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Characterization of substance P binding sites in intact and solubilized rat cortical membranes
132
CHARACTERIZATION SOLUBILIZED RAT Y.
NAKATA,
Dept. Med.,
OF SUBSTANCE P CORTICAL MEMBRANES
C. H I R A O K A ,
Y. M O R I S H I M A
BINDING
and
T.
Pharmacol., Inst. Pharmaceut. Sci., Kasumi I-2-3, H i r o s h i m a 734, J A P A N
SITES
IN
INTACT
AND
Univ.
Sch.
SEGAWA Hiroshima
The regulatory mechanisms of s u b s t a n c e P (SP) b i n d i n g to r a t cerebral cortical membranes were examined. Recently, the s p e c i f i c binding protein for SP w'as s u c c e s s f u l l y solubilized in an a c t i v e form from bovine brainstem with I0 m M 3-[(3-cholamidopropyl) dimethylammonio]l-propane sul f o n a t e (CHAPS) and I00 m M N a C I (Nakata et al., J. N e u r o c h e m . 50, 5 2 2 - 5 2 7 , 1988). In t h i s s t u d y , the effects of G T P a n d c a t i o n s on 3 H - S P b i n d i n g activity in a CHAPS-solubilized fraction from rat c e r e b r a l cortical membranes were investigated and compared w i t h t h o s e in i n t a c t m e m b r a n e s . After incubation of r a t c e r e b r a l cortical membranes with 10 m M C H A P S a n d I00 m M N a C I ( I0 m g p r o t e i n / m l ) at 0 % for 30 m i n , 3 H - S P binding to t h e s u p e r n a t a n t fraction (lO0,000 x g for 60 m i n ) w a s determined by a g l a s s f i b e r f i l t r a t i o n m e t h o d ( B r u n ~ et al., Anal. Biochem., 132, 74-81, 1983). MgCI 2 increased ~H-SP binding activity while GTP and NaCI decreased it in b o t h the CHAPSsolubilized and the m e m b r a n e fraction. The s t i m u l a t o r y effect of MgCI 2 and the inhibitory effects of G T P a n d N a C I w e r e g r e a t e r in the C H A P S - s o l u b i l i z e d f r a c t i o n t h a n in the m e m b r a n e fraction. To determine the binding parameters modified by G T P ~ n d cations competition experiments of 3 H - S P b i n d i n g by u n l a b e l e ~ SP (7 p M -2 ~M) were performed. In t h e m e m b r a n e fraction, nonlinear leastsquares regression analysis of c o m p e t i t i o n binding d a t a in t h e presence of I0 m M M g C I 2 i n d i c a t e d that two distinct affinity states for SP b i n d i n g existed; a h i g h (H, K d = O . l 1 n M ) a n d l o w (L, Kd=5.76 riM). By the addition of |00 uM GTP, although the competition curve remained fitted to t h e t w o - s t a t e model, the maximum specific binding was decreased and H - s t a t e disappeared. The L-state ( K d = 1.54 n M ) a n d a n e w s u p e r - l o w state (Kd=355 nM) were observed. The effects of 100 m M N a C I a n d t h e r e m o v a l of M g C I 2 were similar to that of the a d d i t i o n of 100 u M GTP. In the C H A P S solubilized fraction, the competition curve was steeper t h a n in t h e c a s e of t h e m e m b r a n e fraction and fitted to t h e o n e - s i t e m o d e l . T h e K d v a l u e ( I I. I n M ) w a s r e l a t i v e l y c l o s e to t h a t of t h e L-state in the m e m b r a n e fraction. By the a d d i t i o n of GTP or NaCI, or b y the reduction of MgCI 2 concentration to I mM, the competition c u r v e k e p t a f i t t i n g to the o n e - s i t e model. However, the Kd v a l u e w a s c h a n g e d o n l y s l i g h t l y . To d e t e r m i n e the a p p a r e n t molecular weight of t h e s e SP b i n d i n g sites, SP b i n d i n g s i t e s in rat c e r e b r a l cortical membranes were specifically and c o v a l e n t l y l a b e l e d by m e a n s of d i s u c c i n i m i d y l suberate (DSS), a bifunctional cross-linking reagent. Cross-linking of 3 H - S P by l m M D S S to membranes revealed a specifically l a b e l e d s i n g l e p r o t e i n w i t h an apparent molecular weight of 4 6 , 0 0 0 by s o d i u m dodecyl sulfate polyacrylamide gel e l e c t r o p h o r e s i s or gel f i l t r a t i o n analysis. These results suggest that C H A P S c o u l d s o l u b i l i z e the s p e c i f i c SP b i n d i n g site from rat cerebral cortex in a f o r m s e n s i t i v e to G T P and c a t i o n s . However, in the C H A P S - s o l u b i l i z e d fraction, the effects of G T P a n d c a t i o n s on 3 H - S P b i n d i n g were greater and the binding parameters estimated from competition experiments were distinct f r o m t h o s e in t h e m e m b r a n e fraction. These differences between the two preparations might be d u e to a d i f f e r e n c e of lipid-milieu.
