Intestinal Na+K+-ATPase activity in salmonids

Comp. Biochem. Physiol. Vol. 115A, No. 2, pp. 159-168, Copyright 0 1996 Elsevier Science Inc.

ISSN 0300s9629/96/$15.00 PI1 SO300-9629(96)00029.1

1996

ELSEVIER

Intestinal

Na+/K+-ATPase

in Salmonids

Activity

NEESS

Anne-Gerd Gjevre and Lie, Irtger Mudal

DEPARTMENT OF MORPHOLOGY, GENETICSAND AQUATIC BIOLOGY,NORWEGIANCOLLEGEOF VETERINARY MEDICINE,OSLO, NORWAY

ABSTRACT.

The sodium-potassium

dependent

a key enzyme in adaptive processes in euryhaline in intestinal

preparations

L.), examines strength

from rainbow trout (Oncorhynchus

the ion and substrate requirements

mal enzyme activity

was obtained

triphosphatase

at 13 to 16 mM ATP;

in both species. Nat/K+-ATP

of substrate than the corresponding mal incubation

temperature

may be prolonged Na+/K+-ATPase

exhibits

COMP

WORDS.

properties preparations

BIOCHEM

PHYSIOL

Ca’+-ATPase,

in intestinal

presence

that are not substantially

fish, intestinal

salmon (Salmo s&r

pH, and ouabain.

Na:K-ratio

Maxi-

of 4: 1; total ionic

of an intestinal

preparations

Ca*+-ATPase

was

required a higher concentration

Compared

to gill preparations

Moreover,

the opti-

the incubation

time

this work indicates that salmonid intestinal

different from those of the corresponding

seem to require a higher concentration 1 lSA;2:159-168,

and Atlantic

is 20°C instead of 37°C.

is low. In conclusion,

is assumed to be

activity of Na+/K+-ATPase

temperature,

18 to 24 mM M&l,;

in gill preparations.

preparations

to 60 min if enzyme activity

in the gills, but intestinal temperature.

ase activity

enzyme activity

for intestinal

(Na+/K+-ATPase)

mykiss Walbaum)

and effects of incubation

of Na+ and K+ at 185 mM, and pH at 6.5 to 6.6. The

demonstrated

KEY

adenosine

fish. The present study demonstrates

enzyme

of substrate and a lower incubation

1996.

preparation,

Na+/K+-ATPase,

Oncorhynchus mykiss, Salmo s&r

INTRODUCTION

tinal ATPases,

The

tween May and July (27). Bisbal and Specker (3) measured the activity of Na+/K+-ATPase in the gills and intestine of

sodium-potassium

dependent

adenosine

triphospha-

tase (Na+/K+-ATPase is assumed to be a key enzyme in adaptive processes in euryhaline fish. Studies of gills and intestinal mucosa from different fish species have shown that the enzyme activity in these organs is higher in teleosts living in seawater than in those living in fresh water (5,10,16,33). Optimal time for seawater transfer of salmonids coincides with the peak activity of gill Na’/K+-ATPase (7,8,22,28,39). The activity is associated with an excretory function in chloride cells where excess NaCl is eliminated (13). Although the actual site of uptake of these ions from ingested seawater has not been finally determined, intestina1 uptake seems to be most significant and is presumably dependent upon the activity of intestinal Na+/K+-ATPase (1,9,23,29,30,35). Any changes of enzyme activity during Parr-smolt transformation might thus reflect the role of intestinal Na+/K+-ATPase connected with transfer of fish to seawater.

tine. This is in accordance

However, the different responses in enzyme activity from gill and intestinal preparations in the studies referred to above might also reflect different requirements regarding in vitro incubation conditions. The aim of the present study was to assess the importance of incubation conditions on Na+/K+-ATPase activity in intestinal preparations. The conditions examined include ion and substrate requirements, effect of incubation temperature and time, and effects of pH and MgClz:ATP ratios and Na:K ratios, using the method of Zaugg (38) as a starting point.

MATERIALS

Fish

Address reprint requests to: Anne-Gerd Gjevre, Department of Morphology, Genetics and Aquatic Biology, Norwegian College of Veterinary cine, P.O. Box 8146-Dep., N-0033 Oslo, Notway. Received 7 September 1995; accepted 30 January 1996.

Medi-

with the results of Usher et al.

