and De JoJ~. 1972 ; VAIJ Kleef. De Jong untl Hoenders, 1975). In our laboratory we have shown t,hat severa. ~J~YIII~~ n~itv untlnrgo modifications in this tissue.. There is a lowering of the ixoelect~ric\pH of ~lacose-6-P-ciehv~lro~et~~~s~~. t,riosc-P-isomeritseand nucleoside phoy~hor~-la.st it) t tie core of hunian and lv~vinr lens (Skala-Rul)inson, Vivert and Dreyfus. 19i’6). On the cjt’hcr tmntl. we haw fonncl w.“(‘ros5 reading Naterial” anI1 a mthodiz;ttioii dd’ altlolese (’ in t,hc:inner zones of eahltit lens (Bamwques~Grt!gori ad Sclq)ire. 1976). In other (:;wicx.enzptx ale not iliotlitid. Imt their isozyriiic distribut~iw varies. for example, lactic tleh~clrogeniwe (l~ernstein. Kerrigan and Maiscl. 1966: Hours. Xcuhans antI Hockwin. 19’77) xntl hesosilnlinidase (Poenaru. Skalil,. C’orlrtois aid Ijreyfus. 1977). In t’his stutly we have chosen an enzyme which undergoes moclificatimls in red I~lootlcells: pyruvitte kinase (PK). We recall that tllree main f’om~sof PK megencrall> recognized : PK, (“L” form. predonlinant~ in liver and wythrocytes- --Kahn, Marie clc~l~-~s:c~/~Y:loc,1’7 ;-OS pdOl.OO/O

(. 1ViS Acstlcmic I’wss Inc. (London) Limited 1%

1’yruvwt.e kinase activity was measured ;tc,cording to Kkher 31~1 Pflilitleri*r (I !I.%) 7\-it,ll A \-ariable amount of the substrate phosphoe~~ol pyruvate (PEP) with (11’ without t,he itllo~teric wtivator fructose- &phosphate (FDP) 0.1 ~01. using the method of .1Iuroy;r, S:I~‘~O, Miyazaki, Nishikawa aud Horio (1975). Actjivity JV:LSexpressetl in Interu;ttion;tl I’llits (i.11.) per g of prciteiu : qol of substrate nleticholizecl per min at 37°C i~iid per g of lmteilr. Prckeim

were estin&ed

by the method of Lowry,

Lens extracts were incubated at :j?A”C. At. zero time were immediately removed for assay of PK iLctivit!-.

Rosebrough,

itlid

Farr ;tnd Kandall

s7hsequet~t

intervals,

(1951).

samples

Elect.rofocuxinp on acrylamide gel (in a Btultiphor LKR) was performetl according to l’esterberp (1972) with some modifications. We used ampholines of pH rarlges between (i.0 and 8.0, or 3.0 and 9.5. The wicks for the two electrodes were wettell with NaOH ;rnd H,PO, (0.1 M or 1.0 M according to the pH ranges of anrpholines) respectivel)r. After llligratioll PK isozymes were detected by pouring on the gel a gelose cont;G)lillg the specific st,aining solution (Susor and Rutter, 1971) : the reaction wa.s coupled wit.1~ the hesokinase ad glucose-fi-phosphate dehydrogennse reactiolls: nitroblue tetrazoliunl was reduced into blue violet Fornmzarl. Control experiments without substrate were simultaneously performed in order to verify the absence of an interfering adenylate kinase reactioil. Assay with fructose-1,Miphosphate

(FDP)

Lens zones were homogenized, then centrifuged and supernatants were incubated at 4°C for 1 hr with various concentrations of PDP (between 1W M and 1O-4 11). The mixtures were then submitted to isoelectric focusing.

Epithelial cell extracts were mixed m-it’11either cortex taxtract or nucleus extract. Jlisilig was perfomled in such a manner that each tissue extract had the smle final PK activity. %G~chrrkture anal each tissue estract were either inlttit~(liatelv submitted to rlectmfwnsin~ or preincuhated for 3 lir ;lt 87’mC!. 3. Results

JGctrofocusing experiments with aqholines of pH range I)etween 3.0 and 9..5 showed several M type PK hands in muscle, brain anti heart, and L type (with a l)Hi lower than 6.0) in liver and erythrocytes. Lens PK resolved into several 11 ty])?

t)iiJldS.

