A kinetic study of the gill (Na+, K+)-ATPase, and its role in ammonia excretion in the intertidal hermit crab, Clibanarius vittatus.
To better comprehend the role of gill ion regulatory mechanisms, the modulation by Na(+), K(+), NH(4)(+) and ATP of (Na(+), K(+))-ATPase activity was examined in a posterior gill microsomal fraction from the hermit crab, Clibanarius vittatus. Under saturating Mg(2+), Na(+) and K(+) concentrations, two well-defined ATP hydrolyzing sites were revealed. ATP was hydrolyzed at the high-affinity sites at a maximum rate of V=19.1+/-0.8 U mg(-1) and K(0.5)=63.8+/-2.9 nmol L(-1), obeying cooperative kinetics (n(H)=1.9); at the low-affinity sites, hydrolysis obeyed Michaelis-Menten kinetics with K(M)=44.1+/-2.6 mumol L(-1) and V=123.5+/-6.1 U mg(-1). Stimulation by Na(+) (V=149.0+/-7.4 U mg(-1); K(M)=7.4+/-0.4 mmol L(-1)), Mg(2+) (V=132.0+/-5.3 U mg(-1); K(0.5)=0.36+/-0.02 mmol L(-1)), NH(4)(+) (V=245.6+/-9.8 U mg(-1); K(M)=4.5+/-0.2 mmol L(-1)) and K(+) (V=140.0+/-4.9 U mg(-1); K(M)=1.5+/-0.1 mmol L(-1)) followed a single saturation curve and, except for Mg(2+), obeyed Michaelis-Menten kinetics. Under optimal ionic conditions, but in the absence of NH(4)(+), ouabain (K(I)=117.3+/-3.5 mumol L(-1)) and orthovanadate inhibited up to 67% of the ATPase activity. The inhibition studies performed suggest the presence of F(0)F(1), V- and P-ATPases, but not Na(+)-, K(+)- or Ca(2+)-ATPases as contaminants in the gill microsomal preparation. (Na(+), K(+))-ATPase activity was synergistically modulated by NH(4)(+) and K(+). At 20 mmol L(-1) K(+), a maximum rate of V=290.8+/-14.5 U mg(-1) was seen as NH(4)(+) concentration was increased up to 50 mmol L(-1). However, at fixed NH(4)(+) concentrations, no additional stimulation was found for increasing K(+) concentrations (V=135.2+/-4.1 U mg(-1) and V=236.6+/-9.5 U mg(-1) and for 10 and 30 mmol L(-1) NH(4)(+), respectively). This is the first report to detail ionic modulation of gill (Na(+), K(+))-ATPase in C. vittatus, revealing an asymmetrical, synergistic stimulation of the enzyme by K(+) and NH(4)(+), as yet undescribed for other (Na(+), K(+))-ATPases, and should provide a better understanding of NH(4)(+) excretion in pagurid crabs.
Gonçalves
Rúbia R
RR
Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo. Ribeirão Preto 14040-901, SP, Brazil.
Masui
Douglas C
DC
McNamara
John C
JC
Mantelatto
Fernando L M
FL
Garçon
Daniela P
DP
Furriel
Rosa P M
RP
Leone
Francisco A
FA
D000255:Adenosine Triphosphate / Q000378:metabolism; D000641:Ammonia / Q000378:metabolism*; D000818:Animals; D033381:Anomura / Q000201:enzymology / Q000378:metabolism*; D005880:Gills / Q000201:enzymology*; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology
D014638:Vanadates; D010042:Ouabain; D000641:Ammonia; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium