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A study of peptidase and esterase activity in calf lens
Exp. ,Eye Res. (1962) 1, 330-335
A Study of Peptidase and Esterase Activity in C a l f Lens*-~ ABRAHAM
SPECTOR
Howe Laboratory of Ophthalmology Harvard University Medical School Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, U.S.A. T h e first r e p o r t of p r o t e o l y t i c a c t i v i t y in t h e lens w a s m a d e b y C l a p p (1911, 1924). H e s t u d i e d w h o l e lens h o m o g e n a t e s f l o a t e d b e t w e e n l a y e r s of t o l u e n e a n d c h l o r o f o r m a n d f o u n d a v e r y slow i n c r e a s e in soluble n i t r o g e n a f t e r r e m o v a l of p r o t e i n . I n c u b a t i o n p e r i o d s of t h r e e m o n t h s w e r e n e c e s s a r y to a p p r o x i m a t e l y d o u b l e t h e n i t r o g e n values.
K r a u s e (1933) e x t e n d e d C l a p p ' s w o r k a n d s u g g e s t e d t h a t t h e r e are t w o t y p e s of e n z y m e s i n v o l v e d in t h e a u t o l y t i c process: a n e n d o p e p t i d a s e called fl-protease, w h i c h will a t t a c k lens p r o t e i n in a c i d p H a n d a s e c o n d e n z y m e , t h e a - p r o t e a s e , w h i c h a t t a c k s the breakdown products produced by the endopeptidase. The a-protease was reported to be a c t i v e a t b o t h acid a n d a l k a l i p H ' s . T h e s e conclusions w e r e b a s e d u p o n a n a l y s i s of t h e p r o t e i n a n d n o n - p r o t e i n n i t r o g e n of v a r i o u s lens f r a c t i o n s isolated f r o m whole lens h o m o g e n a t e s i n c u b a t e d a t d i f f e r e n t p H . A s w i t h C l a p p ' s w o r k long i n c u b a t i o n p e r i o d s w e r e n e c e s s a r y to d e m o n s t r a t e a u t o l y s i s . D e s p i t e c o n s i d e r a b l e i n t e r e s t in t h e s e o b s e r v a t i o n s little p r o g r e s s h a s b e e n m a d e in c h a r a c t e r i z i n g p r o t e o l y t i c a c t i v i t y in lens p r e p a r a t i o n s . :Recently Zeller a n d D e v i (1957) a n d D e v i (1959) h a v e r e p o r t e d a n e s t e r a s e a c t i v i t y in lens h o m o g e n a t e s w h i c h t h e y believe to be a s s o c i a t e d w i t h c h y m o t r y p s i n . H o w e v e r , we w e r e n o t able to d e m o n s t r a t e c h y m o t r y p t i c a c t i v i t y in lens h o m o g e n a t e s a l t h o u g h a v e r y a c t i v e e s t e r a s e a c t i v i t y w a s o b s e r v e d (Spector, 1961). A s t u d y of t h e s u b s t r a t e specificity of t h e e s t e r a s e a c t i v i t y ( T a b l e I) r e v e a l e d t h a t while L - P h e e t h y l e s t e r S a n d L - T y r e t h y l ester a r e excellent s u b s t r a t e s , b l o c k i n g t h e a m i n o g r o u p s of t h e s e c o m p o u n d s c o m p l e t e l y i n h i b i t e d t h e i r h y d r o l y s i s . F u r t h e r m o r e a r e q u i r e m e n t for m e t a l ion w a s also found. Since c h y m o t r y p s i n will a t t a c k t h e N - s u b s t i t u t e d e t h y l esters a s well as t h e free a m i n o acid e s t e r s a n d does n o t r e q u i r e m e t a l ion, it is d e f i n i t e l y n o t a s s o c i a t e d w i t h lens e s t e r a s e a c t i v i t y . T h e q u e s t i o n of t h e p h y s i o l o g i c a l role of t h i s e s t e r a s e a r o s e since t h e r e a r e no r e p o r t s of a m i n o acid ester b o n d s in lens. A n a p p r o a c h to t h i s p r o b l e m w a s s u g g e s t e d b y t h e w o r k of D e v i a n d L e r m a n (1961). T h e y r e p o r t e d a n a c t i v e lens a m i n o acid i n c o r p o r a t i n g s y s t e m w h i c h is s t i m u l a t e d t w e n t y f o l d b y t h e a d d i t i o n o f Co ++. Since t h e e s t e r a s e is also a c t i v a t e d b y t h i s m e t a l t h e p o s s i b i l i t y t h a t it m i g h t be i n v o l v e d in s u c h a s y s t e m w a s considered. I t w a s f o u n d t h a t t h e a p p a r e n t i n c o r p o r a t i o n of ~4C- labelled leucine into lens p r o t e i n i n - t h e p r e s e n c e of COC12 w a s i n d e p e n d e n t of cofactors, A T P , i n c u b a t i o n t e m p e r a t u r e a n d i n t a c t r i b o n u c l e i c acid ( T a b l e I I ) . * T h e w o r k r e p o r t e d h e r e w a s s u p p o r t e d b y g r a n t B - 1 9 0 0 f r o m t h e N a t i o n a l I n s t i t u t e of N e u r o l o g i c a l D i s e a s e s a n d :Blindness, U.S. P u b l i c H e a l t h Service. A p a r t of t h i s w o r k w a s c a r r i e d o u t a t t h e M a r i n e ]~iologieal L a b o r a t o r y , \.Voods H o l e , M a s s a c h u s e t t s , U.S.A. :~ T h e c o m m o n s h o r t h a n d a b b r e v i a t i o n s of a m i n o a c i d s p r o p o s e d b y E. B r a n d a r e u s e d in t h i s r e p o r t . O t h e r a b b r e v i a t i o n s are: a d e n o s i n e t r i p h o s p h a t e , A T ] ) ; diethylaminoet,b3¢], D E A E ; e a r b o b e n z o x y , CBZ. 330
