Vol. 72, No. 2, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
CRYSTALLIZATION OF C~THEPSIN D OoV. Kazakova a n d V.N° O r e k h o v i c h Institute
Received
o f B i o l o g i c a l ~nd M e d i c a l C h e m i s t r y , USSR Academy o f M e d i c a l S c i e n c e s , Moscow, USSR
July
29,
1976 SUMUARY
Cathepain 9 from chicken liver purified to apparent homogeneity by t h e method o f a f f i n i t y c h r o m a t o g r a p h ~ on p e p s t a t i n - S e p h a r o s e , was crystallized, u p o n g r a d u a l p r e c i p i t a t i o n w i t h e t h a n o l , from 1°5% p r o t e i n s o l u t i o n i n s l i g h t l y a c i d media c o r r e s p o n d i n g to t h e i s o e l e c t r i c p o i n t o f t h e enzyme. A number o f i n t r a c e l l u l a r cathepsins,
proteinases
of animal o r i g i n ,
have b e e n o b t a i n e d i n t h e h i g h l y p u r i f i e d
or
state,
but
so f a r n o n e o f them have b e e n c r y s t a l l i z e d . C a t h e p s i n D (EC 3 . 4 . 4 . 2 3 )
was i s o l a t e d
from a v a r i e t y
of s o u r c e s
by s e v e r a l g r o u p s o f a u t h o r s ( 1 - 4 ) . S i n c e t h e number o f i s o s y m e s i n different
preparations
essentially
solved whether the multiple
varies,
r e m a i n s t o he
autolysis
exist
in vivo or
appearing in the
procedure.
Having synthesized tin,
still
forms obserTed actually
w h e t h e r t h e y a r e t h e p r o d u c t s of p a r t i a l c o u r s e of t h e i s o l a t i o n
it
the biospecific
a competitive inhibitor
a homogeneous p r e p a r a t i o n
s o r b e n t on ~he b a s i s
of a c i d p r o t e i n a s e s ,
of cathepsin
of pepsta-
we managed t o o b t a i n
D from c h i c k e n l i v e r ,
which
was s u b s e q u e n t l y c r y s t a l l i z e d . ~TERIALS M~B METIIODS Pepstatin,
p r o d u c e d b y Banyu Co ( J a p a n ) was k i n d l y g i v e n by
P r o f . Umezawa. A N - S e p h a r o s e was p r o d u c e d by P h a r m a c i a ( S w e d e n ) . The synthesis
of the carrier
w i t h O.5 M NaC1 s o l u t i o n (30 mg) was d i s s o l v e d addition
(5,6):8g
of A~-Sepharose ~
was washed o f f
(500 m l ) a n d w i t h w a t e r (500 m l ) . P e p s t a t i n
i n 3 ml o f d i m e t h y l f o r m a ~ i d e w i t h s u b s e q u e n t
of h y d r o x ~ s u c c i n i m i d e . D i c y c l o h e x y l c a r b o d t i m i d e
d i s s o l v e d i n 1 ml o f f o r m a m i d e . The s o l u t i o n s
(20 mg) was
were c o o l e d up t o 4°C,
poured together
and a l l o w e d t o s t a n d f o r 1 h r i n t h e c o l d room. Added
to the reaction
m i x t u r e were ~ - S e p h a r o s e
Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
747
a n d 8 ml o f s o l u t i o n
consist-
Vol. 72, No. 2, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
i n g of e q u a l v o l u m e s o f 0 . 1 M p h o s p h a t e b u f f e r , f o r m a m i d e . The m i x t u r e was s t i r r e d
pH 6 . 1 ,
and of d i m e t h y l -
a t room t ° f o r 18 h r s ,
the liquid
was
s e p a r a t e d a n d t h e r e m a i n i n g g e l was washed t w i c e w i t h s m a l l v o l u m e s o f d i m e t h y l f o r m a m i d e . The u n f i x e d p e p s t a t i n aliquots
by i n a c t i v a t i o n
of crystalline
with a mixture consisting amide and w i t h l a r g e
was d e t e r m i n e d i n t h e
pepsin.
o f e q u a l v o l u m e s o f d i o x a n and d i m e t h y l f o r m -
amounts o f w a t e r .
The g e l was a p p l i e d t o a col,]m-
o f 20 X 100 mm and w a s h e d w i t h w a t e r and c i t r a t e these conditions carrier. globin.
a b o u t 20 mg o f p e p s t a t i n
The a c t i v i t y One a c t i v i t y
the extinction
buffer,
pH 3 . 9 .
