484
Specialia
Inhibition of liver micros 9
EXPERIENX'IA3314
epoxide hydrase by cyproheptadine epoxide
J. P a c h e c k a ~, M. S a l m o n a ~, G. Belvedere, L. Cantoni, E. Mussini a n d S. G a r a t t i n i
Istituto di Ricerche Farmacologiche 'Mario Negri', via Eritrea 62, 1-20157 Milan 9 (Italy), 17 September 1976 S u m m a r y . C y p r o h e p t a d i n e e p o x i d e is a c o m p e t i t i v e i n h i b i t o r of r a t liver m i c r o s o m a l e p o x i d e h y d r a s e w i t h an a p p a r e n t K i - v a l n e of 0.75 mM. C y c l o b e n z a p r i n e a n d its e p o x i d e s t i m u l a t e in vitro t h e a c t i v i t y of t h i s e n z y m e , w h e r e a s c y p r o h e p t a d i n e , c a r b a l n a z e p i n e a n d c a r b a m a z e p i n e e p o x i d e h a v e no effect. T h e r e is increasing evidence t h a t labile e p o x i d e s f o r m e d d u r i n g m e t a b o l i s m of m a n y chemicals m a y p l a y a n imp o r t a n t role in carcinogenesis 3-~. The c o n c e n t r a t i o n of t h e s e e p o x i d e s in t h e b o d y is t h e result of a b a l a n c e between the amount formed and the amount metabolized t h r o u g h various m e t a b o l i c processes6, 7. A key e n z y m e for c a t a b o l i s m of e p o x i d e s is t h e e p o x i d e h y d r a s e (EC 4.2.1. 64)~ Therefore, it is obviously of i n t e r e s t to k n o w w h i c h f a c t o r s m a y affect t h e a c t i v i t y of this e n z y m e . I t has b e e n s h o w n t h a t some epoxides, e.g. c y c l o - h e x a n e e p o x i d e and t r i c h l o r o p r o p e n e epoxide, are able to decrease t h e a c t i v i t y of e p o x i d e h y d r a s e s; however, t h e effect of o t h e r epoxides, a n d in p a r t i c u l a r t h o s e f o r m e d d u r i n g t h e m e t a b o l i s m o! some drugs, has n o t b e e n studied. I n p r e v i o u s studies we h a v e f o u n d t h a t several tricyclic d r u g s including carb a m a z e p i n e , c y p r o h e p t a d i n e a n d c y c l o b e n z a p r i n e are m e t a b o l i z e d to f o r m 10, 11-epoxides, w h i c h can be detect e d in blood a n d urine 9,~~ This c o m m u n i c a t i o n s u m m a rizes our r e c e n t findings c o n c e r n i n g t h e effects of t h e s e e p o x i d e s as well as t h e i r p a r e n t c o m p o u n d s on t h e a c t i v i t y of e p o x i d e h y d r a s e in r a t liver microsomes.
Effect of some tricyclic drugs and their epoxides on epoxide hydrase activity Drug added
Concentration Specific activity Activity of the drugs (nlnoles phenyl- ~ (raM) ethylene glycol mg protein 1 min-1)
None Cyproheptadine Cyproheptadine epoxide Carbamazepine Carbamazepine epoxide Cyclobenzaprine Cyclobenzaprine epoxide
1 0.75 1 1 1 1
5.42 ~_ 0.22 5.63 4- 0.15 4.10 • 0.10" 5.20 -t- 0.05 5.53 4- 0.08 6.30 4- 0.28** 6.13 4- 0.29**
100 104 76 96 102 116 113
The substrate (styrene epoxide) concentration was always 0.5 raM. The figures represent the mean value ~ SE of 4 i~dividual experiments. *p % 0.01; **p < 0.05.
