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Regional differences in protein synthesis within the lens of the rat Richard W. Young and H. William Fidhorst Utilization of 3SS-methionine by cells of the rat lens was studied by combined autoradiographic and chemical techniques. Part of the methionine teas converted to cysteine, glutathione, and taurine. Protein synthesis was essentially restricted to the lens cortex, and appeared to be largely attributable to growth. One day after injection, over 90 per cent of total lens radioactivity was recovered from the cortex, most of it in water-soluble protein. Eight labeled protein fractions were separated. Continued peripheral addition of new fibers progressively buried the labeled cells within the body of the lens, so that at 4 weeks the proportion of total radioactivity recovered from the deeper fibers was increased. Insoluble protein (albuminoid) appeared to be largely derived from the precipitation of certain soluble proteins in the lens nucleus. Such selective insolubilization may account for the differing patterns of soluble protein observed in the inner and outer parts of the lens. Methods .he ocular lens is unique among mammalian organs in that it is wholly composed of a single, epithelial cell type. Despite continued production of new cells, older cells are not shed. Instead, they are incorporated directly into the body of the lens itself. Thus, it is a population of cells characterized by growth, rather than renewal. The protein metabolism of these cells may be effectively studied with labeled amino acids. An investigation of the utilization of 3r>S-methionine by cells of the lens in young adult rats, analyzed by autoradiographic and radiobiochemical techniques, is reported below. Autoradiography. Female Long-Evans rats, 8 weeks of age, were used. Six animals were injected intraperitoneally with 4 fie per gram of body weight of 35S-L-methionine in aqueous solution at a concentration of 1.83 me. per milliliter, and a specific activity of 0.65 me. per milligram.* The rats, ranging in weight from 140 to 156 Cm. (mean 150 Cm.), were killed 4, 8, 24, and 96 hours, 1 and 4 weeks after injection. The eyes from each animal were fixed in buffered neutral 4 per cent formaldehyde for 2 days. They were then double-embedded in nitrocellulose-paraffin, sectioned parasagittally at 5 ft, and prepared for autoradiography by the dipping technique with Kodak NTB2 liquid emulsion. Sections were stained with periodic acid-Schiff (PAS) before coating with emulsion, and hematoxylin after development, or hematoxylin only, after development of the autoradiograms. The preparations were developed in Kodak Dektol for 2 minutes at 17° C. after exposure in a cold, dry atmosphere for periods ranging from 1 to 3 weeks. The concentration of silver grains was deter- From the Department of Anatomy, Center for the Health Sciences, University of California at Los Angeles, Los Angeles, Calif. 90024. This work was supported by United States Public Health Service Grant NB-03807. 'Radinchemical Centre, Amersham, England. 288 Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933626/ on 06/17/2017 Volume 5 Number 3 mined in several sections taken at or near the midsaggital plane at the 1 day and 4 week intervals, using an ocular grid graduated in squares (each 57 n2 at 1200 x magnification). The number of grains per square was recorded along a continuous strip, extending across the lens at the equator (excluding the epithelium and capsule). The counts from one side were combined with the corresponding counts on the opposite side, then averaged. The averages (after subtraction of background) were then expressed as a percentage of the average concentration in the most heavily labeled square (to eliminate differences due to dosage, development, isotope decay, etc.). Radiobiochemistry. Thirty female Long-Evans rats, 8 weeks of age, were injected intraperitoneally with 4 y.c per gram of body weight of 35 S-L-methionine in aqueous solution (2 me. per milliliter) at a specific activity of 0.51 me. per milligram. The rats were divided into two groups of 15 each. The animals in group I ranged in weight from 143 to 170 Gm. (mean 154 Gm.); those in group II weighed 141 to 171 Gm. (mean 161 Cm.). Group I was killed 1 day after injection; Croup II 27 days later. The rats were killed individually by an overdose of chloroform; both eyes were enucleated, and the lenses dissected free of ciliary body, vitreous, and lens capsule under the dissecting microscope. The dissections were controlled by histologic examination of comparably prepared lenses. In the following procedure, "water" denotes distilled