Kinetic and molecular characterization of the (Na +, K +) - ATPase of the gill tissue of the Cardisoma guanhumi crab (Latreille, 1825).

Author(s)

Daniel Lima de Farias

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (PCARP/BC)
• Defense date: 2017-09-18

Examining board members

Pietro Ciancaglini; Alessandra da Silva Augusto; John Campbell McNamara; Arthur Henrique Cavalcante de Oliveira; João Martins Pizauro Júnior

• Advisor: Francisco de Assis Leone

Abstract

The (Na+,K+)-ATPase is an integral plasma membrane protein that is subject to complex regulation. In the mangrove fauna, the crustaceans include Cardisoma guanhumi crab (Latreille, 1825), a decapod crustacean that plays a significant role in the dynamics of this ecosystem, considered a relevant fishing resource. This study provides the effect of polyamines, oxidative stress enzymes, ammonium toxicity, and also investigates K+-phosphatase activity and (Na+,K+)-ATPase activity by synergistic K+/NH4+ and NH4+/ K+ stimulation in the microsomal fraction of guaiamum gills. The K+-phosphatase activity and (Na+,K+)-ATPase activity were determined continuously at 25°C on a Shimadzu U1800 spectrophotometer equipped with thermostated cells. All experiments were done in duplicate using at least three different preparations (N 3). The PNFFase activity insensitive to ouabain represents 40% of the total PNFFase activity, and KI value was 370,0 18,5mol L-1. The maximum specific activity estimated was 29.30 ± 1.46 nmol Pi min-1 mg-1 and KM = 2.90 ± 0.14 mmol L-1. On the other hand, the use of the physiological substrate (ATP) allowed the determination of kinetic parameters of the activity (Na+,K+)-ATPase in relation to the modulators ATP, potassium, sodium, magnesium, ammonium and ouabain.The total ATPase activity in the microsomal fraction of freshly caught C. guanhumi (16 S) gill tissue was approximately 166 nmol Pi min-1 mg-1 and a 26.55 nmol Pi min-1 mg-1 ouabain ATPase activity, while acclimated at 22 S the total ATPase activity was 303.28 ± 15.16 nmol Pi min-1 mg-1 and the ouabain insensitive activity of 68.60 ± 3.43 nmol Pi min-1 mg-1. The (Na+,K+)-ATPase present in these two preparations, do not present a synergistic stimulation by K+ and NH4+. There were changes in the enzyme affinity for ATP at the three different concentrations of NH4Cl (120 mg / L, 240 mg / L, 360 mg / L) compared to the control without NH4Cl (KM = 0.1 ± 0.005 mmol L-1). No significant effects were observed using biogenic amines. No significant effects were observed using biogenic amines. Our analyzes have also shown that oxidative stress enzymes are acting in these different preparations to combat oxirradicals. Analysis by Western blotting with monoclonal antibody revealed the presence of a band corresponding to sub subunit of (Na+,K+)-ATPase with molecular mass 110 kDa. Immunolocalization showed that the (Na+,K+)-ATPase sub subunit is predominantly distributed throughout the cytoplasm of the gill pillars, including the apical region below the cuticle. We identified the constitutive gene of the nucleotide partial sequence of the cDNA of ribosomal protein L10 (PRL10) of the gills of Cardisoma guanhumi. The study demonstrated that (Na+,K+)-ATPase is an important regulator of osmoregulation in this species, contributing to a better understanding of the roles played by this enzyme in the processes of osmoregulation and excretion of ammonia in crustaceans. (AU)

• FAPESP's process: 13/00976-8 - Kinetic and molecular Characterization of the gill (Na+,K+)-ATPase of crab Cardisoma guanhumi (Latreille).
• Grantee: Daniel Lima de Farias
• Support Opportunities: Scholarships in Brazil - Doctorate