A Kinetic Characterization of (Na+, K+)-ATPase Activity in the Gills of the Pelagic Seabob Shrimp Xiphopenaeus kroyeri (Decapoda, Penaeidae)

Author(s)

Leone, Francisco Assis ^[1] ; Lucena, Malson Neilson ^[1] ; Rezende, Luciana Augusto ^[1] ; Garcon, Daniela Pereira ^[2] ; Pinto, Marcelo Rodrigues ^[1] ; Mantelatto, Fernando Luis ^[3] ; McNamara, John Campbell ^[3]

Affiliation

^[1] Univ Sao Paulo, Dept Quim, Fac Filosofia Ciencias & Letras Ribeirao Preto, BR-14040901 Ribeirao Preto, SP - Brazil

^[2] Univ Fed Paraiba, Dept Biol Mol, Ctr Ciencias Exatas & Nat, BR-58059900 Joao Pessoa, Paraiba - Brazil

^[3] Univ Sao Paulo, Dept Biol, Fac Filosofia Ciencias & Letras Ribeirao Preto, Sao Paulo - Brazil

• Document type: Journal article
• Source: Journal of Membrane Biology; v. 248, n. 2, p. 257-272, APR 2015.
• Web of Science Citations: 6

Abstract

We characterize the kinetic properties of a gill (Na+, K+)-ATPase from the pelagic marine seabob Xiphopenaeus kroyeri. Sucrose density gradient centrifugation revealed membrane fractions distributed mainly into a heavy fraction showing considerable (Na+, K+)-ATPase activity, but also containing mitochondrial F0F1- and Na+- and V-ATPases. Western blot analysis identified a single immunoreactive band against the (Na+, K+)-ATPase alpha-subunit with an M-r of a parts per thousand 110 kDa. The alpha-subunit was immunolocalized to the intralamellar septum of the gill lamellae. The (Na+, K+)-ATPase hydrolyzed ATP obeying Michaelis-Menten kinetics with V-M = 109.5 +/- A 3.2 nmol Pi min(-1) mg(-1) and K-M = 0.03 +/- A 0.003 mmol L-1. Mg2+ (V-M = 109.8 +/- A 2.1 nmol Pi min(-1) mg(-1), K-0.5 = 0.60 +/- A 0.03 mmol L-1), Na+ (V-M = 117.6 +/- A 3.5 nmol Pi min(-1) mg(-1), K-0.5 = 5.36 +/- A 0.14 mmol L-1), K+ (V-M = 112.9 +/- A 1.4 nmol Pi min(-1) mg(-1), K-0.5 = 1.32 +/- A 0.08 mmol L-1), and NH4 (+) (V-M = 200.8 +/- A 7.1 nmol Pi min(-1) mg(-1), K-0.5 = 2.70 +/- A 0.04 mmol L-1) stimulated (Na+, K+)-ATPase activity following site-site interactions. K+ plus NH4 (+) does not synergistically stimulate (Na+, K+)-ATPase activity, although each ion modulates affinity of the other. The enzyme exhibits a single site for K+ binding that can be occupied by NH4 (+), stimulating the enzyme. Ouabain (K-I = 84.0 +/- A 2.1 A mu mol L-1) and orthovanadate (K-I = 0.157 +/- A 0.001 A mu mol L-1) inhibited total ATPase activity by a parts per thousand 50 and a parts per thousand 44 %, respectively. Ouabain inhibition increases a parts per thousand 80 % in the presence of NH4 (+) with a threefold lower K-I, suggesting that NH4 (+) is likely transported as a K+ congener. (AU)

• FAPESP's process: 10/06395-9 - Role of (Na,K)-ATPase in osmorregulation and excretion of ammonium ions in euryhaline crustaceans: identification and influence of FXYD2 on the regulation of gill (Na,K)-ATPase of the crab Callinectes danae
• Grantee: Daniela Pereira Garçon
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 10/17534-0 - Role of (Na,K)-ATPase in osmoregulation and excretion of ammonium ions in euryhaline crustaceans: identification and influence of FXYD2 on the regulation of gill (Na,K)-ATPase of the crab Callinectes danae
• Grantee: Francisco de Assis Leone
• Support Opportunities: Regular Research Grants
• FAPESP's process: 10/16115-3 - Ontogeny of osmoregulation in the freshwater Shrimp Macrobrachium amazonicum (Heller, 1862): the expression of (na +, K +)-ATPase, influence of lipid composition and role of polyamines in the kinetic activity of the enzyme
• Grantee: Malson Neilson de Lucena
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 10/50188-8 - Decapod crustaceans: multi-disciplinary characterization of the sea biodiversity of the State of São Paulo (taxonomy, spermiotaxonomy, molecular biology and population dynamics)
• Grantee: Fernando Luis Medina Mantelatto
• Support Opportunities: BIOTA-FAPESP Program - Thematic Grants