Characterization of (Na+, K+)-ATPase in gill microsomes of the freshwater shrimp Macrobrachium olfersii

Comparative Biochemistry and Physiology Part B 126 (2000) 303 – 315 www.elsevier.com/locate/cbpb

Characterization of (Na+, K+)-ATPase in gill microsomes of the freshwater shrimp Macrobrachium olfersii R.P.M. Furriel a, J.C. McNamara b, F.A. Leone a,* a

Departamento de Quı´mica, Faculdade de Filosofia, Cieˆncias e Letras de Ribeira˜o Preto, Uni6ersidade de Sa˜o Paulo, Ribeira˜o Preto 14040 -901, SP, Brazil b Departamento de Biologia, Faculdade de Filosofia, Cieˆncias e Letras de Ribeira˜o Preto, Uni6ersidade de Sa˜o Paulo, Ribeira˜o Preto 14040 -901, SP, Brazil Received 7 September 1999; received in revised form 10 February 2000; accepted 25 February 2000

Abstract To better understand the adaptive strategies that led to freshwater invasion by hyper-regulating Crustacea, we prepared a microsomal (Na+, K+)-ATPase by differential centrifugation of a gill homogenate from the freshwater shrimp Macrobrachium olfersii. Sucrose gradient centrifugation revealed a light fraction containing most of the (Na+, K+)-ATPase activity, contaminated with other ATPases, and a heavy fraction containing negligible (Na+, K+)-ATPase activity. Western blotting showed that M. olfersii gill contains a single a-subunit isoform of about 110 kDa. The (Na+, K+)-ATPase hydrolyzed ATP with Michaelis–Menten kinetics with K0.5 = 16595 mM and Vmax = 686.19 24.7 U mg − 1. Stimulation by potassium (K0.5 =2.490.1 mM) and magnesium ions (K0.5 = 0.7690.03 mM) also obeyed Michaelis–Menten kinetics, while that by sodium ions (K0.5 = 6.09 0.2 mM) exhibited site – site interactions (n= 1.6). Ouabain (K0.5 =61.6 92.8 mM) and vanadate (K0.5 =3.290.1 mM) inhibited up to 70% of the total ATPase activity, while thapsigargin and ethacrynic acid did not affect activity. The remaining 30% activity was inhibited by oligomycin, sodium azide and bafilomycin A1. These data suggest that the (Na+, K+)-ATPase corresponds to about 70% of the total ATPase activity; the remaining 30%, i.e. the ouabain-insensitive ATPase activity, apparently correspond to F0F1- and V-ATPases, but not Ca-stimulated and Na- or K-stimulated ATPases. The data confirm the recent invasion of the freshwater biotope by M. olfersii and suggest that (Na+, K+)-ATPase activity may be regulated by the Na+ concentration of the external medium. © 2000 Elsevier Science Inc. All rights reserved. Keywords: (Na +, K +)-ATPase; Crustacean gill; Ouabain; Vanadate; ATP; Macrobrachium; Freshwater shrimp; Microsomal fraction

1. Introduction

Abbre6iations: AMPOL, 2-amino-2-methylpropan-1-ol; BCIP, 5-bromo-4-chloro-3-indole; DMSO, dimethylsulfoxide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LDH, lactate dehydrogenase; NBT, nitroblue tetrazolium; PEP, phosphoenolpyruvate; PGK, phosphoglycerate kinase; PK, pyruvate kinase. * Corresponding author. Tel.: + 55-16-6023668; fax: + 5516-6338151. E-mail address: [email protected] (F.A. Leone)

The (Na+, K+)-ATPase (E.C.3.6.1.37) is a Ptype ATPase involved in cation transport by many vertebrate and invertebrate tissues, and is encoded by a multigene family (Skou and Esmann, 1992; Pressley, 1996; Beauge´ et al., 1997). Although the minimum functional unit of this enzyme is a heterodimer consisting of a catalytic a-subunit and a glycosylated b-subunit (Skou and Esmann, 1992; Pressley, 1996; Beauge´ et al.,

0305-0491/00/$ - see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 3 0 5 - 0 4 9 1 ( 0 0 ) 0 0 1 8 4 - X

304

R.P.M. Furriel et al. / Comparati6e Biochemistry and Physiology, Part B 126 (2000) 303–315

1997), multiple isoenzymes of the a- and b-subunits are known (Beauge´ et al., 1997). The ubiquitous distribution of the (Na+, K+)-ATPase has generated an extensive literature on the structurefunction relationships of this important ionotransporting enzyme, particularly in vertebrates (Glynn, 1985; Skou and Esmann, 1992; Pressley, 1996; Beauge´ et al., 1997). Numerous reports have dealt with (Na+, K+) (Pe´queux, 1995; Towle, 1997), and K+- and Na+-stimulated (Moretti et al., 1991; Proverbio et al., 1991; Lima et al., 1997) ATPase activities in crustacean gills. Crustaceans inhabit a wide variety of biotopes. In addition to the ancestral marine environment, various groups are successfully established in fresh and brackish waters. The intertidal, estuarine and freshwater ecosystems are among the most osmotically stressful aquatic biotopes, and the establishment of crustaceans in such environments derives from the evolution of adaptive physiological strategies of osmotic and ionic regulation (Pe´queux, 1995; Towle, 1997). The gill epithelium is the primary site of ion uptake in hyper-regulating crustaceans (for revision see Pe´queux, 1995; Towle, 1997). In freshwater brachyuran crabs and crayfishes, the basolateral (Na+, K+)-stimulated ion pump (Pe´queux, 1995; Towle, 1997), acting in series with an apical Na+/H+ (or Na+/NH+ 4 ) antiporter, and/or a sodium channel and/or a V-ATPase (Towle, 1997; Onken and Riestenpatt, 1998; Zare and Greenaway, 1998) provides the driving force for the active transport of sodium ions up the steep concentration gradient from the aqueous medium into the hemolymph. Indeed, (Na+, K+)-ATPase activity increases in hyperosmoregulating crustaceans subjected to reduced salinity, and decreases in those exposed to elevated salinities (Pe´queux, 1995; Lima et al., 1997; Towle, 1997). The palaemonid shrimp Macrobrachium olfersii is a recent invader of the freshwater biotope and is still dependent on brackish water for larval development and metamorphosis (Freire and McNamara, 1995). The adult shrimp is a strong hyper-regulator in freshwater where it maintains about 150 mEq sodium l − 1 in the hemolymph (Lima et al., 1997). In M. olfersii, the gill epithelium consists of pillar cells and mitochondriarich, intralamelar septal cells that exhibit membrane surfaces highly amplified by apical microvilli and deep invaginations, respectively (Freire and McNamara, 1995; McNamara and

