8/2, 40120 Bologna, Italy (lel: O~l 24301~); ana "lStltUtO (

A~traet--1. The expected higher gill (Na + + K+)-ATPase

water (BW) with respect to fresh water (FW) is accompanied
the enzyme sensitivity to ouabaln is unaffected.
2. Maximal activation is attained under the optimal tend
Na +, 2.5raM K +, pH 7.0 in FW, and 3raM ATP, 10m/~
in BW.
3. The change of the enzyme activation kinetics by i g 2+
in FW to cooperativity in BW and other habitat-dependent
BW are hypothetically related to an adaptive significance t
4. Gill total lipids and phospholipids are 30% lower in E
some differences in gill total lipid fatty acid composition bet
the unsaturation parameters.
tubul',
Le NaC1 uptake from and the extrusion into the abun(
freshwater and marine teleosts, accou
mechanisms required t o
mpensate for salt osmotic loss and accumulation. Co:
be two processes occur via the gills and imply the isolat,
. . . . . . . . . . . . . . . . . . . . . .
tubular reticulum whose membran(
(Na + K )-ATPase units
abundance and features of salt-wa
account for the currently found higt
in marine teleosts.
Conversely, in freshwater fish, ra
isolated chloride cells, often cut off
)ase (Payan e t a l . , 1984). and containing poor mi t ochondri a
the role of the enzyme, K )-ATPase complexes are separ~
5 by its spec. act., has epithelial cells by unpermeable tight
ice. Most literature tends 1982; Payan e t a l . , 1984).
~ill (Na + + K +)-ATPase The salinity-dependent appearal
tter hyporegulators and thelium is a clear sympt om of the
marine morphological logical mechanisms involved in tele
;ration of chloride cells and sea-water. The model of the Na(
the gills in sea-water proposed by S
nd Thorpe, 1984). These and still fundamentally accepted (E(
a gill pri mary lamellae e t a l . , 1984) is focused on the chlorid(
it between two adjacent uphill C1- extrusion from the cell ((
indirectly activated by the (Na+ -~
)rding t o environmental matched t o the paracellular passive
the leaky junctions. Conversely, in
and mi t ochondri a rich mechanisms involved as well as tl
vhole gill epithelium are uptake by the gills are still undel
mutually separated by evidence indicates t hat salt absorpti(
y are connected by ion- secondary lamellae. The process, lin
t hat make the internal regulation and up t o now repeat(
external milieu. Chloride respiratory cells (Payan e t a l . ,
n an extremely developed recently attributed t o peculiar chic
out in the secondary lamellae (Ave
The role played in Na uptake by t
hydroxytoluene; ATPase . . . . . . is so far uncertain, as the
INTRODUCTION
Th
environment by
respectively, are active
com'
The
activity of (Na + + K +)-ATPase
However in the two habitats
at least as far as suggested
apparent l y a different relevance. Mos
t o demonst rat e t hat a high gill
activity is typical of salt-water
parallels the onset of gill
features, namely the proliferation
where the enzyme accumulates
Bornancin, 1984; Langdon and Thor
ones, typically localized on
in the inteflamellary segment
secondary lamellae, undergo
morphol ogi cal changes accordin
salinity.
I n salt-water fish large
chloride cells spanning the whole
clustered in bunches and
accessory cells t o which the
permeable leaky junctions
fluids communi cat e with the exterm
cell basolateral infoldings form an extr
A b b r e v i a t i o n s uaed--BHT, butylated
EDTA, ethylenediaminetctraacctic ac
hydroxyethyi-piperazine-N'-2-ethane-st
Tris (hydroxy-methyl)-aminomethane
pp. 229-236, 1991
~NDENCE OF THE PRO]
K +)-ATPase IN RAINBO~
ONCORHYNCHUS MYK1
ELLA, R. BALLESTRAZZI,* F. TROMBET
Sezione di Bioehimica Veterinaria, Unive
el: 051 243019); and *Istituto di Produzic
Universit~ di Udine, Italy
( R e c e i v e d 16 A p r i l 1991)
-ATPase activity in r
by some ch~
conditions of 4 t
10 mM Mg 2+, 10
;2+, ATP, Na ~
endent variations
to the differ
BW than in
between FW a
4ost
( D e R e n z i s a n d
q u a n t i t a t i v e a n d
OF GILL
T
and G, TRIGARI
la, Via Belmeloro,
~acoltfi di Agraria,
dapted to brackish
~yme kinetics while
n M M g 2 + , 5 0 m M
) r a M K + , p H 7.5
i s i m p l e saturation
) H alkaline shift in
ntal salinity.
