Saroshi Horo

Seiji Hayasaka

Takashi Shiono

Tokahide Fukuyo

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Partial Purification of Cathepsin B in the
Bovine Ciliary Body and Iris
Seiji Hayasaka, Takashi Shiono, Saroshi Horo, and Tokahide Fukuyo
Cathepsin B in the bovine ciliary body and iris was studied biochemically using a-N-benzoyl-D,L-
arginine-2-naphthylamide as substrate. The enzyme was purified to 210-fold from the autolyzed
extract. The partially purified enzyme had a pH optimum at 6.0 and a molecular weight of 27,000.
The apparent Km value for the substrate was 1.6 mM. The enzyme was activated by disodium
ethylenediamine tetraacetate, cysteine, and dithiothreitol. The enzyme activity was inhibited strongly
by leupeptin and partially by hyaluronate and chondroitin sulfate A. Invest Ophthalmol Vis Sci
24:682-686, 1983
Cathepsin B is known to be a thiol-dependent ly-
sosomal proteinase1 that can degrade collagens,2'3
connective tissue proteins,4 and some native en-
zymes.5 Using a-N-benzoyl-D,L-arginine amide as
substrate, we previously demonstrated that the ciliary
body and iris had the highest activity of cathepsin B
among bovine ocular tissues.6 A more specific sub-
strate, a-N-benzoyl-D,L-arginine-2-naphthylamide,
was introduced recently and is now widely used for
the assay of cathepsin B.1
The present study was undertaken to examine the
enzyme properties in the partially purified prepara-
tion from the bovine eye.
Materials and Methods
Chemicals
a-N-benzoyl-D,L-arginine-2-naphthylamide, mcr-
salyl acid, concanavalin-A Sepharose, pepstatin, and
human umbilical cord hyaluronate were obtained
from Sigma Chemical Company, St. Louis, MO; 4-
amino-2',3-dimethylazobenzene and a-naphthyl-
amine hydrochloride from Wako Pure Chemical
Company (Japan); leupeptin from Protein Research
Foundation (Japan); dithiothreitol and chondroitin
sulfate A from Seikagaku-Kogyo Company (Japan);
cysteine from Kyowahakko Company (Japan); ri-
bonuclease A, chymotrypsinogen A, ovalbumin, Se-
From the Department of Ophthalmology, Tohoku University
School of Medicine, Sendai, Japan.
Submitted for publication June 16, 1982.
Reprint requests: Seiji Hayasaka, MD, Department of Oph-
thalmology, Tohoku University School of Medicine, Sendai, Mi-
yagi 980, Japan.
phadex G-100, and DEAE-Sephadex A-25 from
Pharmacia Fine Chemicals (Sweden).
Purification of Cathepsin B
About 60 adult bovine eyes were maintained at 4
C from the time of slaughter through all subsequent
steps. The ciliary body and iris were dissected in ice-
cold 20 mM potassium phosphate buffer (pH 6.8)
containing 1 mM disodium ethylenediamine tetra-
acetic acid (EDTA), frozen and thawed five times,
and homogenized in a Waring blender. The homog-
enate was centrifuged at 9,000 g for 15 min to obtain
the supernatant (crude extract).
The crude extract was adjusted to pH 4.5 with 1
M acetic acid, incubated at 37 C for 2 hrs, and then
centrifuged at 20,000 g for 15 min. The pellet was
discarded and the supernatant was adjusted to pH 6.8
wilhJ_ M potassium phosphate buffer (pH 7.0) (au-
tolyzed extract). To the autolyzed extract, solid am-
monium sulfate was added to 30% saturation (176
g/1), with constant mechanical stirring. After centrif-
ugation, the precipitate was discarded, and the su-
pernatant was brought to 60% saturation by adding
solid ammonium sulfate (198 g/1). The precipitate
was collected by centrifugation, dissolved in a small
amount of 20 mM potassium phosphate buffer (pH
6.8) containing 1 mM disodium EDTA, and dialyzed
overnight against the same buffer-EDTA solution,
and ammonium sulfate fraction was prepared. To the
ammonium sulfate fraction was added acetone to
40% (v/v), with constant mechanical stirring. After
centrifugation, the precipitate was discarded and the
supernatant was brought to 65% acetone (v/v) by the
addition of acetone. The precipitate was collected by
centrifugation, dissolved in a small amount of 20 mM
0146-0404/8 3/0600/682/$ 1.05 © Association for Research in Vision and Ophthalmology
682
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No. 6 CATHEPSIN D IN THE BOVINE CILIARY BODY AND IRIS / Hoyosako er al. 683
Table 1. Purification of Cathepsin B from Bovine Ciliary Body$
Fraction
Autolyzed extract
Ammonium sulfate fraction
(30-60% sat.)