CHARACTERIZATION SOLUBILIZED RAT Y.
NAKATA,
Dept. Med.,
OF SUBSTANCE P CORTICAL MEMBRANES
C. H I R A O K A ,
Y. M O R I S H I M A
BINDING
and
T.
Pharmacol., Inst. Pharmaceut. Sci., Kasumi I-2-3, H i r o s h i m a 734, J A P A N
SITES
IN
INTACT
AND
Univ.
Sch.
SEGAWA Hiroshima
The regulatory mechanisms of s u b s t a n c e P (SP) b i n d i n g to r a t cerebral cortical membranes were examined. Recently, the s p e c i f i c binding protein for SP w'as s u c c e s s f u l l y solubilized in an a c t i v e form from bovine brainstem with I0 m M 3-[(3-cholamidopropyl) dimethylammonio]l-propane sul f o n a t e (CHAPS) and I00 m M N a C I (Nakata et al., J. N e u r o c h e m . 50, 5 2 2 - 5 2 7 , 1988). In t h i s s t u d y , the effects of G T P a n d c a t i o n s on 3 H - S P b i n d i n g activity in a CHAPS-solubilized fraction from rat c e r e b r a l cortical membranes were investigated and compared w i t h t h o s e in i n t a c t m e m b r a n e s . After incubation of r a t c e r e b r a l cortical membranes with 10 m M C H A P S a n d I00 m M N a C I ( I0 m g p r o t e i n / m l ) at 0 % for 30 m i n , 3 H - S P binding to t h e s u p e r n a t a n t fraction (lO0,000 x g for 60 m i n ) w a s determined by a g l a s s f i b e r f i l t r a t i o n m e t h o d ( B r u n ~ et al., Anal. Biochem., 132, 74-81, 1983). MgCI 2 increased ~H-SP binding activity while GTP and NaCI decreased it in b o t h the CHAPSsolubilized and the m e m b r a n e fraction. The s t i m u l a t o r y effect of MgCI 2 and the inhibitory effects of G T P a n d N a C I w e r e g r e a t e r in the C H A P S - s o l u b i l i z e d f r a c t i o n t h a n in the m e m b r a n e fraction. To determine the binding parameters modified by G T P ~ n d cations competition experiments of 3 H - S P b i n d i n g by u n l a b e l e ~ SP (7 p M -2 ~M) were performed. In t h e m e m b r a n e fraction, nonlinear leastsquares regression analysis of c o m p e t i t i o n binding d a t a in t h e presence of I0 m M M g C I 2 i n d i c a t e d that two distinct affinity states for SP b i n d i n g existed; a h i g h (H, K d = O . l 1 n M ) a n d l o w (L, Kd=5.76 riM). By the addition of |00 uM GTP, although the competition curve remained fitted to t h e t w o - s t a t e model, the maximum specific binding was decreased and H - s t a t e disappeared. The L-state ( K d = 1.54 n M ) a n d a n e w s u p e r - l o w state (Kd=355 nM) were observed. The effects of 100 m M N a C I a n d t h e r e m o v a l of M g C I 2 were similar to that of the a d d i t i o n of 100 u M GTP. In the C H A P S solubilized fraction, the competition curve was steeper t h a n in t h e c a s e of t h e m e m b r a n e fraction and fitted to t h e o n e - s i t e m o d e l . T h e K d v a l u e ( I I. I n M ) w a s r e l a t i v e l y c l o s e to t h a t of t h e L-state in the m e m b r a n e fraction. By the a d d i t i o n of GTP or NaCI, or b y the reduction of MgCI 2 concentration to I mM, the competition c u r v e k e p t a f i t t i n g to the o n e - s i t e model. However, the Kd v a l u e w a s c h a n g e d o n l y s l i g h t l y . To d e t e r m i n e the a p p a r e n t molecular weight of t h e s e SP b i n d i n g sites, SP b i n d i n g s i t e s in rat c e r e b r a l cortical membranes were specifically and c o v a l e n t l y l a b e l e d by m e a n s of d i s u c c i n i m i d y l suberate (DSS), a bifunctional cross-linking reagent. Cross-linking of 3 H - S P by l m M D S S to membranes revealed a specifically l a b e l e d s i n g l e p r o t e i n w i t h an apparent molecular weight of 4 6 , 0 0 0 by s o d i u m dodecyl sulfate polyacrylamide gel e l e c t r o p h o r e s i s or gel f i l t r a t i o n analysis. These results suggest that C H A P S c o u l d s o l u b i l i z e the s p e c i f i c SP b i n d i n g site from rat cerebral cortex in a f o r m s e n s i t i v e to G T P and c a t i o n s . However, in the C H A P S - s o l u b i l i z e d fraction, the effects of G T P a n d c a t i o n s on 3 H - S P b i n d i n g were greater and the binding parameters estimated from competition experiments were distinct f r o m t h o s e in t h e m e m b r a n e fraction. These differences between the two preparations might be d u e to a d i f f e r e n c e of lipid-milieu.