Apparent Coordination of Plasma Membrane Component Synthesis in the Lens

Transcription

1 Apparent Coordnaton of Plasma Membrane Component Synthess n the Lens Rchard J. Cenedella Purpose. Ths study explores the order of assembly of the lens fber cell plasma membrane. Because the lens must synthesze most of ts membrane components, our approach was to map drectly the spatal dstrbuton of cholesterol, fatty acd (reflectng phospholpd), and the man ntrnsc proten, MP26, synthess n the lens and, thereby, determne the extent to whch membrane component synthess was coordnated durng fber cell elongaton. Methods. Young rat lenses were ncubated wth ether trtated water as the substrate for cholesterol and fatty acd synthess or trtated leucne as the substrate for MP26 synthess. We developed a smple technque for unformly dssolvng the decapsulated lens nto small fractons by ncubatng the lens wth gentle strrng n sodum dodecyl sulfatecontanng buffer. Based on the proten content of each fracton and avalable nformaton on the radal dstrbuton of proten n the young rat lens, each fracton was equated to a specfc percentage of the lens radus. Cholesterol was precptated from each fracton by dgtonn; fatty acds were extracted and solated by thnlayer chromatography. The MP26 was recovered both by mmunoprecptaton from each fracton wth antmp26 polyclonal antbody and from sodum dodecyl sulfate polyacrylamde gel electrophoress gels of ntact crude membrane, whch was solated from lens fractons by dssolvng the lens n a urea contanngbuffer. Results. The spatal dstrbuton of ncorporaton of cholesterol, fatty acd, and MP26 was vrtually supermposable, wth essentally all (he ncorporaton occurrng n the outer 0% of the lens radus and peak ncorporaton occurrng n approxmately the outer 36% of the radus. Conclusons. These results ndcate that the synthess of lens membrane components s hghly coordnated and mply that the plasma membrane accumulates constant proportons of cholesterol, phospholpd, and MP26 throughout the course of fber cell elongaton. Invest OphthalmolVsSc. 993;34: What s the order of assembly of plasma membranes? As membranes are assembled, are the ndvdual components ncorporated n proporton to ther ultmate relatve composton or could, for example, protens be nserted nto a preexstng lpdrch blayer? The From the Department of Bochemstry, Krkwlle College of Osleopathc Medcne, Krksvlle, Mssour. Supported by Natonal Insttutes of Health (Ilethesda, MD) grant EY Submtted for publcaton: Aprl 6, 992; accepted August 20, 992. Propretary nterest category: N. Reprnt requests: Rchard /. Cenedella, PhD, Department of Bochemstry, Krksvlle College of Osleopathc Medcne, Krksvlle, MO ocular lens can provde a useful model to address these nterestng questons. The dfferentaton of lens epthelal cells nto fber cells nvolves great cellular elongaton and the formaton of large amounts of plasma membrane. Fber cells elongate to reach both the anteror and posteror sutures of the lens and then are dsplaced nward toward the center by everyounger cells. 2 Thus, at any age, the entre hstory of the lens s dsplayed. The composton of the lens fber cell plasma membrane (essentally the only subcellular organelle of the lens ) s comparatvely smple, beng largely com 286 stgutve Ophthalmology & Vsual Sc Copyrght Assocaton for Reseate.Jnne 993, Vol. 34, No. 7 u Vson and Ophthalmology

2 Coordnaton of Lens Membrane Synthess 287 posed of cholesterol, phospholpd, and a prncpal 26klodalton proten called the man ntrnsc proten (MP26). 3 " 6 We beleved that determnng the spatal dstrbuton n the lens of the synthess of these membrane components could provde nsght nto the order of membrane assembly because the lens lkely syntheszes most, f not all, of ts membrane components (e, cholesterol, 7 fatty acds for phospholpds, 8 and obvously, ts protens) and because t appeared reasonable to assume that, once syntheszed, these components would be rapdly ncorporated nto the membrane rather than enterng a statc, nonmembrane pool. Trtated water was used to measure the synthess by ntact rat lenses of cholesterol and fatty acd (taken to reflect phospholpd synthess), and trtated leucne was used as the substrate for