Lima, 1997). The intralamellar septal cell membranes are bathed by the hemolymph and house the (Na+, K+)-ATPase (McNamara and Torres, 1999). This efficient uptake mechanism maintains an elevated concentration of sodium ions in the hemolymph and renders M. olfersii a convenient model in which to examine the role of the (Na+, K+)-ATPase in ion transport across the gill epithelium. Several species of palaemonid shrimp commonly occur in the coastal waters and streams which discharge into the Atlantic Ocean along the coast of the state of Sa˜o Paulo. Palaemon northropi is restricted to seawater; Palaemon pandaliformis and Macrobrachium heterochirus are found in streams close to the sea while Macrobrachium acanthurus and M. olfersii are distributed several kilometers inland (Moreira et al., 1983). All are dependent on brackish or seawater for complete larval development. In contrast, hololimnetic species like Macrobrachium potiuna and Macrobrachium brasiliense inhabit continental waters in which they spend their entire life cycles, being totally independent of seawater for reproduction (Moreira et al., 1983; Moreira and McNamara, 1984; Lima et al., 1997). Given the broad distribution of the palaemonids, the use of the gill (Na+, K+)-ATPase as a molecular marker provides a convenient tool with which to evaluate the adaptation of this group to biotopes of different salinities. In this report, we characterize the (Na+, K+)ATPase in a microsomal fraction from the gill tissue of M. olfersii and emphasize that a systematic comparison of the kinetic and structural properties of this enzyme in the gills of these different palaemonid shrimps affords a better comprehension of the differential physiological and biochemical adaptations of this group to the osmotic challenges of life in freshwater.

2. Materials and methods

2.1. Materials All solutions were prepared using Millipore MilliQ ultrapure, apyrogenic water and all reagents were of the highest purity commercially available. Tris, vanadium-free ATP dibarium salt, PEP, NAD+, NADH, imidazole, Hepes, LDH,

R.P.M. Furriel et al. / Comparati6e Biochemistry and Physiology, Part B 126 (2000) 303–315

PK, GAPDH, PGK, NBT, alamethicin, BCIP, oligomycin, ouabain, 3-phosphoglyceraldehyde diethyl acetal, sodium orthovanadate, ethacrynic acid and the H6 molecular weight standard kit were purchased from Sigma Chem Co (St Louis, MO). Triethanolamine and DMSO were from Merck. The protease inhibitor cocktail (1 mM benzamidine, 5 mM antipain, 5 mM leupeptin and 1 mM pepstatin A), bafilomycin A1 and thapsigargin were from Calbiochem. The alpha-5 monoclonal antibody against the a-subunit of the (Na+, K+)-ATPase (all isoforms) was purchased from Developmental Studies Hybridoma Bank (Iowa, USA). Antimouse IgG, alkaline phosphatase conjugate was purchased from Promega Corporation (USA). Crystalline suspensions of LDH, PK, PGK and GAPDH were centrifuged at 10 000× g and 4°C for 30 min, and passed through a Sephadex G-25 column (15×1.4 cm) equilibrated and eluted with 50 mM triethanolamine buffer, pH 7.5, just before use. The dibarium salt of ATP (100 mg in 1.0 ml water) was converted to the free acid form using a BioRad AG50W-X8 ion exchanger (400 mg) and neutralized to pH 7.5 with 50 ml triethanolamine. Glyceraldehyde-3-phosphate was prepared by hydrolysis of 3-phosphoglyceraldehyde diethyl acetal with 150 ml HCl (d =1.18 g ml − 1) in a boiling-water bath for 2 min, and neutralized with 50 ml triethanolamine (d =1.12 g ml − 1). Sodium orthovanadate solution was prepared according to Gordon (1991). When necessary, enzyme solutions were concentrated on Amicon Centriflo cones or Microcon microconcentrators.

2.2. Gill dissection Adult, intermolt M. olfersii (Crustacea, Decapoda) were collected from the marginal vegetation of the Pau´ba river ( B0.5 ‰ salinity, 23°C) in Sa˜o Paulo State (Brazil). The shrimps were transported to the laboratory, maintained in large tanks containing Pau´ba river water (25°C) for 2 – 7 days and fed on alternate days with chopped beef or carrot. For each homogenate prepared, 15 – 20 shrimps were anesthetized by chilling on ice immediately before dissection. The gills were rapidly dissected and placed in 10 ml of ice-cold, 20 mM imidazole homogenization buffer, pH 6.8, containing 250 mM sucrose, 6 mM EDTA and the protease inhibitor cocktail.

305

2.3. Preparation of the gill microsomal fraction The gills were rapidly diced and homogenized in homogenization buffer (20 ml g − 1 wet tissue) using a Potter homogenizer. After centrifuging the crude extract at 10 000×g and 4°C for 30 min, the supernatant was placed on crushed ice and the pellet was resuspended in an equal volume of homogenization buffer. After further centrifugation under the same conditions, the two supernatants were pooled and centrifuged at 100 000× g and 4°C for 2 h. The resulting pellet was resuspended in 20 mM imidazole buffer, pH 6.8, containing 250 mM sucrose (15 ml buffer g − 1 wet tissue); 0.5-ml aliquots were rapidly frozen in liquid nitrogen and stored at −20°C. No appreciable loss of activity was seen after 2 months. When required, the aliquots were thawed, placed on crushed ice and used immediately.

2.4. Continuous-density sucrose gradient centrifugation An aliquot (280 mg protein 0.5 ml − 1) of the (Na+, K+)-ATPase-rich microsomal fraction was layered into a 20 to 50% (w/w) continuous-density sucrose gradient in 20 mM imidazole buffer, pH 6.8, and centrifuged at 180 000×g and 4°C for 2 h using a PV50T2 Hitachi vertical rotor. Fractions (0.5 ml) collected from the bottom of the gradient were then assayed for protein, ATPase activity and refractive index.

2.5. Ultrastructural examination A pellet of the microsomal fraction (346 mg protein) obtained by centrifugation at 100 000× g was prepared for electron microscopy according to Freire and McNamara (1995) and examined in a Philips EM 208 electron microscope.

2.6. Measurement of ATPase acti6ity in the gill microsomal fraction Total ATPase activity was routinely assayed at 25°C using a PK/LDH linked system in which the hydrolysis of ATP was coupled to the oxidation of NADH (Rossi et al., 1979). The oxidation of NADH was monitored at 340 nm (o340 nm, pH 7.5= 6200 M − 1 cm − 1) in a Hitachi U-3000 spectrophotometer equipped with thermostated cell holders. Standard conditions were: 50 mM Hepes

306

R.P.M. Furriel et al. / Comparati6e Biochemistry and Physiology, Part B 126 (2000) 303–315

buffer, pH 7.5, containing 2 mM ATP, 5 mM MgCl2, 10 mM KCl, 50 mM NaCl, 0.14 mM NADH, 2.0 mM PEP, 205 mg PK (123 U) and 275 mg LDH (236 U) in a final volume of 1.0 ml. Alternatively, ATPase activity was quantified using a GAPDH/PGK linked system coupled to the reduction of NAD+ at 340 nm (Rossi et al., 1979). Standard conditions were: 50 mM Hepes buffer, pH 7.5, containing 2.0 mM ATP, 5.0 mM MgCl2, 10 mM KCl, 50 mM NaCl, 1.0 mM NAD+, 0.5 mM sodium phosphate, 1.0 mM G3P, 150 mg GAPDH (12 U) and 20 mg PGK (9 U) in a final volume of 1.0 ml. ATPase activity was also measured as above in the presence of 1 mM ouabain. The difference in measured activity in the absence or presence of ouabain was considered to represent the (Na+, K+)-ATPase activity. ATPase activity was also assayed after 10 min pre-incubation of the preparation with alamethicin (1 mg mg − 1 protein), at 25°C to demonstrate the presence of leaky and/or disrupted vesicles (Bonnafous et al., 1982; Tieleman et al., 1998). Controls without added enzyme were included in each experiment to quantify the non-enzymatic hydrolysis of substrate. The initial velocities were constant for at least 15 min provided that less than 5% of NADH (or NAD+) was oxidized (reduced). Assays were performed on duplicate aliquots; each experiment was repeated using at least three different gill homogenates. One enzyme unit (U) is defined as the amount of enzyme that hydrolyzes 1.0 nmol of ATP min − 1, at 25°C. Except for the dependence of enzyme activity on potassium ions, the PK/ LDH linked system was used to assess (Na+, K+)-ATPase activity. The two coupling systems gave equivalent results with a difference of less than 10%.