Acetone fraction (40-65%)
Sephadex G-100 fraction
DEAE-Sephadex fraction
Volume
(ml)
120
26
4
60
100
Total Protein
(mg)
1644
553.8
80
16.8
2.2
Total*
Activity
26.6
34.6
23.7
13.3
7.4
Specific^
Activity
0.016
0.062
0.30
0.80
3.36
Recovery
(%)
100
130
89
50
27.8
Purification
(nfold)
1
3.9
18.8
50
210
* n moles 2-naphthylamine released/37° C, 30 min.
t n moles 2-naphthylamine released/37°C, 30 min/mg protein.
% Enzyme assay and partial purification procedures were described in
Materials and Methods.
potassium phosphate buffer (pH 6.8) containing 1
mM disodium EDTA, dialyzed overnight against the
same buffer-EDTA solution, and the acetone fraction
was obtained.
Two milliliters of acetone fraction were applied to
a column (1.5 by 96 cm) of Sephadex G-100 equil-
ibrated with 20 mM potassium phosphate buffer (pH
6.8) containing 1 mM disodium EDTA. The column
was eluted with the same buffer-EDTA solution at a
flow rate of 18 ml per h, and the fractions of 3.3 ml
were collected. The fractions containing enzyme ac-
tivity (fraction nos. 13-21) were combined to prepare
the Sephadex G-100 fraction. To 15 ml of Sephadex
G-100 fraction, 10 ix\ of 2-mercaptoethanol was
added and applied to a column (0.9 by 27 cm) of
DEAE-Sephadex A-25 equilibrated with 20 mM po-
tassium phosphate buffer (pH 6.8) containing 1 mM
disodium EDTA and 0.1 mM 2-mercaptoethanol.
The column was washed with 50 ml of the same
buffer-EDTA-mercaptoethanol solution and eluted
Elullon Profile of Cltheptln B on • Sephidex Q-100 Column
Fig. 1. Elution profile of cathepsin B in the ciliary body and iris
on a Sephadex G-100 column: 100 /*1 of each fraction were in-
cubated at 37 C for 30 min in 0.1 M potassium phosphate buffer
(pH 6.0) containing 1.4 mM cysteine and 0.7 mM disodium EDTA
with 0.5 mg a-N-benzoyl-D,L-arginine-2-naphthylamide as sub-
strate in a total volume of 0.5 ml (O). Twenty to 50 n\ of fraction
were used for protein determination (•). Fraction nos. 13-21 were
combined as the Sephadex G-100 fraction. Blue dextran was eluted
at No. 3 (1), and pyridoxal phosphate was eluted at No. 36 (I).
with 80 ml of sodium chloride at a continuous linear
gradient established between 0 and 0.5 M, containing
the same buffer—EDTA-2-mercaptoethanol solu-
tion. The fractions of 2 ml were collected. The frac-
tions containing enzyme activity (fractions nos. 10-
22) were combined and dialyzed overnight against 20
mM potassium phoshphate buffer (pH 6.8) contain-
ing 1 mM disodium EDTA, obtaining DEAE-Se-
phadex fraction.
Enzyme Assay
Cathepsin B [E.C. 3.4.22.1.] activity was deter-
mined by a slight modification of the method de-
scribed by Barrett.1 Samples of enzyme solution were
incubated in a total volume of 0.5 ml for 30 min at
37°C in 0.1 M potassium phosphate buffer at pH 6.0,
containing 1.4 mM cysteine and 0.7 mM disodium
EDTA, with 0.5 mg a-N-benzoyl-D,L-arginine-2-
Elution Profile of Cathepsin B on a DEAE-Sephadex Column
1 60 §
- 20 E
Fig. 2. Elution profile of cathepsin B in the ciliary body and iris
on a DEAE-Sephadex column: 200 (A of each fraction were in-
cubated at 37 C for 30 min in 0.1 M potassium phosphate buffer
(pH 6.0) containing 14 mM cysteine and 0.7 mM disodium EDTA
with 0.5 mg a-N-benzoyl-2-naphthylamide as substrate in a total
volume of 0.5 ml (O). One hundred microliters of fraction were
used for protein determination (•). Fraction nos. 10-22 were com-
bined as the DEAE-Sephadex fraction.