r ratio is c o n s t a n t ;
significantly affect
~mbrane contains many
(Laurent, 1984). The
salt-water chloride cells
high enzyme activity
rare and generally
from the outside
and few (Na + +
)arated from other
junctions (Eddy,
marance of gill epi-
different physio-
cost gills in fresh
21 extrusion from
Silva e t a l . (1977)
(Eddy, 1982; Payan
chloride cell system. The
(cellular pat hway)
+ K + ) - A T P a s e is
)assive N a + e x i t t h r o u g h
f r e s h w a t e r , t h e
t h e s i t e o f N a C I
e 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 e
l 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 o
1 9 8 4 ) , h a s b e e n
a r i d c c e l l s f o u n d
A v c l l a e t al., 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 a l 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
" s a l i ni t y- modul a t e d gill ( Na + K +) - ATPa s e ma y possil
:ist ( Gal l i s et al . , 1979). Mor e ove r , me mb r a n e l i pi ds, in BY
bot h q
l own t o be s us cept i ve t o e n v i r o n me n t a l s al i ni t y modit
~eray et al . , 1984) a n d t o af f ect me mb r a n e - b o u n d P3 w~
a ns por t ATPa s e s ( De ut i c ke a n d Haes t , 1987), ma y
e n be i nvol ve d i n t he s al i ni t y- dr i ven mo d u l a t i o n 55,00(
' gi l l ( Na + + K +) - ATPa s e . pellet
On t hes e bas es we devi s ed a c h a r a c t e r i z a t i o n s t udy layere
I gill ( Na + K ) - ATPa s e i n t wo gr oups o f r ai n- 10 ml
~w t r out , c o mi n g f r o m t he s a me p o p u l a t i o n a n d spun
',pt i n wel l f r es h wa t e r or i n br a c ki s h wa t e r at ca The t~
the ur
', p p t sal i ni t y, in me,
yieldil
P4 w
MATERIALS AND METHODS
:perimental f i sh All
The acclimatization of rai nbow t rout (Oncorhynchus The p:
~kiss) to fresh water (FW) and brackish water (BW) was the bil
rformed as follows. Fresh water adapt ed rai nbow t rout (Gorn
tained from a t rout farm were randoml y divided i nt o two by PC
aups. The first group was reared in concrete t anks sup- tubes
ed with well fresh water. The second group was directly
msferred i nt o brackish water, namely to a canal connected
a l agoon nearby the Nor t h Adriatic sea and placed in net
ges. BW average salinity was 21 ppt and it ranged from 18 The
24 ppt. The wat er t emperat ure varied nat ural l y around tratioJ
~C in bot h eases. The pH was ca 7.8 in FW and ranged meth
steps were carried out under cold
~rotein concent rat i on of each fract k
bi uret met hod using bovine serum al l
Gornal l et aL, 1949) or by the met hod ot
Peterson (1977). All fractions we1
of 1.5 ml in liquid nitrogen until
(Na + K )-ATPase activity.
(Na + K )-A TPase activity
The enzyme activity was tested by me~
t rat i on of Pi released according to t h|
met hod of Fiske and Subbarow (1925)
d oxygen was always higher carried out at 30C as reported by Tr
BW. FW and BW groups Required amount s of fractions were inc
diet and were mai nt ai ned t i on mixture detailed in Tabl e 1 and the I
| di t i ons for a mont h from experiments showed, under our conditior
kt the end of the acclimatiz- ship between the P~ released and bot h the
sampled from bot h FW and i ncubat ed in the range 0.1-1.0 mg and t
480 g, were decapitated the up to 10 min, t hat was used in the experil
source. Immediately after was started by the addi t i on of ATP and sl
oved and divided i nt o three by t he addi t i on of 1 ml of ice-cold 15%
of each pool was stored in (w/w). The (Na + + K )-ATPase was
dyses, and t he rest, destined difference between the P~ liberated in the p:
tctions, was subjected to the of KCI in the assay medium. ATPase acti
nts were trimmed from gill as #mol P~ mg protein -~ hr -~. Each deter~
e-cold medi um A (0.25 M activity was carried out in triplicate. A1
,ris-HC1, pH 7. 4)accordi ng run twice unless differently stated. Th
and stored in small port i ons enzyme activation and inhibition promo
ATP was tested by a Hill plot. Appare
calculated by double reciprocal plots
solateral membrane fractions concent rat i on was raised to a power /~
nbetti et al., 1990; Ventrella coefficient (Segel, 1975). The linearity
liquid nitrogen does not witnessed by an absolute value of the coJ
['Pase with respect to the r never lower t han 0.980. The chara
ediately after thawing, t he (Na + + K +)-ATPase activity was carriec
lm A and homogenized by in bot h habitats.
; homogenat e (H) t hat was
lod of Ventrella et al. (1990) Li pi d analyses
t obt ai ned from an initial Immediately after thawing, the gills we
0 mi n was carefully resus- paper, separated from gill arches, weighe
Tile
myki ss )
performed
obt ai n
grout
plied with
t r an
to
cages.
to
10q
from 8.2 to 8.7 in BW. Dissolved
t han 7 ppm bot h in FW and
received the same commercial
under the above described conditions
mi d-January to mid-February. At the
at i on phase, 36 t rout , randoml y
BW groups and weighing 200-480 g,
same day and used as t he gill
deat h t he gills were quickly removed and
pools. Approximately one fourt h of q
liquid nitrogen until the lipid anal~
for preparat i on of subcellular fractions.
following procedure: the filaments
arches, repeatedly rinsed in ice-coh
sucrose, 5 mM EDTA, 16 mM Tris-HC1
to Mur phy and Houst on (1974) and storet
in liquid nitrogen.
Preparation o f subcellular and basolater
As previously described (Trombet t i
et a l . , 1990) tissue storage in
inactivate t he (Na + + K +)-ATPase
freshly prepared material. Immediatel
gills were resuspended in medium
Ul t rat urrax blender t o yield the
centrifuged according to the method of Ven
modified as follows. The pellet
cent ri fugat i on of H at 900g/ 10mi n
pended, rehomogenized and agai n centrit
speed, t hus yielding a new sediment (P1)
put together with t hat obt ai ned from
A. PAGLIARANI et al.
ase act i vi t y hi ghe r fugation, was f~
l t er was r epeat edl y centrifuged at 9(
t i es ( J o h n s t o n a n d namely the mito
3, 1986; De Renzi s supernat ant furtl
Na a ma n s e n , 1989), last supernat ant
,~ i nvol ve d i n Na + and recentrifugec
ing a final pellet
a Na + u p t a k e ma y a supernat ant v
Yeatures pos s i bl y re- previously remo'
n t sal i ni t y, ha s be e n (Na + K+) . A' [
ggest s t h a t i n F W a made it necessar
l er a n d Ki r s c hne r , discontinuous gr,
e f unc t i ona l f o r m arations enriched
aossible to comp
BW, the puril
bot h cases. The r
modified as descl
were diluted
EDTA, 16 mM
55,000 g for 60 r~
pellet (PY), resus
ered over a di
ml 31% (w/w)
at 90,000 g
fraction coll
tpper phase (
medi um A ant
yielding after ren
which repre~
fraction.
and divided i nt o
ored in sin,
a double gauze layer and
n. The pellet produced (P2),
tion, was removed and the
at 50,000g for 90 min. This
r he sediment was suspended
~eed for 60 min, thus obt ai n-
he microsomal fraction, and
~ther with the supernat ant
t ct i on S1. The low specific
bund in fraction P3 in FW
lrify it on a sucrose density
basolateral membrane prep-
:tivity. In order to make it
:ts of ATPase in FW to t hat
:tion P3 was carried out in
~ella et al. (1979) adequately
ts used. Aliquots of fraction
m B (0.1 M sucrose, 5 mM
7.4) and centrifuged at
mt ant was removed and the
1 amount s of medium B, was
crose gradient comprised of
(w/w) sucrose solutions and
a Beckman SW 25.1 rotor.
sucrose interface between
wer phase (LP), was diluted
t 100,000g for 60 min, thus
mpernat ant ($2), the pellet
fled basolateral membrane
conditions (0-4C).