Materials and methods. The liver m i c r o s o m e s y~,erep r e p a r e d from Charles R i v e r CD male r a t s (body w e i g h t 150-180 g) a c c o r d i n g to t h e m e t h o d of K a t o 11, w i t h slight m o d i f i c a t i o n in t h e c o m p o s i t i o n of t h e solution utilized for liver h o m o g e n i z a t i o n w h i c h c o n s i s t e d of Tris-HC1 b u f f e r 0.05 M, p H 7 . 4 c o n t a i n i n g NaC10.15 M a n d MgC125 mM. C a r b a m a z e p i n e was a k i n d gift of Ciba-Geigy, c a r b a m a z e p i n e e p o x i d e was d o n a t e d b y Italseber, Milan9 c y p r o h e p t a d i n e and cyclob e n z a p r i n e were o b t a i n e d b y Merck S h a r p and D o h m e , while t h e i r epoxides were s y n t h e s i z e d as p r e v i o u s l y described 10,12. The a c t i v i t y of e p o x i d e h y d r a s e was d e t e r m i n e d b y utilizing s t y r e n e e p o x i d e as a s u b s t r a t e . The e n z y m a t i c r e a c t i o n was carried out as follows: 4.3 ml buffer was a d d e d to 0.1 m l m e t h a n o l solution of t h e comp o u n d in o r d e r to o b t a i n a final c o n c e n t r a t i o n r a n g i n g f r o m 0.35 t o 2 mM. A t t h e s a m e t i m e 0.5 nil of a liver micros 9 s u s p e n s i o n c o n t a i n i n g a b o u t 5 m g of p r o t e i n were a d d e d . A f t e r 15 m i n Of p r e i n c u b a t i o n a t 37~ 0.1 ml of a n a c e t o n e solution of s t y r e n e e p o x i d e (Merck) was a d d e d to o b t a i n a final s u b s t r a t e c o n c e n t r a t i o n of 0.5 m M or 0.75 mM. The r e a c t i o n was s t o p p e d a f t e r 5 min b y t h e a d d i t i o n of 1 ml of 0.6 M N a O H . The s a m p l e s were imm e d i a t e l y e x t r a c t e d t w i c e w i t h 5 ml of e t h y l acetate. The c o m b i n e d e x t r a c t s were d r i e d u n d e r v a c u u m a t r o o m t e m p e r a t u r e . I n o r d e r t o m e a s u r e p h e n y l e t h y l e n e glycol, t h e diol f o r m e d d u r i n g t h e e n z y m a t i c reaction, a simple gas c h r o m a t o g r a p h i c m e t h o d w a s developed. The diol was q u a n t i t a t i v e l y d e t e r m i n e d t h r o u g h an esterification process w i t h b u t y l b o r o n i c acid 13. 5 txl of b u t y l b o r o n i c acid (100 m g / m l in d i m e t h y l f o r m a m i d e ) were a d d e d to t h e s a m p l e residue dissolved in 200 ~tl of a n a c e t o n e solution of m - d i n i t r o b e n z e n e (0.5 ~xg/tzl) used as a n i n t e r n a l s t a n d ard. i bd of t h i s solution w a s i n j e c t e d in a Model GI gas c h r o m a t o g r a p h a p p a r a t u s (Carlo E r b a , Milan9 e q u i p p e d w i t h a h y d r o g e n flame ionization d e t e c t o r . The s t a t i o n a r y p h a s e was OV-17 3% on Gas C h r o m Q (100-200 mesh) p a c k e d into a 2.5 m glass c o l u m n (internal d i a m e t e r 2 m m , e x t e r n a l d i a m e t e r 4 m m ) . T h e flow rate of t h e carrier gas (nitrogen) was 30 m l / m i n a n d t h e c o l u m n t e m p e r a t u r e was 170~ The r e c o v e r y of p h e n y l e t h y l e n e glycol was 1 2 3 4 5 6 7 8
- Ki
0,57
0.75
1.5 rnM
Inhibitor concentration
Dixon plot of the inhibitory effect of cycloheptadine epoxide on the activity of epoxide hydrase. The concentration of the substrate (styrene epoxide) utilized were 0.5 mM ( 9 9 and 0.75 mM (O--Q). V is given as nmoles of phenylethylene glycol formed mg protein-t rain -1.