2.7. Western blot analysis SDS-PAGE was performed in 5 – 20% gels according to Laemmli (1970), using 20 mg protein per slot. After the run, the gel was split: one half was stained with silver nitrate and the other electroblotted using a Hoefer SE200 system, employing nitrocellulose membranes according to Towbin et al. (1979). The nitrocellulose membrane was incubated for 1 h at 25°C in a 1:10 dilution of alpha-5 monoclonal antibody. After washing (3 times) in 50 mM Tris – HCl buffer, pH 8.0, containing 150 mM NaCl and 0.1% Tween

20, the membrane was incubated for 1 h at 25°C in a 1:7500 dilution of antimouse IgG, alkaline phosphatase conjugate. The specific antibody incorporation was developed in 100 mM Tris–HCl, pH 9.5, containing 100 mM NaCl, 5 mM MgCl2, 0.2 mM NBT and 0.8 mM BCIP.

2.8. Measurement of protein Protein concentration was measured according to Read and Northcote (1981), using bovine serum albumin as the standard.

2.9. Estimation of kinetic parameters The kinetic parameters Vmax, K0.5 (apparent dissociation constant) and the n value (Hill coefficient) for ATP hydrolysis were calculated according to Leone et al. (1992). The curves presented are those which best fit the experimental data. The kinetic parameters provided in the tables are calculated values and represent the mean9SD derived from at least three different gill preparations.

3. Results Sucrose density gradient centrifugation of the gill microsomal fraction revealed two protein fractions, both showing ATPase activity, in the range between 30 and 40% sucrose (Fig. 1). In addition to exhibiting (Na+, K+)-ATPase activity, the light fraction was apparently contaminated by other ATPases. The heavy fraction displayed negligible (Na+, K+)-ATPase activity. Protein recovery in the gradient was greater than 90%. No significant loss of (Na+, K+)-ATPase activity was observed when the microsomal fraction was maintained at 4°C for periods up to 6 h (data not shown). Up to 90%, well-formed, semispherical, closed membrane vesicles of 1799 12 nm diameter (N= 46) abound in thin sections of the pellet (Fig. 2). The electron-lucent vesicle interiors occasionally contained one or two smaller vesicles, myelin figures or electron dense deposits. The high percentage of closed vesicles obtained may derive from the absence of detergent in the homogenization buffer. The (Na+, K+)-ATPase activity unequivocally represents that of leaky and/or disrupted vesicles rather than inside-out vesicles

R.P.M. Furriel et al. / Comparati6e Biochemistry and Physiology, Part B 126 (2000) 303–315

Fig. 1. Sucrose density gradient centrifugation of the microsomal fraction from the gill tissue of Macrobrachium olfersii. A microsomal fraction containing 280 mg protein was layered into a 20 – 50% (w/v) continuous sucrose density gradient in 20 mM imidazole buffer, pH 6.8, and centrifuged for 2 h at 180 000 × g and 4°C. Fractions (0.5 ml) were collected from the bottom of the gradient and analyzed for total ATPase activity ( ); (Na+, K+)-ATPase activity (); ouabain-insensitive ATPase activity ( ); protein concentration () and sucrose concentration ( ). The ATPase activity of each fraction was assayed continuously at 25°C in 50 mM Hepes buffer, pH 7.5, containing 2 mM ATP, 5 mM MgCl2, 10 mM KCl, 50 mM NaCl, 0.14 mM NADH, 2.0 mM PEP and the PK/LDH linked system as described in Section 2. The experiment was performed using duplicate aliquots from at least three different gill homogenates; a representative curve obtained from one homogenate is given.

Fig. 2. Well-formed membrane vesicles in the microsomal fraction from the gill tissue of Macrobrachium olfersii. After centrifugation at 100 000 ×g and 4°C for 2 h, an aliquot containing 346 mg protein was fixed in 0.1 M sodium cacodylate-buffered 0.25 M glutaraldehyde/0.3 M paraformaldehyde, post-fixed in 1% OsO4, dehydrated in a graded ethanol series and embedded in Araldite 502 resin. Magnification: 45 000 ×. Scale bar: 0.2 mm.

307

given that alamethicin, well known to permeabilize membranes to cations and ATP, had no effect on the activity (894.39 26.8 U mg − 1 with, and 875.39 35.0 U mg − 1 without alamethicin) of the microsomal fraction. Western blot analysis identified a single, strongly immunoreactive band of the alpha-5 monoclonal antibody against the a-subunit of the (Na+, K+)-ATPase coincident with a 110 kDa protein band (Fig. 3). The effect of ATP concentration on the (Na+, + K )-ATPase activity of the gill microsomal fraction is shown in Fig. 4. In the presence of 10 mM K+ and 50 mM Na+, the variation in reaction rate with ATP concentration followed a typical Michaelis–Menten (inset B of Fig. 4) curve with Vmax = 686.1924.7 U mg − 1 and K0.5 = 1659 5 mM. When excess free ATP was present, (Na+, K+)-ATPase activity decreased significantly (not shown). Given that the dissociation constant of the ATP-Mg2 + complex is very low compared to the Mg2 + concentration used (5 mM), and that this concentration was higher than the highest ATP concentration used (2 mM), the concentration of free ATP would be negligible compared to that of the ATP-Mg2 + complex. ATP also stimulated the ouabain-insensitive ATPase activity over the same concentration range (inset A of Fig. 4), suggesting the presence of other ATPases in the gill microsomal fraction. The magnesium concentration dependence of the (Na+, K+)-ATPase activity of the gill microsomal fraction under saturating concentrations of ATP and sodium and potassium ions is shown in Fig. 5. Increasing concentrations of magnesium ions from 10 mM to 5 mM stimulated (Na+, K+)-ATPase activity up to 632.5928.5 U mg − 1 following Michaelis–Menten kinetics (inset B of Fig. 5), with a K0.5 value of 0.769 0.03 mM. Excess magnesium ions inhibited (Na+, K+)-ATPase activity (not shown). Importantly, the stimulation of (Na+, K+)-ATPase activity by magnesium ions as shown in Fig. 5 is the sum of two effects: the binding of magnesium ions both to the enzyme and to ATP to form the ATPMg2 + complex. However, while magnesium also stimulated the ouabain-insensitive ATPase activity, the optimal magnesium ion concentration for (Na+, K+)-ATPase activity partially inhibited the ouabain-insensitive ATPase activity (inset A of Fig. 5).