' action was measured by
~rum al bumi n as a st andard
tethod of Lowry as modified
were stored in small
until evaluation of the
measuring the concen-
the principles of the
(1925). The reaction was
Trigari et al. (1985).
were i ncubat ed in the reac-
and the figures. Preliminary
:onditions, a linear relation-
the amount of protein
: and the i ncubat i on time
~eriments. The reaction
stopped after 10 rain
trichloracetic acid
determined as the
~resence and absence
activities are expressed
etermination of ATPase
All experiments were
The cooperativity of
promot ed by cations and
ppar ent Km values were
where the substrate
h, h being the Hill
of all plots was
correlation coefficient
characterization of the
ts carried out on fraction P4
ills were dried on bl ot t i ng
hed and subjected to
out in duplicate according
.nts cont ai ned 0.01% (w/v)
val uat i on of phosphol i pi d
19.2 5:3.0 19.9 + 4.2
rations are named as in Materials and Methods.
: spec. act. expressed as #mol Pi mg protein -~ hr-L
~: percentage of activity of the homogenate recovered in
A: relative spec. act., calculated as the ratio of SA of any fraction to that
rays of the (Na + + K+)-ATPase activity were carried out under the respec
17 mM Tris (pH 7.0), 4.0 mM MgATP, 3.5 mM MgCI 2, 50 mM NaCI, 2.5
3.0mM MgATP, 7.0mM MgCI 2, 100raM NaCI, 10.0raM KCI in BW.
lues given are means 5: SE from three determinations. Statistically sight
* (P < 0.01), or f (P <0.05).
nt ent of lipid extracts, the saponification of t ot al lipids, t hi s t
." eXtraction and met hyl at i on of fatty acids, t he gas- me ml
romat ographi c analysis and identification of fatty acids is c ot
;thylesters were carried out as report ed in a previous l ocal i
per (Pagliarani et al., 1986). The fatty acids separated by
s-liquid chr omat ogr aphy are expressed as weight percent-
es of t he t ot al fatty acids. The unsat urat i on paramet ers I
d I x 100/% SFA were calculated according to Bloj et al.
pH, t
t r o u t
St udent ' s t-test was used for determining the significance act i vi
the differences between FW and BW t rout .
viz fr
MgATP, Tr i s- ATP (all vanadium-free), ouabai n (stro- i mpl i
Lantin-G), EDTA, HEPES and Tris were purchased from
gma Chemical Co., St. Louis, MO. Solvents, BHT, acid
, ~ h ~ , 4 g ' ~ h v n m n e n r h W ~ . . ~ 1 ~ m~ , ~ h a n d d i ~ t h v l ~ n e o l i e n l -
( Na + + K+ ) - ATP a s e depet
t es t ed i n t he r a nge 4. 5- 9. 0, f or t
i s s h o wn i n Fi g. 1. Whi l e i n
act i vi t y cl ear l y pe a ks a t p H 7. 0, i n
c o mp a t i b l e wi t h mo r e t h a
f r o m 6. 8 t o 7.5 whe r e 100% act
i mpl i es a gr eat er t ol e r a nc e o f t h(
mesh and diethylene glicol- Mg 2+ and A T P dependence
Carlo Er ba (Milano, Italy); The e nz yme de pe nde nc e o n Mg 2
licic acid 100 mesh from c e n t r a t i o n s is cl ear l y di f f er ent i n l
,ride in anhydrous met hanol 2 s hows t h a t i n F W t he
~mical Division and from ma x i mu m at 7.5 mM Mg 2+, wi t h c(
t i on by excess Mg 2 (h = 4. 8 + 2. 0)
lde. Quart z double-distilled
utions in the preparat i on of
a +. + K +)-ATPase assays.
s0
K + ) - ATPas e activity ~. is
" ~ .
"Pase act i vi t y, s t i mul a t e d ~-
race o f Na + E
o f r el at i ve spec. act s a n d . ~
of r a i n b o w t r o u t a d a p t e d >
~rackish wat er . As s h o wn ~ s
at s t he e nz yme is equal l y
t c t i ons a n d pr ef er ent i al l y ~- t,"
aely t he mi c r o s o ma l f r ac- ~ 0 " ~ 6
vas pr evi ous l y r e por t e d i n
td ki dneys s ubj ect ed t o a n
~cedure ( Pa gl i a r a ni et al., Fig. 1. Effect of pH on (Na + K+ ) -
rai nbow t rout adapt ed t o FW ( O- - O1
; Tr o mb e t t i et al., 1990; The pH was varied by addi ng appropri a
Tris--HC1 to the reaction medi um
(1973).
Statistics
of
Chemicals
phant i n-G),
Sic
washed Chr omosor b W 60-80
suecinate were obt ai ned from Carl o
silver ni t rat e from Merck; silicic
Mal l i nkrodt ; 14% bor on trifluoride in
from BDH, Labor at or y Chemical
Applied Science Laborat ori es Inc.,
All chemicals were reagent grade.
wat er was used for reagent solutions
fractions P3 and P4 and in ( NaL
RESULTS
Distribution o f the ( Na + K
Th e Mg 2 - de pe nde nt ATPa s e
by t he s i mu l t a n e o u s pr es ence
exhi bi t s a par al l el b e h a v i o u r o f r el at i
r e c ove r y pe r c e nt a ge s i n gills of rl
e i t he r t o f r es h wa t e r or t o br a c ki s h
i n Ta b l e 1, i n t he t wo h a b i t a t s
di s t r i but e d i n t he va r i ous f r a c t i ons
l ocal i zed i n f r a c t i on P3, n a me l
t i on. A s i mi l ar d i s t r i b u t i o n wa s
sea ba s s a n d gi l t he a d gills a n d
a n a l o g o u s f r a c t i o n a t i n g p r o c e d u r e
1986; Ve nt r e l l a et aL, 1987;
Ve nt r e l l a et al., 1990).
