772
Specialia
a d e c r e a s e is seen of t h e c o n t e n t of A T P a n d G-6-P, a n d
a n i n c r e a s e of t h e A D P a n d A M P c o n t e n t in t h e c a r d i a c muscle. T h e s e r e s u l t s are c o n s i s t e n t w i t h a n i n c r e a s i n g p h o s p h o f r u c t o k i n a s e a c t i v i t y in t h e cell. T a b l e I1 shows a serie of e x p e r i m e n t s in w h i c h all t h e h e a r t s were p e r f u s e d for 10 min. T h e zero t i m e r e p r e s e n t s t h e c o n t r o l s in w h i c h t h e h e a r t s were p e r f u s e d for 10 r a i n w i t h t h e D N P - f r e e buffer. T h e periods of 1x/2 a n d 5.0 m i n r e p r e s e n t d e t e r m i n a t i o n s in w h i c h t h e h e a r t s were p r e l i m i n a r y p e r f u s e d for 81/2 a n d 5.0 m i n r e s p e c t i v e l y w i t h D N P - f r e e buffer, a f t e r w h i c h t h e y were r a p i d l y s w i t c h e d t o a b u f f e r c o n t a i n i n g D N P . T i m e 10 r a i n r e p r e s e n t s d e t e r m i n a t i o n s i n w h i c h t h e h e a r t s were p e r f u s e d for 10 rain w i t h a D N P - c o n t a i n i n g buffer. As c a n b e seen, a f t e r lz/2 m i n of p e r f u s i o n w i t h D N P t h e c o n t e n t of G - 6 - P in t h e m u s c l e was f o u n d t o b e lower, a n d a t t h e s a m e t i m e o n l y a s m a l l i n c r e a s e in t h e c o n t e n t of p h o s p h o r y l a s e a could b e noticed. F r o m t h e s e results, i t was possible t o asgume t h a t t h e g l y c o g e n o l y t i c effect of D N P is n o t e x p l a i n e d b y t h e ' p u s h m e c h a n i s m ' , w h e r e t h e G - 6 - P level is f o u n d t o b e h i g h e r t h a n in t h e case of c a t e c h o l a m i n e s 4. T h e G - 6 - P a n d n u c l e o t i d e s levels f o u n d a f t e r a s h o r t p e r i o d of e x p o s i t i o n of t h e h e a r t s t o t h e d r u g (11/2 min), suggest t h a t t h e D N P m a y cause p r i m a r y a c t i v a t i o n of t h e p h o s p h o f r u c t o k i n a s e , a n d in c o n s e q u e n c e a h i g h e r r a t e of glycogenolysis. O n t h e o t h e r h a n d , c o n s i d e r i n g t h a t G - 6 - P is a n i n h i b i t o r of p h o s p h o r y l a s e b k i n a s e a c t i v i t y 12, t h e low levels of t h i s
EXPERIEN`TIA31/7
m e t a b o l i t e w o u l d b e r e s p o n s i b l e for t h e h i g h e r a c t i v i t y of t h e p h o s p h o r y l a s e b k i n a s e f o u n d in t h e h e a r t s p e r f u s e d with DNP. S t u d i e s to b e d e s c r i b e d elsewhere s h o w t h a t t h e G - 6 - P c o n c e n t r a t i o n o b s e r v e d i n t o t h e c a r d i a c cell in n o r m a l c o n d i t i o n s of p e r f u s e d h e a r t s causes s i g n i f i c a n t i n h i b i t i o n of t h e p h o s p h o r y l a s e b kinase.
Summary. To e x p l a i n t h e m e c h a n i s m of D N P a c t i o n t h e c o n t e n t s of A T P , A D P , A M P a n d G - 6 - P were d e t e r m i n e d in p e r f u s e d r a t h e a r t s in a p e r i o d of t i m e in w h i c h t h e r a t e of t h e glycogenolysis w a s increased. T h e levels of t h e s e m e t a b o l y t e s in t h e e x t r a c t were c o n s i s t e n t w i t h a n increase of t h e p h o s p h o f r u c t o k i n a s e a c t i v i t y . On t h e o t h e r h a n d , t h e f i n d i n g of h i g h e r a c t i v i t y of p h o s p h o r y l a s e b k i n a s e in D N P - p o i s o n e d a n i m a l s could b e e x p l a i n e d as d u e to t h e low c o n t e n t of G - 6 - P i n t h e p e r f u s e d h e a r t s subj e c t e d t o t h e a c t i o n of t h e drug. A. E. VERCESI a n d A. F o c E s I JR.
