Distribution of acid lipase in the bovine retinal pigment epithelium

E.rl~. &ye Rm. (1977) 24, l-6

Distribution of Acid Lipase in the Bovine Retinal Pigment Epithelium

(Received 2 March

1976, Xew

York)

Acid lipase in the retinal pigment epit,helium of the bovine eye was studied biochemically rising triglyceride as substrate. The retinal pigment epithelium revealed the highest specific activity, the activity being highest in the lysosomal fraction. The lysosomal distribution of’ acid lipase was supported by the evidence that other hydrolytic enzymes were also localized \+ith highest specific activity in the lysosomal fraction prepared by differential pelleting. The optimal pH of the enzyme was 4-5. Acid lipase in the retinal pigment epithelium had maximum activity with glycerol tridecanoate as subst,rate. Specific activity of ret.inal pigment epit,helium was about lo-fold higher than that of liver. The possible role of a.cid lipase in the retinal pigment epithelium was discussed.

1. Introduction Phagolvsosomal systems in the retinal pigment epithelium (RPE) play a significant role in-the degradation of engulfed rod outer segment (ROS) discs (Young, 1967: lshikawa and Yamada, 1970). Several acid hydrolases in the RPE have been biochemically studied and characterized to be localized mainly in lysosomes; /Igalactosidase (EC 3.2.1.23) and N-acetyl-/%glucosaminidase (EC 3.2.1.30) were demonstrated by Berman (1971), phospholipase A, and A, (EC 3.1.1.4; 3.1.1.5) b! Swartz and Mitchell (1973), and eathepsin D (EC 3.4.23.5) by Hayasaka, Hara and Mizuno (1975). Rrcently Rothman, Feeney and Berman (1976) reported on the acid lipase of the RPE. and emphasized that the enzyme was localized mainly in the cytosol. They. however, used a synthetic fluorescent compound, 4-methylumbelliferyl(4-MU)-palmitate assubstrate for estimation of the enzyme activity. Since acid lipase (EC 3.1.1.3) 1by definition catalyzes the hydrolysis of glycerol-ester bonds, triacylglycerol is normally used as substrate for determination of the enzyme activity. WC, therefore, studied the distribution of acid lipase in the RPE using triglyceride as s&trate. 2. Materials and Methods All common chemicalsused in the preeent experiments were reagent grade. Glycerol triglycerides

were obtained

from Eastman.

The isolation, homogenization and subcellular fractionation of the bovine eye tissues were performed in the same way as described previously (Hayasaka, 1974a). RPE cells were brushed out of the eye cup in 0.25 M-sucrose and washed four times with centrifugation at 1lOxg for 10 min. The isolated materials were examined histologically. Homogenization was made using a Potter-Elvehjem homogenizer. Subcellular fractions were prepared from the homogenate by differential pelleting (Hayasaka et al., 1975). The liver, kidney and blood were homogenized using a Waring blender. A

1

Triglyceride dispersions were prepared by a slight modification of the methocl tlrscril~tl by Hayase and Tappel (1969); all of the suhptrates uPed were tlisperseci in l”,, !l’rit OII .\-I~I~ 1 instead of 10°/A gum arabic solution, so that the final concentration WHS 25 nlb~. Assay

of enzymes

Acid lipase (EC 3.1.1.3) lipolytic activity was measured by a slight modification oj t’he method described by Mahadevan and Tappel (1968) ; the assay system usually (*ontained 0.2 ml of the triglyceride dispersion, 0.1 ml of 1 Iw-citrate-sodium citrat.e in&~ (pH 4*3), 0.1 ml of 10 mM fi-mercaptoethanol, and suitably diluted tissue fraction in a total volume of 1.0 ml. Incubations were performed for 60 min at 37°C. The reaction was stopped by the addition of 1.0 ml of 99%” ethanol and 0.1 ml of 1.0 s-HCl. ln control tubes, the buffer, /3-mercaptoethanol and tissue fraction were incubated, and the substrate dispersion was added after stopping the reaction. The reaction mixtures were vigorously shaken with 6.0 ml of petroleum ether (b.p. 30-60°C). An aliquot (3.0 ml) of the pet’roleutn ether layer was evaporated to dryness, and the residue was dissolved in 3.0 ml of chloroform. The liberated fatty acids in the chloroform solution were estimated colo~i~nct.~~icnll~ by the method of Duncombe (1963). Acid phosphatase (EC 3.1.3.2), cathepsin D (EC 3.4.23.5) and lactic dehytlrogenase (EC 1.1.1.2’7) were assayed in the same wayx described previously (Hayasaka, 1974a,b; Hayasaka et al., 1975), using as substrate I’-nitrophenyl phosphate, bovine serum :~lbumin and pyruvate, respectively. Protein

determination

Protein content was determined by the method of Lowry, Rosebrough, Farr and Randall (1951). Specific activity was expressed as the enzyme activity per mg protein.