Bo t h i n F W a n d BW t he pur i
o b t a i n e d f r o m f r a c t i on P3 is t he ri
K+ ) - AT P a s e act i vi t y. Th e h i g h e n
9ut gill (Na + + K+) - ATPase salinity-depe
+)-ATPase activity recoveries in fractions obts
(Na + +K
SA R'
FW BW FW
0.7 5:0.1 1.7 5: 0.2t 100.0
0.15:0.0 0.55:0.2* 6.25:1.5
1.25:0.1 1.55:0.2 18.35:3.5
0.65:0.1 0.85:0.4 16.3+5.4
2.95:0.5 12.6___3.1"[" 36.65:5.6
2.7 5:0.3 11.0 + 1.6" 100.0
1.2 5:0.7 1.2 5:0.4 16.9 + 3.0
3.65:0.7 8.2+2.8 18.15:2.0
0.7 5:0.4 2.7 5:0.9 5.5 5:0.5
11.1 + 1.2 28.05:0.6* 51.05:0.7
the fraction.
that of the homq
)ctive optimal
2.5 mM KC1 in
aificant diffcr
f r act i on, tl
me mb r a n e vest(
, nsi st ent wil
) cal i zat i on o f
et al., 1988).
p H dependence
The
r a nge
al kal i ne r ange.
Fi gur e
State College, PA.
a n d K +,
l at i ve s
omogenate and fraction P3
RSA
FW BW
1.0 _+ 0. 0 1 . 0 5 : 0 . 0
0.25:0.1 0.35:0.1
1 . 9 5 : 0 . 3 1. 25: 0. 1
0.6+0.1 0.3+0.1
4.6 5:0.7 8.5 5:0.5
1.0 5: 0. 0 1.0 5: 0. 0
0.45:0.2 0.1 5:0.1
1.35:0.3 0.85:0.1
0.3 + 0.1 0.2 5:0.0
2.9 5:0.6 2.6 5:0.2
on P3.
: two habitats: 75 mM HEPES,
HEPES, 57mM Tris (pH 7.5),
W and BW are indicated by
) r r e s pond t o ba s ol a t e r a l
t o t he me t h o d f ol l owed,
al l y r e por t e d ba s ol a t e r a l
a pol a r i z e d cel l s ( Ca p l a n
~endence o n as s ay
f or b o t h F W a n d BW
F W t he e nz yme
BW a wi de r p H
t h a n 9 0 % act i vi t y,
act i vi t y is obs e r ve d,
t he e nz yme i n t he
z+ a n d AT P c on-
t he t wo ha bi t a t s .
t he r e is a c l e a r c ut
c oope r a t i ve i nhi bi -
I), whe r e a s i n BW,
7 8 g
pH
-ATPase activity of
( O- - O) and BW ( 0 - - 0 ) .
mat e concent rat i ons of
cont ai ni ng 75 mM
5 mM MgC12, 50 mM NaCI,
M MgATP, 7 mM MgC12,
aM KC1 in BW.
r
0- 2 4 6 S 10 12 14
Mg ++ (mM)
g. 2. Effect of Mg 2 + concent rat i on on (Na + + K +)-ATP-
e activity of rai nbow t rout adapt ed to F W ( O- - ) and ATPa
V ( O- - Q) . The reaction media cont ai ned the MgCI 2 and lq
ncent rat i ons reported plus HEPES, NaC1, KC1 as detailed conce
r FW and BW in legend to Fig. 1. Four mM ATP in FW as det
d 3 mM ATP in BW was added as Tris salt. The pH
ts adjusted to 7.0 in FW and 7.5 in BW by addi ng Tris,
as obt ai ni ng the final concent rat i ons reported in Table 1.
Th
Table 2. Kinetic parameters of the (Na + + K+)-ATPase coop~
X,.. h K m CC
FW BW FW BW f or 1~
"P 0.6+0.1 0.6+0.1 1.9 i t
y2+ 1.8+0.2 2.0_+0.1
+ 9.8_+0.9
Th,
and h are calculated as described in Materials and Methods;
values are expressed as mean _+ SE from two determinations, cat i oi
~tistically significant differences between FW and BW are indicated
Na +
100 rr
t he
) r oa d p e a k r esul t s f r o m t he ma i n t e n a n c e o f a t l east i n B~/
% act i vi t y i n t he r a nge 5 - 1 5 mM Mg 2+ wi t h a obs e r
ca 9 5
0. 9_ 0.1 In P W t o 3.3 m BW.
1.2+0.2
1.5+0.1 Na + a n d K + d e p e n d e n c e
1.2 + 0.1 The e nz yme was f o u n d t o be difl
a n d K i n t he t wo
c a t i ons t he opt i ma l c o n c e n t r a t i o n s
a n d 2 . 5 mM K , ar e l ower t
1 0 0 mM Na a n d 1 0 . 0 mM K (Fi~
Na / K r a t i o c ha nge s f r o m 20
BW. As a ma t t e r of fact , t h o u g h
obs e r ve d at 1 0 mM K , t he BW e
9 5 % a c t i va t e d at 5. 0 mM K (Fi
Lximal act i vi t y is a t t a i n e d g r e a t e r - t h a n - o p t i ma l c onc e nt r a t i ons
VI i n BW. The a c t i va t i on a ny a ppr e c i a bl e e nz yme i nhi bi t i on.
i n F W a n d c oope r a t i ve Ap p a r e n t K~ val ues f or b o t h c~
lged K~ a n d no i nhi bi t i on i n BW t h a n i n F W ( Ta bl e 2). F
a c t i va t i on ki net i cs ar e, agai n, non- c (
a n d c oope r a t i ve i n BW.
