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Cytidylate cyclase activity is stimulated via activation of a guanine nucleotide-binding protein
L i f e S c i e n c e s , Vol. P r i n t e d in t h e U S A
CYTIDYLATE
52, pp.
13-20
Pergamon
C Y C L A S E A C T I V I T Y IS S T I M U L A T E D V I A A C T I V A T I O N GUANINE NUCLEOTIDE-BINDING PROTEIN
Press
OF
A
N o r i o Muto, Department
Masatoshi
K a n o h and
Itaru Y a m a m o t o *
of I m m u n o c h e m i s t r y , F a c u l t y of P h a r m a c e u t i c a l O k a y a m a U n i v e r s i t y , O k e y a m a 700, J a p a n (Received
in final
form October
26,
Sciences,
1992)
Summary C y t i d y l a t e c y c l a s e is a m e m b r a n e - b o u n d e n z y m e w h i c h c a t a l y z e s the biosynthesis of cytidine 3',5'-cyclic monophosphate (cCMP) from CTP. By u s i n g a s e n s i t i v e and s p e c i f i c e n z y m e i m m u n o a s s a y method, we evaluated the participation of guanine nucleotide-binding protein (G-protein) in the regulation of cytidylate cyclase a c t i v i t y in rat b r a i n and o t h e r tissues. AIF4-, an a c t i v a t o r of G-proteins, effectively elevated the cyclase activity. The stimulation by GTPTS, a nonhydrolyzable GTP analogue, was a l s o observed in t i m e and c o n c e n t r a t i o n - d e p e n d e n t manner during the preincubation and t h i s e f f e c t w a s c o m p e t i t i v e l y i n h i b i t e d by the a d d i t i o n of GDPBS. However, islet-activating p r o t e i n and c h o l e r a t o x i n w h i c h a f f e c t e d a d e n y l a t e c y c l a s e a c t i v i t y h a d no e f f e c t on cytidylate cyclase activity. These results indicate that c y t i d y l a t e c y c l a s e is a s s o c i a t e d w i t h a c e r t a i n G - p r o t e i n and its a c t i v i t y is r e g u l a t e d by the m o d e d i f f e r e n t from t h a t of a d e n y l a t e cyclase. Cytidine 3',5'-cyclic monophosphate (cCMP) w a s first found as a t h i r d c y c l i c n u c l e o t i d e in m a m m a l i a n t i s s u e s and l e u k e m i a L - 1 2 1 0 c u l t u r e s b y B l o c h et al. (1-3). A l t h o u g h its p h y s i o l o g i c a l f u n c t i o n r e m a i n s to be c l a r i f i e d , possible roles in regulating cell proliferation and biological processes h a v e b e e n r e p o r t e d (4-9). We h a v e a l s o d e m o n s t r a t e d its i n h i b i t o r y e f f e c t on the c e l l g r o w t h of rat h e p a t o c y t e s in v i t r o (i0) and its s t i m u l a t o r y e f f e c t on the n e u r i t e o u t g r o w t h of PC12 c e l l s in v i t r o (ii). B o t h e f f e c t s of c C M P h a v e b e e n o b s e r v e d to be less e f f e c t i v e t h a n those of a d e n o s i n e 3 ' , 5 ' - c y c l i c monophosphate (cAMP), but to be more efficient than those of guanosine 3',5'-cyclic monophosphate (cGMP). T h e s e f i n d i n g s s u g g e s t t h a t c C M P m a y be a s e c o n d m e s s e n g e r as w e l l as w e l l - s t u d i e d c y c l i c p u r i n e n u c l e o t i d e s . c C M P is k n o w n to be s y n t h e s i z e d f r o m C T P by c y t i d y l a t e c y c l a s e (12,13). However, the e n z y m o l o g i c a l and b i o c h e m i c a l properties of c y t i d y l a t e c y c l a s e h a v e n o t b e e n e l u c i d a t e d a p p r e c i a b l y , as c o m p a r e d w i t h t h o s e of a d e n y l a t e and guanylate cyclases. This was mainly due to the lack of a reliable and sensitive assay method for cCMP. We h a v e d e v e l o p e d a h i g h l y s e n s i t i v e and s p e c i f i c e n z y m e i m m u n o a s s a y (EIA) m e t h o d by w h i c h 0.5 fmol of c C M P p e r a s s a y t u b e c a n be d e t e r m i n e d (14). By u s i n g this EIA, c y t i d y l a t e c y c l a s e a c t i v i t y w a s d e t e r m i n e d in v a r i o u s t i s s u e h o m o g e n a t e s from rats and m i c e at the level of 0.i-i0 pmol/min/mg protein, with the highest level in b r a i n (15,16). Cytidylate cyclase was characterized by the localization on membranes, M n Z ÷ - d e p e n d e n c e and u n i q u e pH o p t i m u m at a r o u n d 9 and, m o r e o v e r , its a c t i v i t y w a s not i n f l u e n c e d by f o r s k o l i n and l a n t h a n u m chloride, a s p e c i f i c s t i m u l a t o r *To w h o m c o r r e s p o n d e n c e
Copyright
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s h o u l d be a d d r e s s e d .
0024-3205/93 $6.00 + .00 1992 P e r g a m o n P r e s s Ltd. All rights
reserved.
14
Cytidylate
Cyclase
and G - p r o t e i n
Vol.
52, No.
i, 1993
and inhibitor, respectively, of adenylate cyclase (16). These findings i n d i c a t e t h a t t h i s e n z y m e is d i s t i n c t f r o m a d e n y l a t e and g u a n y l a t e c y c l a s e s . Although we have demonstrated the modification of c y t i d y l a t e cyclase a c t i v i t y by c h o l i c a c i d s and p h o s p h o l i p i d s (17), no o t h e r e n d o g e n o u s l i g a n d s or p r o t e i n s a f f e c t i n g the e n z y m e a c t i v i t y h a v e b e e n r e p o r t e d . However, s e v e r a l f i n d i n g s on its d i s t r i b u t i o n , enzymological p r o p e r t i e s a n d f u n c t i o n s of the e n z y m a t i c p r o d u c t a l l o w us to e x p e c t t h e p a r t i c i p a t i o n of a c e r t a i n g u a n i n e n u c l e o t i d e - b i n d i n g p r o t e i n ( G - p r o t e i n ) in the r e g u l a t i o n of c y t i d y l a t e c y c l a s e activity. In this study, we found that this enzyme activity was also stimulated via the activation of G-protein as w e l l as adenylate cyclase activity, but it a p p e a r e d to be r e g u l a t e d by a c e r t a i n G - p r o t e i n d i f f e r e n t from some well-characterized G-proteins. Materials
and M e t h o d s
Materials: ATP, cAMP, NAD, d i t h i o t h r e i t o l (DTT), b o v i n e s e r u m a l b u m i n (BSA, C o h n fr.V), B - g a l a c t o s i d a s e (E. coli) and 4 - m e t h y l u m b e l l i f e r y l - 8 - D - g a l a c t o s i d e (4-MUG) w e r e p u r c h a s e d f r o m S i g m a (St. Louis, MO). GTP, GMP, C T P and c C M P w e r e o b t a i n e d from Y a m a s a S h o y u Co. (Choshi, Japan). G u a n o s i n e 5 ' - O - ( 2 - t h i o d i p h o s phate) (GDPBS) and g u a n o s i n e 5 ' - O - ( 3 - t h i o t r i p h o s p h a t e ) (GTPTS) w e r e p u r c h a s e d from Boehringer Mannheim Yamanouchi (Tokyo, Japan). Islet-activating protein (IAP) a n d c h o l e r a t o x i n (CT) w e r e o b t a i n e d f r o m F u n a k o s h i (Tokyo, Japan) and sodium fluoride was from Nakalai Tesque (Kyoto, Japan). Aluminium chloride w a s a p r o d u c t of I s h i z u P h a r m a c e u t i c a l Co. (Osaka, Japan). All o t h e r r e a g e n t s w e r e of a n a l y t i c a l g r a d e c o m m e r c i a l l y a v a i l a b l e . R a b b i t a n t i s e r a s p e c i f i c to s u c c i n y l c C M P and s u c c i n y l c A M P a n d g o a t a n t i s e r u m s p e c i f i c to r a b b i t IgG w e r e p r e p a r e d in o u r l a b o r a t o r y , as d e s c r i b e d p r e v i o u s l y (14). E a c h a n t i b o d y (IgG fraction) was purified by a ~ m o n i u m sulfate fractionation and ion-exchange chromatography. S u c c i n y l c C M P a n d s u c c i n y l c A M P c o u p l e d w i t h B-galactosidase w e r e s y n t h e s i z e d as d e s c r i b e d p r e v i o u s l y (14). P r e p a r a t i o n of E n z y m e Fraction: M a l e W i s t a r rats ( 2 0 0 - 2 5 0 g) o b t a i n e d f r o m J a p a n SLC (Hamamatsu, Japan) w e r e s a c r i f i c e d by d e c a p i t a t i o n . W h o l e b r a i n was r a p i d l y excised, r i n s e d in c o l d s a l i n e and h o m o g e n i z e d in 9 v o l u m e s of i0 mM T r i s - H C l b u f f e r (pH 7.5) c o n t a i n i n g 1 m M DTT and 0.32 M s u c r o s e at 4°C u s i n g g l a s s - T e f l o n h o m o g e n i z e r . T h i s w a s c e n t r i f u g e d at 3 0 0 x g for i0 m i n at 4°C and the r e s u l t i n g s u p e r n a t a n t was u s e d as t h e h o m o g e n a t e s . In a n o t h e r e x p e r i m e n t , the h o m o g e n a t e s w e r e f u r t h e r c e n t r i f u g e d at 1 0 , 0 0 0 x g for I0 m i n at 4°C and the p r e c i p i t a t e o b t a i n e d was u s e d as the m e m b r a n e p r e p a r a t i o n . Other tissues w e r e p r o c e s s e d in the s a m e w a y to p r e p a r e the h o m o g e n a t e s . The p r o t e i n c o n t e n t w a s d e t e r m i n e d by the m e t h o d of S m i t h et al. (18) u s i n g B S A as a standard. C y c l a s e Assay: Cytidylate ~clase a c t i v i t y was d e t e r m i n e d at pH 8.0, but not at its o p t i m u m pH, to e s t i m a t e its p h y s i o l o g i c a l involvement, as d e s c r i b e d p r e v i o u s l y (15). T h e r e a c t i o n m i x t u r e c o n s i s t e d of 50 m M T r i s - H C l b u f f e r (pH 8.0), 1 mM CTP, 4 m M MnCI2 and an a l i q u o t of e n z y m e s a m p l e in a t o t a l v o l u m e of i00 NI. A f t e r 3 m i n - p r e i n c u b a t i o n , the r e a c t i o n w a s i n i t i a t e d by a d d i n g e n z y m e s o l u t i o n and i n c u b a t e d at 3 0 ° C for i0 min. It w a s t e r m i n a t e d by the a d d i t i o n of 0.9 ml of 50 mM a c e t a t e b u f f e r (pH 4.0), f o l l o w e d by h e a t i n g at 9 0 ° C for 3 min. T h e r e a c t i o n m i x t u r e w a s c e n t r i f u g e d at 5 , 0 0 0 X g for 15 m i n and 50 N1 of the s u p e r n a t a n t was s u b j e c t e d to s u c c i n y l a t i o n r e a c t i o n p r i o r to the q u a n t i f i c a t i o n of c C M P formed. A h e a t e d e n z y m e (90°C for 3 min) was i n c l u d e d as a blank. A d e n y l a t e c y c l a s e a c t i v i t y w a s d e t e r m i n e d in a s i m i l a r m a n n e r by e m p l o y i n g the r e a c t i o n m i x t u r e c o n s i s t e d of 50 mM T r i s - H C l b u f f e r (pH 7.4), 50 N M ATP, 4 mM MnCI2, i0 m M t h e o p h y l l i n e and an e n z y m e s o l u t i o n . Determination of method described method is based
Cyclic Nucleotide: Succinyl cCMP was quantified by the by us (14) w i t h s o m e m o d i f i c a t i o n s . This simplified assay on the direct competitive EIA. In brief, the antibody
Vol.