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684 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / June 1983 Vol. 24
E f f e c t o f p H on C a t h e p s i n B A c t i v i t y
Z E
u c
< o
m <M
in
n CO
0 . 2
0. 1
I '••
4 . 0 5 . 0 6 . 0
p H
7 . 0 8 . 0
Fig. 3. Effect of pH on partially purified cathepsin B of DEAE-
Sephadex fraction from the bovine ciliary body and iris: 2.2 ng
protein of partially purified cathepsin B was incubated at 37 C for
30 min in 1.4 mM cysteine and 0.7 mM disodium EDTA with 0.5
mg a-N-benzoyl-D,L-arginine-2-naphthylamide as substrate. Buff-
ers used were 100 mM citrate-sodium citrate buffer (pH 4.1-5.8)
(O), and 100 mM potassium phosphate buffer (pH 5.8-7.5) (•).
naphthylamide as substrate. Incubation was
terminated by the addition of mersalyl acid. The 2-
naphthylamide released was colorimetrically deter-
mined after coupling with 4-amino-2',3-dimethyla-
zobenzene.
Protein Determination
Protein content was determined by the method of
Lowry et al.7 Specific activity was expressed as the
enzyme activity per milligram of protein.
Catheps in B Act iv i ty in the C i l i a ry Body
and Incubat ion Time
0.8
0.6
» - 0.4
0.2
A 4.4 jug of protein
O 2.2 jug of protein
A 1.1 jug of protein
Fig. 4. Cathepsin B activity in the ciliary body and iris and
incubation time: 4.4 tig protein (A), 2.2 /ig protein (O), and 1.1
Hg protein (A) of partially purified cathepsin B of DEAE-Sephadex
fraction were incubated at 37°C in 0.1 M potassium phosphate
buffer (pH 6.0) containing 1.4 mM cysteine and 0.7 mM disodium
EDTA, with 0.5 mg a-N-benzoyl-D,L-arginine-2-naphthylamide
as substrate.
n 0 2 4 « B • 0 O S t 1.S 2
Concentration ol N-*-Benioyl- Km=1.6mM 1 / 8 (concentration of H-* -Banioyl-
L-Arglnlne-2-Naphthylamid* L-Arglnlne-2-N»phthyl«mld«> ImM'1)
(mM)
Fig. 5. Concentration of N-a-benzoyl-L-arginine-2-naphthyl-
amide and cathepsin B activity in the ciliary body and iris: 2.2
Mg protein of partially purified cathepsin B of DEAE-Sephadex
fraction were incubated at 37 C for 30 min in 0.1 M potassium
phosphate buffer (pH 6.0) containing 1.4 mM cysteine and 0.7
mM disodium EDTA.
Molecular Weight Determination
The apparent molecular weight of cathepsin B was
determined according to the method of Andrews.8
The acetone fraction was applied on a Sephadex G-
100 column (1.5 by 96 cm) previously equilibrated
with 20 mM potassium phosphate buffer (pH 6.8)
containing 1 mM disodium EDTA, and the column
was eluted with same bufFer-EDTA solution. Ribo-
nuclease A (molecular weight, 13,700), chymotryp-
sinogen A (molecular weight, 25,000) and ovalbumin
(molecular weight, 43,000) were used as reference
standards.
The Response to Concanavalin A
The Sephadex G-100 fraction (0.1 mg protein) was
dialyzed for 3 hours against 4 mM CaCl2-4 mM
MnCl2-4 mM MnCl2-10 mM potassium phosphate
buffer (pH 6.8) and applied on a concanavalin A-
Sepharose column (1.5 by 20 cm) previously equili-
brated with 4 mM CaCl2-4 mM MnCl2-4 mM MnCl2-
10 mM potassium phosphate buffer (pH 6.8). The
column was washed with the starting buffer until the
A 280 fell nearly to the starting value, and then the
absorbed fraction was eluted with 250 mM a-methyl-
D-mannoside in the same buffer.
Results
The results of partial purification are summarized
in Table 1. The final preparation, DEAE-Sephadex
fraction, was purified 210-fold over the autolyzed ex-
tract, with an overall recovery of 27.8%. Fig. 1 shows
the elution profile of cathepsin B on Sephadex G-100
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No. 6 CATHEP5IN D IN THE BOVINE CILIARY BODY AND IRIS / Hoyosako er al. 685
column chromatography, and Fig. 2 shows the elu-
tion profile of cathepsin B on DEAE-Sephadex col-
umn chromatography.
The effect of pH on the DEAE-Sephadex fraction
is shown in Fig. 3. The optimal pH was near 6.0-6.2
when N-«-benzoyl-D,L-arginine-2-naphthylamide
was used as substrate.
The enzyme activity was proportional to both en-
zyme concentration and incubation time (Fig. 4).
The apparent Km value of DEAE-Sephadex frac-
tion for N-a-benzoyl-D,L-arginine-2-naphthylamide
was estimated to be 1.6 mM (Fig. 5).
The molecular weight of the enzyme appeared to
be 27,000 by the estimation of a gel filtration method
(Fig. 6).