9 10 11 12 13
Present address: Medical Academy, Banacha 1, Warsaw, Poland. To whom enquiries should be addressed. H.L. Gurtoo and C. V. Dave, Cancer Res. 35, 382 (1975). E. Huberman, T. Kuroki, H. Marquardt, J. K. Selkirk, C. Heidelberger, P. L. Grover and P. Sims, Cancer Res. 32, 1391 (1972). D.M. jerina and J. W. Daly, Science, N, Y. 785, 573 (1974). F. Oesch, D. M. j'erina and J. Daly, Biochim. biophys. Acta 227, 685 (1971). E . Boyland andK. Williams, Biochem. J. 94, 190 (1965). F. Oesch, N. Kaubisch, D. M. Jerina and J. Daly, Biochemistry 10, 4858 (1971). A. Frigerio, R. Fanelli, P. Biandrate, G. Passerini, P. L. Morselli and S. Garattini, J. pharm. Sci. 61, 1144 (1972). A. Frigerio, N. Sossi, G. Belvedere, C. Pantarotto and S. Garattini, J. pharm. Sei., 63, 1536 (1974). R. Kato and M. Takayanaghi, Jap. J. Pharmac. 16, 380 (1966). G. Belvedere, C. Pantarotto, V. Rovei and A. Frigerio, J. pharm. Sei. 65, 815 (1976). C . J . W . Brooks and I. Maclean, J. Chromat. Sci. 9, 18 (1971).
15.4. 1977
485
Specialia
m o r e t h a n 90%. To c o n f i r m t h e n a t u r e of t h e d e r i v a t i v e of p h e n y l e t h y l e n e glycol w i t h b u t y l b o r o n i c acid, m a s s s p e c t r a were carried carried o u t b y using an L K B 9000 i n s t r u m e n t a t t h e following c o n d i t i o n s : ion source t e m p e r a t u r e 290~ i o n i z a t i o n e n e r g y 70 eV; t r a p c u r r e n t 60 btA. S a m p l e i n t r o d u c t i o n was carried o u t e i t h e r b y d i r e c t inlet s y s t e m (DIS) or b y GC a t t h e c o n d i t i o n s d e s c r i b e d above, e x c e p t for t h e use of h e l i u m i n s t e a d of n i t r o g e n as carrier gas. The 2 m a s s s p e c t r a were c o m p a r a b l e . The e n z y m i c a c t i v i t y of epoxide h y d r a s e was e x p r e s s e d in n m o l e s of diol f o r m e d per min of i n c u b a t i o n per m g of m i c r o s o m a l p r o t e i n . The s p o n t a n e o u s h y d r a t i o n of s t y r e n e e p o x i d e occurring in our c o n d i t i o n s was lower t h a n 5% w i t h res p e c t to t h e e n z y m a t i c a n d it was s u b s t r a c t e d for t h e c a l c u l a t i o n of e n z y m a t i c activity. P r o t e i n c o n c e n t r a t i o n was d e t e r m i n e d b y t h e m e t h o d of Lowry*~. Results and discussion. The t a b l e s u m m a r i z e s t h e effects of t h e tricyclic d r u g s a n d t h e i r e p o x i d e s on t h e a c t i v i t y of e p o x i d e h y d r a s e . Only c y p r o h e p t a d i n e e p o x i d e h a d a m a r k e d i n h i b i t o r y effect on t h e e n z y m e , while c y l o b e n zaprine a n d its e p o x i d e s h o w e d a slight s t i m u l a t o r y effect. On t h e o t h e r h a n d , c y p r o h e p t a d i n e , c a r b a m a z e p i n e a n d c a r b a m a z e p i n e e p o x i d e did n o t show a n y influence on t h e a c t i v i t y of t h e e n z y m e . As s h o w n in t h e figure, c y p r o -
h e p t a d i n e e p o x i d e is a c o m p e t i t i v e i n h i b i t o r of e p o x i d e h y d r a s e ; t h e a p p a r e n t K , - v a l u e is 0.75 mM. I t m a y be suggested that styrene epoxide and cyproheptadine epoxide are s u b s t r a t e s for t h e s a m e e n z y m e s , a l t h o u g h t o d a t e t h e r e is no e v i d e n c e t h a t c y p r o h e p t a d i n e epoxide is m e t a b olized to t h e c o r r e s p o n d a n t diol ~5. The significance of t h e s e in vivo findings r e m a i n s to be established. H o w e v e r , t h e h y p o t h e s i s m a y be a d v a n c e d t h a t c y p r o h e p t a d i n e e p o x i d e could interfere w i t h t h e biot r a n s f o r m a t i o n process of some carcinogenic a g e n t s b y affecting t h e d e g r a d a t i o n of t h e i r e p o x i d e s into m e t a b o lites devoid of t o x i c effects. Conversely, it m a y be suggested on t h e basis of d a t a f r o m studies in progress t h a t tricyclic a g e n t s m a y also affect t h e f o r m a t i o n of t h e e p o x i d e s of carcinogenic agents. Therefore, it seems possible to m o d u l a t e t h e a c c u m u l a t i o n of e p o x i d e s f r o m s u c h agents, p e r m i t t i n g us to assess t h e significance of e p o x i d e f o r m a t i o n in c h e m i c a l carcinogenesis.