Departamento de Bioquimica, Instituto de Biologia da Universidade Est. de Campinas, C.P. 1170, Campinas (S. Paulo, Brazil), 7 January ]975. I{REBS~D. S. LovE, G. E. BRATVOLD,K. A. TRAYSER,W. L. ~I/[EYERand t~. H. FISCHER, Biochemistry 3, 1022 (1964).
I2 E. G.
P r e p a r a t i o n and P r o p e r t i e s of a S o l u b l e T r y p s i n - D e x t r a n C o n j u g a t e T h e p r o p e r t i e s of insoluble, i m m o b i l i z e d e n z y m e s h a v e b e e n e x t e n s i v e l y i n v e s t i g a t e d a n d d o c u m e n t e d z. B y c o m p a r i s o n , h o w e v e r , d a t a a v a i l a b l e for e n z y m e s i m m o bilized in s o l u t i o n are l i m i t e d , a l t h o u g h e a r l y i n v e s t i g a t i o n s b y MITZ a n d SUMMARIA 2 i n d i c a t e d t h a t soluble c h y m o t r y p s i n - c e l l u l o s e c o n j u g a t e s e x h i b i t e d h i g h e r cata l y t i c a c t i v i t i e s t h a n t h e i r insoluble c o u n t e r p a r t s . More r e c e n t l y several c h y m o t r y p s i n - d e x t r a n g r a f t c o - p o l y m e r s h a v e b e e n s h o w n t o possess i n c r e a s e d s t a b i l i t y w i t h u n a l t e r e d c a t a l y t i c p r o p e r t i e s 3, 4. T r y p s i n (EC 3.4.31.4) c o v a l e n t l y a t t a c h e d to n e u t r a l p o l y a l d e h y d e d e x t r a n is also m o r e s t a b l e t h a n t h e free e n z y m e b u t its a c t i v i t y t o w a r d s p o l y m e r i c s u b s t r a t e s a n d
10C %
/
>. 80 ,>_
70
6(]
5C
9
1'0
11pH
pH-aetivity profiles for trypsin, I = 0,17 (9 acetaldehydetrypsin, I = 0.17 (O); PADT, I = 0.17 (~)); PADT, I = 1.17 (@). All reaction mixtures contained 0.1 mg/ml protein and 1.27 • 10-8 M BAPNA.
its pI-I-rate profile h a v e b o t h b e e n a l t e r e d b y i m m o b i lization. D e x t r a n TT0 ( P h a r m a c i a , G.B., L t d ) was oxidized b y a n acidic s o l u t i o n of s o d i u m p e r i o d a t e for 16 h, 25 ~ in t h e d a r k . T h e p o l y a l d e h y d e d e x t r a n ( P A D ) so f o r m e d was r e c o v e r e d b y dialysis a n d l y o p h i l i z a t i o n . T r y p s i n (Boer i n g e r C o r p o r a t i o n , L o n d o n , Ltd.) was stirred w i t h a n excess of P A D for 24 h a t 5~ in 0.1 M c i t r a t e b u f f e r pI-I 7.0. No insoluble m a t e r i a l was f o r m e d d u r i n g conj u g a t i o n a n d t h e soluble p o l y a l d e h y d e d e x t r a n - t r y p s i n c o n j u g a t e ( P A D T ) was i s o l a t e d b y gel c h r o m a t o g r a p h y . R e c h r o m a t o g r a p h y of t h e i s o l a t e d c o m p l e x a t h i g h ionic s t r e n g h t h a d n o effect o n t h e p r o t e i n c o n c e n t r a t i o n i n t h e complex. A t y p i c a l p r e p a r a t i o n of t h e P A D T c o m p l e x c o n t a i n e d 0.41% n i t r o g e n e q u i v a l e n t t o a p p r o x i m a t e l y 3 % t r y p s i n (w/w). T r y p t i c a c t i v i t y was a s s a y e d s p e c t r o p h o t o m e t r i c a l l y a t 30 ~ w i t h benzoyl-D, L-arginine p - n i t r a n i l i d e h y d r o c h l o ride ( B A P N A ) (Boeringer C o r p o r a t i o n , L o n d o n , Ltd.) A p p a r e n t M i c h a e l i s - M e n t e n p a r a m e t e r s for B A P N A were e s t i m a t e d f r o m L i n e w e a v e r - B u r k p l o t s a n d were r e f i n e d u s i n g a p r o c e d u r e s i m i l a r t o t h a t of WILKINSON 5. T h e p r o t e o l y t i c a c t i v i t y of t r y p s i n was e s t i m a t e d b y its c a t a l y z e d release of T C A soluble p r o d u c t s f r o m 0.5% casein s o l u t i o n 6. 1 E. KATCHALSKI,I. H. SILMAN and R. GOLDMAN',Advances in Enzymology (Ed. F. F. NORD; Academic Press, New York 1971), vol. 34, p. 465. 2 M. A. MITZ and L. J. SUMMARIA,Nature, Lond. 189, 576, (1961). 3 S. P. O'NEILL, J. R. WYKES, P. DONNILand iV[.D. LILLY, Biotech. Bioeng. 73, 319 (1971). 4 R. AxEN, P.-A. MYRIN and J.-C. JANSEN, Biopolymers 9, 401 (1970). 5 j . WILKINSON, Biochem. J. 80, 324 (1961). 6 H. BERGMEYI~R,Methods o/Enzymatic Analysis (Academic Press, New York 1963), p. 811.
