Characterization of antennary gland Na,K-ATPase in the freshwater crayfish, Procambarus clarki girard

Comp. Bioehem. Physiol.. Vol. 65B. pp. 391 to 394

0305-0491/80/0201-0391102.00/0

© Pergamon Press Ltd 1980. Printed in Great Britain

CHARACTERIZATION OF ANTENNARY GLAND NA,K-ATPase IN THE FRESHWATER CRAYFISH, PROCAMBARUS CLARKI GIRARD SHIRO HORIUCHi Life Science Institute, Sophia University, Kioicho 7, Chiyoda-ku, Tokyo, 102, Japan

(Received 30 April 1979) Abstract--1. Na, K-ATPase activity was demonstrated in a heavy microsomal fraction of antennary

gland preparations of the freshwater crayfish, Procambarus clarki. 2. The highest activity was found in 100 mM of sodium and 4 mM of potassium in the presence of 1 mM of magnesium at pH 8.5. 3. Ks of Na,K-ATPase for ATP was 9.0 x 10 -4 M. 4. A complete inhibitory effect on Na,K-ATPase by ouabain was found with 10 -2 M ouabain. The plso was 4.t. 5. Both renal Na, K-ATPase and Mg-ATPase were inhibited by POMB. The plso was 6.3 and 5.0, respectively. Inhibitory effect of POMB was restored by cysteine. 6. The activity of gill Na, K-ATPase was increased only when the animals were maintained in 50% seawater.

INTRODUCTION Na,K-ATPase (ATP phosphohydrolase, EC 3.6.1.3) was first demonstrated in the crab's nerves, which is activated by Na +, K + and Mg 2+ (Skou, 1957). The suggestion that this enzyme plays an integral role in ion transport is supported by the observation that (1) active transport of Na + and K + and (2) the Na, KATPase activity essentially require Na +, K + and Mg 2+ and (1) and (2) above are both inhibited by ouabain (Skou, 1957, 1965). The freshwater crayfish has a relatively higher hemolymph osmoconcentration than other freshwater animals (Prosser, 1973). The presence of Na, KATPase has been reported in the gill of a freshwater crayfish, Procambarus clarki, and properties of Na, KATPase were previously characterized by Horiuchi (1977). The antennary gland is a renal organ in crustaceans. It is suggested that this organ plays an important role for osmotic and ionic regulation in both freshwater and marine crustaceans (Potts & Parry, 1964). Because of the analogy of the crustacean antennary gland with the mammalian kidney, its function has been reported to be sodium reabsorption, water absorption and nitrogen excretion by a number of investigators (Binns, 1969; Binns & Peterson, 1969; Denne, 1968; Peterson & Loizzi, 1974; Riegel, 1966). An attempt has been made to show the presence of Na,K-ATPase in the antennary gland of freshwater crayfish, since this enzyme seems to be concerned with the active absorption of sodium from the hemolymph at the time of the production of urine. This present communication describes some properties of Na,K-ATPase in Procambarus clarki. MATERIALS AND METHODS Freshwater crayfish, Procambarus clarki Girard, collected on the outskirts of Tokyo was employed in the pre-

sent study. The animals were kept in tap water at room temperature and without feeding for 1 week before use. Antennary gland (green gland) containing coelomic sac, labyrinth, nephridial canal and bladder were taken out and washed with 0.25 M sucrose (the pH being adjusted to 7.5 with Tris-HCl buffer) and were then cut into pieces with scissors. Antennary gland pieces were homogenized in 10 vol of the above-mentioned 0.25 M sucrose with a Teflon Potter-Elvejehem homogenizer. Cell fractionation of the antennary gland was done according to the modified method of Davis (1970). Procedures of the fractionation in detail were described in the previous report (Horiuchi, 1977). The partially purified fraction (ppt-3) which corresponds to the heavy microsomal fraction was suspended in 0.25 M sucrose and used as the enzyme solution (see Horiuchi, 1977).

Assay of the enzyme activity The Na,K-activated, Mg-dependent ATPase (Total ATPase) was determined by using the reaction mixture containing 100mM NaCI, 4mM KC1, l mM MgCI2, 20 mM Tris-HCl (pH 8.4) and 1 mM Tris-ATP (Sigma) in 0.8 ml. The Mg-ATPase was measured in the presence of 0.25 M sucrose in place of NaCI and KC1. The reaction mixture was preincubated for 5 rain at 37°C before 0.2ml of the heavy microsomal suspension (10-60 #g protein/ml) was added. Enzyme reaction was carried out at 37°C for 15 min. The Na,K-ATPase activity was calculated in terms of the difference of the total ATPase and Mg-ATPase values. Under these conditions, a linear relationship was obtained between the ATP hydrolysis and protein concentration of the enzyme. The inorganic phosphate (Pi) liberated from ATP was measured by the Fiske-SubbaRow method (1925) using a Hitachi spectrophotometer (Type 124) at 660nm. The specific activity Na,K-ATPase was defined as #mols of P~ per mg of the enzyme protein during 1 hr. The protein was determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard. For the seawater adaptation of crayfish, the animals were maintained in artificial sea water (Aqua Salz, Nippon Jisei Sangyo Co., Tokyo). The concentration of sea water was gradually increased from 10 up to 50~o. Within each 391