14 O.H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall, J. biol. Chem. 193, 265 (1951). 15 R.L. Hintze, J. S. Wold and L. J. Fisher, Drug Metab. Dispos. 3, 1 (1975).
A n e w a p p r o a c h to p r e p a r a t i o n and purification of c o l o s t r o k i n i n f r o m b o v i n e c o l o s t r u m * M. M. Casellato, G. Imgaro, P. Pasta, E. Manera, P. O r m a s and C. B e r e t t a
Laboratorio di Chimica degli Ormoni, Consiglio Nazionale delle Ricerche, via Mario Bianco 9, 1-20131 Milano (Italy), and Islituto di Farmacologia e Tossicologia Veterinaria, Universits di Milano, via Viotli 3/5, 1-20133 Milano (Italy), 2 August 1976 Summary. A simple a n d r a p i d m e t h o d is r e p o r t e d for p r e p a r a t i o n a n d p a r t i a l p u r i f i c a t i o n of colostrokinin f r o m b o v i n e colostral whey. The active s u b s t a n c e o b t a i n e d a t t h e described stage of p u r i f i c a t i o n a p p e a r s to be a basic p o l y p e p t i d e of low molecular weight. This p e p t i d e p r o v e d to be u n d e s t r o y e d b y pepsin, o n l y p a r t i a l l y b y t r y p s i n a n d c o m p l e t e l y by papain. The first r e p o r t a b o u t some p r o p e r t i e s of colostrokinin d a t e s f r o m 1959 b y G u t h 1. The crude s u b s t a n c e was obt a i n e d b y i n c u b a t i n g kallikrein from urine or saliva w i t h colostruln 2,a. A m e t h o d for colostrokinin p r e p a r a t i o n w i t h a 15-20fold p u r i f i c a t i o n was later p e r f o r m e d b y Werle a n d T r a u t s c h o l d 4, who f o u n d some biological a n d b i o c h e m i c a l differences b e t w e e n colostrokinin a n d p l a s m a kinins, b u t c o n c l u d e d t h a t t h e 2 t y p e s of kinins were c h e m i c a l l y a n d p h a r m a c o l o g i c a l l y v e r y closely related. A b o u t 10 y e a r s later, t h i s s u b j e c t of r e s e a r c h d r e w t h e a t t e n t i o n of some J a p a n e s e i n v e s t i g a t o r s , w h o p r o p o s e d a m e t h o d for isolation a n d p u r i f i c a t i o n of colostrokinin
064
0.5
* Acknowledgment. This research was partially supported by the Italian Ministry of Health. 1 P.S. Guth, Br. J. I)harmac. 14, 549 (1959). 2 E. Werle, in : Polypeptides which affect smooth nmscle and blood vessels, p. 208. Ed. M. Schachter. Pergamon Press, London 1960. 3 P. S. Guth, in: Polypeptides which affect smooth nmscle and blood vessels, p. 268. Ed. M. Schachter. Pergamon Press, London 1960. 4 E. Werle and I. Trautschold, Z. Biol. 112, 169 (1960). 5 K. Yamazaki and H. Moriya, Biochem. Pharmac. 18, 2303 (1969). 6 K. Yamazaki and H. Moriya, Bioehem. Pharmac. 18, 2313 (1969).
o
0.40.30.2._o
~_ 0.1o
250
500
750
1000 1250 1500 1750 2000 2250 2500 2750m[ Eiuti0n volume
Fig. 1. Gel filtration pattern of crude colostrokinin (step 3), prepared from 2000 ml of natural bovine colostrum, on a Sephadex G-25 fine column (5.5 em• cm), equilibrated with 0.1 M ammonium acetate buffer (ph 5). Flow rate 125 ml]h. Fraction size 25ml. Void volume measured with Blue Dextran 2000. Shaded area represents the presence of biological activity. The separation pattern was recorded at 278 nm.
