Exl’. E,ve Rrs. (1955) 21, 307-313
Distribution and Some Properties of Cathepsin D in the Retinal Pigment Epithelium SEIJI HAYASAKA, Ueprtme~~t
SATOSHI HARA AND KATSUVOSHI Mczv~o
of Ophthalmology,
Tohoku University, Sena?& 980, Jnp~
School of Me&&e,
(Received 28 Ja~wuary 1975, Boston) Cathepsin D in the retinal pigment epithelium of the bovine eye was studied using standard enzymological techniques. In the bovine eye the retinal pigment epithelium revealed the highest) specific activity, the activity being highest in the lysosomal fraction. The enzyme to both activity in the lysosomal extract of the retinal pigment epithelium was proportional the enzyme concentration and incubation time. \Vhen the enzyme was heated, the enzyme activity was lost completely. The initial rate of the tyrosine release was dependent on the concentration of t,he bovine serum albumin, exhibiting a hyperbolic saturation curve. The optimal pH of the enzyme was about 4.0. The enzyme act,lvit,y was not affected by tbiol agents.
1. Introduction In a previous paper, Hayasaka (1974) demonstrated that using acid phosphatase antt P-glucuronidase as lysosome markers, the most significant activity of lysoson~al enzymes was found in the uvea and the retina. Acid proteases in animal tissues. mamely cathepsins, have also been used as lysosome markers in other tissues (My&. 1970; Greenbaum. 1971). Proteins and peptides enveloped in phagolysosomes are llydrolyzed by cathepsins. Even cells or tissues can be affected when the lysosomes burst under some conditions. Degradation or turnover of proteins stch as removal of rod outer segments within the pigment epit,helial cell and pathological processes such as choroidal atrophy or pigmentary retinal dystrophy cannot be discussed without considering lysosomal cathepsins. c!at,hepsins have been studied in various organs of animals, and designated cathepsin A. 8. C: D and E: according to the substrate specificity or optimal pH (Mycek, 1970 ; Greenbaum, 1971). Cathepsin D and E split only proteins but not synthetic substrates. The optimal pH of cathepsin D is 3.0-4.5. There are a few reports in the literature of thest enzymes acting on proteins in ocular tissue (Vent0 and Cacioppo, 1973; hrya, illannagh and Irvine, Jr, 1972), but the properties of the enzyme,s have not. been well characterized. The present paper, therefore. deals with the distribution and some properties of cathepsin D in order to clarify its role in cell physiology and pathology in the ret’inal pigment epithelium. 2. Materials and Methods Bovine serum albumin was obtained from Sanka Pure Chemical Co. ; Folio-Cioealteu reagent and cl-nitroso-p-naphthol and glutathioue from Wako Pure Chemical Co. : tyrosine and cysteine from Byowa Hal&o Kogyo Co. : /3-mercaptoethanol from Nakarai Chemical CO.