'PEPTII)ASE
AND
ESTERASE
ACTIVITY
IN
331
LENS
F u r t h e r m o r e , w a s h i n g t h e p r o t e i n p r e c i p i t a t e with versene before d r y i n g a n d c o u n t i n g r e d u c e d t h e a c t i v i t y sixfold.* I t t h e r e f o r e seems clear t h a t the c o b a l t o u s ions are i n v o l v e d in b i n d i n g t h e labelled leucine to t h e lens protein. This is not a n e n z y m a t i c process a n d u n d e r these conditions little or no e n z y m a t i c incorporation of a m i n o acid into lens p r o t e i n occurs. TABLE I
Enzymatic hydrolysis of amino acid esters by a calf lens extract Amino ~cid ester
Relative activity
L-Try ethyl ester L-GIu d i e t h y l e s t e r y-Glu ethyl ester a-Phe ethyl ester L-Tyr ethyl ester N-aeetyl-L-Phe ethyl ester N-aeetyl-L-Tyr ethyl ester L-Leu ethyl ester L-Lys ethyl ester L-Ser ethyl ester
56 100 0 52 40 0 0 28 6 4
S p e c t o r (1961)
TABLE
II
Effect of Co ++ upon amino acid incorporation into calf lens protein Speeifie-a~tivity* zero time 1 hr
Additions
1 2 3 4 5 6 7
None 5 t~ m o l e s A T P , I /~ m o l e G T P , 5 / x m o l e s MgC12 5 /x m o l e s CoC12 a s 3 b u t p r o t e i n p p t wmshed 3 t i m e s w i t h 0. I M v e r s e n e a s 3 b u t p r o t e i n p p t w a s h e d 6 t i m e s w i t h 0.1 hi v e r s e n e a s 3 b u t i n c u b a t e d a t 0°C. a s 3 b u t h o m o g e n a t e w a s f i r s t t r e a t e d w i t h 1.0 m g of r i b o n u e l e ~ s e p e r m l h o m o g e n a t e a t 37°C f o r 1 h r
1 1
2 4
2 2 3
101 30 18 92
2
108
1
* Counts min -1 rag-' T h e r e a c t i o n s w e r e p e r f o r m e d ilx 1 m l v o l u m e s c o n t a i n i n g 0 . 5 m l of a 1 0 % l e n s h o m o g e n a g e in m e d i u m A , 0 . 1 5 m l , 0 . I ~ t r i s , p i t 8.5; 50 /xl [ 1 4 C ] - l e u e i n e , 2.3 × 106 c o u n t s t~ m o l e -1 r a i n - l ; a n d 0.3 ml. H 2 0 . M e d i u m A c o n t a i n s 2 . 5 x 1 0 - 3 N I KC1, 3.5 × 1 0 - 3 M K I - I C O a a n d 2.1 x 1 0 - 2 M s u c r o s e . T h e r e a c t i o n s w e r e s t o p p e d b y 1 0 % T C A a n d t h e p r e c i p i t a t e s p r e p a r e d f o r c o u n t i n g b y a m o d i f i c a t i o n of t h e m e t h o d of S i e k e v i t z (1952).
P e p t i d a s e a c t i v i t y has been p r e v i o u s l y observed in lens h o m o g e n a t e s (Zeller, D a i l y , W ' a k i m a n d H e r r i e k , 1951; K i n o s h i t a a n d B'[asurat, 1957). Since m a n y p e p t i d a s e s are m e t a l d e p e n d e n t , it was of i n t e r e s t to follow this a c t i v i t y d u r i n g the purification of tile esterase from calf lens. B y a c o m b i n a t i o n of zinc precipitation of * Lerm~m has now made si,nilar observations
( p r i v a t e communica.t.ion).
332
A]:~ I £ A H A M
S P]:] C T O l.~
e x t r a n e o u s p r o t e i n f o l l o w e d b y DEA:Ig c h r o m a t o g r a p h y , a p u r i f i c a t i o n o f m o r e t h a n 300-fold was a c h i e v e d ( T a b l e I l I ) . ] ) e t e r m i n a t i o n o{" tile ]?eptidasc a n d e s t e r a s e a c t i v i t i e s of t h e d i f t e r e n t f r a c t i o n s i n d i c a t e s t h a t b o t h a c t i v i t i e s are p u r i f i e d to t h e s a m e e x t e n t . T h i s is i l l u s t r a t e d b y t h e r e l a t i v e l y c o n s t a n t r a t i o o f p e p t i d a s e / e s t e r a s e a c t i v i t y d e t e r m i n e d a t d i f f e r e n t s t a g e s of p u r i f i c a t i o n . S u c h r e s u l t s s u g g e s t t h a t tll(.,. t w o a c t i v i t i e s are r e l a t e d . ~['A~LE I I I
Purification of calf lens esterase Specitic activity (~z m o l e s h y d r o l y s e d p e r m g p r o t e i n p e r rain) Esterase* Peptidase* Crude enzyrne Zinc t r e a t e d enzyme Column fractions ~47 #48 #50 #52
I)eptidase J:,sterv*s(
0.06
0.25
4.2
1.54
5.5
3.6
21.3 18. I. 11.5 9.5
107 80.0 47.0 44.0
5.0 4.4 4. l 4.6
* S u b s t r a t e s : L-Glu diethyl ester, L - L e u - G I y .
