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1 Conversion of soluble lens protein to albuminoid H. William Fulhorst and Richard W. Young The origin of lens albuminoid was investigated by in vivo experiments using radioisotope techniques. Auto radio graphic analysis of lenses from rats injected with labeled niethionine at 1 week of age showed that protein synthesis occurred throughout the lens at this age, but was most intense near the periphery. In succeeding weeks, the radioactive protein was retained within the lens. Seven weeks after injection, it was buried deep within the lens nucleus. In companion radiobiochemical studies, labeled niethionine was administered, to an additional group of 1-week-old rats. Half the animals were killed the following day; the remainder were killed 7 weeks later. One day after injection, practically all of the protein-bound radioactivity was in the water-soluble fraction. Seven weeks later, nearly 40 per cent was recovered in the albuminoid. These findings indicate that water-soluble lens proteins are synthesized directly from aniino acids in growing lens fibers, whereas albuminoid arises through a subsequent conversion of soluble protein to an insoluble form. D.espite the recognized importance of albuminoid in the physiology of the normal and pathologic lens, the mechanism of origin of this water-insoluble protein fraction remains obscure. 1 Indirect evidence suggests that it probably arises (1) by synthesis from amino acid precursors, or (2) by insolubilization of previously soluble lens proteins. However, available information is inadequate to unequivocally support either of these alternatives. A previous study, 2 designed in part to explore this question, revealed that practically all of the protein-bound radioactivity in the lens could be recovered in the From the Department of Anatomy, Center for the Health Sciences, University of California at Los Angeles, Los Angeles, Calif This work was supported by United States Public Health Service Grant NB soluble protein fraction 1 day after injection of 3fi S-methionine in young adult rats. Less than 3 per cent occurred in the insoluble protein. The albuminoid activity was so low that direct synthesis appeared questionable. In the lenses of rats maintained 4 weeks after injection, the proportion of protein radioactivity attributable to albuminoid had risen slightly. This increase was consistent with the contention that soluble protein had been converted to albuminoid, but was too small to be convincing. Additional studies were therefore planned to provide a more decisive experimental situation, in which any shift in labeling from the soluble to the insoluble protein would be accentuated, if it were to occur at all. Labeled methionine was administered to rats at an earlier age, when growth was more rapid. Furthermore, the animals were maintained for a longer interval after injection, This experimental

Volume 5 Number 3 Lens protein conversion 299 design (1) allowed time for appreciable albuminoid formation, and (2) resulted in the initially labeled fibers being buried deep within the older lens in