The bovine eye was maintained at 4’C from the time of slaughter. The cornea. :IC~UWIIW humor, iris plus ciliary body, lens, vit)reous body and neuro-sensory retina were JIWJNLWI ill the same way as described previously (Hayasaka, 1974). Th e retinal pigment epitheliutu was suspended, after removing the neurosensory retina, by carefully washing the iriside of the eyeball with small amounts of ice-cold 0.25 >I-sucrose containing 0.02 >I-Trip- HC”I buffer (pH 7.4), and centrifuged at IlO;cg for 10 min. The pellet was mashed four titncs with 0*25 >I-sucrose containing 0.02 ar-Tris-HCl buffer (pH 7.4). The final pellet, WJ usecl BS the retinal pigment epithelium. Aft’er removing the retinal pigment epitheliunl. the remaining pigmented tissue was used as the choroid. Tissue homogenization was performed using a Waring blender. Specific enzyme activities of ocular tissues were measured in homogenates after dialysis against, 041 mr-potnssiun I phosphate buffer (pH 7.4) following t,he addition of Triton X-1OU to a final concent,ration of 0.1%. In order to measure t,he specific activity of subcellular fractions, the homogenate of the retinal pigment epithelium was made using a Potter-Elverhjem homogenizer in 0.25 &f-sucrose containing 0*02 ;\l-Tris-HCl buffer (pH 7.4). Subcellular fractions were prepared from the homogenate by centrifugation as described previously (Hayasaka, 1974). . Specific enzyme actlvltles of the subcellular fractions were measured after dialysis against 0.01 bI-potassium phosphate buffer (pH 7.4) f o11owing the addition of Triton S -100 to :I final concentration of WlO,. In order to aa~!- the enzyme activity of the Illelrl~)~:rtle-f1.e~ lysosomal fraction from the retinal pigment epit.helium, the lysoxor~~al fract,iotl WI!: t’rozert and t.hawed seven times; after removal of the membranes. the (:leitr superntititnt VXP use11 as the lysosomal extract. ,111manipulations mere done at O-4°C. Standard assay of cathepsin D Cathepsin D [E.C. 3.4.23.51 activity was determined by a modification of t,he method described by Burden, Yates and Reading (1971). Sampl es of the enzyme solutiorl (O.l-O-3 ml) were incubated in a total volume of 1 ml for 1 hr at 37°C in 0.1 ~~-citrate--sclcliunl citrate buffer at pH 4.0 with bovine serum albumin (20 mg/ml) as substrate. Albumin blanks, which contained no enzyme, u-ere treated likewise. The reaction was stopped bp acid. Tissue blanks were prepared by adding trladding 1 ml of 10 96 trichloroacetic chloroacetic acid immediately after addition of the enzyme to the medium. The precipitated protein was removed by centrifugation, and the tyrosme released was determined spectrophotometrically by the method of Ceriotti and Spandrio (1957). Optical density at ,510 nm was read using a Hitachi 101 spectrophotometer and values compared with those obtained using concentrations of authent,ic t.yrosine. Protein detemzin.ation~ Protein content Randall (1951).
was determined
by the method
of Lowry,
Rosebrough,
Farr
and
3. Results By this preparation of the retinal pigment epithelium, the material obtained was suitable histologically (Fig. I). The distribution of cathepsin D in ocular tissues is shown in Table I. The standard deviation of each value was less than lO’$& respectively. The vitreous body revealed no enzyme activity. The cornea and the lens exhibited a low enzyme activity. On the other hand, the retina and the uvea showed relatively high enzyme activity. The most significant specific activity was found in the retinal pigment epithelium. No
CATHEPSIN
D IN
THE
PIGMENT
309
EPITHELIUM
enzyme activity was detected in bovine blood by this assay method, although its presence in white blood cells has been reported (Lapresle and Webb, 1962). Hence, it is apparent that enzyme activities determined in the present study are not due to blood contamination.
i . .
. FIG. 1. Ketinal
pigment epithelium
preparation.
Hematoxylin-eosin
staining.
In the subcellular fractions of the retinal pigment epithelinm, t,he specific activity was highest in the lysosomal fraction, sedimenting 400 000 x g/min (Fig. 2). The enzyme activity in the lysosomal extract of the retinal pigment epithelium was proportional
Distribution
of cnthgsin
C~~rIEa Aqueous humor Iris and ciliary body Lens Vitreous body Seurosensory retina Retinal pigment epithelium Clhoroitl
D in the hmogenclte
of the eyf timues
310
S. HdYASAKiA,
S. HARA
AA-I)
Ii. UI%l.NO
to both enzyme concentration (Fi g. 3) and incut)ation time (Fig, 4). \\‘tren the (~i~zyit1~’ was heated at 100°C for 2 min the enzyme wtivit? was lost completely (Fig. .4). ‘I’hra initial rate of the tyrosine release was dependent on bhe concentration of that twvi w serum albumin; its satnration curve WLShyperbolic (Fig. 5).