(36) and may indicate different mechanisms for regulation of gill and intestinal Na+/K+-ATPase activity in salmonids.

monids. In freshwater-adapted

mykiss), there are seasonal variations in the activity of intes-

activity peaking be-

cortisol-treated, juvenile Atlantic salmon (S&no s&r) in fresh water. They found that cortisol induced an increased activity of Na+/K+-ATPase in the gills, but not in the intes-

Little attention has been paid to changes in the activity of intestinal Nat/K+-ATPase during smoltification of salrainbow trout (Oncorhynchus

with Na+/K+-ATPase

AND

METHODS

Rainbow trout and Atlantic salmon were obtained from local hatcheries, kept in dechlorinated tapwater in indoor facilities for about one year, and fed a commercial salmonid diet. Average water temperature was 4°C from November to April, increasing to maximum 12°C during the summer months. Four Atlantic salmon ranging in weight from 59

160

A.-G. Gjevre and L. 1. Madal

to 233 g and six rainbow trout (351 to 1003 g) were sacrificed in February.

Tissue

Sampling

Procedure

The fish were stunned with a blow to the head, after which filaments from the right side of the gills were removed. A midline cut was made from the pectoral

fins to the anus

and the entire intestine, including the pyloric cecae, was removed. Intestinal content was evacuated by firm compression. Perivisceral fat, pancreatic tissue, and connective tissue with blood vessels were removed. The entire intestines of all trout were diced into small pieces and mixed together, as were intestines of the salmon. Aliquots of intestinal and gill tissue (200 to 400 mg) were transferred to plastic tubes containing 1.0 ml SE1 buffer (0.3 M sucrose, 0.02 M Na2EDTA, and 0.1 M imidazole at pH 7.1) and immediately frozen in liquid nitrogen. The material was stored at - 70°C until tested.

Assay of Na+/K+-ATPase

in three different

tissue preparations

Nzess

in each experiment.

The activities of Nat/K+-ATPase or total ATPase are given in pmol inorganic phosphate hydrolysed from ATP per hour and mg protein (I’, t (mg protein h)-‘), in percent of maximal Na+/K+-ATPase activity, or in percent of total ATPase activity. Optimal range is defined as the range showing enzyme activities exceeding 90% of the maximum. Peak activity or maximal activity are defined as 100% activity. Mean values with corresponding sented in Fig. 1.

95% confidence

intervals are pre-

RESULTS Ion Requirements for Intestinal Na+/K+-ATPase in Rainbow Trout and Atlantic Salmon Intestinal homogenates rainbow trout contained

from both Atlantic salmon and ATPases activated by Mg’+alone,

by Mg2+, Na+, and K+ in combination, and by Cal’ alone (Fig. 1). Addition of either Nat or K’ in combination with Mg’+ gave no further enzyme activation than addition of Mg’+ alone, but addition of all three ions in combination

Activity

Enzyme activity was estimated according to Zaugg (38), using a modification described by Finstad et al. (10). The intestinal tissue preparations were diluted 1: 10 with

resulted in a markedly higher activity of total ATPases. This increase in enzyme activity was sensitive to ouabain. Ammonium ions could replace potassium ions, but not sodium

and

ions in the activation of this enzyme. Addition of C&I: to an incubation solution without other ion supplementation

carefully homogenized with 30 strokes of a Potter Elvehjem homogenizer at ca. 6500 rpm. The homogenate was centrifuged for 20 minutes at 4700 X g and 4”C, and the superna-

stimulated ATPase activity. This activity was inhibited by addition of Na’ and K+ ions and was not sensitive to ouabain. In both rainbow trout and Atlantic salmon, Ca2’-

tant fraction was carefully decanted and used as the enzyme source. Of enzyme preparation, 100 ,~l (about 0.8 mg pro-

activated

SEID-buffer

(SE1 + 0.1 g/100 ml natriumdeoxycholate)

tein/ml) was transferred

to test tubes cooled on ice. Half

of the tubes contained 650 ~1 of the incubation solution consisting of 23 mM MgCll 6 HLO, 155 mM NaCl, 75 mM KCl, and 115 mM imidazole. The rest of the tubes contained 650 ~1 of the incubation solution plus 0.5 mM ouabain. In both solutions, pH wab adjusted to 7.1 with 1.0 M HCI at 37°C. The tubes were transferred to a 37°C water bath, and 100 ~1 of a 100 mM Nal-ATP-solution (Sigma, No. A-5394,. vanadium free) was added. After 20 minutes incubation, the reaction was stopped by addition of 1.5 ml cooled (4°C)