Figure 1 showsthe electrofocusing pattern of PK in ra.hhit tissuesusing ampholines of pH ranges between 6.0 and 84. In lens we found several $1 l)ands, t,he pHi of these tlifkred according to the zones. In epithelial cell extract,, four major IXUA are 11au~ls

risil)k with pHi ljetween 8.6 ant1 6.9. Because t’hcl l)Hi of the most anodic I~ntl ccnwspontls to t,hat of the preponderant t)antl in spleen and in heart? w-eshall call it “MI, ” . ‘l’hr most cathodic hand correq;l,oudsto the Xwcle hand and we shall call it i’N,‘.. We ~11x11 call the two ot’her intermediary bands “1113”and “M4” according to the nonlenclat,nre of Marie et, al. (1976) for human t,issues.The PK pattern of cortex wncl mrcleus fifw cells is different : the two anodic I~andsthat it-e have called “M2’.’ ailcl “M,” are ncjtSvisible ant1 only “54,” ant1 “M,” persist. It must, he noted t,hat the “Nl” I IiLtltl resolred in certain experiments into several hands. The inculcation (3 hr at 37°C:)of adult extracts of ccbrtex or nucleus alone did not changtl t,hc electrofocusing pattern of the PK from t~hese&sues (Fig. 2). By contrast, t,he incuhat’ion of epithelium extract, alone led t,o t#hcJdisappearance of the “JI,” and .‘;113”tj!+peisozymes -and gave the smnepattrrn as that of cortex and nucleus. IZy inculcating a. mixture of cortex or nucleus extract, with epithelium extract? we alscbotwmwl the disappeara~nceof “M,” ant1 “JI,” with the persistence of ‘W1”

(b)

(a)

: II II

(I)

(a)

(b)

i

(2)

(a)

: ,I

I

(b) (3)

: II II I

(a)

(b) (4)

IQo. 2. Modification of elect~roforusing pattern of YK by incubation anti mixture of tiiffem~t~ esbraets (adult rabbit). (a) non-incubated cstract: (h) after incubation for 3 hr at SC’. (1) C’ortcx: (2) IIW~~US: (4) cortex mixed with epithelium. (31 epithelium:

Pk activity

(i.uJ

Pk actiwty

25 -

V,,, = 24 i. u.

l/r--‘-/0’// t/ / 0

S,-,.s=0.9

d

,v 0

(Id

12 I(,,,=9

iu.

-8-

../. f

rn~ ~!p+K,=025m~ 4

,L5mu 4 I

7 J 1 I

I 2

-

Epithelial

I 3 ~MPEP ceils

0

I I _ Nucleus

I 2 fiber

I 3 n-IMP :P cells

t,hat the hiding wit2l FDP could motlif,v the isozymic pattern. We fo7mtl no change in the PK pat,tern of epithelium ur nucleus fiber cells. We have performed similar experiments on lensof 1%clay-old rabbits. The epithelial zone cannot he isolated; Fig. 1 shon-st’he result+ which we have obtained with the imier zone. It is seen that “MvI,” and “X3” I)ands are present, consequently the isozymic pattern of the core of very young rabbit is similar to t’he pattern of the epithelial zone of adult rahhit.

h-irwtk

results

Figure 3 shows the kinetics of the PK activity in of rabllit lens. This kinetics is Michaelian for the inrwr FDP), anal ident,ical with the well-known Michaelian the contrary, t,hc PK of the epithelial cell.3 gives a h!p’erl~c~lic \vhen FDP is added.

Sucltus (Imtes Epithelium

15 35 Sl.Kl

t&e epithelium and the nucleus part of the lens (with or wit’hout kinetics of muscle enzyme. On sigmtlidal curve, which I)ecnlw

o.1a 0~6.5 \Vithout FDI’: 0.90 \\Yth FI)l’: 0.25

TaiJe I show the PK activity per g of protein am1 the apparent & or S,., values (concentration of substrate at half maximum velocity): it is seen that there is a decrease from epithelium to the inner zone. The differences between the h’, values (without FDP) for the epithelium and nucleus enzymes are statistically significant: 0.05 -; I> < 0.02. It is to he noted that protein concentrat8ion is greater in the nucleus and the cortex than in the epithelium. That, is to sap the total PK activit!in the nucleus is relatively high.