15. 7. 1975
Specialia
The single stage coupling of t r y p s i n to t h e p r e - a c t i v a t e d d e x t r a n m a t r i x is c o m p l e t e w i t h i n 24 h a t 5 ~ as s h o w n b y t h e a b s e n c e of free e n z y m e on gel c h r o m a t o g r a p h y . T h e r e s u l t a n t g r a f t is s t a b l e a t r o o m t e m p e r a t u r e in a q u e o u s solutions of n e u t r a l p H . The Figure shows p H - r a t e profiles for t h e h y d r o l y s i s of B A P N A c a t a l y z e d b y free a n d b o u n d t r y p s i n a t ionic s t r e n g t h s of 0.17 a n d 1.17. The p H o p t i m u m is displaced b y a p p r o x i m a t e l y 1 p H u n i t t o w a r d s m o r e alkaline values b y c o n j u g a t i o n . A similar shift t o w a r d s h i g h e r pI-I values h a s b e e n o b s e r v e d p r e v i o u s l y for t r y p s i n - p o l y e l e c t r o l y t e d e r i v a t i v e s a n d in t h a t case was a t t r i b u t e d to t h e neighb o u r i n g carrier b o u n d c h a r g e d g r o u p s 7. F o r t h i s s y s t e m such a n e x p l a n a t i o n is i n a d e q u a t e . O x i d a t i o n of polysaccarides b y s o d i u m m e t a p e r i o d a t e , u n d e r the a b o v e c o n d i t i o n s t e r m i n a t e s a t t h e a l d e h y d e level of o x i d a t i o n , a n d t h e p H shift is n o t r e d u c e d b y t h e large c h a n g e in ionic s t r e n g t h s The r e s u l t a n t p H - a c t i v i t y profile a f t e r t r e a t m e n t of t r y p s i n w i t h excess a c e t a l d e h y d e was d e t e r m i n e d in o r d e r t o s e p a r a t e t h e effects due t o t h e m a t r i x f r o m t h o s e due to chemicaI m o d i f i c a t i o n of t h e p r o t e i n molecule
Table I. Apparent Michaelis constants for trypsin catalyzed hydrolysis of BAPNA in 0.1 M verona1 buffers, plus 0.02 M calcium chloride, 30 ~
Trypsin PADT
K(~,v~) ( • 104 M) pH 8.0
pH 8.85
7.40 5.60
5.80 5.95
Table If. Percentage retention of tryptie activity towards BAPNA, at 30~ in 0.1 M citrate buffer pH 7.0 (protein concentration, 1 rag/ ml). 3 days Trypsin Trypsin plus 0.02 M CaCle PADT PADT plus 0.02 M CaC12
2 14 46 53
773
(Figure). I n a g r e e m e n t w i t h LABOUESSE a n d GERVAIS 9 a small alkaline s h i f t was observed, b u t smaller t h a n t h a t caused b y t h e p o l y a l d e h y d e d e x t r a n . The m a t r i x h a d no effect, w i t h i n e x p e r i m e n t a l error, on t h e s u b s t r a t e d e p e n d a n t kinetics of t r y p s i n . B o t h free a n d a t t a c h e d enzyule o b e y e d t h e M i c h a e l i s - M e n t e n e q u a t i o n for t h e h y d r o l y s i s of B A P N A o v e r t h e r a n g e 0.1-1.27 • l 0 -a M ; t h e p a r a m e t e r s d e t e r m i n e d a t each p H o p t i m u m are s h o w n in T a b l e I. I m m o b i l i z a t i o n i m p a r t s increased r e s i s t a n c e to a u t o lysis (Table II). T r y p s i n loses 98% of its a c t i v y w i t h i n 72 h at 30 ~ p H 7.0, b u t s o m e w h a t less (86 %) in t h e presence of 0.2 M calcium chloride. B y c o n t r a s t t h e conjug a t e r e t a i n s 42% of its initial a c t i v i t y e v e n a f t e r 34 d a y s u n d e r t h e s a m e c o n d i t i o n s ; calcium ions also h a v e a small stabilizing effect on t h e conjugate, T a b l e I (care w a s t a k e n to a v o i d b a c t e r i a l c o n t a m i n a t i o n ) . This r e d u c e d a u t o l y t i c c a p a b i l i t y of t h e carrier b o u n d e n z y m e is t h e result of t w o m a i n factors. B y a n a l o g y w i t h t h e usual r e a c t i o n p r o d u c t s of a l d e h y d e s w i t h p r o t e i n s , t h e e x p e c t e d sites of r e a c t i o n w o u l d be to t h e E - a m i n o g r o u p s of Iysine so p r e v e n t i n g t h e l a t t e r f r o m b i n d i n g t o t h e active sites of o t h e r t r y p s i n molecules. I n a d d i t i o n , t h e p r e s e n c e of t h e a t t a c h e d d e x t r a n will sterically h i n d e r t h e m u t u a l a p p r o a c h of t r y p s i n molecules. This effect is p r o b a b l y also r e s p o n s i b l e for t h e d e c r e a s e d d e g r a d a t i o n of casein b y t h e c o n j u g a t e w h i c h is only 4% of t h a t of t h e native enzyme. As a r e q u i s i t e t o f u r t h e r e l u c i d a t i o n of t h e o b s e r v e d kinetic p r o p e r t i e s of t h e c o n j u g a t e , its s t r u c t u r e is u n d e r investigation.