308

R.P.M. Furriel et al. / Comparati6e Biochemistry and Physiology, Part B 126 (2000) 303–315

Fig. 3. SDS-PAGE and Western blot analysis of the microsomal fraction from the gill tissue of Macrobrachium olfersii. Electrophoresis was performed in a 5–20% polyacrylamide gel using 20 mg microsomal protein. Lane 1, silver nitrate-stained SDS-PAGE; lane 2, Western blotting.

Fig. 4. Effect of ATP concentration on (Na+, K+)-ATPase activity in the microsomal fraction from the gill tissue of Macrobrachium olfersii. Activity was assayed continuously at 25°C using 14 mg protein in 50 mM Hepes buffer, pH 7.5, containing 5 mM MgCl2, 10 mM KCl, 50 mM NaCl, 0.14 mM NADH, 2.0 mM PEP and the PK/LDH linked system, as given in Section 2. Inset A: effect of ATP concentration on ouabain-insensitive () and total ATPase activity ( ). Inset B: Hill plot for (Na+, K+)-ATPase activity. The experiment was performed using duplicate aliquots from at least three different gill homogenates; a representative curve obtained from one homogenate is given.

The effect of potassium ions on the (Na+, K )-ATPase activity of the gill microsomal fraction is shown in Fig. 6A. Under saturating conditions of ATP, sodium and magnesium ions, the (Na+, K+)-ATPase activity was stimulated by potassium ions following Michaelis – Menten kinetics; ATP hydrolysis exhibited Vmax =556 9 27.8 U mg − 1 and K0.5 =2.4 90.1 mM. The K+-unstimulated activity was negligible (less than 5%) compared to the stimulated activity. The lack +

Fig. 5. Effect of magnesium ions on (Na+, K+)-ATPase activity in the microsomal fraction from the gill tissue of Macrobrachium olfersii. Activity was assayed continuously at 25°C using 14 mg protein in 50 mM Hepes buffer, pH 7.5, containing 2 mM ATP, 10 mM KCl, 50 mM NaCl, 0.14 mM NADH, 2.0 mM PEP and the PK/LDH linked system, as given in Section 2. Inset A: effect of magnesium ion concentration on ouabain-insensitive () and total ATPase activity ( ). Inset B: Hill plot for (Na+, K+)-ATPase activity. The experiment was performed using duplicate aliquots from at least three different gill homogenates; a representative curve obtained from one homogenate is given.

Fig. 6. Effect of potassium and sodium ions on (Na+, K+)ATPase activity in the microsomal fraction from the gill tissue of Macrobrachium olfersii. For sodium ions, the activity was assayed continuously at 25°C using 14 mg protein in 50 mM Hepes buffer, pH 7.5, containing 2 mM ATP, 5 mM MgCl2, 10 mM KCl, 0.14 mM NADH, 2.0 mM PEP and the PK/ LDH linked system. For potassium ions, the activity was assayed as above in 50 mM Hepes buffer, pH 7.5, containing 2.0 mM ATP, 5.0 mM MgCl2, 50 mM NaCl, 1.0 mM NAD+, 0.5 mM sodium phosphate, 1.0 mM G3P and the GAPDH/ PGK linked system. A, potassium ions. B, sodium ions. Insets: effect of ion concentration on ouabain-insensitive () and total ATPase activity ( ). The experiment was performed using duplicate aliquots from at least three different gill homogenates; a representative curve obtained from one homogenate is given.

R.P.M. Furriel et al. / Comparati6e Biochemistry and Physiology, Part B 126 (2000) 303–315 Table 1 Kinetic parameters for the stimulation by sodium, potassium and magnesium ions and ATP of the (Na+, K+)-ATPase activity from the gills of Macrobrachium olfersii a Effector

Vmax (U mg−1)

K0.5 (mM)

n

Na+ K+ Mg2+ ATP

653.8 926.1 556.1 927.8 632.5 928.5 686.1 9 24.7

6.09 0.2 2.49 0.1 0.76 90.03 0.165 9 0.005

1.6 0.8 0.8 1.0

Assays were performed using 14 mg protein in 50 mM Hepes buffer, pH 7.5, containing 2 mM ATP and variable metal ion concentrations in a final volume of 1.0 ml. The effect of each ion was evaluated under optimal concentrations of the others, as given in Section 2. Data are the mean 9 S.D. from at least three different gill preparations. The GAPDH/PGK linked system was used for the study of K+ only. a

of stimulation of the ouabain-insensitive ATPase activity (212.69 7.6 U mg − 1) over the range from 10 − 5 to 10 − 1 M KCl suggests the absence of K+-stimulated ATPases (inset of Fig. 6A). Under saturating concentrations of ATP, and K+ and Mg2 + , the (Na+, K+)-ATPase activity of the gill microsomal fraction was also stimulated by sodium ions, following a single titration curve, with Vmax=653.8 9 26.1 U mg − 1 and K0.5=6.0 9 0.2 mM (Fig. 6B); however site – site interactions

309

(n=1.6) were observed. The Na+-unstimulated activity was nearly negligible (less than 9%) compared to the stimulated activity. As observed for K+ ions, the lack of stimulation of the ouabaininsensitive ATPase activity (218.19 8.3 U mg − 1) over the range from 10 − 4 to 10 − 1 M NaCl suggests the absence of Na+-stimulated ATPases (inset of Fig. 6B). Table 1 summarizes the kinetic parameters for the modulation of the (Na+, K+)ATPase activity by these effectors. Table 2 shows the effect of various inhibitors on ATP hydrolysis by the gill microsomal fraction. Under saturating conditions of ATP and metal ions, a residual activity of about 30% was found in the presence of 1 mM ouabain. Similar effects were also observed in the absence of sodium plus potassium ions, and in the presence of ouabain plus vanadate or vanadate alone. These data suggest that 70% of the total ATPase activity corresponds to the (Na+, K+)-ATPase. Further, the 30%, residual, ouabain-insensitive ATPase activity seen with ouabain plus vanadate probably resulted from the absence of neutral phosphatases and P-type ATPases other than the (Na+, K+)-ATPase in the microsomal fraction. The insensitivity to thapsigargin and ethacrynic acid of the ouabain-insensitive ATPase activity suggests that the preparation was free of Ca-AT-

Table 2 Effect of various inhibitors on the ATPase activity of the microsomal fraction from Macrobrachium olfersii gillsa Effector Na+ (mM)

K+ (mM)