H o w e v e r , n o t all t h e p e p t i d a s e a c t i v i t y f o u n d in lens h o m o g e n a t e s is a s s o c i a t e d w i t h t h i s esterase. F o r e x a m p l e , o n l y t w o p e r c e n t of t h e i n i t i a l a c t i v i t y t o w a r d s G l y - L - L e u is f o u n d w i t h t h e c h r o m a t o g r a p h i c a l l y p u r i f i e d esterase. T h u s t h e r e is a t l e a s t o n e o t h e r p e p t i d a s e i n c a l f lens. T h e p e p t i d a s e as well as t h e e s t e r a s e h a s a n a b s o l u t e r e q u i r e m e n t for m e t a l ion. F u r t h e r m o r e t h e e x t e n t o f r e a c t i v a t i o n of t h e t w o a c t i v i t i e s a f t e r t r e a t m e n t w i t h E D T A w a s a p p r o x i m a t e l y t h e s a m e a n d in t h e o r d e r M n + + > M g + + > Co ++ ( T a b l e IV). TABLE I V
Reactivation after treatment with E19 T A MetM a d d e d
None ~ I n ++ Mg ++ Co ++
~/o O r i g i n a l a c t i v i t y l'Jsterase Peptidase 4.0 67 .~6 26
2.5 65 39 18
O t h e r m e t a l ions h a d l i t t l e or n o e f f e c t . " I t was f o u n d t h a t n e i t h e r t h e estera~se n o r t h e p e p t i d a s e a c t i v i t y w a s a f f e c t e d b y t h e u s u a l s u l p h y d r y l i n h i b i t o r s s u c h as i o d o a c e t a m i d e , N - e t h y l m a l e i m i d e or p - h y d r o x y m e r c u r i b e n z o a t e . All of t h e s e o b s e r v a t i o n s s u p p o r t t h e i d e a o f one e n z y m e b e i n g r e s p o n s i b l e for b o t h e s t e r a s e a n d p e p t i d a s e activity. I t w a s of i n t e r e s t to e x a m i n e t h e r e a c t i v i t y of t h e p u r i f i e d e n z y m e t o w a r d s a s e l e c t e d g r o u p of p e p t i d e s a n d a m i d e s ( T a b l e V). A l m o s t all of t h e c o m p o u n d s i n v e s t i g a t e d were h y d r o l y s e d to s o m e e x t e n t . T h e i n a b i l i t y of t h e e n z y m e to h y d r o l y s e
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gltds(~thiono in [)rol)al)ly d u e t o tim y c,a r l ) o x y l liill(a.ge o f tl,u glllt, nrni( +. a.(-id. 'l;tlis :q?i?i:~t'ent r e q , t i r e r n e n t for a,n a,m i n o groul)(+, t() t h e l)on(t t() I)e attm;l<{.xl is a,lso f(,In(l w i t h lJho ester s l i l ) s t r a t e s ('l?aMe ]). y-i,-Cllu, c b l l y l ester is n o t .hy(Irolys,(t w h i l e the -t I J,-(,l:t ( l i e t } i y l est;el: i8 all ex(Jell.er~b sill)st;rate. J V l ' o r o o v e l ; , l)he lli(>,k (:)f re,.x:tivity wit, ll (Yi3Z J,-Lett--(31y a g a i n c o n f i r m s tile r e q u i r e r n e t l t for a, f r e e a m i n o g r o u p ; P, ut, t,h~, e n z y m e d o e s n o t r e q l l i r e a, f r e e (J~trlJoxyl grOl.l[) sin("e a.rnides a r e g o o d Slli)strat, es. .it c i m also be co//cltlde,(l l,h~l,t t h e enzyrn(; is n o t s i m p l y a d i l ) e p t M a , se sillee l,-l,eli (Jly G l y is t h e best sut)stJrat;e s t u d i e d t.il) i,o tJio p r e s e n t t i n i e . T h e l.)road rang(: or fl.(.,tivit,.7 'FA II1,1'; V
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r e m o v a l o f a s i n g l e a m i n o a c i d a t a t i m e , T. h i s h y p o t h e s i s w a s c o n f i r m e d b y ;~ d e t a i l e d s t u d y of' t h e e n z y m e ,' s a c t i o n u p o n t h e o x i d i z e d A c h a i n o f insfilin (Fig, 1.). T h e +& c h a i n is p a r t i c u l a r l y s u i t e d f o r t h i s t y p e o f e x p e r i m e n t , s i n c e it, e o n t M n s o n l y o n e * W e w i s h t o e x p r r : a s o u r a, p p r e e i a t i o n t o D r . O t t o B e h r e n s o f t, h e E l i L i l l y ( ? o m p } ~ n y ( t r ) n a t i r , n o f the+" gl J e e r , o n Z n i n s u l i n a n d t h e o x i d i z P d A a r m P~ (:hMn+~ o f in.~utin.
