Paglariani Et Al 1991

8/12/2019 Paglariani Et Al 1991

1/8

8/2, 40120 Bolog na, Italy (lel : O~l 24301~); an a lStltUtO (

A~ trae t - -1 . The expected higher gil l (Na + + K +)-ATPasewater (BW ) with respect to fresh water (FW ) is accompaniedthe enzyme sensitivity to ouabaln is unaffected.2. Maxim al activation is attained under the optimal ten dNa +, 2 .5raM K +, pH 7.0 in FW , and 3raM ATP, 10m/~in BW.3. The change o f the enzyme activation kinetics by i g 2+in F W to cooperativity in BW an d other habitat-dependentBW are hypothetically related to an adaptive significance t4. Gill total l ipids and phospholipids are 30% lower in Esom e differences in gill total lipid fatty acid compo sition b etthe unsaturation parameters.

tubul',Le N a C 1 u p t a k e f r o m a n d t h e e x t r u s i o n i n t o t h e a b u n (f re shw a te r and m a r ine t e leos t s , a ccoum e c h a n i s m s r e q u i r e d t om p e n s a t e f o r s a lt o s m o t i c l o s s a n d a c c u m u l a t io n . C o :be two pro cesses occ ur via the gil ls an d imply the i sola t,

t u b u l a r r e t i c u l u m w h o s e m e m b r a n (( N a + K ) - A T P a s e u n i t sa b u n d a n c e a n d f e a tu r e s o f s al t- w aa c c o u n t f o r th e c u r r e n t l y f o u n d h i g ti n m ar ine t e l eos t s .Conve r se ly , i n f r e shwa te r f i sh , r ai so l a t ed ch lo r ide ce ll s, o f t en cu t o f f) a se ( P a y a n e t a l . , 1 9 84 ). a n d c o n t a i n i n g p o o r m i t o c h o n d r i at h e r o l e o f t h e e n z y m e , K ) - A T P a s e c o m p l e x e s a r e s ep ar ~5 by i t s spec . a c t . , ha s ep i t he li a l c e ll s by unp e rm eab le t i gh ti ce . M os t l i t e r a tu re t ends 1982; Pa ya n e t a l . , 1984).~ ill (N a + + K +) -A T P ase T h e sa l i n i t y -dependen t appea ra lt t e r h y p o r e g u l a t o r s a n d t h e l iu m i s a c l e ar s y m p t o m o f t h em a r i n e m o r p h o l o g i c a l l o g ic a l m e c h a n i s m s i n v o l v e d i n t el e; r a ti o n o f c h l o r i d e c el ls a n d s ea - w a te r . T h e m o d e l o f t h e N a (the g i l l s i n sea -wa te r p roposed by Snd T ho rpe , 1984) . T hese and st il l fund am enta l l y accep t ed (E (a g i l l p r im a ry l am e l l ae e t a l . , 1984) i s focused o n t he ch lo r id (i t be tween two ad j ace n t uph i l l C1- ex t rus ion f rom the ce ll ( (i nd i r ec t l y ac t i va t ed by t he (Na+ -~) r di n g t o e n v i r o n m e n t a l m a t c h e d t o t h e p a r a c e l lu l a r p a s s iv et h e l e a k y j u n c t io n s . C o n v e r s e l y , i na n d m i t o c h o n d r i a r i c h m e c h a n i s m s i n v o l v e d a s w e ll a s tlvhole gi ll epi the l ium are up tak e b y the gi ll s a re s t il l undelm u tua l l y sepa ra t ed by ev idence i nd i ca t e s t ha t sa l t abso rp t i (y a re conn ec t ed by i on - secon da ry l am e l l ae . T h e p rocess , l int h a t m a k e t h e i n t e rn a l r e g u l a t io n a n d u p t o n o w r e pe at (ex t e rna l m i l i eu . Ch lo r ide r e sp i r a to ry ce ll s (Pay an e t a l . ,n an ex t r em e ly deve loped r ecen t ly a t t r i bu t ed t o pecu l i a r ch i cou t i n t he seconda ry l am e l l ae (AveT h e r o l e p l a y e d i n N a u p t a k e b y thydroxytoluene; ATPase. . . . .s so f a r unce r t a in , a s t he

INTRODUCTIONT he n v i r o n m e n t b yrespect ive ly , a re ac t ivec o m 'T h ea c t iv i t y o f ( N a + + K + ) - A T P a s eH o w e v e r i n t h e t w o h a b i t a t sa t l e a s t a s f a r a s sugges t edappa ren t ly a d i f f e ren t r e l evance . Mosto dem ons t ra t e t ha t a h igh g i l la c t i v i t y i s t yp i ca l o f sa l t -wa te rpa ra l l e ls t he onse t o f g i llf e a t u r e s , n a m e l y t h e p r o l i f e r a t i o nw h e r e t h e e n z y m e a c c u m u l a t e sB o r n a n c i n , 1 9 84 ; L a n g d o n a n d T h o rones , t yp i ca l l y l oca l i z ed oni n t h e i n t e f l a m e l l a r y s e g m e n ts e c o n d a r y l a m e l l a e , u n d e r g om o r p h o l o g i c a l c h a n g e s a c c o r d insa l ini ty .In sa l t -wa te r f i sh l a rgech lo r ide ce l l s spann ing t he who lec l u s t e r e d i n b u n c h e s a n daccesso ry ce l l s t o wh ich t hep e r m e a b l e l e a k y j u n c t i o n sf lu i d s c o m m u n i c a t e w i t h t h e e x t e r mcel l ba so l a te ra l i n fo ld ings fo rm an ex t r

A b b r e v i a t i o n s uaed--BHT, butyla tedEDTA, ethylenediaminetctraacctic achydroxyethyi-piperazine-N'-2-ethane-stTris (hydroxy-m ethyl)-aminomethane

pp. 229-236 , 1991

~ N D E N C E O F T H E P R O ]K + - A T P a se I N R A I N B O ~O N C O RH Y N CH U S M YKELLA, R . BALLESTRAZZI,*F. TROMBETSezione di B ioehimica Veterinaria, Univeel: 051 243019 ); and *Istituto di Prod uzicUniversit~ di Udine, Italy

R e c e i v e d 16 A p r i l 1991)-ATPase activity in rby som e ch~

conditions of 4 t10 m M Mg 2+, 10;2+, ATP , N a ~endent variationsto the differBW than inbetween FW a

4 o s t

D e R e n z i s a n d

q u a n t i t a t i ve a n d

O GILLT

and G, T RIGARIla, Via Belmeloro,~acoltfi di Agraria,

dapted to brackish~yme kinetics w hilen M M g 2 , 5 0 m M) r a M K +, p H 7 .5i s i m p l e s a t u r a t i o n) H a l k a l i n e h i f t nn t a l s a l i n it y .r r a t i o s c o n s t a n t ;significantly affect

~ m b r a n e c o n t a i n s m a n y(L auren t , 1984) . T hesa l t -water chlor ide ce l l sh i g h e n z y m e a c t iv i t y

ra re and gene ra l l yf r o m t h e o u t s i d ea n d f e w ( N a + +) a r a t e d f r o m o t h e rj u n c t i o n s ( E d d y ,m arance o f g i l l ep i -d i f f e ren t phys io -cost g i l l s in f resh21 ex t rus ion f ro m

Si lva e t a l . (1977)( E d d y , 1 9 8 2 ; P a y a nch lo r ide ce ll sys t em . T h e(ce l l u l a r pa thway)+ K + ) - A T P a s e i s

) a ss i ve a + e x i t h r o u g hf r e s h wa t e r , t h e

t h e s i t e o f N a C Ie f i n e d . I n c r e a s i n g

) t i o n o c c u r s v i a t h el i n k e d t o a c i d - b a s e

; p e a t e d l y a s c r i b e d t o1 9 8 4 ) , h a s b e e na r i d c c e l l s f o u n d