4. Discussion First. we show that PK type of ml)l)it leus is 77nicl77elyof “11” I vpe. It is to lw noted that, on the contrary, PK in rrcl cells is of “L” type (with a pHi alwrit 53). Moreoverl me found a loss of PK isozyllles in the inner zones of the lenh: that ‘--\I,” and

“i&"

tp?s

Are

NJt

ViSihk

011

the

e~eCtrOf~xL7Si7lg

gel.

hr

ek!ct7W~~hCtic

I't'sllh

are confirnwd i)y the kinetic findings: the curve of act,ivit,y 14th PEP as s77l)st,ratc* is sig7noidal in e$thelium while it is Mchaelian in tlrc core of the atl77k lms \\-lm~: ttie “111,” and “i&” t’ypes havt: tlisa~~l~raretl. Apparent Ii,,, (or ,S(,.J oonfirt77 il,tSO t,lris (~vohttion: we have f'o~ound a $,,.5 for PEP of 0.9 7m7 iI7 rl)itheli77777 (where the> fo77r types are present). and 0.15 in47 in the 7777clelis(n-it’17 only SI, ant1 Jr, t,ypw).‘h latter K,,, is intermedia.ry between the Ii,,, of the t+theli77777 mtl that, of thcx tt7rlsrtc enzyme (K,,, = 046 niiu). The incubation of nucleos 01’ tortes extract, aIO77C(lid not, change their cbl(~c:t7~ofocusing pattern. whereas we ohservetl the lo 5s rif t,he 777ost,anodic isozytues iti qitl7eliiiiti extract incubated under the same conditions. It seems that iii Atro. tht! Jr, atid M, PK types are more ea.sily inuot~ivnted and iilore sensitive to wrioiis efkct,c~rs. than t,hc ot,her types. This innctivatiw might, occur in vivo tluring t,he long lit’ta of titc wzyt7e in the lens n77cIe77s.Mowover. t,here was a thermolal)ile fraatiot7 (~1’ I’K at, 52”(’ in Dhe epitheli77n7 which tlisal,ppared in thr CiJre of the lens. The following hypotmheses may tw snggestetl. 110 there hypothetical factors ICWI to the transforn7at’ion of 11, iSOZVllle ht,o 11, in the core of the lens. as descri~~et~ t’o7 several ot#her human tiss77es. I& Marie et al. (1976). ()77r experiiurnt~s do not confirm this h,vputliesis. By mixing epitheliun7 eXt,rilCt wit17 nucleus extract: we have found the same elecCrofoc7ising patter77 as with epithelial cells alone. No hand has disappeared. Siniilarlp. our experiments of arltlit~ioi7 of FDP do not confirm t.hc 17,vpothesis 01’ a role of this effector in t#he different isozvttle patt’erns of le77s zones;. All o77r experiments show t,he tlisappkwanee of the anodic types i71the i7mer zot7cs of aclult~ rat)hit iens. This disappearant:t~ seems to l)e due to aging: ttgiilg of t,he Icns (the oldest, components l)eing it7 ehc nticletis) and aging of the organisnl -as confirnietl h\- the l)resence of the four II types in the core of very young ra\)ljit lens. Tl7we rt7odifications are surclv l’ust-translatir,77;tl . since they occ77r in zones without lmkei77 517nt,hesis. The life-span of “i&” and “MS” t)ypes may be shorter than that of “&I,” and *‘RI,“. In fact. “MS” and “MS” tqTpes seen7 t,cj I)e more labile. 8o7ne other examples are known of isozynes with a difference life-span i77 the same tissue (Weber. Hatzfeltl and Schapira, 19’72-73). It seems bhat each isozyue turns partly for its proper co77t7t. partly 77nder environmental influences. Anyway, the rnodificat,ion of pyrurat,e base kinetics in the iiiner ~017~sof ral)l Iit, lens may have some physiological or pathological consequences. We plan to search for such modifications in human and experimktal cataracts.

REFEREP\‘C!ES Ba.nrtxprcs. J., Gregori. C. and S&pira, F. (1976). Post-synt~hetit modifications of aldolase isosymes in rabbit, lens during aging. E’EK3 1,eetters 65, 204 7. Bernstein. L., Kerr&an, 31. and Maisel. H. (19AA). Lactic tlehydrogenase isozynes in lens and cortte~~. hp. K,I/P Res. 5, X19-14. Blncmendxl, H., Rcrns, A. J. M., Van Drr Ouderaa. F. J. and l)e Jong, \V. \V. (ISi?). Evidence for a “non genetic“ origin of the A1 chains of x-crystallin. Ezra. E;y Rrs. 14, SO-~1. Roars, -. R. and ‘l’urrant, (:. M. (I 97”). Altered enzymes in h~~marr fihrol,lasis. SU~UW. ~,ouc/o)~

238, Jti--30. Ibsen.