3. Results All values were expressed as means of three experiments. The distribution of acid lipase in ocular tissues is shown in Table I. The cornea, the aqueous humor and the vitreous body had no enzyme activity. The lens exhibited a low enzyme activity. On TABLE

Distribution

of acid

lipase

Cornea Aqueous humor ciliary

retina

Choroid

Preparation as substrate.

and

enzyme

the eye tissues

not

body

Retinal pignlent epithelium

used

of

not detectable

Vitreous body Neurosensory

the homogen.ate

Specific activity* (pg decanoic acid relessed/37”C!, hr/mg protein)

Eye tissues

Iris and Lens

in

I

assay

were

as described

detectable 2YT&6.1 4.2+2.5 not detectable 35.355.3 420~4+3w 20.3+F3

in Materials

and

Methods.

Glycerol

tridccanoate

was

ACID

LIPASE

IN

RETINAL

PIGMENT

EPITHELIVJI

3

the other hand. the retina and the uvea showed relatively high enzyme activity. The highest specific activity was found in the RPE. The intraocular distribution of acid lipase is similar to that of cathepsin D (Hayasaka et al., 1975). In the subcellular fractions of the RPE (Fig. l), the specific activity of acid lipasr was hjghest in the lysosomal fraction and lowest in the soluble fraction (cytosol). This finding was confirmed by the subcellular distribution of marker enzymes (Table II).

Cc

n.

ij

20 Per

cent

40 of

cc total

80

loo

protein

s

FIG. 1. Distribution of acid lipase in subcellular fractions of retinal pigment epithelium. Preparatioll and enzyme assay were as described in Materials and Methods. Glycerol tridecanoate wa6 used as Nub&rate. K, nuclear fraction: Mt, mitochondrial fraction: Lys, lysosomal fraction; MC, microsomal fraction: S, soluble fraction. Each fraction is represented in t,he ordinate by its specific activity. On the abscissa, each fraction is represented by its percentage of protein.

The two acid hydrolases used as lysosomal markers, namely acid phosphatase and cathepsin D were highest in the lysosomal fraction prepared by differential pelleting. The effects of pH on acid lipase in the homogenate and lysosomal fraction were shown in Fig. 2. to be similar to each other. The optimal pH of the enzyme in RPE

2

3

4

5

6

7

8

9

PH

FIG. 2. Effect of pH on acid lipase in the homogenate and lysosomal fraction of RPE. Glycerol tridecanoate was used as substrate. 0, Homogenate (1.5 mg protein) was incubated at 37Y for 60 min; 0. lysosomal fraction (0.71 mg protein) was incubated at 37°C for 60 min. Buffers wed were 0.2 .Mcitrate+sodium citra.te (pH 2.0-6.7) and 0.2 v-Tris-HC’I (pH 7.2-94).

4

S. HAVASAkKA

ET

AL.

cdls w;~s 4-d. and there was almost no lieutrsl lipase in t,hesc~ tolls. The hytlrolysis 4 difkrcwt triacylglycerols 1)~ the enzynle in the homogenatc~ of’RPI1: exllibitetl uli~xinral activity with gljwrol triclecanoatc. which is in agreenicnt with tlata for ixt li\-cxr at111

Subcellztlur distributior~ of enqmes in retinctl pigment c~pitldiu ttt Increase Acid phosphatase

Fraction

Kuclear Mitochondrial Lysosomal Microsomal Soluble

Preparation bovine serum

of specific

C’athepsin

0.5 0.9 3.0 0.8 0.7

activity

over homogenate

(-fold)

Acid lipasr

D

O-X 70 34 I4 Il.6

0.7 I.7 2.1 0.9 0.5

not, cletectahlc not, detectable not detectahic 0.1 2.8

and enzyme assay were as described in Materials and Methods. p-Nitrophenyl phosphate, albumin, glycerol tridecanoate and pyruvate were used as substrate, respectively.