water adjusted to pH 7 with a minimum of phosphate bufFer. All centrifugation was carried out at 2,500 r.p.m. in the cold (4° C.). The method initially devised by Krause,1 and used in a number of subsequent investigations, was employed for separating the superficial (cortical) and central (nuclear) portions of the lens. The lenses were collected in a capped centrifuge tube containing 4 ml. of water, and maintained in an ice bath. After all the lenses had been dissected, the centrifuge tube was shaken by hand for 3 minutes. It was then centrifuged for 2.5 hours. The supernatant was taken to 5 ml. with water (cortical extract). The residue could be seen under the dissecting microscope to consist of lens nuclei and a few sedimented free fibers. This material was transferred to a Potter-Elvehjem homogenizing flask. The centrifuge tube was then washed twice with the 1.5 ml. of water, the washes being added to the homogenizing flask. The residue was next homogenized in the cold for 5 minutes, then transferred to a capped centrifuge tube. The homogenizing flask was then washed with 1.4 ml. of water; the wash was added to the homogenate, and the homogenate centrifuged for 1.5 hours. The supernatant was then taken to 5 ml. with water (nuclear extract). Regional differences in protein synthesis 289 The homogenizing flask was then washed again with 0.8 ml. of water, and the washings added to the residue, which was suspended by gentle shaking and stored at 4° C. overnight, to assure the complete extraction of water-soluble material. It was then centrifuged for 2 hours, and the supernatant (albuminoid wash) taken to 2 ml. with water. The residue was lyophilized, then taken up as a suspension in 10 ml. of chloroform: methanol (2:1), and incubated overnight at 4° C. This solution was centrifuged for 0.5 hours, after the addition of 0.2 volumes of methanol to sediment the residue. The supernatant was removed, and the extraction repeated with 5 ml. of chloroform:methanol (lipid extract). The residue was dried overnight in a vacuum desiccator, taken up as a suspension in 9 ml. of 6N HC1, and hydrolyzed under reflux for 20 hours at 107° C. The hydrolysate was then taken to 10 ml. with water (albuminoid hydrolysate). Four aliquots from each fraction were plated on preweighed copper planchets. The planchets were reweighed after drying, the difference in weight representing the weight of the sample. The weight of the hydrolysate obtained in this manner included the weight of the associated HC1. Accordingly, the net weight of the hydrolysate was obtained by subtracting from the gross weight the weight of a comparable volume of 6N HC1 solution. Radioactivity was analyzed in a thin end-window, gas-flow Geiger-Muller counter. Counts were corrected for background, for selfabsorption (using a constant activity sodium 35 Ssulfate correction curve), and for isotope decay. The means and standard errors were averaged. Four 10 fi\. aliquots of the aqueous extracts were taken for a microbiuret determination of protein concentration,* using a bovine serum albumin standard. The four values were averaged. Continuous flow electrophoresis was performed at 4° C. on S & S 598 paper curtains in a Misco electrochromatography chamber, \ at 774 V., 4 to 5 ma., in pH 8.6 veronal buffer, ionic strength 0.019, using a 3.8 mm. wide internal wick. Aliquots of 1 ml. from the cortical and nuclear extracts were run for 18 to 19 hours. The curtains were stained with bromphenol blue after heat fixation (110° C. for 30 minutes). The unbound dye and nonprotein sample constituents were then removed by prolonged washing in 6 per cent acetic acid. After analysis by autoradiography with x-ray film, a 3 cm. wide horizontal strip was cut from the curtain near its lower edge. The amount of protein-bound dye on the strip was then measured with a densitometer equipped with an external galvanometer, \ using a 510 m/i nar"Coleman Instruments, Maywood, 111. fMicrochemical Specialties, Berkeley, Calif. JPhotovolt model 425, Photovolt Corporation, New York. N. Y. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933626/ on 06/17/2017 Investigative Ophthalmology Juno 1966 290 Young and Fulhorst Fig. 1. Outer cortical zone at the equator of the lens of an 8-week-old rat killed 1 day after injection of Hr>S-methionme. Silver grains are concentrated at the periphery, in the region of lens fiber formation. (Autoradiogram, hematoxylin, x200.) row-band interference filter. The strips were then assayed for radioactivity in a chromatogram strip counter. Areas under the dye-binding and radioactivity curves were determined by planimetry. Extraction with 10 per cent trichloroacetic acid (TCA) was used to determine the partition of radioactivity between protein and nonprotein constituents in two aliquots from each of the three aqueous extracts. An equal volume of 20 per cent cold TCA was added, the solution incubated at 4° C. for 15 minutes, the supernatant obtained by centrifugation, and the residue (precipitate) washed three times with cold 10 per cent TCA. The combined supernatants (representing the nonprotein fraction) were then extracted three times with equal volumes of ether (which removed most of the TCA, but none of the radioactivity), taken to 2 ml. with water, and two 0.1 ml. aliquots of each plated, weighed, and counted in the C-M counter. The TCA precipitates were washed twice with ethanol, then three times with ether, and hydrolyzed with 6N HC1. The free amino acid fraction of the aqueous extracts obtained at the 1 day interval was prepared by ultrafiltration through dialysis tubing under vacuum in the cold. This fraction, the albuminoid hydrolysates, and the hydrolysates of the TCA precipitates were lyophilized, then taken up in a small volume of water or 80 per cent ethanol, and analyzed by thin-layer chromatography (TLC). Aliquots were spotted on 20 by 20 cm. glass plates coated with a 250 fi layer of silica gel G.° Butanol (normal):acetic acid;water •Brinknwnn Instruments, Westbury, N. Y, (3:1:1) was used for development in the first dimension, and phenol:water (4:1) in the second dimension. The plates were sprayed with ninhydrin (0.2 per cent in n-butanol), and the spots identified by comparison with standard maps derived from running known reagents under comparable conditions. Radioactivity was detected by apposition of Kodak Blue Brand Medical x-ray film for periods ranging from 4 days to 1 month. Results Autoradiography. Some labeling was observed in all ocular structures. Most heavily reactive at the early intervals were the lens, corneal epithelium, retina, and ciliary epithelium. At later intervals (1 to 4 weeks), the lens exhibited a greater retention of labeling than these other tissues. In the lens, at 4 and 8 hours after injection, the autoradiographic reaction was almost entirely restricted to the cortex, within which it was particularly heavy in the more superficial fibers. The most intense labeling occurred at the equator, in the region of lens fiber information. Deeper in the lens, the reaction decreased progressively, and was negligible over the center of the lens. The lens epithelium was moderately labeled. The reaction was weaker, but significant, over the capsule. There was little apparent change at 1 Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933626/ on 06/17/2017 Volume 5 Number 3 Regional differences in protein synthesis 291 Fig. 2. A, lens of a 12-week-old rat killed 28 days after injection of 35S-methionine. Peripheral addition of lens fibers during the 4 week interval has buried the heavily labeled fibers within the lens body, yielding a reaction band (arrow) which outlines the limits of the lens as it existed at 8 weeks. The black areas over the lens nucleus ate artifacts. (Autoradiogram, PASheniatoxylin. x20.) B, details of the equatorial region of the same lens. Note the progressive decline in labeling intensity in new lens fibers at the periphery. {x50.) day (Fig. 1). By 4 days, the first few of the most superficial lens fibers in the equatorial region were labeled somewhat less intensely than the immediately subjacent fibers, in which labeling was greatest. From 1 through 4 weeks, a continuation of this process had yielded a complete, superficial coating of progressively less radioactive lens fibers, thickest at the equator, and thinnest at the poles (Fig. 2). Capsular labeling appeared not to have decreased, but the epithelium was now only slightly reactive at the exposures used. The central portion of the lens nucleus remained unlabeled. An analysis of the concentration of silver grains along an equatorial axis extending from the center of the lens to the periphery at 1 day and 4 weeks is given in Fig. 3. It is evident that most of the bound radioactivity ascribed to the lens nucleus at 4 weeks had actually been utilized by cells initially located in the cortex. These labeled fibers were subsequently buried by the peripheral apposition of more superficial lens fibers. As a result, an increased Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933626/ on 06/17/2017 292 Young and Fulhorst Invcsligativc Oiththalmology June 1966 proportion of radioactivity was recovered in the second or "nuclear" extract, as described below. Radiobiochemistry. Autoradiographic examination of the 35S-L-methionine solution, separated by two-dimensional