the membrane proten synthess. A smple technque based on the gradual dssoluton of the lens n sodum dodecyl sulfate (SDS)contanng buffer provded the opportunty to "dssolve" the lens unformly nto small fractons. Each fracton was then equated to a precse segment of the lens radus, based on the percent of total lens proten n each fracton and the avalable knowledge about the spatal dstrbuton of proten n the young rat lens. 9 The results of ths study ndcate that synthess of all major components of the lens fber cell plasma membrane s hghly coordnated. A precedent for such coordnated synthess of membrane components was provded prevously. 0 The earler report demonstrated that cholesterol and phospholpd synthess was coordnated n prolferatng L 6 myoblasts. MATERIALS AND METHODS Radal Fractonaton of Lenses Lenses (n = 06) from 2025dayold Sprague Dawley rats (male and female), whch had been ncubated as descrbed subsequently, were carefully decapsulated and placed nto a 5cm plastc culture dsh, whch was parttoned nto a large (3.5 cm) and small (.5 cm) compartment by a nylon mesh screen ( mm 2 ) glued across the dsh. The lenses were placed nto the larger compartment and suspended n 3 ml of ether 5 mmol/ Trs buffer (ph 8) contanng mmol/ ethylenedamnetetracetc acd, 5 mmol/ 2mercaptoethanol (Buffer A), and 0.2% SDS (wt/vol) or n the 0 mmol/ phosphate cell lyss buffer (ph 7.5) descrbed earler, whch contaned % Trton X00 and 0.% SDS. The lenses were mmedately agtated at room temperature on a gyrorotatory shaker (Model G76, New Brunswck Scentfc, Edson, NJ) at approxmately 80 oscllatons/mn. The buffer was quckly asprated from the small compartment at ntervals of mn or more and mmedately replaced wth 3 ml of fresh buffer, whch was added to the small compartment. The buffer was typcally replaced at, 2, 4, 6, 8, 0, 2, 5, 20, and mn. The lens regon remanng after mn (the nucleus) was homogenzed n 3 ml of the buffer. The volume of each fracton was estmated by weght, and each was assayed for proten content by a modfed Lowry assay. 2 Ths procedure provded a smple method for the unform and rapd dssoluton of the lens (Fg. A) at a rate whch appeared to follow frstorder knetcs (Fg. IB). Because no partculate matter was seen by lowpower mcroscopy n the buffer or at the lens perphery durng dssoluton, we assume that the plasma membrane was beng "solublzed" along wth the other components of the fber cell. Furthermore, ultracentrfugaton of the solublzed lens fractons yelded no pellet. Each fracton obtaned (0 mn, 2 mn, and so forth) was eventually equated to a specfc percentage of the lens radus, based on the percent of the lens total proten removed wth each fracton. Usng publshed data for the proten dstrbuton versus lens radus n the dayold rat, 9 we constructed a plot of the cumulatve percent of the lens proten removed, "solublzed" versus the percent of lens radus remanng (Fg. C). The calculated relatonshp was well approxmated by drect expermental observaton (Fg. C). Although ths method can fractonate the lens nto small arcs of radus, t cannot dstngush between equatoral, anteror, or posteror regons of the lens. The anmals were klled by carbon doxde nhalaton, and all procedures adhered to the ARVO Statement for the Use of Anmals n Ophthalmc and Vson Research. The lenses were collected and used mmedately after death. Measurement of Lens Sterol and Fatty Acd Synthess Intact lenses (n = 04) from 2425dayold rats were ncubated for 3 hr n Dulbecco's modfed Eagle's medum (DMEM, mnus calf serum) contanng ether 8 or 6 mc/ml of trtated water (25 mc/g, New England Nuclear, Boston, MA) at ether 0 C or 37 C. The lenses were ncubated n 5cm culture dshes, whch were sealed wth Paraflm (Amercan Can Co., Greenwch, CT) to prevent contamnaton of the ncubator. The lenses were then rnsed several tmes wth phosphatebuffered salne (ph 7.4), carefully decapsulated, and dssocated n 3ml alquots of Buffer A plus 0.2% SDS as descrbed. Each 3ml fracton was lyophlzed and saponfed n 2 ml of 0.67 N KOH n 67% ethanol (00 C for 24 hr). Carrer cholesterol ( mg) and trolen (0.2 mg) were added before saponfcaton. The carrers were also added to the blank samples, whch were dentcally treated. After dluton wth 2 ml of water, the cholesterol was ex