TABLE

III

Hydrolysis of different triglycerides by acid lipase in homogenate of retinal pigment epithelium

Glycerol

Relative rates of hydrolysis

triester

Decanoate Palmitate Oleate

1.5 mg protein

100 38 31

of homogenate

was incubated

TABLE

at 37°C for 60 min.

IV

Comparisonof acid lipnse in the bovine organa

Tissues

(pg decanoic

Specific activity acid released/37”C,

hr/mg

protein)

Retinal Liver Kidney Blood

pigment

Preparation and enzyme was used as substrate.

epithelium

assay were

420 43 38 not detectable

aa described

in Materials

and Methods.

Glycerol

tridecanoate

ACID

LIYASE

IN

RETINAL

PIGMENT

.i

EPITHELICM

kidney acid lipase (Mahadevan and Tappel, 1968). Specific activity of bovine RPE was about IO-fold higher than those of liver and kidney, whilst blood revealed no activit,y (Table IV). 4. Discussion From the present results, it appears that RPE has a very high acid lipase activity, and that the specific activity is highest in the lysosomal fraction. It, is possible that the discrepancies between the results by Rothman et al. (1976) and ours may be due to the difference of t,he substrate. Triglyceride, which we usccl, is considered to be more appropriate as substrate for lipase than 4-MU or p-nitrophenol derivatives. The use of 4-MU and p-nitrophenol derivatives for cletermiuation of lipase is practical but unphysiolo$cal, Ijecause both have different, physico~heritiaal prol)erties from lipid (Imai, 1966). We used fl-mercaptoethanol additionally in tlw incubation medium according to the method dwcribed by Slahadevan and ‘I’app~l (1968), because of the fact that partially purified acid lipase of RPE was activated I)! thiol compounds (Hayasaka, Hara and Mizuno ; in preparation). Although BerTuan and Feeney (1976) recommended to wash about six cycles by centrifqations at’ 140 150 xg for 10 min in 0.32 M-sucrose for isolation of RPE cells. t,ho it~atwial ol)tained in the present study was suitable histologically (Hayasaka et al.. 1975). Alt~hoqh we did not use morphological criteria to ascertain the purit)y of t,hrb SI~IJwllul;~r fractions of RPE, our fractionation method did show acid hydrola~es to IN! hi~&wt in the lvsosomal fraction. The optimal pH of lysosomal Iipase of rat lil-w anti h kitlllcxy is reported to be 4.3 (Rlahadevan and Tappel, 1968), which is similar, to the> valrw of t)he enzyme obtain& in RPE. If acid lipaw is localizetl mainl\- in cytosol. ;I’: rtbported by Rothman et al. (1976) it remains unclear how the cwzyme cntclrs t Iw ph:~~olysosomal systeiu. It is possible that acid lipasc, with itCXOlllp~~tl~Tillg [lllOS~)ll0lil):lse. ill the lysosomes of RPE may l)lay an essential rolkb in the di,cwt,ion of lil~iti cnn~~mwnts lnwent in the ROS discs, which arc’ well known to b(l riclb iI1 lil)itl (Anrlrrson. l?‘c~l~lmanand Feldmq 1950; Borggreven. Dar~tuen and Bontilq. l!G’O; L’c~il~wlot nntl _41)r;t h amson, 1970). RPE cells are I)elievcd to recvclc manv Illltri(tllts to tlw retina (Hogan, 1952). Therefore, a&i lipase in lysosome~ of .KPE IINJ- ~)I;I!- ~1 ~i~:l~ifiicil~llt rolr not onlv in lipid catalA5nl I)nt, also imlircctlr Li in tlw ;uial~olistI~cd tlw l
The authors thank Nrs Sagara for her technical assistance, and Xiss Oyamadx fur 11f.r excellent typewriting. We also thank Drs Berman and Feeney for sendingus copiesof their papers before publication, and for helpful suggestions. Th& work has been supporwtl by a grant for Pigmentary Retinal Dystrophy Research from the Xnistry of Health ant1 N elfare of Japan. REFERENCES Anderson,

It.

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E.,

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Clean

start

for

the

retinal

pigment

epithelium.

Invest.

6

S. H:ZYAS.IKA

ET

AI,.

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