Comparative Biochemistry and Physiology Part C 132 (2002) 471–482

Modulation by ammonium ions of gill microsomal (Naq,Kq)ATPase in the swimming crab Callinectes danae: a possible mechanism for regulation of ammonia excretion D.C. Masuia, R.P.M. Furriela, J.C. McNamarab, F.L.M. Mantelattob, F.A. Leonea,* a

´ ˆ ˜ Preto, Universidade de Sao ˜ Paulo, Departamento de Quımica, Faculdade de Filosofia, Ciencias e Letras de Ribeirao Ribeirao Preto 14040-901, SP, Brazil b ˆ ˜ Preto, Universidade de Sao ˜ Paulo, Departamento de Biologia, Faculdade de Filosofia, Ciencias e Letras de Ribeirao Ribeirao Preto 14040-901, SP, Brazil Received 4 February 2002; received in revised form 13 June 2002; accepted 1 July 2002

Abstract q q The modulation by Naq, Kq, NHq 4 and ATP of the (Na ,K )-ATPase in a microsomal fraction from Callinectes danae gills was analyzed. ATP was hydrolyzed at high-affinity binding sites at a maximal rate of Vs35.4"2.1 U mgy1 and K0.5s54.0"3.6 nM, obeying cooperative kinetics (nH s3.6). At low-affinity sites, the enzyme hydrolyzed ATP obeying Michaelis–Menten kinetics with KM s55.0"3.0 mM and Vs271.5"17.2 U mgy1 . This is the first demonstration of a crustacean (Naq ,Kq )-ATPase with two ATP hydrolyzing sites. Stimulation by sodium (K0.5s 5.80"0.30 mM), magnesium (K0.5s0.48"0.02 mM) and potassium ions (K0.5 s1.61"0.06 mM) exhibited site–site interactions, while that by ammonium ions obeyed Michaelis–Menten kinetics (KM s4.61"0.27 mM). Ouabain (KIs 147.2"7.2 mM) and orthovanadate (KIs11.2"0.6 mM) completely inhibited ATPase activity, indicating the absence of contaminating ATPase andyor neutral phosphatase activities. Ammonium and potassium ions synergistically stimulated the enzyme, increasing specific activities up to 90%, suggesting that these ions bind to different sites on the molecule. The presence of each ion modulates enzyme stimulation by the other. The modulation of (Naq ,Kq )-ATPase activity by ammonium ions, and the excretion of NHq 4 in benthic crabs are discussed. 䊚 2002 Elsevier Science Inc. All rights reserved.

Keywords: Ammonium ion; ATP; Crustacean gill; Microsomal fraction; (Naq,Kq)-ATPase; Ouabain; Vanadate; Callinectes

1. Introduction The ubiquitous (Naq,Kq)-ATPase (E.C. 3.6.1.37), a member of the P2 ATPase family, Abbreviations: G3P, glyceraldehyde-3-phosphate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DTT, Dithiothreitol; LDH, lactate dehydrogenase; PGK, phosphoglycerate kinase; PEP, phosphoenolpyruvate; PK, pyruvate kinase; Hepes, N-(2-hydroxyethyl) piperazine-N9-ethanesulfonic acid. *Corresponding author. Tel.: q55-16-602-3668; fax: q5516-633-8151. E-mail address: [email protected] (F.A. Leone).

couples the hydrolysis of ATP to the translocation of two Kq ions into, and three Naq ions out of the cell. The electrochemical gradient generated is critical to maintaining cell osmotic equilibrium, in establishing the resting membrane potential, and in determining the excitable properties of nerve and muscle cells (Hu and Kaplan, 2000; Geering 2000). Crustacean gills play a central role in active ´ osmo- and iono-regulatory processes (Pequeux, ¨ 1995), respiratory gas exchange (Bottcher and Siebers, 1993), hemolymph acid–base regulation

1532-0456/02/$ - see front matter 䊚 2002 Elsevier Science Inc. All rights reserved. PII: S 1 5 3 2 - 0 4 5 6 Ž 0 2 . 0 0 1 1 0 - 2

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(Henry and Wheatly, 1992), and the excretion of nitrogenous metabolic end products (Cameron and Batterton, 1978; Weihrauch et al., 1998, 1999). In hyperosmoregulating animals, the gills act as a selective interface that actively absorbs Naq and Cly ions from dilute external media; the (Naq,Kq)-ATPase is a key molecule in this pro´ cess (Pequeux, 1995; Furriel et al., 2000). Regardless of whether they occupy marine or freshwater habitats, most crustaceans excrete their nitrogenous end products largely as ammonia (NH3qNHq 4 ). This process is still poorly understood both in osmoconformers and in hyperegulators, although recent evidence suggests that the (Naq,Kq)-ATPase is also important in the active excretion of ammonium ions across the gill epithelia of crustaceans (Weihrauch et al., 1998, 1999) and fish (Mallery, 1983). The swimming crab Callinectes danae Smith, 1869, is a large, euryhaline, portunid crab of commercial interest distributed in the Western Atlantic from Florida to the south of Brazil (Melo, 1996). It inhabits estuaries, mangroves, bays and adjacent coastal waters, but is more commonly found on sand andyor silt bottoms, occurring from the intertidal zone up to 75 m depth (Williams, 1974). Like most crabs of the genus, C. danae moves among areas of varying salinity regimes ˜ (Guerin and Stickle, 1997). In Ubatuba Bay, Sao Paulo State, Brazil, most adult crabs are found throughout the year in waters of moderate salinity (28–35‰), but are influenced by freshwater during the Southern summer (Mantelatto and Fransozo, 2000, 1999). Studies on the osmoregulatory behavior of C. danae are still unavailable; however, the related species, C. sapidus and C. similis, hyperegulate well above isosmoticity in low salinities, and hyporegulate slightly or are hypoconformers in salinities above 35‰ (Guerin and Stickle, 1997). C. danae possesses eight laterally disposed gill pairs, arranged as two arthrobranchiae and six pleurobranchiae. The gill epithelium in crabs of the genus Callinectes consists of a single layer of cells lining the cuticle on either side of the gill lamellae with occasional pillar cells forming connections across the hemolymph space (Copeland and Fitzjarrell, 1968; Towle and Kays, 1986). Thin epithelial cells, which presumably function in gas exchange, predominate in the anterior gills, while thick cells, possessing membrane surfaces highly amplified by apical folds and deep basolateral