Per cent of total protein
FIG. 3. Distribution of cathepsin D in subcellular fractions of itnd enzyme assay were as described in Materials and Methods. fraction; Lys, lysosomal fraction; MC, microsomal fraction ; S, sented in the ordinate by its specific activity. On the abscissa, centage of protein.
retinal pigment epithelium. Preparation IL’. nuclear fraction; &It, mitochondrial soluble fraction. Each fraction is rcprceach fraction is represented by its per-
These data strongly suggested that tyrosine released from the bovine serum albumin under incubation conditions proceeded as an enzymic reaction. The optimal pH of the enzyme was about 4.0 (Fig. 6), which agreed with the value of cathepsin D obtained in other tissues (Greenbaum, 1971). It has been reported that the optimal pH of cathepsin A, B, C’and E are 5.0.4.0-6.5, 5.0 and ‘2.5. respectively (Mycek, 1970 ;
Enzyme concentration (mq protein)
FIG. 3. Enzyme activity and enzyme concentration. Varied amounts of the enzyme which were prepared from the lysosomal extract of the retinal pigment epithelium were incubated at 37°C’ for 30 min.
FIG. 4. Enzyme activity and incubation time. 0.9 mg protein of lysosomal extract of the retinal pigment epithelium was incubated at 37°C at pH 4.0. O-0, Lysosomal extract untreated; 0-0, lysosomal extract which was heated at 100°C for 2 min.
CATHEPSIN
D IN
THE
PIGMEKT
311
EPITHELIUN
E, 0.20 i? G 0.15 0 0 z 0.10 z 2 E 0.05 El u, P i c 0
20
40
60
Concentration of bovine serum olbumln krq)
PIG. 5. Relationship between the concentration of the bovine serum albumin and the initial rate of t.yrosine release. 1.8 mg Protein of lysosomal extract of the retinal pigment epithelium was incubated at 37°C for 5 min st pH 4.0.
z c 0 E
z z 1 2 2 E c 15
0.8
0.6 0.4 0.2 0
2345670
FIG. 6. Effect of pH on cathepsin D activity. 0.7 mg protein of lysosomal extract of the retinal epit~helium was incubated at 37°C for 60 min. 0.3 M-citrate-sodium citrate buffer was used.
TABLE
11
Effect of thiol agents, metal iorz or detergent 011cathepsin D actidy the lysosomal extract of retinal pigment epithelium
Xddit~ion
from
Relative sctivitv (76)
under incubation
---1u04
8.mercaptoethanol
lo-" 10.-'iI
PCBIB JlgSO* E DTA Triton
10 -4 10 -6 IO-1 10 -6 10-4
X-100
lOk3 10-S 10-a 10-6 O.lY10
k5.6 !JY,S x9.5
1014 82.2 934 110.1 88.6 96d.J
113.1 109~1 rJ6.o
0.75 mg Protein of lysosomal extract was incubstc,d at 37°C for 60 min with or without
the addition.
31”