2 Volume 5 Number 3 Lens protein conversion 299 design (1) allowed time for appreciable albuminoid formation, and (2) resulted in the initially labeled fibers being buried deep within the older lens in the nuclear site of preferential albuminoid formation. A report of the findings of these new investigations is presented below. Methods Autoradiography. Ten 1-week-old Long-Evans rats were injected intraperitoneally with 5 ^c. per gram of body weight of H H-L-methionine in aqueous solution, 0 and killed at 1 and 8 hours, 1, 2, and 4 days, 1, 2, 4, 7, and 11 weeks after injection. Autoradiograms of eye sections were prepared,- and exposed for 5 to 9 weeks. The concentration of developed silver grains in the autoradiograms was determined along a continuous strip extending across the lens in the equatorial plane 2 in animals killed at the 1 day, 1, 4, 7, and 11 week intervals. Radiobiochemistry. Twenty 1-week-old Long- Evans rats were injected subcutaneously with 5 fie. per gram of body weight of 35 S-L-methionine in aqueous solution.! The rats were divided into two groups of ten animals each. Croup I was killed 1 day after injection; Croup II, 7 weeks later. The lenses were dissected as described previously, 2 collected in 2.5 ml. of water (ph 7 at 4 C.) in a Potter-Elvehjem homogenizing flask, and homogenized in the cold for 5 minutes. The homogenate was then transferred to a capped centrifuge tube, and the flask washed with 2.4 ml. of water, which was added to the tube. The homogenate was then centrifuged for 2 hours, 2 following which the supernatant (first aqueous extract) was removed and taken to 5 ml. with water. The residue was suspended in 0.9 ml. of water, and stored at 4 C. overnight, to ensure the extraction of all water-soluble material. It was then centrifuged for 1 hour, and the supernatant (second aqueous extract) taken to 1 ml. with water. The residue (albuminoid) was solubilized by heating in a glass-stoppered 10 ml. volumetric flask in 4 ml. of 6N IIC1 for 12 hours at 110 C. in an autoclave. The hydrolysate was then taken to 10 ml. with water. After determination of the total radioactivity in each of these fractions, 2 the partition of radioactivity between protein and nonprotein in the aqueous extracts was determined by extraction with cold 10 per cent trichloracetic Specific activity, 160 me. per millimole, Volk Biochemical Co., Skokie, 111. :l H-methionine was used, rather than :lr> S-methionine, because of the improved autoradiographic resolution obtained with tritium, and because of its appreciably lower cost. Pilot experiments indicated that there was no difference between the two in the general distribution of the autoradiographic reaction, t Specific activity, 45 me. per millimole, Volk Biochemical Co., Skokie, 111. acid (TCA), by methods described elsewhere. 2 All radioactivity determinations were corrected for background, self-absorption, and isotope decay. Results Autoradiography. Within 1 hour after injection, there was an appreciable concentration of radioactivity throughout the lens, including its central portion. The reaction was considerably more intense towards the periphery, in the younger lens fibers. There was no apparent change within the first 24 hours. However, at 2 days it was evident that the newly-formed superficial fibers in the equatorial zone were less heavily labeled than the immediately subjacent fibers, which had been located at or near the surface the preceding day. 3 This process was further advanced at 4 days. By 1 week, a surface coating of more weakly labeled fibers encircled the lens. Fig. 1. Diagram of the lenses from rats killed 1 day (left) and 7 weeks (right) after injection of 3 H-methionine at 1 week of age. One half of each lens is shown, drawn to scale, and apposed at the midline, with the anterior surface below. The distribution of the autoradiographic reaction is indicated by stippling. Note the initial concentration of labeling near the periphery of the lens, and its subsequent localization near the center, associated with a compaction of the labeled fibers. The regions designated by the letters A through F are depicted in Fig. 2.

300 Fulhorst and Young At 2 weeks, traces of labeling were still detectable in the most superficial fibers. There was no diminution of the reaction over the more intensely reactive, deeper fibers.

3 300 Fulhorst and Young At 2 weeks, traces of labeling were still detectable in the most superficial fibers. There was no diminution of the reaction over the more intensely reactive, deeper fibers. At 4 weeks and thereafter, no further incorporation of radioactivity into the surface layers of new lens fibers was observed. As late as 7 and 11 weeks after injection, there was still no evidence of any depletion of radioactivity from the previously labeled regions. The heavily reactive fibers were now buried deep within the lens nucleus (Figs. 1 and 2). These observations were confirmed by grain counts, which also revealed that, with increasing time after injection, the Inocstigatioc Ophthalmology June 1966 region of most intense labeling was progressively displaced towards the center of the lens. This process of "compaction" was also reflected in a gradual narrowing of the region of peak labeling. These findings are depicted in the grain-count distributions given in Fig. 3. Radiobiochemistry. One day after injection, the vast majority of total lens radioactivity was recovered in the first aqueous extract (95.6 per cent). Only a small, additional increment (1.7 per cent) was obtained by a second extraction with water. Five per cent of total radioactivity in this combined water-soluble material was not precipitated with TCA; and was considered ', Fig. 2. Autoracliograms of lenses from rats killed 1 day (A, B, C) and 7 weeks (D, E, F) after injection of 3H-methionme at 1 week of age. The regions shown are indicated in Fig. 1. (Periodic acid-schiff and hematoxylin x800.)