invaginations, apparently involved in ion transport, predominate within the posterior gill lamellae (Copeland and Fitzjarrell, 1968; Towle and Kays, ´ 1986; Pequeux, 1995). The basolateral surface of the epithelial cells is bathed by the hemolymph and houses the (Naq,Kq)-ATPase (Towle and Kays, 1986). Details of the life cycle of C. danae are not well known and it is uncertain to what extent the species depends on estuaries for reproduction, either as recruitment sites for post-larvae or as nursery grounds for juveniles (Weber and Levy, 2000). Apparently, while ovigerous females copulate in waters of low salinity, spawning occurs in high salinities where larval survival and floatability are maximal (Paul, 1982). In this report, we characterize the (Naq,Kq)ATPase in a microsomal fraction from the gill tissue of C. danae held in seawater of 33‰ salinity. In particular, we focus on the direct effect of ammonium ions on the hydrolytic activity of the (Naq,Kq)-ATPase to better comprehend the role of this enzyme in the active excretion of this nitrogenous end product by the crustacean gill. This is the first report showing that (Naq,Kq)ATPase activity can be modulated by ammonium ions independently of the presence of Kq ions, a mechanism that may be of considerable relevance for ammonium excretion. 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, NADq, NADH, imidazole, Hepes, LDH, PK, GAPDH, PGK, alamethicin, ouabain, 3-phosphoglyceraldehyde diethyl acetal and sodium orthovanadate were purchased from Sigma Chemical Co. (USA). Triethanolamine was from Merck (Germany), and the protease inhibitor cocktail (1 mM benzamidine, 5 mM antipain, 5 mM leupeptin and 1 mM pepstatin A) was from Calbiochem (USA). Crystalline suspensions of LDH, PK were centrifuged at 14 000 rpm at 4 8C for 15 min in an Eppendorf Model 5810 refrigerated centrifuge. The pellet was resuspended in 300 ml of 50 mM Hepes buffer, pH 7.5, transferred to a YM-10 Microcon filter and washed five times in the same buffer at

D.C. Masui et al. / Comparative Biochemistry and Physiology Part C 132 (2002) 471–482

14 000 rpm and 4 8C for 15 min each until complete removal of ammonium ions (tested using the Nessler reagent). Finally, the pellet was resuspended in the original volume. For PGK and GAPDH, the suspension was treated as above in 50 mM triethanolamine buffer, pH 7.5, containing 1 mM DTT. The dibarium salt of ATP (100 mgy 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 (ds1.12 g mly1). G3P was prepared by hydrolysis of 3-phospho-glyceraldehyde diethyl acetal with 150 ml HCl (ds1.18 g mly1) in a boiling-water bath for 2 min, and neutralized with 50 ml triethanolamine. Sodium orthovanadate solution was prepared according to Gordon (1991). When necessary, enzyme solutions were concentrated on YM-10 Amicon Centriflo cones or Microcon filters. 2.2. Gill excision Adult, intermolt specimens of C. danae were collected from Ubatuba Bay (238269S, 458029W), ˜ Paulo State (Brazil) using double rig trawl Sao nets. The crabs were transported to the laboratory, maintained in tanks containing aerated seawater (33‰ salinity, 25 8C) for 2–7 days and fed with shrimp on alternate days. Crab fresh body weight was 73.9"17.3 g while carapace width, measured between the penultimate and last lateral spines, was 82.0"5.65 mm. For each homogenate prepared, 5–8 crabs were anesthetized by chilling in a freezer at y20 8C; the entire carapace was then quickly removed and the animals sacrificed. Posterior gill pairs 6, 7 and 8 were rapidly excised and placed in 10 ml of ice-cold 20 mM imidazole buffer, pH 6.8, 250 mM sucrose, 6 mM EDTA, containing the protease inhibitor cocktail (homogenization buffer). 2.3. Preparation of the gill microsomal fraction The gills were rapidly diced and homogenized in the homogenization buffer (20 mlyg wet tissue) using a Potter homogenizer. After centrifuging the crude extract at 20 000 g for 35 min at 4 8C, the supernatant was placed on crushed ice, and the pellet was resuspended in an equal volume of homogenization buffer. After further centrifugation as above, the two supernatants were pooled and centrifuged at 100 000 g for 2 h at 4 8C. The

