Acid hydrolases of the retinal pigment epithelium E. R. Berman Two enymes, P-galactosidase and N-acetyl-fi-glucosaminidase, have been detected in pigment epithelial cells of cattle. The optimum activity for N-acetyl-P-glucosaminidase is at ph 5; in contrast, /3-galactosidase exhibits a broad activity maximum from approximately ph 4 to 6, with a "shoulder" in the latter range. Differential centrifugation of.25m sucroseethylenediamine tetraacetic acid (EDTA) homogenates showed that the highest specific activities were in the mitochondrial + lysosomal (M+L) fraction and in the fraction sedimenting at 6,000 g-minutes, i.e., the pigment granules + nuclei (PG+N). Agents, such as sonication, freeing-and-thawing, or homogeniation with Triton X-100, known to induce osmotic shock or membrane rupture, restdted in the release of nearly all the enymes from the particulates in the PG+N fraction. Key words: retina, 8-galactosidase, N-acetyl-/3-glucosaminidase, lysosomal enymes, pigment epithelial cells, acid hydrolases, mitochondria. A,.part from secretory and nutritive functions, the pigment epithelial cells also play an important role in the visual cycle through the storage and conversion of retinol (Vitamin A) to the biologically active esterified 11-cis form. 1 ' 2 In addition, another function, namely phagocytosis and breakdown of peripherally shedded outer rod segments, has been demonstrated in many animal species. 3 " s Failure of this From the Department of Ophthalmology, Biochemistry Research Laboratory,* Hebrew University-Hadassah Medical School, Jerusalem, Israel. These investigations were supported in part by the Michael Polak Research Fellowships in Ophthalmology. Manuscript submitted Nov. 3, 1970; manuscript accepted Nov. 30, 1970. "The Sir Isaac and Lady Wolfson Ophthalmic Research Laboratories. 64 mechanism to operate may underlie certain hereditary retinal dystrophies in rats, 7 ' 9 which are in many respects the animal counterpart of human retinitis pigmentosa. The phagocytic activities of pigment epithelial cells appear to be localied in, or associated with, lysosome-like organelles that have been called "lammellar inclusion bodies," 3 ' 4> 10 ' 12 or "phagosomes." s> 9 Lysosomes in other tissues are rich in acid hydrolases, 13 but there is no information concerning the presence of these enymes in the lysosome-like structures of the pigment epithelial cells. Apart from their potential role in the degradation of outer rod segments, these enymes may also function in the metabolism of the mucopolysaccharides (glycosaminoglycans) and glycoproteins found in the extracellular matrix surrounding the photoreceptor
Volume 10 Number 1 Acid hydrolases in retinal pigment epithelium 65 cells. 14 ' 15 These substances appear to be synthesied by the pigment epithelial cells, 8-1G but little is known regarding their catabolism. Abnormalities or dysfunctions of specific lysosomal enymes are believed to be the major defect in a large number of inherited metabolic diseases characteried by the intracellular accumulation of either lipids or complex carbohydrates, or both." In view of the fundamental role of lysosomal enymes in the catabolic processes of normal cells and their functional deficiencies in many disease processes, it seemed important to know, if, in fact, these hydrolytic enymes were present in pigment epithelial cells. Materials and methods Preparation of pigment cells. Fresh cattle eyes were brought to the laboratory on ice and dissected immediately. The anterior portions of the eyes were discarded and after removal of the retinas, approximately 0.5 ml. of 0.25M sucrose-0.005m ethylenediaminetetraacetic acid (EDTA) was added to the exposed pigment cells. The surface was then rubbed gently with a soft brush and the cells were collected by aspiration. Pigment cells from 15 cattle eyes were pooled and the volume was adjusted to 15 ml. with the sucrose-edta solution. The cell suspension was then homogenied with three upand-down strokes with a loose-fitting Teflon pestle in a motor-driven glass-teflon tissue homogenier (Arthur H. Thomas Co., Philadelphia, Pa.). This preparation could be stored at -25 C. for periods up to two months with no loss in activity. Enyme assays. For experiments to determine ph optima, the homogenate, either fresh or after storage at -25 C, was used directly. /3-Galactosidase (EC 3.2.1.23) was assayed in an incubation mixture consisting of 0.2 ml. of homogenate, 0.6 ml. of 0.1M citrate-phosphate or phosphate buffer (ph 3-8), and 0.2 ml. of 0.005M p-nitrophenyl-/3-d-galactopyranoside (Sigma Chemical Co., St. Louis, Mo.). N-Acetyl-/?