" " 20
.>_
g.
8 10
c~ 5 10
nt r at i on on ( Na + + K +)-
at adapted t o FW ( - - ) Fig. 5. Effect o f K + concent rat i on on ( N
ion media cont ai ned the activity of rai nbow t rout adapt ed to F~
ed plus HEPES, NaCl, KC1 ( O- - Q) . The reaction media containec
Na* 21.9 _+ 1.2"
K + 0.4+0.1 1.1 _+0.0"
K.
Statisticall'
by * (P < 0.01).
a b r o a d
9 0 %
ma x i mu m at 10.0 mM Mg 2+.
As s h o wn i n Fi g. 3, t he ma x i m
at 4. 0 mM i n F W a n d 3.0 mM i n
ki net i cs ar e n o n - c o o p e r a t i v e
i n BW ( Tabl e 2), wi t h unchav
by excess s ubs t r a t e i n b o t h cases.
20
10
F--
ATP (mMI
Fig. 3. Effect of ATP concent rat i on
ATPase activity of rai nbow t rout
and BW ( O- - Q) . The reaction
Tr i s- ATP concent rat i ons reported I
as detailed for FW and BW in legend
(7.5 mM in FW and 10 mM in BW) was
salt. The pH was adjusted as detail
A. PAOUARAN] et al.
~ 20,
._>
to
t~
(3_
I -
Fig. 4. Effect (
ATPase activity
BW ( o - - o
concent rat i ons re
as detailed for F~
a d.
e c ha nge
) pe r a t i vi t y ir
c o n s t a n c y a]
f or Mg 2+ . Fi n a
2.t + 0.2. i n F W 3
2.6 _ 0.3*
2.4 + 0.4*
2.0-t-0.1"
by Na
O0 150 200
Na (raM)
~ntration on (Na + + K +)-
ut adapt ed to FW ( O- - O)
media cont ai ned the NaC1
~PES, MgATP, MgCI2, KC1
,~gend to Fig. 1. The pH was
led in Fig. 2.
t i on ki net i cs f r o m n o n -
) er at i vi t y i n BW a n d t he
ed f or AT P ar e al so val i d
/ AT P r a t i o c ha nge s f r om
di f f er ent l y a c t i va t e d
ha bi t a t s . F o r t hes e
i n FW, 50 mM
t h a n t h a t i n BW,
( Fi gs 4, 5); mo r e o v e r
20. 0 i n F W t o 10.0
100% act i vi t y is
e nz yme is al r eady
(Fi g. 5). I n al l cases
at i ons do not r es ul t i n
c a t i ons ar e hi gher
Fu r t h e r mo r e , t he
n o n - c o o p e r a t i v e i n F W
15 20
K* (mM)
Na + + K +)-ATPase
FW ( O- - O) or BW
ont ai ned the KC1 concen-
NaC1, MgATP, MgC12 as
end to Fig. I. The pH was
ed in Fig. 2.
-~u -9 - 8 -7 - 6 -5 - 4 -3
0uabain (log M )
g. 6. Effect of ouabain on rainbow trout (Na + +K+) -
['Pase. The ouabain concentrations reported were added
the reaction media detailed in Table 1 for FW ( O- - O)
d BW ( O- - Q) . The BW enzyme sensitivity was further
ted in a modified BW medium containing 2.5 mM KCI
~tead of 10.0 mM KC1 ( 1 ~ 1 ) . Each point is the average
from three determinations.
Tect o f ouabain
At odds wi t h t he ( Na + + K +) - ATPase dependence
pH, ATP and cations, t he enzyme sensitivity t o
tabain is subst ant i al l y si mi l ar in t he two habi t at s as Resnlu
monst r at ed by t he common/ 50 (5 10 -7 M) s h o wn Ot her s
the two act i vi t y curves (Fig. 6). The appar ent l y
Net sensitivity of t he BW enzyme in t he range statisti
5 1 0 - 7 M ouabai n shown by t he upper profile of
curve as well as t he higher 100% i nhi bi t or y
ncent r at i on wi t h respect to F W (5 x 10- 3M vs t hat l
< 10 -4 M) are pr obabl y rel at ed t o t he higher K t hat i
ncent rat i on in t he assay medi um cor r espondi ng to and (
hi gher K opt i mum in BW wi t h respect t o FW. act i vi
a mat t er of fact, i f t he BW enzyme sensitivity is basol~
t ed in a 2.5 mM K -cont ai ni ng medi um as in FW, t hat il
," 50% and 100% i nhi bi t i on occur at 2 x 10 -7 M
17:1, 18: 0 iso, 22: 1 a nd ot her mi nor unl
SFA: s at ur at ed f at t y acids; MUFA:
St at i st i cal l y si gni fi cant differences bet ween FW
by * ( P < 0.01) a nd ~" ( P < 0.05).
the enzyme act i vi t y in BW, a,,
in FW, is at least 4-fol d great
doubl e in fract i on P4. The d
act i vi t y r at i o in t he t wo fract i ons is
basol at er al membr ane cont ent in
implies a l ower enri chment in A
fract i on P4 wi t h respect t o FW.
)ctively, namel y at even
ns t han in FW. Gill lipid composition
The cont ent of t ot al lipids (TL) al
(PL) of gill tissue is shown in Tabl
e act i vi t y in BW is expressed as percent ages of wet wt
tat in FW. The maxi mal BW t han in FW; as t he PL/ TL rat i o (
respective opt i mal assay any vari at i on, t he TL decrease even
,'sent experi ment (4 mM change of phosphorus-free lipids.