S. HAYASAKA.
S. H.4IlP-a XXI)
K. JlI%~SO
Greenbaum, 1971 ; &latsuda and Misake, 19i4). The enzyme activity was not, atit~ctecl by cysteine, glutathione, P-mercaptoethanol. p-chloromercuribenzoic acid. rnagnc:hiun: sulfate, ethylenediamine tetra-acetic acid, or Triton X- 100 (Table II). It ha5 Iwn reported that cathepsin B and C’ are activated by thiol agems (Jlycek. 1970: Greenbaum, 1971). Consistent wit,11the data obtained in the reticuloentlothelial system by (treenbuurn (1971), cathepsin D in the retinal pigment epithelium was not affected l)y thiol agems. 4. Discussion Prom the present results, it appears that the distribution of cathepsin -II in the bovine eye is similar to that of acid phosphatase and P-glucuronidase (Hayasaka, 1974), and that the retinal pigment epithelium exhibits a high specific activity of cathepsin D. Cathepsin D and E are the major catheptic endopeptidases, especiall! in the reticuloendothelial system. Cathepsin A. B and C seem to have only exopeptidase activity in that they attack either the carboxyl terminus or the amino terminus of protein. The existence of isozymes of cathepsin D has been reported in various organs (Natsuda and Nisaka, 1974); it is conceivable that cathepsin D may act at the first step of protein degradation in the cell. Young (1967) reported that the photoreceptor outer segment was continuously renewed at its base, in conjunction with a balanced removal of material at its apex. Ishikawa and Tamada (1970) suggested that the pigment epithelial cell of rat retina played an active role in the intracellular degradation of engulfed outer segment, removed from its apex. Since then, the highly developed phagolysosomal system of the pigment epithelium has received increasing attention, and acid hydrolases of the pigment epithelium has been studied by several aut,hors. Berman (1971) showed P-galactosidase and N-acetyl-/3-glucosaminidase in pigment epithelial cell. The proteases utilized in the hydrolysis of degenerating rhodopsin molecules have not, been characterized as yet. Feeney (1973) pointed out that the phagolysosomal system of the pigment epithelium might be a key to retinal disease, while Srya, Mannagh and Irving, Jr (1972) demonstrated that detachment of cornea1 endothelium cells in tissue culture was due to cathepsin, but not to p-glucuronidase. aryl sulfatase or acid phosphatase. There is thus a strong possibility that cathepsin D may play a role in the hydrolysis of rhodopsin in the pigment epithelium and even in protein hydrolysis of neighboring tissues under some pathological conditions. Berman, Schwell and Feeney (1974) indicated the high content of retinol in the retinal pigment epithelium, whilst Vento and Cacioppo (1973) demonstrated that acid phosphatase, /3-glucuronidase and cathepsins were released through the action of retinol on the pigment epithelium. It is therefore possible that lysosomal enzymes may participate in t,he etiology of pigmentary retinal dystrophy. Ansell and Marshall (1974) reported the presence and distribution of extra-cellular acid phosphatase in the retinas of retinitis pigmentosa rats. Burden, Yates and Reading (1971) and Herron, Riegel and Myers (1969) proposed that some abnormality of the photoreceptor renewal-removal mechanism by the pigment epithelium could account for retinal dystrophies. It is requisite to study whether cathepsin D in the retinal pigment epithelium specifically hydrolyzes rodopsin and other rod outer segment proteins.
(‘ATHEPSIK
D IN
THE
PIC:NEST
EPITHELIVM
3 13
ACKNOWLEDGNENTS
The authors thank Xr K. Sate for his technicwl assistance. REFERESCES ht~sell. I’. L. and Marshall, J. (1974). The distribut’ion of extra-cellular acid phosphat’ase in the retinas of retinitis pigmentosa rats. Exp. Eye Kes. 19, ‘373. AIrya. I). V.. Mannagh. J. and Irvine, Jr, A. R. (1972). Effect of lysosomes on cultured rabbit cornea1 mdothelial cells. Invest. OphthaZmoZ. 11, 66% Berman, E. R., Schwell, H. and Feeney, L. (1974). The retina.1 pigment epithelium-chemical composition and structure. Invest. Ophthnlmol. 13, 675. Berma,n, E. R. (1971). Acid hydrolases of the retinal pigment epithelium. Inrenf. Ophthnlrwl.
10, 6-l. Burden. E. M., Yates, C. M. and Reading, H. W. (1971). Investigation into the structural integrity of lysosomes in the normal and dystrophic retina. Es-p. Q/e Kes. 19, 1273. t’eriotti. G. and Spandrio, L. (1957). Calorimetric determination of tyrosine. Rio&em. 1.66, 6~7. Feeney. I,. (1973). The phagolysosomal system of the pigment epithrlium, a key to retinal disease. ItLwst.
Ophthnlrnol.
12, 635.
(:reenbarrm, L. 11. (1971). Cathepsins and kinin-forming and -dest,roying enzymes. The E/qn/rr (Ed. P. D. Boper). Vol. 3, p. 475. Academic Press, Sex York and London. Hapssaka, S. (1974). Distribution of lysosomal enzymes in the bovine eye. Jnp. J. Ophtholnrol.