-glucosaminidase (EC 3.2.1.30) was assayed with 0.05 ml. of homogenate, 0.75 ml. of buffer, and 0.2 ml. of 0.00375M p-nitrophenyl-n-acetyl-/?-dglucosaminide (Sigma Chemical Co.). Appropriate sample blanks and substrate blanks were used in all experiments. After incubation for 1 hr. at 37 C, 0.4 ml. of 0.1M glycine buffer was added and the preparations were centrifuged at 10,000 r.p.m. for 20 minutes. The p-nitrophenol released was read at 410 nm. and calculated against standard solutions of p-nitrophenol. Results of ph activity studies are expressed as per cent of maximum activity. Tissue fractionation. Freshly prepared homogenates in 0.25M sucrose-0.005m EDTA were fractionated by a modification of the procedure of de Duve and associates. 18 The homogenate was centrifuged at 0 C. for 6,000 g-minutes. The resulting sediment, which contained all of the pigment granules by visual inspection, was re-homogenied in sucrose-edta and centrifuged as above. The resulting sediment (PG+N) contained pigment granules, nuclei, and undoubtedly some cell debris. The supernatants were combined and centrifuged for 320,000 g-minutes. The resulting pellet (M+L) contained, according to present definitions, 18 ' 19 both heavy and light mitochondria, as well as lysosomes. The supernatant was then centrifuged at 0 C. in a Type 40 rotor (Beckman Model L-2 preparative ultracentrifuge) for 6,000,000 g-minutes to yield the microsomal pellet (R) and the cell supernatant (S). Definitions of the fractions as PG+N, M+L, R, and S should be considered as "operational," as they were not examined microscopically. Moreover, this is the first report, as far as is known, describing the subcellular fractionation of pigment epithelial cells. It is well known that the heterogeneous nature of all the cellular inclusion bodies precludes the isolation of either morphologically or biochemically pure fractions. 19 Hence, the procedure used here would be expected to yield subcellular fractions of approximately the same degree of purity as those isolated from other tissues. All fractions were assayed in 1 ml. of incubation mixture at ph 4.8 as described above. Protein was estimated by the Lowry procedure 14 and data are reported as specific activities of the enymes, i.e., millimicromoles of substrate hydrolysed per hour per milligram of protein. Results Optimum ph. The ph activity curves (Fig. 1) indicate a single maximum for N-acetyl-/?-glucosaminidase at ph 5, which is typical for this enyme. In contrast, /?-galactosidase exhibits a broad maximum from approximately ph 4 to 6 (within this range there is a small "shoulder" at ph 5.5 to 6.0). Similar activity curves have been observed in cell homogenates from many tissues and reflect the presence of at least two, possibly three, isoenymes of /8-galactosidase 20 with differing and
66 Berman Investigative Ophtlialmology January 1971 100 x LL O 50 < Fig. 1. Influence j3-galactosidase ( saminidase ( homogenates. ph ph on the activity of ) and N-acetyl-/?-glucoin pigment epithelial cell Table I. Release of acid hydrolases from the PG+N fraction of pigment epithelial cells Treatment PG+N fraction before treatment Triton X-100* Sonicationf Freeing-thawing \ overlapping ph optima. This suggests that pigment epithelial cell homogenates also contain varying proportions of the individual isoenymes, with different cell particulates contributing in an as yet undetermined proportion to the over-all activity. All further experiments on the subcellular distribution of both /?-galactosidase and N-acetyl-/?-glucosaminidase were performed at ph 4.8, notwithstanding the probable existence of different ph optima for the isoenymes, especially those of /?-galactosidase. Subcellular distribution. The distribution of the two acid hydrolases after differential centrifugation is shown in Fig. 2. It should be noted from the ordinate scales that all the cell fractions contain two to three times more N-acetyl-/?