Download Distribution of acid phosphatase, beta-glucuronidase, and

Distribution of acid phosphatase,
/3-glucuronidase, and lysosomal
hyaluronidase in the anterior segment of
the rabbit eye
Seiji Hayasaka and Marvin L. Sears
Distribution of acid phosphatase, (3-glucuronidase, and lysosomal hyaluronidase in the anterior segment of the rabbit eye was studied biochemically. Acid phosphatase activity was
higher in the anterior uvea and cornea but lower in the sclera. /3-Glucumnidase activity was
higher in the anterior uvea but lower in the corneoscleral tissues. Lysosomal hyaluronidase
activity was higher in the anterior uvea. The inner layer of the corneoscleral junction showed
the highest specific activity offt-glucuronidaseand lysosomal hyaluronidase among the corneoscleral tissues. Lysosomal hyaluronidase activity was detected in all corneoscleral tissues.
Key words: acid phosphatase, /3-glucuronidase, lysosomal hyaluronidase, iris,
cornea, sclera, acid mucopolysaccharides, distribution
* 3 ince Barany and Scotchbrook1 reported
that after treatment with bovine testicular
hyaluronidase the resistance of filtering angle
of excised cattle eyes dropped to about onehalf the initial value, much attention has
been devoted to the hyaluronidase-sensitive
mucopolysaccharides in the outflow apparatus. Several investigators have confirmed the
presence of acid mucopolysaccharides in the
trabecular meshwork of primates as well as
other species 2 " 7 and the reduction in aqueous
outflow resistance after intracameral testicuFrom the Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, Conn.
This work was supported in part by U.S. Public Health
Service grants EY-00237 and EY-00785, Research to
Prevent Blindness, Inc., and the Connecticut Lions
Eye Research Foundation, Inc.
Submitted for publication Dec. 16, 1977.
Reprint requests: Marvin L. Sears, M.D., Professor and
Chairman, Department of Ophthalmology & Visual
Science, Yale University School of Medicine, 333
Cedar St., New Haven, Conn. 06510.
982
lar hyaluronidase. 8 10 The biosynthesis of
acid mucopolysaccharides in tissue culture of
trabecular meshwork cells has recently been
demonstrated. 11 ' 12 In spite of the presence
and biosynthesis of acid mucopolysaccharides
in the trabecular meshwork, the degradation
mechanism remains unclear.
Many mucopolysaccharide-degrading enzymes are thought to be localized in lysosomes of mammalian tissues. 13 In another
study using a sensitive method based on carbocyanine dye binding, 14 we found that the
rabbit iris has a lysosomal hyaluronidase. 15 In
this study we therefore examined the distribution of lysosomal enzymes, particularly
lysosomal hyaluronidase, in the anterior
segment of the rabbit eye.
Materials and methods
Chemicals. Para-nitrophenyl phosphate was
purchased from Dai-ichi Pharmaceutical Co., Tokyo; p-nitrophenyl glucuronide from Nakarai
Pharmaceutical Co., Tokyo; hyaluronicacid (grade
I, human umbilical cord), testicular hyaluronidase
0146-0404/78/100982+06$00.60/0 © 1978 Assoc. for Res. in Vis. and Ophthal., Inc.
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Volume 17
Number 10
Enzymes in anterior segment
983
Table I. Effect of rabbit eye tissues on absorbance of hyaluronic acid—carbocyanine
dye complex
Rabbit eye tissues
Concentration
(fig protein 10.1 ml)
None
Iris:
Homogenate
Supernatant
Lysosomal extract
Tips of ciliary process:
Homogenate
Lysosomal extract
Ciliary body:
Homogenate
Lysosomal extract
Cornea (tissue extract):
Outer layer
Middle layer
Inner layer
Corneoscleral junction (tissue extract):
Outer layer
Middle layer
Inner layer
Sclera (tissue extract):
Outer layer
Inner layer
Aqueous humor
Absorbance
at 640 nm
0.348
22
5
22
5
5
0.000
0.143
0.000
0.126
0.340
5
5
0.015
0.339
5
5
0.020
0.342
1
1
1
0.342
0.349
0.338
1
0.345
0.343
0.338
1
1
1
1
4
0.339
0.345
0.115
Two micrograms of hyaluronic acid were suspended with or without tissue extract in 0.1 ml of 0.02M sodium acetate buffer (pH 3.