TLC, demonstrated the presence of several SILVER GRAIN CONCENTRATION Fig. 3. Distribution of silver grains in autoradiograms of lenses from rats sacrificed 1 day (above) and 28 days (below) after injection of 35S-methionine. Grain concentration was analyzed in a zone extending from the equatorial surface (left) to the center of the lens nucleus (C, arrow). The shaded area under the curve represents the approximate proportions of total radioactivity recovered in the cortical extract at each interval (cf. Tables I and II). Note that most of the radioactivity recovered in the nuclear extract at 28 days was present in the cortex at 1 day. weakly labeled contaminants. Analysis of one-dimensional separations in the strip counter indicated that these represented less than 3 per cent of the total radioactivity. Histologic examination of lenses dissected by the procedures used in this part of the study revealed that adherence of the lens epithelium to the capsule resulted in removal of the epithelium during decapsulation. Consequently, the following results are based on analysis of the lens proper, free of both capsule and epithelium. The residue of the first or "cortical" extract was also examined histologically, in 5 M paraffin sections stained by a variety of procedures. This material indicated that loss of protein from the lens nuclei was negligible, and that the cortical extract contained only the outermost lens fibers. The distribution of radioactivity among the several lens fractions isolated from animals killed 1 day after injection is given in Table I. The major portion (by weight) of the lens material is soluble in water, and most of this material is protein. The second most prominent fraction is water-insoluble protein, termed albuminoid. (The albuminoid weight includes the water taken up during peptide bond hydrolysis.) The total amount of water-soluble protein recovered in the cortical and nuclear extracts was roughly comparable. Subsequent overnight extraction of the residue yielded only a small, additional amount of water-soluble protein. Lipid constitutes a minor fraction of the lens material. Most of the radioactivity was recovered Table I. Distribution of radioactivity among different lens constituents 1 day after administration of 3fiS-methionine Weight (ma.) Protein Lens fraction Counts per minute (mean +. S.E.) Per cent total c.p.m. Per cent c.p.m. in protein Cortical extract Nuclear extract Albuminoid wash Lipid extract Albuminoid hydrolysate Total 178.7 121.6 17.1 5.3 123.7 446.4 138.0 112.0 12.4 — — 262.4 995,965 ± 3705 58,615+ 480 5,998 ± 64 479 ± 11 17,290+ 166 1,078,347 ± 3740 92.4 5.4 0.6 <0.1 1.6 100.0 80.2 77.4 70.2 — — — Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933626/ on 06/17/2017 Regional differences in protein synthesis 293 Volume 5 Number 3 Table II. Distribution of radioactivity among different lens constituents 28 days after administration of 3fiS-methionine Weight (ma.) Protein Lens fraction Counts per minute (mean ± S.E.) Per cent total c.p.m. Per cent c.p.m. in protein Cortical extract Nuclear extract Albuminoid wash Lipicl extract Albuminoid hydrolysate Total 170.6 187.0 28.2 6.7 177.7 570.2 132.7 147.7 15.4 1,025,846 ± 3589 327,945+1130 27,742+ 187 1,361 + 19 49,545+ 305 1,432,439 + 3782 71.6 22.9 2.0 <0.1 3.5 100.0 89.9 90.8 89.9 — — — — 295.8 in the aqueous extract of the superficial fibers (92.4 per cent). Only 5.4 per cent occurred in the subsequent aqueous extract of the lens nuclei, and less than 1 per cent in the albuminoid wash. Labeling of the lipid fraction was negligible. The insoluble protein contained veiy little 3r>S, accounting for only 1.6 per cent of the total radioactivity in the lens. Extraction of the aqueous extracts with 10 per cent TCA removed some of the water-soluble radioactivity. This fraction, considered to represent nonprotein, ranged from 20 to 30 per cent of the total. Table II presents comparable data derived from animals killed 28 days after injection. The amount of radioactivity was greater (by about 25 per cent). In the nuclear fraction, the percentage of total radioactivity had increased markedly, constituting 22.9 per cent of the total (compared to 5.4 per cent at 1 day). About 10 per cent of the water-soluble radioactivity was extracted with TCA, and was therefore ascribed to nonprotein. The proportion of total radioactivity occurring in the albuminoid had increased over the 1 day figure (3.5 per cent compared to 1.6 per cent). Two-dimensional TLC separations of the free amino acid fraction of the lens, 1 day after injection, yielded up to 30 ninhydrinpositive spots. No effort was made to systematically identify all of