title: A kinetic characterization of the gill V(H(+))-ATPase in juvenile and adult Macrobrachium amazonicum, a diadromous palaemonid shrimp.
abstract: 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 Mg(2+) 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.
authors.affiliation: Departamento de Química, Faculdade de Filosofia, Ciências e Letras da Universidade de São Paulo, Ribeirão Preto 14040-901, SP, Brazil.
authors.forename: Malson N
authors.initials: MN
authors.lastname: Lucena
authors.affiliation: Departamento de Química, Faculdade de Filosofia, Ciências e Letras da Universidade de São Paulo, Ribeirão Preto 14040-901, SP, Brazil.
authors.forename: Marcelo R
authors.initials: MR
authors.lastname: Pinto
authors.affiliation: Departamento de Biologia Molecular, Centro de Ciências Exatas e da Natureza, Universidade Federal da Paraíba, PB, Brazil.
authors.forename: Daniela P
authors.initials: DP
authors.lastname: Garçon
authors.affiliation: Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras da Universidade de São Paulo, Ribeirão Preto 14040-901, SP, Brazil.
authors.forename: John C
authors.initials: JC
authors.lastname: McNamara
authors.affiliation: Departamento de Química, Faculdade de Filosofia, Ciências e Letras da Universidade de São Paulo, Ribeirão Preto 14040-901, SP, Brazil. Electronic address: fdaleone@ffclrp.usp.br.
authors.forename: Francisco A
authors.initials: FA
authors.lastname: Leone
mesh_terms: D000818:Animals; D005618:Fresh Water; D005880:Gills / Q000201:enzymology*; D033541:Palaemonidae / Q000201:enzymology* / Q000254:growth & development; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D025262:Vacuolar Proton-Translocating ATPases
keywords: Biochemical characterization; Crustacea; Freshwater shrimp gill; V(H(+))-ATPase kinetics