Absorbance at 640 nm was read after addition of 0.9 ml of the carbocyanine dye solution.
(bovine, 415 NF units/mg protein), and Coomassie brilliant blue G-250 from Sigma Chemical Co.,
St. Louis; carbocyanine dye (l-ethyl-2-[3-(l-ethylnaphtho[l,2d]-thiazolin-2-ylidene)-2- methyl-propenyl]naphtho[l,2d]thiazolium bromide) from
Eastman Organic Chemicals, Rochester, N. Y. All
other reagents were of analytical grade or the best
grade available.
Tissue preparation. Two albino rabbits, weighing 2 to 3 kg, were anesthetized with intravenous
nembutal (100 mg of pentobarbital per kilogram
body weight) for each experiment. The eyes were
enucleated and placed in ice-cold 0.25M sucrose
containing 0.02M Tris-HCl buffer (pH 7.4). Aqueous humor was obtained by aspirating the anterior
chamber contents with a 30-gauge needle and a
tuberculin syringe. Sclera was radially cut off from
the posterior pole to the equator. After the vitreous and lens were removed, the tissues shown in
Fig. 1A were separated under a Zeiss surgical microscope. The iris, tips of ciliary process, and
ciliary body were homogenized in 1 ml 0.25M sucrose containing 0.02M Tris-HCl buffer (pH 7.4)
and the "homogenate," "supernatant," and "lysosomal extract" were prepared by centrifugation
previous described.lo This preparation is outlined
in Fig. IB.
The iris, tips of ciliary process, ciliary body,
cornea, corneoscleral junction, and sclera were
minced in 0.5 ml of 0.02M sodium acetate buffer
(pH 3.8), homogenized with a Potter-Elvehjem
homogenizer, treated five times with freezing and
thawing, dialyzed for 6 hr against 0.02M sodium
acetate buffer (pH 3.8), and centrifuged at
12,500 x g for 20 min. The resulting supernatant
was used as "tissue extract." This preparation is
outlined in Fig. 1C.
Enzyme assay. Hyaluronidase (EC 3.2.1.35)
activity was determined in the same way as previously described.15 Briefly, enzyme source was incubated at 37° C for 1 to 3 hr at pH 3.8 with
hyaluronic acid (2 /u,g) as substrate in a total volume of 0.1 ml. Incubations were terminated by
addition of 0.9 ml of the carbocyanine dye solution, and the mixture was spectrophotometrically
read at 640 nm. Enzyme activity was expressed as
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Invest. Ophthalmol. Visual Sci.
October 1978
984 Hayasaka and Sears
cornea outer layer
cornea middle layer
cornea inner layer
corneoscleral junction outer layer
corneoscleral junction middle layer
corneoscleral junction inner layer
sclera outer layer
clera inner layer
tips of ciliary
process
ciliary body
Fig. 1A. Schematic representation of tissues dissected.
Table II. Distribution of acid phosphatase, /3-glucuronidase, and lysosomal hyaluronidase
in the anterior segment of rabbit eye
Tissue
Iris
Tips of ciliary process
Ciliary body
Cornea:
Outer layer
Middle layer
Inner layer
Corneoscleral junction:
Outer layer
Middle layer
Inner layer
Sclera:
Outer layer
Inner layer
Acid
phosphatase*
[$-Glucuronidase *
89
100
77
15.5
22.0
18.0
102|
69$
54
49
78
4.8
3.6
5.1
18
12
28
61
62
7.7
8.5
24
32
70
35
42
10.5
6.7
6.5
Lysosomal
hyaluronidase]
96J
34
10
10
*Specific activities of acid phosphatase and /3-glucuronidase are represented as /xg of p-nitrophenol released per hour per milligram of
protein. Acid phosphatase and /3-glucuronidase were measured in the tissue extract.
tSpecific activity of hyaluronidase was represented as A A640 per hour per milligram of protein.