-glucosaminidase than ^-galactosidase. Considering the individual fractions, very little activity was found in either the R or S fractions. The latter indicates that little or no enymes had been released from the structural components during their isolation in the osmotically protected medium used. Most of the /3-galactosidase and N-ace- N-Acetyl-Pglucosaminidase Galactosidase Total activity (nifimoles of substrate hi) drolyed) 1,340 660 280 920 96 1,068 198 3,150 2,150 600 3,020 155 3,510 188 "Homogenied in 0.1 per cent Triton X-100 and centrifuged at 10,000 r.p.m. for 10 minutes. f9 Kc sonic oscillations for 90 seconds at 8 C. Centrifuged as above. tfroen at -50 C. and thawed at 6 to 8 C. six times. Centrifuged as above. tyl-/3-glucosaminidase activities were in the PG+N and M+L fractions. The specific activity in the latter was approximately twice as high as in the PG+N fraction. The enrichment of activity in the M+L fractions is in accord with the high concentration of acid hydrolases found in lysosomes in other tissues. However, the finding of so much activity in the PG+N fraction suggests some unique properties of these granules in pigment epithelial cells. Latency. It was of interest, therefore, to determine whether the enymes in the PG+N fraction displayed the same type of structure-linked latency that characteries these hydrolases in other tissues. The results of treatment with various agents known to induce osmotic shock are shown in Table I. Freeing-and-thawing, as well as sonication, were the most effective, resulting in the release of nearly all the activities in soluble form; Triton X-100 was somewhat less efficient. Whether the enymes are actually enclosed within the membranous sac observed on the surface of pigment granules 21 or whether they are
Volume 10 Number 1 Acid hydrolases in retinal pigment epithelium 67 p-galactosidase N-Acetyl-p-gluco&aminidase 100 h 200 r > > M+L h- < 50 P6 + N 100 u UJ Q. 0 L 0 50 100 % OF TOTAL PROTEIN 50 100 % OF TOTAL PROTEIN Fig. 2. Distribution of /?-galactosidase and N-acetyl-/?-glucosaminidase in subcellular fractions of pigment epithelial cell homogenates. Results are expressed according to de Duve and colleagues 18 and represent, on the ordinate, the mean specific activity of the enyme in the individual fraction. The abscissa gives the relative proportion of protein in the fractions, in order of their isolation. These are PG+N, M+L, R, and S (see text). nuclear in origin remains to be established. It is also possible that the observed activity may be associated with another type of inclusion body as yet unidentified that sediments together with the granules and nuclei in this tissue. Discussion Two hydrolytic enymes, /?-galactosidase and N-acetyl-/?-glucosaminidase, heretofore unknown in pigment epithelial cells, have been studied and their intracellular distribution determined. Both have optimum activity at acid ph values and almost no activity at neutral ph. The highest specific activity is found in the particulate fraction (M+L) sedimenting at 320,000 g-minutes. This fraction, by analogy with other tissues, should consist of a mixture of heavy and light mitochondria together with lysosomes. Further separation of these organelles is difficult to achieve by differential centrifugation alone. 19 Considerable activity is also associated with the PG+N fraction. These particles sediment at 6,000 g-minutes in.25m sucrose medium and it is reasonable to assume that the enymes are in a soluble form within one (or more) of the membrane-bound particles present in this fraction. This is based on their nearly quantitative release by agents that would be expected to rupture cell membranes. Of those tested, sonication and freeingand-thawing were the most effective; Triton X-100 was less effective. The present work is the first, as far as is known, describing the fractionation of the intracellular organelles of pigment epithelial cells. Although it has been tacitly assumed that most of the cell participates in pigment cells have the same sedimentation characteristics as their counterparts in other tissues, verification of this requires further investigation. On the basis of presently available morphologic evidence, the following organelles are present in these cells: nuclei, pigment granules, mitochondria, "lysosome-like particles," Golgi apparatus and endoplasmic reticulum (both smooth and rough). 3 ' * s > 10 ~ 12 Two other types of particles, apparently unique to pigment epithelial cells, have also been observed. One, a lipid inclusion body that probably represents retinol (Vitamin A) and its derivatives, has been termed "lipo-