A v c l l a e t a l . 1 9 8 7 ) .b y t h e N a + + K + ) -t h e i n v o l v e m e n t o f

t e r al e x t r u s i o n o f N a +: o n s i d e r e d P f c i l e r a n d


2/8

s a l i n i t y - m o d u l a t e d g i ll ( N a + K + ) - A T P a s e m a y p os si l: i s t ( G a l l i s e t a l . , 1 9 7 9) . M o r e o v e r , m e m b r a n e l i p id s , i n B Yb o t h ql o w n t o b e su s c e p t iv e t o e n v i r o n m e n t a l s al i n it y m o d it~eray e t a l . , 1 9 84 ) a n d t o a f fe c t m e m b r a n e - b o u n d P 3 w~a n s p o r t A T P a s e s ( D e u t i c k e a n d H a e s t , 1 9 8 7) , m a ye n b e i n v o l v e d i n t h e s a li n i t y - d r iv e n m o d u l a t i o n 55 ,0 0(' g i l l ( N a + + K + ) - A T P a s e . p e ll e tO n t h e s e b a s e s w e d e v i s e d a c h a r a c t e r i z a t i o n s t u d y l ay er eI g i ll ( N a + K ) - A T P a s e i n t w o g r o u p s o f r a i n - 1 0 m l~w t r o u t , c o m i n g f r o m t h e s a m e p o p u l a t i o n a n d s p un',p t i n w e l l f re s h w a t e r o r i n b r a c k i s h w a t e r a t c a The t~t h e u r', p p t s a l i n i t y , i n m e ,

yieldilP 4 wMATERIALS AND METHODS:per imen ta l f i sh AllT h e a c c l i m a t i z a t i o n o f r a i n b o w t r o u t O n c o r h y n c h u s The p :~kiss) t o f r e s h w a t e r ( F W ) a n d b r a c k i s h w a t e r (B W ) w a s t h e b i lr f o r m e d a s f o ll o w s. F r e s h w a t e r a d a p t e d r a in b o w t r o u t ( G o r nt a i n e d f r o m a t r o u t f a r m w e r e r a n d o m l y d i v i d e d i n t o t w o b y PCa u ps . T h e f i rs t g r o u p w a s r e a r e d i n c o n c r e t e t a n k s s u p - t u b e se d w i t h w e l l fr e s h w a t e r . T h e s e c o n d g r o u p w a s d i r e c t lym s f e r r e d i n t o b r a c k i s h w a t e r , n a m e l y t o a c a n a l c o n n e c t e da l a g o o n n e a r b y t h e N o r t h A d r i a t i c s ea a n d p l a c ed i n n e tg e s. B W a v e r a g e s a l i n it y w a s 2 1 p p t a n d i t r a n g e d f r o m 1 8 T h e2 4 p p t . T h e w a t e r t e m p e r a t u r e v a r i e d n a t u r a l l y a r o u n d t r a ti o J~ C i n b o t h e a s e s . T h e p H w a s ca 7 .8 i n F W a n d r a n g e d m e t h

s t e p s w e r e c a r r i e d o u t u n d e r c o l d~ ro te in c o n c e n t r a t i o n o f e a c h f r a c t kb i u r e t m e t h o d u s i n g b o v i n e s er u m a l lG o r n a l l e t a L , 1 9 49 ) o r b y t h e m e t h o d o tPe te rson (1977) . Al l f rac t ions we1of 1 .5 ml in l iqu id n i t rog en u nt i l( N a + K ) - A T P a s e a c t iv i t y.( N a + K - A T P a s e a c t iv i ty

The enzyme ac t iv i ty was tes ted by me~t r a t i o n o f P i r e l e a s e d a c c o r d i n g t o t h |m e t h o d o f F i s k e a n d S u b b a r o w ( 1 9 2 5 )d o x y g e n w a s a l w a y s h i g h e r c a r r i e d o u t a t 3 0 C a s r e p o r t e d b y T rB W . F W a n d B W g r o u p s R e q u i r e d a m o u n t s o f f r a c ti o n s w e r e i n cd i e t a n d w e r e m a i n t a i n e d t i o n m i x t u r e d e t a i l e d i n T a b l e 1 a n d th e I| d i ti o n s f o r a m o n t h f r o m e x p e r im e n t s s h o w e d , u n d e r o u r c o n d i t io rk t t h e e n d o f t h e a c c li m a t i z - s h i p b e t w e e n t h e P~ r e l e as e d a n d b o t h t h es a m p l e d f r o m b o t h F W a n d i n c u b a t e d i n t h e ra n g e 0. 1 -1 . 0 m g a n d t4 8 0 g , w e r e d e c a p i t a t e d t h e u p t o 1 0 m i n , t h a t w a s u s e d i n t h e e x p e r i ls o u r c e . I m m e d i a t e l y a f t e r w a s s t a r t e d b y t h e a d d i t i o n o f A T P a n d slo v e d a n d d i v i d e d i n t o th r e e b y t h e a d d i t i o n o f 1 m l o f i c e - co l d 1 5 %o f e a c h p o o l w a s s t o r e d i n ( w /w ) . T h e ( N a + + K ) - A T P a s e w a sdyses , and the res t , des t ined d i ffe rence be tween the P~ l ibera ted in the p:t c t io n s , w a s s u b j ec t e d t o th e o f K C I i n t h e a s s a y m e d i u m . A T P a s e a c t in t s were t r im me d f rom g i l l as #m ol P~ mg pro te in -~ hr -~ . Each de te r~e-co ld me dium A (0 .25 M ac t iv i ty was car r ied ou t in t r ip l ica te . A1, ri s -H C 1 , p H 7 . 4 ) a c c o r d i n g r u n t w i c e u n l e s s d i f fe r e n t ly s ta t e d . T ha n d s t o r e d i n s m a l l p o r t i o n s e n z y m e a c t i v a ti o n a n d i n h i b i t i o n p r o m oA T P w a s t e s t e d b y a H i l l p l o t . A p p a r ec a l c u l a t e d b y d o u b l e r e c i p r o c a l p l o t sso la tera l mem brane f rac t ions c o n c e n t r a t i o n w a s r a i s e d t o a p o w e r / ~

n b e t t i et a l . , 1990; Ve ntre l la coefficient (Segel, 1975). Th e l inea ri tyl i q u i d n i t r o g e n d o e s n o t w i t n e s s e d b y a n a b s o l u t e v a l u e o f t h e c oJ[ 'Pase wi th respec t to the r neve r lower than 0 .980 . The charae d i a t e l y a f t e r t h a w i n g , t h e ( N a + + K +) - A T P a s e a c t i v i ty w a s c a r r ie cl m A a n d h o m o g e n i z e d b y i n b o t h h a b i t a t s.; h o m o g e n a t e ( H ) t h a t w a sl o d o f V e n t r e l l a et al. (1990) L i p i d a n a l y se st o b t a i n e d f r o m a n i n i t i a l I m m e d i a t e l y a f t e r t h a w i n g , t h e g il ls w e0 min was care fu l ly resus- paper , separa ted f rom g i l l a rches , weighe

ilem y k i s s )p e r f o r m e do b t a i ng r o u tp l i e d w i t ht r a ntocages.to10qf r o m 8 . 2 t o 8 .7 i n B W . D i s s o l v e dt h a n 7 p p m b o t h i n F W a n dr e c e i v e d t h e s a m e c o m m e r c i a lu n d e r t h e a b o v e d e s c r i b e d c o n d i t i o n sm i d - J a n u a r y t o m i d - F e b r u a r y . A t t h ea t i o n p h a s e , 3 6 t r o u t , r a n d o m l yB W g r o u p s a n d w e i g h in g 2 0 0 - 4 8 0 g ,s a m e d a y a n d u s e d a s t h e g i l ld e a t h t h e g i ll s w e r e q u ic k l y r e m o v e d a n dp o o ls . A p p r o x i m a t e l y o n e f o u r t h o f ql i q u i d n i t r o g e n u n t i l t h e l i p i d a n a l ~f o r p r e p a r a t i o n o f s u b c e l l u l a r f ra c t io n s .f o l l o w i n g p r o c e d u r e : t h e f i l a m e n t sarches , repea ted ly r insed in ice -cohs u c ro s e , 5 m M E D T A , 1 6 m M T r i s - H C 1t o M u r p h y a n d H o u s t o n ( 1 97 4 ) a n d s t o r e ti n l i q u i d n i t r o g e n .Prepa ration o f subcellu lar and basolater

A s p r e v i o u s l y d e s c r ib e d ( T r o m b e t t ie t a l . 1990 t i s sue s torage ini n a c t i v a t e t h e ( N a + + K + - A T P a s ef r e s h l y p r e p a r e d m a t e r i a l . I m m e d i a t e lg i l l s w e r e r e s u s p e n d e d i n m e d i u mU l t r a t u r r a x b l e n d e r t o y i e l d t h ecent r i fuged accord ing to the m ethod of Venmodi f ied as fo l lows . The pe l le tc e n t r i f u g a t i o n o f H a t 9 0 0 g / 1 0 m i np e n d e d , r e h o m o g e n i z e d a n d a g a i n c e n t r i ts p e e d , t h u s y i e l d i n g a n e w s e d i m e n t ( P 1 )p u t t o g e t h e r w i t h t h a t o b t a i n e d f r o m