{Specific activities of hyaluronidase in the iris, tip of ciliary process, and ciliary body were measured in the lysosomal extract.
Hyaluronidase in the corneoscleral tissues was measured in the tissue extract.
A A640. The hyaluronidase assay method based on
carbocyanine dye binding has a limitation.15 In
other words, the absorbance of hyaluronic acidcarbocyanine dye complex is affected by various
compounds such as the rabbit iridial homogenate.
Hence the effect of rabbit eye tissues on the absorbance must be examined first.
Acid phosphatase (EC 3.1.3.2) activity was determined as previously described,16 with p-ni-
trophenyl phosphate used as substrate and the
p-nitrophenol released measured spectrophotometrically.
/8-Glucuronidase (EC 3.2.1.31) activity was determined as previously described,16 with p-nitrophenyl glucuronide used as substrate and the
p-nitrophenol released spectrophotometrically
measured.
Protein. Protein was determined by the method
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Volume 17
Number 10
'Enzymes in anterior segment 985
rabbit iris, tips of ciliary process or ciliary body
4
minced
4
homogenized
filtered
4
4
4
at 1000 g for 10 min
dialyzed
4
| homogenate
4
4
sup.
4
pellet
4
suspended
4
homogenized
4
at 1000 g for 10 min
4
4
~4
sup.
pellet
I
4
at 1 2,500 g for 20 min
4
4
4
sup.
pellet
4-
4
dialyzed
suspended
4
4
supernatant|
freezing and thawing
dialyzed
at 12,500 n for 20 min
4
i
sup.
i
pellet
4
lysosomal extract)
Fig. IB. Preparation of homogenate, supernatant, and lysosomal extract. (Method from
Hayasaka, S., and Sears, M. L.: INVEST. OPHTHALMOL. VISUAL SCI. 17:639, 1978.)
of Bradford,17 using bovine serum albumin as
standard. Specific enzyme activity was expressed
as the enzyme activity per milligram of protein.
Proteins (1 to 2 fig) in the different tissue extracts
or in the lysosomal extracts were incubated for
hyaluronidase assay; 5 to 20 fig of proteins were
incubated for acid phosphatase and /3-glucuronidase assays.
Results
The averages of values in triplicate experiments are shown.
Preliminary observation: effect of tissues
on absorbance. Because the absorbance of
hyaluronic acid-carbocyanine dye complex is
known to be affected by various compounds,
the effect of rabbit eye tissues was examined
(Table I). The homogenate and supernatant
rabbit i r i s , tips of c i l i a r y process, c i l i a r y body,
cornea, corneo-scleral j u n c t i o n , or sclera
4
minced
4
homogenized
4-
freezing & thawing
4dialyzed
4at 12,500 g for 20 min
4- .
I
pellet
sup.
["tissue extract]
Fig. 1C. Preparation of tissue extract.
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Invest. Ophthalmol. Visual Sci.
October 1978
986 Hayasaka and Sears
of iris, tips of ciliary process, and ciliary body
strongly disturbed the absorbance, as did
aqueous humor. The tissue extracts of these
uveal tissues also disturbed absorbance (not
shown in the table). On the other hand,
lysosomal extracts of the iris, tips of ciliary
process, and ciliary body and tissue extracts
of the cornea, corneoscleral junction, and
sclera did not affect the absorbance. Therefore these extracts were thought to be suitable as enzyme source for estimation of
hyaluronidase.
The absorbance of p-nitrophenol was not
affected by the tissue extracts of iris, tips of
ciliary process, ciliary body, cornea, corneoscleral junction, or sclera. Therefore tissue extracts were used as enzyme source for
estimation of activities of acid phosphatase
and /3-glucuronidase.