3 Investgatve Ophthalmology & Vsual Scence, June 993, Vol. 34, No A. o oooo I oo Tme (mn.) 2.0 r B..8.6 E.4.2 fcaton mxture. The hexane extracts were washed wth water and evaporated; the fatty acds were separated by thnlayer chromatography on slca gel G usng a solvent of hexanedethyletherglacal acetc acd (73:25:2, vol/vol/vol). The fatty acd bands were scraped from the plates after locaton by bref exposure to odne vapor and suspended n ml of methanol; the trtum content measured by scntllaton countng. The samples, ncludng the backgrounds, were counted for 50 mn for a maxmum countng error (<r) of 3%. The ncorporaton of trtated water nto the lens sterols and fatty acds s a measure of the absolute rates of sterol and fatty acd synthess.7 Rad 3 Measurement of the Lens Regonal Dstrbuton of Cholesterol and Phospholpd Mass.9.7 I I III Dssoluton Tme (mn.) _c / / / / / / / / %*> V/? / // // / ' // //o V I % Lens Radus Remonng FIGURE. Dssoluton of the rat lens n SDScontanng buffer. (A) Decapsulated lens (2dayold rat) was ncubated wth strrng for varyng tmes (mnutes) n 3.0 ml of Buffer A contanng 0.2% SDS. The lens was photographed at the ndcated tmes, and alquots of the suspended soluton were removed for proten assay (approxmately 0 sec requred per tme). Bar = ] mm. (B) Frstorder plot of tmedependent changes n lens radus. (C) Relatonshp between lens proten dstrbuton and radus (calculated from publshed data, sold lne, and expermentally determned from the lens n A and B). tracted by three 4ml alquots of hexane, and precptated by dgtonn as dglondeprecptable sterols. The radoactvty was measured by scntllaton countng as descrbed prevously.7 The fatty acds were then extracted nto hexane after acdfcaton of the sapon Decapsulated lenses (n = 6) from 24dayold rats were fractonated by dssocaton n Buffer A wth 0.2% SDS as descrbed. After removng small alquots for proten determnaton, the fractons were lyophlzed, and the total lpds were extracted nto two 3ml alquots of chloroformmethanol (2:, vol/vol). The lpd resdue remanng after solvent evaporaton was redssolved n chloroformmethanol (2:, vol/vol) and dvded nto two portons. One was used for measurement of total phospholpd phosphorus by colormetrc assay after ashng the samples at 500 C,3 and the other was used for total cholesterol determnaton by gaslqud chromatography (after saponfcaton) as descrbed prevously.6 The molar cholesterolphospholpd ratos for the ndvdual lens fractons were calculated and related to specfc percentages of the lens radus based on the proten dstrbuton. Measurement of the Dstrbuton of Lens Membrane Proten Synthess Immunoprecptaton of MP26 Sxteen rat lenses (from 20dayold rats) were ncubated at 37 C n 3 ml of leucnedefcent DMEM contanng 50 /C/ml of added trtated 3,4,5leucne (58 C/mmol, New England Nuclear). The lenses were decapsulated and dssolved n 3 ml of the SDS cell lyss buffer descrbed earler," whch addtonally contaned 0. mol/l of unlabeled Lleucne. Dthothretol was omtted. The 26klodalton man ntrnsc proten (MP26) of the lens membrane was mmunoprecptated usng rabbt antbovne lens MP26 antserum (furnshed by Dr. Isaac Bekhor, Unversty of Southern Calforna, Los Angeles, CA) by a procedure smlar to one recently descrbed for the mmunoprecptaton of 3hydroxy3methylglutaryl coenzyme A reductase, an ntrnsc proten of the endoplasmc retculum membrane. The antbovne lens MP26 antbody had a hgh reactvty toward rat lens MP26,

4 Coordnaton of Lens Membrane Synthess 289 even when tested at a dluton of :50,000 by western blot (data not shown). Others also measured the synthess of lens MP26 by quanttatve mmunoprecptaton of sulfur35labeled MP26 usng a polyclonal antbody to ths proten. 