Specialia
Inhibition of liver micros 9
EXPERIENX'IA3314
epoxide hydrase by cyproheptadine epoxide
J. P a c h e c k a ~, M. S a l m o n a ~, G. Belvedere, L. Cantoni, E. Mussini a n d S. G a r a t t i n i
Istituto di Ricerche Farmacologiche 'Mario Negri', via Eritrea 62, 1-20157 Milan 9 (Italy), 17 September 1976 S u m m a r y . C y p r o h e p t a d i n e e p o x i d e is a c o m p e t i t i v e i n h i b i t o r of r a t liver m i c r o s o m a l e p o x i d e h y d r a s e w i t h an a p p a r e n t K i - v a l n e of 0.75 mM. C y c l o b e n z a p r i n e a n d its e p o x i d e s t i m u l a t e in vitro t h e a c t i v i t y of t h i s e n z y m e , w h e r e a s c y p r o h e p t a d i n e , c a r b a l n a z e p i n e a n d c a r b a m a z e p i n e e p o x i d e h a v e no effect. T h e r e is increasing evidence t h a t labile e p o x i d e s f o r m e d d u r i n g m e t a b o l i s m of m a n y chemicals m a y p l a y a n imp o r t a n t role in carcinogenesis 3-~. The c o n c e n t r a t i o n of t h e s e e p o x i d e s in t h e b o d y is t h e result of a b a l a n c e between the amount formed and the amount metabolized t h r o u g h various m e t a b o l i c processes6, 7. A key e n z y m e for c a t a b o l i s m of e p o x i d e s is t h e e p o x i d e h y d r a s e (EC 4.2.1. 64)~ Therefore, it is obviously of i n t e r e s t to k n o w w h i c h f a c t o r s m a y affect t h e a c t i v i t y of this e n z y m e . I t has b e e n s h o w n t h a t some epoxides, e.g. c y c l o - h e x a n e e p o x i d e and t r i c h l o r o p r o p e n e epoxide, are able to decrease t h e a c t i v i t y of e p o x i d e h y d r a s e s; however, t h e effect of o t h e r epoxides, a n d in p a r t i c u l a r t h o s e f o r m e d d u r i n g t h e m e t a b o l i s m o! some drugs, has n o t b e e n studied. I n p r e v i o u s studies we h a v e f o u n d t h a t several tricyclic d r u g s including carb a m a z e p i n e , c y p r o h e p t a d i n e a n d c y c l o b e n z a p r i n e are m e t a b o l i z e d to f o r m 10, 11-epoxides, w h i c h can be detect e d in blood a n d urine 9,~~ This c o m m u n i c a t i o n s u m m a rizes our r e c e n t findings c o n c e r n i n g t h e effects of t h e s e e p o x i d e s as well as t h e i r p a r e n t c o m p o u n d s on t h e a c t i v i t y of e p o x i d e h y d r a s e in r a t liver microsomes.
Effect of some tricyclic drugs and their epoxides on epoxide hydrase activity Drug added
Concentration Specific activity Activity of the drugs (nlnoles phenyl- ~ (raM) ethylene glycol mg protein 1 min-1)
None Cyproheptadine Cyproheptadine epoxide Carbamazepine Carbamazepine epoxide Cyclobenzaprine Cyclobenzaprine epoxide
1 0.75 1 1 1 1
5.42 ~_ 0.22 5.63 4- 0.15 4.10 • 0.10" 5.20 -t- 0.05 5.53 4- 0.08 6.30 4- 0.28** 6.13 4- 0.29**
100 104 76 96 102 116 113
The substrate (styrene epoxide) concentration was always 0.5 raM. The figures represent the mean value ~ SE of 4 i~dividual experiments. *p % 0.01; **p < 0.05.
