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Aminopeptidase activity is asymmetrically distributed in selected zones of rat brain
L i f e S c i e n c e s , Vol. 43, pp. 935-939 Printed in the U.S.A.
Pergamon Press
K\HNOPEPTIDASE ACTIVITY IS ASYIJt~.~I'RICALLYDISTRIBUTE33 1N SELECTED ZONES OF RAT BRAIN
F r a n c i s c o Alba, Manuel Ramirez, E m i l i o 5. C a n t a l e j o and Concepci6n I r i b a r Department of B i o c h e m i s t r y and M~lecular B i o l o g y , F a c u l t y of Medicine, U n i v e r s i t y of Granada. Granada. S p a i n . (Received i n f i n a l form J u l y 26, 1988) Sunma ry L e v e l s of s o l u b l e a m i n o p e p t i d a s e (AP), measured a s a r y l a m i d a s e a c t i v i t y u s i n g L - L r u c i n e - 2 - N a p h t h y l a m i d e (Leu-2-NA) a s s u b s t r a t e , were d e t e r m i n e d in t h e s o l u b l e f r a c t i o n of e l e v e n zones of r a t b r a i n . R e s u l t s showed t h a t AP a c t i v i t y i s a s y r r m e t r i c a l l y d i s t r i b u t e d i n f r o n t a l c o r t e x and hypothalamus with both l e f t s i d e s h a v i n g s i g n i f i c a n t l y h i g h e r l e v e l s of AP a c t i v i t y , r e s p e c t i v e l y , than t h e r i g h t sides. S i m u l t a n e o u s l y , the a c t i v i t i e s of l a c t a t e dehydrogenase (LDH) and g l u t a m a t e - o x a l a c e t a t e a m i n o t r a n s f e r a s e (GOT) were measured in t h e saJ~ c e r e b r a l r e g i o n s ; no s i g n i f i c a n t d i f f e r e n c e was recorded i n t h e s e a c t i v i t i e s between e i t h e r s i d e of t h e r a t b r a i n in any of the zones s t u d i e d . P r o v i d e d t h a t a m i n o p e p t i d a s e s a r e i n v o l v e d i n t h e d e g r a d a t i o n of some endogenously r e l e a s e d n e u r o p e p t i d e s , the r e : u l t s suKqest a new mode of e x p r e s s i o n of c e r e b r a l lateral ization. Anatomical and f u n c t i o n a l c e r e b r a l a s y m m e t r i e s have been e x t e n s i v e l y d e s c r i b e d i n man and a n i m a l s ( 1 ) , and m u l t i p l e approaches have been used i n order to study b r a i n l a t e r a l i z a t i o n ( 2 ) . However, the n e u r o c h e m i c a l b a s i s underlying left-right d i f f e r e n c e s a r e s c a r c e l y known and l i t t l e evidence e x i s t s t o imply c l a s s i c a l n e u r o t r a n s m i t t e r s in t h i s m a t t e r . Some of them have been r e p o r t e d t o be a s y r m a e t r i c a l l y d i s t r i b u t e d in c e r t a i n c e r e b r a l l o c a t i o n s i n man and o t h e r nmnrrals ( 3 , a , 5 , 6 , 7 , 8 , 9 ) . N e v e r t h e l e s s , t h e r e i s no e v i d e n c e of a s y r m l e t r i c a l d i s t r i b u t i o n of n e u r o p e p t i d e s in the c e n t r a l n e r v o u s system e i t h e r in humans or ir. a n i m a l s (10, 11). B r a i n t~Ps have been i m p l i c a t e d in the mechanism of enzymmtic h y d r o l y sis of several neurc~eptides (12). Some of these p e p t i d e s seem t o p a r t i c i p a t e in the mechanism of synaptic transmission and have been desiRnated as neuromodulators. Provided that p e p t i d e s are presumably s y n t h e t i z e d as bi.~£er precursors by riboscmes and t r a n s p o r t e d v i a synaptic t e r m i n a l s , where they a c t , i t would be reasonable to assume that the a c t i v i t y of those neuromodulat o r s could be c o n t ~ o l l e d by the l e v e l s or the a c t i v i t i e s of p r o t e o l y t i c enzymes l o c a t e d near the s i t e where n e u r o p e p t id e s a c t . T h e r e f o r e , b r a i n APs, the rr~)st abundant p r o t e o l y t i c enzymes found in CNS, could be one way of c o n t r o l l i n ~ n e u r o p e p l i d e a c t i v i t y , dK)reover, i f AP a c t i v i t y were asynTr~tricaf l y d i s t r i b u t e d in b r a i n hemispheres, n e u r o p e p t i d e a c t i v i t y presunTablv would be a f f e c t e d in an as}~rmetrical way.