A. PAGLIARANI et a l .a s e a c t i v i t y h i g h e r f u g a ti o n , w a s f~l t e r w a s r e p e a t e d l y c e n tr i fu g e d a t 9 (t i e s ( J o h n s t o n a n d n a m el y t h e m i to3 , 1 9 8 6 ; D e R e n z i s s u p e r n a t a n t f u rt lN a a m a n s e n , 1 9 8 9) , l as t s u p e r n a ta n t,~ i n v o l v e d i n N a + a n d r e c e nt r if u g e cing a f ina l pe l le ta N a + u p t a k e m a y a s u p e r n a ta n t vY e a t u re s p o s s i b l y r e - p r e v i o u s l y r e m o 'n t s a l i n it y , h a s b e e n ( N a + K + ) . A ' [g g e s t s t h a t i n F W a m a d e i t n e c e ss a rl e r a n d K i r s c h n e r , d i s c o n ti n u o u s g r,e f u n c t i o n a l f o r m a r a ti o n s e nr ic he da o s si b le t o c o m pB W , t h e p u r i lbo th cases . The rmod i f ied as desc lw e r e d i l u t e dE D T A , 1 6 m M55,000 g for 60 r~pe l le t (PY) , resuse r e d o v e r a d im l 3 1 % ( w /w )a t 90 ,000 gf r a c t i o n c o l ltpper phase (m e d i u m A a n ty ie ld ing a f te r renwhich repre~f rac t ion .

a n d d i v i d e d i n t o

ored in s in ,

a d o u b l e g a u z e l a y e r a n dn . The pe l le t p roduced (P2) ,t i o n , w a s r e m o v e d a n d t h ea t 5 0 , 0 0 0 g f o r 9 0 m i n . T h i sr h e s e d i m e n t w a s s u s p e n d e d~ e ed f o r 6 0 m i n , t h u s o b t a i n -h e m i c r o s o m a l f r a c t i o n , a n d~ t h e r w i t h t h e s u p e r n a t a n ttc t ion S1 . The low spec i f icb u n d i n f r a c t i o n P 3 i n F Wlr i fy i t on a sucrose dens i tyb a s o l a t e r a l m e m b r a n e p r e p -: t iv i ty . In order to make i t: ts o f A T P a s e i n F W t o t h a t: t i o n P 3 w a s c a r r i e d o u t i n~ella et a l . ( 1 9 7 9 ) a d e q u a t e l yt s u s e d . A l i q u o t s o f f r a c t i o nm B (0 .1 M sucrose , 5 m M7 . 4 ) a n d c e n t r i f u g e d a tm t a n t w a s r e m o v e d a n d t h e1 a m o u n t s o f m e d i u m B , w a sc r o s e g r a d i e n t c o m p r i s e d o f( w / w ) s u c r o s e s o l u t i o n s a n da B e c k m a n S W 2 5 . 1 r o t o r .s u c r o s e i n t e r f a c e b e t w e e nw e r p h a s e ( L P ) , w a s d i l u t e dt 1 00 ,000g for 60 min , thu sm p e r n a t a n t ( $ 2 ) , t h e p e l l e tf l e d b a s o l a t e r a l m e m b r a n ec o n d i t i o n s ( 0 - 4 C ) .' a c t io n w a s m e a s u r e d b y~ r u m a l b u m i n a s a s t a n d a r dt e t h o d o f L o w r y a s m o d i f i edw e r e s t o r e d i n s m a l lu n t i l e v a l u a t i o n o f t h e

m e a s u r i n g t h e c o n c e n -t h e p r i n c i p l e s o f t h e(1925) . The reac t ion wasT r i g a r i et a l . (1985).w e r e i n c u b a t e d i n t h e r e a c -and the f igures . Pre l iminary:ondi t ion s , a l inear re la t ion-t h e a m o u n t o f p r o te i n: a n d t h e in c u b a t i o n t i m e~eriments . T he reac t io ns t o p p e d a f t e r 1 0 r a int r ich lorace t ic ac idd e t e r m i n e d a s t h e~resence and absenceac t iv i t i es a re exp ressede t e r m i n a t io n o f A T P a s eA l l e x p e r i m e n t s w e r eT h e c o o p e r a t i v i t y o fp r o m o t e d b y c a ti o n s a n dp p a r e n t K m v a l u e s w e r ew h e r e t h e s u b s t r a t eh , h be ing the Hi l lo f a l l p l o t s w a scorrelat ion coefficientc h a r a c t e r i z a t i o n o f t h et s c a r r i e d o u t o n f r a c t i o n P 4

i ll s were dr ied on b lo t t in gh e d a n d s u b j e c t e d too u t i n d u p l i c a t e a c c o r d i n g.n ts c o n t a i n e d 0 . 0 1 % ( w / v )v a l u a t i o n o f p h o s p h o l i p i d


3/8

19.2 5:3.0 19.9 + 4.2rations are nam ed as in Materials and M ethods.: spec. act. expressed as #m ol P i mg protein -~ hr- L~: percentage of activity of the hom ogenate recovered inA: relative spec. act., calculated as the ratio o f SA of any fraction to thatrays of the (N a + + K+)-AT Pase activity were carried out under the respec17 mM Tris (pH 7.0) , 4 .0 mM MgATP, 3.5 mM MgCI , 50 mM NaCI, 2.53 .0mM MgATP, 7 .0mM MgCI2, 100raM N aCI, 10.0raM KCI in BW .lues given are means 5: SE from three d eterminations. Statistically sight* (P < 0.01) , or f (P


4/8

r0- 2 4 6 S 10 12 14

M g++ mM)g . 2 . E f fec t o f M g 2+ c o n c e n t r a t i o n o n ( N a + + K + ) - A T P -e a c ti v it y o f r a in b o w t r o u t a d a p t e d t o F W ( O - - ) a n d A T P aV ( O - - Q ) . T h e r e a c t io n m e d i a c o n t a i n e d th e M g C I 2 a n d lqn c e n t r a t i o n s r e p o r t e d p l u s H E P E S , N a C1 , K C 1 a s d e t a i l e d c o n c er F W a n d B W in l e g en d t o F i g . 1. F o u r m M A T P i n F W a s d e td 3 m M A T P i n B W w a s a d d e d a s T r is s a lt . T h e p Ht s a d j u s t e d t o 7 .0 i n F W a n d 7 .5 i n B W b y a d d i n g T r i s ,a s o b t a i n i n g t h e f i n a l c o n c e n t r a t i o n s r e p o r t e d i n T a b l e 1 .

T hTable 2. Kinetic parameters of the (Na + + K+)-ATPase coop ~X,.. h K m CCF W B W F W B W f o r 1 ~

P 0 .6+0 .1 0 .6+0 .1 1 .9 i ty2+ 1.8+ 0.2 2.0_ +0. 1+ 9 .8 _ + 0 .9T h ,and h are calculated as described in Materials and Methods;values are expressed as mean _+ SE from two determinations, ca ti o i~tistically ignifican tdifferencesbetween FW and BW are indicated N a +

100 r rt h e) r o a d p e a k r e s u lt s f r o m t h e m a i n t e n a n c e o f a t l e a st i n B~/% a c t iv i t y i n t h e r a n g e 5 - 1 5 m M M g 2+ w i t h a o b s e r

c a 9 5

0 .9 _ 0 .1 I n P W t o 3 .3 m B W .1 .2+0 .21 .5+0 .1 N a + a n d K d e p e n d e n c e1.2 + 0 .1 T h e e n z y m e w a s f o u n d t o b e d i fl

a n d K i n t h e t w oc a t i o n s t h e o p t i m a l c o n c e n t r a t i o n s

a n d 2 . 5 m M K , a re l o w e r t1 0 0 m M N a a n d 1 0 . 0 m M K (F i~

N a / K r a t i o c h a n g e s f r o m 2 0B W . A s a m a t t e r o f f ac t , t h o u g h

o b s e r v e d a t 1 0 m M K , th e B W e9 5 % a c t i v a t e d a t 5 .0 m M K ( F i

L xim al a c t i v it y i s a t t a i n e d g r e a t e r - t h a n - o p t i m a l c o n c e n t r a t i o n sVI i n B W . T h e a c t i v a t i o n a n y a p p r e c i a b l e e n z y m e i n h i b i t i o n .

i n F W a n d c o o p e r a ti v e A p p a r e n t K ~ v a lu e s f o r b o t h c~l ge d K ~ a n d n o i n h i b i t io n i n B W t h a n i n F W ( T a b l e 2 ). F

a c t i v a t i o n k i n e t i c s a r e , a g a i n , n o n - c (a n d c o o p e r a t i v e in B W .