Zusammen[assung. D u r c h k o v a l e n t e B i n d u n g a n P o l y a l d e h y d - d e x t r a n wird T r y p s i n immobilisiert. Die polym e r i s c h e M a t r i x stabilisiert das E n z y m u n d b e w i r k t eine p H - E r h 6 h u n g der m a x i m a l e n Aktivit/it, b e e i n f l u s s t a b e r d e n M i c h a e l i s - M e n t e n P a r a m e t e r fiir die I-tydrolyse des S u b s t r a t e s B A P N A nicht. 1~. L. FOSTER 10 School o/ Pharmacy, Liverpool Polytechnic, Liverpool L3 3 A F (England), 26 March 7975.
34 days 0 0 42 55
T. L. WESTMAN, Biochem. biophys. Res. Commun. 35, 313 (1969). 8 M. PECHT and Y. LEVlN, Biochem. biophys. Res. Commun. d6, 2054 (1972). J. LA~OUESSEand 1~{.GERVAIS, Eur. J. Biochein. 2, 215 (1967). 10 The author gratefully acknowledges the valuable discussions with Dr. A. R. THOMSON, A.E.R.E., Harwell.
Alteration of Erythrocyte (Na + + K+)-ATPase by Replacement of Cholesterol by Desmosterol in the Membrane I t was s h o w n b y us t h a t t r e a t m e n t of r a t s w i t h a n i n h i b i t o r of d e s m o s t e r o l r e d u c t a s e , i.e. 20.25-diazacholesterol, results in a n increased specific (Na+ + K+)A T P a s e a c t i v i t y in m e m b r a n e s of d i f f e r e n t tissues. S u c h a n effect was s h o w n for t h e s a r c o l e m m a of skeletal a n d cardiac muscle 1, 2 a n d e r y t h r o c y t e g h o s t s 3, ,. P r e l i m i n a r y s t u d i e s s suggest t h a t t h e a l t e r a t i o n in (Na+ + K+)A T P a s e a c t i v i t y is due to t h e r e p l a c e m e n t of cholesterol b y d e m o s t e r o l in t h e m e m b r a n e s of a n i m a l s t r e a t e d w i t h this substance. Male ~ ' i s t a r r a t s were daily t r e a t e d w i t h 10 m g 20.25d i a z a c h o l e s t e r o l d i h y d r o c h l o r i d e in 0.2 m l of w a t e r given b y a n oesophageal c a n n u l a for a p e r i o d of 8 weeks. Control
a n i m a l s were s u b j e c t e d to t h e s a m e p r o c e d u r e using 0.2 m l of w a t e r only. Blood was d r a w n f r o m one t r e a t e d a n d one c o n t r o l a n i m a l into h e p a r i n i z e d syringes b y aortic p u n c t u r e of t h e a n e s t h e t i z e d animal. P l a s m a a n d red t j. B. PETER and W. FIEHN, Science 179, 910 (1973). 2 W. F1EHN, D. SEILER, E. KUHN and DOROTHEA BARTELS, Eur. J. elin. Invest., in press. 3 W. FIEnN, E. Kumr and I. GELDMACHER, FEBS Left. 3d, 163 (1973). J. B. PETER, R. M. ANmMAN, R. L. BOWMANSand T. NAGAMOTO, Expl. Neurol. dl, 738 (1973). 5 D. SELLER,W. FIEHr~ and E. K~JnN, Z. klin. Chem., in press.