0024-3205/88 $3.00 + .00 Copyright (c) 1988 Pergamon Press ple
936
Aminopeptldase and Brain Asynmetry
Vol. 43, No. ii, 1988
i , . i a t e r i a l s and ~ i e t h o d s iJale albino rats (avera%e wetqht: 250 ~=,) w e r e a n e s t h e t i z e d with ]~uithensin and their brains perfused with saline through the cardiac left ventriculum. Brains were quickly r e m o v e J, c o o l e d in d r y i c e , and s l i c e d ~n coronal sections of 1 rrm t h i c k . Tissue sar0ples (average weight: 20 ~ , ) , disseetcM from these slices in a£reefnent w i t h t h e s t e r e o t a x t c a t l a s of XL~n~q a n d K l i p p e l (13) w e r e o b t a i n e d , a s s ~ r ~ r . e t r i c a l l y a s p o s s i b l e , from the ri,iht and left frontal cortex (FC), occipital c o r t e x (CYg), p a r i e t o t e m p o r a l cortex (PC), caudateputamen (CP), thalamus (TA), h y p o l h a l a m u s (TIY) m e s e t ~ c c p h a l o n (!,~), m e d u l l a C.|)), cerebellum iCE); h i p p o c a m o u s (HC) a n d c e r v i c a l spinal c o r d ( S C ) , Each s a m p l e was h o r r o ~ e n i z e d in lO v o l u m e s of 59 n$', Tris!-~Cl b n f f e r . ptt 7 . 4 and u l t r a c e n t r i f u g e d ( l O 0 , O 0 0 £ , 30 r,l i n ) . The s n p e r n n t a n t ~,~s d e c a n t e d a n d i m m e d i a t e l y a n a l v z e d f o r AP, iT)T, LDH a c t ~ . v i t i e s and p r o t e i n s . AP ( a r y l a m i d a s e ) a c t i v i t y was m e a s u r e d f l u o r c ~ ~ t r i c a l l y , r~er t r i p l i c a t e , u s i n g Leu-2-NA a s s u b s t r a t e , a c c o r d i n g t o t h e .aetllorl o f Greet.her!,, i t / , ) , n ~ c l i f i e d a s f o l l o w s : 10 u l o f s u p e r n a t a n t was i n c u b a t e c i 30 rain w~th I ml of substrate solution ( 0 . 8 ~_~/lO0 rnl l.eu-2-~IA; lO n~,/lO0 ml b o v i n e a l b u m i n ; If) r ~ / l O 0 ml d i t h i o t h r e i t o l ; i n lO rnl p h o s p h a t e b u f f e r , pll 7./~) a t 2.5 -°C. Ttle e n z y n n t i c r e a c t i o n was s t o p p e d b y a d d i n . c 1 m l , 0.1 ;, a c e t a t e h u f f e r , pH / , . 2 , The f l u o r e s c e n c e intensity of 2-naphthylarrine released ~¢as ~r~asurc~] a t a n excitation wavelength o f 345 nm a n d a n e n f i s s l o n waw, l e n ~ h t of 411 nm. Proteins were measured, per tri'.)licate, b y t i e m e t h o t o f ~3radford ( 1 5 ) . R e s u l t s w e r e e x p r e s s e d a s u n i t s o f AP p e r m i l l i q r a m o f p r o t e i n . One u n i t of AP was c o n s i d e r e d a s t h e amount o f e n ~ e that hydrolyzed 1 nano~)I of Leu-2-NA p e r m i n u t e . LDII and ~ activities were detennined spectrophotometrically, per triplicate, as previously described (16,17) and e x p r e s s e d a s milliunits of enzymatic activity p e r m i l l i q r a m of o r o t e i n , l)ata were test~.] u s i n ~ a 3-way a n a l y s i s of v a r i a n c e '^'ith two ~'ixed a n d one random f a c t o r . To detect indtviduaI asy~m~,etries, r r u l t i l ~ l e c o m p a r i s o r ~ s w e r e 'rode usin,~ t h e v a r i a n c e and T u r k e y ' s t e s t . !~esul ! s The l e v e l s o f AP, LDIt and COT s p e c i f i c activities in both the left and riuht sides of the eleven zones studied are shown in T a b l e ] . :~o significant differences w e r e found b e t w e e n t h e n,ean v a l u e s o f t h e l e f t s i d e a n d t h e r i g h t s i d e f o r LDH and $~T a c t i v i t i e s f o r ~4~ole b r a i n o r f o r z o n e s individually considered. H o w e v e r , t h e l e v e l s e f Ar~ a c t i , l l y w e r e s i o n i f i c a n tty different ( p < O . O 5 ) b e t w e e n t h e l e f t and r i ~ h t s i d e of ti~e r n t ) r a i n . Of t h e s e o n l y FC and '.IY w e r e s i g n i f i c a n t l y a s y r n n . e t r i c a l a n d in ',~olh c a s e s w e r e h i ~ h e s t i n t h e l e f t s i d e . AP a c t i v i t y was n o t s i g n i f i c a n t l y different belg.,con the left and the right side in t h e re~v'ainino n i n e zones studied. "l'l;o statist,cal analysis also revealed intrahemisiJheric differences in t h e l e v e l s o f AP, I.Dfl ,~md GOT, a l t h o u q h n o c o r r e l a t i o n c o u l d be f o a n d a~0on~] t h e s e t h r e e enzyr~tic activities..L;oreover, ~,~hen n e u r o c h o ~ i c a l l e v e l s of t h e t h r e e e a z y rues w e r e e x p r e s s e d a s a p e r c e n t i n c r e a s e of one s i d e o v e r t h e o t h e r (t:i,~. 1 ), the number of left-biased z o n e s and t h e . n e r c e n t a ¢ e o f t h e i r i n c r e a s e s w e r e I l i q h e r f o r AP a c t i v i t y t h a n f o r I,DI.I and GOT' a c t i v i t i e s .
