A kinetic characterization of the gill V(H+)-ATPase in juvenile and adult Macrobrachium amazonicum, a diadromous palaemonid shrimp

Novel kinetic properties of a microsomal gill V(H+)-ATPase from juvenile and adult Amazon River shrimp, Macrobrachium amazonicum, are described. While protein expression patterns are markedly different, Western blot analysis reveals a sole immunoreactive band, suggesting a single V(H+)-ATPase subunit isoform, distributed in membrane fractions of similar density in both ontogenetic stages. Immunofluorescence labeling locates the V(H+)-ATPase in the apical regions of the lamellar pillar cells in both stages in which mRNA expression of the V(H+)-ATPase B-subunit is identical. Juvenile (36.6 ± 3.3 nmol Pi min−1 mg−1) and adult (41.6 ± 1.3 nmol Pi min−1 mg−1) V(H+)-ATPase activities are similar, the apparent affinity for ATP of the adult enzyme (K0.5 = 0.21 ± 0.02 mmol L−1) being 3-fold greater than for juveniles (K0.5 = 0.61 ± 0.01 mmol L−1). The K0.5 for Mg2+ interaction with the juvenile V(H+)-ATPase (1.40 ± 0.07 mmol L−1) is ≈6-fold greater than for adults (0.26 ± 0.02 mmol L−1) while the bafilomycin A1 inhibition constant (KI) is 45.0 ± 2.3 nmol L−1 and 24.2 ± 1.2 nmol L−1, for juveniles and adults, respectively. Both stages exhibited residual bafilomycin-insensitive ATPase activity of ≈25 nmol Pi min−1 mg−1, suggesting the presence of ATPases other than the V(H+)-ATPase. These differences may reflect a long-term regulatory mechanism of V(H+)-ATPase activity, and suggest stage-specific enzyme modulation. This is the first kinetic analysis of V(H+)-ATPase activity in different ontogenetic stages of a freshwater shrimp and allows better comprehension of the biochemical adaptations underpinning the establishment of palaemonid shrimps in fresh water.