Delta crystallin synthesis during chick lens differentiation

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(1973) 15, 353-360.

Delta Crystallin Synthesis During Chick Lens Differentiation ARTHITR

KATOH

ASD KAZVO

T~SHIDA

Antiswum to delta crystallin was prepared by absorption of total c,hi&en lens antiserum with bovine lens extracts. This yielded an antiserum specific to delta crystallin based on immunoelectrophoretic a.nalysis. The developing lens rudiments of chick embryos betSweet 24 and 96 hr of inrubation were pulse-labeled with W”-reconst,ituted prot,ein hydrolpsatc. and the soluble extract react,ed with delta crystallin antiserum. The radioactivity precipitated by t’he specific antiserum shows delta crystallin synthesis initiated between 54 a,nd 60 hr. A similar series of pulse-labeled lens tissues were examined by cellulose acetate membrane electrophoresis. By this method, the initial synthesis of delta crystallin was shown to occur in 54 hr samples. Thus, these two methods corroborate each other with respect to the initial synthesis of delta crystallin. and also provide data on the rates of delta

c.rystallin synthesis during the first seven days of embryonic developmmt.

1. Introduction Wilt (1962) and Schreiber and Feigelson (1967) have described a general method for precipit,ating radioactive proteins with specific antisera. thus endowing highly specific immunologic assayswith quantitative pot.ential. Wilt (1962) incubated chick blastodermsin radioactive precursorsof the material he wished to assay and then preripated an extract, of the blastodcrms with a specific ant,iserum.washedthe precipit,at,e t.horoughly by centrifugation, and assayed the radioactivity in the precipitate in a. scintillation counter. Schreiber and Feigelson (1967) described a technique for collecting the precipitate on filter paper. washing it thoroughly. and counting the radioact#ivity in the precipitate in scintillation fluid. The general method of precipitating specific radioactive prot,ein promises to Irw H powerful tool for investigating the ontogeny of tissue+pecific proteins, especially in tissueswch as lens. which contain unique and characteristic prot,ein species.We have applied this technique to determine the rates of delta crystallin synthesis during lens differentiation in the early chick rmhryo. Expanding upon the method of assaying selective radioactivity incorporated during a short pulse. we have previously demon.ctrat,eclthat chick lens proteins can be separa,tedand ident,ified t)y cellulose acetat,+, membrane electrophoresis (Yoshitla and Katoh. 1971a). ‘Il’e have ubilized these two techniques. augmenting and corroborating each other. t,o track the appearance anal rates of’ s,vntheRisof delta crpstallin during the first oven days of chick development. 2. Materials and Methods Total adult lens antiserum was prepared from adult’ chicken lenses, lvhich had heel1 cleanly dissected from the surrounding tissues. A 15 9 a0 (w/v) extract prepared in Howard’s saline containing approximately 1 mg protein, was mixed in a ratio of 2:3 with Freund’s complete adjuvant (Difco. Detroit, Michigan, V.S.A.). Subcutaneous injections of such a prepar&on were made at weekly intervals into t,hrer adult female albino rabbits for x