2.5,271,4 24,1-i ,6
2~.772.2
26.072.2 24.672.2
2".171.8 26.771.9 21.771.3
CP TA
tW
:~g i I)
CL HC 5.2
24.1+2.l 2,5.372.4 20.7"gl .3
24.3~1.7 23.572.6
22. ,671.8e
25,0~1,5 24,671,7
19.O+1.4e 21 . I + 1 . 2 22. S'~l .5
R
4.18+O.a 3.06"70.3 3.1670.3
3.35+0.4 3.9g+0.5
2.7/,+0.3
3,16"/0,2 3.7070,4
3.1 ~+0.3 3.32+0.3 3.417"0.4
1.
LDI~
of AP, l.r);{and '~T~F a c t i v i t i e s
z. 22~o.4 3.0170.3 ,3.0670.3
3.16"70.3 3.8(~'0.6
2.58"$0.3
3.2470.3 3,8070,3
3.22+0.3 3.26~+0.3 3.1470.3
R
in l e f t
L
C~T
l . 2970.06 O.83+0.0a O.qa70.05
1.26"70.06 1.2 ~g70.05
0 • o,2+O. 10
l, 1170,o5 l, 1.570,O4
1.30T0.07 0.83~0.05 0.8970.03
1.16"70.05 1.1 770.05
O. 9670.05
1,0770,05 1,1970,06
0.,¢'7+0.U5 1.0770.05 O. f,O'20. O5
(R) s e l e c t e d zones of
0.53+0.05 1.0570.05 O. 8(~70.04
(I,) and r i g h t
V a l u e s r e p r e s e n t mean (+S.E.}'..) l e v e l s o f , r e s p e c t i v e l y , u n i t s of AP/,~E, of p r o t e i n (n=21), m u n i l s of LI)H/rr~. of p r o t e i n (n=18) and n t m i t s of CCrl'/mg., of p r o t e i n (n=lO). A b b r e v i a t i o n s a s in t i e t e x t . e ( p ( O . 0 5 ) .
23.2+2.0 22.g7+1.4 24.~1.7
:'C PC v'K]
L
AP
l istribution }nt hra in.
Table I
t,o
¢'t
I:u
I1
O
F.4 ~O Oo
O
O
938
Aminopeptidase and 5raln Asymmetry
Vol. 43, No. ii, 1988
L20
o0 i '~
I
R10
, 10t
~
¢~
|
RIO
'
L.J
1
LIOt
i_~
f i R10
'
~_~
, j.~ GOT
LLJ
, FC, pC, OC, Cp= TA, HY,iEllVl~ CEi HC, SCI
F1G. 1 Reo~ic~at percentaooe d i f f e r e n c e s f o r each one of t h e e n z y me a c t i v i t i e s s t u d i e d , h / l , h i g h e r mean v a l u e / l o w e r mean v a l u e ; RlO: l(Y/o p r o p o r t i o n a l i n c r e a s e of r i g h t p r ed o m i n an c e : LIO: 10% p r o p o r t i o n a l i n c r e a s e of l e f t predom.inance. A b b r e v i a t i o n s a s in t h e t e x t . Discussion Over t h e p a s t two d e c a d e s t h e r e has been g r e a t i n t e r e s t the o r g a n i z a t i o n of t h e c e r e b r a l h e m i s p h e r e s of nonhuman s p e c i e s b e c a u s e of t h e possibility of p r o v i d i n g a nonhuman a n a l o g u e of c e r e b r a l a n a t o m i c a l and f u n c t i o n a l o r g a n i z a t i o n . U n t i l r e c e n t l y man was c o n s i d e r e d t h e only manrnal shc~cing b r a i n l a t e r a l i z a t i o n (10). However, s e v e r a l l i n e s of e v i d e n c e show t h a t a n a t o m i c a l , f u n c t i o n a l and biocJ~eJrical asyn~netries can a l s o be found in some a n i m a l s (1,2). S e v e r a l n e u r o t r a n s m i t t e r s have been r e p o r t e d t o be lateralized in some c e r e b r a l n u c l e i (6,7,8,9), but t h e r e seems t o be no d i f f e r e n c e among any of t h e n e u r o p e p t i d e s s t u d i e d (11). Our work shows t h a t AP a c t i v i t y i s a s y r r m e t r i c a l l y d i s t r i b u t e d in I~ and HY of t h e r a t b r a i n , w i t h th