0.8 M H1S04.

Inorganic

phosphate

released

from ATP was determined by the method of Fiske and Subbarow ( 1 I), and protein determinations were performed according to Lowry et al. (21), using bovine serum albumin as standard. The chemicals were obtained from Chemical Company (St Louis, Missouri, USA) Merck (Darmstadt, Germany). With these incubation solutions used as a base incubation time of 20 minutes at 37”C, the various were tested as described in the figure legends.

Sigma and E. and an factors

ATPases

phosphate equivalent

appeared

to release

more

inorganic

from ATP than Mg’+-activated ATPases concentrations of the divalent ions.

at

The results presented in Fig. 1 indicate that there may be a difference between the salmonid species concerning intestinal activities of Na+/K+-ATPase and Ca’+-activated ATPase. ference

However, more data is needed to verify if this difis significant.

Effect of Temperature The experiments

indicated that the rainbow trout intestinal

preparations could be divided into two groups-one group with relatively high enzyme activity (Fig. 2a) and another group exhibiting

clearly lower enzyme activity (Fig. 2b). In-

cubation at 20°C resulted in a close to linear release of phosphate in both groups during 60 min incubation (Fig. 2a and b, broken lines). However, at 37”C, the release of phosphate levelled off after 20 min (Fig. 2a and b, solid lines). This was most pronounced for the preparations showing lowest enzyme activity. The enzyme activities after 20 min incubation in both groups were higher at 37°C than at 20°C.

Data Presentation

Effect of NazATP

Concentration

Each value given is the mean of 3 to 4 parallel measurements. With few exceptions, enzyme activity was assayed

The optimal concentration of Na?ATP for determining activity of Na+/K+-ATPase in intestinal homogenates from

intestinal Na+/K+-ATPase in Salmonids

161

8 FIG. 1. Ion requirements for intestinal Nat/K+-ATPase in rainbow trout and Atlantic salmon. A: Basic solution without ion supplementation (11.8 mM Tris-ATP, 101.5 mM imidazole, and 0.1 ml 4700 X g supernatant in a final volume of 0.85 ml) (N = 2). B: A + 17.6 mM MgClz (N = 2) C: A + 17.6 mM M&l, + 57.4 mM KC1 (IV= 2) D: A + 17.6 mM MgClz + 118.5 mM NaCl (N= 2) E: A + 17.6 mM MgCl, + 57.4 mM KC1 + 118.5 mM NaCl (N= 3) F: A + 17.6 mM MgC& + 57.4 mM KC1 + 118.5 mM NaCl + 0.38 mM ouabain (N = 3) G: A + 17.6 mM MgCIZ + 57.4 mM NH&l (N = 2) H: A + 17.6 mM MgCl, + 57.4 mM NH&l + 57.4 mM KC1 (N= 2) I: A + 17.6 mM MgCl* + 57.4 mM NH&l + 118.5 mM NaCl (N= 2) J: A + 17.6 mM CaCIZ (N = 3) K: A + 17.6 mM CaClz + 57.4 mM KC1 + 118.5 mM NaCl (N= 2) L: A + 17.6 mM CaCll + 0.38 mM ouabain ( N = 1) The height of the bars represents the mean of the total ATPase activity expressed as amount of inorganic phosphate released per mg protein and hour. Vertical lines show 95% confidence interval of the mean. Open bars-raingreyhatched bow trout; bars-Atlantic salmon. “N” refers to the number of intestinal tissue aliquots tested in each species.

7

CI

x =

.

6

1

0 B

C

D

E

F

G

H

I

J

K

L

Ion supplementation rainbow trout was in the range of 12 to 23 mM, with maxi-

and a pH optimum similar to that of the intestinal prepara-

mum activity at 13 to 16 mM NazATP

tions was found (Fig. 4b). The highest activities for both gill and intestinal preparations were obtained at pH 6.5 to 6.6.

(Fig. 3).