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glycine a n d isoleucine residue in tile N - t e r m i n a l a n d adjaeet~t positions respectively. A similar a p p r o a c h was used b y Hill a n d S m i t h (1.957) in studies w i t h leucine a m i n o p e p t i d a s e . T h e Piez a n d Morris p r o c e d u r e (1960) was followed with a P]~oenix precision a m i n o acid analyser. Dr. Gerald Mechanic k i n d l y r a n the ana, lyses. The e x p e r i m e n t s i n d i c a t e t h a t t h e action of tile e n z y m e u p o n t h e A chain did n o t p r o d u c e a n y oligopeptides or u n e x p e c t e d a,mino acids. F u r t h e r m o r e t h e q u a n t i t i e s of a m i n o acids f o u n d ag~'eed in all cases wittl those e x p e c t e d f r o m t h e a m i n o acid sequence. [['here was no i n d i c a t i o n t h a t t h e i n t e r n a l b o n d s of the insulin chain were broken. I t was t h e r e f o r e e v i d e n t t h a t t h e e n z y m a t i c h y d r o l y s i s b e g a n a t the N - t e r m i n a l residue a n d r e m o v e d a single amino acid a t a time. In five hours 41 °/o of the A chain molecules were a t t a c k e d a n d as m a n y as 14 of t h e 21 residues h y d r o l y s e d . H o w e v e r , t h e a m o u n t s of each a m i n o acid do n o t decrease in t h e order of t h e i r sequence b u t are a r r a n g e d in groups sepa, r a t e d b y eysteic acid. T h u s t h e a m i n o acids in g r o u p A (Fig. 1) are p r e s e n t a t a level 2-~-t i m e s t h a t in g r o u p B a n d 17 t i m e s t h a t in group C.* These o b s e r v a t i o n s suggest t h a t eysteic acid b o n d s are h y d r o l y s e d slowly c o m p a r e d to the o t h e r b o n d s in t h e molecule. T h u s t h e h y d r o l y s i s would eo~nmence a t the N - t e r m i n a l glyeine a n d proceed swiftly to cysteie acid. H e r e t h e r e a c t i o n would be r e t a r d e d b u t once t h e cysteic acid residues were r e m o v e d , h y d r o l y s i s would again proceed rapidly. Since cysteie acid p e p t i d e s are n o t n o r m a l c o n s t i t u e n t s of p r o t e i n t h e i r a p p a r e n t resistance to e n z y m a t i c h y d r o l y s i s is n o t surprising. F r o m t h e o b s e r v a t i o n s r e p o r t e d here it can be c o n c l u d e d t h a t t h e e n z y m e u n d e r i n v e s t i g a t i o n is an a m i n o p e p t i d a s e w h i c h has esterase a c t i v i t y a s s o c i a t e d w i t h it. R e c e n t l y t h e r e h a v e b e e n r e p o r t s of leucine a m i n o p e p t i d a s e a c t i v i t y in lens (I~esniek, 1962; F i t t k a u , Gl~isser a n d H a n s o n , 1961). H o w e v e r , in n e i t h e r i n v e s t i g a t i o n were sufficient s u b s t r a t e s s t u d i e d to i n d i c a t e t h a t t h e a c t i v i t y o b s e r v e d wa,s a s s o c i a t e d w i t h a t r u e e x o p e p t i d a s e a n d in o n l y one s t u d y was t h e work done w i t h a purified e n z y m e . T h e a m i n o p e p t i d a s e is a c t i v a t e d b y Co ++ a n d has esterase a c t i v i t y w h e r e a s t h e r e are no r e p o r t s of such b e h a v i o u r w i t h leucine a m i n o p e p t i d a s e . H o w e v e r , t h e c o n d i t i o n s of t h e leueine a m i n o p e p t i d a s e i n v e s t i g a t i o n s were n o t t h e s a m e as t h o s e c o n d u c t e d in this l a b o r a t o r y (Hill a n d Smith,&957). Since t h e esterase a c t i v i t y v a r i e s m a r k e d l y w i t h different s u b s t r a t e s a n d since m a x i m u m a c t i v a t i o n b y different m e t a l ions m a y be p r o d u c e d a t different pIK v a l u e s no definite conclusion c o n c e r n i n g t h e r e l a t i o n s h i p of t h e t w o e n z y m e s can be m a d e a t this time. P r e l i m h l a r y a t t e m p t s to h y d r o l y s e lens p r o t e i n w i t h a m i n o p e p t i d a s e h a v e b e e n unsuccessful. I t m a y be t h a t large p r o t e i n s m u s t first be a l t e r e d p e r h a p s b y d e n a t u r a tion, o x i d a t i o n of disulphides, or even b y t h e action of e n d o p e p t i d a s e s , before becoming susceptible to a t t a c k . F o r e x a m p l e , even n a t i v e insulin is n o t n e a r l y as good a subs t r a t e as t h e oxidized A or B chain. T h e N - t e r m i n a l residues of n a t i v e ]proteins are p r o b a b l y n o t r e a d i l y accessible to t h e e n z y m e since such groups are m a s k e d or b u r i e d in t h e s u p e r s t r u c t u r e of t h e molecule. I t is now clear t h a t m u c h of t h e p e p t t d a s e a c t i v i t y of calf lens can be a t t r i b u t e d to t h e a m i n o p e p t i d a s e r e p o r t e d in this paper. A r e p o r t of e n d o p e p t i d a s e a c t i v i t y a p p e a r s elsewhere in this s y m p o s i u m . T h u s t h e original concept-of lens a u t o l y s i s as c o n c e i v e d b y K rause m a y r e s e m b l e t h e t r u e s i t u a t i o n . H o w e v e r , it is p r o b a b l e t h a t some basic s t r u c t u r a l change in t h e lens p r o t e i n m u s t first occur before it will be s u s c e p t i b l e to a t t a c k b y either endopept, i dases or e x o p e p t i d a s e s . * A full r e p o r t of tbi-s w o r k is in p r e s s .