20

.>_

g8 10 ~ 5 10n t r a t i o n o n N a + + K + ) -a t a d a p t e d t o F W - - ) F i g . 5 . E f f e ct o f K + c o n c e n t r a t io n o n Ni o n m e d i a c o n t a i n e d t h e a c t iv i ty o f r a i n b o w t r o u t a d a p t e d t o F ~e d p l u s H E P E S , N a C l , K C 1 ( O - - Q ) . T h e r e a ct i o n m e d i a c o n t a in e c

Na * 21.9 _+ 1.2K + 0.4 +0 .1 1.1 _+0.0K .Statist icall'by * (P < 0.01).

a b r o a d9 0 %m a x i m u m a t 1 0. 0 m M M g 2 + .

A s s h o w n i n F i g . 3, t h e m a x i ma t 4 . 0 m M i n F W a n d 3 . 0 m M i nk i n e t i c s a r e n o n - c o o p e r a t i v ei n B W ( T a b l e 2 ), w i t h u n c h a vb y e x c e s s s u b s t r a t e i n b o t h c a s e s .

20

10

F--

ATP mMIF i g . 3 . E f f e c t o f A T P c o n c e n t r a t i o nA T P a s e a c t iv i ty o f r a i n b o w t r o u ta n d B W ( O - - Q ) . T h e r e a ct io nT r i s - A T P c o n c e n t r a t io n s r e p o r t e d Ia s d e t a i l e d f o r F W a n d B W i n l e g e n d( 7. 5 m M i n F W a n d 1 0 m M i n B W ) w a ss a l t . T h e p H w a s a d j u s t e d a s d e t a i l

A. PAOUARAN] e t a l .

20,

._>

tot~3_I -

Fig. 4. Effect (A T P a s e a c t i v i t yBW o - - oc o n c e n t r a t i o n s r eas de ta i l ed fo r F~ad .

e c h a n g e) p e r a t i v i t y i rc o n s t a n c y a ]f o r M g 2 + . F i n a

2 . t + 0 .2. in F W 32.6 _ 0.3*2.4 + 0.4*2.0-t-0.1b y N a

O 15 2Na raM)

~ n t r at i on o n ( N a + + K + ) -u t ad a pt e d t o F W ( O - - O )m e d i a c o n t a i n e d t h e N a C 1~PES, MgATP, MgCI2 , KC1,~gend to F ig. 1 . Th e pH wasled in Fig. 2 .

t i o n k i n e t i c s f r o m n o n -) e r a ti v i ty in B W a n d t h ee d f o r A T P a r e a l s o v a li d/ A T P r a t io c h a n g e s f r o m

d i f f e r e n t l y a c t i v a t e dh a b i t a t s . F o r t h e s e

i n F W , 5 0 m Mt h a n t h a t i n B W ,

( F i g s 4 , 5 ) ; m o r e o v e r2 0 . 0 i n F W t o 1 0 . 0

1 0 0 % a c t i v i t y i se n z y m e i s a l r e a d y

( F i g . 5 ) . I n a l l c a s e sa t i o n s d o n o t r e s u l t i n

c a t i o n s a r e h i g h e rF u r t h e r m o r e , t h e

n o n - c o o p e r a t i v e in F W

1 5 2 0K* mM)

N a + + K + ) - A T P a s eF W O - - O ) o r B Wo n t a i n e d t h e K C 1 c o n c e n -N a C 1, M g A T P , M g C 1 2 a se n d t o F i g . I . T h e p H w a sed in F ig . 2 .


5/8

- ~ u - 9 - 8 - 7 - 6 - 5 - 4 - 30uabain logg. 6. Effect o f ouabain on rainbow trout Na + +K+) -[ 'Pase. The ouab ain concentrat ions repor ted were adde dthe r eac tion media de tai led in Table 1 f or FW ( O - - O )d BW (O--Q) . The BW enzyme sensi t ivi ty was fur therted in a modif ied BW medium containing 2.5 mM KCI~tead of 10.0 mM KC1 ( 1 ~ 1 ) . Each poin t i s the aver age

from three determinations.Tect o f ouabainA t o d d s w i t h t h e ( N a + + K + ) - A T P a s e d e p e n d e n c ep H , A T P a n d c a t i o n s , t h e e n z y m e s e n s i ti v i ty t ot a b a i n i s s u b s t a n t i a l l y s i m i l a r i n t h e t w o h a b i t a t s a s R e sn lum o n s t r a t e d b y t h e c o m m o n / 5 0 ( 5 1 0 - 7 M ) s h o w n O t he rs

t h e t w o a c t i v i t y c u r v e s ( F i g . 6) . T h e a p p a r e n t l yN e t s e n s i t i v i t y o f t h e B W e n z y m e i n t h e r a n g e s ta ti st i5 1 0 - 7 M o u a b a i n s h o w n b y t h e u p p e r p r o f il e o fc u r v e a s w e l l a s t h e h i g h e r 1 0 0 % i n h i b i t o r yn c e n t r a t i o n w i t h r e s p e c t t o F W ( 5 x 1 0 - 3 M v s t h a t l< 1 0 - 4 M ) a r e p r o b a b l y r e l a t e d to t h e h i g h e r K t h a t in c e n t r a t i o n i n t h e a s s a y m e d i u m c o r r e s p o n d i n g t o a n d (h i g h e r K o p t i m u m i n B W w i t h r e s p e c t t o F W . a c t i v ia m a t t e r o f f a c t , i f t h e B W e n z y m e s e n s i t i v i ty i s b as ol ~t e d i n a 2 .5 m M K - c o n t a i n i n g m e d i u m a s i n F W , t h a t il, 5 0 % a n d 1 0 0 % i n h i b i t i o n o c c u r a t 2 x 1 0 - 7 M

1 7 : 1 , 1 8 : 0 i s o , 2 2 : 1 a n d o t h e r m i n o r u n lS F A : s a t u r a t e d f a t t y a c i d s ; M U F A :S t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s b e t w e e n F Wb y * ( P < 0 . 0 1 ) a n d ~ ( P < 0 . 0 5 ) .

t h e e n z y m e a c t i v i t y i n B W , a ,,i n F W , i s a t l e a s t 4 - f o l d g r e a td o u b l e i n f r a c t i o n P 4 . T h e da c t i v i t y r a t i o i n t h e t w o f r a c t i o n s i sb a s o l a t e r a l m e m b r a n e c o n t e n t i ni m p l i e s a l o w e r e n r i c h m e n t i n Af r a c t io n P 4 w i t h r e s p e c t to F W .) c t iv e l y , n a m e l y a t e v e nn s t h a n i n F W . Gill lipid composition

T h e c o n t e n t o f t o t a l l i p id s (T L ) a l( P L ) o f g i ll t i s s u e is s h o w n i n T a b le a c t i v i t y i n B W i s e x p r e s s e d a s p e r c e n t a g e s o f w e t w tt a t i n F W . T h e m a x i m a l B W t h a n i n F W ; a s t h e P L / T L r a t i o (r e s p e ct i v e o p t i m a l a s s a y a n y v a r ia t i o n , t h e T L d e c r e a s e e v e n,'s en t e x p e r i m e n t ( 4 m M c h a n g e o f p h o s p h o r u s - f r e e l i p i d s .[ N a + , 2 . 5 r a M K + , p H G i l l f a t t y a c i d c o m p o s i t i o n a n d re10 m M M g ~+ , 1 0 0 m M a r e r e p o r t e d i n T a b l e 4 . B W t r o u t sint B W ) , a n d e x p re s s e d a s t o F W t r o u t , a h ig h e r c o n t e n t o f t h, i s 3 .8 i n c r u d e m i c r o - a c i d s 1 6 : 0 a n d 1 8 : 0 a n d o f t h e ul1 5 .1 i n p u r i f i e d b a s o - 1 8 : 2 n - 6 a n d 1 8 : 3 n - 3 a n d l o w e r p e r (: W , a n d 1 8.6 a n d 2 9. 0, c h a i n p o l y u n s a t u r a t e d f a t t y a c i d s (Lge ( N a + + K + ) - A T P a s e 2 0 : 4 n - 6 , 2 0 : 5 n - 3 a n d 2 2 : 4 n - 6 . S u (W a n d 1 3 .9 + 1 .5 i n B W n o t i m p l y s i g n i f i c a n t d if f e re n c e s b[ a n d 2 2 .4 _+ 1 .0 r e s p e c t - h a b i t a t s i n t h e u n s a t u r a t i o n p a r a mt f r o m t h e v a r i o u s t e s t s n 3 / n 6 r a t i o .t a b l e 1 , F i g s 1 - 6 ) , s h o w