Volume 5 Number 3 Lens protein conversion 301 loor DISTANCE:EQUATOR-CENTER Fig. 3. Distribution of developed silver grains in autoradiograms of lenses from rats killed 1 day, 7 and 11 weeks after injection of a H-methionine at 1 week of age.

4 Volume 5 Number 3 Lens protein conversion 301 loor DISTANCE:EQUATOR-CENTER Fig. 3. Distribution of developed silver grains in autoradiograms of lenses from rats killed 1 day, 7 and 11 weeks after injection of a H-methionine at 1 week of age. Grain count is expressed as a percentage of the concentration of silver grains in the region of heaviest labeling. C, center of the lens; E t E?, En, indicate the equatorial surface of the lens at 1 day, 7 and 11 weeks, respectively. Compare with Figs. 1 and 2. to represent nonprotein. The water-insoluble albuminoid fraction contained only 2.7 per cent of the radioactivity recovered from the lens. The total amount of radioactivity recovered from the lenses of rats killed 7 weeks after injection had increased by about 18 per cent (760,314 c.p.m., compared to 643,559 c.p.m. obtained at 1 day). Of this total, 58.3 per cent was recovered in the first, and 2.4 per cent in the second aqueous extract. Less than 1 per cent of this activity was extracted with TCA. About 40 per cent of total lens radioactivity was recovered in the albuminoid. Thus, the absolute amount of radioactivity recovered in the albuminoid rose by a factor of more than 17 (from about 17,000 c.p.m. at 1 day to about 300,000 c.p.m. at 7 weeks). These findings are summarized in Table 1 and Fig. 4. Discussion Synthesis of protein from amino acid precursors, as determined autoradiographically,' 1 occurs throughout the lens of the 1-week-old rat, in contrast with the situation at 8 weeks of age, in which it is largely restricted to the superficial fibers of the 1 DAY 7 WEEKS POST-INJECTION INTERVAL Fig. 4. Distribution of radioactivity in soluble and insoluble (albuminoid) protein fractions of the rat lens 1 clay and 7 weeks after injection of 3r 'Smethionine at 1 week of age. Note the marked shift of radioactivity from soluble to insoluble protein at the 1 week interval. lens cortex. 2 However, even at 1 week there are gradients within the lens, the rate of protein synthesis being higher in the rapidly developing fibers situated near the surface. One day after injection in these young animals, practically all of the protein-bound radioactivity was in water-soluble protein. Less than 3 per cent was recovered in albuminoid. 0 The same relationship is maintained in 8-week-old rats. 2 These findings suggest that (1) the watersoluble proteins of the lens are synthesized from amino acid precursors, and (2) this process occurs predominantly in immature, growing lens fibers. The absence of significant albuminoid labeling 1 day after injection in both age groups indicates that direct synthesis of albuminoid from amino "The probable presence of trace amounts of lipid and carbohydrate in the albuminoid fraction is considered irrelevant, since the sulfur content of both is negligible or nil.-- " The albuminoid fraction may also have been slightly contaminated with a soluble protein fraction which precipitates in the cold. 7 Since this fraction predominates in the cortex, and is greatly reduced in amount at 8 weeks, compared to 1 week of age, any such contamination would preferentially increase the albuminoid labeling at the 1 day interval.

$Distribution of total radioactivity in different lens fractions 1 day and 7 weeks after a single injection of 3r> S-methionine Postinjection interval 7 weeks Lens fraction Water-soluble protein*$