[ Na +, 2.5 r aM K +, p H Gi l l f at t y aci d composi t i on and re
10 mM Mg ~ +, 100 mM are r epor t ed in Tabl e 4. BW t r out sin
t BW), and expressed as t o F W t r out , a higher cont ent of t h
, is 3.8 in crude mi cro- aci ds 16:0 and 18:0 and of the ul
15.1 in purified baso- 18: 2n-6 and 18:3n-3 and l ower per(
: W, and 18.6 and 29.0, chai n pol yunsat ur at ed fat t y aci ds (
Lge (Na + + K +)-ATPase 20: 4n-6, 20: 5n-3 and 22: 4n-6. Su(
W and 13.9 + 1.5 in BW not i mpl y significant differences b
[ and 22.4 _+ 1.0 respect- habi t at s in t he unsat ur at i on pa r a m
t from t he vari ous tests n-3/n-6 rat i o.
t a bl e 1, Fi gs 1-6), show
DI SCUSSI ON
phosphol i pi d cont ent
Phosphol i pi ds The differences in t he pr epar at i ve
( % of t ot al lipids) in t he met hods o f ( Na + + K +)-,
43.61 + 0 . 9 0 eval uat i on in l i t erat ure make it diffi
43.53 _+ 0.80 the spec. act. values presented here wi
I UW~, I
0- 5
the
concent r at i o
5
conc~
t he
As
tested in
the
and 1 x lO -4 M ouabai n respectively
l ower i nhi bi t or concent rat i ons
Specific activity
The ( Na + K *) - ATPase
const ant l y far hi gher t han t hat
spec. act. obt ai ned under t he res
condi t i ons f ound in t he present
ATP, 7. 5r aM Mg 2+, 50mM
7.0 in F W and 3 mM ATP,
Na , 10mM K , pH 7.5 in
#mol s P i mg pr ot ei n -1 hr -J
somal pr epar at i ons (P3) and
l at eral membr anes (P4) in FW,
respectively in BW. The avera
act i vi t y values, 2.9 +_ 0.5 in F W
in fract i on P3 and 11.5 +0 . 4
ively in fract i on P4 obt ai ned
carri ed out on this fract i on ("
Tabl e 3. Gi l l t ot al lipid a nd
Tot al lipids
( % o f wet wt ) ( % o f wet wt )
F W 3.00 0.03 1.31 0.03
BW 2.10 + 0.05* 0.91 0.02*
Val ues expressed as mean SE f r om t wo det er
St at i st i cal l y si gni fi cant differences bet ween FW a
b y * ( P < 0.01).
out gill (Na + + K +)-ATPase salinity-delx
.o Tabl 4. Fl
Fa t t y aci d
1 4 : 0
15: 0
o ~ 16: 0
v 16:1
~0 ~ 18:0
..-. 1 8 : 1
18: 2n- 6
18: 3n- 6
18: 3n- 3
20:1
20: 2
loo 20: 3n- 6
20: 4n -6
20: am-3
2 0 : 5 n - 3
2 2 : 4 n - 6
2 2 : 5 n - 6
2 2 : 5 n - 3
2 2 : 6 n - 3
O t h e r s
~ S F A
Z MUF A
Zn - 6
Z n - 3
n - 3/ n - 6
I
I x 100/ %SFA
Resul t s ar e express~
Ot her s ar e t he sum (
,ely,
~tion of gill t ot al lipids
BW
2 . 4 + 0 . 1
0. 2 0. 0
20.0 0.3':
5.0 0. !
7.5 -,I- 0.1 *
19.9.4- O.Ot
4.3+0.17
0.20.1
0.9+0.17
2.1 + 0.1
0.3+0.1
0 . 6 + 0 . 1
3.4 + 0 . 0 "
0. 4 0.0
5.1 0.2*
0.3 0.0'
0.4 0.0
1.30.1
23.2 0.4
2.5 o.o
30.1 0 . 4 ?
2 7 . 0 0 . 0 7
9 . 3 + 0 . 2
3 0 . 9 0 . 2 *
3 . 3 + 0 . 4
2 . 3 + 0 . I
8 . 6 0 . 3
f r o m t w o d e t e r m i n a t i o n s .
~ttty a 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 i d s .
F W a n d B W a r e i n d i c a t e d
as c o m p a r e d w i t h
reater in fraction P 3
different B W / F W
is d u e to the higher
B W m i c r o s o m e s
.~nt in A T P a s e activity in
( T L ) a n d phospholipids
T a b l e 3. T L a n d P L
are 30/'0 l o w e r in
d o e s n o t u n d e r g o
~e even reflects a paral l el
rel at ed par amet er s
s h o w , w i t h respect
t he sat ur at ed fat t y
unsat ur at ed 1 8 : 1 ,
percent ages of l ong-
( PUFA) such as
~h variations d o
b e t w e e n the t w o
)arametcrs a n d in the
procedures a n d
) - A T P a s e activity
ficult to c o m p a r e
vith t hat elsewhere
sed on gill homogenat e,
on purified fract i ons
vithout search for t he
The hi gher ( Na + K+) - ATPas e act i vi t y in BW influe
t h respect t o FW is in perfect agreement with and
~rature dat a (De Renzis and Bor nanci n, 1984; in sa
Lngdon and Thorpe, 1984). As st at ed by the Pross
: rat ure, the sal t -wat er enzyme act i vi t y increase optirr
:lects the higher ( Na + + K +)-ATPase cont ent in resp~
1 epi t hel i um due to sal i ni t y- pr omot ed increase of hi gh(
chl ori de cell number, compl exi t y of basol at er al of co
,' mbranes and densi t y of enzyme units (Eddy, 1982; of tha
Lurent, 1984; Payan e t a l . , 1984). A mor phol ogi cal the e
Terentiation between FW and BW t r out consi st ent hyper
th l i t erat ure was poi nt ed out by el ect ron micros- subst~
py even in t he present experi ment (unpubl i shed zyme
ta). Physi ol ogi cal l y, the hi gher act i vi t y of (Na + ar our
)-ATPase in BW may be rel at ed t o t he funct i on in B$
t he Na pump in NaC1 ext rusi on from t he cell in obser