473

resulting pellet was homogenized in 20 mM imidazole buffer, pH 6.8, 250 mM sucrose (10 mlyg wet tissue). Finally, 0.5 ml aliquots were rapidly frozen in liquid nitrogen and stored at y20 8C. No appreciable loss of activity was seen after 2months storage. When required, the aliquots were thawed, placed on crushed ice and used immediately. 2.4. Western blot analysis SDS-PAGE and Western blot analyses were performed as described by Furriel et al. (2000). 2.5. ATPase activity in the gill microsomal fraction ATPase activity was routinely assayed at 25 8C using a PKyLDH linked system in which the hydrolysis of ATP was coupled to the oxidation of NADH according to Furriel et al. (2000). The oxidation of NADH was monitored at 340 nm (´340 nm, pH 7.5s6200 My1 cmy1) in a Hitachi U-3000 spectrophotometer equipped with thermostatted cell holders. Standard conditions were: 50 mM Hepes buffer, pH 7.5, 2 mM ATP, 3 mM MgCl2, 10 mM KCl, 100 mM NaCl, 0.14 mM NADH, 2.0 mM PEP, containing 82 mg PK (49 U) and 110 mg LDH (94 U) in a final volume of 1.0 ml. Alternatively, ATPase activity was estimated using a GAPDHyPGK linked system coupled to the reduction of NADq at 340 nm (Furriel et al., 2000). Standard conditions were: 50 mM triethanolamine buffer, pH 7.5, 2 mM ATP, 3 mM MgCl2, 10 mM KCl, 100 mM NaCl, 1 mM NADq, 0.5 mM sodium phosphate, 1 mM G3P, containing 150 mg GAPDH (12 U) and 20 mg PGK (9 U) in a final volume of 1 ml. ATPase activity was also assayed at 25 8C after 10 min pre-incubation of the preparation with alamethicin (1 mgymg protein) to demonstrate the presence of leaky andyor disrupted vesicles (Furriel et al., 2000). Controls without added enzyme were included in each experiment to quantify the nonenzymatic hydrolysis of substrate. The initial velocities were constant for at least 15 min provided that less than 5% of NADH (or NADq) 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 per minute, at 25 8C. Except for experiments on the

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derived from preparations.

at

least

three

different

gill

3. Results

Fig. 1. SDS-PAGE and Western blot analysis of the microsomal fraction from the posterior gill tissue of C. danae. Electrophoresis was performed in a 5–20% polyacrylamide gel using 45 mg microsomal protein. Lane A: silver nitrate-stained SDSPAGE; lane B: Western blotting against the a-subunit of the (Naq,Kq)-ATPase as revealed by an antimouse IgG, alkaline phosphatase conjugate.

Western blot analysis identified a single, strongly immunoreactive band of the alpha-5 monoclonal antibody against the a-subunit of the (Naq,Kq)ATPase coincident with a 100-kDa protein band, suggesting the presence of a single a-subunit isoform (Fig. 1). The effect of increasing ATP concentration on the (Naq,Kq)-ATPase activity of the gill microsomal fraction from C. danae is shown in Fig. 2. Under saturating Kq, Naq and Mg2q concentrations, ATP hydrolysis followed a well-defined biphasic curve. At the high-affinity sites (10y8 – 10y6 M), ATP was hydrolyzed obeying cooperative kinetics (nHs3.6) with Vs35.4"2.1 U mgy1 and K0.5s0.054"0.004 mM (inset to Fig. 2). Typical Michaelis–Menten kinetics was seen for the low-affinity sites (10y6 –10y3 M) at which ATP was hydrolyzed with Vs271.5"17.2 U mgy1 and KMs55.0"3.0 mM). This is the first demonstration of two ATP hydrolyzing sites on a crustacean (Naq,Kq)-ATPase. No significant differences in specific activity of the enzyme from

dependence of enzyme activity on Kq and NH4q ions, the PKyLDH linked system was used to assay (Naq,Kq)-ATPase activity throughout. The two coupling systems gave equivalent results with a difference of less than 10%. 2.6. Protein Protein concentration was measured according to Read and Northcote (1981) using bovine serum albumin as the standard. 2.7. Estimation of kinetic parameters The kinetic parameters V (maximum velocity), K0.5 (apparent dissociation constant), KM (Michaelis–Menten constant), nH (Hill coefficient) and the enzyme-inhibitor apparent dissociation constant (KI) for ATP hydrolysis were calculated according to Furriel et al. (2000). The curves presented are those which best fit the experimental data. The kinetic parameters provided in the tables are calculated values and represent the mean"S.D.

Fig. 2. Effect of ATP concentration on (Naq,Kq)-ATPase activity in the microsomal fraction from the gill tissue of C. danae. Activity was assayed continuously at 25 8C using 9.4 mg protein samples in 50 mM Hepes buffer, pH 7.5, 3 mM MgCl2, 10 mM KCl, 100 mM NaCl, 0.14 mM NADH, 2.0 mM PEP, containing 82 mg PK (49 U) and 110 mg LDH (94 U). The experiment was performed using duplicate aliquots from at least three different gill homogenates; a representative curve obtained from one homogenate is given. Inset: high-affinity sites.