18, %33. Herron. LV. L., Biegel, B. IV\‘. and Myers, D. E. (1969). Retinal dystrophy in the rat--a pigment epithelial disease. Invest. Ophthalmol. 8, 595. lshikawa. T. and Yamada, E. (1970). The degradation of the phot,orereptor outer segment within the pigment epithelial cell of rat retina. J. Elrc.. Microsc. 19, 85. Laprwlr and Webb (1962). The purification and properties of a proteolytic enzyme. rabbit ctathepsin 1: and further studies on rabbit cathepsin D. Hiochem. J. 84, 455. Ir,nry. 0. H.. Rosebrough. N. J., Farr, A. L. and Randall. R. .J. (1951). Protein measurement \vith the Folin phenol reagent. J. Biol. Chem. 193, 265. Ptlats\lda, K. and Misaka, E. (1974). Studies on cathrpsins of rat liver lpsosome. 1. pwification and multiple forms. J. Biochem. 76, 639. Myce!;. 31. J. (1970). Cathepsins. Nefhods in EnzymoZogy (Ed. Gertrude, E. P. and Laszlo. L.J. Vol. 19, p. 685. Academic Press, Kew York and London. Vento, K. and Cacioppo. F. (1973). The effect of retinol on the lysosomal enzymes of hovine retina and pigment epithelium. Esp. Eye Res. 15, 43. Young. tt. \V. (1967). The renewal of photoreceptor cell outer segments. J. C’eZZBiol. 33, (il.
Distribution and Some Properties of Cathepsin D in the Retinal Pigment Epithelium SEIJI HAYASAKA, Ueprtme~~t
SATOSHI HARA AND KATSUVOSHI Mczv~o
of Ophthalmology,
Tohoku University, Sena?& 980, Jnp~
School of Me&&e,
(Received 28 Ja~wuary 1975, Boston) Cathepsin D in the retinal pigment epithelium of the bovine eye was studied using standard enzymological techniques. In the bovine eye the retinal pigment epithelium revealed the highest) specific activity, the activity being highest in the lysosomal fraction. The enzyme to both activity in the lysosomal extract of the retinal pigment epithelium was proportional the enzyme concentration and incubation time. \Vhen the enzyme was heated, the enzyme activity was lost completely. The initial rate of the tyrosine release was dependent on the concentration of t,he bovine serum albumin, exhibiting a hyperbolic saturation curve. The optimal pH of the enzyme was about 4.0. The enzyme act,lvit,y was not affected by tbiol agents.
1. Introduction In a previous paper, Hayasaka (1974) demonstrated that using acid phosphatase antt P-glucuronidase as lysosome markers, the most significant activity of lysoson~al enzymes was found in the uvea and the retina. Acid proteases in animal tissues. mamely cathepsins, have also been used as lysosome markers in other tissues (My&. 1970; Greenbaum. 1971). Proteins and peptides enveloped in phagolysosomes are llydrolyzed by cathepsins. Even cells or tissues can be affected when the lysosomes burst under some conditions. Degradation or turnover of proteins stch as removal of rod outer segments within the pigment epit,helial cell and pathological processes such as choroidal atrophy or pigmentary retinal dystrophy cannot be discussed without considering lysosomal cathepsins. c!at,hepsins have been studied in various organs of animals, and designated cathepsin A. 8. C: D and E: according to the substrate specificity or optimal pH (Mycek, 1970 ; Greenbaum, 1971). Cathepsin D and E split only proteins but not synthetic substrates. The optimal pH of cathepsin D is 3.0-4.5. There are a few reports in the literature of thest enzymes acting on proteins in ocular tissue (Vent0 and Cacioppo, 1973; hrya, illannagh and Irvine, Jr, 1972), but the properties of the enzyme,s have not. been well characterized. The present paper, therefore. deals with the distribution and some properties of cathepsin D in order to clarify its role in cell physiology and pathology in the ret’inal pigment epithelium. 2. Materials and Methods Bovine serum albumin was obtained from Sanka Pure Chemical Co. ; Folio-Cioealteu reagent and cl-nitroso-p-naphthol and glutathioue from Wako Pure Chemical Co. : tyrosine and cysteine from Byowa Hal&o Kogyo Co. : /3-mercaptoethanol from Nakarai Chemical CO.