50 50 50 50 50 50 50 50 50 50 – – – – –

10 10 10 10 10 10 10 10 10 – 10 – – – –

Inhibitor

% Vmax

Ouabain (1 mM) Vanadate (50 mM) Ouabain (1 mM)+sodium azide (100 mM) Ouabain (1 mM)+vanadate (50 mM) Ouabain (1 mM)+bafilomycin A1 (0.4 mM) Ouabain (1 mM)+thapsigargin (0.5 mM) Ouabain (1 mM)+ethacrynic acid (2 mM) Ouabain (1 mM)+oligomycin (1 mg ml−1) Ouabain (1 mM)+DMSO (20 ml ml−1) Ouabain (1 mM) Ouabain (1 mM) Ouabain (1 mM) Oligomycin (1 mg ml−1) Sodium azide (100 mM) –

30.2 9 1.1 28.6 9 1.2 8.4 9 0.4 31.1 9 1.2 11.6 90.6 36.2 91.3 32.99 1.1 7.1 90.4 34.8 91.2 27.9 91.1 28.2 91.2 29.4 91.0 6.19 0.3 8.6 9 0.4 32.29 1.1

Initial rates were measured continuously using the PK/LDH linked system and 14 mg protein in 50 mM Hepes buffer, pH 7.5, containing 2 mM ATP, 5 mM MgCl2 and the given concentrations of sodium and potassium ions in a final volume of 1.0 ml. 100% specific ATPase activity corresponds to 870.1 934.8 U mg−1. Data are the mean 9 S.D. from at least three different gill preparations. a

310

R.P.M. Furriel et al. / Comparati6e Biochemistry and Physiology, Part B 126 (2000) 303–315

Fig. 7. Effect of ouabain on total ATPase activity in the microsomal fraction from the gill tissue of Macrobrachium olfersii. Activity was assayed continuously at 25°C using 14 mg protein in 50 mM Hepes buffer, pH 7.5, containing 2 mM ATP, 5 mM MgCl2, 10 mM KCl, 50 mM NaCl, 0.14 mM NADH, 2.0 mM PEP and the PK/LDH linked system, as given in Section 2. One-hundred percent specific activity corresponds to 870.19 34.8 U mg − 1. Inset: Dixon plot for the estimation of Ki, the enzyme-inhibitor complex dissociation constant, in which 6c is the reaction rate corresponding to (Na+, K+)-ATPase activity only. The experiment was performed using duplicate aliquots from at least three different gill homogenates; a representative curve obtained from one homogenate is given.

Fig. 8. Effect of vanadate on total ATPase activity in the microsomal fraction from the gill tissue of Macrobrachium olfersii. Activity was assayed continuously at 25°C using 14 mg protein in 50 mM Hepes buffer, pH 7.5, containing 2 mM ATP, 5 mM MgCl2, 10 mM KCl, 50 mM NaCl, 0.14 mM NADH, 2.0 mM PEP and the PK/LDH linked system as given in Section 2. A total of 100% specific activity corresponds to 870.19 34.8 U mg − 1. Inset: Dixon plot for the estimation of Ki, the enzyme-inhibitor complex dissociation constant in which 6c is the reaction rate corresponding to (Na+, K+)-ATPase activity only. The experiment was performed using duplicate aliquots from at least three different gill homogenates; a representative curve obtained from one homogenate is given.

Pase, and Na+- or K+-ATPases, respectively. However, the inhibition of this ouabain-insensi-

tive ATPase activity by oligomycin, sodium azide and bafilomycin A1 suggests the presence of F0F1and V-ATPase in the microsomal fraction. The effect of a wide range of ouabain concentrations on the ATPase activity of the gill microsomal fraction is shown in Fig. 7. The preparation showed considerable sensitivity to ouabain, and the inhibition pattern apparently corresponds to that for a single binding site. The apparent Ki value (ouabain concentration at which activity is 50% inhibited) calculated from the Dixon plot was 61.69 2.8 mM (inset of Fig. 7). The inhibition of the gill microsomal ATPase by vanadate was time dependent (not shown) and up to 70% of the ATPase activity was depleted by increasing concentrations of vanadate up to 50 mM (Fig. 8). Under steady state conditions, half maximum inhibition was achieved at about 3 mM vanadate. This value is in close agreement with the Ki (3.29 0.1 mM) calculated from the Dixon plot (inset of Fig. 8).

4. Discussion Osmotic and ionic equilibrium in hyper-regulating decapod Crustacea from estuarine or freshwater habitats is achieved through the coordinated activity of specialized ion transporting cells in the gills, renal organs, and gut. However, the gills, with their high levels of (Na+, K+)-ATPase activity, constitute the primary site of ion uptake from the medium to the hemolymph in these organisms (Pe´queux, 1995; Towle, 1997). Consequently, the enzyme in this tissue is a suitable choice in which to examine the adaptive responses of the Crustacea to such habitats. While the gill microsomal fraction from M. olfersii is contaminated by up to 30% F0F1- and V-type ATPase activities, this detergent-free preparation constitutes a convenient material to study the in vitro properties of the (Na+, K+)ATPase since the native interactions between the enzyme and the membrane bilayer are apparently preserved in the absence of detergent. A further advantage is that the microsomal fraction, which contains up to 90% sealed vesicles, also provides a suitable system to evaluate ion transport kinetics when an isosmotic assay medium is used. Although detergent was not used to disrupt the vesicle membranes, the hydrolytic activity was not necessarily measured in inside-out vesicles. The

R.P.M. Furriel et al. / Comparati6e Biochemistry and Physiology, Part B 126 (2000) 303–315

similar ATPase activities obtained in the presence and absence of alamethicin that permeabilizes membranes to cations and ATP (Bonnafous et al., 1982; Tieleman et al., 1998), suggest that leaky and/or disrupted vesicles predominate during activity measurements. Western blot analysis suggests that M. olfersii gill tissue contains a single a-subunit isoform of (Na+, K+)-ATPase of around 110 kDa. While this value is very similar to those reported for the vertebrate enzyme (Pressley, 1996), it is higher than those found for the brine shrimps Artemia salina (Cortas et al., 1991), A. franciscana (Garcia-Saez et al., 1997) and the crab Cancer pagurus (Balerna et al., 1975). The specific activity of ATP hydrolysis (686.1924.7 U mg − 1) by the (Na+, K+)-ATPase from the gill tissue of M. olfersii is elevated compared to the range (6 – 300 U mg − 1) known for the gill tissue of various freshwater crustaceans (Horiuchi, 1977; Wheatly and Henry, 1987; Harris and Bayliss, 1988; Zare and Greenaway, 1998). For the genus Macrobrachium, specific activities of from 70 to 100 U mg − 1 for gill homogenates (Moretti et al., 1991; Proverbio et al., 1991) and 120 – 200 U mg − 1 for microsomal fractions (Stern et al., 1984; Lima et al., 1997) have been found. Although such variation may result from differences in substrate, ionic conditions and/or preparation procedures and from species-specific characteristics and the salinity of the external medium, the high specific (Na+, K+)-ATPase activity of M. olfersii gill tissue may reflect the recent freshwater invasion by this palaemonid shrimp (Freire and McNamara, 1995). Many species of the genus Macrobrachium are still in the process of penetrating the freshwater biotope, and exhibit high hemolymph osmo-ionic concentrations. This contrasts with the well established freshwater crustaceans that have minimized the osmotic and ionic gradient by lowering hemolymph osmolality and osmotic and ionic permeability, reducing the energetic cost of ionic transport mediated by the gill (Na+, K+)-ATPase (Pe´queux, 1995; Onken and Riestenpatt, 1998). The characterization of ATP binding sites has attracted considerable attention (Beauge´ et al., 1997). Kinetic studies describing high-affinity (K0.5 between 0.1 and 1.0 mM) and low-affinity hydrolyzing sites (K0.5 between 0.01 and 0.2 mM) have been reported in vertebrates (Glynn, 1985; Ward and Cavieres, 1998) where the maximal