D I S C U S S I O Np h o s p h o l i p i d c o n t e n tP h o s p h o l i p i d s T h e d i ff e re n c e s i n t h e p r e p a r a t i v e

( % o f t o ta l l ip ids) in the m eth od s o f ( N a + + K +) - ,43.61 + 0 . 9 0 e v a l u a t i o n i n l i t e r a t u r e m a k e i t d i ff i43.53 _+ 0.80 the spec. act . valu es pre sen ted here wi

IUW~ I0 - 5t h ec o n c e n t r a t i o5 conc~t h eA st e s t e d i nt h ea n d 1 x l O - 4 M o u a b a i n r e s p e c ti v e l yl o w e r i n h i b i t o r c o n c e n t r a t i o n sSpecif ic act ivi ty

T h e ( N a + K * ) - A T P a s ec o n s t a n t l y f a r h i g h e r t h a n t h a ts p e c . a c t . o b t a i n e d u n d e r t h e r e sc o n d i t i o n s f o u n d i n t h e p r e s e n tA T P , 7 . 5 r a M M g 2 + , 5 0 m M7 .0 in F W a n d 3 m M A T P ,N a , 1 0 m M K , p H 7 .5 i n# m o l s P i m g p r o t e i n -1 h r - Js o m a l p r e p a r a t i o n s ( P 3 ) a n dl a t e r a l m e m b r a n e s ( P 4 ) i n F W ,r e s p e c ti v e l y i n B W . T h e a v e r aa c t i v i t y v a l u e s , 2 . 9 + _ 0 . 5 i n F Wi n f r a c ti o n P 3 a n d 1 1.5 + 0 . 4i v e ly i n f r a c t i o n P 4 o b t a i n e dc a r r i e d o u t o n t h i s f r a c t i o n (T a b l e 3 . G i l l t o t a l l i p i d a n d

T o t a l l i p i d s( % o f w e t w t ) ( % o f w e t w t )F W 3 . 0 0 0 . 0 3 1 .3 1 0 . 0 3B W 2 . 1 0 + 0 . 0 5 * 0 . 9 1 0 . 0 2 *V a l u e s e x p r e s se d a s m e a n S E f r o m t w o d e t e rS t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s b e t w e e n F W a

b y * ( P < 0 . 0 1 ) .

ou t gill (Na + + K +)-ATPase salinity-delx.o Tab l 4 . F l

F a t t y a c i d1 4 : 01 5 : 0

o 1 6 : 0v 16 :1~ 0 ~ 18:0. . - . 1 8 : 11 8 : 2 n - 6

1 8 : 3 n - 61 8 : 3 n - 32 0 : 12 0 : 2

l o o 2 0 : 3 n - 62 0 : 4 n - 62 0 : a m - 32 0 : 5 n - 32 2 : 4 n - 62 2 : 5 n - 62 2 : 5 n - 32 2 : 6 n - 3O t h e r s~ S F AZ M U F AZ n - 6Z n - 3n 3 / n 6

I x 1 0 0 / % S F AResu l t s a re exp res s~O t h e r s a r e t h e s u m (

,ely,

~t ion o f g i l l t o t a l l i p id sBW

2 . 4 + 0 . 10 . 2 0 . 020.0 0 .3 :5 . 0 0 .7.5 -, I-0.1 *

19.9 .4- O .O t4 . 3 + 0 . 1 70 . 2 0 . 10 . 9 + 0 . 1 72.1 0.10.3+0.10 . 6 + 0 . 13 . 4 + 0 . 00 . 4 0.05.1 0.2*0.3 0.0'0.4 0.01.30.123.2 0.42.5 o.o3 0 . 1 0 4 ?

2 7 . 0 0 . 0 79 . 3 + 0 . 23 0 . 9 0 . 2 *3 . 3 + 0 . 42 . 3 + 0 . I8 . 6 0 . 3

f r o m t w o d e t e r mi n a t i on s .~ t t ty c i d s : 1 2 : 0 , 1 6 : 0 i s o , 1 7 : 0 ,

u n k n o w n c o m p o n e n t s .m o n o u n s a t u r a t e d f a t t y a c id s .

F W a n d B W a r e i n di c a t e d

a s c o m p a r e d w i t hr e a t e r i n f r a c t i o n P 3

d if fe re nt B W / F Wi s d u e t o t h e h i g h e rB W m i c r o s o m e s

.~n t n A T P a s e a c ti v i ty n

( T L ) a n d p h o s p h o l i pi d sT a b l e 3. T L a n d P L

a r e 30 / 0 o w e r i nd o e s n o t u n d e r g o~e even r e f lec t s a pa r a l le l

r e l a t e d p a r a m e t e r ss h o w , w i t h r e s p e c tt h e s a t u r a t e d f a t t yu n s a t u r a t e d 1 8 : 1 ,p e r c e n t a g e s o f l o n g -( P U F A ) s u c h a s

~ h v a r i a t io n s d ob e t w e e n t h e t w o

) a r a m e tc r s a n d i n t h e

p r o c e d u r e s a n d) - A T P a s e a c t i v i t yf ic ul t t o c o m p a r evi th tha t e l sew her es e d o n g i l l h o m o g e n a t e , o n p u r i f i e d f r a c t i o n sv i t h o u t s e a r c h f o r t h e


6/8

T h e h i g h e r ( N a + K + ) - A T P a s e a c t iv i t y i n B W i nf lu et h r es p e c t t o F W i s i n p e r f e c t a g r e e m e n t w i t h a n d~ r a t u r e d a t a ( D e R e n z i s a n d B o r n a n c i n , 1 9 84 ; i n s aL n gd o n a n d T h o r p e , 1 9 8 4 ). A s s t a t e d b y t h e P r o s s: r a t u r e , t h e s a l t - w a t e r e n z y m e a c t i v i t y i n c r e a s e o p t i r r: le c ts t h e h i g h e r ( N a + + K + ) - A T P a s e c o n t e n t in r e s p ~1 e p i t h e l i u m d u e t o s a l i n i t y - p r o m o t e d i n c r e as e o f h i g h (c h l o r i d e c e ll n u m b e r , c o m p l e x i t y o f b a s o l a t e r a l o f c o, ' m b r a n e s a n d d e n s i t y o f e n z y m e u n i t s ( E d d y , 1 98 2 ; o f t haLurent , 1984; Payan e t a l . 1 9 84 ). A m o r p h o l o g i c a l t h e eT e r e n ti a ti o n b e t w e e n F W a n d B W t r o u t c o n s i s t e n t h y p e rt h l i t e r a t u r e w a s p o i n t e d o u t b y e l e c t r o n m i c r o s - s ub st ~p y e v e n i n t h e p r e s e n t e x p e r i m e n t ( u n p u b l i s h e d z y m et a ) . P h y s i o l o g i c a l l y , t h e h i g h e r a c ti v i t y o f (N a + a r o u r) - A T P a s e i n B W m a y b e r e l a t e d t o t h e f u n c t i o n i n B $t h e N a p u m p i n N a C 1 e x t r u s i o n f r o m t h e c e ll i n o b s e rL t- w at er , w h e r e i t m i g h t p l a y a m o r e r e m a r k a b l e i nc re ~l e a c c o r d i n g t o t h e m o d e l o f S i l v a e t a l . (197 7), in th~i n i n t h e a ct i v e N a u p t a k e i n f r e s h w a t e r b y a r o u respeciA p a r t f r o m t h e d i f f e r e n t e n z y m e s p e c . a c t . , t h eo h a b i t a t s e v e n i m p l y a d i f f e re n t e n z y m e r e s p o n s e O nv a r i o u s e f f e c t o r s, t h a t i n m o s t c a s e s , i s a p p a r e n t l y i n B Ye r ab l e t o t h e d i f f e r e n t e n v i r o n m e n t a l r e q u i r e - t o th ~; n ts . T h e d i f f e r e n t p H d e p e n d e n c e c u r v e s o f t h e e x e r tcz y m e , a s w e l l a s t h e d i f f er e n t F W a n d B W p H a s s a y