D.C. Masui et al. / Comparative Biochemistry and Physiology Part C 132 (2002) 471–482

Fig. 3. Effect of magnesium and sodium ions on (Naq,Kq)ATPase activity in the microsomal fraction from the gill tissue of C. danae. Activity was assayed continuously at 25 8C in samples containing 9.4 mg protein. The experiments were performed using duplicate aliquots from at least three different gill homogenates; representative curves obtained from one homogenate are given. (a) Magnesium ions. Assay performed in 50 mM Hepes buffer, pH 7.5, 2 mM ATP, 10 mM KCl, 100 mM NaCl, 0.14 mM NADH, 2 mM PEP, containing 82 mg PK (49 U) and 110 mg LDH (94 U). (b) Sodium ions. Assay performed in 50 mM Hepes buffer, pH 7.5, 2 mM ATP, 10 mM KCl, 3 mM MgCl2, 0.14 mM NADH, 2 mM PEP, containing 82 mg PK (49 U) and 110 mg LDH (94 U). Insets: respective Hill plots.

adult males or females were found. However, microsomal fractions from the anterior gills exhibited a twofold lower specific activity than posterior gills (not shown). Alamethicin had no effect on the (Naq,Kq)-ATPase activity of the microsomal fraction, suggesting that only leaky andyor disrupted vesicles were present. Magnesium ions were essential for the (Naq,Kq)-ATPase activity of the gill microsomal fraction (Fig. 3a). Under saturating ATP, Naq and Kq concentrations, increasing concentrations of

475

Mg2q from 10 mM to 3 mM stimulated (Naq,Kq)-ATPase activity up to Vs309.7"15.7 U mgy1 with a K0.5s0.48"0.02 mM. Cooperative effects (nHs1.3) were observed for the interaction of Mg2q ions with the enzyme, suggesting multiple binding sites (inset to Fig. 3a). As seen for ATP, (Naq,Kq)-ATPase activity was significantly inhibited by excess free Mg2q (not shown). A typical curve demonstrating the Naq ion concentration dependence of (Naq,Kq)-ATPase activity under saturating ATP, Kq and Mg2q concentrations is shown in Fig. 3b. The Naqdependence of the stimulation was characterized by Vs302.1"14.1 U mgy1 and K0.5s5.80"0.30 mM, with multiple binding sites (nHs1.6) being observed (inset to Fig. 3b). The Naq-unstimulated activity was nearly negligible (f5% of Naqstimulated activity). Table 1 summarizes the kinetic parameters for the modulation of (Naq,Kq)-ATPase activity by ATP, Mg2q and Naq ions. The effect of Kq and NHq 4 ions on the hydrolysis of ATP by the gill microsomal (Naq,Kq)ATPase is shown in Fig. 4. Under saturating ATP, Naq and Mg2q concentrations, and in the absence of NHq 4 ions, stimulation of enzyme activity by Kq ions occurred through site–site interactions (nHs2.5), suggesting multiple binding sites. The kinetic parameters calculated for the hydrolysis of ATP were Vs294.0"11.8 U mgy1 and K0.5s 1.61"0.06 mM (Fig. 4a). The Kq-unstimulated activity was negligible (f5% of Kq-stimulated activity). Stimulation of the phosphohydrolytic activity by Kq ions in the presence of fixed NHq 4 ion concentrations gave interesting results. q At 1–50 mM NHq 4 concentrations, increasing K Table 1 Kinetic parameters for the stimulation by sodium and magnesium ions and ATP of (Naq,Kq)-ATPase activity in the gills of C. danae Effector

V (U mgy1)

K0.5 (mM)

KM (mM)

nH

q

302.1"14.1 309.7"15.7 35.4"2.1 271.5"17.2

5.80"0.30 0.48"0.02 0.054"0.004 –

– – – 55.0"3.0

1.6 1.3 3.6 1.0

Na Mg2q ATP

Initial rates were measured using 9.4 mg protein in 50 mM Hepes buffer, pH 7.5, 2 mM ATP and containing 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. Data are the mean"S.D. from at least three different gill preparations.

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Fig. 4. Stimulation of the (Naq,Kq)-ATPase activity in the microsomal fraction from the gill tissue of C. danae by potassium and ammonium ions. The activity was assayed continuously at 25 8C using 7 mg protein in 50 mM triethanolamine buffer, pH 7.5, 2 mM ATP, 3 mM MgCl2, 100 mM NaCl, 1 mM NADq, 0.5 mM sodium phosphate, 1 mM G3P, containing 150 mg GAPDH (12 U) and 20 mg PGK (9 U). The experiments were performed using duplicate aliquots from at least three different gill homogenates; representative curves obtained from one homogenate are given. (a) Stimulation by potassium ions at a fixed concentration of ammonium ions: (%) OmM; (d) 1 mM; (m) 5 mM; (j) 20 mM; (⽧) 50 mM. (b) Stimulation by ammonium ions at a fixed concentration of potassium ions: (%) OmM; (d) 3 mM; (m) 5 mM; (j) 7 mM; (⽧) 10 mM.

ion concentrations stimulated the (Naq,Kq)-ATPase activity following a single titration curve, up to Vs557.0"28.3 U mgy1, a value 89% greater than that estimated in the absence of NHq 4 ions. Further, site–site interactions were also observed for the interaction of Kq ions with the enzyme, independently of the presence of NHq 4 ions. The synergistic stimulation of (Naq,Kq)-ATPase by q NHq ions suggests that these ions bind 4 plus K to different sites. A very different situation pertains q for the stimulation by NHq ion 4 ions at fixed K concentrations (Fig. 4b). Under saturating ATP, Mg2q, and Naq concentrations, and in the absence