311

rates for the high-affinity sites correspond to only 1–10% of the total specific activity of the enzyme (Glynn, 1985; Ward and Cavieres, 1998). The K0.5 of about 0.2 mM calculated for the ATP binding sites of the (Na+, K+)-ATPase from M. olfersii gills is very similar to that estimated for the enzyme from axon membranes of Cancer pagurus (Balerna et al., 1975; Gache et al., 1977) and the gills of Macrobrachium rosenbergii (Stern et al., 1984). This value is a little lower than those for the salt gland and intestinal tissues of A. salina (Cortas et al., 1989), the gills of the crayfish Procambarus clarkii (Horiuchi, 1977) and the crab Potamon potamios (Tentes and Stratakis, 1991). Similar values have been reported for estuarine crabs (Corotto and Holliday, 1996). Evidently, among the crustaceans, the apparent affinity of the (Na+, K+)-ATPase for ATP is virtually independent of the tissue of origin and of the environment inhabited. The lack of evidence for a high-affinity ATP hydrolyzing site for the (Na+, K+)-ATPase from the gill tissue of M. olfersii may derive from the spectrophotometric method used which does not provide good resolution of ATPase activity at very low ATP concentrations. Given this limitation, also reported by various authors using the same methodology (Glynn, 1985), only a single family of ATP-hydrolyzing sites has been described for the enzyme from crustaceans (Gache et al., 1976; Cortas et al., 1989) and other sources (Glynn, 1985). The affinity for magnesium ions (K0.5 = 0.6 mM) of the (Na+, K+)-ATPase from M. olfersii gill tissue is very similar to that reported for the vertebrate enzyme (Glynn, 1985; Robinson and Pratap, 1991), crab axon membranes (Gache et al., 1976; Rossi et al., 1978), and the gill enzymes of other crustaceans (Tentes and Stratakis, 1991; Corotto and Holliday, 1996). However, recent studies show that each a-subunit isoform has a different apparent affinity for sodium and potassium ions when ATP is used as a substrate. These differences depend on the organism and tissue, membrane factors and post-translational modifications (Therien et al., 1996; Beauge´ et al., 1997). The apparent affinity for sodium ions of the (Na+, K+)-ATPase from diverse sources varies widely, ranging from 4 to 25 mM. That for potassium ions is significantly higher and ranges from 0.5 to 2.5 mM (Robinson and Pratap, 1991; Vilsen, 1995; Therien et al., 1996; Beauge´ et al., 1997; Specht et al., 1997). The affinity of the enzyme in

312

R.P.M. Furriel et al. / Comparati6e Biochemistry and Physiology, Part B 126 (2000) 303–315

M. olfersii gills for sodium ions (K0.5 =6 mM) is lower than that reported for the gill enzymes of decapod crustaceans already well established in freshwater, which lie around 0.06 to 0.26 mM (Harris and Bayliss, 1988). Again, this difference may reflect the recent invasion of this biotope by M. olfersii, a notion reinforced by the fact that the K0.5 values for sodium ions reported for estuarine crabs are very similar to that for M. olfersii (Specht et al., 1997). The evolutionary selection of a (Na+, K+)-ATPase showing a high Na+ affinity in the gill epithelium may be an important requirement for the establishment of an organism in freshwater, contributing to the development of an efficient system of ion capture. In contrast to Na+, the apparent affinity of the (Na+, K+)-ATPase from M. olfersii gill tissue for K+ is similar to the values for the gill enzyme from other crustaceans, independent of the salinity of the medium these inhabit (Tentes and Stratakis, 1991; Corotto and Holliday, 1996). The 30% residual ATPase activity in the microsomal fraction observed in the presence of 1.0 mM ouabain or 50 mM vanadate suggests that the (Na+, K+)-ATPase represents up to 70% of the total activity. Further, the very similar values for ATPase activity in the presence of 1.0 mM ouabain plus 50 mM vanadate suggests that the microsomal fraction is free of phosphatase and other P-type ATPase contaminants. This is a relevant finding since (Na+, K+)-ATPase preparations from the gill tissues of crustaceans are usually contaminated by neutral (Lovett et al., 1994), and/or acid and alkaline phosphatases (Reddy and Rao, 1990) and Ca-ATPase (Morris and Greenaway, 1992; Flik et al., 1994). The apparent Ki for ouabain (61.6 92.8 mM) is in the same range (10–500 mM) known for the (Na+, K+)ATPase in the Crustacea (Horiuchi, 1977; Stern et al., 1984; Tentes and Stratakis, 1991; Corotto and Holliday, 1996; Postel et al., 1998), but is higher than those reported for the vertebrate enzyme (Pressley, 1996; Beauge´ et al., 1997). Like the (Na+, K+)-ATPase from most sources (Holleland and Towle, 1990; Skou and Esmann, 1992), that in the microsomal fraction of M. olfersii gill tissue is strongly inhibited by vanadate (Ki =3.2 90.1 mM). Such vanadate inhibition was time-dependent as reported elsewhere (Glynn, 1985). The partial inhibition of the ouabain-insensitive ATPase activity by 100 mM sodium azide or 1 mg ml − 1 oligomycin strongly suggests the presence of

an F-type ATPase in the microsomal fraction. Since the gill tissue of M. olfersii contains abundant mitochondria (Freire and McNamara, 1995; McNamara and Lima, 1997), mitochondrial membrane fragments may contaminate the preparation. The presence of a heavy protein fraction with negligible (Na+, K+)-ATPase activity observed in the sucrose density gradient corroborates this idea. That ouabain-insensitive ATPase activity was also inhibited by 0.4 mM bafilomycin A1 indicates the presence of a V-ATPase (Bowman et al., 1988) in the microsomal fraction of M. olfersii gill tissue. This finding is in agreement with the recent demonstration of a V-ATPase in the gill tissue of brachyuran crabs (Onken and Riestenpatt, 1998) and the crayfish Cherax destructor (Zare and Greenaway, 1998). The detection of a V-ATPase in the microsomal fraction has physiological relevance, since the transporters involved in the uptake of sodium ions from the dilute environment to the intracellular medium are not well characterized in hyper-regulating crustaceans, particularly the freshwater shrimps (Pe´queux, 1995; Towle, 1997; Onken and Riestenpatt, 1998; Zare and Greenaway, 1998). The insensitivity to thapsigargin and ethacrynic acid of the ouabain-insensitive ATPase activity suggests that Ca2 + - (Thastrup et al., 1989) and Na+- or K+-ATPase (Moretti et al., 1991; Proverbio et al., 1991; Lima et al., 1997) were not present in the microsomal fraction of the gill tissue. The present results corroborate the model proposed for ion movements across the gill epithelium of M. olfersii (McNamara and Torres, 1999). The (Na+, K+)-ATPase is located in the intralamelar septal cells, in which the K+ binding sites are bathed by the hemolymph, while the Na+ binding sites face the cytosol. The concentration of Na+ in the gill epithelial cells of freshwater-adapted crustaceans is around 6 mM and that of K+ in the hemolymph is around 5 mM (McNamara et al., 1990; Zare and Greenaway, 1998). Given that maximal activity in vitro resulted at 50 mM Na+ and 10 mM K+, the enzyme activity may be directly modulated by the concentrations of these ions. Does the model adequately explain modulation of enzyme activity with respect to the salinity of the surrounding medium? In freshwater, the hemolymph Na+ concentration in M. olfersii is around 150 mM, while that of K+ in the cytosol is about 120 mM (Skou and Esmann, 1992). During acute exposure to moderate salinity