Under
was f o u n d t o be f i x e d t o t h e
o f c a t h e p s i n D was d e t e r m i n e d b y s p l i t t i n g
o f hemo-
u n i t was d e f i n e d as t h e amount of enzyme i n c r e a s i n g
of t r i c h l o r o a c e t i c
t h e sample c o n t a i n i n g :
filtrates
a t 280 n m b y 1 . 0 p e r h r , w i t h
2 ml o f 1% h e m o g l o b i n s o l u t i o n
of Reanal) i n 0°I M c i t r a t e tion;
The g e l was washed
buffer,
2 ml o f 5% t r i c h l o r o a c e t i c
pH 3 . 0 ;
(the preparation
0.1 ml o f t h e enzyme i n s o l u -
acid.
RESULTS AND DISCUSSIO~
Chicken livers were extracted with 0.05 M citrate buffer, pH 2,7, and f r a c t i o n a t e d 40-7~
w i t h ammonium s u l f a t e .
saturation
The f r a c t i o n
solution
of c a t h e p s i n
washed w i t h 0ol M a c e t a t e
buffer,
Table 1. P u r i f i c a t i o n
containing
and with a small
o f c a t h e p s i n D on p e p s t a t i n - S e p h a r o s e
16 000
activity
0 . 5 M NaC1 ( s o l u t i o n
adsorbed proteins,
Total fraction (40-70% s a t u r a t i o n )
Protein
Preparation obtained from pepstatin-Sepharose 30
0,8
300
100
70
(units) Yield from total activity
column,
D o c c u r e d . The column was t h e n
pH 4 . 0 ,
A) i n o r d e r t o remove u n s p e c i f i c a l l y
(%)
748
to
was removed, t h e r e m a i n i n g
was p a s s e d t h r o u g h a p e p s t a t i n - S e p h a r o s o
and c o m p l e t e a d s o r b t i o n
Specific
at
was d e s a l t e d i n S e p h a d e x G-25 c o l u m n s and a d j u s t e d
pH 4o0 w i t h 1 M CH3COOH; t h e p r e c i p i t a t e transparent
precipitating
Vol. 72, No. 2, 1976
volume o f w a t e r .
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
As d e m o n s t r a t e d e a r l i e r
complex c o m p l e t e l y d i s s o c i a t e s was e l u t e d from t h e c o l u ~ w i t h 0o5 M ~aC1 ( s o l u t i o n cific
(?),
in slightly
the cathepsin D-pepstatin
alkaline
media° The enzyme
0°1 M Na b i c a r b o n a t e
solution
~)o The y i e l d of t h e enzyme b $ p r o t e i n
containi~
and b$ s p e -
a c t i v i t y , i s p r e s e n t e d i n T a b l e 1o
uf~
--t
^ F i g . 1 . I s o l a t i o n o f c a t h e p s i n D on p e p s t a t i n - S e p h a r o s e 1 - o p t i c a l d e n s i t y a t 280 2 - proteoly~ic activity For c o n d i t i o n s and o t h e r d e t a i l s s e e t h e t e x t
The p r e p a r a t i o n 8
o b t a i n e d had one b a n d upon e l e c t r o p h o r e s i s
acrylamide gel (Figo2). Their activity hog p e p s i n t
as measured by splitting
ever~ t h e s l i g h t
traces
c o l o u r to the s o l u t i o n ;
was 5 0 - 7 ~
that
of c r y s t a l l i n e
o f h e m o g l o b i n . I n some c a s e s ~ how-
o f p i g m e n t were s e e n , i m p a r t i n g a y e l l o w i s h to eliminate
these,
the affinity
r e p e a t e d twice i n t h e course of the c r y s t a l l i z a t i o n
s o r p t i o n was
p r o c e d u r e . The s o l u -
t i o n of c a t h e p s i n D o b t a i n e d f~om t h e p e p s t a t i n - S e p h a r o s e d i a l y z e d f o r 3 - 4 h r s on a m a g n e t i c s t i r r e r ,
its
to the pepstatin-Sepharose 0ol M c i t r a t e
c o m p l e x wa~
pH was a d j u s t e d
b y g r a d u a l a d d i n g 0°5 ~ CH3COOH, and t h e r e s u l t i n g
solution
t o ~o0
was a p p l i e d
columu t p r e v i o u s l y washed w i t h ~ M u r e a a n d
b u f f e r ~ pH 3 . 9 .