The enzyme activity in DEAE-Sephadex fraction
was activated by disodium EDTA, cysteine, and di-
thiothreitol, and inhibited by leupeptin, which is an
inhibitor of thiol proteinase. The enzyme activity was
not affected by pepstatin, an inhibitor of carboxyl
proteinase. The enzyme activity was partially inhib-
ited by hyaluronate and chondroitin sulfate A (Table
2). The enzyme was unadsorbed by concanavalin A-
Sepharose.
Discussion
We previously showed the high specific activity of
cathepsin B in the bovine ciliary body and iris using
a-N-benzoyl-L-arginine amide as substrate.6 The
present study clarified some properties of the enzyme:
the optimal pH (6.0), Km values for N-a-benzoyl-L-
arginine-2-naphthylamide (1.6 mM), and molecular
weight (27,000) in the bovine ciliary body and iris are
Molecular Weight of Cathepsin B
Table 2. Effect of Various Compounds on
Cathepsin B from Bovine Ciliary Body*
0.8
0.6
0.4
0.2
Ribonuclease A (13,700)
Chymotrypsinogen A (25,000)
Cathepsin B (27,000)
Ovalbumin (43,000)
10* 2X10* 5X104 10s
Molecular Weight
Fig. 6. Molecular weight of cathepsin B from the bovine ciliary
body and iris. A column (1.5 by 96 cm) of Sephadex G-100 equil-
ibrated with 20 mM potassium phosphate buffer (pH 6.8) con-
taining 1 mM disodium EDTA was used to determine the molec-
ular weight.
Compounds
Absorbance
at 520 nm\
Disodium EDTA (0.7 mM) 0.040 ± 0.010
Cysteine (1.4 mM) 0.280 ± 0.030
Disodium EDTA (0.7 mM) + cysteine (1.4
mM) 0.385 ± 0.035
Disodium EDTA (0.7 mM) + DTT (0.7 mM) 0.370 ± 0.035
Disodium EDTA (0.7 mM) + cysteine (1.4
mM) + pepstatin (10 nM) 0.375 ± 0.030
Disodium EDTA (0.7 mM) + cysteine (1.4
mM) + leupeptin (10 nM) 0.020 ± 0.010
Disodium EDTA (0.7 mM) + cysteine (1.4
mM) + human umbilical cord hyaluronate
(0.17mg/ml) 0.250 ±0.035
Disodium EDTA (0.7 mM) + cysteine (1.4
mM) + chondroitin sulfate A (0.2 mg/ml) 0.210 ± 0.030
* DEAE-Sephadex fraction was dialyzed for 4 hours against 20 mM po-
tassium phosphate buffer (pH 6.8). Dialyzed DEAE-Sephadex fraction (4.4
/i protein) was incubated at 37°C for 30 min in 0.1 M potassium phosphate
buffer (pH 6.0) with 0.5 mg a-N-benzoyl-D,L-arginine-2-naphthylamide and
with compound.
t Mean ± S.D. (N = 4)
quite similar to these in bovine liver and spleen, as
reported by others.1 The substrate used in the present
study is active with cathepsin H as well as cathepsin
B. Barrett and Kirschk9 reported that cathepsin B
does not bind to concanavalin A, while cathepsin H
does. The Sephadex G-100 fraction was unadsorbed
by concanavalin A-Sepharose, indicating cathepsin
B. It is of particular interest that the enzyme was
partially inhibited by exogenous hyaluronate and
chondroitin sulfate A (Table 2). This finding suggests
that the activation of the enzyme during autolysis
may be partly due to the degradation of endogenous
by hyaluronate and chondroitin sulfate. Hence, the
enzyme released from the ciliary body may be par-
tially inhibited by the vitreous body, as the ciliary
body is in close contact with vitreous body hyaluro-
nate.
Cathepsin B, a proteinase that degrades collagens
and proteins,1 should be considered in some patho-
physiologies of the uvea, because (1) the enzyme is
found in the aqueous humor from patients with Bah-
cet's disease110; (2) the enzyme may have an impor-
tant role in tissue injury in uveitis; and (3) the enzyme
may have potential to degrade not only native protein
but also synthetic polymers, such as nylon.11
Key words: cathepsin B, bovine ciliary body and iris, partial
purification
Acknowledgments
The authors would like to thank Professor K. Mizuno
for advice, Ms. Y. Sagara for her technical assistance, and
Miss Y. Miura for her secretarial assistance.
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686 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / June 1983 Vol. 24
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Partial Purification of Cathepsin B in the Bovine Ciliary Body and Iris

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Partial Purification of Cathepsin B in the Bovine Ciliary Body and Iris

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