these. However, the distribution appeared to be similar to that previously described in the rat- and other mammalian lenses.3' ' The largest spots were due to reduced glutathione (GSH), taurine, and glutamic acid. Methione was present in small amounts. No cysteine, cystine, or oxidized glutathione were detected. Several radioactive constituents were revealed by autoradiographic analysis of Gly (TluSer Fig. 4. Map of the thin-layer chromatographic separation of albuminoid hydrolysate. The origin is shown by a black dot. The direction of the second development (phenol:water) is indicated by an arrow. P, phenylalanine; L, I, leucine, isoleucine; T, tyrosine; M, methionine (radioactive); V, valine; Pro, proline; A, alanine; Gly, glycine; Th, threonine; Ser, serine; GA, glutamic acid; AA, aspartic acid; H, histidine; C, cysteine (radioactive); Ar, arginine; Ly, lysine. Cystine was not detected, perhaps due to its destruction during hydrolysis. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933626/ on 06/17/2017 294 Investigative Ophthalmology June 1966 Young and Fulhorst Table III. Distribution of protein and radioactivity at 1 day (cortex) and 28 days (nucleus) after injection of 8r'S-methionine Protein fraction Source Protein" Cortex Nucleus Radioactivity! Cortex Nucleus a P, P* & Pt 1 ft y< Y* 32.2 22.1 8.7 6.3 15.8 8.9 7.3 6.8 4.8 9.9 5.0 7.1 14.5 24.2 11.7 14.7 24.7 20.7 1.6 2.4 27.0 9.9 5.2 6.9 3.1 6.0 1.6 5.1 20.6 29.5 16.2 19.5 "Per cent of total protein-bound dye. tPer cent of total radioactivity. The variations in per cent protein/per cent radioactivity probably reflect differing proportions of sulfur-containing amino acids in the several protein fractions. these separations. The labeled components have been identified as methionine (including sulfone-sulfoxide), taurine, GSH, and three additional spots with relatively low Rt values in both solvents, tentatively identified as cystathionine, cysteic acid, and cysteinesulfinic acid. Autoradiographic analysis of TLC separations of the hydrolysates of water-soluble and water-insoluble proteins in both experimental groups indicated that some of the 35S had been incorporated into protein as a constituent of cysteine, as well as in methionine. A typical map of the albuminoid hydrolysate is given in Fig. 4. Eight water-soluble protein fractions were resolved by continuous flow electrophoresis of cortical and nuclear extracts from both groups of rats. Two additional fractions, one with high anodal and the other high cathodal mobility, temporarily bound the dye, then faded and disappeared in the staining bath. The extracts also contained several components, probably nonprotein in nature, which fluoresced in ultraviolet light before, but not after staining.5 The pattern of distribution of the eight stable protein fractions* differed in the inner and outer portions of the lens in a similar fashion in both groups of animals "Classically, the soluble lens proteins have been divided into three groups (a, /?, and 7). In paper strip electrophoresis (pH 8.6) rat lenses yield four fractions, the middle two of which are considered to be ft components. By comparing densitometric analysis of such strips with similarly analyzed continuous flow separations, the eight fractions separated by the latter technique have been classified. (Fig. 5). The a component (highest net negative charge) and the /?2 fraction were stronger in the cortex; /?1} /3;,, and /?5 were low and similar in both cortex and nucleus. Both y components (lowest net negative charge) were stronger in the nucleus of the lens. The relative distribution of total soluble protein among these fractions is given in Table III. One day after injection, there was very little radioactivity in the soluble proteins of the more centrally located lens fibers. However, all proteins derived from the more superficial fibers of the cortex showed appreciable activity at this interval. Over 25 per cent of the soluble protein radioactivity was in the (32 fraction, almost 25 per cent in the a component, and about 36 per cent in the two y fractions. The remaining four (3 fractions, constituting about 25 per cent of the total protein, contained less than 12 per cent of the total protein radioactivity. No evidence of a minor, highly labeled fraction was obtained.0 Of particular interest was the distribution of radioactivity in the nuclear fraction at 4 weeks, in the light of the above evidence that these proteins are synthesized in the cortex. The nuclear pattern of radioactivity, like that of the proteins with which it was associated, differed significantly from that of the cortex. The a and /?2 fractions together contained only about 