52, No.
I, 1 9 9 3
Cytidylate Cyclase and G-protein
15
specific to cCMP was adjusted to the protein concentration at 3 N g / m l in 0.05 M phosphate buffer (pH 6.8)-0.5 M NaCI-5 mM MgCI2 (buffer A). Polystyrene tubes ( F a l c o n 2052) w e r e c o a t e d w i t h 2 0 0 N 1 a n t i b o d y s o l u t i o n o v e r n i g h t at 4°C. A f t e r r e m o v a l , t h e r e s i d u a l b i n d i n g c a p a c i t y of t u b e s w a s b l o c k e d w i t h 500 N 1 o f 0 . 5 % B S A i n b u f f e r A ( b l o c k i n g s o l u t i o n ) at 3 7 ° C for 1 hr. T h e t u b e s w e r e r i n s e d t w i c e w i t h 0 . 1 % B S A in b u f f e r A ( w a s h i n g s o l u t i o n ) , f o l l o w e d b y a d d i n g I 0 0 N 1 of s e r i a l l y d i l u t e d s t a n d a r d o r t e s t s o l u t i o n a n d i00 N1 of cCMP-B-galactosidase conjugate which were both diluted with blocking buffer. T h e m i x t u r e w a s i n c u b a t e d at 2 6 0 C for 4 hr, followed by washing twice with washing buffer. T h e t u b e s w e r e a d d e d 3 0 0 N 1 o f 0.2 m M 4-MUG in buffer A and incubated under shaking for 90 m i n at 37°C. The r e a c t i o n w a s t e r m i n a t e d b y a d d i t i o n o f 1.5 ml o f 0.i M s o d i u m c a r b o n a t e a n d t h e f l u o r e s c e n c e i n t e n s i t y w a s m o n i t o r e d in S h i m a d z u f l u o r o s p e c t r o m e t e r w i t h a n e x c i t a t i o n at 3 6 0 n m a n d a n e m i s s i o n at 4 6 0 nm. T h e a m o u n t of c C M P f o r m e d in the test solution was quantified by interpolation onto the inhibition curve generated with serial dilutions of a standard cCMP solution. In t h i s assay, the limit of detection was 3 fmol/assay tube. Succinyl cAMP was q u a n t i f i e d b y E I A d e s c r i b e d p r e v i o u s l y (14). In b o t h a s s a y s , t h e c r o s s r e a c t i v i t y b y o t h e r n u c l e o t i d e s w a s c o n f i r m e d to be l e s s t h a n 0 . 0 1 % . E f f e c t of AIF4- o n C y c l a s e A c t i v i t y : T h e h o m o g e n a t e s w e r e p r e i n c u b a t e d at 4 ° C for 1 h r w i t h a c o m b i n a t i o n of N a F a n d A I C I 3 at t h e c o n c e n t r a t i o n s o f i - i 0 a n d 0 . 2 - 2 mM, r e s p e c t i v e l y , a n d d e t e r m i n e d f o r b o t h c y c l a s e a c t i v i t i e s . Effect of GTP and GDP Analogues on Cyclase Activity: Brain membrane preparat i o n (1-2 m g p r o t e i n ) w a s p r e i n c u b a t e d w i t h v a r i o u s c o n c e n t r a t i o n s o f G T P T S a t 3 0 ° C for 3 0 m i n i n a t o t a l v o l u m e o f 4 0 0 N1 o f 30 m M T r i s - H C l b u f f e r (pH 7.5)-1 m M DTT. After dilution with the same volume of cold buffer, the membranes were washed twice by centrifugation at 1 5 , 0 0 0 x g for 5 min. The m e m b r a n e p e l l e t s w e r e s u s p e n d e d in i0 m M T r i s - H C l b u f f e r (pH 7 . 5 ) - 1 m M D T T
200
~0
~ ~
O
100
0 0
!
I
I
I
2 0.4
4 0.8
6 1.2
10 2.0
FIG.
10 0
O
0 NaF (mM) 2.0 AICI3(mM)
1
Stimulation of cyclase activity of rat brain homogenates by AIF4-. Homogenates incubated with NaF and AICI 3 were measured for c y t i d y l a t e (e) a n d a d e n y l a t e (0) c y c l a s e a c t i v i t i e s . T h e c o n t r o l values for cytidylate and adenylate cyclases were 5.76 and 198.5 pmol/min/mg protein, respectively. Each point represents the mean of triplicate determinations. These results are representative of three experiments.
16
Cytidylate
Cyclase
and G - p r o t e i n
Vol.
52, No.
i, 1993
and d e t e r m i n e d for b o t h c y c l a s e a c t i v i t i e s . The e f f e c t of G D P B S or G M P w a s e s t i m a t e d in t h e s a m e w a y d e s c r i b e d a b o v e a f t e r i n c u b a t i o n of b r a i n m e m b r a n e s w i t h v a r i o u s c o n c e n t r a t i o n s of e a c h c o m p o u n d in the p r e s e n c e of i0 NM GTPTS. E f f e c t of B a c t e r i a l T o x i n on C y c l a s e A c t i v i t y : B r a i n m e m b r a n e p r e p a r a t i o n (1-2 mg p r o t e i n ) w a s p r e i n c u b a t e d w i t h v a r i o u s c o n c e n t r a t i o n s of a c t i v a t e d IAP at 3 0 ° C for 30 m i n in a t o t a l v o l u m e of 500 N1 of 30 m M T r i s - H C I b u f f e r (pB 7.5)2mM D T T - 6 0 NM N A D - 0 . 6 mM A T P - 6 0 N M GTP. A c t i v a t i o n of IAP w a s d o n e by its i n c u b a t i o n at 3 0 ° C for 20 m i n in i00 ram D T T - 0 . 1 m M A T P - 5 0 mM T r i s - H C I b u f f e r (pH 7.5). A f t e r d i l u t i o n w i t h the s a m e v o l u m e of c o l d buffer, the m e m b r a n e s w e r e w a s h e d t w i c e by c e n t r i f u g a t i o n at 1 5 , 0 0 0 X g for 5 m i n and d e t e r m i n e d for both cyclase activities. The p r e t r e a t m e n t of b r a i n m e m b r a n e p r e p a r a t i o n (1-2 mg p r o t e i n ) w i t h v a r i o u s c o n c e n t r a t i o n s of a c t i v a t e d CT w a s c o n d u c t e d at 3 0 ° C for 60 m i n in a t o t a l v o l u m e of 500 N1 of 30 mM T r i s - H C l b u f f e r (pH 7 . 5 ) - 2 mM D T T - 6 0 N M N A D - 0 , 6 m M A T P - 1 2 N M G T P T S - 2 m M MgClz. CT w a s a c t i v a t e d by its i n c u b a t i o n at 3 7 ° C for 20 m i n in 50 mM D T T - 5 0 mM s o d i u m p h o s p h a t e b u f f e r (pH 7.5). A f t e r washing, the m e m b r a n e s w e r e d e t e r m i n e d for b o t h c y c l a s e a c t i v i t i e s . Results Brain homogenates and m e m b r a n e preparation used in t h i s study showed c y t i d y l a t e c y c l a s e a c t i v i t i e s of 5 . 0 3 ± 0 . 6 5 and 6 . 5 5 ± 1 . 4 9 p m o l / m i n / m g protein, respectively, which were much lower than adenylate cyclase activities. Effect of AIF4-, a s t i m u l a t o r of a d e n y l a t e c y c l a s e v i a a c t i v a t i o n of G - p r o t e i n (19), on b o t h c y c l a s e a c t i v i t i e s was f i r s t d e t e r m i n e d . The c o m b i n e d a d d i t i o n of 2-4 mM N a F and 0.4-0.8 mM AICI 3 resulted in m o r e than 2-times elevation of
300 A
200 (D O
"-6 o
~
lOO
0
I
0
10
I
20 Time (rain) FIG.
I
I
30
0.1
I
I
i
I
1 10 100 1000 GTP'¢S (gM)
2
S t i m u l a t i o n of c y c l a s e a c t i v i t y of rat b r a i n m e m b r a n e s by GTPTS. (A) T i m e d e p e n d e n c y . B r a i n m e m b r a n e s w e r e i n c u b a t e d at 30°C for v a r i o u s t i m e s w i t h (circle) or w i t h o u t ( t r i a n g l e ) i0 NM G T P T S and determined for cytidylate (o,&) and adenylate (0,4) cyclase activities. (B) C o n c e n t r a t i o n d e p e n d e n c y . M e m b r a n e p r e p a r a t i o n w a s i n c u b a t e d w i t h v a r i o u s c o n c e n t r a t i o n s of G T P T S at 3 0 ° C for 30 m i n a n d d e t e r m i n e d for c y t i d y l a t e (o) and a d e n y l a t e (o) c y c l a s e a c t i v ities. The control values for c y t i d y l a t e and a d e n y l a t e c y c l a s e s were 5.1 and 193 p m o l / m i n / m g protein, respectively. Each point represents the m e a n of t r i p l i c a t e determinations. These results are r e p r e s e n t a t i v e of t h r e e e x p e r i m e n t s .