I.'I,]I"I.'II)ASE ANI) .I']~T!!;I~AHI,; A C T I V I T Y IN I, Io;NS
335
1'~ E V E I'~ENCES Clapp, C. A. (19 l I). J. A m,e.r, mc'd. Ass. 56, 808. Clapp, C. A. (]924). Amer. J. Ophlha,l. 7, 131. Devi, A. (1959). Amcr. J. Ophthal. 48, 2!). Devi, A. and ] , e r m a , , S. (1961). Fed. Proc. 20, 378. ~'ittkau, S., GlEsser, D. and Hanson, H. (1961). lloplm S,;?]l. g. 322, 101. I-Ii]l, ]~. L. and Smith, iE. L. (1957). J. biol. Chem. 228, 577. Kinoshita, J. }I. and Masurat, T. (1957). Arch. OphH~al., Chicago 57, 266. Krause, A. C. (1933). Arch. Ophthal. Chicago, 10, 631. I)iez, K. A. and Morris, L. (1960). Anal. ]3iochem. l, 187. Resnick, It. (1962). Sixth Conferenco on Ophthalmic Biochemistry, Bosf, on, M~J,s:~ehu~..tt~, U.S.A. Siekevitz, ]?. (1952). J. biol. Chem. 195, 549. Spaekman, ]2). ]-I., Smith, E. L. and Brown, D. M. (1955). J. biol. Chem. 212, 271. Speetor, A. (]961). Exp. Eye ges. 1, 60. Zeller, E. A. and Devi, A. (1957). Amer. J. Ophthal. 44, 281. Zeller, I~. A., Daily, L., Wakin, K. G. ~nd Herriek, J. ].0. (1.951). Proc. Staff Meet., Mayo Clin. 26, 194.
A Study of Peptidase and Esterase Activity in C a l f Lens*-~ ABRAHAM
SPECTOR
Howe Laboratory of Ophthalmology Harvard University Medical School Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, U.S.A. T h e first r e p o r t of p r o t e o l y t i c a c t i v i t y in t h e lens w a s m a d e b y C l a p p (1911, 1924). H e s t u d i e d w h o l e lens h o m o g e n a t e s f l o a t e d b e t w e e n l a y e r s of t o l u e n e a n d c h l o r o f o r m a n d f o u n d a v e r y slow i n c r e a s e in soluble n i t r o g e n a f t e r r e m o v a l of p r o t e i n . I n c u b a t i o n p e r i o d s of t h r e e m o n t h s w e r e n e c e s s a r y to a p p r o x i m a t e l y d o u b l e t h e n i t r o g e n values.
K r a u s e (1933) e x t e n d e d C l a p p ' s w o r k a n d s u g g e s t e d t h a t t h e r e are t w o t y p e s of e n z y m e s i n v o l v e d in t h e a u t o l y t i c process: a n e n d o p e p t i d a s e called fl-protease, w h i c h will a t t a c k lens p r o t e i n in a c i d p H a n d a s e c o n d e n z y m e , t h e a - p r o t e a s e , w h i c h a t t a c k s the breakdown products produced by the endopeptidase. The a-protease was reported to be a c t i v e a t b o t h acid a n d a l k a l i p H ' s . T h e s e conclusions w e r e b a s e d u p o n a n a l y s i s of t h e p r o t e i n a n d n o n - p r o t e i n n i t r o g e n of v a r i o u s lens f r a c t i o n s isolated f r o m whole lens h o m o g e n a t e s i n c u b a t e d a t d i f f e r e n t p H . A s w i t h C l a p p ' s w o r k long i n c u b a t i o n p e r i o d s w e r e n e c e s s a r y to d e m o n s t r a t e a u t o l y s i s . D e s p i t e c o n s i d e r a b l e i n t e r e s t in t h e s e o b s e r v a t i o n s little p r o g r e s s h a s b e e n m a d e in c h a r a c t e r i z i n g p r o t e o l y t i c a c t i v i t y in lens p r e p a r a t i o n s . :Recently Zeller a n d D e v i (1957) a n d D e v i (1959) h a v e r e p o r t e d a n e s t e r a s e a c t i v i t y in lens h o m o g e n a t e s w h i c h t h e y believe to be a s s o c i a t e d w i t h c h y m o t r y p s i n . H o w e v e r , we w e r e n o t able to d e m o n s t r a t e c h y m o t r y p t i c a c t i v i t y in lens h o m o g e n a t e s a l t h o u g h a v e r y a c t i v e e s t e r a s e a c t i v i t y w a s o b s e r v e d (Spector, 1961). A s t u d y of t h e s u b s t r a t e specificity of t h e e s t e r a s e a c t i v i t y ( T a b l e I) r e v e a l e d t h a t while L - P h e e t h y l e s t e r S a n d L - T y r e t h y l ester a r e excellent s u b s t r a t e s , b l o c k i n g t h e a m i n o g r o u p s of t h e s e c o m p o u n d s c o m p l e t e l y i n h i b i t e d t h e i r h y d r o l y s i s . F u r t h e r m o r e a r e q u i r e m e n t for m e t a l ion w a s also found. Since c h y m o t r y p s i n will a t t a c k t h e N - s u b s t i t u t e d e t h y l esters a s well as t h e free a m i n o acid e s t e r s a n d does n o t r e q u i r e m e t a l ion, it is d e f i n i t e l y n o t a s s o c i a t e d w i t h lens e s t e r a s e a c t i v i t y . T h e q u e s t i o n of t h e p h y s i o l o g i c a l role of t h i s e s t e r a s e a r o s e since t h e r