The bovine eye was maintained at 4’C from the time of slaughter. The cornea. :IC~UWIIW humor, iris plus ciliary body, lens, vit)reous body and neuro-sensory retina were JIWJNLWI ill the same way as described previously (Hayasaka, 1974). Th e retinal pigment epitheliutu was suspended, after removing the neurosensory retina, by carefully washing the iriside of the eyeball with small amounts of ice-cold 0.25 >I-sucrose containing 0.02 >I-Trip- HC”I buffer (pH 7.4), and centrifuged at IlO;cg for 10 min. The pellet was mashed four titncs with 0*25 >I-sucrose containing 0.02 ar-Tris-HCl buffer (pH 7.4). The final pellet, WJ usecl BS the retinal pigment epithelium. Aft’er removing the retinal pigment epitheliunl. the remaining pigmented tissue was used as the choroid. Tissue homogenization was performed using a Waring blender. Specific enzyme activities of ocular tissues were measured in homogenates after dialysis against, 041 mr-potnssiun I phosphate buffer (pH 7.4) following t,he addition of Triton X-1OU to a final concent,ration of 0.1%. In order to measure t,he specific activity of subcellular fractions, the homogenate of the retinal pigment epithelium was made using a Potter-Elverhjem homogenizer in 0.25 &f-sucrose containing 0*02 ;\l-Tris-HCl buffer (pH 7.4). Subcellular fractions were prepared from the homogenate by centrifugation as described previously (Hayasaka, 1974). . Specific enzyme actlvltles of the subcellular fractions were measured after dialysis against 0.01 bI-potassium phosphate buffer (pH 7.4) f o11owing the addition of Triton S -100 to :I final concentration of WlO,. In order to aa~!- the enzyme activity of the Illelrl~)~:rtle-f1.e~ lysosomal fraction from the retinal pigment epit.helium, the lysoxor~~al fract,iotl WI!: t’rozert and t.hawed seven times; after removal of the membranes. the (:leitr superntititnt VXP use11 as the lysosomal extract. ,111manipulations mere done at O-4°C. Standard assay of cathepsin D Cathepsin D [E.C. 3.4.23.51 activity was determined by a modification of t,he method described by Burden, Yates and Reading (1971). Sampl es of the enzyme solutiorl (O.l-O-3 ml) were incubated in a total volume of 1 ml for 1 hr at 37°C in 0.1 ~~-citrate--sclcliunl citrate buffer at pH 4.0 with bovine serum albumin (20 mg/ml) as substrate. Albumin blanks, which contained no enzyme, u-ere treated likewise. The reaction was stopped bp acid. Tissue blanks were prepared by adding trladding 1 ml of 10 96 trichloroacetic chloroacetic acid immediately after addition of the enzyme to the medium. The precipitated protein was removed by centrifugation, and the tyrosme released was determined spectrophotometrically by the method of Ceriotti and Spandrio (1957). Optical density at ,510 nm was read using a Hitachi 101 spectrophotometer and values compared with those obtained using concentrations of authent,ic t.yrosine. Protein detemzin.ation~ Protein content Randall (1951).
was determined
by the method
of Lowry,
Rosebrough,
Farr
and
3. Results By this preparation of the retinal pigment epithelium, the material obtained was suitable histologically (Fig. I). The distribution of cathepsin D in ocular tissues is shown in Table I. The standard deviation of each value was less than lO’$& respectively. The vitreous body revealed no enzyme activity. The cornea and the lens exhibited a low enzyme activity. On the other hand, the retina and the uvea showed relatively high enzyme activity. The most significant specific activity was found in the retinal pigment epithelium. No
CATHEPSIN
D IN
THE
PIGMENT
309
EPITHELIUM
enzyme activity was detected in bovine blood by this assay method, although its presence in white blood cells has been reported (Lapresle and Webb, 1962). Hence, it is apparent that enzyme activities determined in the present study are not due to blood contamination.
i . .