Vol.
52, No.
i, 1993
Cytidylate
Cyclase
and G - p r o t e i n
c y t i d y l a t e c y c l a s e a c t i v i t y of b r a i n h o m o g e n a t e s as well a c t i v i t y (Fig. i). In this e x p e r i m e n t , a s i n g l e a d d i t i o n no a c t i v a t i o n of b o t h c y c l a s e s .
as of
adenylate compounds
17
cyclase induced
The pretreatment of b r a i n m e ~ r a n e p r e p a r a t i o n w i t h GTPTS, a nonhydrol y z a b l e G - p r o t e i n a c t i v a t o r (20), at i0 N M i n d u c e d a t i m e - d e p e n d e n t a c t i v a t i o n of b o t h c y c l a s e s , t h o u g h c y t i d y l a t e c y c l a s e was a c t i v a t e d m h ~ h ~ f a s t e r t h a n adenylate cyclase (Fig. 2A). T h e i n c u b a t i o n of m e m b r a n e p r e p a r a t i o n in the a b s e n c e of G T P T S s h o w e d no c h a n g e in t h e i r c y c l a s e a c t i v i t i e s . In addition, GTPTS-induced activation of c y t i d y l a t e c y c l a s e as w e l l as a d e n y l a t e c y c l a s e w a s o b s e r v e d in a c o n c e n t r a t i o n - d e p e n d e n t f a s h i o n at 0 . i - i 0 0 0 NM, r e a c h i n g a p l a t e a u of 3 - f o l d i n c r e a s e at the h i g h e s t c o n c e n t r a t i o n u s e d (Fig. 2B). G D P ~ S is an i n a c t i v a t o r the a c t i v a t e d protein (21). NM) e f f e c t i v e l y a b o l i s h e d the (Fig. 3). This i n h i b i t i o n by in a s i m i l a r m a n n e r . However,
of G - p r o t e i n w h i c h c a n r e p l a c e G T P or G T P T S in The increasing concentrations of G D P ~ S (i-i000 a c t i v a t i o n of c y t i d y l a t e c y c l a s e by i0 N M G T P T S G T P ~ S w a s a l s o d e m o n s t r a t e d for a d e n y l a t e c y c l a s e GMP did not s h o w s u c h an e f f e c t .
To estimate a G-protein component in the regulation of cytidylate cyclase activity, two well-characterized bacterial toxins which regulate a d e n y l a t e c y c l a s e a c t i v i t y (22-25) w e r e e m p l o y e d in t h i s study. T r e a t m e n t w i t h IAP r e s u l t e d in an i n c r e a s e of a d e n y l a t e c y c l a s e a c t i v i t y at the c o n c e n t r a t i o n s of 0 . 0 1 - 0 . i Ng/ml, w h e r e a s it h a d no i n f l u e n c e on c y t i d y l a t e c y c l a s e activity (Fig. 4A). In addition, CT m a r k e d l y a c t i v a t e d adenylate c y c l a s e at 20 Ng/ml, but c y t i d y l a t e c y c l a s e w a s not a f f e c t e d by this t r e a t m e n t (Fig. 4B). Cytidylate cyclase a c t i v i t y has b e e n d e t e c t e d in v a r i o u s tissues (15). We f i n a l l y d e t e r m i n e d w h e t h e r c y t i d y l a t e cyclase a c t i v i t i e s in t i s s u e s b e s i d e s b r a i n a r e a f f e c t e d by AIF4-. W h e n a d d e d w i t h 4 mM N a F a n d 0.8 mM AICIs, the
..=Z'~- 100 7~ "6 (1)._
8o so
0
I
0
1
I
I
I
10 100 1000 GDPyS or GMP (mM) FIG.
3
E f f e c t of G D P ~ S and G M P o n G T P T S - s t i m u l a t e d c y c l a s e a c t i v i t y of rat b r a i n m e m b r a n e s . Membrane preparation used in FIG. 2 were i n c u b a t e d at 3 0 ° C for 30 m i n w i t h v a r i o u s c o n c e n t r a t i o n s of G D P ~ S (circle) or G M P (triangle) in the p r e s e n c e of i0 N M G T P T S and d e t e r m i n e d for c y t i d y l a t e (e,A) a n d a d e n y l a t e (0,4) c y c l a s e a c t i v ities. E a c h p o i n t r e p r e s e n t s the m e a n of t r i p l i c a t e d e t e r m i n a t i o n s . rhese r e s u l t s a r e r e p r e s e n t a t i v e of two e x p e r i m e n t s .
18
Cytidylate
Cyclase
and G - p r o t e i n
A
300
Vol.
i, 1993
B
1100 1000
Y
8 O 200 tn O
o
52, No.
300
10C
200 100 C
0
I
0.01
I
I
I
0.1 1 lAP (~g/ml)
I
10
0
FIG.
0.2
I
I
2 20 CT (~g/ml)
I
200
4
E f f e c t of t r e a t m e n t w i t h IAP and CT on c y c l a s e a c t i v i t y of rat brain membranes. (A) Membrane preparation was incubated with various concentrations of a c t i v a t e d IAP at 3 0 ° C for 30 m i n and determined for c y t i d y l a t e (e) and a d e n y l a t e (o) c y c l a s e a c t i v i ties. (B) M e m b r a n e p r e p a r a t i o n w a s i n c u b a t e d w i t h v a r i o u s c o n c e n t r a t i o n s of a c t i v a t e d CT at 3 0 ° C for 60 m i n and d e t e r m i n e d for cytidylate (e) and a d e n y l a t e (0) c y c l a s e a c t i v i t i e s . The c o n t r o l values for c y t i d y l a t e and a d e n y l a t e c y c l a s e a c t i v i t i e s w e r e 6.4 and 340 p m o l / m i n / m g protein, respectively. Each point represents t h e m e a n of t r i p l i c a t e d e t e r m i n a t i o n s . T h e s e v a l u e s are r e p r e s e n t a t i v e of two e x p e r i m e n t s .
TABLE Effect
of A I F 4- on C y t i d y l a t e
Tissue
Cyclase
Activity
in Rat T i s s u e s
Cytidylate cyclase activity (pmol c C M P f o r m e d / m i n / m g p r o t e i n ) None
Brain Lung Adrenal Spleen Kidney Liver
I
4.84 1.09 0.581 0.544 0.459 0.266
~ ~ ~ ~ ~ ±
4 mM NaF + 0.8 mM AICI 3 0.074 0.137 0.009 0.060 0.045 0.003
16.79 3.40 1.73 1.03 0.838
~ ~ ~ ~ ~
0.318
~
0.935 0.377 0.009 0.065 0.027 0.002
V a r i o u s t i s s u e h o m o g e n a t e s w e r e i n c u b a t e d w i t h 4 m M N a F and 0.8 mM AICI3 a n d d e t e r m i n e d for c y c l a s e a c t i v i t y . Each value repres e n t s the m e a r ~ S D of t h r e e animals, e a c h a s s a y e d in t r i p l i c a t e . h o m o g e n a t e s f r o m lung, spleen, k i d n e y a n d a d r e n a l s h o w e d a 2 - 3 - t i m e s increase, resembling the p h e n o m e n o n o b s e r v e d in b r a i n h o m o g e n a t e s (Table I). However, this treatment induced little enhancement of c y t i d y l a t e c y c l a s e a c t i v i t y in l i v e r h o m o g e n a t e s w h i c h p o s s e s s e d the l o w e s t v a l u e a m o n g t h e t i s s u e s e x a m i n e d .
Vol.
52, No.