e l e f t s i d e s h a v i n g h i g h e r a c t i v i t y than t h e r i g h t s i d e s . Such l a t e r a l i z a t i o n i s not d e t e c t e d in t h e r e m e i n i n g n i n e zones s t u d i e d . Moreover, the a c t i v i t i e s of two c l a s s i c a l m e t a b o l i c enzymes, LDI{ and C~T, were not l a t e r a l t z e d in any of t h e e l e v e n zones s t u d i e s . The reason f o r such a s p e c i f i c d i s t r i b u t i o n i s , o b v i o u s l y , unknown; but i t could he hy'pothesized that differences in d e g r a d a t i o n (or a c t i v a t i o n ) r a t e s have t h e same f u n c t i o n a l s i g n i f i c a n c e as d i f f e r e n c e s in t h e c o n t e n t of a p a r t i c u l a r neuro-p e p t i d e w i t h i n a ~ iven zone of th e b r a i n .
Vol. 43, No. ii, 1988
Aml-opeptidase and Brain Asy-,,etry
939
The b r a i n p r o b a b l y c o n t a i n s s e v e r a l s o l u b l e APs ( 1 8 ) a b l e t o h y d r o l y ze the substrate Leu-2~NA. T h e r e f o r e , left-right differences i n AP a c t i v i t y i n FC and HY c o u l d b e t h e r e s u l t of differences i n AP c o n c e n t r a t i o n in these zones or it could be due to a different interhemispherical distribution o f AP i s o e n z y m e s i n t h e s e two z o n e s o f t h e r a t b r a i n . References
1. N. GESCI{WIND and A.M. GALABURDA. Cerebral Dominance. The Biological Fundat i o n s , p. 232, Harvard University Press. Cambridge (1984). 2. S.D. GLICK. Cerebral l a t e r a l i z a t i o n in Nonhuman Species, p. 287. Academic Press, London, (1985). 3. S.D. GLICK, D.A. IK)SSand L.S. ~XIGH. Brain Res. 234, 53-64 (1982) 4. A. OKE; ,~. KELLER, I. }.EFFORD and R.N. ADAMS. Science, 200, 14111413 (1981). 5. L. AMADU(3CI, S. ff3RBI, A. ALBANESE and C,AI}K)TTI. Neurology; 31, 799-805 (l 981 ). 5. A. C~(E, R. LEWISand R.N. ADA~. Brain Res. 188, 268-272 (1980). 7. G.D. ROSEN, S. FINKLESTEIN, A.L. STOLL, D.a. YUTZEY and V.H. DENENBERG. Life Sci. 34, I143-I148 (1984). ~$. h.S. $~I'ARR'-'~nd I.C. KILPATRICK. Neurosci. Lett. 25, 167-172 (1981) 9. ] . S . VALDES, C.F. ~,iACTUTUS, R.N. OORI and W.RT"-CAMERON. Physiol. Behav. 27, 381-388 (1981). lO.--tZ. }[ANSEN, T.L. PERRY and J .A. WADA.Brain Res. 45, 318-320 (1972). I I . C.R. }lUNG; J .S. H~qG and S.C. BONDY. Neurosci. ,7-~-2295-2298 (1982). 12. K. BAUER, INSERt,',, llO, 475-494 (1982). 13. J.F.R. KONIG and T . A . KLIPPEL. The Rat Brain, Krieger Huntington, New York, (1967). 14. L.J. GREEE,~BERG. Biochem. Biophys. Res. Com'oun. 9, 430-435 (1962). 15. M.M. BRADFORD. Anal. Biochem. 72, 248-254 (19767. 16. H.U. BERG~EYER and E. BERNT.-'~,{ethods of Enz~,matic Analysis, Bergmeyer, H.U. ed. 2, 574-582. Academic Press, New York, (197&). 17. A. KAP~,EN; F. WROBLESKI and T.S. LAUDE. J. Clin. Invest. 34, 129 (1955). 18. II.J. BERG and N. ~¥~RKS. J. Neurosci. Res. II. 313-321 (19~2[).