of Kq ions, the enzyme was stimulated by NHq 4 ions obeying typical Michaelis–Menten kinetics, with Vs377.8"22.7 U mgy1 and K0.5s 4.61"0.27 mM. The NHq 4 -unstimulated activity was nearly negligible (f7% of NHq 4 -stimulated activity). When stimulation of enzyme activity by NHq 4 ions was estimated at fixed concentrations of Kq ions (3–10 mM), a maximal value of Vs 529.6"23.8 U mgy1 was recorded; however, under these conditions, ATP hydrolysis occurred according to Michaelis–Menten kinetics. The simultaneous presence of Kq and NHq ions 4 caused a synergistic stimulation of the enzyme, suggesting that these ions bind to different sites. Control experiments performed using choline chloride to substitute each ion independently showed no synergistic effect (not shown), suggesting that rather than ionic strength effects, the stimulation of enzyme activity was a consequence of ion binding. Table 2 summarizes the kinetic parameters calculated for the stimulation of (Naq,Kq)-ATPase activity in the gill microsomal fraction by Kq and NHq 4 ions. Fig. 5 shows the dependence of V, nH and KM (or K0.5) estimated for the stimulation of the (Naq,Kq)-ATPase activity by NH4q (or Kq) ions at fixed Kq (or NHq 4 ) ion concentrations. The changes in V and KM (or K0.5) in response to q stimulation of enzyme activity by NHq 4 (or K ) q q ions at a fixed concentration of K (or NH4 ) ions suggest that each ion modulates the dependence of ATP hydrolysis on the other (Fig. 5b, c, e, f). However, while cooperative effects were observed for the modulation by NHq 4 ions of the stimulation of ATP hydrolysis by Kq ions (Fig. 5a), Michaelis–Menten kinetics were seen for the modulation by Kq ions of the stimulation of ATP hydrolysis by NHq 4 ions (Fig. 5d). These data suggest that multiple binding sites for Kq but not for NHq 4 are present, independently of the presence of NHq 4 ions (Fig. 5a and d). The effect of a wide range of ouabain concentrations on the ATPase activity of the gill microsomal fraction is shown in Fig. 6a. ATPase activity was completely inhibited by 1 mM ouabain, both in the absence and in the presence of saturating concentrations of NHq 4 ions. The inhibition patterns appear to correspond to those for a single inhibitor-binding site and, according to the Dixon plot, the calculated KI values were 147.2"7.2 and 53.9"3.3 mM for ouabain inhibition in the

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477

Table 2 Kinetic parameters for the stimulation by potassium andyor ammonium ions of (Naq,Kq)-ATPase activity in the gills of C. danae wKqx (mM)

wNHq 4 x (mM)

V (U mgy1)

K0.5 (mM)

KM (mM)

nH

VyK

Variable Variable Variable Variable Variable 0 3 5 7 10

0 1 5 20 50 Variable Variable Variable Variable Variable

294.0"11.8 340.9"11.9 413.8"18.6 482.9"18.3 557.0"28.3 377.8"22.7 464.3"23.2 468.8"16.4 501.8"22.5 529.6"23.8

1.61"0.06 2.11"0.07 2.85"0.13 2.92"0.11 5.50"0.30 – – – – –

– – – – – 4.61"0.27 8.92"0.44 7.85"0.27 7.91"0.35 4.74"0.20

2.5 1.6 2.0 2.4 1.4 1.1 1.2 1.1 1.1 1.2

182.6 161.5 145.2 165.4 101.2 81.9 52.6 59.7 63.4 125.0

Initial rates were measured using 7 mg protein in 50 mM triethanolamine buffer, pH 7.5, 2 mM ATP, 3 mM MgCl2 , 100 mM NaCl, and containing the given concentrations of NH4Cl and KCl, in a final volume of 1.0 ml. Data are the mean"S.D. from at least three different gill preparations.

absence or presence of NHq 4 ions, respectively (inset to Fig. 6a). Orthovanadate also completely inhibited the (Naq,Kq)-ATPase at concentrations up to 0.1 mM (Fig. 6b). The inhibition patterns appear to correspond to those for a single inhibitorbinding site and, according to the Dixon plot, the calculated KI values were 11.2"0.6 and 2.9"0.1 mM for orthovanadate inhibition in the absence or presence of NHq 4 ions, respectively (inset to Fig.

6b). These data reveal that other ATPase andyor neutral phosphatase activities are not present in the gill microsomal preparation. 4. Discussion The present study discloses two important findings. The (Naq,Kq)-ATPase from posterior gills of C. danae is the first crustacean enzyme to

q Fig. 5. Dependence of the kinetic parameters calculated for the stimulation of the (Naq,Kq )-ATPase activity by NHq 4 (or K ) ions q q in the presence of fixed concentrations of K (or NH4 ) ions. The activity was assayed continuously at 25 8C using 7 mg protein samples in 50 mM triethanolamine buffer, pH 7.5, 2 mM ATP, 3 mM MgCl2 , 100 mM NaCl, 1 mM NADq , 0.5 mM sodium phosphate, 1 mM G3P, containing 150 mg GAPDH (12 U) and 20 mg PGK (9 U). Each data point represents the corresponding kinetic parameter calculated from a representative saturation curve comprising at least 12 experimental substrate concentrations. The experiments were performed using duplicate aliquots from at least three different gill homogenates.