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period of :i-8 weeks. At the ettd of this time, serw were poo1etl ilIlt tested itt Ouc*hterlott! plates for poteucy. Three distinct bands were observed against a ntittintuttt of 2.7 pg of thea immunizing antigen. Sera from t#hese rabbits were heated to 5Ci’Cfor I 11r:ml thett pcmlc~l. Delta crystallitt antiserum was prepa,red by absorbittg the total atlult. 1e11santiseruttl clescribed above with lyophilized, decapsulated adult bovine lens. The Iyophilizetl powtlet was added to the atttiseruttt until inttttuttoelectrt,phoresis tletttottst,ratetl the disappear:~ttc~r of the c.haract,eristic alpha and beta crvstallin arcs. Itttntunoelectrophoresis of adult chicken lens atrt~igetts was conducted on 1 ,.\ $in ttticroscope slides coated wit,21 2.1 Jtnl of 0%5”,, Iotiagar tto. 2 (&lab, Chicago Heights, Illinois. I.3.A.) in barbital buffer, pH 8%. The clectrophoreais was run at 150 V for 90 mitt. >tttd the pattern developed overnight wit’h antisera in it tttoist. chatnber at 4 C. The slides were rinsed in 0.15 Ii NaCl to retttove utt boutitl nonspecific proteins. iltld then were stained with Ponceau S. Heginning at 24 hr of ittcubatiott attd at every 6 hr t,hereaRer until 96 hr, the presumptive lens areas and lenses were harvested front chick embryos, and pulsed for 6 hr in b c h warz-Mann, Orangeburg. l() &i of 14C-reconstituted protein hydrolysate ( 14C-RPH) (‘3 K.Y., IY.S.$.) in Eagle’s tttinitttuttt essential rttediuttt (MENI) supplemented lvith lO(l,, 100 units/ml each (Difco, Detroit. chicken serum and penicillin ant1 streptomycin. Michigan, U.S.A.). After the pulse, the tissues were thoroughly rinsed in three separate dishes of Hanks balanced salt solution (FM), and hotnogetiized in 0% ml of TBS. The homogenized sample was then transferred by Pasteur pipet to a l-1111 conical, Pyres centrifuge tube. The homogenizer was rinsed twice with HSS, and the rinses Were pooled with the original homogenate itt the cetttrifuge tube. Thr samples were then stored in t,ltcB freezer at, -WC until all were collected. The samples were thawed, and centrifuged at lO.O(JO g for 20 mitt. The superttstartt was withdrawn and protein concentrations of each sample was detertttined by the method described by Lowry, Rosebrouyh, Farr and Randall (1951). Labeled atttigen and delta crystallin antiseruttt were cornbitted itt the experiments in proportiotts such that antihod! was in slight excess, as determined b; t,he equivalence titration. Such antigen-antibod! complexes were then handled as tlescrtbetl for the antiserum t.itrution. Titration of delta crpstallitt atrtiseruttt was carried out with extracts of 12-day-old itt chick embryo letts fiber cell s, which hatI been pulsed for 5 hr itt 14C-RPH (25 &i/tttl Titration of antiserutn .5 1111of Eagle’s MEM plus 10”,, c~hickett serunt ittld antibiotics). apitittst a constant quatttitv of labele(l antigen, to determine atitigen-:l,ntihodv equivalence \vits carried out, as follows: a series of t,uhes were set up with increasing amounts of atttiseruttt, rangittg frottt 10 to .%I() ~1. To rach t,ube. I5 p g of radioactive lens protein was added. The tubes were thett sealed with p:Lrafilttt. itlc&at~etl at 37°C for 30 mitt, anti then stored at) 4°C overnight. The following ttrortting all tubes mere centrifuged at Io,ocJO g for 20 tttin. The superttatattts were carefully witltdrawtt witlt Pasteur pipet,s, and saved for t,he superttatant t)ests in Ouchterlotty plates. Tlte pellet was washed with BSS, usittg a fine capillary t)ubittg sealed at one etttl as :I st,irrittg rod. The rrsuspetrtled pellet was c*etttrifuge(l at lO.OlNl g for 20 min. This washing procedure was repeated three titttes. After the final wash and cetttrifugatiott. the superttatattt was discartled, and the tube drained. At this point, O.:i tttl of srintillatiott fluid (Brutto and Christian, 1961) was added, and the pellet solubilized with t,hr aid of t.he capillarv st’irrittg rod. The contents were then yuantitativelv transferred to scintillat,iott vials, &h the c,etttrifuge tube being rinsed ;i tot,al of five t&es. Radioactivitv was couttted in a Packard Tri-Garb sc.itttillatiott counter, after l(J ml of scitttillatiott fluid’ were added to t,he vials. Supernatattt tests for the detertttination of regions of antigen and antibody excess were conducted on intrttuttodiffusiott plates (Hvlattd Laboratories, Los Attgeles, California. 1r.S.A.). The peripheral wells in both series ivere loaded with 10 ~1 of superttatant samples. For antigen excess, the central well was loaded with 10 yl of delta crystallin antiserum; for antibody excess, the central well bvaa loaded with 1( I ~1 of t4C-labeled lens antigen which

