Modulation by K+ Plus NH4+ of microsomal (Na+, K+)-ATPase activity in selected ontogenetic stages of the diadromous river shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae)

Abstract

We investigate the synergistic stimulation by K(+) plus NH4 (+) of (Na(+), K(+))-ATPase activity in microsomal preparations of whole zoea I and decapodid III, and in juvenile and adult river shrimp gills. Modulation of (Na(+), K(+))-ATPase activity is ontogenetic stage-specific, and particularly distinct between juveniles and adults. Although both gill enzymes exhibit two different sites for K(+) and NH4 (+) binding, in the juvenile enzyme, these two sites are equivalent: binding by both ions results in slightly stimulated activity compared to that of a single ionic species. In the adult enzyme, the sites are not equivalent: when one ion occupies its specific binding site, (Na(+), K(+))-ATPase activity is stimulated synergistically by ≈ 50% on binding of the complementary ion. Immunolocalization reveals the enzyme to be distributed predominantly throughout the intralamellar septum in the gill lamellae of juveniles and adults. Western blot analyses demonstrate a single immunoreactive band, suggesting a single (Na(+), K(+))-ATPase α-subunit isoform that is distributed into different density membrane fractions, independently of ontogenetic stage. We propose a model for the modulation by K(+) and NH4 (+) of gill (Na(+), K(+))-ATPase activity. These findings suggest that the gill enzyme may be regulated by NH4 (+) during ontogenetic development in M. amazonicum.

Figure 1. Sucrose density gradient centrifugation of a microsomal fraction from gill tissue of juvenile and adult M. amazonicum.

Aliquots containing 4.5% (w/w) continuous sucrose density gradients. Fractions (0.5 mL) collected from the bottom of each gradient were analyzed for total ATPase activity (○), (Na⁺, K⁺)-ATPase activity (•), ouabain-insensitive ATPase activity (▵), protein concentration (▴) and sucrose concentration (□). Inset: Adult gill tissue.

Figure 2. SDS-PAGE and Western blot analyses of microsomal fractions from whole decapodid III, and juvenile and adult M. amazonicum gills.

Electrophoresis was performed in a 5–20% polyacrylamide gel using 4 μg microsomal protein for silver staining and 160 μg for Western blotting. The analysis was repeated three times (N = 3) using aliquots from different homogenates prepared from each ontogenetic stage. Silver nitrate-stained gels: A- Decapodid III. B- Juvenile. C- Adult. Western blots: D- Decapodid III. E- Juvenile. F- Adult.

Figure 3. Immunolocalization of the gill (Na⁺, K⁺)-ATPase α-subunit in juvenile and adult M. amazonicum.

Frozen cross sections taken transversely to the gill lamellae long axes were incubated with mouse monoclonal IgG α-5 antibody raised against chicken (Na⁺, K⁺)-ATPase α-subunit then incubated in donkey anti-mouse IgG secondary antibody conjugated with Alexa-fluor 488. Phase contrast/DAPI/α-5 images demonstrating typical lamellar structure. Immunofluorescence labeling (Alexa-fluor 488, 495/519 nm) showing distribution of the (Na⁺, K⁺)-ATPase α-subunit (green) located predominantly in the intralamellar septal cells identified by their DAPI-stained nuclei (blue). A- Juvenile gill lamellae. B- Adult gill lamellae. Scale bars  = 50 µm.

Figure 4. Effect of NH₄ ⁺ concentration on modulation by K⁺ of microsomal (Na⁺, K⁺)-ATPase activity in whole M. amazonicum zoea I and decapodid III.

Data are the mean ± SEM (N = 3) obtained using duplicate aliquots containing 13.4 μg protein (zoea I) and 7.2 μg protein (decapodid III) from three different homogenates. Activity was assayed at 25°C in 50 mmol L⁻¹ triethanolamine buffer (pH 7.5), containing 2 mmol L⁻¹ ATP, 5 mmol L⁻¹ MgCl₂, 1.0 mmol L⁻¹ NAD⁺, 0.5 mmol L⁻¹ sodium phosphate, 1.0 mmol L⁻¹ G3P, 150 μg GAPDH (12 U), 20 μg PGK (9 U) and NaCl (50 mmol L⁻¹ for zoea I and 20 mmol L⁻¹ for decapodid III) in a final volume of 1 mL. A- Zoea I. B- Decapodid III. NH₄ ⁺ concentration: (•) none, (○) 30 mmol L⁻¹.