e a r e no r e p o r t s of a m i n o acid ester b o n d s in lens. A n a p p r o a c h to t h i s p r o b l e m w a s s u g g e s t e d b y t h e w o r k of D e v i a n d L e r m a n (1961). T h e y r e p o r t e d a n a c t i v e lens a m i n o acid i n c o r p o r a t i n g s y s t e m w h i c h is s t i m u l a t e d t w e n t y f o l d b y t h e a d d i t i o n o f Co ++. Since t h e e s t e r a s e is also a c t i v a t e d b y t h i s m e t a l t h e p o s s i b i l i t y t h a t it m i g h t be i n v o l v e d in s u c h a s y s t e m w a s considered. I t w a s f o u n d t h a t t h e a p p a r e n t i n c o r p o r a t i o n of ~4C- labelled leucine into lens p r o t e i n i n - t h e p r e s e n c e of COC12 w a s i n d e p e n d e n t of cofactors, A T P , i n c u b a t i o n t e m p e r a t u r e a n d i n t a c t r i b o n u c l e i c acid ( T a b l e I I ) . * T h e w o r k r e p o r t e d h e r e w a s s u p p o r t e d b y g r a n t B - 1 9 0 0 f r o m t h e N a t i o n a l I n s t i t u t e of N e u r o l o g i c a l D i s e a s e s a n d :Blindness, U.S. P u b l i c H e a l t h Service. A p a r t of t h i s w o r k w a s c a r r i e d o u t a t t h e M a r i n e ]~iologieal L a b o r a t o r y , \.Voods H o l e , M a s s a c h u s e t t s , U.S.A. :~ T h e c o m m o n s h o r t h a n d a b b r e v i a t i o n s of a m i n o a c i d s p r o p o s e d b y E. B r a n d a r e u s e d in t h i s r e p o r t . O t h e r a b b r e v i a t i o n s are: a d e n o s i n e t r i p h o s p h a t e , A T ] ) ; diethylaminoet,b3¢], D E A E ; e a r b o b e n z o x y , CBZ. 330
'PEPTII)ASE
AND
ESTERASE
ACTIVITY
IN
331
LENS
F u r t h e r m o r e , w a s h i n g t h e p r o t e i n p r e c i p i t a t e with versene before d r y i n g a n d c o u n t i n g r e d u c e d t h e a c t i v i t y sixfold.* I t t h e r e f o r e seems clear t h a t the c o b a l t o u s ions are i n v o l v e d in b i n d i n g t h e labelled leucine to t h e lens protein. This is not a n e n z y m a t i c process a n d u n d e r these conditions little or no e n z y m a t i c incorporation of a m i n o acid into lens p r o t e i n occurs. TABLE I
Enzymatic hydrolysis of amino acid esters by a calf lens extract Amino ~cid ester
Relative activity
L-Try ethyl ester L-GIu d i e t h y l e s t e r y-Glu ethyl ester a-Phe ethyl ester L-Tyr ethyl ester N-aeetyl-L-Phe ethyl ester N-aeetyl-L-Tyr ethyl ester L-Leu ethyl ester L-Lys ethyl ester L-Ser ethyl ester
56 100 0 52 40 0 0 28 6 4
S p e c t o r (1961)
TABLE
II
Effect of Co ++ upon amino acid incorporation into calf lens protein Speeifie-a~tivity* zero time 1 hr
Additions
1 2 3 4 5 6 7
None 5 t~ m o l e s A T P , I /~ m o l e G T P , 5 / x m o l e s MgC12 5 /x m o l e s CoC12 a s 3 b u t p r o t e i n p p t wmshed 3 t i m e s w i t h 0. I M v e r s e n e a s 3 b u t p r o t e i n p p t w a s h e d 6 t i m e s w i t h 0.1 hi v e r s e n e a s 3 b u t i n c u b a t e d a t 0°C. a s 3 b u t h o m o g e n a t e w a s f i r s t t r e a t e d w i t h 1.0 m g of r i b o n u e l e ~ s e p e r m l h o m o g e n a t e a t 37°C f o r 1 h r
1 1
2 4
2 2 3
101 30 18 92
2
108
1
* Counts min -1 rag-' T h e r e a c t i o n s w e r e p e r f o r m e d ilx 1 m l v o l u m e s c o n t a i n i n g 0 . 5 m l of a 1 0 % l e n s h o m o g e n a g e in m e d i u m A , 0 . 1 5 m l , 0 . I ~ t r i s , p i t 8.5; 50 /xl [ 1 4 C ] - l e u e i n e , 2.3 × 106 c o u n t s t~ m o l e -1 r a i n - l ; a n d 0.3 ml. H 2 0 . M e d i u m A c o n t a i n s 2 . 5 x 1 0 - 3 N I KC1, 3.5 × 1 0 - 3 M K I - I C O a a n d 2.1 x 1 0 - 2 M s u c r o s e . T h e r e a c t i o n s w e r e s t o p p e d b y 1 0 % T C A a n d t h e p r e c i p i t a t e s p r e p a r e d f o r c o u n t i n g b y a m o d i f i c a t i o n of t h e m e t h o d of S i e k e v i t z (1952).
P e p t i d a s e a c t i v i t y has been p r e v i o u s l y observed in lens h o m o g e n a t e s (Zeller, D a i l y , W ' a k i m a n d H e r r i e k , 1951; K i n o s h i t a a n d B'[asurat, 1957). Since m a n y p e p t i d a s e s are m e t a l d e p e n d e n t , it was of i n t e r e s t to follow this a c t i v i t y d u r i n g the purification of tile esterase from calf lens. B y a c o m b i n a t i o n of zinc precipitation of * Lerm~m has now made si,nilar observations
( p r i v a t e communica.t.ion).