Effect of pH

The incubation mixture of the trout intestinal Na+/K+ATPase-assay showed optimal activity at pH values from about 6.3 to 6.8 (Fig. 4a). Gill homogenates from Atlantic salmon were tested according to the method of Finstad et al. (10) at different pH values in the incubation mixture,

Effect of

Mg : ATP Ratio

The effect of varying the ratio between Mg’+ and Na?ATP was examined by varying both the Mg’+ and the ATE’ concentrations. The optimal range was observed at MgClz concentrations between 15 and 27 mM at 11.8 mM ATE’ and

A.-G. Gjevre and L. I. Masdal NZSS

a

b

I

I

I

I

I

20

30

40

50

60

I

10

0

0

IO

Time (min.)

20

30

40

50

60

Time (min.)

FIG. 2. Accumulation of inorganic phosphate from rainbow trout intestinal ouabain-sensitive ATPase at increasing incubation times at 20°C and 37°C. The final concentrations of the incubation mixture were 101.5 mM imidazole; 20.3 mM MgClr 6 H,O; 66.2 mM KCl; 136.8 mM NaCl. In the ouabain~containing mixture, the final concentration of ouabain was 0.38 mM ouabain. The results from three assays at each temperature are presented with different symbols. a) High activity enzyme preparations. b) Low activity enzyme preparations. Solid line, 37°C; broken line, 20°C.

between

15 and 58 mM at 16.5 mM ATP

(Fig. 5). There

was little difference between 16.5 and 11.8 mM ATP in the peak range (18 to 24 mM Mg’+) for the Na+/K+-ATPase activity. However, at 16.5 mM the enzyme activity did not appear to be inhibited by increasing magnesium concentrations to the same extent

as at the lower ATP

concentra-

tions.

Effect of No+ Constant

: K+ Ratio at a

Ionic Strength

of Na+

and K+

The total ionic strength of Na+ and K’ was kept at ca. 200

mM by varying the Na’ : K’ ratio. Different Na+ : K+ ratios at substrate concentrations of 11.8 and 16.5 mM NazATP were tested. As expected, lack of potassium supplementation gave no ouabain sensitivity ATP activity. The highest activities were observed when [Na+] > [K’]. Optimal Nat : K’ ratio was in the range from about 2 : 1 to about 11: 1 for

16.5 mM ATP and from about 2: 1 to about 10: 1 for 11.8 mM ATP. The highest enzyme activity was observed around a ratio of 4: 1 for both ATP concentrations (Fig. 6). Figure 6 illustrates an experiment that includes a “high” activity homogenate and a “low” activity homogenate in the same experimental condition (circles). The first figure shows that the range of Na:K that produces optimal activity is not affected by the activity level in the homogenates. Our conclusion is that the activity level in the homogenate does not seem to affect the shape of the activity curve in experiments other than the temperature experiment.

Effect of Different of Na+

Ionic Strengths

and K+ at a Constant

Ratio

The ionic strength of sodium and potassium in different incubation mixtures used in the determination of Na+/K+ATPase varied (Table 1). In the present study, the ratio of

Intestinal Nat/K+-ATPase in Salmonids

t:

163

rainbow trout and Atlantic salmon this concentration was found to be lower than 8 X 10-j mM ouabain for intestinal

4sI’:,

I

Na+/K+-ATPase.

DISCUSSION

n n

m

:

A

0

Notable characteristics of the Na+/K+-ATPase include requirements for Na+ and K’ in addition to Mg2+ ions for activation and inhibition by the cardiac glycoside ouabain. Our study shows that salmonid intestinal Nat/K+-ATPase also has these characteristics. Moreover, we observed that K+ but not Na+ can be replaced by NH4+. This is consistent with studies of ouabain-sensitive,

Mg’+-dependent

ATPase

in homogenates from salmonid and goldfish gills (Car&us nurutus) and from seabass (Dicentrarchus l&ax) and goldfish kidneys (17,4,24). In the present study, we also demonstrate the presence of an intestinal Ca’+-activated ATPase. In both rainbow trout and Atlantic salmon, this enzyme was inhibited by the addition of K+ and Na+. Studies of freshwater tilapia, Oreochromis mosambicus, indicated that a probable basolaterally located Ca2+-ATPase was involved in intestinal calcium absorption in this species (12). Ca’+activated ATPases have also been demonstrated in salmonid and goldfish gills and in kidneys from goldfish (17,4). The present study showed that determination

of Nat/K+-

ATPase activity in supernatant fractions from whole intestinal homogenates required about four times higher concentration of substrate than that used in the corresponding

gill

assay (Table 1). Finstad et al. (10) used a final concentration of 3.5 mM ATP for determination of gill Nat/K+-ATPase FIG. 3. Effect of Na2ATP concentration on rainbow trout intestinal Na+/K+-ATPase. The enzyme assays were carried out as described in Materials and Methods, but increasing amounts of NazATP were added to the test tubes, and enzyme preparation was added thereafter. The final concentrations of the incubation mixture were as given in Fig. 2, except for Na+-ions, which varied from 141.6 to 183.8 mM. The results from three assays are shown with different symbols.