Pergamon Press
K\HNOPEPTIDASE ACTIVITY IS ASYIJt~.~I'RICALLYDISTRIBUTE33 1N SELECTED ZONES OF RAT BRAIN
F r a n c i s c o Alba, Manuel Ramirez, E m i l i o 5. C a n t a l e j o and Concepci6n I r i b a r Department of B i o c h e m i s t r y and M~lecular B i o l o g y , F a c u l t y of Medicine, U n i v e r s i t y of Granada. Granada. S p a i n . (Received i n f i n a l form J u l y 26, 1988) Sunma ry L e v e l s of s o l u b l e a m i n o p e p t i d a s e (AP), measured a s a r y l a m i d a s e a c t i v i t y u s i n g L - L r u c i n e - 2 - N a p h t h y l a m i d e (Leu-2-NA) a s s u b s t r a t e , were d e t e r m i n e d in t h e s o l u b l e f r a c t i o n of e l e v e n zones of r a t b r a i n . R e s u l t s showed t h a t AP a c t i v i t y i s a s y r r m e t r i c a l l y d i s t r i b u t e d i n f r o n t a l c o r t e x and hypothalamus with both l e f t s i d e s h a v i n g s i g n i f i c a n t l y h i g h e r l e v e l s of AP a c t i v i t y , r e s p e c t i v e l y , than t h e r i g h t sides. S i m u l t a n e o u s l y , the a c t i v i t i e s of l a c t a t e dehydrogenase (LDH) and g l u t a m a t e - o x a l a c e t a t e a m i n o t r a n s f e r a s e (GOT) were measured in t h e saJ~ c e r e b r a l r e g i o n s ; no s i g n i f i c a n t d i f f e r e n c e was recorded i n t h e s e a c t i v i t i e s between e i t h e r s i d e of t h e r a t b r a i n in any of the zones s t u d i e d . P r o v i d e d t h a t a m i n o p e p t i d a s e s a r e i n v o l v e d i n t h e d e g r a d a t i o n of some endogenously r e l e a s e d n e u r o p e p t i d e s , the r e : u l t s suKqest a new mode of e x p r e s s i o n of c e r e b r a l lateral ization. Anatomical and f u n c t i o n a l c e r e b r a l a s y m m e t r i e s have been e x t e n s i v e l y d e s c r i b e d i n man and a n i m a l s ( 1 ) , and m u l t i p l e approaches have been used i n order to study b r a i n l a t e r a l i z a t i o n ( 2 ) . However, the n e u r o c h e m i c a l b a s i s underlying left-right d i f f e r e n c e s a r e s c a r c e l y known and l i t t l e evidence e x i s t s t o imply c l a s s i c a l n e u r o t r a n s m i t t e r s in t h i s m a t t e r . Some of them have been r e p o r t e d t o be a s y r m a e t r i c a l l y d i s t r i b u t e d in c e r t a i n c e r e b r a l l o c a t i o n s i n man and o t h e r nmnrrals ( 3 , a , 5 , 6 , 7 , 8 , 9 ) . N e v e r t h e l e s s , t h e r e i s no e v i d e n c e of a s y r m l e t r i c a l d i s t r i b u t i o n of n e u r o p e p t i d e s in the c e n t r a l n e r v o u s system e i t h e r in humans or ir. a n i m a l s (10, 11). B r a i n t~Ps have been i m p l i c a t e d in the mechanism of enzymmtic h y d r o l y sis of several neurc~eptides (12). Some of these p e p t i d e s seem t o p a r t i c i p a t e in the mechanism of synaptic transmission and have been desiRnated as neuromodulators. Provided that p e p t i d e s are presumably s y n t h e t i z e d as bi.~£er precursors by riboscmes and t r a n s p o r t e d v i a synaptic t e r m i n a l s , where they a c t , i t would be reasonable to assume that the a c t i v i t y of those neuromodulat o r s could be c o n t ~ o l l e d by the l e v e l s or the a c t i v i t i e s of p r o t e o l y t i c enzymes l o c a t e d near the s i t e where n e u r o p e p t id e s a c t . T h e r e f o r e , b r a i n APs, the rr~)st abundant p r o t e o l y t i c enzymes found in CNS, could be one way of c o n t r o l l i n ~ n e u r o p e p l i d e a c t i v i t y , dK)reover, i f AP a c t i v i t y were asynTr~tricaf l y d i s t r i b u t e d in b r a i n hemispheres, n e u r o p e p t i d e a c t i v i t y presunTablv would be a f f e c t e d in an as}~rmetrical way.