o b s e r v e d k i n e t i c c h a n g e s i n B We a s e d m o d u l a b i l i t y o f t h e ( N a t h e p r e s e n c e o f N a c o n c e n t r aa r o u n d h i g h e r v a l u e s . T h i s a s p e~ p e c i a l l y n o t e w o r t h y i n t h e m a i nt h e c o n t r a r y , t h e l o w e r s u s c e pB W w i t h r e s p e c t t o F W i s o n l y at h e k n o w n a n t a g o n i s m t o w a r d se x e r t e d b y K ( F o r b u s h I I I , 1 9 82' m e d i u m a t h i g h e r o p t i m a l c o n~~t c o m m o n l y u s e d t e l e o s t B e s id e s t h e i n t e r n a l c o m p a r i s o n IR e n z is a n d B o r n a n c i n , B W t r o u t , w e h a v e c o m p a r e d t h e p) e , 1 98 4; M a d s e n a n d M g 2 , A T P , K a n d N a o p t i m a vn i c k e t a l . 1 98 9) t h a t r e p o r t e d c o n c e n t r a t i o n v a l u e s o f t: d a s o p t i m a l i n o t h e r t e le o s t g i ll ( N a + K ) - A T P a s e a s s ai le r a n d K i r s c h n e r , 1 97 2; t h e o p t i m a l c o n c e n t r a t i o n s o b t a i n ea y b e t e n t a t i v e l y r e l a t e d c h a r a c t e r i z a t i o n i n t e l e o s t e a n o s m o r~ o r d in g l y, t h e h i g h e r p H p a y i n g a t t e n t i o n t o o u r p r e v i o u s) - A T P a s e a n d t h e m o r e s u r v e y p o i n t s o u t A T P a n d M g 2~v i ty p r o f i le i n B W w i t h g e n e r a l ly i n t h e r a n g e 3 - 5 m M w i tla c e r t a i n e n z y m e t o l e r - r a t i o o f 1.0 ( L a n g d o n a n d T h o r p e , 1(~t o p H c h a n g e s i n t h e 1 9 85 ; F e n w i c k a .n d L a m , 1 9 8 8 ; V e n tJ' , t h u s g u a r a n t e e i n g t h e s e l d o m b e t w e e n 1 .0 a n d 2 . 0 ( P f e i lee p i t h e l i u m d i r e c t l y i n 1 9 72 ; P a g l i a r a n i e t a l . 1988; Mads~v i r o n m e n t , s e n , 1 9 89 ), a n d o c c a s i o n a l l y l o w e rd i f fe r e n ce s b e t w e e n F W e t a l . 1 9 7 9 ) o r g r e a t e r t h a n 2 . 0 ( Mr a c t i o n k i n e t i c s o f t h e 1 9 89 ). N a a n d K o s c i l l a t e a r o ug A T P a n d th e a c t i v a t i n g 2 0 - 2 5 m M , r e s p e c t i v e ly , w i t h N a /m o s t s t r i k in g d i s p a r i t y o r 5 .0 ( D e R e n z i s a n d B o r n a n c i n , 19i n t h e a c t i v a t i o n k i n e t i c s 1 9 85 ; P a g l i a r a n i e t a l . 1988) . On th~ + t h a t a r e M i c h a e l i a n d a t a o n t h e o p t i m a l c o n c e n t r a ti o n s

W . W h i l e i n t h e c a s e o f h a b i t a t s a n d o f N a i n B W a r ec c h a n g e i s a c c o m p a n i e d l i t e r a t u r e , a n d e v e n t h e a f f i n i t y co~p o i n t s b e t w e e n F W a n d a n d c a t i o n s d o n o t s u b s t a n t i a l ly_ 1 I k K _ ~ I - iAe . l r ~ l r~ _ _ ~ , _ _ J i . . . . . . . . . . . . ~ . . . . . Z l - - L I - / h - - 1 I i . . . _ ~_

~ I L - W ~ , L ~ Ir o l et h a nh y p e r r e g u l a t o r s .At w otor e f e r a b l em e n t s .e n zo p t i m a , b o t h l y i n g in t h e m o s t c o m m (g i ll ( N a + K ) - A T P a s e p Ha n d S a u n d e r s , 1 9 8 1 ; D e R e n z i s1 9 8 4 ; L a n g d o n a n d T h o r p e ,N a a m a n s e n , 1 98 9; M c C o r m i c ki n c l u d e s p H v a l u e s r e p o r t e dc h a r a c t e r i z a t i o n s t u d i es ( P f e il e rF e n w i c k a n d L a m , 1 98 8) , m at o a f u n c t i o n a l m e a n i n g . A c c o r d i no p t i m u m o f t h e ( N a + K a l k a l i n e - s h i ft e d e n z y m e a c t i v i tr e sp e c t t o F W m a y i n d i ca t ea n c e t o a l k a l i n e p H s a n da l k a l i n e f i e l d t y p i c a l o f B Wa c t i v i t y h o m e o s t a s i s i n a nc o n t a c t w i t h t h e e x t e r n a l e n w r o n m eO n l y p a r t o f t h e o b s e r v e d d i ff e r en ta n d B W t r o u t i n t h e i n t e r a c t i o ne n z y m e w i t h t h e s u b s t r a t e Mc a t i o n s a r e n o t e w o r t h y . T h eb e t w e e n t h e t w o h a b i t a t s l i e s i n t h e a cb y A T P , M g 2 , N a + a n d K i n F W a n d c o o p e r a t iv e in B W .M g 2 a n d A T P s u c h a k i n e t i cb y o n l y p o o r d i f f e r e n t i a t i n gB W ( n a m e l y a h i g h er o p t i m a l M g 2 a g r e a t e r s u s c e p t i v i t y t o e x c e s s M gr e s p e c t t o F W ) , f o r N a a n d K

A PAGLIARANI t a l .t i o n s. A n y w a y , o u r w i t h v a r i a t io n sv a l u e s i n F W a n d a n d K m v a l u e s ie l o w e r e d g e o f t h e d i f f e r e n t s u sc ee i t h e r i n e u r y h a l i n e a d a p t i v e si g n if io v a r i o u s s a l i n i ti e s , c o o p e r a t i v e a c tl g h l y s i m i l a r t o t h e o t h e r c h a r a c t e ra c h l o r i d e c e l l p r e p - e u r y h a l i n e t e le ,, 1981; J i i rss e t a l . e t a l . 1988; Tn c i n , 1 9 84 ; M a d s e n s o m e h o w l i n k ea l t - w a t e r , h o w e v e r , o s m o l a r i t y a sn t r i b u t i o n t o b a s o - o f s a l t- w a t e rb o d y f l u i d s ( E 'Luenced, eve~g e n e r a l l ys a l t - w a t e r 1)sser, 1986).~ t imal ac t ivi t3~ ec t t o F Wc a t i o n i c c oc o o p e r a t i v et h e a f f i n i t y qe n v i r o n m e r9 e r b o l i c t os u b s t r a t e c o n c es us cep t ixa r o u n d t h e K m (B W t h e K m

h o m e o s t a s i s .

n m o n l ' t h a n i n F W .r a n g e ( J o h n s t o n

m e n t .

o p t i m a l c o n c e n t r a t i o n sF W , th a t m a y m a k e t h et a n d K + o f p o s s i b l ei n c r e a s e d s a l i n i t y . T h ea + , a l r e a d y o b s e r v e d i ne s o n s e a - w a t e r - a d a p t e de t a L 1 9 8 5 ; P a g l i a r a n id . , 1 9 9 0 ) a p p e a r s t o b ea r i n e e n v i r o n m e n t . T h et n d N a + c o n c e n t r a t i o n sb a n t h a t o f t e le o s t e a nT h e s e o n e s i n t u r n a r eb y t h e e x t e r n a l m i l i e ua e r s a l t c o n c e n t r a t i o n sh wa te r (E vans , 1979 ;i f t h e s h i f t o f t h e B W) n c e n t r a t i o n v a l u e s w i thi g h e n z y m e a c t i v i t i e s t oi n p a r a l l e l w i t h t h e o n s e ti n e t i c s a n d t h e i n c r e a s ey b e m o r e a d e q u a t e f o re n t s . T h e c h a n g e f r o ma a p e i n t h e a c t i v i t y v sv e i m p l i e s a h i g h e r e n -c o n c e n t r a t i o n c h a n g e s1 9 77 ). I f w e c o n s i d e r t h a th i g h e r t h a n i n F W , t h em a y i n d i c a t e a nN a + K ) - A T P a s en c e n t r a t i o n f l u c t u a t i o n s9ec t s eems to bem a i n t e n a n c e o f N a