Materials and methods. The liver m i c r o s o m e s y~,erep r e p a r e d from Charles R i v e r CD male r a t s (body w e i g h t 150-180 g) a c c o r d i n g to t h e m e t h o d of K a t o 11, w i t h slight m o d i f i c a t i o n in t h e c o m p o s i t i o n of t h e solution utilized for liver h o m o g e n i z a t i o n w h i c h c o n s i s t e d of Tris-HC1 b u f f e r 0.05 M, p H 7 . 4 c o n t a i n i n g NaC10.15 M a n d MgC125 mM. C a r b a m a z e p i n e was a k i n d gift of Ciba-Geigy, c a r b a m a z e p i n e e p o x i d e was d o n a t e d b y Italseber, Milan9 c y p r o h e p t a d i n e and cyclob e n z a p r i n e were o b t a i n e d b y Merck S h a r p and D o h m e , while t h e i r epoxides were s y n t h e s i z e d as p r e v i o u s l y described 10,12. The a c t i v i t y of e p o x i d e h y d r a s e was d e t e r m i n e d b y utilizing s t y r e n e e p o x i d e as a s u b s t r a t e . The e n z y m a t i c r e a c t i o n was carried out as follows: 4.3 ml buffer was a d d e d to 0.1 m l m e t h a n o l solution of t h e comp o u n d in o r d e r to o b t a i n a final c o n c e n t r a t i o n r a n g i n g f r o m 0.35 t o 2 mM. A t t h e s a m e t i m e 0.5 nil of a liver micros 9 s u s p e n s i o n c o n t a i n i n g a b o u t 5 m g of p r o t e i n were a d d e d . A f t e r 15 m i n Of p r e i n c u b a t i o n a t 37~ 0.1 ml of a n a c e t o n e solution of s t y r e n e e p o x i d e (Merck) was a d d e d to o b t a i n a final s u b s t r a t e c o n c e n t r a t i o n of 0.5 m M or 0.75 mM. The r e a c t i o n was s t o p p e d a f t e r 5 min b y t h e a d d i t i o n of 1 ml of 0.6 M N a O H . The s a m p l e s were imm e d i a t e l y e x t r a c t e d t w i c e w i t h 5 ml of e t h y l acetate. The c o m b i n e d e x t r a c t s were d r i e d u n d e r v a c u u m a t r o o m t e m p e r a t u r e . I n o r d e r t o m e a s u r e p h e n y l e t h y l e n e glycol, t h e diol f o r m e d d u r i n g t h e e n z y m a t i c reaction, a simple gas c h r o m a t o g r a p h i c m e t h o d w a s developed. The diol was q u a n t i t a t i v e l y d e t e r m i n e d t h r o u g h an esterification process w i t h b u t y l b o r o n i c acid 13. 5 txl of b u t y l b o r o n i c acid (100 m g / m l in d i m e t h y l f o r m a m i d e ) were a d d e d to t h e s a m p l e residue dissolved in 200 ~tl of a n a c e t o n e solution of m - d i n i t r o b e n z e n e (0.5 ~xg/tzl) used as a n i n t e r n a l s t a n d ard. i bd of t h i s solution w a s i n j e c t e d in a Model GI gas c h r o m a t o g r a p h a p p a r a t u s (Carlo E r b a , Milan9 e q u i p p e d w i t h a h y d r o g e n flame ionization d e t e c t o r . The s t a t i o n a r y p h a s e was OV-17 3% on Gas C h r o m Q (100-200 mesh) p a c k e d into a 2.5 m glass c o l u m n (internal d i a m e t e r 2 m m , e x t e r n a l d i a m e t e r 4 m m ) . T h e flow rate of t h e carrier gas (nitrogen) was 30 m l / m i n a n d t h e c o l u m n t e m p e r a t u r e was 170~ The r e c o v e r y of p h e n y l e t h y l e n e glycol was 1 2 3 4 5 6 7 8
- Ki
0,57
0.75
1.5 rnM
Inhibitor concentration
Dixon plot of the inhibitory effect of cycloheptadine epoxide on the activity of epoxide hydrase. The concentration of the substrate (styrene epoxide) utilized were 0.5 mM ( 9 9 and 0.75 mM (O--Q). V is given as nmoles of phenylethylene glycol formed mg protein-t rain -1.