I<. H. (197;). Interrclationsll~pn and functions of pyruvate kinase isozymes and thrsir vwiant fi,rtns: a review. Crr~acer I?ri;errrch 37, 341-X%. Ibsen, Ii. 11.. Murr:iy, I,. and Marles. S. \\‘. (197(i). Electrotbcusinp and kinetic stutlics of ;ttlult A 116I vrnbryonic, chicken pyruvatc kinases. Hioclcetrc i&y 15, 1 CM%-~79. ln~a~trr~ra. I<. and Tanaka, B. (1972). Multimolecular fornls of PK from rat and other ulamnraliarl tiwlw. 1. Elec,trophoretic studio. .J. Biochem. 71, 104% .?I, fi~11111..\. . Jlarie, .r. and Hoivin, P. (19ili). Pyruratr kinwr isozymes ill man. 1 I. I,-typ(a alld cr.~tIn.oc~tr-t?-f)e isozgmes. Elv~.trofornsinp and i,t~l,rlll~olo~i~~~~l stllclier. Ifrc~. //em+. 33. 3.5 Iii. Lowr:\-. 0. H., R.owbrough, X. ,J.. Yaw. ;1. J,. and Randall. 1:. .J. (19.51). Protein nw~s~ux~tncnt \\ith the folin phenol reagent. J. Kiol. Chew. 193, 265 75. Jlarie, .I.. Kahn. .\. and Roirin. P. (IWO). Pyruvate kinuw isozymrs iu 11~1n. I. -\I-t~ypv isozymcri ill ildlilt and Wal tisslles, elect rofocusinp and in~mr~nolo~iral stud& Ilu~~~~ (:~ilptil, 31, 35 45. 3Jarie. .I.. (Arrreau. H. and Kahn, A. (1977). Eridence for a post-synthetic prot,eolytic transfornrntioll of human crythrocyte pyruvate kinase into r,-type enzpmc. FEB8 Letters 78, 91-1. 3Iuroya. Iv., Xagao. Y., Miyazaki. I<., Sishikawa. K. and Horio. T. (1976). Pyruvatr kinnw isozynws in v;wious Cssues of rat, and increase of spleen-type pyruratc kinasr ill liver b\ injtbc+ing chromatins from spleen and tumor. J. Biochrw. 79, W-1 5. Orgc4. I.. IE. (19K31. The maintenanw of the accurac>of protein synthrsis and its relt~oauc~~ tr, :iying. Proc. .Vfrtl. Acrid. Sri. I’.S.A. 49, 517. Cl. Pagli;L. 1). and Valentine, 11’. (1970). Evidence for molecular alteration of pyvmtr kinasr as ;I cw~seq~~erwe of erythrocyte aging. J. Lrtb. (‘tin. Xed. 76? 205 12. Pa.lnwr. \\‘. C. and Papaconstantinou. J. (1969). Aging of’x-rqx%llins during derrlopment of t IN* 1~~s. J’roc. Srrt. Acml. Sci. li.S.,-i 64, -Qk-410. Papac,otlnt,aIlt.inol~. .J. (1967). Molec~~lar aspects of lens cell differentiation. A’cience 156, 38X-4(i. Poer~itr~~. l,.. Skala. H.. Courtois, T. and Dreyfus, J. C. (1977). Lens glycosidases in human and s in llnrnan and bovine Irns. Exp. F;!yc, bovine species. II. The isozpme:: of P-hexosaminidase I:~.s. 25, S9-45. Schnpira. F. and C:regori, C. (1971). I’yruvate kinase de I’bbpatorne, dn placenta et dllfoie fwtal de 171t. c’. 8. ..ld. ski. ship D. 272, 1169-72. Sk&-l:ubinson. 11,. \‘ibert,, %I. and Dre>Tfus, J. C. (1976). Electrophoretic modificatio:w ot thw: enzyme5 in extracts of tillman and bovine lrnr. Pilst-transl:LtionRl “agving” of Ieus enz~,,,rs. C’liw. Chit,?. detrc 70, 3X5-91 I. Strandllc~lm. J. .J.. Dyson, R. D. and (.‘ardenas. J. 31. (1976). Bovine pywwte kinwse isozymrs atl(l hybrid isozynlw. Electrophorctic~ stIltlies and tissue distribution. .-lwh. Riorhrn~. Hiophys. 173, 1’7.5-:31.