Figure 5. Effect of NH₄ ⁺ concentration on modulation by K⁺ of microsomal (Na⁺, K⁺)-ATPase activity in gill tissue from juvenile and adult M. amazonicum.

Data are the mean ± SEM (N = 3) obtained using duplicate aliquots containing 9.5 μg protein (juveniles) and 10.7 μg protein (adults) from three different gill homogenates. Activity was assayed at 25°C in 50 mmol L⁻¹ triethanolamine buffer (pH 7.5), containing 2 mmol L⁻¹ ATP, 5 mmol L⁻¹ MgCl₂, 1.0 mmol L⁻¹ NAD⁺, 0.5 mmol L⁻¹ sodium phosphate, 1.0 mmol L⁻¹ G3P, 150 μg GAPDH (12 U), 20 μg PGK (9 U) and NaCl (50 mmol L⁻¹ for juveniles and 20 mmol L⁻¹ for adults), in a final volume of 1 mL. A- Juveniles. Inset: Variation in K_0.5 with NH₄ ⁺ concentration. B- Adults. Variation in V_M (inset a) and K_0.5 (inset b) with NH₄ ⁺ concentration. NH₄ ⁺ concentration: (•) none, (○) 0.3 mmol L⁻¹, (▪) 1 mmol L⁻¹, (□) 2 mmol L⁻¹, (▵)3 mmol L⁻¹, (▴) 5 mmol L⁻¹, (▹)10 mmol L⁻¹, (▸) 30 mmol L⁻¹.

Figure 6. Effect of K⁺ concentration on modulation by NH₄ ⁺ of microsomal (Na⁺, K⁺)-ATPase activity in whole M. amazonicum zoea I and decapodid III.

Data are the mean ± SEM (N = 3) obtained using duplicate aliquots containing 13.4 μg protein (zoea I) and 7.2 μg protein (decapodid III) from three different homogenates. Activity was assayed at 25°C in 50 mmol L⁻¹ triethanolamine buffer (pH 7.5), containing 2 mmol L⁻¹ ATP, 5 mmol L⁻¹ MgCl₂, 1.0 mmol L⁻¹ NAD⁺, 0.5 mmol L⁻¹ sodium phosphate, 1.0 mmol L⁻¹ G3P, 150 μg GAPDH (12 U), 20 μg PGK (9 U) and NaCl (50 mmol L⁻¹ for juveniles and 20 mmol L⁻¹ for adults), in a final volume of 1 mL. A- Zoea I. B- Decapodid III. K⁺ concentration (•) none, (○) 20 mmol L⁻¹ K⁺.

Figure 7. Effect of K⁺ concentration on modulation by NH₄ ⁺ of microsomal (Na⁺, K⁺)-ATPase activity in gill tissue from juvenile and adult M. amazonicum.

Data are the mean ± SEM (N = 3) obtained using duplicate aliquots containing 9.5 μg protein (juveniles) and 10.7 μg protein (adults) from three different gill homogenates. Activity was assayed at 25°C in 50 mmol L⁻¹ triethanolamine buffer (pH 7.5), containing 2 mmol L⁻¹ ATP, 5 mmol L⁻¹ MgCl₂, 1.0 mmol L⁻¹ NAD⁺, 0.5 mmol L⁻¹ sodium phosphate, 1.0 mmol L⁻¹ G3P, 150 μg GAPDH (12 U), 20 μg PGK (9 U) and NaCl (50 mmol L⁻¹ for juveniles and 20 mmol L⁻¹ for adults), in a final volume of 1 mL. A- Juveniles. Inset: Variation in K_0.5 with K⁺ concentration. B- Adults. Variation in V_M (inset a) and K_0.5 (inset b) with K⁺ concentration. K⁺ concentration (•) none, (○) 0.4 mmol L⁻¹, (□) 0.5 mmol L⁻¹, (▪) 2 mmol L⁻¹, (▵) 5 mmol L⁻¹, (▴) 10 mmol L⁻¹, (▹) 20 mmol L⁻¹.