Lt-water, where it mi ght pl ay a mor e r emar kabl e incre~
le accordi ng t o t he model of Silva e t a l . (1977), in th~
i n in t he active Na upt ake in freshwat er by ar our
especi
Apar t f r om t he different enzyme spec. act. , the
o habi t at s even i mpl y a different enzyme response On
vari ous effectors, t hat in most cases, is appar ent l y in BY
"erable t o the different envi ronment al require- to th~
;nts. The different pH dependence curves of the exertc
zyme, as well as the different F W and BW pH assay
observed kinetic changes in BW
eased modul abi l i t y of t he (Na
the presence of Na concent ra
ar ound higher values. This aspe
~pecially not ewor t hy in t he mai n
the cont r ar y, the l ower suscep
BW wi t h respect to F W is onl y a
the known ant agoni sm t owar ds
exerted by K ( For bush III, 1982
' medi um at higher opt i mal con~
~t commonl y used teleost
Besides t he i nt ernal compar i son I
Renzis and Bornanci n, BW t rout , we have compar ed the p
)e, 1984; Madsen and Mg 2, ATP, K and Na opt i ma v
ni ck e t a l . , 1989) t hat r epor t ed concent r at i on values of t
:d as opt i mal in ot her teleost gill (Na + K )-ATPase assa
iler and Ki rschner, 1972; the opt i mal concent rat i ons obt ai ne
ay be t ent at i vel y rel at ed char act er i zat i on in t el eost ean osmor
~ordingly, t he higher pH payi ng at t ent i on t o our previ ous
)-ATPase and t he mor e survey poi nt s out ATP and Mg 2~
vity profile in BW with generally in the range 3-5 mM witl
a cert ai n enzyme t ol er- rat i o of 1.0 (Langdon and Thorpe, 1(~
to pH changes in the 1985; Fenwi ck a.nd Lam, 1988;VentJ
', t hus guarant eei ng the sel dom between 1.0 and 2.0 (Pfeile
epi t hel i um di rect l y in 1972; Pagl i arani e t a l . , 1988; Mads~
vi ronment , sen, 1989), and occasi onal l y l ower
differences between FW e t a l . , 1979) or great er t han 2.0 (M
ract i on kinetics of the 1989). Na and K oscillate ar ou
gATP and the act i vat i ng 20-25 mM, respectively, with Na /
most st ri ki ng di spar i t y or 5.0 (De Renzis and Bornanci n, 19
in t he act i vat i on kinetics 1985; Pagl i arani e t a l . , 1988). On t h
~+ t hat are Mi chael i an dat a on the opt i mal concentrations
W. Whi l e in the case of habi t at s and of Na in BW are
c change is accompani ed l i t erat ure, and even t he affinity co~
poi nt s between F W and and cat i ons do not subst ant i al l y
_ 1 I k K _ ~ I - i Ae . l r ~l r ~ _ _ ~ , _ _ J i . . . . . . . . . . . . ~ . . . . . Z l - - L I - / h - - 1"Ii . . . . _~_
~ I L - W ~ , L ~ I "
role
t han
hyperregul at ors.
A
t wo
t o
referabl e
ments.
enz
opt i ma, bot h l yi ng in the most comm(
gill ( Na + K ) - ATPase pH
and Saunders, 1981; De Renzis
1984; Langdon and Thor pe,
Naamansen, 1989; McCor mi ck
includes pH values r epor t ed
char act er i zat i on studies (Pfeiler
Fenwi ck and Lam, 1988), ma
t o a funct i onal meani ng. Accor di n
opt i mum of the (Na + K
al kal i ne-shi ft ed enzyme act i vi t
respect to F W may i ndi cat e
ance t o al kal i ne pHs and
al kal i ne field t ypi cal of BW
act i vi t y homeost asi s in an
cont act with the ext ernal enwr onme
Onl y par t of t he observed different
and BW t r out in t he i nt eract i on
enzyme with the subst rat e M
cat i ons are not ewort hy. The
between the two habi t at s lies in t he ac
by ATP, Mg 2, Na + and K
in F W and cooper at i ve in BW.
Mg 2 and ATP such a kinetic
by onl y poor di fferent i at i ng
BW (namel y a higher opt i mal Mg 2
a great er susceptivity t o excess Mg
respect to FW) , for Na and K
A. PAGLIARANI e t a l .
tions. Anyway, our with vari at i ons
values in FW and and K m values i
e l ower edge of t he different susce t
either in euryhal i ne adapt i ve signifi
o vari ous salinities, cooperat i ve act
l ghl y similar to the ot her charact er
a chl ori de cell pr ep- euryhal i ne tele,
, 1981; Jiirss e t a l . , e t a l . , 1988; T
ncin, 1984; Madsen somehow linke
al t -wat er, however, osmol ar i t y as
nt r i but i on to baso- of sal t -wat er
body fluids (E'
Luenced, eve~
generally
sal t -wat er 1
)sser, 1986).
~timal activit3
~ect to FW
cat i oni c co
cooperat i ve
t he affinity q
environmer
9erbolic t o
subst rat e conce
susceptix
ar ound t he Km(
BW the Km
homeost asi s.
nmonl' t han in FW.
range ( Johnst on
ment.