5 302 Fulhorst and Young Investigative Ophthalmology Juno 1966 Table I. Distribution of total radioactivity in different lens fractions 1 day and 7 weeks after a single injection of 3r> S-methionine Postinjection interval 7 weeks Lens fraction Water-soluble protein* Water-soluble non-protein f Albuminoid Total 1 day C.p.m. (mean ± S E.) 594,891 ± 1,630 31,146 ± ,522 ± ,559 ± 1,684 Per cent total c.p.m C.p.m. (mean ± S 460,334 ± 1,289 ± 298,691 ± E.) Per cent total c.p.m ,314 ± 1,154 "Combined aqueous extracts, TCA supernatant. ftca supernatant. acid precursors, if it occurs at all, is not the primary mode of formation of the insoluble protein fraction. In the lens of the 8-\veek-old rat, about 18 per cent of the total protein is albuminoid. 5 What is the source of this insoluble protein, if not by direct synthesis from amino acids? The answer to this question appears to lie in the results of the following experiments. If rats are killed at 8 weeks of age, 1 clay after injection of 35 S- methionine, less than 2 per cent of total lens radioactivity is recovered in albuminoid. 2 However, if rats are killed at 8 weeks of age, 7 weeks after injection, some 40 per cent of the radioactivity in the lens is recovered in the albuminoid. The source of this markedly elevated albuminoid labeling (and thus the source of the albuminoid) must be the soluble protein, which is the only other fraction containing significant levels of radioactivity. However, alternative interpretations should be considered. Labeled protein precursors are apparently available (at steadily decreasing specific activity) for as long as 2 weeks after a single in vivo injection. Could these be the source of the augmented albuminoid labeling at 7 weeks? The answer must be negative, since the findings in animals injected at both 1 and 8 weeks of: age demonstrate that the incorporation of labeled amino acids into protein is almost entirely restricted to the soluble fraction. Therefore, the continued availability of labeled protein precursors could not account for the shift in the balance of labeling toward the insoluble protein. Nor could the increased percentage of total labeling in albuminoid be due simply to a preferential loss (removal or disappearance) from the lens of the soluble protein. This hypothesis is incompatible with the finding that the radioactivity in albuminoid increases strikingly during the 7-week interval in an absolute sense. Furthermore, there is in this material no evidence of any loss of radioactive protein. On the contrary, the protein, once labeled, appears to be retained within the lens.* With age, however, it is increasingly recovered in insoluble form. The results are completely consistent with the conclusion that the albuminoid arises through a conversion of soluble to insoluble protein. It has long been known that albuminoid preferentially accumulates deep within the lens nucleus. s In the 8- week-old rat, the center of the lens nucleus is comprised of the fibers which constituted the entire lens at 1 week of age. At 1 week, all of the cells are metabolically active, and are engaged in the synthesis of soluble protein. In subsequent weeks, however, these fibers are progressively compacted, dehydrated, and deprived of an adequate nutrient source, by the apposition of increasing layers of newer fibers peripherally. This drastically altered microenvironment in- The increase in total radioactivity at 7 weeks, compared to 1 day, is believed to be due to the retention of initial labeling, and the continued incorporation of low levels of radioactivity for several days after injection.

6 Vf>/.M»IC 5 Number 3 Lens protein conversion 303 hibits the synthesis of soluble protein, and accelerates its conversion to an insoluble form. The technical assistance of Mrs. Mirdza Berzins is gratefully acknowledged. REFERENCES 1. Nordmann, J.: Present state and perspectives in research of the lens, INVEST. OPHTH. 4: 384, Young, R. W., and Fulhorst, H. W.: Regional differences in protein synthesis within the lens of the rat, INVEST. OPHTH. 3. Mikulicich, A. G., and Young, R. W.: Cell proliferation and displacement in the lens epithelium of young rats injected with tritiated thymidine, INVEST. OPHTH. 2: 344, Droz, B., and Warshawsky, II.: Reliability of the radioautographic technique for the detection of newly synthesized protein, J. Histochem. & Cytochem. 11: 426, Dische, Z., Borenfreund, E., and Zelmenis, C: Changes in lens proteins of rats during aging, Arch. Ophth. 55: 471, Dische, Z.: Clycoproteins of the lens, INVEST. OPHTH. 4: 592^ Lerman, S., and Zigman, S.: The metabolism of the lens as related to aging and experimental cataractogenesis, INVEST. OPHTH. 4: 643, 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.

Metabolic pathways in experimental diabetic cataract. Sidney Lerman

Metabolic pathways in experimental diabetic cataract Sidney Lerman Specific areas in carbohydrate and protein metabolism were studied in the lenses of alloxandiabetic rats. The studies were performed at