Distribution of enzymes. Specific enzyme
activities in the tissues of rabbit eye are
shown in Table II. Acid phosphatase activity
was higher in the anterior uvea and cornea
but lower in the sclera. The inner layer of the
corneoscleral junction, containing trabecular
meshwork, did not necessarily show the high
activity. Our findings on the distribution of
acid phosphatase are similar to those previously determined with histochemical techniques.18' 19 Distribution of/3-glucuronidase
was different from that of acid phosphatase.
/3-Glucuronidase activity was higher in the
anterior uvea but lower in the corneoscleral
tissues. Among the corneoscleral tissues the
inner layer of the corneoscleral junction
showed the highest specific activity of
/3-glucuronidase. The notable difference between the distribution of/3-glucuronidase in
our study and that described by others using
histochemistry20 is our finding of lower activity in the cornea. These discrepancies may be
due to technical variations such as substrate
use or incubation conditions.
Lysosomal hyaluronidase activity was determined and detected in the lysosomal extracts of the anterior uvea and in tissue extracts of corneoscleral tissues. Among the
corneoscleral tissues the inner layer of corneoscleral junction showed the highest specific activity of lysosomal hyaluronidase.
Discussion
From this experiment it becomes apparent
that the inner layer of the corneoscleral junction shows the high activities of /3-glucuronidase and hyaluronidase. The inner layer
of the corneoscleral junction was histologically examined to include the trabecular
meshwork. These enzymes play an important
role in the catabolism of acid mucopolysaccharides. Hyaluronidase splits hyaluronic
acid, chondroitin, chondroitin-4- and -6-sulfate and, in part, dermatan sulfate into oligosaccharides, from which terminal glucuronosyl groups are removed by the action of
/3-glucuronidase. Therefore it is possible
that the rabbit trabecular meshwork can degrade the acid mucopolysaccharides. The
main distinction between testicular and
lysosomal hyaluronidases is their different
pH optima. Testicular hyaluronidase has a
broad pH optimum. When measured by viscosity reduction or by turbidity methods, the
testicular enzyme is active at neutral pH.21
Therefore it is conceivable that the testicular
enzyme can be active after the intracameral
injection. On the other hand, the rabbit iris
lysosomal hyaluronidase had a pH optimum
of 3.8 and no activity above pH 5.0.15 Therefore it is probable that the lysosomal hyaluronidase can be active only within the
phagolysosomal system under a physiological
condition. Since the trabecular meshwork is
reported to have a phagocytic activity,22 the
lysosomal enzymes present in this tissue may
degrade the engulfed acid mucopolysaccharides.
It is of interest that hyaluronidase activity
was detected in all corneoscleral tissues examined in this study. Hyaluronic acid is reported to be absent in the cornea.23*24 Therefore it is likely that the enzyme plays a role in
the degradation of chondroitin and chondroitin-4-sulfate in the cornea.
One must consider which cells are responsible for the lysosomal enzyme activity—
whether they are corneal epithelial cells,
keratocytes in the stroma, corneal endothelial cells, trabecular endothelial cells,22 goniocytes (fibroblasts of the trabeculum11) or
scleral cells (fibrocytes). Further work is in
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Volume 17
Number 10
progress to examine which cells produce the
lysosomal enzymes.
We thank Mr. D. Keller for his assistance.
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testicular hyaluronidase on the resistance to flow
through the angle of the anterior chamber, Acta
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2. Berggren, L., and Vrabec, F.: Demonstration of a
coating substance in the trabecular meshwork of the
eye, Am. J. Ophthalmol. 44:200, 1957.
3. Zimmerman, L.: Demonstration of hyaluronidasesensitive acid mucopolysaccharide, Am. J. Ophthalmol. 44:1, 1957.
4. Segawa, K.: The localization of acid mucopolysaccharides in the human trabecular meshwork, Rinsho-ganka 24:363, 1971 (in Japanese).
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Enzymes in anterior segment 987
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