SYNTHESIS

OF

DELTA

(!RYSTALLIS

355

was shown by cellulose acetat’e electrophoresis (Fig. 3) to he composed only of delt;i crystallin. The specificity of the delta crystalliu antibody in precipitating radioactivity from ot,her non-lens Gssues was monitored in the following manner. Equal amounts, as judged b! visual inspection, of neural retina. heart, brain, limb bud, and lenses were taken from ‘i-day-oh1 chick embryos, and pulsed for 5 hr in 14C-RPH. The tissues were homogenized ;htid their protein concentrations determined. The labeler1 soluble protein from these estracts was incubated with delta crystallin antiserum : equivalent amounts of each component~, ~4swas used in the experimental series, were used. The reaction t,ubes were then inruhate(1 and processed for scintillation counting as described above. C’ellul~~r acetate elect’rophoresis was performed as previously tlevril)ed (Yoshida atid <otl. 1971a1. 3. Results

The three arcs characteristic of chick lens crystallins are shown in a typical innnunoelect’rophoretic analysis of our total lens antiserum in Fig. l(a). Absorbing this antiserum w&h bovine lens extract produces a serum demonstrating but one arc hv imnmnoelectrophoresis [Fig. l(b)]. Th is arc is produced by delta crystallin, which is unique t,o birds and reptiles. and sometimescalled F.I.S.C. (First Important Soluble Cryst~allin; Rabaey, 1962).

Titration of a constant quantity of radioactive lens antigen from l%clay-old chick embryo lens fiber cells. with increasing quantities of delta crystallin antiserum, demonstrates a major broad peak and equivalence zone in the 100-200~1 region. where neither ant,igen nor antibody was demonstrable in the supernatants. This description is based upon the results of three experiments with virtually identical result*s.Figure 2 showsthe results of one of these.

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Figure 3 shows the results of electrophoresis on cellulose acetate of t,he lahelecl ant,igen used in the antiserum t,itration. We have shown previously (Yoshida and Katoh. 1971a) that the three crystallins of the chick lens can he separated and identitied hy this method. The pattern shown in Fig. 2 demonstrates a single dominant’ peak of radioactivity which is identified as delta crystallin (by its mobility aml comparison with other samples showing all three crystallin species: see Yoshida amI Katotl. 197la).

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FIG. 3. cellulose acetate elect~rophoretic profilr of 1%day-old embryo lens fiber cells which had been incubated for 5 hr in I’(‘-RPH. Major peak at fract,ions 22-36 is that, of delta rryst,allin. Anode is to the right.

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FIG. 1. Radioactivity precipitated from labeled chick embryo preuumptivr lens areas and lenses I)> delta crystallin antiserum. Lens rudiments were excised at 34 hr. and at every 6 hr t,hereafter uutil96 hl,. and incubat,ed for 6 hr in W-RPH. Each sample was adjusted to 30 119 protein. and incubated with IW ml of delta rryst.allin antiserum. Radioactivity precipitatc4 by p”-irnrnlcnization se1’um has l,pcw naht.raci.ecl.