4 The dssolved lens fractons (each equated to a specfc percent of the lens radus, based on ts proten content) were ncubated for mn at 37 C and then soncated for 0 sec on ce (Probe SonferCell Dsruptor 200, Branson Sonc Power, Danbury, CT). Both steps were done to ensure solublzaton of MP26. We added ml of each 3ml fracton to a.5ml Mcrofuge tube (Beckman Mcrofuge B, Fullerton, CA)) and precleared t by addng 40 /ul of a 25% suspenson of agarosebound Proten A (Sgma, St. Lous, MO). The samples were spun for 2 mn, and 40 /A of antmp26 antserum was added to the recovered supernatant and ncubated wth rotatory mxng for 90 mn at room temperature. We added 80 jl of the Proten A suspenson, and the ncubaton contnued for an addtonal 90 mn at room temperature wth mxng. The antgenantbodyproten A complex was sedmented by centrfugaton, and the resultng pellet was washed fve tmes wth ml alquots of the SDScontanng lyss buffer wth 0. mol/ unlabeled Lleucne. The antgen was released from Proten A by addng 45 jul of Laemml 5 sample buffer contanng 8 mol/ urea. The protens were separated by SDS polyacrylamde gel electrophoress (SDSPAGE) usng 2% gels by the Laemml 5 method and subjected to fluorography usng EN 3 HANCE (New England Nuclear), accordng to the manufacturer's nstructons. An estmate of the relatve contrbuton of each lens fracton to total MP26 synthess was obtaned by denstometrc scannng of the gel lanes n the exposed flm (Hoefer GS 0 scannng denstometer, San Francsco, CA) and comparng the area under the absorbance curves for MP26 n each fracton to the total absorbance of MP26. We attempted to test the completeness of the mmunoprecptaton of MP26 by western blottng alquots of the supernatant recovered from centrfugalon of the antgenantbodyproten A complex usng the anlmp26 antserum. However, the presence of trace amounts of unprecptated mmunoglobuln G n ths supernatant resulted n mmunostan appearng at molecular weghts correspondng both to mmunoglobuln G heavy and lght chans (approxmately 25 klodaltons). Although the concentratons of MP26 n the dssolved lens fractons were smlarly low (estmated at 24 ng for each of the frst ten fractons) and the concentraton of antserum was hgh, we could not ensure that the newly syntheszed MP26 was quanttatvely precptated from each fracton. Thus, we sought to confrm the dstrbuton of MP26 synthess n the lens usng an addtonal approach. Drect Estmaton of MP26 Synthess Because the lens membrane s nsoluble n 8 mol/ urea, 3 ' 6 we recovered MP26 from the lens fractons by dssolvng the lenses n Buffer A contanng 8 mol/ urea. Incluson of SDS n the dssoluton buffer would solublze the plasma membrane and, thereby, prevent the separaton of ntrnsc membrane proten from the great concentraton of the lens's soluble proten. Although the ureacontanng buffer s useful n separatng the decapsulated lens nto numerous fractons, dssoluton by strrng n such a buffer was a less precse method for fractonatng the lens than dssoluton n SDScontanng buffers. The lens fber cells unraveled n bundles and clumps n the ureacontanng buffer. Also, the rate of dssoluton was very rapd, wth 60% or more of the total lens proten "solublzed" n 20 mn. Between 46 lenses from 2425dayold rats were ncubated for 3 hr at ether 0 C or 37 C n 5cm culture dshes contanng 3 ml of leucnedefcent DMEM wth 50 /C/ml of trtated leucne. The lenses were then washed wth phosphatebuffered salne, decapsulated, and dssocated n 3ml alquots of Buffer A contanng 8 mol/ urea. After removng small alquots for proten determnaton, each fracton was centrfuged (00,000 X g X 60 mn, SW 60 Beckman rotor), and the pellet was washed once wth 2.5 ml of the ureacontanng buffer. The ureansoluble fracton (crude lens membrane) was washed once wth Buffer A (wthout urea), and the pellet was dssolved n Laemml sample buffer. The protens were separated by SDSPAGE and staned; the bands correspondng to MP26 were excsed. The proten n the excsed bands was solublzed accordng to a publshed method, 6 and the radoactvty was measured. Specfcally, the bands were homogenzed n.0 ml of 0.% SDS, 50 mmol/ NH 4 HCO 3, and 5% 2mercaptoethanol (vol/vol); heated for 5 mn at 00 C; and ncubated for 8 hr at 37 C wth trypsn (250 fg/m\, TLCK free, Sgma). We added 5 ml of aqueous countng soluton (Ready Solv HP, Beckman), and the trtum content was measured by scntllaton countng. Lens Dstrbuton of Trtated Leucne The dstrbuton of substrate for MP26 synthess was examned n lenses from 9dayold rats, whch were ncubated at 37 C for up to 8 hr n leucnedefcent DMEM contanng 50 jtc/ml of trtated 3,4,5leucne. Lenses, n groups of two or four, were removed at 3, 8, and 8 hr; quckly washed twce wth Buffer A contanng 0 mmol/ unlabeled Lleucne; blotted dry; decapsulated; and physcally separated by teasng nto the cortex and nucleus. Here the cortex ac