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Fig. 6. Effect of ouabain and orthovanadate on (Naq,Kq)ATPase activity in the microsomal fraction from the gill tissue of C. danae, in the presence and absence of ammonium ions. Activity was assayed continuously at 25 8C using 7 mg protein in 50 mM Hepes buffer, pH 7.5, 2 mM ATP, 3 mM MgCl2, 10 mM KCl, 100 mM NaCl, 0.14 mM NADH, 2.0 mM PEP, containing 82 mg PK (49 U), 110 mg LDH (94 U) and (s) 50 mM NH4Cl or (d) no NH4Cl. 100% specific activity corresponds to 555.4"28.8 U mgy1 and 308.2"12.3 U mgy1 in the presence or absence of ammonium ions respectively. The experiments were performed using duplicate aliquots from at least three different gill homogenates; representative curves obtained from one homogenate are given. (a) Ouabain. (b) Orthovanadate. Insets: Dixon plots for the estimation of the values of KI.

possess two distinct sites of ATP hydrolysis. This (Naq,Kq)-ATPase is synergistically stimulated by q NHq ions. 4 and K Since both the osmoregulatory and excretory processes in the aquatic Crustacea occur mainly ´ via the gill epithelium (Pequeux, 1995; Weihrauch et al., 1999), a comprehensive kinetic characterization of this (Naq,Kq)-ATPase in a typical brachyuran crab like C. danae will provide a better understanding of the biochemical mechanisms that underlie such processes. Further, the preparation

employed is a useful system in which to study the properties of the enzyme in vitro without further purification since the entire ATPase activity of the gill microsomal fraction appears to derive from the (Naq,Kq)-ATPase. This contrasts with the gill tissues of other crabs that are often contaminated with Ca2q-ATPase (Wheatly, 1999), Naq-ATPase (Proverbio et al., 1991) andyor neutral phosphatase activities (Lovett et al., 1994). The specific activity of Vs306.9"17.2 U mgy1 for the (Naq,Kq)-ATPase from the gill tissue of C. danae with respect to ATP hydrolysis is significantly greater than that reported for gill homogenates (35–225 U mgy1) from other species of Callinectes (Lovett and Watts, 1995; Lucu et al., 2000). However, despite some discrepant values (Harris and Bayliss, 1988), the specific activity for C. danae is similar to that for other euryhaline brachyurans acclimated to seawater (D’Orazio and Holliday, 1985; Lucu et al., 2000). Significantly greater specific activities (907–1380 U mgy1) have been reported after treatment of microsomal fractions with deoxycholate, but the Hill coefficient of approximately 1.0 suggests that ATP hydrolysis in the range of 10y6 –3=10y3 M occurred without site–site interactions (Savage and Robinson, 1983). Although high- (KM between 0.1 and 1.0 mM) and low-affinity hydrolyzing sites (KM between 0.01 and 0.2 mM) have been reported for the vertebrate enzyme (Glynn, 1985; Ward and Cavieres, 1998), the characterization of ATP binding sites for the (Naq,Kq)-ATPase is still controversial (Beauge´ et al., 1997; Martin and Sachs, 2000) since maximal rates for the high-affinity sites correspond to only 1–10% of the total specific activity (Glynn, 1985; Ward and Cavieres, 1998). In the case of (Naq,Kq)-ATPase from gill tissue of C. danae, ATP is hydrolyzed by a family of low-affinity sites (KMs55.0"3.0 mM) that obey Michaelis–Menten kinetics, and high-affinity sites (K0.5s0.054"0.004 mM), which contrasts with the data reported for the enzyme from Cancer pagurus axon membranes (Gache et al., 1977), and the gills of the freshwater shrimp M. olfersii (Furriel et al., 2000), and C. sapidus (Wheatly and Henry, 1987) and certain euryhaline crabs (Holliday, 1985; D’Orazio and Holliday, 1985; Corotto and Holliday, 1996). However, this highaffinity site, apparent at 10y8 –10y6 M ATP concentrations is consistent with the general Post-Albers model of a (ab)4 tetraprotomer for

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the functional unit of the enzyme in the membrane. According to this model, each reaction intermediate reflects half of the site property of the intermediate while the other half binds ATP (Taniguchi et al., 2001). In the absence of magnesium ions, the (Naq,Kq)-ATPase of C. danae does not hydrolyze ATP, which agrees with data reported elsewhere (Furriel et al., 2000; Glynn, 1985). As for the crab axon enzyme (Gache et al., 1976), the increase in hydrolysis rate with Mg2q ion concentration is cooperative (nHs1.3), suggesting multiple binding sites for Mg2q ions. However, in addition to its binding effects, the Mg2q ion is also essential for forming the true enzyme substrate. With respect to Mg2q ion affinity under saturating ATP concentrations, the C. danae gill enzyme has a K0.5 (0.48"0.02 mM) very similar to that reported for the vertebrate enzyme (Glynn, 1985; Robinson and Pratap, 1991), crab axon membranes (Gache et al., 1976), and gill tissue of M. olfersii (Furriel et al., 2000). Higher K0.5 values have been reported for other euryhaline crabs (Corotto and Holliday, 1996; Holliday, 1985; D’Orazio and Holliday, 1985; Neufeld et al., 1980). The apparent affinity of the C. danae gill (Naq,Kq)-ATPase for Naq ions (K0.5s 5.80"0.30 mM) is in the range reported for various euryhaline crabs (Specht et al., 1997) and for M. olfersii, a recent invader of the freshwater biotope (Furriel et al., 2000). However, this apparent affinity is almost 10- to 20-fold lower compared to the shore crab Carcinus maenas and the gill enzymes of decapod crustaceans well established in freshwater (Harris and Bayliss, 1988). That the gill enzyme of crabs collected from Ubatuba Bay shows a K0.5 for sodium ions similar to estuarine crabs may derive from the fact that C. danae migrates to inshore and shallow waters to reproduce, mainly in those areas of Ubatuba Bay that receive considerable freshwater input (Mantelatto and Fransozo, 2000). C. danae showed monophasic inhibition by ouabain in contrast to the biphasic response reported for Uca pugnax (Holliday, 1985), Potamon potamios (Tentes and Stratakis, 1991) and C. sapidus (Neufeld et al., 1980). While the KI value (147.2"7.2 mM) is significantly greater than that for the vertebrate enzyme (Beauge´ et al., 1997), it is similar to that for the enzyme from the gill tissue of C. sapidus (Neufeld et al., 1980), certain crabs (Corotto and Holliday, 1996; Tentes and