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The rate of synthesis of delta crystallin. as measured by the precipitation of raclioactivity by delta crystallin antiseruttt. is shown in Fig. 4. Lens anlage and lens tisstw were excised frotn ct~l~r~os beginning at 24 hr of incttbat~ion. and every 6 hr t herrafter until 96 hr, t,hen incubated for 6 hr in lY’-RPH. The t,issues were lwocessecl t,o assa) the amount of radioactivit,v incorporated into the attt,ibotiy-l-‘rrcipitable t~l~t~(kl. C!otttrol values, i.e. radioactivky precipitated bp pre-inttnune seruttt (averaging at, each point c. ~$30 counts/tttin) have been subtracted frottt each poitk Three exporittlents dentonstrated the same pattern and relative quantities of precipita,tiott by t,ltc delta crystallin antiserum. d tlist,ittct increase in delta crystallin synthesis begirts frottt 54 hr in ova, and continues t,o rise sharply for t’he duration of the period exatninctl, to 96 hr.

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FIG. 5. (a-f) Electrophoretic profiles of labeled chick embryo presumptive lens areas and lenses. 3 /% protein of labeled extracts were applied onto cellulose acetate membrane electrophoresis. Anode is to the right. Delta crystallin is located in fractions 22-26. (a) 48 hc; (b) 54 hr; (c) 60 hr: (d) 4 days; (P) 6 days; (f) 7 days.

SYNTHESIS

OF

DELTA

CRYSTALLIN

339

In order to check the specificity of the delta crystal&n antibody, tissue extracts from ‘i-day-old chick embryos which had been pulsed in 14C-RPH, were incubated with antiserum and the extent of radioimmunoprecipitation determined. Table I gives the data of such a control experiment, and indicates that there is an ubiquitous non-lens component which complexes with the antiserum, and is responsible for precipit)ating an average of 140 counts. Thus. by this crit’eria. the low level of activity shown in the experimental samples (Fig. 4) taken between 36 and 54 hr appears t.o be of a non-lens crystallin nature. The synthesis of delta crystallin in the presumptive lens and lens areas of early embryos was also followed by cellulose acetate membrane electrophoresis. Figure 5 shows electrophoretic profiles of lens areas taken from 48, 54, and 60 hr, and from lenses of 4. 5: 6? and 7 days of development. The tissues were pulsed in 14C-RPH for 6 hr. and t,he specific act,ivities of the extracted proteins were determined before application onto cellulose acetate memhranes for electrophoresis. The results b> elect#rophoresis corroborate that, ohtained by radioimmunoprecipitation, in that the synt’hesis of delta crystallin is detected from 54 hr samples, and followed by sharply increasing rates of synthesis thereafter. Delta crystallin was not’ detected from samples taken earlier t.han 54 hr and examined by electrophoresis. 4. Discussion Numerous procedures have utilized the specificity of the immune reaction and the sensitivity of radioassays for measuring specific prot’eins. A number of these depend upon conjugating the antiserum with radioactive material, and thus, they measure the total amount of antigen present at any given time (Berson. Yalow, Glick and Roth, 1964; Wide, Axen and Porath. 1967; Catt, Niall, Tregear and Burger, 1968; Rodbard, Rayford, Cooper and Ross, 1968; Wust, 1968; Miles and Hales. 1968). Other methods introduce radioactivity as precursors of the antigen, as in this study, so that precipitating activity measures incorporation during the pulse period (Campbell and Stone. 1957 ; Wilt, 1962). It. is now feasible to prepare antisera to lens crystallins purified t,o desired electrophoretic or chrornatographic criteria. We have explored in this investigation the use of ant,iserum to delta crystallin and its ability to precipitate radioactivity in its corresponding homologous antigen. The data shown in Fig. 4 shows a distinct spurt in synt,hesis of delta crystallin heginning at 54 hr of incubat’ion. Experiments in which lens &sues. beginning from 48 hr and extending up to 7 days of development. were pulsed in 14(J-RPH and examined by cellulose acet,ate membrane electrophoresis. are presented in Fig. 5. The results indicate that t’he earliest, detection of delta crystallin synthesis is at 54 hr. Thus. t’he two techniques of radioimmunoprecipitation and cellulose acetate membrane electrophoresis corroborate each other. and are in agreement. wit.h the irnmunofluorescent studies of Zwaan and Ikeda (1968). in which the earliest. detection of delta cryst,allin by that method was at, 51! hr. Various investigations in the past, have failed to agree regarding the onset of Ienx crystallin synthesis. Burke. Sulli\-an. Petersen and Weed (1944) did not demonstrate lens antigens until after 96 hr of incubation. In contrast: Langman (1959), Clarke an0 Fowlcr (1960), and Perlmann and De Vincentiis (1961) found positive indications as early as 40-44 hr. This report’ appears to be the first to corroborate the immunofluorescence studies of Zwaan and Ikeda (1968), and is in agreement with the nlore rcscent work of Brahma and van Doorenmaalen (197 1). using the imtnurlofluorescenc~