0024-3205/88 $3.00 + .00 Copyright (c) 1988 Pergamon Press ple
936
Aminopeptldase and Brain Asynmetry
Vol. 43, No. ii, 1988
i , . i a t e r i a l s and ~ i e t h o d s iJale albino rats (avera%e wetqht: 250 ~=,) w e r e a n e s t h e t i z e d with ]~uithensin and their brains perfused with saline through the cardiac left ventriculum. Brains were quickly r e m o v e J, c o o l e d in d r y i c e , and s l i c e d ~n coronal sections of 1 rrm t h i c k . Tissue sar0ples (average weight: 20 ~ , ) , disseetcM from these slices in a£reefnent w i t h t h e s t e r e o t a x t c a t l a s of XL~n~q a n d K l i p p e l (13) w e r e o b t a i n e d , a s s ~ r ~ r . e t r i c a l l y a s p o s s i b l e , from the ri,iht and left frontal cortex (FC), occipital c o r t e x (CYg), p a r i e t o t e m p o r a l cortex (PC), caudateputamen (CP), thalamus (TA), h y p o l h a l a m u s (TIY) m e s e t ~ c c p h a l o n (!,~), m e d u l l a C.|)), cerebellum iCE); h i p p o c a m o u s (HC) a n d c e r v i c a l spinal c o r d ( S C ) , Each s a m p l e was h o r r o ~ e n i z e d in lO v o l u m e s of 59 n$', Tris!-~Cl b n f f e r . ptt 7 . 4 and u l t r a c e n t r i f u g e d ( l O 0 , O 0 0 £ , 30 r,l i n ) . The s n p e r n n t a n t ~,~s d e c a n t e d a n d i m m e d i a t e l y a n a l v z e d f o r AP, iT)T, LDH a c t ~ . v i t i e s and p r o t e i n s . AP ( a r y l a m i d a s e ) a c t i v i t y was m e a s u r e d f l u o r c ~ ~ t r i c a l l y , r~er t r i p l i c a t e , u s i n g Leu-2-NA a s s u b s t r a t e , a c c o r d i n g t o t h e .aetllorl o f Greet.her!,, i t / , ) , n ~ c l i f i e d a s f o l l o w s : 10 u l o f s u p e r n a t a n t was i n c u b a t e c i 30 rain w~th I ml of substrate solution ( 0 . 8 ~_~/lO0 rnl l.eu-2-~IA; lO n~,/lO0 ml b o v i n e a l b u m i n ; If) r ~ / l O 0 ml d i t h i o t h r e i t o l ; i n lO rnl p h o s p h a t e b u f f e r , pll 7./~) a t 2.5 -°C. Ttle e n z y n n t i c r e a c t i o n was s t o p p e d b y a d d i n . c 1 m l , 0.1 ;, a c e t a t e h u f f e r , pH / , . 2 , The f l u o r e s c e n c e intensity of 2-naphthylarrine released ~¢as ~r~asurc~] a t a n excitation wavelength o f 345 nm a n d a n e n f i s s l o n waw, l e n ~ h t of 411 nm. Proteins were measured, per tri'.)licate, b y t i e m e t h o t o f ~3radford ( 1 5 ) . R e s u l t s w e r e e x p r e s s e d a s u n i t s o f AP p e r m i l l i q r a m o f p r o t e i n . One u n i t of AP was c o n s i d e r e d a s t h e amount o f e n ~ e that hydrolyzed 1 nano~)I of Leu-2-NA p e r m i n u t e . LDII and ~ activities were detennined spectrophotometrically, per triplicate, as previously described (16,17) and e x p r e s s e d a s milliunits of enzymatic activity p e r m i l l i q r a m of o r o t e i n , l)ata were test~.] u s i n ~ a 3-way a n a l y s i s of v a r i a n c e '^'ith two ~'ixed a n d one random f a c t o r . To detect indtviduaI asy~m~,etries, r r u l t i l ~ l e c o m p a r i s o r ~ s w e r e 'rode usin,~ t h e v a r i a n c e and T u r k e y ' s t e s t . !~esul ! s The l e v e l s o f AP, LDIt and COT s p e c i f i c activities in both the left and riuht sides of the eleven zones studied are shown in T a b l e ] . :~o significant differences w e r e found b e t w e e n t h e n,ean v a l u e s o f t h e l e f t s i d e a n d t h e r i g h t s i d e f o r LDH and $~T a c t i v i t i e s f o r ~4~ole b r a i n o r f o r z o n e s individually considered. H o w e v e r , t h e l e v e l s e f Ar~ a c t i , l l y w e r e s i o n i f i c a n tty different ( p < O . O 5 ) b e t w e e n t h e l e f t and r i ~ h t s i d e of ti~e r n t ) r a i n . Of t h e s e o n l y FC and '.IY w e r e s i g n i f i c a n t l y a s y r n n . e t r i c a l a n d in ',~olh c a s e s w e r e h i ~ h e s t i n t h e l e f t s i d e . AP a c t i v i t y was n o t s i g n i f i c a n t l y different belg.,con the left and the right side in t h e re~v'ainino n i n e zones studied. "l'l;o statist,cal analysis also revealed intrahemisiJheric differences in t h e l e v e l s o f AP, I.Dfl ,~md GOT, a l t h o u q h n o c o r r e l a t i o n c o u l d be f o a n d a~0on~] t h e s e t h r e e enzyr~tic activities..L;oreover, ~,~hen n e u r o c h o ~ i c a l l e v e l s of t h e t h r e e e a z y rues w e r e e x p r e s s e d a s a p e r c e n t i n c r e a s e of one s i d e o v e r t h e o t h e r (t:i,~. 1 ), the number of left-biased z o n e s and t h e . n e r c e n t a ¢ e o f t h e i r i n c r e a s e s w e r e I l i q h e r f o r AP a c t i v i t y t h a n f o r I,DI.I and GOT' a c t i v i t i e s .