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Stratakis, 1991; Holliday, 1985; D’Orazio and Holliday, 1985), and other crustaceans (Horiuchi, 1997; Furriel et al., 2000). The KI value for enzyme inhibition by orthovanadate (11.2"0.6 mM) is also very similar to those for the enzymes from most sources (Furriel et al., 2000; Skou and Esmann, 1992). Given that in the presence of NHq 4 ions, the KI values estimated for ouabain and orthovanadate inhibition were almost fourfold lower than those estimated in the absence of these ions, the most likely explanation is that the E1 – E2 conformational equilibrium favors the vanadatesensitive (or ouabain-sensitive) E2 conformation. Thus, the stable trigonal bipyramidal structure of vanadate may compete with the transition state analog of inorganic phosphate forming a stable intermediate with the E2 conformation of the enzyme, resulting in enzyme inhibition (Pick, 1982). While the mechanistic basis for the alterations in apparent KI values is quite complex, several phosphoenzyme intermediates between E1P and E2P (Yoda and Yoda, 1988) and subconformations of the E2P phosphorylated form of the enzyme (Fedosova et al., 1998) may account for such alterations. The isolated effects of Kq and NHq 4 ions on (Naq,Kq)-ATPases from different sources are well documented and significantly higher affinities (0.5–2.5 mM) for Kq ions than for Naq ions have been reported for various (Naq,Kq)-ATPases (Robinson and Pratap, 1991; Beauge´ et al., 1997; Specht et al., 1997). Further, NHq 4 ions can replace Kq ions in sustaining the hydrolysis of ATP by the (Naq,Kq)-ATPase of vertebrate (Robinson, 1970; Mallery, 1983; Skou and Esmann, 1992; Wall, 1996) and crustacean gill tissues (Holliday, 1985). There is also evidence that like Kq ions, NHq 4 ions are also actively transported by the vertebrate (Naq,Kq)-ATPase (Mallery, 1983; Wall, 1996), and that NHq 4 ions can substitute for Kq ions in the transport of Naq ions by the (Naq,Kq)-ATPase in membrane vesicles from C. sapidus (Towle and Holleland, 1987). To our knowledge, this is the first report demonstrating synergistic stimulation of the (Naq,Kq)-ATPase from a crustacean gill tissue q by NHq 4 (or K ) ions in the presence of fixed concentrations of Kq (or NHq 4 ) ions. In the absence of NHq 4 ions, the apparent affinity of the enzyme from C. danae gill tissue for Kq ions (K0.5s1.61"0.06 mM) is very similar to that for the enzyme from crab nerve (Skou, 1960), and for

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the gill enzyme from various crabs (Corotto and Holliday, 1996; Holliday, 1985; D’Orazio and Holliday, 1985; Tentes and Stratakis 1991) and other crustaceans (Furriel et al., 2000), independently of the salinity inhabited. The Hill coefficient (nHs 2.5) suggests multiple binding sites for Kq ions as also reported for the crab axon membrane enzyme (Gache et al., 1976), but contrasts with the Michaelis–Menten behavior reported for the (Naq,Kq)-ATPase from U. pugnax (Holliday, 1985) and U. pugilator (D’Orazio and Holliday, 1985). In the absence of Kq ions, the KM of the enzyme from C. danae gill tissue for NHq 4 ions (KMs4.61"0.27 mM) is very similar to crab nerve (Skou, 1960) and vertebrate enzymes (Robinson, 1970). However, the Hill coefficient of approximately 1.0 suggests that in addition to Kq sites there is only a single binding site for NHq 4 ions, responsible for the synergistic stimulation observed. The lower affinity of the gill enzyme q from C. danae for NHq 4 ions compared to K ions is similar to vertebrate and crab nerve enzymes (Robinson, 1970; Skou, 1962). Further, the 20–30% higher specific activity observed for NHq 4 stimulation of the C. danae gill enzyme is in close agreement with data reported for the above enzymes. The present findings are of significant physiological relevance. With few exceptions, crustaceans are ammoniotelic (Weihrauch et al., 1998, 1999). Most ammonia excretion occurs via the gills (Weihrauch et al., 1999), and less than 2% is eliminated via the urine in C. sapidus (Cameron and Batterton, 1978). Much of the ammonia content is excreted as NHq 4 (Weihrauch et al., 1998, 1999), and the fact that active transepithelial NHq 4 ion fluxes are sensitive to basolateral ouabain, strongly suggests the involvement of the (Naq,Kq)-ATPase (Weihrauch et al., 1998, 1999; Lucu et al., 1989). According to Weihrauch et al. (1998), both the basolateral (Naq,Kq)-ATPase and Kq channels participate directly in the translocation of NHq 4 from the hemolymph into the epithelial cells. The increasing intracellular NHq 4 concentration then generates a gradient across the apical membrane that drives the outward flux of q NHq 4 from the epithelial cells via an apical Na y q NH4 antiporter. The ambient ammonia concentration in the aquatic environment is usually low as a consequence of bacterial nitrification of ammonia to nitrite and nitrate followed by absorption by auto-

