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

Crystallization of cathepsin D.

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...
1MB Sizes 0 Downloads 0 Views