i,
1993
Cytidylate
Cyclase
and G - p r o t e i n
19
Discussion G - p r o t e i n s h a v e b e e n c h a r a c t e r i z e d to m o d i f y the s i g n a l t r a n s d u c t i o n from r e c e p t o r s to e f f e c t o r e n z y m e s in v a r i o u s b i o l o g i c a l r e s p o n s e s (26). A d e n y l a t e cyclase system is t h e representative of m e m b r a n e reactions in w h i c h such G-proteins a r e i n v o l v e d as r e g u l a t o r y c o m p o n e n t s (27). O u r p r e v i o u s s t u d i e s h a v e r e v e a l e d t h a t c C M P a l s o p l a y s an i m p o r t a n t r o l e in c e l l p r o l i f e r a t i o n and d i f f e r e n t i a t i o n as w e l l as cAMP. T h e r e f o r e , a p r o o f of the c o u p l i n g of G-protein in the cytidylate cyclase system is i m p o r t a n t to e v a l u a t e the p h y s i o l o g i c a l r o l e of t h i s e n z y m e in m e m b r a n e signal t r a n s d u c t i o n . Cytidylate c y c l a s e a c t i v i t y in rat b r a i n m e m b r a n e s was m a r k e d l y e l e v a t e d by the p r e t r e a t m e n t of AIF4- and G T P T S and the a c t i v a t i o n by G T P T S was e f f e c t i v e l y a b o l i s h e d b y GDP~S, s u g g e s t i n g this p o s s i b i l i t y strongly. A l t h o u g h the same p h e n o m e n o n was observed in the c a s e of a d e n y l a t e cyclase, the s y n t h e s i s of c C M P w a s s h o w n not to r e s u l t f r o m a s i d e r e a c t i o n by a d e n y l a t e c y c l a s e (15). S u c h an a c t i v a t i o n of c y t i d y l a t e c y c l a s e by AIF4- was d e m o n s t r a t e d in o t h e r t i s s u e s except liver. These results indicate that cytidylate cyclase is also associated with a certain G-protein in the m e m b r a n e . However, it r e m a i n s u n c l e a r w h e t h e r the r e g u l a t i o n m o d e of c y t i d y l a t e c y c l a s e a c t i v i t y by t h e s e agents is different among tissues, as speculated on the activation of a d e n y l a t e c y c l a s e by AIF4- and f o r s k o l i n (28,29). The e l e v a t i o n of c y t i d y l a t e c y c l a s e a c t i v i t y v i a G - p r o t e i n a c t i v a t i o n was considerably different from t h a t of a d e n y l a t e c y c l a s e in s e v e r a l respects, s u c h as r a p i d a c t i v a t i o n by i0 N M G T P ~ S and u n r e s p o n s i v e n e s s to t h e s e two k i n d s of G - p r o t e i n - m o d i f y i n g bacterial toxins. The f i n d i n g m a y s u g g e s t the d i f f e r e n c e in the s p e c i e s of G - p r o t e i n a c t i n g in b o t h c y c l a s e systems. As IAP and CT are k n o w n to r e g u l a t e the f u n c t i o n s of Gi and G., r e s p e c t i v e l y , t h r o u g h their ADP-ribosylation (22-25), G-protein which functions in cytidylate c y c l a s e s y s t e m m a y be at least n e i t h e r Gi nor Gs- However, f u r t h e r i n v e s t i g a t i o n is r e q u i r e d for c h a r a c t e r i z i n g t h i s p r o t e i n in t e r m s of the n e c e s s i t y of A D P - r i b o s y l a t i o n , b e c a u s e the e x p e r i m e n t a l c o n d i t i o n s u s e d in the p r e s e n t s t u d y are s u i t a b l e o n l y for the A D P - r i b o s y l a t i o n of G - p r o t e i n s in a d e n y l a t e c y c l a s e system. In o u r p r e v i o u s studies, c C M P and c A M P w e r e s h o w n to e x h i b i t s i m i l a r e f f e c t s o n the i n h i b i t i o n of c u l t u r e d h e p a t o c y t e g r o w t h and on the s t i m u l a t i o n of n e u r i t e o u t g r o w t h of PC12 cells, a l t h o u g h b o t h a c t i v i t i e s by c C M P w e r e w e a k e r t h a n t h o s e by c A M P (I0,ii). T h e s e f i n d i n g s s u g g e s t t h a t c C M P a n d c A M P w h o s e b i o s y n t h e s i s is c o n t r o l l e d by d i s t i n c t r e g u l a t i o n s y s t e m s m a y c o o p e r a t e in s o m e b i o l o g i c a l r e s p o n s e s . B o t h h o m o g e n a t e s and m e m b r a n e p r e p a r a t i o n from rat b r a i n u s e d as e n z y m e sources had the comparable cytidylate cyclase activities. However, the occurrence of cCMP phosphodiesterase which hydrolyzes cCMP has been demonstrated in v a r i o u s t i s s u e e x t r a c t s (30-32). T h i s e n z y m e is i n s e n s i t i v e to t h e o p h y l l i n e and 3 - i s o b u t y l - l - m e t h y l - x a n t h i n e which are potent inhibitors of c A M P and c G M P p h o s p h o d i e s t e r a s e s . In fact, c y t i d y l a t e c y c l a s e a c t i v i t y w a s not a f f e c t e d by t h e a d d i t i o n of t h e s e c l a s s i c a l inhibitors in the p r e s e n t study (data not shown). Therefore, no i n h i b i t o r was a d d e d to the r e a c t i o n m i x t u r e for c y t i d y l a t e c y c l a s e assay. A l t h o u g h this m a y c a u s e an u n d e r e s t i m a t e of t h i s e n z y m e a c t i v i t y , it has no i n f l u e n c e on the p r o f i l e of e n z y m e a c t i v a tion. Recently, there appeared a report describing surprisingly high specific a c t i v i t i e s of c y t i d y l a t e c y c l a s e in v a r i o u s tissues, w h i c h w e r e c o m p a r a b l e to t h o s e of a d e n y l a t e c y c l a s e (33). T h e s e d i d not a r i s e f r o m the i n h i b i t i o n of d e g r a d a t i o n of cCMP, b e c a u s e no i n h i b i t o r w a s used. T h i s d i s c r e p a n c y o b s e r v e d in e n z y m e a c t i v i t i e s is not y e t solved, but it m a y be d u e in part to the difference in a n a l y t i c a l factors including the s e n s i t i v i t y and s p e c i f i c i t y . A l t h o u g h s e v e r a l r e p o r t s h a v e d e s c r i b e d the p r o p e r t i e s of c y t i d y l a t e c y c l a s e (12,13,15,16,33-35), the fact of e n z y m e p r o t e i n is still n o t f u l l y e l u c i d a t e d . To clarify the substantial association of a G-protein and enzymological a s p e c t s in this e n z y m e system, it is n e c e s s a r y to p u r i f y the e n z y m e to h i g h l y
20
homogeneous
Cytidylate Cyclase and G-protein
s t a t e a n d to s e a r c h f o r an e n d o g e n o u s
Vol.
52, No.
i, 1993
agonist to this enzyme system.
Acknowledgement T h i s w o r k w a s s u p p o r t e d in p a r t b y G r a n t s - i n - A i d for S c i e n t i f i c R e s e a r c h (No. 0 2 6 7 0 9 9 8 ) f r o m t h e M i n i s t r y of E d u c a t i o n , S c i e n c e a n d C u l t u r e of Japan. References i. A. BLOCH, B i o c h e m . B i o p h y s . Res. C o m m u n . 58 6 5 2 - 6 5 9 (1974). 2. A. BLOCH, G. D U T S C H M A N a n d R. MAUE, B i o c h e m . B i o p h y s . Res. C o m m u n . 59 9 5 5 959 (1974). 3. A. BLOCH, A d v a n c e s in C y c l i c N u c l e o t i d e R e s e a r c h , v o l . 5 , G.I. D r u m m o n d , P. G r e e n g a r d a n d G.A. R o b i n s o n (eds), 3 3 1 - 3 3 8 , R a v e n Press, N e w Y o r k (1975). 4. T.R. A N D E R S O N , Mol. Cell. E n d o c r i n o l . 28 3 7 3 - 3 8 5 (1982). 5 L.G. S H E F F I E L D , C e l l . Biol. Int. Rep. Ii 5 5 7 - 5 6 2 (1987). 6 J. KOWAL, E n d o c r i n o l o g y 98 4 6 1 - 4 6 8 (1973). 7 M.A. P I S A R E V a n d D . L . K . P I S A R E V , A c t a E n d o c r i n o l . 84 2 9 7 - 3 0 2 (1977). 8 P.E. C A N A S a n d L.F. C O N G O T E , Can. J. B i o c h e m . 60 1-7 (1982). 9 J. E R V E N S a n d R. S E I F E R T , B i o c h e m . B i o p h y s . Res. C o m m u n . 174 2 5 8 - 2 6 7 (1991). i0 T. KUBIN, M. Y A N A G I D A , S. MORI, Y. H A Y A S H I , E. G O H D A a n d I. Y A M A M O T O , C e l l Biol. I n t e r n a t . Rep. 13 9 0 7 - 9 1 7 (1989). ii M. Y A N A G I D A , E. G O H D A a n d I. Y A M A M O T O , N e u r o s c i . Lett. 114 3 2 3 - 3 2 8 (1990). 12 S.Y. C E C H a n d L.J. IGNARR0, S c i e n c e 198 1 0 6 3 - 1 0 6 5 (1977). 13 S.Y. C E C H a n d L.J. IGNARR0, B i o c h e m . B i o p h y . Res. C o m m u n . 80 1 1 9 - 1 2 5 (1978). 14 I. Y A M A M O T O , T. T A K A I a n d J. TSUJI, I m m u n o p h a r m a c o l o g y 4 3 3 1 - 3 4 0 (1982). 15 I. Y A M A M O T O , T. T A K A I a n d S. MORI, B i o c h i m . B i o p h y s . A c ~ a 933 1 9 1 - 1 9 8 (1989). 16 S. MORI, M. Y A N A G I D A , K. K U B O T U a n d I. Y A M A M O T O , Sec. Mess. P h o s p h o p r o t . 13 1-12 (1990). 17. S. MORI, N. M U T O a n d I. Y A M A M O T O , B i o c h e m . B i o p h y . Res. C o m m u n . 162 15021507 (1989). 18. P.K. SMITH, R.I. KROHN, G.T. H E R M A N S O N , A.K. M A L L I A , F.H. G A R T N E R , M.D. P R O V E N Z A N 0 , E.K. F U J I M O T O , N.M. GOEKE, B.J. O L S O N a n d D.C. KLENK, Anal. B i o c h e m . 150 7 6 - 8 5 (1985). 19. P.C. S T E R N W E I S a n d A.G. G I L M A N , Proc. Natl. Acad. Sci. U S A 79 4 8 8 8 - 4 8 9 1 (1982). 20. T. P F E U F F E R a n d E . J . M . H E L M R E I C H , J. Biol. Chem. 250 8 6 7 - 8 7 6 (1975). 21. F. E C K S T E I N , D. C A S S E L , H. L E V K O V I T Z , M. L O W E and Z. S E L I N G E R , J. Biol. C h e m . 254 9 8 2 9 - 9 8 3 4 (1979). 22. T. K A T A D A a n d M. UI, J. Biol. Chem. 257 7 2 1 0 - 7 2 1 6 (1982). 23. T. K A T A D A a n d M. UI, Proc. Natl. Acad. Sci. U S A 79 3 1 2 9 - 3 1 3 3 (1982). 24. D. C A S S E L a n d T. P F E U F F E R , Proc. Natl. A c a d . Sci. U S A 75 2 6 6 9 - 2 6 7 3 (1978). 25. D.M. G I L L a n d R. M E R E N , Proc. Natl. A c a d . Sci. U S A 75 3 0 5 0 - 3 0 5 4 (1978). 26. A. GILMAN, Annu. Rev. B i o c h e m . 56 6 1 5 - 6 4 9 (1987). 27. M. R O D B E L L , H . M . J . KRANS, S.L. P O H L a n d L. B I R N B A U M E R , J. Biol. Chem. 246 1 8 7 2 - 1 8 7 6 (1971). 28. E.W. S U T H E R L A N D , T. W. R A L L a n d T. MENON, J. Biol. Chem. 237 1 2 2 0 - 1 2 2 7 (1962). 29. K. B. S E A M O N , W. P A D G E T T a n d J.W. DALY, Proc. Natl. Acad. Sci. U S A 78 3 3 6 3 - 3 3 6 7 (1981). 30. Y.C. C H E N G a n d A. BLOCH, J. Biol. Chem. 253 2 5 2 2 - 2 5 2 4 (1978). 31. J.F. KU0, N . L . B R A C K E T T , M. S H O J I a n d J. TSE, J. Biol. Chem. 253 2 5 1 8 - 2 5 2 1 (1978). 32. R.P. N E W T O N a n d S.G. SALIH, Int. J. B i o c h e m . 18 7 4 3 - 7 5 2 (1986). 33. R.P. N E W T O N , B.J. S A L V A G E a n d N.A. H A A K E E M , B i o c h e m . J. 265 5 8 1 - 5 8 6 (1990). 34. R.M. G A I R O N a n d G. K R I S H I N A , B i o c h e m . B i o p h y . Res. C o m m u n . 86 1 0 5 - 1 1 1 (1979). 35. R.M. G A I R O N a n d G. K R I S H I N A , S c i e n c e 203 672 (1979).