. FIG. 1. Ketinal
pigment epithelium
preparation.
Hematoxylin-eosin
staining.
In the subcellular fractions of the retinal pigment epithelinm, t,he specific activity was highest in the lysosomal fraction, sedimenting 400 000 x g/min (Fig. 2). The enzyme activity in the lysosomal extract of the retinal pigment epithelium was proportional
Distribution
of cnthgsin
C~~rIEa Aqueous humor Iris and ciliary body Lens Vitreous body Seurosensory retina Retinal pigment epithelium Clhoroitl
D in the hmogenclte
of the eyf timues
310
S. HdYASAKiA,
S. HARA
AA-I)
Ii. UI%l.NO
to both enzyme concentration (Fi g. 3) and incut)ation time (Fig, 4). \\‘tren the (~i~zyit1~’ was heated at 100°C for 2 min the enzyme wtivit? was lost completely (Fig. .4). ‘I’hra initial rate of the tyrosine release was dependent on bhe concentration of that twvi w serum albumin; its satnration curve WLShyperbolic (Fig. 5).
Per cent of total protein
FIG. 3. Distribution of cathepsin D in subcellular fractions of itnd enzyme assay were as described in Materials and Methods. fraction; Lys, lysosomal fraction; MC, microsomal fraction ; S, sented in the ordinate by its specific activity. On the abscissa, centage of protein.
retinal pigment epithelium. Preparation IL’. nuclear fraction; &It, mitochondrial soluble fraction. Each fraction is rcprceach fraction is represented by its per-
These data strongly suggested that tyrosine released from the bovine serum albumin under incubation conditions proceeded as an enzymic reaction. The optimal pH of the enzyme was about 4.0 (Fig. 6), which agreed with the value of cathepsin D obtained in other tissues (Greenbaum, 1971). It has been reported that the optimal pH of cathepsin A, B, C’and E are 5.0.4.0-6.5, 5.0 and ‘2.5. respectively (Mycek, 1970 ;
Enzyme concentration (mq protein)
FIG. 3. Enzyme activity and enzyme concentration. Varied amounts of the enzyme which were prepared from the lysosomal extract of the retinal pigment epithelium were incubated at 37°C’ for 30 min.
FIG. 4. Enzyme activity and incubation time. 0.9 mg protein of lysosomal extract of the retinal pigment epithelium was incubated at 37°C at pH 4.0. O-0, Lysosomal extract untreated; 0-0, lysosomal extract which was heated at 100°C for 2 min.
CATHEPSIN
D IN
THE
PIGMEKT
311
EPITHELIUN
E, 0.20 i? G 0.15 0 0 z 0.10 z 2 E 0.05 El u, P i c 0
20
40
60
Concentration of bovine serum olbumln krq)
PIG. 5. Relationship between the concentration of the bovine serum albumin and the initial rate of t.yrosine release. 1.8 mg Protein of lysosomal extract of the retinal pigment epithelium was incubated at 37°C for 5 min st pH 4.0.
z c 0 E
z z 1 2 2 E c 15
0.8
0.6 0.4 0.2 0
2345670
FIG. 6. Effect of pH on cathepsin D activity. 0.7 mg protein of lysosomal extract of the retinal epit~helium was incubated at 37°C for 60 min. 0.3 M-citrate-sodium citrate buffer was used.