5 290 Investgatve Ophthalmology & Vsual Scence, June 993, Vol. 34, No. 7 counted for approxmately 38% of the total lens proten. The samples were homogenzed n 3 nl of Buffer A, and alquots were removed for proten assay. The total proten was precptated wth trchloroacetc acd (brought to 0%). The proten was separated by ultracent rfugaton (00,000 X g X mn), and alquots of the supernatant were assayed for trtum content, both before and after lyophlzaton. Approxmately 7% of the radolabel was lost durng lyophlzaton, presumably as trtated water. When checked by twodmensonal paper chromatography, 7 99% of the trchloroacetc acdsoluble and nonvolatle radolabel recovered from the chromatograph comgrated wth Lleucne, and 88% of the theoretc amount of trtum label appled to the paper was recovered (data not shown). The concentraton of trtated leucne n the cortex and nucleus after varyng perods of ncubaton was expressed as dsntegratons per mnute of trchloroacetc acdsoluble and nonvolatle trtum label per mcroller of lens water. The concentraton of trtated leucne n the lens nuclear water reached approxmately 55% of that n the cortex (Table ). RESULTS The spatal dstrbuton of de novo cholesterol and fatty acd synthess n the lens appeared dentcal, wth 90% of the total of each occurrng n the outer 0% of the lens radus (00% to 90% of the radus) and peak synthess occurrng n approxmately the outer 36% of the radus (97% to 94% of the radus, Fg. 2). Fatty acd synthess was equated wth phospholpd synthess because essentally all lentcular fatty acd s present n the fber cell plasma membrane 8 " 20 and we assume that newly syntheszed fatty acds are rapdly esterfed nto phospholpds. Between 9095% of the carbon 4labeled acetate ncorporated nto fatty acds by the lens was recovered n phospholpds. 82 The molar rato of cholesterol to phospholpd throughout the outer 25% of the young rat lens radus tended to be unform (Fg. 2). The rato was approxmately 0.36 from 97% to 64% of the radus and slghtly less, approxmately from 00% to 97% of the radus. The nner 64% of the lens radus corresponds to the nucleus and possessed a cholesteroltophospholpd molar rato of approxmately 0.83, a value very smlar to that seen earler. 22 The hgh cholesterol to phospholpd rato n the nucleus reflects a loss of phospholpd from ths regon, rather than an ncrease n cholesterol. 22 ' 23 The constancy of the cholesteroltophospholpd rato throughout most of the cortex supports the possblty that the lens stes for cholesterol and phospholpd synthess are smlar. The apparent slghtly lower cholesteroltophospholpd rato n the extremely superfcal cortex (outer 2% of the radus) mght reflect a carry over of phospholpds from epthelal cells durng dfferentaton. The epthelal cells from the embryonc chcken lens possess a lower cholesteroltophospholpd rato than that of ts fber cells (Fleschner CR, unpublshed observatons, 992). Rabbt antbovne MP26 antbody precptated four trtated leucnelabeled protens of approxmately 2028 klodaltons from the solublzed rat lens fractons (Fg. 3A). The prncpal proten was 26 klodaltons and, therefore, taken as MP26. The dentty of the other mmunoprecptated protens s unknown. However, because MP26 s degraded to lower molecular weght forms, 24 ncludng a 9.5klodalton form, 25 t appeared possble that proteolyss of MP26 occurred durng the solaton procedures. In fact, others also observed some proteolyss of MP26 durng ts solaton by mmunoprecptaton. 4 TABLE l. Dstrbuton of 3 HLeucne Substrate (TCASoluble and Nonvolatle Radolabel) n the Lens* Sample Incubaton No. of Proten Tme (hr) Lens (mg) Estmated Water} dpm X 0~ 6 / dpm X 0~ 5 / Sample p Water / Cortex Nucleus Cortex Nucleus Cortex Nucleus J 2.8].326J 3.467} * Lenses (9dayold ras) were ncubated n DMEVl + 50 tc\/m\ s Hleucne for the ndcated lmes. Lenses were dvded nto corlex and nucleus and [he TCAsoluble and nonvolatle radolabel ( s Illeucne) recovered and measured. fthe water conlen of 2to22dayold rat lens was prevously determned to be 52% n cortex and 46% n nucleus.