Specialia
a d e c r e a s e is seen of t h e c o n t e n t of A T P a n d G-6-P, a n d
a n i n c r e a s e of t h e A D P a n d A M P c o n t e n t in t h e c a r d i a c muscle. T h e s e r e s u l t s are c o n s i s t e n t w i t h a n i n c r e a s i n g p h o s p h o f r u c t o k i n a s e a c t i v i t y in t h e cell. T a b l e I1 shows a serie of e x p e r i m e n t s in w h i c h all t h e h e a r t s were p e r f u s e d for 10 min. T h e zero t i m e r e p r e s e n t s t h e c o n t r o l s in w h i c h t h e h e a r t s were p e r f u s e d for 10 r a i n w i t h t h e D N P - f r e e buffer. T h e periods of 1x/2 a n d 5.0 m i n r e p r e s e n t d e t e r m i n a t i o n s in w h i c h t h e h e a r t s were p r e l i m i n a r y p e r f u s e d for 81/2 a n d 5.0 m i n r e s p e c t i v e l y w i t h D N P - f r e e buffer, a f t e r w h i c h t h e y were r a p i d l y s w i t c h e d t o a b u f f e r c o n t a i n i n g D N P . T i m e 10 r a i n r e p r e s e n t s d e t e r m i n a t i o n s i n w h i c h t h e h e a r t s were p e r f u s e d for 10 rain w i t h a D N P - c o n t a i n i n g buffer. As c a n b e seen, a f t e r lz/2 m i n of p e r f u s i o n w i t h D N P t h e c o n t e n t of G - 6 - P in t h e m u s c l e was f o u n d t o b e lower, a n d a t t h e s a m e t i m e o n l y a s m a l l i n c r e a s e in t h e c o n t e n t of p h o s p h o r y l a s e a could b e noticed. F r o m t h e s e results, i t was possible t o asgume t h a t t h e g l y c o g e n o l y t i c effect of D N P is n o t e x p l a i n e d b y t h e ' p u s h m e c h a n i s m ' , w h e r e t h e G - 6 - P level is f o u n d t o b e h i g h e r t h a n in t h e case of c a t e c h o l a m i n e s 4. T h e G - 6 - P a n d n u c l e o t i d e s levels f o u n d a f t e r a s h o r t p e r i o d of e x p o s i t i o n of t h e h e a r t s t o t h e d r u g (11/2 min), suggest t h a t t h e D N P m a y cause p r i m a r y a c t i v a t i o n of t h e p h o s p h o f r u c t o k i n a s e , a n d in c o n s e q u e n c e a h i g h e r r a t e of glycogenolysis. O n t h e o t h e r h a n d , c o n s i d e r i n g t h a t G - 6 - P is a n i n h i b i t o r of p h o s p h o r y l a s e b k i n a s e a c t i v i t y 12, t h e low levels of t h i s
EXPERIEN`TIA31/7
m e t a b o l i t e w o u l d b e r e s p o n s i b l e for t h e h i g h e r a c t i v i t y of t h e p h o s p h o r y l a s e b k i n a s e f o u n d in t h e h e a r t s p e r f u s e d with DNP. S t u d i e s to b e d e s c r i b e d elsewhere s h o w t h a t t h e G - 6 - P c o n c e n t r a t i o n o b s e r v e d i n t o t h e c a r d i a c cell in n o r m a l c o n d i t i o n s of p e r f u s e d h e a r t s causes s i g n i f i c a n t i n h i b i t i o n of t h e p h o s p h o r y l a s e b kinase.
Summary. To e x p l a i n t h e m e c h a n i s m of D N P a c t i o n t h e c o n t e n t s of A T P , A D P , A M P a n d G - 6 - P were d e t e r m i n e d in p e r f u s e d r a t h e a r t s in a p e r i o d of t i m e in w h i c h t h e r a t e of t h e glycogenolysis w a s increased. T h e levels of t h e s e m e t a b o l y t e s in t h e e x t r a c t were c o n s i s t e n t w i t h a n increase of t h e p h o s p h o f r u c t o k i n a s e a c t i v i t y . On t h e o t h e r h a n d , t h e f i n d i n g of h i g h e r a c t i v i t y of p h o s p h o r y l a s e b k i n a s e in D N P - p o i s o n e d a n i m a l s could b e e x p l a i n e d as d u e to t h e low c o n t e n t of G - 6 - P i n t h e p e r f u s e d h e a r t s subj e c t e d t o t h e a c t i o n of t h e drug. A. E. VERCESI a n d A. F o c E s I JR.
