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

作者：Lucena Malson N; Pinto Marcelo R; Garcon Daniela P; McNamara John C; Leone Francisco A^*

来源：Comparative Biochemistry and Physiology B-Biochemistry & Molecular Biology, 2015, 181: 15-25.

DOI：10.1016/j.cbpb.2014.11.002

• V(H+)-ATPase kinetics
• Freshwater shrimp gill
• Crustacea
• Biochemical characterization

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 +/- 33 nmol Pi min(-1) mg(-1)) and adult (41.6 +/- 13 nmol P-i min(-1) mg(-1)) V(H+)-ATPase activities are similar, the apparent affinity for ATP of the adult enzyme (K-0.5 = 0.21 +/- 0.02 mmol L-1) being 3-fold greater than for juveniles (K-0.5 = 0.61 +/- 0.01 mmol L-1). The K-0.5 for Mg2+ interaction with the juvenile V(H+)-ATPase (1.40 +/- 0.07 mmol L-1) is 6-fold greater than for adults (026 +/- 0.02 mmol L-1) while the bafilomycin A1 inhibition constant (K-I) is 45.0 +/- 2.3 nmol L-1 and 24.2 +/- 12 nmol L-1, for juveniles and adults, respectively. Both stages exhibited residual bafilomycin-insensitive ATPase activity of approximate to 25 nmol P-i, 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.

• 出版日期2015-3