332
A]:~ I £ A H A M
S P]:] C T O l.~
e x t r a n e o u s p r o t e i n f o l l o w e d b y DEA:Ig c h r o m a t o g r a p h y , a p u r i f i c a t i o n o f m o r e t h a n 300-fold was a c h i e v e d ( T a b l e I l I ) . ] ) e t e r m i n a t i o n o{" tile ]?eptidasc a n d e s t e r a s e a c t i v i t i e s of t h e d i f t e r e n t f r a c t i o n s i n d i c a t e s t h a t b o t h a c t i v i t i e s are p u r i f i e d to t h e s a m e e x t e n t . T h i s is i l l u s t r a t e d b y t h e r e l a t i v e l y c o n s t a n t r a t i o o f p e p t i d a s e / e s t e r a s e a c t i v i t y d e t e r m i n e d a t d i f f e r e n t s t a g e s of p u r i f i c a t i o n . S u c h r e s u l t s s u g g e s t t h a t tll(.,. t w o a c t i v i t i e s are r e l a t e d . ~['A~LE I I I
Purification of calf lens esterase Specitic activity (~z m o l e s h y d r o l y s e d p e r m g p r o t e i n p e r rain) Esterase* Peptidase* Crude enzyrne Zinc t r e a t e d enzyme Column fractions ~47 #48 #50 #52
I)eptidase J:,sterv*s(
0.06
0.25
4.2
1.54
5.5
3.6
21.3 18. I. 11.5 9.5
107 80.0 47.0 44.0
5.0 4.4 4. l 4.6
* S u b s t r a t e s : L-Glu diethyl ester, L - L e u - G I y .
H o w e v e r , n o t all t h e p e p t i d a s e a c t i v i t y f o u n d in lens h o m o g e n a t e s is a s s o c i a t e d w i t h t h i s esterase. F o r e x a m p l e , o n l y t w o p e r c e n t of t h e i n i t i a l a c t i v i t y t o w a r d s G l y - L - L e u is f o u n d w i t h t h e c h r o m a t o g r a p h i c a l l y p u r i f i e d esterase. T h u s t h e r e is a t l e a s t o n e o t h e r p e p t i d a s e i n c a l f lens. T h e p e p t i d a s e as well as t h e e s t e r a s e h a s a n a b s o l u t e r e q u i r e m e n t for m e t a l ion. F u r t h e r m o r e t h e e x t e n t o f r e a c t i v a t i o n of t h e t w o a c t i v i t i e s a f t e r t r e a t m e n t w i t h E D T A w a s a p p r o x i m a t e l y t h e s a m e a n d in t h e o r d e r M n + + > M g + + > Co ++ ( T a b l e IV). TABLE I V
Reactivation after treatment with E19 T A MetM a d d e d
None ~ I n ++ Mg ++ Co ++
~/o O r i g i n a l a c t i v i t y l'Jsterase Peptidase 4.0 67 .~6 26
2.5 65 39 18
O t h e r m e t a l ions h a d l i t t l e or n o e f f e c t . " I t was f o u n d t h a t n e i t h e r t h e estera~se n o r t h e p e p t i d a s e a c t i v i t y w a s a f f e c t e d b y t h e u s u a l s u l p h y d r y l i n h i b i t o r s s u c h as i o d o a c e t a m i d e , N - e t h y l m a l e i m i d e or p - h y d r o x y m e r c u r i b e n z o a t e . All of t h e s e o b s e r v a t i o n s s u p p o r t t h e i d e a o f one e n z y m e b e i n g r e s p o n s i b l e for b o t h e s t e r a s e a n d p e p t i d a s e activity. I t w a s of i n t e r e s t to e x a m i n e t h e r e a c t i v i t y of t h e p u r i f i e d e n z y m e t o w a r d s a s e l e c t e d g r o u p of p e p t i d e s a n d a m i d e s ( T a b l e V). A l m o s t all of t h e c o m p o u n d s i n v e s t i g a t e d were h y d r o l y s e d to s o m e e x t e n t . T h e i n a b i l i t y of t h e e n z y m e to h y d r o l y s e
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glycine a n d isoleucine residue in tile N - t e r m i n a l a n d adjaeet~t positions respectively. A similar a p p r o a c h was used b y Hill a n d S m i t h (1.957) in studies w i t h leucine a m i n o p e p t i d a s e . T h e Piez a n d Morris p r o c e d u r e (1960) was followed with a P]~oenix precision a m i n o acid analyser. Dr. Gerald Mechanic k i n d l y r a n the ana, lyses. The e x p e r i m e n t s i n d i c a t e t h a t t h e action of tile e n z y m e u p o n t h e A chain did n o t p r o d u c e a n y oligopeptides or u n e x p e c t e d a,mino acids. F u r t h e r m o r e t h e q u a n t i t i e s of a m i n o acids f o u n d ag~'eed in all cases wittl those e x p e c t e d f r o m t h e a m i n o acid sequence. [['here was no i n d i c a t i o n t h a t t h e i n t e r n a l b o n d s of the insulin chain were broken. I t was t h e r e f o r e e v i d e n t t h a t t h e e n z y m a t i c h y d r o l y s i s b e g a n a t the N - t e r m i n a l residue a n d r e m o v e d a single amino acid a t a time. In five hours 41 °/o of the A chain molecules were a t t a c k e d a n d as m a n y as 14 of t h e 21 residues h y d r o l y s e d . H o w e v e r , t h e a m o u n t s of each a m i n o acid do n o t decrease in t h e order of t h e i r sequence b u t are a r r a n g e d in groups sepa, r a t e d b y eysteic acid. T h u s t h e a m i n o acids in g r o u p A (Fig. 1) are p r e s e n t a t a level 2-~-t i m e s t h a t in g r o u p B a n d 17 t i m e s t h a t in group C.