CYTIDYLATE
52, pp.
13-20
Pergamon
C Y C L A S E A C T I V I T Y IS S T I M U L A T E D V I A A C T I V A T I O N GUANINE NUCLEOTIDE-BINDING PROTEIN
Press
OF
A
N o r i o Muto, Department
Masatoshi
K a n o h and
Itaru Y a m a m o t o *
of I m m u n o c h e m i s t r y , F a c u l t y of P h a r m a c e u t i c a l O k a y a m a U n i v e r s i t y , O k e y a m a 700, J a p a n (Received
in final
form October
26,
Sciences,
1992)
Summary C y t i d y l a t e c y c l a s e is a m e m b r a n e - b o u n d e n z y m e w h i c h c a t a l y z e s the biosynthesis of cytidine 3',5'-cyclic monophosphate (cCMP) from CTP. By u s i n g a s e n s i t i v e and s p e c i f i c e n z y m e i m m u n o a s s a y method, we evaluated the participation of guanine nucleotide-binding protein (G-protein) in the regulation of cytidylate cyclase a c t i v i t y in rat b r a i n and o t h e r tissues. AIF4-, an a c t i v a t o r of G-proteins, effectively elevated the cyclase activity. The stimulation by GTPTS, a nonhydrolyzable GTP analogue, was a l s o observed in t i m e and c o n c e n t r a t i o n - d e p e n d e n t manner during the preincubation and t h i s e f f e c t w a s c o m p e t i t i v e l y i n h i b i t e d by the a d d i t i o n of GDPBS. However, islet-activating p r o t e i n and c h o l e r a t o x i n w h i c h a f f e c t e d a d e n y l a t e c y c l a s e a c t i v i t y h a d no e f f e c t on cytidylate cyclase activity. These results indicate that c y t i d y l a t e c y c l a s e is a s s o c i a t e d w i t h a c e r t a i n G - p r o t e i n and its a c t i v i t y is r e g u l a t e d by the m o d e d i f f e r e n t from t h a t of a d e n y l a t e cyclase. Cytidine 3',5'-cyclic monophosphate (cCMP) w a s first found as a t h i r d c y c l i c n u c l e o t i d e in m a m m a l i a n t i s s u e s and l e u k e m i a L - 1 2 1 0 c u l t u r e s b y B l o c h et al. (1-3). A l t h o u g h its p h y s i o l o g i c a l f u n c t i o n r e m a i n s to be c l a r i f i e d , possible roles in regulating cell proliferation and biological processes h a v e b e e n r e p o r t e d (4-9). We h a v e a l s o d e m o n s t r a t e d its i n h i b i t o r y e f f e c t on the c e l l g r o w t h of rat h e p a t o c y t e s in v i t r o (i0) and its s t i m u l a t o r y e f f e c t on the n e u r i t e o u t g r o w t h of PC12 c e l l s in v i t r o (ii). B o t h e f f e c t s of c C M P h a v e b e e n o b s e r v e d to be less e f f e c t i v e t h a n those of a d e n o s i n e 3 ' , 5 ' - c y c l i c monophosphate (cAMP), but to be more efficient than those of guanosine 3',5'-cyclic monophosphate (cGMP). T h e s e f i n d i n g s s u g g e s t t h a t c C M P m a y be a s e c o n d m e s s e n g e r as w e l l as w e l l - s t u d i e d c y c l i c p u r i n e n u c l e o t i d e s . c C M P is k n o w n to be s y n t h e s i z e d f r o m C T P by c y t i d y l a t e c y c l a s e (12,13). However, the e n z y m o l o g i c a l and b i o c h e m i c a l properties of c y t i d y l a t e c y c l a s e h a v e n o t b e e n e l u c i d a t e d a p p r e c i a b l y , as c o m p a r e d w i t h t h o s e of a d e n y l a t e and guanylate cyclases. This was mainly due to the lack of a reliable and sensitive assay method for cCMP. We h a v e d e v e l o p e d a h i g h l y s e n s i t i v e and s p e c i f i c e n z y m e i m m u n o a s s a y (EIA) m e t h o d by w h i c h 0.5 fmol of c C M P p e r a s s a y t u b e c a n be d e t e r m i n e d (14). By u s i n g this EIA, c y t i d y l a t e c y c l a s e a c t i v i t y w a s d e t e r m i n e d in v a r i o u s t i s s u e h o m o g e n a t e s from rats and m i c e at the level of 0.i-i0 pmol/min/mg protein, with the highest level in b r a i n (15,16). Cytidylate cyclase was characterized by the localization on membranes, M n Z ÷ - d e p e n d e n c e and u n i q u e pH o p t i m u m at a r o u n d 9 and, m o r e o v e r , its a c t i v i t y w a s not i n f l u e n c e d by f o r s k o l i n and l a n t h a n u m chloride, a s p e c i f i c s t i m u l a t o r *To w h o m c o r r e s p o n d e n c e
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s h o u l d be a d d r e s s e d .
0024-3205/93 $6.00 + .00 1992 P e r g a m o n P r e s s Ltd. All rights
reserved.
14
Cytidylate
Cyclase
and G - p r o t e i n
Vol.
52, No.
i, 1993
and inhibitor, respectively, of adenylate cyclase (16). These findings i n d i c a t e t h a t t h i s e n z y m e is d i s t i n c t f r o m a d e n y l a t e and g u a n y l a t e c y c l a s e s . Although we have demonstrated the modification of c y t i d y l a t e cyclase a c t i v i t y by c h o l i c a c i d s and p h o s p h o l i p i d s (17), no o t h e r e n d o g e n o u s l i g a n d s or p r o t e i n s a f f e c t i n g the e n z y m e a c t i v i t y h a v e b e e n r e p o r t e d . However, s e v e r a l f i n d i n g s on its d i s t r i b u t i o n , enzymological p r o p e r t i e s a n d f u n c t i o n s of the e n z y m a t i c p r o d u c t a l l o w us to e x p e c t t h e p a r t i c i p a t i o n of a c e r t a i n g u a n i n e n u c l e o t i d e - b i n d i n g p r o t e i n ( G - p r o t e i n ) in the r e g u l a t i o n of c y t i d y l a t e c y c l a s e activity. In this study, we found that this enzyme activity was also stimulated via the activation of G-protein as w e l l as adenylate cyclase activity, but it a p p e a r e d to be r e g u l a t e d by a c e r t a i n G - p r o t e i n d i f f e r e n t from some well-characterized G-proteins. Materials
and M e t h o d s
Materials: ATP, cAMP, NAD, d i t h i o t h r e i t o l (DTT), b o v i n e s e r u m a l b u m i n (BSA, C o h n fr.V), B - g a l a c t o s i d a s e (E. coli) and 4 - m e t h y l u m b e l l i f e r y l - 8 - D - g a l a c t o s i d e (4-MUG) w e r e p u r c h a s e d f r o m S i g m a (St. Louis, MO). GTP, GMP, C T P and c C M P w e r e o b t a i n e d from Y a m a s a S h o y u Co. (Choshi, Japan). G u a n o s i n e 5 ' - O - ( 2 - t h i o d i p h o s phate) (GDPBS) and g u a n o s i n e 5 ' - O - ( 3 - t h i o t r i p h o s p h a t e ) (GTPTS) w e r e p u r c h a s e d from Boehringer Mannheim Yamanouchi (Tokyo, Japan). Islet-activating protein (IAP) a n d c h o l e r a t o x i n (CT) w e r e o b t a i n e d f r o m F u n a k o s h i (Tokyo, Japan) and sodium fluoride was from Nakalai Tesque (Kyoto, Japan). Aluminium chloride w a s a p r o d u c t of I s h i z u P h a r m a c e u t i c a l Co. (Osaka, Japan). All o t h e r r e a g e n t s w e r e of a n a l y t i c a l g r a d e c o m m e r c i a l l y a v a i l a b l e . R a b b i t a n t i s e r a s p e c i f i c to s u c c i n y l c C M P and s u c c i n y l c A M P a n d g o a t a n t i s e r u m s p e c i f i c to r a b b i t IgG w e r e p r e p a r e d in o u r l a b o r a t o r y , as d e s c r i b e d p r e v i o u s l y (14). E a c h a n t i b o d y (IgG fraction) was purified by a ~ m o n i u m sulfate fractionation and ion-exchange chromatography. S u c c i n y l c C M P a n d s u c c i n y l c A M P c o u p l e d w i t h B-galactosidase w e r e s y n t h e s i z e d as d e s c r i b e d p r e v i o u s l y (14). P r e p a r a t i o n of E n z y m e Fraction: M a l e W i s t a r rats ( 2 0 0 - 2 5 0 g) o b t a i n e d f r o m J a p a n SLC (Hamamatsu, Japan) w e r e s a c r i f i c e d by d e c a p i t a t i o n . W h o l e b r a i n was r a p i d l y excised, r i n s e d in c o l d s a l i n e and h o m o g e n i z e d in 9 v o l u m e s of i0 mM T r i s - H C l b u f f e r (pH 7.5) c o n t a i n i n g 1 m M DTT and 0.32 M s u c r o s e at 4°C u s i n g g l a s s - T e f l o n h o m o g e n i z e r . T h i s w a s c e n t r i f u g e d at 3 0 0 x g for i0 m i n at 4°C and the r e s u l t i n g s u p e r n a t a n t was u s e d as t h e h o m o g e n a t e s . In a n o t h e r e x p e r i m e n t , the h o m o g e n a t e s w e r e f u r t h e r c e n t r i f u g e d at 1 0 , 0 0 0 x g for I0 m i n at 4°C and the p r e c i p i t a t e o b t a i n e d was u s e d as the m e m b r a n e p r e p a r a t i o n . Other tissues w e r e p r o c e s s e d in the s a m e w a y to p r e p a r e the h o m o g e n a t e s . The p r o t e i n c o n t e n t w a s d e t e r m i n e d by the m e t h o d of S m i t h et al. (18) u s i n g B S A as a standard. C y c l a s e Assay: Cytidylate ~clase a c t i v i t y was d e t e r m i n e d at pH 8.0, but not at its o p t i m u m pH, to e s t i m a t e its p h y s i o l o g i c a l involvement, as d e s c r i b e d p r e v i o u s l y (15). T h e r e a c t i o n m i x t u r e c o n s i s t e d of 50 m M T r i s - H C l b u f f e r (pH 8.0), 1 mM CTP, 4 m M MnCI2 and an a l i q u o t of e n z y m e s a m p l e in a t o t a l v o l u m e of i00 NI. A f t e r 3 m i n - p r e i n c u b a t i o n , the r e a c t i o n w a s i n i t i a t e d by a d d i n g e n z y m e s o l u t i o n and i n c u b a t e d at 3 0 ° C for i0 min. It w a s t e r m i n a t e d by the a d d i t i o n of 0.9 ml of 50 mM a c e t a t e b u f f e r (pH 4.0), f o l l o w e d by h e a t i n g at 9 0 ° C for 3 min. T h e r e a c t i o n m i x t u r e w a s c e n t r i f u g e d at 5 , 0 0 0 X g for 15 m i n and 50 N1 of the s u p e r n a t a n t was s u b j e c t e d to s u c c i n y l a t i o n r e a c t i o n p r i o r to the q u a n t i f i c a t i o n of c C M P formed. A h e a t e d e n z y m e (90°C for 3 min) was i n c l u d e d as a blank. A d e n y l a t e c y c l a s e a c t i v i t y w a s d e t e r m i n e d in a s i m i l a r m a n n e r by e m p l o y i n g the r e a c t i o n m i x t u r e c o n s i s t e d of 50 mM T r i s - H C l b u f f e r (pH 7.4), 50 N M ATP, 4 mM MnCI2, i0 m M t h e o p h y l l i n e and an e n z y m e s o l u t i o n . Determination of method described method is based