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Fig. 1. Effect of various concentrations of Na+ on P. clarki antennary gland Na,K-ATPase. Incubation medium contained 4 mM KCI, 1 mM MgCI2, 20 mM Tris-HCl (pH 8.4) and 1 mM Tris-ATP in 0.8 ml. 0.2 ml of antennary gland microsomal suspension was added to start the reaction, which was run for 15 min at 37°C. step of the adaptation, the animals were kept at least for 1 week or more. RESULTS The effect of Na + on Procambarus antennary gland Na,K-ATPase was determined. The highest activity was observed in the presence of 4 m M KCI in the range of a sodium concentration of between 100 and 200mM, as shown in Fig. 1. In the presence of 100mM NaC1, the highest activity was observed in the concentration of 4 m M KC1 (7.3 ___0.6/~mol Pi/mg protein-hr). Therefore, the following experiments were carried out in a combination consisting of a concentration of 100mM sodium and of 4 m M potassium. Crayfish antennary gland Na, K-ATPase was determined at different pH. The highest activity was found at pH 8.5, and no activity was revealed at pH 7.0 (Fig. 2). Both Na, K-ATPase and Mg-ATPase were also influenced by the concentration of magnesium ion. The highest activity of Na,K-ATPase was found at the concentration of 1 mM MgC12, and the MgATPase activity reached its highest at 0.5 m M MgClz

(Fig. 3). Both enzyme activities decreased when the concentration of MgC12 increased from 1 to 6 mM. The features of the effect of ATP concentration on the two ATPases are striking. The highest Na,KATPase activity was observed approximately at 2 mM ATP, whereas the Mg-ATPase activity increased as the concentration of ATP increased (Fig. 4). As shown in Fig. 5, the Lineweaver-Burk plot of Na,K-ATPase was obtained. From this figures, the K,, (Michaelis constant) of the crayfish antennary gland Na,K-ATPase for ATP was calculated as 9.0 x 10-4M. The inhibitory effect of an ouabain (cardiac glycoside) was demonstrated on antennary gland Na,KATPases. Figure 6 shows the inhibition curve for Na,K-ATPase obtained by adding graded amounts of ouabain (10-1°-10-2 M). A complete inhibition was caused by 10 -2 M, and a 50~o inhibition was caused by approx 10 -4 M. The plso (the negative logarithm of the molar ouabain concentration causing 50~o inhibition) was 4.1 in the presence of 4 mM KC1, which is almost the same in the case of gill Na,K-ATPase in Procambarus clarki (Horiuchi, 1977). Such an inhibitory effect in the Mg-ATPase of crayfish was not seen with any range of ouabain concentration examined. Both these renal ATPases of crayfish were inhibited by P O M B (sodium p-hydroxymercuribenzoate) as shown in Fig. 7. Na,K-ATPase was highly inhibited over 10 -6 M concentration of POMB. The plso was 6.3 At 10 -4 M POMB, the Na,K-ATPase was almost inhibited, whereas the inhibitory effect on MgATPase varied from that of Na,K-ATPase. The plso

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Fig. 4. Effect of ATP concentration on crayfish antennary gland ATPase. Na,K-ATPase (O ©), Mg-ATPase (=

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Characterization of antennary gland Na, K-ATPase

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Fig. 5. Lineweaver-Burk plot of crayfish antennary gland Na,K-ATPase.