Departamento de Bioquimica, Instituto de Biologia da Universidade Est. de Campinas, C.P. 1170, Campinas (S. Paulo, Brazil), 7 January ]975. I{REBS~D. S. LovE, G. E. BRATVOLD,K. A. TRAYSER,W. L. ~I/[EYERand t~. H. FISCHER, Biochemistry 3, 1022 (1964).
I2 E. G.
P r e p a r a t i o n and P r o p e r t i e s of a S o l u b l e T r y p s i n - D e x t r a n C o n j u g a t e T h e p r o p e r t i e s of insoluble, i m m o b i l i z e d e n z y m e s h a v e b e e n e x t e n s i v e l y i n v e s t i g a t e d a n d d o c u m e n t e d z. B y c o m p a r i s o n , h o w e v e r , d a t a a v a i l a b l e for e n z y m e s i m m o bilized in s o l u t i o n are l i m i t e d , a l t h o u g h e a r l y i n v e s t i g a t i o n s b y MITZ a n d SUMMARIA 2 i n d i c a t e d t h a t soluble c h y m o t r y p s i n - c e l l u l o s e c o n j u g a t e s e x h i b i t e d h i g h e r cata l y t i c a c t i v i t i e s t h a n t h e i r insoluble c o u n t e r p a r t s . More r e c e n t l y several c h y m o t r y p s i n - d e x t r a n g r a f t c o - p o l y m e r s h a v e b e e n s h o w n t o possess i n c r e a s e d s t a b i l i t y w i t h u n a l t e r e d c a t a l y t i c p r o p e r t i e s 3, 4. T r y p s i n (EC 3.4.31.4) c o v a l e n t l y a t t a c h e d to n e u t r a l p o l y a l d e h y d e d e x t r a n is also m o r e s t a b l e t h a n t h e free e n z y m e b u t its a c t i v i t y t o w a r d s p o l y m e r i c s u b s t r a t e s a n d
10C %
/
>. 80 ,>_
70
6(]
5C
9
1'0
11pH
pH-aetivity profiles for trypsin, I = 0,17 (9 acetaldehydetrypsin, I = 0.17 (O); PADT, I = 0.17 (~)); PADT, I = 1.17 (@). All reaction mixtures contained 0.1 mg/ml protein and 1.27 • 10-8 M BAPNA.
its pI-I-rate profile h a v e b o t h b e e n a l t e r e d b y i m m o b i lization. D e x t r a n TT0 ( P h a r m a c i a , G.B., L t d ) was oxidized b y a n acidic s o l u t i o n of s o d i u m p e r i o d a t e for 16 h, 25 ~ in t h e d a r k . T h e p o l y a l d e h y d e d e x t r a n ( P A D ) so f o r m e d was r e c o v e r e d b y dialysis a n d l y o p h i l i z a t i o n . T r y p s i n (Boer i n g e r C o r p o r a t i o n , L o n d o n , Ltd.) was stirred w i t h a n excess of P A D for 24 h a t 5~ in 0.1 M c i t r a t e b u f f e r pI-I 7.0. No insoluble m a t e r i a l was f o r m e d d u r i n g conj u g a t i o n a n d t h e soluble p o l y a l d e h y d e d e x t r a n - t r y p s i n c o n j u g a t e ( P A D T ) was i s o l a t e d b y gel c h r o m a t o g r a p h y . R e c h r o m a t o g r a p h y of t h e i s o l a t e d c o m p l e x a t h i g h ionic s t r e n g h t h a d n o effect o n t h e p r o t e i n c o n c e n t r a t i o n i n t h e complex. A t y p i c a l p r e p a r a t i o n of t h e P A D T c o m p l e x c o n t a i n e d 0.41% n i t r o g e n e q u i v a l e n t t o a p p r o x i m a t e l y 3 % t r y p s i n (w/w). T r y p t i c a c t i v i t y was a s s a y e d s p e c t r o p h o t o m e t r i c a l l y a t 30 ~ w i t h benzoyl-D, L-arginine p - n i t r a n i l i d e h y d r o c h l o ride ( B A P N A ) (Boeringer C o r p o r a t i o n , L o n d o n , Ltd.) A p p a r e n t M i c h a e l i s - M e n t e n p a r a m e t e r s for B A P N A were e s t i m a t e d f r o m L i n e w e a v e r - B u r k p l o t s a n d were r e f i n e d u s i n g a p r o c e d u r e s i m i l a r t o t h a t of WILKINSON 5. T h e p r o t e o l y t i c a c t i v i t y of t r y p s i n was e s t i m a t e d b y its c a t a l y z e d release of T C A soluble p r o d u c t s f r o m 0.5% casein s o l u t i o n 6. 1 E. KATCHALSKI,I. H. SILMAN and R. GOLDMAN',Advances in Enzymology (Ed. F. F. NORD; Academic Press, New York 1971), vol. 34, p. 465. 2 M. A. MITZ and L. J. SUMMARIA,Nature, Lond. 189, 576, (1961). 3 S. P. O'NEILL, J. R. WYKES, P. DONNILand iV[.D. LILLY, Biotech. Bioeng. 73, 319 (1971). 4 R. AxEN, P.-A. MYRIN and J.-C. JANSEN, Biopolymers 9, 401 (1970). 5 j . WILKINSON, Biochem. J. 80, 324 (1961). 6 H. BERGMEYI~R,Methods o/Enzymatic Analysis (Academic Press, New York 1963), p. 811.