Cyclic Nucleotide: Succinyl cCMP was quantified by the by us (14) w i t h s o m e m o d i f i c a t i o n s . This simplified assay on the direct competitive EIA. In brief, the antibody
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Cytidylate Cyclase and G-protein
15
specific to cCMP was adjusted to the protein concentration at 3 N g / m l in 0.05 M phosphate buffer (pH 6.8)-0.5 M NaCI-5 mM MgCI2 (buffer A). Polystyrene tubes ( F a l c o n 2052) w e r e c o a t e d w i t h 2 0 0 N 1 a n t i b o d y s o l u t i o n o v e r n i g h t at 4°C. A f t e r r e m o v a l , t h e r e s i d u a l b i n d i n g c a p a c i t y of t u b e s w a s b l o c k e d w i t h 500 N 1 o f 0 . 5 % B S A i n b u f f e r A ( b l o c k i n g s o l u t i o n ) at 3 7 ° C for 1 hr. T h e t u b e s w e r e r i n s e d t w i c e w i t h 0 . 1 % B S A in b u f f e r A ( w a s h i n g s o l u t i o n ) , f o l l o w e d b y a d d i n g I 0 0 N 1 of s e r i a l l y d i l u t e d s t a n d a r d o r t e s t s o l u t i o n a n d i00 N1 of cCMP-B-galactosidase conjugate which were both diluted with blocking buffer. T h e m i x t u r e w a s i n c u b a t e d at 2 6 0 C for 4 hr, followed by washing twice with washing buffer. T h e t u b e s w e r e a d d e d 3 0 0 N 1 o f 0.2 m M 4-MUG in buffer A and incubated under shaking for 90 m i n at 37°C. The r e a c t i o n w a s t e r m i n a t e d b y a d d i t i o n o f 1.5 ml o f 0.i M s o d i u m c a r b o n a t e a n d t h e f l u o r e s c e n c e i n t e n s i t y w a s m o n i t o r e d in S h i m a d z u f l u o r o s p e c t r o m e t e r w i t h a n e x c i t a t i o n at 3 6 0 n m a n d a n e m i s s i o n at 4 6 0 nm. T h e a m o u n t of c C M P f o r m e d in the test solution was quantified by interpolation onto the inhibition curve generated with serial dilutions of a standard cCMP solution. In t h i s assay, the limit of detection was 3 fmol/assay tube. Succinyl cAMP was q u a n t i f i e d b y E I A d e s c r i b e d p r e v i o u s l y (14). In b o t h a s s a y s , t h e c r o s s r e a c t i v i t y b y o t h e r n u c l e o t i d e s w a s c o n f i r m e d to be l e s s t h a n 0 . 0 1 % . E f f e c t of AIF4- o n C y c l a s e A c t i v i t y : T h e h o m o g e n a t e s w e r e p r e i n c u b a t e d at 4 ° C for 1 h r w i t h a c o m b i n a t i o n of N a F a n d A I C I 3 at t h e c o n c e n t r a t i o n s o f i - i 0 a n d 0 . 2 - 2 mM, r e s p e c t i v e l y , a n d d e t e r m i n e d f o r b o t h c y c l a s e a c t i v i t i e s . Effect of GTP and GDP Analogues on Cyclase Activity: Brain membrane preparat i o n (1-2 m g p r o t e i n ) w a s p r e i n c u b a t e d w i t h v a r i o u s c o n c e n t r a t i o n s o f G T P T S a t 3 0 ° C for 3 0 m i n i n a t o t a l v o l u m e o f 4 0 0 N1 o f 30 m M T r i s - H C l b u f f e r (pH 7.5)-1 m M DTT. After dilution with the same volume of cold buffer, the membranes were washed twice by centrifugation at 1 5 , 0 0 0 x g for 5 min. The m e m b r a n e p e l l e t s w e r e s u s p e n d e d in i0 m M T r i s - H C l b u f f e r (pH 7 . 5 ) - 1 m M D T T
200
~0
~ ~
O
100
0 0
!
I
I
I
2 0.4
4 0.8
6 1.2
10 2.0
FIG.
10 0
O
0 NaF (mM) 2.0 AICI3(mM)
1
Stimulation of cyclase activity of rat brain homogenates by AIF4-. Homogenates incubated with NaF and AICI 3 were measured for c y t i d y l a t e (e) a n d a d e n y l a t e (0) c y c l a s e a c t i v i t i e s . T h e c o n t r o l values for cytidylate and adenylate cyclases were 5.76 and 198.5 pmol/min/mg protein, respectively. Each point represents the mean of triplicate determinations. These results are representative of three experiments.
16
Cytidylate
Cyclase
and G - p r o t e i n
Vol.
52, No.
i, 1993
and d e t e r m i n e d for b o t h c y c l a s e a c t i v i t i e s . The e f f e c t of G D P B S or G M P w a s e s t i m a t e d in t h e s a m e w a y d e s c r i b e d a b o v e a f t e r i n c u b a t i o n of b r a i n m e m b r a n e s w i t h v a r i o u s c o n c e n t r a t i o n s of e a c h c o m p o u n d in the p r e s e n c e of i0 NM GTPTS. E f f e c t of B a c t e r i a l T o x i n on C y c l a s e A c t i v i t y : B r a i n m e m b r a n e p r e p a r a t i o n (1-2 mg p r o t e i n ) w a s p r e i n c u b a t e d w i t h v a r i o u s c o n c e n t r a t i o n s of a c t i v a t e d IAP at 3 0 ° C for 30 m i n in a t o t a l v o l u m e of 500 N1 of 30 m M T r i s - H C I b u f f e r (pB 7.5)2mM D T T - 6 0 NM N A D - 0 . 6 mM A T P - 6 0 N M GTP. A c t i v a t i o n of IAP w a s d o n e by its i n c u b a t i o n at 3 0 ° C for 20 m i n in i00 ram D T T - 0 . 1 m M A T P - 5 0 mM T r i s - H C I b u f f e r (pH 7.5). A f t e r d i l u t i o n w i t h the s a m e v o l u m e of c o l d buffer, the m e m b r a n e s w e r e w a s h e d t w i c e by c e n t r i f u g a t i o n at 1 5 , 0 0 0 X g for 5 m i n and d e t e r m i n e d for both cyclase activities. The p r e t r e a t m e n t of b r a i n m e m b r a n e p r e p a r a t i o n (1-2 mg p r o t e i n ) w i t h v a r i o u s c o n c e n t r a t i o n s of a c t i v a t e d CT w a s c o n d u c t e d at 3 0 ° C for 60 m i n in a t o t a l v o l u m e of 500 N1 of 30 mM T r i s - H C l b u f f e r (pH 7 . 5 ) - 2 mM D T T - 6 0 N M N A D - 0 , 6 m M A T P - 1 2 N M G T P T S - 2 m M MgClz. CT w a s a c t i v a t e d by its i n c u b a t i o n at 3 7 ° C for 20 m i n in 50 mM D T T - 5 0 mM s o d i u m p h o s p h a t e b u f f e r (pH 7.5). A f t e r washing, the m e m b r a n e s w e r e d e t e r m i n e d for b o t h c y c l a s e a c t i v i t i e s . Results Brain homogenates and m e m b r a n e preparation used in t h i s study showed c y t i d y l a t e c y c l a s e a c t i v i t i e s of 5 . 0 3 ± 0 . 6 5 and 6 . 5 5 ± 1 . 4 9 p m o l / m i n / m g protein, respectively, which were much lower than adenylate cyclase activities. Effect of AIF4-, a s t i m u l a t o r of a d e n y l a t e c y c l a s e v i a a c t i v a t i o n of G - p r o t e i n (19), on b o t h c y c l a s e a c t i v i t i e s was f i r s t d e t e r m i n e d . The c o m b i n e d a d d i t i o n of 2-4 mM N a F and 0.4-0.8 mM AICI 3 resulted in m o r e than 2-times elevation of
300 A
200 (D O
"-6 o
~
lOO
0
I
0
10
I
20 Time (rain) FIG.
I
I
30
0.1
I
I
i
I
1 10 100 1000 GTP'¢S (gM)
2
S t i m u l a t i o n of c y c l a s e a c t i v i t y of rat b r a i n m e m b r a n e s by GTPTS. (A) T i m e d e p e n d e n c y . B r a i n m e m b r a n e s w e r e i n c u b a t e d at 30°C for v a r i o u s t i m e s w i t h (circle) or w i t h o u t ( t r i a n g l e ) i0 NM G T P T S and determined for cytidylate (o,&) and adenylate (0,4) cyclase activities. (B) C o n c e n t r a t i o n d e p e n d e n c y . M e m b r a n e p r e p a r a t i o n w a s i n c u b a t e d w i t h v a r i o u s c o n c e n t r a t i o n s of G T P T S at 3 0 ° C for 30 m i n a n d d e t e r m i n e d for c y t i d y l a t e (o) and a d e n y l a t e (o) c y c l a s e a c t i v ities. The control values for c y t i d y l a t e and a d e n y l a t e c y c l a s e s were 5.1 and 193 p m o l / m i n / m g protein, respectively. Each point represents the m e a n of t r i p l i c a t e determinations. These results are r e p r e s e n t a t i v e of t h r e e e x p e r i m e n t s .