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technique. which clet,ect)e(l tttr rarlirst clentotlst,ratiort of’cfelta cryst,allirt itrotttttl 60 111’. Intnlunofluorescrncr stutlies itr(* ttowvttr. accuutulation products king visualizrvl ilt tttcb tinte of santpliny. ant1 gi\xs no tlrfinitivtb inforutitt,ion ilhOUt ratez ol’ svntttwi.\ uor when that synthesis was initiutwl. It is t,o thcsr two crucial poiuts wit It rywct to clvlta crvstjallin. to which WC ttaw ~~~l~lrc~sse~lourwlvt~~ in this stttclv.

This investigation was supported in pxrt by ;t coutr>rct AT (31 I) ~65 front the I’.S. Atomic Energy Commission. We gratefully ;tclirtcwledge the tectittic~al assistatiw of Linda Engelbrecht and Surin Charoerlsiri. K l3F’ERES(‘ES Berson.

S. A.. Yalow,

R. S., Glick.

S. RI. and Roth, J. (1964). JIetnbolisnl 13, 11%. \V. J. (1971). Ophthaln~ic. fles. 2, M4. Bruno. C. and Christian, J. (1961). Annlyt. <‘he//r. 33, 1212. Burke, V.. Sullivan, N. P.. Petersen, H. and \Veed. R. (1944). .1. Infect. Ui.r. 74. L’2.5. ~‘a~mpbell. P. N. and Stone. S. E. (1957). Riochern. J. 66, 19. (‘a,tt, K. J.. Kiall. H. I).. Tregear. C:. \Y. and Burger. H. (I. (1968). .I. (‘litl. h’nrlocriuol. Metub. 28, 121. C’larke. \\T. $1. and Fowler. 1. (1960). f)ec.. Z:iol. 2, 155. Langman, J. (1959). J. h’v~bryol. Exp. Morphol. 7, 19::. Lowry. 0. H.. Rosebrough, N. J., Parr, d. L. and Randall, A. .1. (19.51). J. Biol. Chew. 193, 36.5. Miles, L. E. &I. and Hales. C. N. (1968). NU~UX (London) 219, 186. Perlmann, P. and De Vincentiis, M. (1961). E.rp. Call. Kpb. 23, 612. Rabaey, M. (1962). Exp. Eye fles. 1, 310. J. C’lin. &uiocrinol. Metab. Rodbard, D., Rayford, P. L.. Cooper. ,I. .I. and ROW, (G. T. (1968). 28, 1412. Srhreiber. G. and Feigelson, P. (1967j. Annlyt. f3ioch.m. 21, Sk. W’ide, L., Axen, R. and Porath, J. (1967). I,///r,runoc.he)/ti.stry4, Ml. \\‘ilt, F. (1962). Proc. Natl. dcad. Rri. 48, 1582. \Vust, C. S. (1968). Arch. Biochem. Biophys. 126, 524. Toshida, K. and Katoh. A. (1971a). Exp. fi;yp Z?es. 11, ICI. Yoshida, K. and Katoh, A. (197lb). Exp. Eye Res. 11, IsA. %waan, J. and Ikeda,, A. (196X). F:.c/l. Evp Kr.9. 7, SOI

Brahma, S. K. and van Doorenmaalen,