Caracterização cinética da (Na+,K+)-ATPase da fração microsomal do tecido branquial do siri Callinectes ornatus ordway, 1863 (Crustacea, Portunidae) / A kinetic characterization of the (Na+,K+)-ATPase in gill microsomes from the crab Callinectes ornatus.

AUTOR(ES)
DATA DE PUBLICAÇÃO

2007

RESUMO

(Na+, K+)-ATPase present on branchial tissue osmoregulatory crustaceans is an essential component of their osmotic and ionic regulation system. Apparently, this enzyme also have a relevant role in the active excretion de NH4+ through the branchial crustacean tissue. A (Na+, K+) ATPase-rich microsomal fraction was prepared by differential centrifugation from Callinectes ornatus homogenized branchial tissue. The sucrose gradient sucrose centrifugation showed the presence of a single protein peak with ATPase activity, and SDS-PAGE revealed the presence of several proteins bands. The use of the 5 monoclonal antibody, against the ? subunit, revealed the presence of a unique protein band of 110 kDa corresponding to the (Na+, K+) ATPase. (Na+, K+) ATPase hydrolyzed the PNPP (V= 52.0 2.0 U/mg and K0.5= 1.1 0.1 mM) through the site-site interactions (nH= 1.6). The modulation of (Na+, K+) ATPase by magnesium (V= 52.3 1.3 U/mg and K0.5= 1.1 ? 0.05 mM), potassium (V= 51.4 1.5 U/mg and K0.5= 2.3 0.1 mM) and ammonia ions (V= 56.7 2.7 U/mg and K0.5= 9.8 ? 0.4 mM) followed cooperative kinetics. However, sodium ions inhibited PNPPase activity of (Na+, K+)?ATPase with Ki= 1.7 0.1 mM. Ouabain also inhibited up to 80% the total activity PNPPase independent of the presence of ammonium ions. The hydrolysis of ATP by (Na+, K+) ATPase followed Michaelis-Menten kinetics with Km= 0.16 0.01 mM and V= 116.3 5.6 U/mg, while enzyme modulation by magnesium (V= 111.0 5.4 U/mg and K0.5= 0.54 0.03 mM), sodium (V= 110.6 5.3 U/mg and K0.5= 6.3 0.3 mM), potassium (V= 116.0 5.5 U/mg and K0.5= 1.5 ? 0.1 mM) and ammonium ions (V= 173.3 . Interestingly, the stimulation of (Na+, K+)-ATPase activity by potassium ions in the presence of increasing concentration of ammonium ions to K+ resulted in a 50% higher specific activity. Ouabain inhibited approximately 86% the activity (Na+, K+) ATPase with (Ki= 74.5 M), suggesting the presence of about 14% of phosphatases and/or other ATPases. This is the first work showing synergistic stimulation of crustacean (Na+, K+) ATPase by potassium and ammonium ions when PNPP is used a substrate. The results reported herein for Callinectes ornatus branchial (Na+, K+) ATPase might open new perspectives concerning the physiological adaption and the survival of these animals in different environmental.

ASSUNTO(S)

(na+ nh4+ excretion k+)-atpase nh4cl k+)-atpase osmoregulation (na+ brânquia excreção

ACESSO AO ARTIGO

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