2.5,271,4 24,1-i ,6
2~.772.2
26.072.2 24.672.2
2".171.8 26.771.9 21.771.3
CP TA
tW
:~g i I)
CL HC 5.2
24.1+2.l 2,5.372.4 20.7"gl .3
24.3~1.7 23.572.6
22. ,671.8e
25,0~1,5 24,671,7
19.O+1.4e 21 . I + 1 . 2 22. S'~l .5
R
4.18+O.a 3.06"70.3 3.1670.3
3.35+0.4 3.9g+0.5
2.7/,+0.3
3,16"/0,2 3.7070,4
3.1 ~+0.3 3.32+0.3 3.417"0.4
1.
LDI~
of AP, l.r);{and '~T~F a c t i v i t i e s
z. 22~o.4 3.0170.3 ,3.0670.3
3.16"70.3 3.8(~'0.6
2.58"$0.3
3.2470.3 3,8070,3
3.22+0.3 3.26~+0.3 3.1470.3
R
in l e f t
L
C~T
l . 2970.06 O.83+0.0a O.qa70.05
1.26"70.06 1.2 ~g70.05
0 • o,2+O. 10
l, 1170,o5 l, 1.570,O4
1.30T0.07 0.83~0.05 0.8970.03
1.16"70.05 1.1 770.05
O. 9670.05
1,0770,05 1,1970,06
0.,¢'7+0.U5 1.0770.05 O. f,O'20. O5
(R) s e l e c t e d zones of
0.53+0.05 1.0570.05 O. 8(~70.04
(I,) and r i g h t
V a l u e s r e p r e s e n t mean (+S.E.}'..) l e v e l s o f , r e s p e c t i v e l y , u n i t s of AP/,~E, of p r o t e i n (n=21), m u n i l s of LI)H/rr~. of p r o t e i n (n=18) and n t m i t s of CCrl'/mg., of p r o t e i n (n=lO). A b b r e v i a t i o n s a s in t i e t e x t . e ( p ( O . 0 5 ) .
23.2+2.0 22.g7+1.4 24.~1.7
:'C PC v'K]
L
AP
l istribution }nt hra in.
Table I
t,o
¢'t
I:u
I1
O
F.4 ~O Oo
O
O
938
Aminopeptidase and 5raln Asymmetry
Vol. 43, No. ii, 1988
L20
o0 i '~
I
R10
, 10t
~
¢~
|
RIO
'
L.J
1
LIOt
i_~
f i R10
'
~_~
, j.~ GOT
LLJ
, FC, pC, OC, Cp= TA, HY,iEllVl~ CEi HC, SCI
F1G. 1 Reo~ic~at percentaooe d i f f e r e n c e s f o r each one of t h e e n z y me a c t i v i t i e s s t u d i e d , h / l , h i g h e r mean v a l u e / l o w e r mean v a l u e ; RlO: l(Y/o p r o p o r t i o n a l i n c r e a s e of r i g h t p r ed o m i n an c e : LIO: 10% p r o p o r t i o n a l i n c r e a s e of l e f t predom.inance. A b b r e v i a t i o n s a s in t h e t e x t . Discussion Over t h e p a s t two d e c a d e s t h e r e has been g r e a t i n t e r e s t the o r g a n i z a t i o n of t h e c e r e b r a l h e m i s p h e r e s of nonhuman s p e c i e s b e c a u s e of t h e possibility of p r o v i d i n g a nonhuman a n a l o g u e of c e r e b r a l a n a t o m i c a l and f u n c t i o n a l o r g a n i z a t i o n . U n t i l r e c e n t l y man was c o n s i d e r e d t h e only manrnal shc~cing b r a i n l a t e r a l i z a t i o n (10). However, s e v e r a l l i n e s of e v i d e n c e show t h a t a n a t o m i c a l , f u n c t i o n a l and biocJ~eJrical asyn~netries can a l s o be found in some a n i m a l s (1,2). S e v e r a l n e u r o t r a n s m i t t e r s have been r e p o r t e d t o be lateralized in some c e r e b r a l n u c l e i (6,7,8,9), but t h e r e seems t o be no d i f f e r e n c e among any of t h e n e u r o p e p t i d e s s t u d i e d (11). Our work shows t h a t AP a c t i v i t y i s a s y r r m e t r i c a l l y d i s t r i b u t e d in I~ and HY of t h e r a t b r a i n , w i t h