6 Coordnaton of Lens Membrane Synthess % Radus FIGURE 2. Dstrbuton of cholesterol and fatty acd synthess and cholesterollophospholpd molar ratos n 2425dayold rat lens. After ncubaton at 37 C n meda contanng (vtated water (8 mc/ml), ten ntact rat lenses were decapsulated and dssocated n Buffer A plus 0.2% SDS. The dstrbuton of proten n the fractons was determned. After saponfcaton, cholesterol was solated as dgtondeprecptable sterols and the fatty acds, by thnlayer chromatography. The dstrbuton of synthess was related to specfc segments of the lens radus, based on the known dstrbuton of proten n the lens (Fg. C). Fatty acd total counts above background = 236 dpm; dgtondeprecptable sterols total counts above background = 66 dpm. All samples were counted to an error (<r) of less than 3%. When checked, ncorporaton was decreased by more than 98% by ncubaton at 0 c C. The ntensty of MP26 labelng was greatest n fractons 2 mn to 68 mn (Fg. 3A). These fractons collectvely accounted for 9% of the lens total proten and corresponded to approxmately the outer 6% of the lens radus (estmated from Fg. C). Only trace labelng of MP26 was seen n fracton 45 mn and n the nucleus that represented 85% to 78% and the nner 78% of the radus. The ncrease n MP26 labelng n fracton 20 mn s partally the result of the greater proten content of ths fracton (5.8% of total lens proten) compared wth the early fractons (.42.2%of total proten). The selectve precptaton of MP26 by the antmp26 antserum from the total lens proten s made obvous by comparson of the fluorographs of the mmunoprecptated MP26 (34 day flm exposure) wth the ntense labelng of total proten from an equvalent fracton of the lens (3day flm exposure, Fg. 3B). We attempted to quantfy the spatal dstrbuton of MP26 synthess n the lens by () estmatng the ntensty of MP26 labelng n each fracton by denstometrc scannng of the exposed flm and (2) relatng the area under the scan curves of MP26 to ts synthess. Wth the excepton of one pont, 85% of the total radus, the plot of the spatal dstrbuton of MP26 synthess n the lens (Fg. 4A) was very smlar to that descrbng the spatal dstrbuton of cholesterol and fatty acd (phospholpd) synthess (Fg. 2). In both, the outer 0% of the lens accounted for approxmately 90% of the total synthess, wth the peak synthess occurrng n the outer 5% of the radus. When the relatve MP26 synthess was expressed per mllgram of proten, the major synthetc actvty was confned to approxmately the outer 35% of the lens radus (97% to 95% of the total radus, Fg. 4B). Because of uncertanty about the extent of quanttatve mmunoprecptaton of the newly syntheszed MP26, a second approach was used to measure the dstrbuton of trtated MP26 n lenses ncubated wth trtated leucne. Intact crude lens membrane was recovered from lenses dssolved nto several fractons by strrng n ureacontanng buffer. The protens extracted from the 26klodalton band of the SDSPAGE gels of the crude membrane were assayed for trtum content. Although the MP26 band was ntensely mmunostaned by the antmp26 antserum (data not shown), we cannot exclude the presence of other labeled protens underlyng these bands. However, others recently demonstrated that only one polypeptde from lens membrane mgrated at 26 klodaltons, MP26. 5 Drect measurement of the trtum content of the 26klodalton ntrnsc membrane proten ndcated that membrane proten synthess was largely confned to the outer 0% of the lens radus (Fg. 5).

7 Investgatve Ophthalmology & Vsual Scence, June 993, Vol. 34, No Dssocaton Interval n Mnutes and % Total Lens Proten/Interval ( ) kd f mn. (2 2) (l.ahi.7hl4h2.j>wl9wlql % The somewhat broader spatal dstrbuton of membrane proten synthess seen wth the ureadssocated lenses lkely reflects the lesser capacty of the ureacontanng buffer to dssolve the lens unformly. However, by permttng recovery of the ntact membrane, the ureadssocaton approach enabled us to show that the spatal dstrbuton of MP26 synthess essentally paralleled that seen when radolabeled MP26 was solated by mmunoprecptaton. 2C Nucleus (.8)(2. ) (3.5)(5.8)(I2.2)(6I.5) 2C 4.4" B. FIGURE 3. Dstrbuton of MP26 synthess n the lens: fluorographc analyss. Sxteen ntact lenses from 20dayold rats were ncubated for 8 hr at 37 C n 3.0 ml of leucnedefcent DMEM contanng 50 ^C/ml of trtated leucne. (A) Decapsulated lenses were dssolved n 3.0ml portons of Buffer A plus 0.2% SDS. The proten dstrbuton n the fractons was determned. The MP26 was nmunoprecptated from ml alquots usng antserum to MP26, and the recovered proten was subjected to SDSPAGE wth detecton of the radolabel by (luorography. The gels were exposed to flm for 34 days. The upper numbers n the fgure (0, 2, and so forth) are the lens dssoluton ntervals (fractons) n mnutes. The lower numbers n parentheses show the percent of the total lens proten that each nterval (fracton) contaned. (B) Fluorography (F) of an alquot of total proten from the 0 mn sample, 3day flm exposure % Radus FIGURE 4. Quanttatve estmaton of the dstrbuton of MP26 synthess n the lens: fluorographc analyss. An optcal densty scan of the fluorographs n Fgure 3 was made, and the areas under the MP26 absorbance curves were determned. (A) The area under each MP26 band (Fg. 3, sample 0 to nucleus) was expressed as a percent of the total synthess (total area) and related to a specfc percentage of the lens radus on the bass of proten dstrbuton. (B) The dstrbuton of synthess was expressed on the bass of the proten content per fracton (the area under MP26 absorbance curve per fracton dvded by the mllgrams of proten per fracton).