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Cytidylate
Cyclase
and G - p r o t e i n
c y t i d y l a t e c y c l a s e a c t i v i t y of b r a i n h o m o g e n a t e s as well a c t i v i t y (Fig. i). In this e x p e r i m e n t , a s i n g l e a d d i t i o n no a c t i v a t i o n of b o t h c y c l a s e s .
as of
adenylate compounds
17
cyclase induced
The pretreatment of b r a i n m e ~ r a n e p r e p a r a t i o n w i t h GTPTS, a nonhydrol y z a b l e G - p r o t e i n a c t i v a t o r (20), at i0 N M i n d u c e d a t i m e - d e p e n d e n t a c t i v a t i o n of b o t h c y c l a s e s , t h o u g h c y t i d y l a t e c y c l a s e was a c t i v a t e d m h ~ h ~ f a s t e r t h a n adenylate cyclase (Fig. 2A). T h e i n c u b a t i o n of m e m b r a n e p r e p a r a t i o n in the a b s e n c e of G T P T S s h o w e d no c h a n g e in t h e i r c y c l a s e a c t i v i t i e s . In addition, GTPTS-induced activation of c y t i d y l a t e c y c l a s e as w e l l as a d e n y l a t e c y c l a s e w a s o b s e r v e d in a c o n c e n t r a t i o n - d e p e n d e n t f a s h i o n at 0 . i - i 0 0 0 NM, r e a c h i n g a p l a t e a u of 3 - f o l d i n c r e a s e at the h i g h e s t c o n c e n t r a t i o n u s e d (Fig. 2B). G D P ~ S is an i n a c t i v a t o r the a c t i v a t e d protein (21). NM) e f f e c t i v e l y a b o l i s h e d the (Fig. 3). This i n h i b i t i o n by in a s i m i l a r m a n n e r . However,
of G - p r o t e i n w h i c h c a n r e p l a c e G T P or G T P T S in The increasing concentrations of G D P ~ S (i-i000 a c t i v a t i o n of c y t i d y l a t e c y c l a s e by i0 N M G T P T S G T P ~ S w a s a l s o d e m o n s t r a t e d for a d e n y l a t e c y c l a s e GMP did not s h o w s u c h an e f f e c t .
To estimate a G-protein component in the regulation of cytidylate cyclase activity, two well-characterized bacterial toxins which regulate a d e n y l a t e c y c l a s e a c t i v i t y (22-25) w e r e e m p l o y e d in t h i s study. T r e a t m e n t w i t h IAP r e s u l t e d in an i n c r e a s e of a d e n y l a t e c y c l a s e a c t i v i t y at the c o n c e n t r a t i o n s of 0 . 0 1 - 0 . i Ng/ml, w h e r e a s it h a d no i n f l u e n c e on c y t i d y l a t e c y c l a s e activity (Fig. 4A). In addition, CT m a r k e d l y a c t i v a t e d adenylate c y c l a s e at 20 Ng/ml, but c y t i d y l a t e c y c l a s e w a s not a f f e c t e d by this t r e a t m e n t (Fig. 4B). Cytidylate cyclase a c t i v i t y has b e e n d e t e c t e d in v a r i o u s tissues (15). We f i n a l l y d e t e r m i n e d w h e t h e r c y t i d y l a t e cyclase a c t i v i t i e s in t i s s u e s b e s i d e s b r a i n a r e a f f e c t e d by AIF4-. W h e n a d d e d w i t h 4 mM N a F a n d 0.8 mM AICIs, the
..=Z'~- 100 7~ "6 (1)._
8o so
0
I
0
1
I
I
I
10 100 1000 GDPyS or GMP (mM) FIG.
3
E f f e c t of G D P ~ S and G M P o n G T P T S - s t i m u l a t e d c y c l a s e a c t i v i t y of rat b r a i n m e m b r a n e s . Membrane preparation used in FIG. 2 were i n c u b a t e d at 3 0 ° C for 30 m i n w i t h v a r i o u s c o n c e n t r a t i o n s of G D P ~ S (circle) or G M P (triangle) in the p r e s e n c e of i0 N M G T P T S and d e t e r m i n e d for c y t i d y l a t e (e,A) a n d a d e n y l a t e (0,4) c y c l a s e a c t i v ities. E a c h p o i n t r e p r e s e n t s the m e a n of t r i p l i c a t e d e t e r m i n a t i o n s . rhese r e s u l t s a r e r e p r e s e n t a t i v e of two e x p e r i m e n t s .
18
Cytidylate
Cyclase
and G - p r o t e i n
A
300
Vol.
i, 1993
B
1100 1000
Y
8 O 200 tn O
o
52, No.
300
10C
200 100 C
0
I
0.01
I
I
I
0.1 1 lAP (~g/ml)
I
10
0
FIG.
0.2
I
I
2 20 CT (~g/ml)
I
200
4
E f f e c t of t r e a t m e n t w i t h IAP and CT on c y c l a s e a c t i v i t y of rat brain membranes. (A) Membrane preparation was incubated with various concentrations of a c t i v a t e d IAP at 3 0 ° C for 30 m i n and determined for c y t i d y l a t e (e) and a d e n y l a t e (o) c y c l a s e a c t i v i ties. (B) M e m b r a n e p r e p a r a t i o n w a s i n c u b a t e d w i t h v a r i o u s c o n c e n t r a t i o n s of a c t i v a t e d CT at 3 0 ° C for 60 m i n and d e t e r m i n e d for cytidylate (e) and a d e n y l a t e (0) c y c l a s e a c t i v i t i e s . The c o n t r o l values for c y t i d y l a t e and a d e n y l a t e c y c l a s e a c t i v i t i e s w e r e 6.4 and 340 p m o l / m i n / m g protein, respectively. Each point represents t h e m e a n of t r i p l i c a t e d e t e r m i n a t i o n s . T h e s e v a l u e s are r e p r e s e n t a t i v e of two e x p e r i m e n t s .
TABLE Effect
of A I F 4- on C y t i d y l a t e
Tissue
Cyclase
Activity
in Rat T i s s u e s
Cytidylate cyclase activity (pmol c C M P f o r m e d / m i n / m g p r o t e i n ) None
Brain Lung Adrenal Spleen Kidney Liver
I
4.84 1.09 0.581 0.544 0.459 0.266
~ ~ ~ ~ ~ ±
4 mM NaF + 0.8 mM AICI 3 0.074 0.137 0.009 0.060 0.045 0.003
16.79 3.40 1.73 1.03 0.838
~ ~ ~ ~ ~
0.318
~
0.935 0.377 0.009 0.065 0.027 0.002
V a r i o u s t i s s u e h o m o g e n a t e s w e r e i n c u b a t e d w i t h 4 m M N a F and 0.8 mM AICI3 a n d d e t e r m i n e d for c y c l a s e a c t i v i t y . Each value repres e n t s the m e a r ~ S D of t h r e e animals, e a c h a s s a y e d in t r i p l i c a t e . h o m o g e n a t e s f r o m lung, spleen, k i d n e y a n d a d r e n a l s h o w e d a 2 - 3 - t i m e s increase, resembling the p h e n o m e n o n o b s e r v e d in b r a i n h o m o g e n a t e s (Table I). However, this treatment induced little enhancement of c y t i d y l a t e c y c l a s e a c t i v i t y in l i v e r h o m o g e n a t e s w h i c h p o s s e s s e d the l o w e s t v a l u e a m o n g t h e t i s s u e s e x a m i n e d .
Vol.
52, No.