trophs (Weihrauch et al., 1999). In unpolluted, oxygenated seawater, NHq ion concentrations 4 rarely exceed 5 mM (Koroleff, 1983), in contrast to hemolymph concentrations of approximately 100 mM in various brachyuran species adapted to different salinities (Weihrauch et al., 1999). In pelagic animals this situation facilitates ammonia excretion mainly by the passive diffusion of NHq 4 down its concentration gradient. C. danae is a benthic crab that buries in sediments that may contain ammonia concentrations up to 2.8 mM (Weihrauch et al., 1999; Rebelo et al., 1999). While buried, such crabs continuously excrete ammonia that may contribute to a local increase in concentration (Weihrauch et al., 1999). Although exposure to elevated ammonia concentrations may cause substitution of Kq by NHq 4 ions, leading to a decrease in intracellular potassium (Towle and Holleland, 1987), acute exposure of the crab Chasmagnathus granulata to ammonia shows that hemolymph ammonia concentration is lower than ambient ammonia (Rebelo et al., 1999). It thus seems likely that under specific environmental circumstances, benthic crabs may encounter ambient ammonia concentrations that exceed hemolymph levels facilitating the influx of ammonia, particularly in estuarine and marine animals, owing to the higher permeability of their gill epithelia (Weihrauch et al., 1999). This may have led to the evolutionary selection of a mechanism for the excretion of NHq 4 against its gradient (Weihrauch et al., 1998, 1999). The active excretion of NHq 4 across the gill epithelium against elevated external ammonia concentrations reported for Carcinus maenas, Eriocheir sinensis and Cancer pagurus (Weihrauch et al., 1998, 1999) supports the view that the (Naq,Kq)-ATPase is directly involved in the translocation of NHq 4 from the hemolymph. The synergistic stimulation of the gill (Naq,Kq)q ATPase by NHq ions may constitute an 4 and K important component of the physiological response of C. danae to elevated external ammonia levels. At 33‰ salinity, hemolymph wKqx measures 10– 15 mM (Masui D.C., unpublished data), a level that provides maximal (Naq,Kq)-ATPase activity in vitro. At the habitually low hemolymph ammonia levels, Kq transport thus would be greatly favored. However, the synergistic stimulation of the enzyme by NHq 4 ions above 100 mM concentration, even at saturating Kq concentrations (Fig. 4), would ensure the transepithelial excretion of ammonia against its gradient.

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Despite evidence that NHq 4 excretion and active ion absorption are not directly coupled in crabs (Weihrauch et al., 1999), the future kinetic characterization of the (Naq,Kq)-ATPase from the gill tissue of C. danae when acclimated to low salinity may provide new insights into the biochemical mechanisms underlying osmoregulatory and excretory processes in hyperegulating Crustacea. Acknowledgments This work is part of an M.Sc. thesis by DCM and was supported by research grants from FAPESP and CNPq. FAL, JCM and FLMM received research scholarships from CNPq, and DCM received an M. Sc. scholarship from CAPES. The authors thank R.B. Garcia and R.A. Christofoletti for gill dissection. The antibodies used were obtained from the Developmental Studies Hybridoma Bank maintained by The University of Iowa (USA). References ´ L.A., Gadsby, D.C., Garrahan, P.J., 1997. NayKBeauge, ATPase and related transport ATPases. Structure, mechanism and regulation. Ann. NY Acad. Sci. 834, 1–694. ¨ Bottcher, K., Siebers, S., 1993. Biochemistry, localization, and physiology of carbonic anhydrase in the gills of euryhaline crabs. J. Exp. Zool. 265, 398–409. Cameron, J.N., Batterton, C.V., 1978. Antennal gland function in the freshwater crab Callinectes sapidus: water, electrolyte acid–base and ammonia excretion. J. Comp. Physiol. 123B, 143–148. Copeland, D.E., Fitzjarrell, A.T., 1968. The salt absorbing cells in gills of blue crab (Callinectes sapidus Rathbun) with notes on modified mitochondria. Z. Zellforsch. Mik. Ana. 92, 1–22. 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. D’Orazio, S.E., Holliday, C.W., 1985. Gill NaK-ATPase and osmoregulation in the sand fiddler crab Uca pugilator. Physiol. Zool. 58, 364–373. Fedosova, N.U., Cornelius, F., Klodos, I., 1998. E2P phosphoforms of Na,K-ATPase. I. Comparison of phosphointermediates formed from ATP and Pi by their reactivity toward hydroxylamine and vanadate. Biochemistry 37, 13634–13642. Furriel, R.P.M., McNamara, J.C., Leone, F.A., 2000. Characterization of (Naq,Kq )-ATPase in gill microsomes of the freshwater shrimp Macrobrachium olfersii. Comp. Biochem. Physiol. 126B, 303–315. Gache, C., Rossi, B., Lazdunski, M., 1976. (Naq,Kq)-activated adenosinetriphosphatase of axonal membranes, cooperativity and control. Steady-state analysis. Eur. J. Biochem. 6, 293–306.

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