8 Coordnaton of Lens Membrane Synthess Exp / \ / \ % Radus FIGURE 5. The dstrbuton of MP26 synthess n the lens: drect analyss. Fourteen to sxteen ntact lenses from 24 25dayold rats were ncubated for 3 hr at 0 C or 37 C n 3.0 ml of leucneclefcent DMEM contanng 50 /tc/ml of trtaed leucne. The results of two experments are shown. Decapsulated lenses were dssocated n Buffer A plus 8 mol/ urea. The crude membrane was recovered by ultracentrfugaton of each fracton, and the membrane proten was separated by SDSPAGE. The polypeptde band at 26 klodaltons was excsed, and the proten was solublzed and counted. Exp : MP26 total counts above background (37 C) = 6,980 dpm. Incorporaton at 0 C = 0 dpm. Exp 2: MP26 total counts above background (37 C) = 6,650 dpm. DISCUSSION Trtated water s an excellent substrate for measurng the dstrbuton of lpd synthess n the lens because t provdes hydrogen atoms for the synthess of both cholesterol and fatty acds 7 and because t rapdly equlbrates wth the total lens water. 26 Thus, the specfc actvty of the substrate remans constant throughout the lens. Because we were unable to detect ncorporaton of trtum from trtated water nto MP26 n prelmnary studes, a radolabeled amno acd (trtated leucne) was used as the substrate. Ths represents a compromse because, after 3, 8, and 8hr ncubatons of ntact lenses wth trtated leucne, the concentraton of labeled leucne n the lens nuclear water was only 37%, 47%, and 55%, respectvely, of that n the cortex. However, because ncorporaton of trtated leucne nto MP26 was shown to be confned to approxmately the outer 05% of the lens radus (and largely to the outer 67%), t appears reasonable to assume that the specfc actvty of the substrate was unform n the outer cortex, partcularly n the longer term ncubaton. Measurng the relatve dstrbuton of the synthess of cholesterol, fatty acd, and MP26 along the lens radus should dentfy the stes of synthess of these components and assembly nto the fber cell membrane. We assume that membrane components are assembled nto membrane at the place where the components are syntheszed. Our measurements ndcate that the dstrbuton of synthess for all major components of the lens membrane s vrtually dentcal and confned to a small regon, one whch approxmately occupes the outer 28% of the lens radus. Peak synthess appeared to occur n a narrow band representng the outer 36% of the lens radus. If the average crosssectonal dameter of a lens fber cell s taken to be.0 jtm 27 and the radus of the 2dayold rat lens to be.5 mm, the regon of maxmum membrane synthess (the outer 36% of the radus) would span fber cell layers 4590 below the capsule. Dr. Kuszak (Rush Presbyteran, Chcago, IL) estmated that rat lens fber cells are fully elongated, extendng from suture to suture, by approxmately 0050 cell layers below the capsule (personal communcaton). Ths would correspond to approxmately the outer 0% of the lens radus, the ste at whch we observed synthess of all membrane components to be essentally complete. Earler work located lens cholesterol and fatty acd synthess to the cortex, but no more specfcally. 26 Detaled measurements of the lens dstrbuton of the messenger RNA for MP26, the other major membrane component, provded nformaton on the dstrbuton of MP26 synthess n the lens f we assume that all of the messenger RNA dentfed was translatonally actve. 28 Usng n stu hybrdzaton wth an antsense messenger RNA, the dstrbuton of MP26 messenger RNA was mapped n young rat lenses. 28 The messenger RNA was mmedately dentfed n elongatng fber cells, found n the hghest concentraton n the bow regon, and dstrbuted throughout the cortex. It was present also n low concentratons n the lens nucleus. Thus, the messenger RNA for MP26 seemed more broadly dstrbuted n the lens than dd the MP26 synthess observed n the current study. In concluson, ths study dentfed a narrow shell of the lens radus where the bulk of lens membrane synthess occurred. Wthn ths dscrete regon, whch reflects the zone of rapd cellular elongaton, the synthess of all membrane components appeared to be hghly coordnated. Ths fndng mples that, as a fber cell elongates, ts membrane accumulates constant proportons of cholesterol, phospholpd, and MP26 throughout ths process. Key Words lens, plasma membrane, cholesterol, fatty acd, man ntrnsc proten