th e l e f t s i d e s h a v i n g h i g h e r a c t i v i t y than t h e r i g h t s i d e s . Such l a t e r a l i z a t i o n i s not d e t e c t e d in t h e r e m e i n i n g n i n e zones s t u d i e d . Moreover, the a c t i v i t i e s of two c l a s s i c a l m e t a b o l i c enzymes, LDI{ and C~T, were not l a t e r a l t z e d in any of t h e e l e v e n zones s t u d i e s . The reason f o r such a s p e c i f i c d i s t r i b u t i o n i s , o b v i o u s l y , unknown; but i t could he hy'pothesized that differences in d e g r a d a t i o n (or a c t i v a t i o n ) r a t e s have t h e same f u n c t i o n a l s i g n i f i c a n c e as d i f f e r e n c e s in t h e c o n t e n t of a p a r t i c u l a r neuro-p e p t i d e w i t h i n a ~ iven zone of th e b r a i n .
Vol. 43, No. ii, 1988
Aml-opeptidase and Brain Asy-,,etry
939
The b r a i n p r o b a b l y c o n t a i n s s e v e r a l s o l u b l e APs ( 1 8 ) a b l e t o h y d r o l y ze the substrate Leu-2~NA. T h e r e f o r e , left-right differences i n AP a c t i v i t y i n FC and HY c o u l d b e t h e r e s u l t of differences i n AP c o n c e n t r a t i o n in these zones or it could be due to a different interhemispherical distribution o f AP i s o e n z y m e s i n t h e s e two z o n e s o f t h e r a t b r a i n . References
1. N. GESCI{WIND and A.M. GALABURDA. Cerebral Dominance. The Biological Fundat i o n s , p. 232, Harvard University Press. Cambridge (1984). 2. S.D. GLICK. Cerebral l a t e r a l i z a t i o n in Nonhuman Species, p. 287. Academic Press, London, (1985). 3. S.D. GLICK, D.A. IK)SSand L.S. ~XIGH. Brain Res. 234, 53-64 (1982) 4. A. OKE; ,~. KELLER, I. }.EFFORD and R.N. ADAMS. Science, 200, 14111413 (1981). 5. L. AMADU(3CI, S. ff3RBI, A. ALBANESE and C,AI}K)TTI. Neurology; 31, 799-805 (l 981 ). 5. A. C~(E, R. LEWISand R.N. ADA~. Brain Res. 188, 268-272 (1980). 7. G.D. ROSEN, S. FINKLESTEIN, A.L. STOLL, D.a. YUTZEY and V.H. DENENBERG. Life Sci. 34, I143-I148 (1984). ~$. h.S. $~I'ARR'-'~nd I.C. KILPATRICK. Neurosci. Lett. 25, 167-172 (1981) 9. ] . S . VALDES, C.F. ~,iACTUTUS, R.N. OORI and W.RT"-CAMERON. Physiol. Behav. 27, 381-388 (1981). lO.--tZ. }[ANSEN, T.L. PERRY and J .A. WADA.Brain Res. 45, 318-320 (1972). I I . C.R. }lUNG; J .S. H~qG and S.C. BONDY. Neurosci. ,7-~-2295-2298 (1982). 12. K. BAUER, INSERt,',, llO, 475-494 (1982). 13. J.F.R. KONIG and T . A . KLIPPEL. The Rat Brain, Krieger Huntington, New York, (1967). 14. L.J. GREEE,~BERG. Biochem. Biophys. Res. Com'oun. 9, 430-435 (1962). 15. M.M. BRADFORD. Anal. Biochem. 72, 248-254 (19767. 16. H.U. BERG~EYER and E. BERNT.-'~,{ethods of Enz~,matic Analysis, Bergmeyer, H.U. ed. 2, 574-582. Academic Press, New York, (197&). 17. A. KAP~,EN; F. WROBLESKI and T.S. LAUDE. J. Clin. Invest. 34, 129 (1955). 18. II.J. BERG and N. ~¥~RKS. J. Neurosci. Res. II. 313-321 (19~2[).