: p t iv i t y t o o u a b a i na p p a r e n t a n d d u eo u a b a i n b i n d i n gi ) p r e s e n t i n t h ec o n c e n t r a ti o n i n B Wb e t w e e n F W a n d~ res en t re s u l t s onw i t h t h e c u r r e n t l yt h e s e e f f e c t o r s i na y m e d i a a n d w i t ho b t a i n e d i n t h e e n z y m eo s m o r e g u l a t o r y t is s u es ,s t u d i e s . S u c h a+ c o n c e n t r a t i o n sw i t h a n M g 2 + / A T P1984; T r iga r i e t a l .V e n t r e l l a e t a l . 1990),P f e i l e r a n d K i r s c h n e r ,M a d s e n a n d N a a m a n -t h a n 1 .0 ( G a l l i sM c C o r m i c k e t a l .a r o u nc F 1 00 m M a n dr K r a t i o s o f 4 . 01984 ; T r iga r i e t a l .t h e s e b a s e s p r e s e n to f A T P i n b o t h

c o n s i s t e n t w i t hc o n s t a n t s f o r A T Pd i f f e r f r o m t h eR e n z i s a n d B o r n a n c i n ,e n w i c k a n d L a m , 1 9 8 8;


7/8

( N a + K ) - A T P a s e p ro p e r t i e s c o u l d b e m o d u - E d dye d b y m e m b r a n e l i p id s ( D e u t i c k e a n d H a e s t , 19 87 ) fi shP h yt t h e d i f f e r e n t e n v i r o n m e n t a l s a l i n i t y ( L e r a y e t a l . , E v a n s8 4 ) a p p a r e n t l y r e m o t e i n t h e p r e s e n t e x p e r i m e n t Osne n i f s o m e c h a n g e s o f s i n gl e f a t t y a c i d s a r e V o lt e c t a b le . F i n a l l y , t h e f a c t t h a t o u r d a t a a r e b a s e d F e nw ia n a l y s e s c a r r i e d o u t o n t h e w h o l e t i ss u e a n d n o t a ir ~

b a s o l a te r a l m e m b r a n e s b e c a u se o f t h e p a u c i ty o f O x ;s u e a v a i l a b l e c o u l d n o t b e n e g l e c te d . T h e o b s e r v e d F is~c r e a s e i n B W o f th e l i p id s u n d e r s t u d y m a y b el e a s t p a r t i a l l y r e l a t e d t o s a l i n i t y - p r o m o t e d g i ll d ote" u ct u ra l c h a n g e s . N e w c h l o r i d e c el l s t y p i c a l o f t h e F o l chb i t a t m a y e x h i b i t a l o w l i p i d / p r o t e i n r a t i o , m e tT h e d if f e r e n t ( N a + K ) - A T P a s e f e a tu r e s i n t h e a niro h a b i t a ts , t h o u g h d i f f e r e n tl y r e m a r k a b l e f o r th er i o u s e f f e c to r s , m a y b e r e l a t e d t o s a l i n i t y - d r iv e n N a l1 m o r p h o l o g i c a l c h a n g e s a n d c o n s i d e r e d t o b e r e - poreo n s i v e t o t h e d i f f e r e n t e n v i r o n m e n t a l r e q u i r e m e n t s . P P.l z y m e s t r u c t u r e s a t l e a s t p a r t i a l l y d i f f e r e n t m a y b e G a l li sv o l v ed i n t h e i n w a r d a n d o u t w a r d t r a n sp o r t o f a daEffe~ C1 . T h e b i o s y n t h e s i s o f n e w e n z y m e u n i t s m a t c h e d D ct h e p r o l i f e r a t i o n o f c h l o r i d e c e ll s i n s a l in e m e d i a i na u r e n t , 1 9 84 ; P a y a n e t a l . , 1 9 84 ) m a y r e s u l t i n t h e G o r mo d u c t i o n o f n e w ( N a + K ) - A T P a s e c o m p l e x e s D o t

t h k i n e t i c f e a t u r e s m o r e s u i t a b l e f o r h y p o r e g u l a t i o n , r ea cl e v a r i o u s ( N a + K + ) - A T P a s e i s o f o r m s , re c e n t l y K in se :

111 [IYO J) L, i I UlOl.OIl lUN a K - A T P a s e . I n Current Topics in M~~ort (E d i t e d b y B ro n n e r F . a n d K le in167-201 . Acade mic Press , New YJ . L . , Lasse rre P . and Be l loc F .adap ta t ion in the euryha l ine te leos tEffec t o f adap ta t ion , p ro lac t in , cor t is~o n p la s m a o s m o t i c b al a n c e a n d (Ngi l l and k idney . Ge n. comp. EndocG o rn a l l G . , B a rd a w i l l C . J . a n d D a ,D e te rm in a t io n o f s e ru m p ro te in s b y rreac t ion . J . biol. Chem. 177, 751-757KinseUa J . L . , Holohan P . D. , Pessah Nd i f f e r i n th e a m in o a c id (19 7 9) I s o la t io n o f l u m in a l a n d a n t i la n d in th e s e n s i t i v i ty to f ro m d o g k id n e y c o r te x . Biochim. iI S w e a d n e r , 1 9 8 9 ) . T h e r e - E . and Saunders R. L .t -w a te r c h lo r id e c e l l s o n t r a n s fo rm a t io n o f y e a r lin g a t l a n t ic s a;n t , 1984) co n t a i n d if f e r- a t several rea r ing tempera tu res . Can.a f o r m s f r o m t h e e n z y m elo c a te d o n th e s e c o n d a ry J fi rs s K . , B i t to r f T h . an d V rk le r T h . Isa l in i ty and food depriva t ion on g ira t io and ce r ta in enzyme ac t iv i t ie srs wish to thank Professor Salmo gairdneri Richardson) . C o m pt i p th is w o rk c o u ld n o t h a v ei is g ra te fu l ly acknow ledged J f irs s K. , Bi t to rf Th . , Vrk le r Th . andof rea r ing and sampling In f luence o f nu tr i t ion on b iochemica l sD r T ib a ld i fo r h i s v a lu a b le o f t h e r a in b o w t ro u t Salm o gairdneri Int. This s tudy was supported Biochem. Physiol. 75B, 713-717. Pub l ic Educa t ion . Lang don J . S . and Tho rpe J . E . (1984) INa +-K + AT Pase ac t iv i ty , succin ic dehand ch lor ide ce l ls to sa l twa te r ada lsa lmon, Salmo salar L. , pa rr and sm323-331.in M. a n d Ma y e r -G o s ta n N . L a u re n t P . (1 9 8 4 ) G i l l i n t e rn a l m o lod ium in flux in the ra inbow Physiology (E d i t e d b y H o a r W. S . aaated to artific ial freshw ater Vol. X, Par t A, pp. 73-183. Acad emitb l l , 159-169 . Leray C. , Chape l le S . , Du port a i l G. andR . M. a n d T ru c c o R . E . C h a n g e s in f lu id i ty a n d 2 2 :6 (n-3) c~y ac ids and regu la t ion o f l ip ids o f t rou t in tes tina l b rush-bor31oste ric behav io ur o f e ry th - re la ted to env iron men ta l sa lin ity . Bio

g i l ls p o n s l v eE n z ,i n v o l v e dN a C 1 . T h e b i o st o( L a u r e n t ,p r o d u c t i o nw i t hT h ed e s c r i b e d i n s e v e r a l t i s s u e ss e q u e n ce o f t h e ~ - s u b u n i t a n do u a b a i n a n d p h o s p h o l i p i d s (f o r e th e h y p o t h e s i s t h a t s a l t - w a t e rt h e p r i m a r y l a m e l l a e ( L a u r e n t ,e n t ( N a + + K + ) - A T P a s e i scf o r m s o p e r a t i n g i n F W a n d l o c a t e d cl a m e l l a e s h o u l d n o t b e d i s c a r d e d .A c k n o w le d g e m e n t s - -T h e a u th o r sA. R. Borga t t i wi thou t whose he lbeen done . Professor D. Lanari i sfo r t rou t ava i lab i l i ty and usefacilities . Thanks are also due tocooperation during the experiment.b y g ra n t s f ro m th e Min i s t ry o f