15. 7. 1975
Specialia
The single stage coupling of t r y p s i n to t h e p r e - a c t i v a t e d d e x t r a n m a t r i x is c o m p l e t e w i t h i n 24 h a t 5 ~ as s h o w n b y t h e a b s e n c e of free e n z y m e on gel c h r o m a t o g r a p h y . T h e r e s u l t a n t g r a f t is s t a b l e a t r o o m t e m p e r a t u r e in a q u e o u s solutions of n e u t r a l p H . The Figure shows p H - r a t e profiles for t h e h y d r o l y s i s of B A P N A c a t a l y z e d b y free a n d b o u n d t r y p s i n a t ionic s t r e n g t h s of 0.17 a n d 1.17. The p H o p t i m u m is displaced b y a p p r o x i m a t e l y 1 p H u n i t t o w a r d s m o r e alkaline values b y c o n j u g a t i o n . A similar shift t o w a r d s h i g h e r pI-I values h a s b e e n o b s e r v e d p r e v i o u s l y for t r y p s i n - p o l y e l e c t r o l y t e d e r i v a t i v e s a n d in t h a t case was a t t r i b u t e d to t h e neighb o u r i n g carrier b o u n d c h a r g e d g r o u p s 7. F o r t h i s s y s t e m such a n e x p l a n a t i o n is i n a d e q u a t e . O x i d a t i o n of polysaccarides b y s o d i u m m e t a p e r i o d a t e , u n d e r the a b o v e c o n d i t i o n s t e r m i n a t e s a t t h e a l d e h y d e level of o x i d a t i o n , a n d t h e p H shift is n o t r e d u c e d b y t h e large c h a n g e in ionic s t r e n g t h s The r e s u l t a n t p H - a c t i v i t y profile a f t e r t r e a t m e n t of t r y p s i n w i t h excess a c e t a l d e h y d e was d e t e r m i n e d in o r d e r t o s e p a r a t e t h e effects due t o t h e m a t r i x f r o m t h o s e due to chemicaI m o d i f i c a t i o n of t h e p r o t e i n molecule
Table I. Apparent Michaelis constants for trypsin catalyzed hydrolysis of BAPNA in 0.1 M verona1 buffers, plus 0.02 M calcium chloride, 30 ~
Trypsin PADT
K(~,v~) ( • 104 M) pH 8.0
pH 8.85
7.40 5.60
5.80 5.95
Table If. Percentage retention of tryptie activity towards BAPNA, at 30~ in 0.1 M citrate buffer pH 7.0 (protein concentration, 1 rag/ ml). 3 days Trypsin Trypsin plus 0.02 M CaCle PADT PADT plus 0.02 M CaC12
2 14 46 53
773
(Figure). I n a g r e e m e n t w i t h LABOUESSE a n d GERVAIS 9 a small alkaline s h i f t was observed, b u t smaller t h a n t h a t caused b y t h e p o l y a l d e h y d e d e x t r a n . The m a t r i x h a d no effect, w i t h i n e x p e r i m e n t a l error, on t h e s u b s t r a t e d e p e n d a n t kinetics of t r y p s i n . B o t h free a n d a t t a c h e d enzyule o b e y e d t h e M i c h a e l i s - M e n t e n e q u a t i o n for t h e h y d r o l y s i s of B A P N A o v e r t h e r a n g e 0.1-1.27 • l 0 -a M ; t h e p a r a m e t e r s d e t e r m i n e d a t each p H o p t i m u m are s h o w n in T a b l e I. I m m o b i l i z a t i o n i m p a r t s increased r e s i s t a n c e to a u t o lysis (Table II). T r y p s i n loses 98% of its a c t i v y w i t h i n 72 h at 30 ~ p H 7.0, b u t s o m e w h a t less (86 %) in t h e presence of 0.2 M calcium chloride. B y c o n t r a s t t h e conjug a t e r e t a i n s 42% of its initial a c t i v i t y e v e n a f t e r 34 d a y s u n d e r t h e s a m e c o n d i t i o n s ; calcium ions also h a v e a small stabilizing effect on t h e conjugate, T a b l e I (care w a s t a k e n to a v o i d b a c t e r i a l c o n t a m i n a t i o n ) . This r e d u c e d a u t o l y t i c c a p a b i l i t y of t h e carrier b o u n d e n z y m e is t h e result of t w o m a i n factors. B y a n a l o g y w i t h t h e usual r e a c t i o n p r o d u c t s of a l d e h y d e s w i t h p r o t e i n s , t h e e x p e c t e d sites of r e a c t i o n w o u l d be to t h e E - a m i n o g r o u p s of Iysine so p r e v e n t i n g t h e l a t t e r f r o m b i n d i n g t o t h e active sites of o t h e r t r y p s i n molecules. I n a d d i t i o n , t h e p r e s e n c e of t h e a t t a c h e d d e x t r a n will sterically h i n d e r t h e m u t u a l a p p r o a c h of t r y p s i n molecules. This effect is p r o b a b l y also r e s p o n s i b l e for t h e d e c r e a s e d d e g r a d a t i o n of casein b y t h e c o n j u g a t e w h i c h is only 4% of t h a t of t h e native enzyme. As a r e q u i s i t e t o f u r t h e r e l u c i d a t i o n of t h e o b s e r v e d kinetic p r o p e r t i e s of t h e c o n j u g a t e , its s t r u c t u r e is u n d e r investigation.