opt i mal concent rat i ons
FW, t hat may make the
t and K + of possi bl e
i ncreased salinity. The
a +, al r eady observed in
es on sea- wat er - adapt ed
e t a L , 1985; Pagl i arani
d., 1990) appear s to be
arine envi ronment . The
tnd Na + concent rat i ons
ban t hat of t el eost ean
These ones in t ur n are
by the ext ernal milieu
aer salt concent rat i ons
h wat er (Evans, 1979;
i f the shift of the BW
)ncent rat i on values with
igh enzyme activities to
in paral l el with the onset
inetics and the increase
y be more adequat e for
ents. The change from
aape in the act i vi t y vs
ve implies a higher en-
concent r at i on changes
1977). I f we consi der t hat
higher t han in FW, t he
may i ndi cat e an
Na + K ) - ATPase
ncent r at i on fl uct uat i ons
9ect seems to be
mai nt enance of Na
:ptivity t o ouabai n
appar ent and due
ouabai n bi ndi ng
i) present in t he
concent r at i on in BW
between FW and
~resent results on
with the current l y
these effectors in
ay medi a and with
obt ai ned in t he enzyme
osmor egul at or y tissues,
studies. Such a
+ concent rat i ons
wi t h an Mg2+/ ATP
1984; Trigari e t a l . ,
Vent rel l a e t a l . , 1990),
Pfei l er and Ki rschner,
Madsen and Naaman-
t han 1.0 (Gal l i s
McCor mi ck e t a l . ,
arouncF 100 mM and
rK rat i os of 4.0
1984; Tri gari e t a l . ,
these bases present
of ATP in bot h
consi st ent with
const ant s for ATP
differ from the
Renzis and Bornanci n,
enwick and Lam, 1988;
( Na + K ) - ATPas e pr oper t i es coul d be modu- Eddy
ed by me mbr a ne l i pi ds ( Deut i cke and Haest , 1987) fish
Phy
t t he di f f er ent envi r onment al sal i ni t y ( Ler ay et al., Evans
84) appar ent l y r emot e in t he pr esent exper i ment Osn
en i f s ome changes o f single f at t y aci ds ar e Vol
t ect abl e. Fi nal l y, t he fact t hat our dat a ar e based Fenwi
anal yses car r i ed out on t he whol e tissue and not air~
basol at er al me mbr a ne s because o f t he pauci t y o f Ox;
sue avai l abl e coul d not be negl ect ed. The obs er ved Fis~
crease i n BW o f t he l i pi ds under st udy ma y be
l east par t i al l y r el at ed t o s al i ni t y- pr omot ed gill dote
"uctural changes. Ne w chl or i de cells t ypi cal o f t he Folch
bi t at ma y exhi bi t a l ow l i pi d/ pr ot ei n rat i o, met
The di f f er ent ( Na + K ) - ATPas e f eat ur es i n t he anir
o habi t at s, t hough di fferent l y r emar kabl e f or t he
r i ous effect ors, ma y be r el at ed t o sal i ni t y- dr i ven Nal
1 mor phol ogi c a l changes and consi der ed t o be re- pore
onsi ve t o t he di fferent envi r onment al r equi r ement s. PP.
l zyme st r uct ur es at l east par t i al l y di fferent ma y be Gallis
vol ved in t he i nwar d and out wa r d t r ans por t o f ada
Effe
~C1. The bi osynt hesi s o f new enzyme uni t s ma t c he d D c
t he pr ol i f er at i on o f chl or i de cells in sal i ne medi a in
aur ent , 1984; Payan et al., 1984) ma y resul t in t he Gorm
oduct i on o f new ( Na + K ) - ATPas e compl exes Dot
t h ki net i c feat ures mor e sui t abl e f or hypor egul at i on, reac
le var i ous ( Na + K +) - ATPas e i sof or ms, r ecent l y Kinse:
111 [ I YOJ ) L, i i I' Ul Ol . OIl l U
NaK-ATPase. In Current Topics in M~
~ort (Edited by Bronner F. and Klein
167-201. Academic Press, New Y
J. L., Lasserre P. and Belloc F.
adaptation in the euryhaline teleost
Effect of adaptation, prolactin, cortis~
on plasma osmotic balance and (N
gill and kidney. Gen. comp. Endoc
Gornall G., Bardawill C. J. and Da,
Determination of serum proteins by r
reaction. J. biol. Chem. 177, 751-757
KinseUa J. L., Holohan P. D., Pessah N
di ffer in t he a mi no aci d (1979) Isolation of luminal and antil
and in t he sensi t i vi t y t o from dog kidney cortex. Biochim. i
ISweadner, 1989). Ther e-
E. and Saunders R. L.
t - wat er chl or i de cells on transformation of yearling atlantic sa
;nt, 1984) cont ai n differ- at several rearing temperatures. Can.
af or ms f r om t he enzyme
l ocat ed on t he secondar y Jfirss K., Bittorf Th. and Vrkler Th. I
salinity and food deprivation on gi
ratio and certain enzyme activities
rs wish to thank Professor (Salmo gairdneri Richardson). Comp
tip this work could not have
i is gratefully acknowledged Jfirss K., Bittorf Th., Vrkler Th. and
of rearing and sampling Influence of nutrition on biochemical s
Dr Tibaldi for his valuable of the rainbow trout (Salmo gairdneri I
nt. This study was supported Biochem. Physiol. 75B, 713-717.
Public Education. Langdon J. S. and Thorpe J. E. (1984) I
Na +-K + ATPase activity, succinic deh
and chloride cells to saltwater adal
salmon, Salmo salar L., parr and sm
323-331.
in M. and Mayer-Gostan N. Laurent P. (1984) Gill internal mol
odium influx in the rainbow Physiology (Edited by Hoar W. S. a
aated to artificial freshwater Vol. X, Part A, pp. 73-183. Academit
bl l , 159-169. Leray C., Chapelle S., Duportail G. and
R. M. and Trucco R. E. Changes in fluidity and 22:6 (n-3) c~
y acids and regulation of lipids of trout intestinal brush-bor
31osteric behaviour of eryth- related to environmental salinity. Bio
gill
sponsl ve
Enz,
i nvol ved
NaC1. The bi os
t o
( Laur ent ,
pr oduc t i on
wi t h
The
descr i bed in several tissues
sequence o f t he ~- s ubuni t and
oua ba i n and phos phol i pi ds (
f or e t he hypot hesi s t hat sal t - wat er
t he pr i ma r y l amel l ae ( Laur ent ,
ent ( Na + + K +) - ATPas e isc
f or ms oper at i ng in F W and l ocat ed c
l amel l ae shoul d not be di scar ded.
Acknowledgements--The authors
A. R. Borgatti without whose hel
been done. Professor D. Lanari is
for trout availability and use
facilities. Thanks are also due to
cooperation during the experiment.
by grants from the Ministry of
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