30 per cent, and the remaining /? components about 20 per cent of the total activ- Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933626/ on 06/17/2017 Volume 5 Number 3 Regional differences in protein synthesis 295 Fig. 5. Continuous flow electrophoresis of soluble lens proteins. The anodal pole is on the left. A, curtain showing distribution of separated proteins in the cortical extract (1 day group). Note the heavy staining of the a fraction (left), fi» fraction (center, arrow), and the two y fractions (right). B, autoradiogram of the separation, depicting the distribution of radioactivity among the several protein components. C, curtain showing the protein pattern obtained from the nuclear extract (4 week group). There is a marked predominance of the y fractions (arrow). D, the corresponding autoradiogram. There are cortical-nuclear differences in the distribution of protein and protein-bound S35. ity. Thus, the two y fractions comprised roughly one-half of the total activity associated with the nuclear water-soluble proteins. Since most of the radioactivity contained in the nucleus at 4 weeks was present in the cortex at 1 day, these data are presented for comparison (Fig. 5 and Table III). (The cortical patterns of protein and radioactivity at 4 weeks were similar to those at 1 day.) Discussion Hf 'S, injected as a component of methionine, was subsequently recovered from the lens in cysteine, glutathione, and taurine, as well as in methionine. Evi- dently, cells of the lens are capable of converting methionine to cysteine which, in turn, is used in the synthesis of protein and the tripeptide, glutathione, and in part is converted to taurine.2' 7 3 Protein synthesis in the lens is almost entirely restricted to the outer, cortical cell layers. 0 ' ao1 Over 90 per cent of total lenticular radioactivity was localized in the superficial fibers at 1 day, most of it in protein. Continued peripheral addition of new lens fibers progressively buried the heavily labeled cells which were present near the surface at 1 day. During succeeding days, the rate of 35S incorporation dropped sharply, as the level of circulat- Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933626/ on 06/17/2017 296 Young and Fulhorst ing amino acid 35S decreased, as shown by the rapid decline in silver grain concentration at the lens periphery in the older animals. As a consequence, at 4 weeks a higher proportion of total radioactivity was recovered in the nuclear protein fraction. Short-term radioisotope studies have suggested that there is a rapid turnover of lens proteins.9-12'13 However, in the current material, more protein radioactivity was recovered from the lens 4 weeks after a single injection of labeled methionine than at 1 day after injection. A significant decrease in activity might be expected, despite the continued availability of very low levels of amino acid 35S, if any considerable breakdown of protein had taken place within the 27-day interval. The lens alone, among several initially labeled ocular structures, retained appreciable concentrations of radioactivity at later intervals. Autoradiograms of lenses from rats killed as late as 11 weeks after injection of 3H methionine failed to show any significant depletion of the radioactivity incorporated in the first few days.14 A relatively low level of protein turnover is certainly not excluded by these data, since radioactivity liberated by degradation of lens proteins might be reutilized in situ in the synthesis of new protein. Nevertheless, the findings appear to indicate that protein synthesis in the lens during the period studied is largely attributable to growth. Such growth is accompanied by a net production of albuminoid, which occurs predominantly in the lens nucleus.1':5> 1C There appear to be two possible mechanisms of albuminoid formation: 1. Direct synthesis from amino acid precursors. Lerman and associates17 apparently observed some direct incorporation of labeled amino acids into the albuminoid of rat lenses in vitro. About 2 per cent of total protein radioactivity was recovered in the albuminoid fraction 1 day after injection in the current work. This figure appears too low, and the interval too long 7»ijcs lhjc Ophthalmology June 1966 after injection, to be ascribed with certainty to direct synthesis. 