NaCl to KC1 was kept at about 2: 1 in the incubation

mix-

in Arctic charr (Salvelinus alpinus L.). Our results show that at this ATP concentration, intestinal Na+/K+-ATPase activity in rainbow trout is approximately 60% of that obtained with 13 mM. The difference may be due to the presence of other enzymes competing for the substrate, such as alkaline phosphatase isoenzymes. The activity of alkaline phosphatase seems to be rather high in the intestinal mucosa of the Atlantic salmon (Gjevre and Landsverk, in preparation) and in other teleost species (18). In the present study, however, less than 1% of the substrate was hydrolysed during the 20 min incubation. Thus, the substrate

ture at various ionic strengths of Nat and K’, ranging from 70 to 480 mM. The optimal Na+/K+-ATPase activity was

seemed to be abundant. Di Costanzo et al. (6) used a final concentration of 1 mM ATP in their determination of Nat/

observed in a wide range from 90 mM to 340 mM, with the highest activity around 185 mM (Fig. 7).

K+-ATPase activity in rainbow trout intestinal mucosa scrapings (Table 1). These authors used microsomal preparations, and differences in the enzyme preparations may be responsible for the increased substrate requirement in our study.

Ouabain was added to the incubation

Increasing the ATP concentration of the incubation mixture from 3.5 mM to 11.8 mM resulted in a reduction of pH from 7.1 to 6.6 in the present study. Although the recommended pH for determination of gill Na+/K+-ATPase in salmonid fish is 7.0 to 7.4 (Table l), our results showed that the relative Na+/K+-ATPase activity in the gill homogenate was only 87% to 88% of the maximum at this

mixture in increasing

concentrations from 8.0 X 10-j mM to about 2 mM. The final concentrations of the incubation mixture were as described in Fig. 2. Although the levels of Na+/K+-ATPase activity in the rainbow trout preparations varied considerably, the inhibitory concentration appeared to be independent of the variation in the Na+/K+-ATPase level. In both

A.-G. Gjevre and L. I. Masdal Naess

164

a

b

60

20

0

I

595

1

6

I

I

6.5

7

0 795

PH

595

6

635

7

795

PH

FIG. 4. Effect of pH on sahnonid intestinal and gill Na+/K+-ATPase. The pH of the incubation solutions (23 mM MgCIZ 6 H,O), 155 mM NaCl, 75 mM KCl, 115 mM imidazole) was adjusted to every 0.1 pHunit from 6.3 to 7.8 with solutions of HCl and NaOH. The pH was not adjusted after the addition of NalATP. The given pH values were measured in the incubation mixture after addition of 11.8 mM ATP. The final concentrations of the incubation mixture were as given in Figure 2. (A) Activity of Na+/K+-ATEase in intestinal preparations from rainbow trout. The results of three assays are shown. (B) Activity of Na+/K+-ATPase in gill preparations from Atlantic salmon. The results of two assays are shown with different symbols.

pH (Fig. 4b). Thus, the Na+/K+-ATPase in both the rainbow trout intestine and the Atlantic salmon gill seem to require a pH lower than 7.0 for maxima1 in vitro activity. This is in accordance with the results of Trigari et al. (34), who found that gill Nat/K+-ATPase of sea bass (Dicentrurthus labrax) had a pH optimum at 6.5, while the pH optimum of kidney Na+/K+-ATPase in this species was 7.0 (24). Katz and Michell (19) reported that the pH optimum depended on the purity of the ATP used as a substrate. They concluded that with purer ATP, renal rat Na+/K+-ATPase had an in vitro pH optimum close to the intracellular pH. However, vanadium-free ATP was used in all studies where the optima1 pH of fish Na+/K+-ATPase was determined (Table 1). Intestinal homogenates with both low and high Na+/K+ATPase activity showed a linear release of phosphate during