68 Berman Investigative Ophthalmology January 1971 fuscin" 10 or "oil droplet." 8 The other has been termed a "lamellar inclusion body" 3 ' 4> 10-12 or "phagosome" s> 9 and may be a specialied kind of lysosomal particle in pigment epithelial cells. Their behavior under the conditions of differential centrifugation used in the present investigation could not be determined, i.e., the "lamellar inclusion bodies" or "phagosomes" could not be localied as such in the fractions isolated. It may indeed be very difficult to concentrate these particules in a single fraction because they appear to be small in number compared to other cell particulates and, in addition, are highly heterogeneous in sie and shape. 3 It is tempting, nevertheless, to speculate that the particles responsible for the phagocytosis of the outer rod segments are in fact lysosomal particles containing acid hydrolases similar to those found in other tissues. This point remains to be established. The author wishes to thank Prof. I. C. Michaelson for his continued interest and encouragement of this work and Mrs. Rosemarie Rut for expert technical assistance. REFERENCES 1. Dowling, J. E.: Chemistry of visual adaptation in the rat, Nature 188: 114, 1960. 2. Krinsky, N. I.: The enymatic esterification of vitamin A, J. Biol. Chem. 232: 881, 1958. 3. Dowling, J. E., and Gibbons, I. R.: The fine structure of the pigment epithelium in the albino rat, J. Cell Biol. 14: 459, 1962. 4. Bairati, A., Jr., and Oralesi, N.: The ultrastructure of the pigment epithelium and of the photoreceptor-pigment epithelium junction in the human retina, J. Ultrastruct. Res. 9: 484, 1963. 5. Meier-Ruge, W.: The pathophysiological morphology of the pigment epithelium and its importance for retinal structures and functions, Mod. Prob. Ophthal. Basel/New York, 1968, S. Karger AG 8: 32. 6. Young, R. W., and Dro, B.: The renewal of protein in retinal rods and cones, J. Cell Biol. 39: 169, 1968. 7. Herron, W. L., Riegel, B. W., Myers, O. E., and Rubin, M. L.: Retinal dystrophy in the rat A pigment epithelial disease, INVEST. OPHTHAL. 8: 595, 1969. 8. Young, R. W., and Bok, D.: Autoradiographic studies on the metabolism of the retinal pigment epithelium, INVEST. OPHTHAL. 9: 524, 1970. 9. Bok, D., and Hall, M. O.: The etiology of retinal dystrophy in RCS rats, INVEST. OPHTHAL. 8: 649, 1969. (Abst.) 10. Kroll, A. J., and Machemer, R.: Experimental retinal detachment in the owl monkey. III. Electron microscopy of retina and pigment epithelium, Amer. J. Ophthal. 66: 410, 1968. 11. Kroll, A. J., and Machemer, R.: Experimental retinal detachment in the owl monkey. V. Electron microscopy of the reattached retina, Amer. J. Ophthal. 67: 117, 1969. 12. Braekevelt, C. R., and Hollenberg, M. J.: Development of the retinal pigment epithelium, choriocapillaris and Bruch's membrane in the albino rat, Exp. Eye Res. 9: 124, 1970. 13. Tappel, A. L.: Lysosomal enymes and other components, in Dingle, J. T., and Fell, H. B., editors: Lysosomes in biology and pathology, Vol. 2, Amsterdam/London, 1969, North- Holland Publishing Co., p. 207. 14. Berman, E. R., and Bach, C: The acid mucopolysaccharides of cattle retina, Biochem. J. 108: 75, 1968. 15. Bach, C, and Berman, E. R.: Characteriation of a sialoglycan isolated from cattle retina, Ophthal. Res. In press. 16. Berman, E. R.: The biosynthesis of mucopolysaccharides and glycoproteins in pigment epithelial cells of bovine retina, Biochim. Biophys. Acta 83: 371, 1964. 17. Hers, H. C, and van Hoof, F.: Genetic abnormalities of lysosomes, in Dingle, J. T., and Fell, H. B., editors: Lysosomes in biology and pathology, Vol. 2, Amsterdam/ London, 1969, North-Holland Publishing Co., p. 19. 18. De Duve, C, Pressman, B. C, Gianetto, R., Wattiaux, R., and Appelmans, F.: Tissue fractionation studies. VI. Intracellular distribution patterns of enymes in rat-liver tissues, Biochem. J. 60: 604, 1955. 19. Beaufay, H.: Methods for the isolation of lysosomes, in Dingle, J. T., and Fell, H. B., editors: Lysosomes in biology and pathology, Vol. 2, Amsterdam/London, 1969, North-Holland Publishing Co., p. 515. 20. Asp, N.-G., and Dahlqvist, A.: Rat smallintestinal /?-galactosidases. Kinetic studies with three separated fractions, Biochem. J. 110: 143, 1968. 21. Shearer, A. C. I.: Morphology of the isolated pigment particle of the eye by scanning electron microscopy, Exp. Eye Res. 8: 122, 1969.