TABLE
11
Effect of thiol agents, metal iorz or detergent 011cathepsin D actidy the lysosomal extract of retinal pigment epithelium
Xddit~ion
from
Relative sctivitv (76)
under incubation
---1u04
8.mercaptoethanol
lo-" 10.-'iI
PCBIB JlgSO* E DTA Triton
10 -4 10 -6 IO-1 10 -6 10-4
X-100
lOk3 10-S 10-a 10-6 O.lY10
k5.6 !JY,S x9.5
1014 82.2 934 110.1 88.6 96d.J
113.1 109~1 rJ6.o
0.75 mg Protein of lysosomal extract was incubstc,d at 37°C for 60 min with or without
the addition.
31”
S. HAYASAKA.
S. H.4IlP-a XXI)
K. JlI%~SO
Greenbaum, 1971 ; &latsuda and Misake, 19i4). The enzyme activity was not, atit~ctecl by cysteine, glutathione, P-mercaptoethanol. p-chloromercuribenzoic acid. rnagnc:hiun: sulfate, ethylenediamine tetra-acetic acid, or Triton X- 100 (Table II). It ha5 Iwn reported that cathepsin B and C’ are activated by thiol agems (Jlycek. 1970: Greenbaum, 1971). Consistent wit,11the data obtained in the reticuloentlothelial system by (treenbuurn (1971), cathepsin D in the retinal pigment epithelium was not affected l)y thiol agems. 4. Discussion Prom the present results, it appears that the distribution of cathepsin -II in the bovine eye is similar to that of acid phosphatase and P-glucuronidase (Hayasaka, 1974), and that the retinal pigment epithelium exhibits a high specific activity of cathepsin D. Cathepsin D and E are the major catheptic endopeptidases, especiall! in the reticuloendothelial system. Cathepsin A. B and C seem to have only exopeptidase activity in that they attack either the carboxyl terminus or the amino terminus of protein. The existence of isozymes of cathepsin D has been reported in various organs (Natsuda and Nisaka, 1974); it is conceivable that cathepsin D may act at the first step of protein degradation in the cell. Young (1967) reported that the photoreceptor outer segment was continuously renewed at its base, in conjunction with a balanced removal of material at its apex. Ishikawa and Tamada (1970) suggested that the pigment epithelial cell of rat retina played an active role in the intracellular degradation of engulfed outer segment, removed from its apex. Since then, the highly developed phagolysosomal system of the pigment epithelium has received increasing attention, and acid hydrolases of the pigment epithelium has been studied by several aut,hors. Berman (1971) showed P-galactosidase and N-acetyl-/3-glucosaminidase in pigment epithelial cell. The proteases utilized in the hydrolysis of degenerating rhodopsin molecules have not, been characterized as yet. Feeney (1973) pointed out that the phagolysosomal system of the pigment epithelium might be a key to retinal disease, while Srya, Mannagh and Irving, Jr (1972) demonstrated that detachment of cornea1 endothelium cells in tissue culture was due to cathepsin, but not to p-glucuronidase. aryl sulfatase or acid phosphatase. There is thus a strong possibility that cathepsin D may play a role in the hydrolysis of rhodopsin in the pigment epithelium and even in protein hydrolysis of neighboring tissues under some pathological conditions. Berman, Schwell and Feeney (1974) indicated the high content of retinol in the retinal pigment epithelium, whilst Vento and Cacioppo (1973) demonstrated that acid phosphatase, /3-glucuronidase and cathepsins were released through the action of retinol on the pigment epithelium. It is therefore possible that lysosomal enzymes may participate in t,he etiology of pigmentary retinal dystrophy. Ansell and Marshall (1974) reported the presence and distribution of extra-cellular acid phosphatase in the retinas of retinitis pigmentosa rats. Burden, Yates and Reading (1971) and Herron, Riegel and Myers (1969) proposed that some abnormality of the photoreceptor renewal-removal mechanism by the pigment epithelium could account for retinal dystrophies. It is requisite to study whether cathepsin D in the retinal pigment epithelium specifically hydrolyzes rodopsin and other rod outer segment proteins.
(‘ATHEPSIK
D IN
THE
PIC:NEST
EPITHELIVM
3 13
ACKNOWLEDGNENTS
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