60 min incubation at 20°C whereas at 37°C the release of phosphate levelled off after 20 minutes incubation (Fig. 2a, b). The differences in the enzyme activity levels probably reflect a variation in the experimental material. This may be due to individual variation of intestinal Nat/K+-ATPase activity as well as variation between the intestinal segments. Gjevre et al. (in preparation) showed that the activity of Na+/K+-ATPase in the pyloric ceca and proximal intestine is higher compared to that in the distal intestine in the Atlantic salmon. It is reported that toad skin Na+/K+-ATPase is less sensitive to ouabain at 23°C than at 37°C and that the enzyme affinity for K + is increased at the lower temperature (25). However, this cannot explain the differences observed in our study. Neither product inhibition of the enzyme nor the presence of proteolytic enzymes can explain the difference in enzyme activity between high and

Intestinal Na+/K+-ATPase

in Salmonids

165

(20). A discontinuity

3

Na+/K+-ATPase corresponding

in the Arrhenius

plot of seabass gill

occurred at a temperature to

the

rearing

approximately

temperature

of the

fish

(17.7”C) (34). This phenomenon is reported in studies of Nat/K+-ATPases from several fish species (25,15,32,31,4). Trigari et al. (34) concluded that the degree of unsaturation of the plasma membrane seems to play a major role in the temperature-dependent activity of membrane-bound enzymes in fish. However, this does not explain the differences between high and low activity homogenates found in the present study. Our findings indicate that optimal incubation temperature for intestinal preparations is ZO”C, and that the incubation

time may be prolonged to 60 min if enzyme ac-

tivity is low. Regarding the Mg:ATP

ratio and the Na:K

ratio, our

0 0

30

20

10

Magnesium

40

50

60

chloride (mM)

FIG. 5. Effect of Mg ‘+:NazATP ratio on rainbow trout intese tinal Na+/K+*ATPase. The final concentrations of NaZATP of 7.1, 11.8, and 16.5 mM were tested in concentrations of MgC& ‘6 Hz0 from 0 to 61.8 mM. The final concentrations of other components of the incubation mixture were as given in Fig. 2. The median value of three assays are given at each concentration: circle, 7.1 mM; triangle, 11.8 mM; square, 16.5 mM Na2ATP.

low activity homogenates

at 20°C and 37°C during 60 min

incubation. It is reported that the thermal stability of alkaline phosphatase may vary in different organs from the same species as well as between species (37). The intestinal alkaline phosphatase of rainbow trout had lower heat stability than that in liver and kidney. Morever, alkaline phosphatase in rainbow trout tissue showed faster thermal degradation than the corresponding enzyme in the eel (Anguilla japonica) and carp (Cyprinus carpio) (37). In the study by Johnson et al. (17), activity of salmonid gill Na+/K+ATPase appeared to level off after 40 min at 37°C. This may indicate that metnbrane-bound enzymes in the salmonid intestine have a low heat stability. It has been reported that Arrhenius plots of membrane-bound enzymes show discontinuities that seem to be related to a temperature-dependent change of the physical state of the membrane lipids

l:o

II:1

lo:1

6:1

%I

4:l

3:,

21

,:,

,:2

1:s

,:a

15

12

Ratio Na:K FIG. 6. Effect of Na+:K+ ratio on rainbow trout intestinal Na+/K+-ATPase. The total ionic strengths of Na+ and K+ were kept at ca. 200 mM by varying the Na+/K+ ratio. Fours teen different Na+/K+ ratios were tested. The final concentrations of other components of the incubation mixture were as given in Fig. 2. The results from one assay of 11.8 mM and two assays of 16.5 mM Na2ATP are shown. The difference between the two assays of 16.5 mM ATP probably reflects a variation in the experimental material. Triangle, 11.8 mM ATP (one assay); circle, 16.5 mM ATI? (two assays).

and Nass,

present

study*

(24)

from chmook

salmon

from salmonids

(0. aura-

tsha-

from sea bass

Intestine from rainhow trout and Atlantic salmon (Salmo snlar)

Kidneys

alpinw )

Intestinal mucosa from rainhow trout (0. mykiss) Gills from sea bass (Dicentnrchus labXlX) Gills from Arctic charr (Saluehnus

Gills

Gills from gold fish (Car&us IUS) Kidneys from gold fish

wytsch)