i,
1993
Cytidylate
Cyclase
and G - p r o t e i n
19
Discussion G - p r o t e i n s h a v e b e e n c h a r a c t e r i z e d to m o d i f y the s i g n a l t r a n s d u c t i o n from r e c e p t o r s to e f f e c t o r e n z y m e s in v a r i o u s b i o l o g i c a l r e s p o n s e s (26). A d e n y l a t e cyclase system is t h e representative of m e m b r a n e reactions in w h i c h such G-proteins a r e i n v o l v e d as r e g u l a t o r y c o m p o n e n t s (27). O u r p r e v i o u s s t u d i e s h a v e r e v e a l e d t h a t c C M P a l s o p l a y s an i m p o r t a n t r o l e in c e l l p r o l i f e r a t i o n and d i f f e r e n t i a t i o n as w e l l as cAMP. T h e r e f o r e , a p r o o f of the c o u p l i n g of G-protein in the cytidylate cyclase system is i m p o r t a n t to e v a l u a t e the p h y s i o l o g i c a l r o l e of t h i s e n z y m e in m e m b r a n e signal t r a n s d u c t i o n . Cytidylate c y c l a s e a c t i v i t y in rat b r a i n m e m b r a n e s was m a r k e d l y e l e v a t e d by the p r e t r e a t m e n t of AIF4- and G T P T S and the a c t i v a t i o n by G T P T S was e f f e c t i v e l y a b o l i s h e d b y GDP~S, s u g g e s t i n g this p o s s i b i l i t y strongly. A l t h o u g h the same p h e n o m e n o n was observed in the c a s e of a d e n y l a t e cyclase, the s y n t h e s i s of c C M P w a s s h o w n not to r e s u l t f r o m a s i d e r e a c t i o n by a d e n y l a t e c y c l a s e (15). S u c h an a c t i v a t i o n of c y t i d y l a t e c y c l a s e by AIF4- was d e m o n s t r a t e d in o t h e r t i s s u e s except liver. These results indicate that cytidylate cyclase is also associated with a certain G-protein in the m e m b r a n e . However, it r e m a i n s u n c l e a r w h e t h e r the r e g u l a t i o n m o d e of c y t i d y l a t e c y c l a s e a c t i v i t y by t h e s e agents is different among tissues, as speculated on the activation of a d e n y l a t e c y c l a s e by AIF4- and f o r s k o l i n (28,29). The e l e v a t i o n of c y t i d y l a t e c y c l a s e a c t i v i t y v i a G - p r o t e i n a c t i v a t i o n was considerably different from t h a t of a d e n y l a t e c y c l a s e in s e v e r a l respects, s u c h as r a p i d a c t i v a t i o n by i0 N M G T P ~ S and u n r e s p o n s i v e n e s s to t h e s e two k i n d s of G - p r o t e i n - m o d i f y i n g bacterial toxins. The f i n d i n g m a y s u g g e s t the d i f f e r e n c e in the s p e c i e s of G - p r o t e i n a c t i n g in b o t h c y c l a s e systems. As IAP and CT are k n o w n to r e g u l a t e the f u n c t i o n s of Gi and G., r e s p e c t i v e l y , t h r o u g h their ADP-ribosylation (22-25), G-protein which functions in cytidylate c y c l a s e s y s t e m m a y be at least n e i t h e r Gi nor Gs- However, f u r t h e r i n v e s t i g a t i o n is r e q u i r e d for c h a r a c t e r i z i n g t h i s p r o t e i n in t e r m s of the n e c e s s i t y of A D P - r i b o s y l a t i o n , b e c a u s e the e x p e r i m e n t a l c o n d i t i o n s u s e d in the p r e s e n t s t u d y are s u i t a b l e o n l y for the A D P - r i b o s y l a t i o n of G - p r o t e i n s in a d e n y l a t e c y c l a s e system. In o u r p r e v i o u s studies, c C M P and c A M P w e r e s h o w n to e x h i b i t s i m i l a r e f f e c t s o n the i n h i b i t i o n of c u l t u r e d h e p a t o c y t e g r o w t h and on the s t i m u l a t i o n of n e u r i t e o u t g r o w t h of PC12 cells, a l t h o u g h b o t h a c t i v i t i e s by c C M P w e r e w e a k e r t h a n t h o s e by c A M P (I0,ii). T h e s e f i n d i n g s s u g g e s t t h a t c C M P a n d c A M P w h o s e b i o s y n t h e s i s is c o n t r o l l e d by d i s t i n c t r e g u l a t i o n s y s t e m s m a y c o o p e r a t e in s o m e b i o l o g i c a l r e s p o n s e s . B o t h h o m o g e n a t e s and m e m b r a n e p r e p a r a t i o n from rat b r a i n u s e d as e n z y m e sources had the comparable cytidylate cyclase activities. However, the occurrence of cCMP phosphodiesterase which hydrolyzes cCMP has been demonstrated in v a r i o u s t i s s u e e x t r a c t s (30-32). T h i s e n z y m e is i n s e n s i t i v e to t h e o p h y l l i n e and 3 - i s o b u t y l - l - m e t h y l - x a n t h i n e which are potent inhibitors of c A M P and c G M P p h o s p h o d i e s t e r a s e s . In fact, c y t i d y l a t e c y c l a s e a c t i v i t y w a s not a f f e c t e d by t h e a d d i t i o n of t h e s e c l a s s i c a l inhibitors in the p r e s e n t study (data not shown). Therefore, no i n h i b i t o r was a d d e d to the r e a c t i o n m i x t u r e for c y t i d y l a t e c y c l a s e assay. A l t h o u g h this m a y c a u s e an u n d e r e s t i m a t e of t h i s e n z y m e a c t i v i t y , it has no i n f l u e n c e on the p r o f i l e of e n z y m e a c t i v a tion. Recently, there appeared a report describing surprisingly high specific a c t i v i t i e s of c y t i d y l a t e c y c l a s e in v a r i o u s tissues, w h i c h w e r e c o m p a r a b l e to t h o s e of a d e n y l a t e c y c l a s e (33). T h e s e d i d not a r i s e f r o m the i n h i b i t i o n of d e g r a d a t i o n of cCMP, b e c a u s e no i n h i b i t o r w a s used. T h i s d i s c r e p a n c y o b s e r v e d in e n z y m e a c t i v i t i e s is not y e t solved, but it m a y be d u e in part to the difference in a n a l y t i c a l factors including the s e n s i t i v i t y and s p e c i f i c i t y . A l t h o u g h s e v e r a l r e p o r t s h a v e d e s c r i b e d the p r o p e r t i e s of c y t i d y l a t e c y c l a s e (12,13,15,16,33-35), the fact of e n z y m e p r o t e i n is still n o t f u l l y e l u c i d a t e d . To clarify the substantial association of a G-protein and enzymological a s p e c t s in this e n z y m e system, it is n e c e s s a r y to p u r i f y the e n z y m e to h i g h l y
20
homogeneous
Cytidylate Cyclase and G-protein
s t a t e a n d to s e a r c h f o r an e n d o g e n o u s
Vol.
52, No.
i, 1993
agonist to this enzyme system.
Acknowledgement T h i s w o r k w a s s u p p o r t e d in p a r t b y G r a n t s - i n - A i d for S c i e n t i f i c R e s e a r c h (No. 0 2 6 7 0 9 9 8 ) f r o m t h e M i n i s t r y of E d u c a t i o n , S c i e n c e a n d C u l t u r e of Japan. References i. A. BLOCH, B i o c h e m . B i o p h y s . Res. C o m m u n . 58 6 5 2 - 6 5 9 (1974). 2. A. BLOCH, G. D U T S C H M A N a n d R. MAUE, B i o c h e m . B i o p h y s . Res. C o m m u n . 59 9 5 5 959 (1974). 3. A. BLOCH, A d v a n c e s in C y c l i c N u c l e o t i d e R e s e a r c h , v o l . 5 , G.I. D r u m m o n d , P. G r e e n g a r d a n d G.A. R o b i n s o n (eds), 3 3 1 - 3 3 8 , R a v e n Press, N e w Y o r k (1975). 4. T.R. A N D E R S O N , Mol. Cell. E n d o c r i n o l . 28 3 7 3 - 3 8 5 (1982). 5 L.G. S H E F F I E L D , C e l l . Biol. Int. Rep. Ii 5 5 7 - 5 6 2 (1987). 6 J. KOWAL, E n d o c r i n o l o g y 98 4 6 1 - 4 6 8 (1973). 7 M.A. P I S A R E V a n d D . L . K . P I S A R E V , A c t a E n d o c r i n o l . 84 2 9 7 - 3 0 2 (1977). 8 P.E. C A N A S a n d L.F. C O N G O T E , Can. J. B i o c h e m . 60 1-7 (1982). 9 J. E R V E N S a n d R. S E I F E R T , B i o c h e m . B i o p h y s . Res. C o m m u n . 174 2 5 8 - 2 6 7 (1991). i0 T. KUBIN, M. Y A N A G I D A , S. MORI, Y. H A Y A S H I , E. G O H D A a n d I. Y A M A M O T O , C e l l Biol. I n t e r n a t . Rep. 13 9 0 7 - 9 1 7 (1989). ii M. Y A N A G I D A , E. G O H D A a n d I. Y A M A M O T O , N e u r o s c i . Lett. 114 3 2 3 - 3 2 8 (1990). 12 S.Y. C E C H a n d L.J. IGNARR0, S c i e n c e 198 1 0 6 3 - 1 0 6 5 (1977). 13 S.Y. C E C H a n d L.J. IGNARR0, B i o c h e m . B i o p h y . Res. C o m m u n . 80 1 1 9 - 1 2 5 (1978). 14 I. Y A M A M O T O , T. T A K A I a n d J. TSUJI, I m m u n o p h a r m a c o l o g y 4 3 3 1 - 3 4 0 (1982). 15 I. Y A M A M O T O , T. T A K A I a n d S. MORI, B i o c h i m . B i o p h y s . A c ~ a 933 1 9 1 - 1 9 8 (1989). 16 S. MORI, M. Y A N A G I D A , K. K U B O T U a n d I. Y A M A M O T O , Sec. Mess. P h o s p h o p r o t . 13 1-12 (1990). 17. S. MORI, N. M U T O a n d I. Y A M A M O T O , B i o c h e m . B i o p h y . Res. C o m m u n . 162 15021507 (1989). 18. P.K. SMITH, R.I. KROHN, G.T. H E R M A N S O N , A.K. M A L L I A , F.H. G A R T N E R , M.D. P R O V E N Z A N 0 , E.K. F U J I M O T O , N.M. GOEKE, B.J. O L S O N a n d D.C. KLENK, Anal. B i o c h e m . 150 7 6 - 8 5 (1985). 19. P.C. S T E R N W E I S a n d A.G. G I L M A N , Proc. Natl. Acad. Sci. U S A 79 4 8 8 8 - 4 8 9 1 (1982). 20. T. P F E U F F E R a n d E . J . M . H E L M R E I C H , J. Biol. Chem. 250 8 6 7 - 8 7 6 (1975). 21. F. E C K S T E I N , D. C A S S E L , H. L E V K O V I T Z , M. L O W E and Z. S E L I N G E R , J. Biol. C h e m . 254 9 8 2 9 - 9 8 3 4 (1979). 22. T. K A T A D A a n d M. UI, J. Biol. Chem. 257 7 2 1 0 - 7 2 1 6 (1982). 23. T. K A T A D A a n d M. UI, Proc. Natl. Acad. Sci. U S A 79 3 1 2 9 - 3 1 3 3 (1982). 24. D. C A S S E L a n d T. P F E U F F E R , Proc. Natl. A c a d . Sci. U S A 75 2 6 6 9 - 2 6 7 3 (1978). 25. D.M. G I L L a n d R. M E R E N , Proc. Natl. A c a d . Sci. U S A 75 3 0 5 0 - 3 0 5 4 (1978). 26. A. GILMAN, Annu. Rev. B i o c h e m . 56 6 1 5 - 6 4 9 (1987). 27. M. R O D B E L L , H . M . J . KRANS, S.L. P O H L a n d L. B I R N B A U M E R , J. Biol. Chem. 246 1 8 7 2 - 1 8 7 6 (1971). 28. E.W. S U T H E R L A N D , T. W. R A L L a n d T. MENON, J. Biol. Chem. 237 1 2 2 0 - 1 2 2 7 (1962). 29. K. B. S E A M O N , W. P A D G E T T a n d J.W. DALY, Proc. Natl. Acad. Sci. U S A 78 3 3 6 3 - 3 3 6 7 (1981). 30. Y.C. C H E N G a n d A. BLOCH, J. Biol. Chem. 253 2 5 2 2 - 2 5 2 4 (1978). 31. J.F. KU0, N . L . B R A C K E T T , M. S H O J I a n d J. TSE, J. Biol. Chem. 253 2 5 1 8 - 2 5 2 1 (1978). 32. R.P. N E W T O N a n d S.G. SALIH, Int. J. B i o c h e m . 18 7 4 3 - 7 5 2 (1986). 33. R.P. N E W T O N , B.J. S A L V A G E a n d N.A. H A A K E E M , B i o c h e m . J. 265 5 8 1 - 5 8 6 (1990). 34. R.M. G A I R O N a n d G. K R I S H I N A , B i o c h e m . B i o p h y . Res. C o m m u n . 86 1 0 5 - 1 1 1 (1979). 35. R.M. G A I R O N a n d G. K R I S H I N A , S c i e n c e 203 672 (1979).