Zusammen[assung. D u r c h k o v a l e n t e B i n d u n g a n P o l y a l d e h y d - d e x t r a n wird T r y p s i n immobilisiert. Die polym e r i s c h e M a t r i x stabilisiert das E n z y m u n d b e w i r k t eine p H - E r h 6 h u n g der m a x i m a l e n Aktivit/it, b e e i n f l u s s t a b e r d e n M i c h a e l i s - M e n t e n P a r a m e t e r fiir die I-tydrolyse des S u b s t r a t e s B A P N A nicht. 1~. L. FOSTER 10 School o/ Pharmacy, Liverpool Polytechnic, Liverpool L3 3 A F (England), 26 March 7975.
34 days 0 0 42 55
T. L. WESTMAN, Biochem. biophys. Res. Commun. 35, 313 (1969). 8 M. PECHT and Y. LEVlN, Biochem. biophys. Res. Commun. d6, 2054 (1972). J. LA~OUESSEand 1~{.GERVAIS, Eur. J. Biochein. 2, 215 (1967). 10 The author gratefully acknowledges the valuable discussions with Dr. A. R. THOMSON, A.E.R.E., Harwell.
Alteration of Erythrocyte (Na + + K+)-ATPase by Replacement of Cholesterol by Desmosterol in the Membrane I t was s h o w n b y us t h a t t r e a t m e n t of r a t s w i t h a n i n h i b i t o r of d e s m o s t e r o l r e d u c t a s e , i.e. 20.25-diazacholesterol, results in a n increased specific (Na+ + K+)A T P a s e a c t i v i t y in m e m b r a n e s of d i f f e r e n t tissues. S u c h a n effect was s h o w n for t h e s a r c o l e m m a of skeletal a n d cardiac muscle 1, 2 a n d e r y t h r o c y t e g h o s t s 3, ,. P r e l i m i n a r y s t u d i e s s suggest t h a t t h e a l t e r a t i o n in (Na+ + K+)A T P a s e a c t i v i t y is due to t h e r e p l a c e m e n t of cholesterol b y d e m o s t e r o l in t h e m e m b r a n e s of a n i m a l s t r e a t e d w i t h this substance. Male ~ ' i s t a r r a t s were daily t r e a t e d w i t h 10 m g 20.25d i a z a c h o l e s t e r o l d i h y d r o c h l o r i d e in 0.2 m l of w a t e r given b y a n oesophageal c a n n u l a for a p e r i o d of 8 weeks. Control
a n i m a l s were s u b j e c t e d to t h e s a m e p r o c e d u r e using 0.2 m l of w a t e r only. Blood was d r a w n f r o m one t r e a t e d a n d one c o n t r o l a n i m a l into h e p a r i n i z e d syringes b y aortic p u n c t u r e of t h e a n e s t h e t i z e d animal. P l a s m a a n d red t j. B. PETER and W. FIEHN, Science 179, 910 (1973). 2 W. F1EHN, D. SEILER, E. KUHN and DOROTHEA BARTELS, Eur. J. elin. Invest., in press. 3 W. FIEnN, E. Kumr and I. GELDMACHER, FEBS Left. 3d, 163 (1973). J. B. PETER, R. M. ANmMAN, R. L. BOWMANSand T. NAGAMOTO, Expl. Neurol. dl, 738 (1973). 5 D. SELLER,W. FIEHr~ and E. K~JnN, Z. klin. Chem., in press.