R.P.M. Furriel et al. / Comparati6e Biochemistry and Physiology, Part B 126 (2000) 303–315

(21‰), hemolymph K+ is maintained fairly constant in contrast to Na+ which increases considerably (McNamara et al., 1990). While the neuroendocrine regulation of gill (Na+, K+)-ATPase activity is well established (McNamara et al., 1990), given the above, and considering the competition between Na+ and K+ for their binding sites, enzyme activity may well be modulated by the Na+ concentration of the external medium. Despite extensive investigations on (Na+, K+)ATPases from crustacean gill tissues (for revision see Pe´queux, 1995), no systematic studies have been conducted on the kinetic properties of the enzyme in gill tissues of hyper-regulating freshwater shrimps. Available data are scanty, incomplete and often limited to comparative studies of enzyme in a single species acclimated to different salinities. The present study better characterizes the (Na+, K+)-ATPase from the gill tissue of a hyper-regulating shrimp and, in the context of further studies on the (Na+, K+)-ATPase of gill tissue in other palaemonids, should contribute to a more comprehensive understanding of the biochemical adaptations involved in the invasion of freshwater.

Acknowledgements This study is part of a Ph.D. Thesis by RPMF and was supported by research grants from FAPESP and CNPq. We thank Dr Hector Barrabin for helpful suggestions and critically reading the manuscript, and Dr Valder de Melo for access to the Electron Microscopy facilities of the Department of Morphology, Faculty of Medicine at Ribeira˜o Preto, USP. FAL and JCM received research scholarships from CNPq. The antibodies used in this study were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242.

References Balerna, M., Fosset, M., Chicheportiche, R., Romey, G., Lazdunski, M., 1975. Constitution and properties of axonal membranes of crustacean nerves. Biochemistry 14, 5500–5511.

313

Beauge´, L.A., Gadsby, D.C., Garrahan, P.J., 1997. Na/K-ATPase and related transport ATPases. Structure, mechanism and regulation. Ann. NY Acad. Sci. 834, 1 – 694. Bonnafous, J.C., Dornand, J., Mani, J.C., 1982. Alamethicin or detergent permeabilization of the cell membrane as a tool for adenylate cyclase determination. Biochim. Biophys. Acta 720, 235 – 241. Bowman, E.J., Siebers, A., Altendorf, K., 1988. Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells. Proc. Natl. Acad. Sci. USA 85, 7972 – 7976. Corotto, F.S., Holliday, C.W., 1996. Branchial Na, K-ATPase and osmoregulation in the purple shore crab Hemigrapsus nudus (Dana). Comp. Biochem. Physiol. 113A, 361 – 368. Cortas, N., Arnaout, M., Salon, J., Edelman, I.S., 1989. Isoforms of Na, K-ATPase in Artemia salina. II Tissue distribution and kinetic characterization. J. Membr. Biol. 108, 187 – 195. Cortas, N., Elstein, D., Markowitz, D., Edelman, I.S., 1991. Anomalous mobilities of (Na+, K+)-ATPase alpha subunit isoforms in SDS-PAGE: identification by N-terminal sequencing. Biochim Biophys. Acta 1070, 223 – 228. Flik, G., Verbost, P.M., Atsma, W., Lucu, C., 1994. Calcium transport in gill plasma membranes of the crab Carcinus maenas: evidence for carriers driven by ATP and a Na+ gradient. J. Exp. Biol. 195, 109 – 122. Freire, C.A., McNamara, J.C., 1995. Fine structure of the gills of the freshwater shrimp Macrobrachium olfersii (Decapoda): effect of acclimation to high salinity medium and evidence for involvement of the lamellar septum in ion uptake. J. Crust. Biol. 15, 103 – 116. Gache, C., Rossi, B., Lazdunski, M., 1977. Mechanistic analysis of the (Na+, K+)-ATPase using new pseudosubstrates. Biochemistry 16, 2957 – 2965. Gache, C., Rossi, B., Lazdunski, M., 1976. Na+, K+activated adenosinetriphosphatase of axonal membranes, cooperativity and control. Eur. J. Biochem. 65, 293 – 306. Garcia-Saez, A., Perona, R., Sastre, L., 1997. Polymorphism and structure of the gene coding for the alpha subunit of the Artemia franciscana. Biochem. J. 321, 509 – 518. Glynn, I.M., 1985. The Na+, K+-transporting adenosine triphosphatase. In: Martonosi, A.N. (Ed.), The Enzymes of Biological Membranes, vol. 3. Plenum Press, New York, pp. 35 – 114. Gordon, J.A., 1991. Use of vanadate as protein-phosphotyrosine phosphatase inhibitor. Methods Enzymol. 201, 477 – 482. Harris, R.R., Bayliss, D., 1988. Gill (Na+/K+)-ATPases in decapod crustaceans: distribution and char-

314

R.P.M. Furriel et al. / Comparati6e Biochemistry and Physiology, Part B 126 (2000) 303–315