9 10 11 12 13
Present address: Medical Academy, Banacha 1, Warsaw, Poland. To whom enquiries should be addressed. H.L. Gurtoo and C. V. Dave, Cancer Res. 35, 382 (1975). E. Huberman, T. Kuroki, H. Marquardt, J. K. Selkirk, C. Heidelberger, P. L. Grover and P. Sims, Cancer Res. 32, 1391 (1972). D.M. jerina and J. W. Daly, Science, N, Y. 785, 573 (1974). F. Oesch, D. M. j'erina and J. Daly, Biochim. biophys. Acta 227, 685 (1971). E . Boyland andK. Williams, Biochem. J. 94, 190 (1965). F. Oesch, N. Kaubisch, D. M. Jerina and J. Daly, Biochemistry 10, 4858 (1971). A. Frigerio, R. Fanelli, P. Biandrate, G. Passerini, P. L. Morselli and S. Garattini, J. pharm. Sci. 61, 1144 (1972). A. Frigerio, N. Sossi, G. Belvedere, C. Pantarotto and S. Garattini, J. pharm. Sei., 63, 1536 (1974). R. Kato and M. Takayanaghi, Jap. J. Pharmac. 16, 380 (1966). G. Belvedere, C. Pantarotto, V. Rovei and A. Frigerio, J. pharm. Sei. 65, 815 (1976). C . J . W . Brooks and I. Maclean, J. Chromat. Sci. 9, 18 (1971).
15.4. 1977
485
Specialia
m o r e t h a n 90%. To c o n f i r m t h e n a t u r e of t h e d e r i v a t i v e of p h e n y l e t h y l e n e glycol w i t h b u t y l b o r o n i c acid, m a s s s p e c t r a were carried carried o u t b y using an L K B 9000 i n s t r u m e n t a t t h e following c o n d i t i o n s : ion source t e m p e r a t u r e 290~ i o n i z a t i o n e n e r g y 70 eV; t r a p c u r r e n t 60 btA. S a m p l e i n t r o d u c t i o n was carried o u t e i t h e r b y d i r e c t inlet s y s t e m (DIS) or b y GC a t t h e c o n d i t i o n s d e s c r i b e d above, e x c e p t for t h e use of h e l i u m i n s t e a d of n i t r o g e n as carrier gas. The 2 m a s s s p e c t r a were c o m p a r a b l e . The e n z y m i c a c t i v i t y of epoxide h y d r a s e was e x p r e s s e d in n m o l e s of diol f o r m e d per min of i n c u b a t i o n per m g of m i c r o s o m a l p r o t e i n . The s p o n t a n e o u s h y d r a t i o n of s t y r e n e e p o x i d e occurring in our c o n d i t i o n s was lower t h a n 5% w i t h res p e c t to t h e e n z y m a t i c a n d it was s u b s t r a c t e d for t h e c a l c u l a t i o n of e n z y m a t i c activity. P r o t e i n c o n c e n t r a t i o n was d e t e r m i n e d b y t h e m e t h o d of Lowry*~. Results and discussion. The t a b l e s u m m a r i z e s t h e effects of t h e tricyclic d r u g s a n d t h e i r e p o x i d e s on t h e a c t i v i t y of e p o x i d e h y d r a s e . Only c y p r o h e p t a d i n e e p o x i d e h a d a m a r k e d i n h i b i t o r y effect on t h e e n z y m e , while c y l o b e n zaprine a n d its e p o x i d e s h o w e d a slight s t i m u l a t o r y effect. On t h e o t h e r h a n d , c y p r o h e p t a d i n e , c a r b a m a z e p i n e a n d c a r b a m a z e p i n e e p o x i d e did n o t show a n y influence on t h e a c t i v i t y of t h e e n z y m e . As s h o w n in t h e figure, c y p r o -
h e p t a d i n e e p o x i d e is a c o m p e t i t i v e i n h i b i t o r of e p o x i d e h y d r a s e ; t h e a p p a r e n t K , - v a l u e is 0.75 mM. I t m a y be suggested that styrene epoxide and cyproheptadine epoxide are s u b s t r a t e s for t h e s a m e e n z y m e s , a l t h o u g h t o d a t e t h e r e is no e v i d e n c e t h a t c y p r o h e p t a d i n e epoxide is m e t a b olized to t h e c o r r e s p o n d a n t diol ~5. The significance of t h e s e in vivo findings r e m a i n s to be established. H o w e v e r , t h e h y p o t h e s i s m a y be a d v a n c e d t h a t c y p r o h e p t a d i n e e p o x i d e could interfere w i t h t h e biot r a n s f o r m a t i o n process of some carcinogenic a g e n t s b y affecting t h e d e g r a d a t i o n of t h e i r e p o x i d e s into m e t a b o lites devoid of t o x i c effects. Conversely, it m a y be suggested on t h e basis of d a t a f r o m studies in progress t h a t tricyclic a g e n t s m a y also affect t h e f o r m a t i o n of t h e e p o x i d e s of carcinogenic agents. Therefore, it seems possible to m o d u l a t e t h e a c c u m u l a t i o n of e p o x i d e s f r o m s u c h agents, p e r m i t t i n g us to assess t h e significance of e p o x i d e f o r m a t i o n in c h e m i c a l carcinogenesis.