* These o b s e r v a t i o n s suggest t h a t eysteic acid b o n d s are h y d r o l y s e d slowly c o m p a r e d to the o t h e r b o n d s in t h e molecule. T h u s t h e h y d r o l y s i s would eo~nmence a t the N - t e r m i n a l glyeine a n d proceed swiftly to cysteie acid. H e r e t h e r e a c t i o n would be r e t a r d e d b u t once t h e cysteic acid residues were r e m o v e d , h y d r o l y s i s would again proceed rapidly. Since cysteie acid p e p t i d e s are n o t n o r m a l c o n s t i t u e n t s of p r o t e i n t h e i r a p p a r e n t resistance to e n z y m a t i c h y d r o l y s i s is n o t surprising. F r o m t h e o b s e r v a t i o n s r e p o r t e d here it can be c o n c l u d e d t h a t t h e e n z y m e u n d e r i n v e s t i g a t i o n is an a m i n o p e p t i d a s e w h i c h has esterase a c t i v i t y a s s o c i a t e d w i t h it. R e c e n t l y t h e r e h a v e b e e n r e p o r t s of leucine a m i n o p e p t i d a s e a c t i v i t y in lens (I~esniek, 1962; F i t t k a u , Gl~isser a n d H a n s o n , 1961). H o w e v e r , in n e i t h e r i n v e s t i g a t i o n were sufficient s u b s t r a t e s s t u d i e d to i n d i c a t e t h a t t h e a c t i v i t y o b s e r v e d wa,s a s s o c i a t e d w i t h a t r u e e x o p e p t i d a s e a n d in o n l y one s t u d y was t h e work done w i t h a purified e n z y m e . T h e a m i n o p e p t i d a s e is a c t i v a t e d b y Co ++ a n d has esterase a c t i v i t y w h e r e a s t h e r e are no r e p o r t s of such b e h a v i o u r w i t h leucine a m i n o p e p t i d a s e . H o w e v e r , t h e c o n d i t i o n s of t h e leueine a m i n o p e p t i d a s e i n v e s t i g a t i o n s were n o t t h e s a m e as t h o s e c o n d u c t e d in this l a b o r a t o r y (Hill a n d Smith,&957). Since t h e esterase a c t i v i t y v a r i e s m a r k e d l y w i t h different s u b s t r a t e s a n d since m a x i m u m a c t i v a t i o n b y different m e t a l ions m a y be p r o d u c e d a t different pIK v a l u e s no definite conclusion c o n c e r n i n g t h e r e l a t i o n s h i p of t h e t w o e n z y m e s can be m a d e a t this time. P r e l i m h l a r y a t t e m p t s to h y d r o l y s e lens p r o t e i n w i t h a m i n o p e p t i d a s e h a v e b e e n unsuccessful. I t m a y be t h a t large p r o t e i n s m u s t first be a l t e r e d p e r h a p s b y d e n a t u r a tion, o x i d a t i o n of disulphides, or even b y t h e action of e n d o p e p t i d a s e s , before becoming susceptible to a t t a c k . F o r e x a m p l e , even n a t i v e insulin is n o t n e a r l y as good a subs t r a t e as t h e oxidized A or B chain. T h e N - t e r m i n a l residues of n a t i v e ]proteins are p r o b a b l y n o t r e a d i l y accessible to t h e e n z y m e since such groups are m a s k e d or b u r i e d in t h e s u p e r s t r u c t u r e of t h e molecule. I t is now clear t h a t m u c h of t h e p e p t t d a s e a c t i v i t y of calf lens can be a t t r i b u t e d to t h e a m i n o p e p t i d a s e r e p o r t e d in this paper. A r e p o r t of e n d o p e p t i d a s e a c t i v i t y a p p e a r s elsewhere in this s y m p o s i u m . T h u s t h e original concept-of lens a u t o l y s i s as c o n c e i v e d b y K rause m a y r e s e m b l e t h e t r u e s i t u a t i o n . H o w e v e r , it is p r o b a b l e t h a t some basic s t r u c t u r a l change in t h e lens p r o t e i n m u s t first occur before it will be s u s c e p t i b l e to a t t a c k b y either endopept, i dases or e x o p e p t i d a s e s . * A full r e p o r t of tbi-s w o r k is in p r e s s .
I.'I,]I"I.'II)ASE ANI) .I']~T!!;I~AHI,; A C T I V I T Y IN I, Io;NS
335
1'~ E V E I'~ENCES Clapp, C. A. (19 l I). J. A m,e.r, mc'd. Ass. 56, 808. Clapp, C. A. (]924). Amer. J. Ophlha,l. 7, 131. Devi, A. (1959). Amcr. J. Ophthal. 48, 2!). Devi, A. and ] , e r m a , , S. (1961). Fed. Proc. 20, 378. ~'ittkau, S., GlEsser, D. and Hanson, H. (1961). lloplm S,;?]l. g. 322, 101. I-Ii]l, ]~. L. and Smith, iE. L. (1957). J. biol. Chem. 228, 577. Kinoshita, J. }I. and Masurat, T. (1957). Arch. OphH~al., Chicago 57, 266. Krause, A. C. (1933). Arch. Ophthal. Chicago, 10, 631. I)iez, K. A. and Morris, L. (1960). Anal. ]3iochem. l, 187. Resnick, It. (1962). Sixth Conferenco on Ophthalmic Biochemistry, Bosf, on, M~J,s:~ehu~..tt~, U.S.A. Siekevitz, ]?. (1952). J. biol. Chem. 195, 549. Spaekman, ]2). ]-I., Smith, E. L. and Brown, D. M. (1955). J. biol. Chem. 212, 271. Speetor, A. (]961). Exp. Eye ges. 1, 60. Zeller, E. A. and Devi, A. (1957). Amer. J. Ophthal. 44, 281. Zeller, I~. A., Daily, L., Wakin, K. G. ~nd Herriek, J. ].0. (1.951). Proc. Staff Meet., Mayo Clin. 26, 194.