2. Insolubilization of initially soluble proteins. Albuminoid accumulates deep within the lens, in a microenvironment in which protein synthesis is negligible. Factors which lower the rate of soluble protein synthesis have little or no effect on albuminoid accumulation.18"20 The observed increase in the proportion of total protein radioactivity recovered in albuminoid over the 4-week period studied is also consistent with the view that albuminoid is formed by the insolubilization of soluble protein. Additional experiments, reported separately,14 strongly support this contention. Albuminoid formation by preferential insolubilization of certain soluble protein fractions could contribute to the differences in the patterns of protein1- -1"-5 and protein-bound 35S in the inner and outer parts of the lens. Such selective precipitation of soluble protein fractions in the lens nucleus would have the effect of leaving a residual pattern of soluble nuclear proteins which differs from that present in the more superficial fibers. The technical assistance of Mrs. Mirdza Berzins is gratefully acknowledged. REFERENCES 1. Krause, A. C : Chemistry of the lens. V. Relation of the anatomic distribution of the lenticular proteins to their chemical composition, Arch. Ophth. 10: 788, 1933. 2. Dardenne, U., and Kirsten, C : Presence and metabolism of amino acids in young and old lenses, Exper. Eye Res. 1: 415, 1962. 3. Malatesta, C : Studio cromatografico degli amino-acidi liberi nei liquidi endoculari, nel cristallino e nel sangue di bue, Boll. d'Oculistica 31: 762, 1952. 4. Recldy, D. V. N., and Kinsey, V. E.: Studies on the crystalline lens. IX. Quantitative analysis of free amino acids and related compounds, INVEST. OPHTH. 1: 635, 1962. 5. Wood, D. C : An electrophoretic study of soluble lens proteins from different species, Am. J. Ophth. 51: 305, 1961. 6. Spector, A., and Kinoshita, J. H.: The incorporation of labeled amino acids into lens protein, INVEST. OPHTH. 3: 517, 1964. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933626/ on 06/17/2017 Volume 5 Number 3 Regional differences in protein synthesis 297 7. Mandel, P., Dardenne, U., and Lessinger, A.: Incorporation et degradation de la methionine par le cristallin de bovides, Compt. rend. Acad. sc. 245: 985, 1957. 8. Kinsey, V. E., and Merriam, F. C.: Studies on the crystalline lens. II. Synthesis of glutathione in the normal and cataractous rabbit lens, Arch. Ophth. 44: 370, 1950. 9. Waley, S. C : Metabolism of amino acids in the lens, Biochetn. J. 91: 576, 1964. 10. Lessinger, A., and Mandel, P.: Incorporation de methionine-SfiS dans le cristallin d'aminaux jeunes et ages, J. de Physio]. (Paris) 52: 150, 1960. 11. Ilanna, C : Changes in DNA, RNA, and protein synthesis in the developing lens, INVEST. 18. 19. 20. OPHTH. 4: 480, 1965. 12. Merriam, F. C , and Kinsey, V. E.: Studies on the crystalline lens. III. Incorporation of glycine and serine in the proteins of lenses cultured in vitro, Arch. Ophth. 44: 651, 1950. 13. Weber, D.: Uber die mittlere Lebensdauer von Linseneiweissen, untersucht am lebendigen Kaninchen, Deutsche Ophth. Gesellschaft, Bericht. 64: 296, 1961. 14. Fulhorst, H. W., and Young, R. W.: Conversion of soluble lens protein to albuminoid, 21. 22. 23. 24. INVEST. OPHTH. Submitted for publication. 15. Leiman, S.: Cataract, Springfield, 111., 1964, Charles C Thomas, Publisher. 16. Dische, Z., Borenfreund, E., and Zelmenis, C : Changes in lens proteins of rats during aging, Arch. Ophth. 55: 471, 1956. 17. Lerman, S., Devi, A., and Hawes, S.: The incorporation of labelled amino acids into 25. lens protein of normal, galactose and xylosefed rats, Am. J. Ophth. 51: 1012, 1961. Dische, Z., Borenfreund, E., and Zelmenis, C : Proteins and protein synthesis in rat lenses with galactose cataract, Arch. Ophth. 55: 633, 1956. Dische, Z., Elliott, J., Pearson, E., and Merriam, G. R.: Changes in proteins and protein synthesis: In tryptophane deficiency and radiation cataracts of rats, Am. J. Ophth. 47: 368, 1959. Dische, Z., and Zelmenis, C : Nutritional and endocrine influence on the synthesis of albuminoid in rat lenses. I. The eftect of restricted diet and L-thyroxine on the albuminoid formation, Am. J. Ophth. 48: 500, 1959. Morner, C. T.: Untersuchung der Proteinsubstanzen in den leichtbrechenden Medien des Auges, Ztschr. Physiol. Chem. 18: 61, 1894. Bjork, I.: Chromatographic separation of bovine a-crystallin, Exper. Eye Res. 2: 239, 1963. Francois, J., and Rabaey, M.: On the existence of an embryonic lens protein, Arch. Ophth. 57: 672, 1957. Kinoshita, J. H., and Merola, L. O.: The distribution of glutathione and protein sulfhydryl groups in calf and cattle lenses, Am. J.' Ophth. 46: 36, 1948. Kleifeld, O., Hockwin, O., and Fuchs, R.: Untersuchungen uber wasserloslichen Eiweisse in Kern und Rinde der Linsen alter und junger Tiere und menschlicher klarer und getriibter Linsen, Deutsche Ophth. Cesellschaft, Bericht. 60: 108, 1956. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933626/ on 06/17/2017