14 O.H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall, J. biol. Chem. 193, 265 (1951). 15 R.L. Hintze, J. S. Wold and L. J. Fisher, Drug Metab. Dispos. 3, 1 (1975).
A n e w a p p r o a c h to p r e p a r a t i o n and purification of c o l o s t r o k i n i n f r o m b o v i n e c o l o s t r u m * M. M. Casellato, G. Imgaro, P. Pasta, E. Manera, P. O r m a s and C. B e r e t t a
Laboratorio di Chimica degli Ormoni, Consiglio Nazionale delle Ricerche, via Mario Bianco 9, 1-20131 Milano (Italy), and Islituto di Farmacologia e Tossicologia Veterinaria, Universits di Milano, via Viotli 3/5, 1-20133 Milano (Italy), 2 August 1976 Summary. A simple a n d r a p i d m e t h o d is r e p o r t e d for p r e p a r a t i o n a n d p a r t i a l p u r i f i c a t i o n of colostrokinin f r o m b o v i n e colostral whey. The active s u b s t a n c e o b t a i n e d a t t h e described stage of p u r i f i c a t i o n a p p e a r s to be a basic p o l y p e p t i d e of low molecular weight. This p e p t i d e p r o v e d to be u n d e s t r o y e d b y pepsin, o n l y p a r t i a l l y b y t r y p s i n a n d c o m p l e t e l y by papain. The first r e p o r t a b o u t some p r o p e r t i e s of colostrokinin d a t e s f r o m 1959 b y G u t h 1. The crude s u b s t a n c e was obt a i n e d b y i n c u b a t i n g kallikrein from urine or saliva w i t h colostruln 2,a. A m e t h o d for colostrokinin p r e p a r a t i o n w i t h a 15-20fold p u r i f i c a t i o n was later p e r f o r m e d b y Werle a n d T r a u t s c h o l d 4, who f o u n d some biological a n d b i o c h e m i c a l differences b e t w e e n colostrokinin a n d p l a s m a kinins, b u t c o n c l u d e d t h a t t h e 2 t y p e s of kinins were c h e m i c a l l y a n d p h a r m a c o l o g i c a l l y v e r y closely related. A b o u t 10 y e a r s later, t h i s s u b j e c t of r e s e a r c h d r e w t h e a t t e n t i o n of some J a p a n e s e i n v e s t i g a t o r s , w h o p r o p o s e d a m e t h o d for isolation a n d p u r i f i c a t i o n of colostrokinin
064
0.5
* Acknowledgment. This research was partially supported by the Italian Ministry of Health. 1 P.S. Guth, Br. J. I)harmac. 14, 549 (1959). 2 E. Werle, in : Polypeptides which affect smooth nmscle and blood vessels, p. 208. Ed. M. Schachter. Pergamon Press, London 1960. 3 P. S. Guth, in: Polypeptides which affect smooth nmscle and blood vessels, p. 268. Ed. M. Schachter. Pergamon Press, London 1960. 4 E. Werle and I. Trautschold, Z. Biol. 112, 169 (1960). 5 K. Yamazaki and H. Moriya, Biochem. Pharmac. 18, 2303 (1969). 6 K. Yamazaki and H. Moriya, Bioehem. Pharmac. 18, 2313 (1969).
o
0.40.30.2._o
~_ 0.1o
250
500
750
1000 1250 1500 1750 2000 2250 2500 2750m[ Eiuti0n volume
Fig. 1. Gel filtration pattern of crude colostrokinin (step 3), prepared from 2000 ml of natural bovine colostrum, on a Sephadex G-25 fine column (5.5 em• cm), equilibrated with 0.1 M ammonium acetate buffer (ph 5). Flow rate 125 ml]h. Fraction size 25ml. Void volume measured with Blue Dextran 2000. Shaded area represents the presence of biological activity. The separation pattern was recorded at 278 nm.