Gills

Buffer

determining

9mM imidazole 30 mM HEPES 30 mM Tris 100 mM unidazoie 100 mM Tris 1OOmM Tris 98 mM imidazole 50 mM TEA-HCI 75 mM HEPES 102 mM imidatole 75 mM HEPES 102 mM imidazole

studies

5

5

5 4

5 20

13-16

1

2

18-24

4

20

5

3

2

10

2

2

10

3

5

20

6

]ATW mM

6

]Mg*+l mM

Na+/K+-ATPase

1:1-2:1

2:1

5:l

1:l

2:1

5:l

2:3

1:1

2:1

1:l

3:5

1:l

165t

100

137

100

100

133

12

48

260

100

100

104

INamM

in fish tissue.

[Mg2+]:[ATPl

activitv

44t

25

66

20

20

64

18

12

120

20

20

18

]K+l mM

To make the table more readable, all concentrations and rates are given in whole numbers. Authors marked with an asterisk have exammed and defined the condition for determination of Imaximal Na’ /K’-ATPase xtivity. The other authors referred paper. tThe values given are from the [Nat]: [K’] ratio experiment. Abbreviations: TEA, triethanolamine; HEPES, N-2-hydr~~,xyrthylpirerazinr-N’-ethanesulh~nlc

Gjevre

et al., 1988*

et al., 1989 (10)

Finstad

Pagliarani

et al., 1985* (34)

et al., 1983 (6)

Trigari

Di Costanzo

1982 (38)

and Chavin,

Busacker

1981* (4)

et al., 1977 (17)

Zaugg,

(26)

1976* (14)

1972*

in different

Organ and fish species

mixtures

Gills, intestinal mucosa, and kidneys from several fish species Gills from seawater rainbow trout (Oncorhynchus mykiss) Gills from who salmon (0. kisutch)

of incubation

1970 (16)

Johnson

and Vanstone,

and Kirchner,

Pfeiler

Giles

and Epstein,

Jampol

References

TABLE 1. Comaarison

acid.

do not give

4:l

4:1

2:l

5:l

5:1

2:1

2:3

4:l

2:1

5:1

5:1

6:1

ahout

185

125

203

120

120

197

30

60

380

120

120

122

this

[Na+] + [KC] mM

informanon

[Na+]:[K+]

m the

6.5

7.0

7.1

6.5

7.4

7.0

7.5

7.0

7.2

7.4

7.1

7.8

PH

i CL LL

!-

L! L

Q X’ c: ;;

P 0

167

Intestinal Na+/K’-ATPase in Salmonids

the gills, but intestinal concentrations

preparations

seem to require higher

of substrate and lower incubation

tempera-

ture. This work was jinuncially supported by the Research Council ofNorway (previously the Nurwegian Fisheries Research Council). The authors thank Dr Birgir Dannewig, Dr Knut El&en, for valuable criticism and discussion.

A

n n

l

n

0 n l

4A l n

n

A

A :

A l

l

1

loo

0

I

I

200

300

I

400

509

Ionic strength of Na and K in mM FIG. 7. Effect of different ionic strengths of Na+ and K+ at a ratio of 2: 1 on rainbow trout intestinal Na+/K+-ATPase. In this experiment 14 total ionic strengths of Na+ and K+ from about 70 mM to 480 mM were examined at a constant Na+/K+ ratio of about 2. The final concentrations of other components of the incubation mixture were as given in Fig. 2. The results of three assays are shown with different symbols.

results correspond

and Dr Kjell M&val

well with what has been reported from

other studies (Table 1). The Mg:ATP ratio used by Zaugg (38) and Finstad et al. (10) is somewhat higher than the ratio producing optimal activity (90% of the highest activity) in our study, though. The range of Na:K ratio producing optimal activity in the present study is wide. Sodium concentrations in this range is from about 70 to 190 mM, indicating that salmonid intestinal Nat/K+-ATPase is not as sensitive to high sodium concentrations as was observed for Tilapia red& intestinal Na+/K+-ATPase (2). These workers showed that the intestinal enzyme in this species was inhibited by 140 mM Na+-ions, whereas the gill enzyme was not inhibited by 250 mM. In conclusion, our results indicate that salmonid intestinal Na+/K+-ATPase does not exhibit properties substantially different from these of the corresponding enzyme in

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