acteristics in relation to Na+ regulation. Comp. Biochem. Physiol. 90A, 303–308. Holleland, T., Towle, D.W., 1990. Vanadate but not ouabain inhibits (Na+, K+)-ATPase and sodium transport in tight inside-out native membrane vesicles from crab gill (Carcinus maenas). Comp. Biochem. Physiol. 96B, 177–181. Horiuchi, S., 1977. Characterization of gill Na, K-ATPase in the freshwater crayfish, Procambarus clarki (Girard). Comp. Biochem. Physiol. 56B, 135–138. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Leone, F.A., Degreve, L., Baranauskas, J.A., 1992. SIGRAF: a versatile computer program for fitting enzyme kinetic data. Biochem. Ed. 20, 94–96. Lima, A.G., McNamara, J.C., Terra, W.A., 1997. Regulation of hemolymph osmolytes and gill Na+/K+ATPase activities during acclimation to saline media in the freshwater shrimp Macrobrachium olfersii (Wiegman, 1863) (Decapoda, Palaemonidae). J. Exp. Mar. Biol. Ecol. 215, 81–91. Lovett, D.L., Towle, D.W., Faris, J.E., 1994. Salinitysensitive alkaline phosphatase activity in gills of the blue crab, Callinectes sapidus (Rathbun). Comp. Biochem. Physiol. 109B, 163–173. McNamara, J.C., Lima, A.G., 1997. The route of ion and water movements across the gill epithelium of the freshwater shrimp Macrobrachium olfersii (Decapoda, Palaemonidae): evidence from ultrastructural changes induced by acclimation to saline media. Biol. Bull. 192, 321–331. McNamara, J.C., Saloma˜o, L.C., Ribeiro, E.A., 1990. The effect of eyestalk ablation on haemolymph osmotic and ionic concentrations during acute salinity exposure in the freshwater shrimp Macrobrachium olfersii (Wiegmann) (Crustacea, Decapoda). Hydrobiologia 199, 193–199. McNamara, J.C., Torres, A.H., 1999. Ultracytochemical location of Na+/K+-ATPase activity and effect of high salinity acclimation in gill and renal epithelia of the freshwater shrimp M. olfersii (Crustacea, Decapoda). J. Exp. Zool. 284, 617–628. Moreira, G.S., McNamara, J.C., 1984. Physiological responses of the early zoeal stages of Palaemon pandaliformis (Stimpson) and Palaemon northropi (Rankin) to salinity variation. Hydrobiologia 113, 165–169. Moreira, G.S., McNamara, J.C., Shumway, S.E., Moreira, P.S., 1983. Osmoregulation and respiratory metabolism in Brazilian Macrobrachium (Decapoda, Palaemonidae). Comp. Biochem. Physiol. 74A, 57–62. Moretti, R., Martin, M., Proverbio, T., Proverbio, F., Marin, R., 1991. Ouabain-insensitive Na-ATPase activity in homogenates from different animal tissues. Comp. Biochem. Physiol. 98B, 623–626.

Morris, M.A., Greenaway, P., 1992. High affinity, Ca2 + specific ATPase and (Na+, K+)-ATPase in the gills of a supralittoral crab Leptograpsus 6ariegatus. Comp. Biochem. Physiol. 102A, 15 – 18. Onken, H., Riestenpatt, S., 1998. NaCl absorption across split gill lamellae of hyperegulating crabs: transport mechanisms and their regulation. Comp. Biochem. Physiol. 119A, 883 – 893. Pe´queux, A., 1995. Osmotic regulation in crustaceans. J. Crust. Biol. 15, 1 – 60. Postel, U., Petrausch, G., Riestenpatt, S., et al., 1998. Inhibition of Na+, K+-ATPase and of active iontransport functions in the gills of the shore crab Carcinus maenas induced by cadmium. Marine Biol. 130, 407 – 416. Pressley, T.A., 1996. Structure and function of the Na, K pump-10 years of molecular biology. Miner. Electrolyte Metab. 22, 264 – 271. Proverbio, F., Marin, R., Proverbio, T., 1991. The ouabain-insensitive sodium pump. Comp. Biochem. Physiol. 99A, 279 – 283. Read, S.M., Northcote, D.H., 1981. Minimization of variation in the response to different proteins of the Coomassie blue G dye-binding assay for protein. Anal. Biochem. 116, 53 – 64. Reddy, M.S., Rao, K.V., 1990. Aldrin and lindane impact on acid and alkaline phosphatase activities of prawn, Metapenaeus monoceros: in vitro study. Biochem. Int. 22, 1033 – 1040. Robinson, J.D., Pratap, P.R., 1991. Na, K-ATPase: modes of inhibition by Mg2 + . Biochim. Biophys. Acta 1061, 267 – 278. Rossi, B., Gache, C., Lazdunski, M., 1978. Specificity and interactions at the cationic sites of the axonal (Na+, K+)-activated adenosinetriphosphatase. Eur. J. Biochem. 85, 561 – 570. Rossi, B., Leone, F.A., Gache, C., Lazdunski, M., 1979. Pseudosubstrates of the sarcoplasmic Ca2 + ATPase as tools to study the coupling between substrates hydrolysis and Ca2 + -transport. J. Biol. Chem. 254, 2302 – 2307. Skou, J.C., Esmann, M., 1992. The (Na+, K+)-ATPase. J. Bioenerg. Biomembr. 24, 249 – 261. Specht, S.C., Rodriguez, C., Quinones, L., Velazquez, S., 1997. Effect of high ionic strength and inhibitors of H, K-ATPase on the ouabain sensitive K-p-nitrophenylphosphatase activity in the sea anemone Stichodactyla helianthus. Comp. Biochem. Physiol. 117B, 217 – 224. Stern, S., Borut, A., Cohen, D., 1984. Characterization of (Na+, K+)-ATPase from the gills of the freshwater prawn Macrobrachium rosenbergii (De Man). Comp. Biochem. Physiol. 79B, 47 – 50. Tentes, I., Stratakis, E., 1991. Partial purification and properties of Na, K-ATPase from Potamon potamios. Comp. Biochem. Physiol. 100C, 619 – 624.

R.P.M. Furriel et al. / Comparati6e Biochemistry and Physiology, Part B 126 (2000) 303–315

Thastrup, O., Dawson, A.P., Scharff, O., et al., 1989. Thapsigargin, a novel molecular probe for studying intracellular calcium release and storage. Agents Actions 27, 17–23. Therien, A.G., Nestor, N.B., Ball, N.J., Blostein, R., 1996. Tissue-specific versus isoform-specific differences in cation activation kinetics of the Na, K-ATPase. J. Biol. Chem. 271, 7104–7112. Tieleman, D.P., Breed, J., Berendsen, H.J.C., Sansom, M.S.P., 1998. Alamethicin channels in a membrane: molecular dynamics simulations. Faraday Disc. 111, 209–223. Towbin, H., Staehelin, T.O., Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354. Towle, D.W., 1997. Molecular approaches to understanding salinity adaptation of estuarine animals. Am. Zool. 37, 575–584. Vilsen, B., 1995. Mutant Glu-781(Ala of the rat kidney Na, K-ATPase displays low cation affinity and cata-

.

315

lyzes ATP hydrolysis at a high rate in the absence of potassium ions. Biochemistry 34, 1455 – 1463. Ward, D.G., Cavieres, J.D., 1998. Affinity labeling of two nucleotide sites on Na, K-ATPase using 2%(3%)O-(2,4,6-trinitrophenyl) 8-azidoadenosine-5%[alphaP-32] diphosphate (TNP-8N(3)-[alpha-P-32] ADP) as a photoactivatable probe-label incorporation before and after blocking the high affinity ATP site with fluorescein isothiocyanate. J. Biol. Chem. 273, 33759 – 33765. Wheatly, M.G., Henry, R.P., 1987. Branchial and antennal gland Na+, K+-dependent ATPase and carbonic anhydrase activity during salinity acclimation of the euryhaline crayfish Pacifastacus leniusculus. J. Exp. Biol. 133, 73 – 86. Zare, S., Greenaway, P., 1998. The effect of moulting and sodium depletion on sodium transport and the activities of Na+, K+-ATPase, and V-ATPase in the freshwater crayfish Cherax destructor (Crustacea: Parastacidae). Comp. Biochem. Physiol. 119A, 739 – 745.