9 294 Investgatve Ophthalmology & Vsual Scence, June 993, Vol. 34, No. 7 Acknowledgments The author thanks Dr. Isaac Bekhor (Unversty of Southern Calforna) for a supply of polyclonal antbody to MP26 and Vck Trutt and Jeanne Mtchell for techncal assstance. References. Broekhuyse RM. Membrane lpds and protens n ageng lens and cataract. In: Ellot K, Ftzsmons DW, eds. The Human Lens In Relatonshp to Cataracts: Cba Foundaton Symposum, vol. 9. Amsterdam: Elsever; 973: Raffety WS. Lens morphology. In: Masel H, ed. The Ocular Lens, Structure and Functon and Pathology. New York: Marcel Dekker; 985: Broekhuyse RM, Kuhlmann ED. Lens membranes: I. Composton of ureatreated plasma membranes from calf lens. Exp Eye Res. 974; 9: AlcalaJ, Masel H. Bochemstry of lens plasma membranes and cytoskeleton. In; Masel H, ed. The Ocular Lens, Structure and Functon and Pathology. New York: Marcel Dekker; 985: Johnson KR, Sas DF, Johnson RG. MP26, a proten of ntercellular junctons n the bovne lens: Electrophoretc and chromatographc characterzaton. Exp Eye Res. 99;52: Fleschner CR, Cenedella RJ. Lpd composton of lens plasma membrane fractons enrched n fber junctons. J Lpd Res. 99;32: Cendella RJ. Sterol synthess by the ocular lens of the rat durng postnatal development. J Lpd Res. 982:23: AlbersJackson B, Farrs BD, Reddan JR, Swndell RT. Incorporaton of l4 Cacetate nto he phospholpds and fatty acds of rabbt lens n organ culture. Curr Eye Res ;2: Phlpson B. Dstrbuton of proten wthn the normal rat lens. Invest Ophthalmol. 969:8: Cornell RB, Horwtz AF. Apparent coordnaton of the bosynthess oflpds n cultured cells: Its relatonshp to the regulaton of the membrane sterokphospholpd rato and cell cyclng. J Cell Bol. 980:86: Feld FJ, Shreves T, Fujwara D, Murthy S, Albrght E, Mathur SN. Regulaton of gene expresson, synthess and degradaton of 3hydroxy3methylgluta"yl coenzyme A reductase by mcellar cholesterol n CaCo2 cells./ Lpd Res. 99;32: Lees MB, Paxman S. Modfcaton of the Lowry procedure for the analyss of proteolpd proten. Anal Bochem. 972;47:84J Pollet S, Ermdou S, Le Saux F, Monge M, Baumann N. Mcroanalyss of bran lpds: Multple twodmensonal thnlayer chromatography. J Lpd Res. 978;9: Hentzen PC, Bessem CC, Sorgente N, Bekhor I. Total poly(a+)messenger RNA from bovne lens cofractonates wth sucrose purfed fber cell plasma membrane. Exp Eye Res. 984;39: Laemml UK. Cleavage of structural protens durng the assembly of the head of bacterophage T4. Nature. 970;227: Iozzo RV, Kovalszky I, Hacoban N, Schck PK, EllngsonJS, Dodge GR. Fatty acylaton of heparan sulfate proteoglycan from human colon carcnoma cells. J Bol Chen. 990:265: Smth I. Amno acds, amnes, and related compounds. Secton I: Paper Chromatography. In: Smth I, ed. Chromatographc and Electrophoretc Technques. New York: John Wley 8c Sons; 969: Anderson RE, Maude MB, Feldman GL. Lpds of ocular tssues:. The phospholpds of mature rabbt and bovne lens. Bochm Bophys Ada. 969; 87: Broekhuyse RM, Bogemann B. Lpds n tssues of the eye: XVI. Uptake oflpds by the rabbt lens n vtro. Exp Eye Res. 978;26: Coller E, Obara Y, Toftness B. Cholesterol and phospholpds n proten fractons of human lens and senle cataract. Bochm Bophys Ada. 978; 5: Culp TW, Hall FF, Jeter J, Ratlff CR. Lens lpds: Bosynthess and hstologcal dstrbuton n rabbt lens. Ophthalmc Res. 970; : Cenedella RJ. Regonal dstrbuton of lpds and phospholpase A 2 actvty n normal and cataractous rat lens. Curr Eye Res. 985;4: L LK, So L, Specor A. Agedependent changes n the dstrbuton and concentraton of human lens cholesterol and phospholpds. Bochm Bophys Ada. ]987;97:2]2O. 24. Roy D. Age dependent changes n the abundance of the major polypeptdes of human lens membrane. Bochem Bophys Res Commun. 979;88: Takemoto LJ, Gorthy WC, Morn CL, Steward DE. Changes n lens membrane major ntrnsc polypeptde durng cataractogeness n aged Hannover Wstar rats. Invest Ophthalmol Vs Sc. 99:32: Cenedella RJ. Regonal dstrbuton of sterol and fatty acd synthess n the ocular lens. Exp Eye Res. 984;38: Kuszak JR, Rae JL. Scannng electron mcroscopy of the frog lens. Exp Eye Res. 982; 35: Bekhor I. MP26 messenger RNA sequences n normal and cataractous lens: A molecular probe for abundance and dstrbuton of a fber cellspecfc gene product. Invest Ophthalmol Vs Sc. 988; 29:80283.