REFERENCESA v e l l a M. , Ma s o n i A . , B o rn a n c in(19 8 7) G i l l m o rp h o lo g y a n d s o d iu m int ro u t Salm o gairdneri) acc l imatedenvironments . J. exp. Zool. 241.B lo j B . , Mo re ro R . D . , F a r i a s(1973) Membrane l ip id fa t tymem brane- boun d enzym es . Al los te r ic 1:ro c i t e Mg 2+ -A T P a s e , (N a + + K ) -Achol ines te rase f rom ra ts fed d iffe ren tdiets. Biochim. biophys. Acta 311 , 67-

au t g i l l (Na + + K +)-AT Pase sa lin ity -dopeB W M g 2 + o p t i m a C a p la n M . J ., A nu n c o m m o n l y h i g h , J . D . ( 19 88 ) S oth a t i n s p i t e o f t h e in p o la r i z ed e l,~ e x c e p t io n a l ly lo w (E d i t e d b y S k c

V t h e K + o p t i m u m a n d E s m a n n lVY o rk .a t p r e v i o u s l y f o u n d D e R e n z is G . a ntn e t e l e o s t s s e a b a s s g i ll A T P a s e s . Id ( V e n t r e l l a e t a l . , a n d R a n d a l l D .d e s w i t h o u r p r e v i - P re ss , N e w Y o( T r o m b e t t i e t a l . , Deuticke B. ando n c o n s t a n c y o f g i l l o f t r a ns p o r t 1t h e p o s s i b i l i t y t h a t A. Roy. PhysioF. B. (1982fish with partie r~siol. 73B, 1D . H . ( l~Osmoregulatio~Vol. 1, pp. 305F e n w ic k J. C. ante x p o s u re o n~eleotris m~

Fish Physiol.F is k e C . a n dd e te rm in a t io n400.Fo lc h J . , Lees M.m e th o d fo r i s oLnimal tissue . ,F o rb u s h B . , I I I

468-477 .J o h n s to n C .

3 8 1 1 8 9 - 9 8 .~ d o n

83B, 425-433 .

Pa lade G . E . a n d J a m ie s o nsyn thes ized Na , K-ATPaseIn The N a + , K + P u m py J. G . M a u n s b a c h A . B .51-58 . Alan R. L is s , New[. (1984) Ion t ransport and7gy (E d i t e d b y H o a r W. S .r t B , pp . 65-104 . Academicq . (1987) L ip id modula t ion~rtebrate cell membranes.ion ic regu la t ion in cap t ivesalmonids. Comp. B iochem.

Comparative Physiology ofdi ted by Malo iy G. M. O. ) ,ic Press, L ond on.}88) Studies on the effect ofA T P a s e o f t h e m a rb le g o b y ,~ultative air-breathing fish.-130 .(1925) The co lor imetr ic~. J. biol. Chem. 66, 3 7 5 -~nley G. H. (1957) A simplef ica t ion o f to ta l l ip ids f rom226, 497-509.o ton ic s te ro id b ind ing toics in Me mbra ne an d Trans-nd K le inze l le r A. ) , Vol . 19 ,Y o rk .(1979) Freshwate rChelon labrosus. I.cort iso l and ac t inomycin(N a + + K + ) -A T P a s eEndocr. 38, 1-10.David M. M. (1949)m e a n s o f t h e b iu re t751-757.~essah N. I . and Ross C . R.a n t i lu m in a l m e m b ra n e sbiophys. Acta 552,

(1981) Parr-smolts a lm o n Salm o salar )Can. J . Fish. Aqua tic Sci.(1986) In f luence o fr o w t h , R N A / D N Ain ra in b o w t ro u t~. Biochem. Physiol.

Wacke R. (1983)lemica l seawate r adap ta t ion~irdneri Richardson) . Comp.Response o f the g i l lc inic dehydrogenase ac t iv i ty~tation in Atlanticand smolt . J . Fish Biol. 24,

m o rp h o lo g y . In Fisha n d R a n d a l l D . J . ) ,kcademic Press , New York.F lore n tz A. (1984)c o n te n t i n p h o s p h o -ru s h -b o rd e r m e m b ra n e a sBiochim. biophys. ActaE. T . (1989) P lasma ion ic+-A T P a s e c h a n g e s d u r in g


8/8

IV I~IIK; Ih e a d b re a m Sparus auratus L.). Com~

72A, 637-642. Trig aig i ia ran i A. , P ir in i M. , Tr iga r i G. and Ventre l la V. (1986) (19Effec t o f d ie ts con ta in ing d iffe ren t o i ls on b ra in fa t ty theac id compos i t ion in sea bass Dicentrarchus labrax L.). Ph.Comp. Biochem. Physiol. 83B, 277-282. Tronc~gl ia ran i A. , Ventre l la V. , Trombet t i F . , Tr iga r i G. andB o rg a t t i A . R . (19 8 8) (N a + + K + ) - a n d N a + -s t imu la t e d a ntMg2+-dependen t ATPase ac t iv i t ie s in k idney of sea bassDicentrarchus labrax L.). Comp. Biochem. Physiol. 90B,41-52. Ventxtyan P . , Gir a rd J . P . and M ayer-G os tan N. (1984) BoBranch ia l ion mov emen ts in te leosts. The ro les o f re sp ir- ca ta to ry and ch lor ide ce i ls . In Fish Physiology (E d i t e d b y N aHo ar W . S . and R anda l l D . J . ), Vol . X, Pa r t B , pp . 39-64 .Acad emic Press, Lond on ., terson G. L. (1977) A simp lification of the prot ein assay antme th o d o f L o w ry et al., which is m ore generally applicable, stirAnalyt. Biochem. 83, 346-356. he~~ei le r E . and Kirschner L . B. (1972) S tud ies on g i l l AT Pase

A PAGLIARANIet al.a r l in g r a in b o w t ro u t , o f r a in b o w t lad seasonal variation. Acta 282, 301Prosse r C. L . (a n d M a c l n t y r e A . D . O r g a n i s m s , pa d N a + , K +- A T P a s e S c r img e o u r K . (parr--smolt transform- Reactions, pp.salar). Fish Physiol. Segel I. H . (1975

and Sons , Ne(1974) Env ironm enta l S i lva P . , So lom cs te m o f th e f r e s hw a te r O u a b a in inh is and b ranch ia l mic ro- ac t ive ch lor idethe rmal ly acc l imated Sweadner K. I~~p. Biochem. Physiol. A T P a s e . Biocigari G. , Bore(1985) Characthe sea bass (ysiol. 80B,T ro mb e t t i F . , VBorga t t i A. 1a n d N a + - A T ISparus aural343-354.Ventre l la V. , PaBorga t t i A Ication s in the. ,Na + -s t imula t t691-695.Ventre l la V. , Iand Borga t t is t imula ted ME

95B, 95-105.

airdneri). Biochim. biophys.ional Biology: Molecules toL i l e y a n d So n s N e w Y o r k .f istry and C ontrol of Enzym edemic Press , London .ics, pp . 365-375 . Joh n W ileyK. and Epstein F. H. (1977)N a K -A T P a s e : r e l a t io n s toexp. ZooL 199 , 419-426 .y m e s o f t h e ( N a + + K + ) -[cta 988, 185-220.gliarani A. and Ventrella V.~ il l (N a + K + ) -A T P a s e inlabrax L.). Comp. Biochem.tgliarani A., Trigari G. and:+ -d e p e n d e n t (N a + + K +) -dneys o f the g i l thead b ream9. Biochem. Physiol. 97B,d g a r i G . , T ro mb e t t i F . a n d-- l ike e ffec t o f monova len tea bass gill Mg 2+ -depen dent~mp. Biochem . Physiol. 88B,Pagl ia ran i A. , Tr iga r i G.; il l ( N a + + K + ) - a n d N a + -ATP ase ac t iv i t ie s in the g i l t -~p. Biochem . Physiol.

Documents

Paglariani Et Al 1991