d e s a l t e d by d i a l y s i s
in poly-
After the second elution~
t h e enzyme was
and c o n c e n t r a t e d by p o l y e t h y l e n e g l y c o l
(mole Wto
20000, p r o d u c e d b~ Merck) t o t h e v o l u m e o f 1 . 5 - 2 ml~ t h e c o n c e n t r a t i o n o f t h e enzyme i n s o l u t i o n were u s e d t o c r y s t a l l i z e As w i l l
be s e e n from t h e e l e c t r o p h o r e t i c
the preparations possible
r e a c h i n g 1.5-1o7%. The s o l u t i o n s
thus prepared
t h e enzyme° patterns
presented
(Figo2)~
c o n t a i n e d no i s o s y m e s of c a t h e p s i n D. I t seew~ h a r d l y
that the isosymes v having similar
749
physico-chemical propertie$~
Vol. 72, No. 2, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
F i g . 2. E l e c t r o p h o r e s i s o f p u r i f i e d c a t h e p s i n D i n p o l y a c r y l a m i d e g e l . E l e c t r o p h o r e s i s was c o n d u c t e d i n T r i s - g l y c i n e b u f f e r , pH 8 o 9 . P o t e n t i a l g r a d i e n t was 5 , 5 mA p e r s a m p l e , t i m e was 120 min
c o u l d be s e p a r a t e d step.
Besides,
low a c t i v i t y ing that authors
and d i s c a r d e d
the fractions
of cathepsin
multiple
a t t h e ammonium s u l f a t e
discarded
~ere all
characterized
D. The d a t a w e r e i n t e r p r e t e d
forms of cathepsin
( 2 ) a r e n o t p r e - f o r m e d i n viwo b u t a r i s e
Before crystallization,
to the protein
v o l u m e ) . The s l i g h t
by centrifugation.
from the e t h e r
film,
containing
autolysis
was added e t h a n o l was r e m o v e d
out in capillaries
LiO mm
w i t h t h e enzyme s o l u t i o n
membranes, f i x e d
end w i t h t h e p a r a f f i n
with solution
solution
was c a r r i e d
The c a p i l l a r i e s
c o v e r e d f r o m one end w i t h d i a l y s i s
were filled
from partial
by o t h e r
procedure.
dimness seen in the solution
Crystallization
l o n g and 2 mm i n d i a m e t e r .
by a v e r y
b y us a s i n d i c a t -
D found in chicken liver
o f t h e enzyme i n t h e c o u r s e o f t h e p r e p a r a t i o n (up t o 3 ~ b y
fractionation
"Parafilm
by e l a s t i c ~'.
rings,
and
Glass vessels
25 ml o f one o f t h e f o l l o w i n g
750
were
0.05 M
Vol. 72, No. 2, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
F i g . 3. C r y s t a l s of e a t h e p s i n D m a g n i f i e d 8 0 0 0 - f o l d .
buffers: 6.0,
citrate
(pR ~ . 0 ) ,
acetate
(pH 4 . 5 ,
6 . 5 , ? . 0 ) and 25 ml of e t h a n o l
capillaries
5.0,
(the final
5 . 5 ) o r p h o s p h a t e (pH
concentration 5~).
The
were p l a c e d i n t o t h e v e s s e l s which were s t o p p e r e d and a l l o w e d
t o s t a n d i n t h e c o l d room f o r s e v e r a l d a y s . In t h e c a p i l l a r y the vessel with the acetate buffer, condition~ closest grew ( F i g . 3 ) .
to its
pH 5 . 0 , where t h e enzyme was u n d e r
isoelectrie
In o t h e r c a p i l l a r i e s
placed into
point,
small rhomb-like crystals
t h e enzyme p r e c i p i t a t e d
a s amorphous
grains. The p r o c e d u r e f o r o b t a i n i n g l a r g e r c r y s t a l s used in X-ray structure
studies is currently
751
of c a t h e p s i n D t o b e
being developed.
VoI. 72, No. 2, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
ACKNOWLEI~IEMENTS We would like ~o thank for helpful discussions.
Dr. K.F. Firfarova and mr. M.B. fiarber
REFERENCES Io Press E.M., Porter R.R., Cebra J. Biochem. J, 74 (1960), 504. 2. lloessner J.F. In: "Tissue Proteinases", North-Iiolland Publ., 1971, p. 294. 3. Keilova H., Markovi~ 0., Keil B, Collect. Czech. Chem. Communs. 34 (1969), 2154. 4. B a r r e t t A . J . Biochem. J . 117 (1970), 601. 5. Corvol P., Oevaux C., Menard J. FEBS L e t t . 34 (1973), 189. 6. Kazako~m O.V., 0rekhovich ¥.N. Siokhimiya 40 (1975), 969. 7. Kazako~ O.Y., Orekhovich ~.~. Int. J. Pept. Prot. Res. 7 (1975), 23.
752