Fig. 7. Effect of POMB on crayfish antennary gland Na,KATPase. Na,K-ATPase (O O), Mg-ATPase (~----O). were 6.3 and 5.0, respectively. The inhibitory effect of POMB on both ATPases was restored with the addition of cysteine (Davis, 1970). In the presence of 2 x 10 -4 M cysteine, 89.4~ of Na,K-ATPase was inhibited, whereas 42.0~o of Mg-ATPase was inhibited. However, the inhibition percentage was lowered to 13.1 and 9.0~, respectively, when 1 × 1 0 - a M cysteine was added to the incubation medium (Table 1). Na,K-ATPase and Mg-ATPase of the gill and antennary gland were demonstrated during the process of seawater adaptation of the crayfish. As shown in Fig. 8, the activity of three ATPases was almost unchanged except for the gill Na,K-ATPase, the ac: tivity of which latter increased when the crayfish was maintained in 50~o seawater from 6-10 days, DISCUSSION

The present studies clearly showed that the Na, KATPase system is contained in the heavy microsomal fraction of the antennary gland homogenate of the crayfish, P. clarki. As for the crustacean Na, KATPase, the presence of the enzyme was reported in the gill of the semi-terrestrial crab, Cardisoma 9uanhumi (Quinn & Lane, 1966), the freshwater crayfish, P. clarki (Horiuchi, 1977), and in the kidney of crayfish (Peterson & Loizzi, 1974).

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The characteristic properties of the antennary gland Na, K-ATPase were similar to those of the enzyme from the crayfish gill with reference to (A) the dependence of Na ÷' K ÷ and Mg 2+, (B) the K,, value and (C) the inhibitory effect of ouabain (Figs l, 3, 5 and 6) (Horiuchi, 1977). Both Na,K-ATPase and Mg-ATPase were inhibited by POMB, the sulfhydryl reagent. At 10 -5 M POMB, the former was almost inhibited, and the latter was inhibited to almost half of the original activity. The presence of cysteine at 2 x 10-4M restored the Na,K-ATPase activity to 89.4~o, and the Mg-ATPase activity to 42.0~, and also the presence of cysteine at 1 x 10-3M restored the activity of both of the ATPases to almost 10~o of the original inhibition rate (Table 1 and Fig. 7), suggesting that the sulfhydryl contents of Na,K-ATPase are different from those of Mg-ATPase in the crayfish antennary gland (Davis, 1970). The freshwater crayfish can survive for extended periods in waters of a relatively high salt content (Sharma, 1966). As shown in Fig. 8, only the gill Na,K-ATPase showed a tendency to increase its activity when the crayfish were kept in 50~o seawater for ten days, suggesting that the organ responsible for the seawater adaptation is the gill, and not the antennary gland. A pair of antennary glands (green gland), is the crustacean renal organ located in the head, and opening to the outside independently near the bases of the second antenae. Each antennary gland consists of four parts; viz. coelomic sac, labyrinth, nephridial canal and bladder in the freshwater form. The renal duct begins with a closed coelomic sac. Following the coelomic sac is the labyrinth, a highly convoluted canal. Table 1. Inhibitory effects of POMB on crayfish antennary gland Na,K-ATPase and Mg-ATPase under the absence or the presence of cysteine

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SH1RO HORIUCHI concluded that the antennary gland Na,K-ATPase found in the present studies is presumably related to the reabsorption of sodium and chloride ions in the nephridial canal of the antennary gland in P. clarki.

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Acknowledgements--The author would like to thank Miss Y. Toishi for her technical assistance and Professor Daniel McCoy, S. J., Sophia University for reviewing this manuscript.

:a, REFERENCES

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Fig. 8. Effect of environmental NaCI concentration on freshwater crayfish ATPase. Gill Na,K-ATPase (A A), gill Mg-ATPase (& A), antennary gland Na, K-ATPase (O O), Mg-ATPase (O------O). The nephridial canal, which is lacking in marine forms, leads from the labyrinth to an expanded bladder (Ramsay, 1952). The decrease in chloride concentration takes place during the passage of the urine along the nephridial canal (Potts & Parry, 1964). The highest level of Na, K-ATPase was seen in the nephridial canal, followed by the labyrinth in the freshwater crayfish kidney; these results supported by cytochemical observations imply that the kidney Na,K-ATPase functions mainly in fluid reabsorption (Peterson & Loizzi, 1974). As for the function of the antennary gland, it has been reported that this organ doesn't play an important role in nitrogen excretion in the spiny lobster, Jasus edwardsi (Binns & Peterson, 1969) and Carcinus maenas (Binns, 1969). It is suggested that the antennary gland, on the contrary, functions in the balancing of ionic components in hemolymph as well as in the passive water flux and in the active reabsorption of glucose and amino acids through urine production in Carcinus (Binns, 1969), in prawn, Macrobrachium (Denne, 1968), although Dall (1967) reported that the water uptake and salt excretion take place in the gut and major excretion doesn't occur via the gill in the hypo-osmoregulatory crustaceans such as marine shrimps and crabs. Thus the function of the antennary gland is so complicated that its function is still not fully known. By micropuncture sampling of the antennary gland of the crayfish, Riegel (1966) revealed the two kinds of distinct particulate materials, viz. spheroids and vesicles in the urine. Considering the above-mentioned findings, it is

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