Hemolymph ion regulation and kinetic characteristics of the gill (Na⁺, K⁺)-ATPase in the hermit crab Clibanarius vittatus (Decapoda, Anomura) acclimated to high salinity.

We examine hemolymph ion regulation and the kinetic properties of a gill microsomal (Na(+), K(+))-ATPase from the intertidal hermit crab, Clibanarius vittatus, acclimated to 45‰ salinity for 10 days. Hemolymph osmolality is hypo-regulated (1102.5 ± 22.1 mOsm kg(-1) H(2)O) at 45‰ but elevated compared to fresh-caught crabs (801.0 ± 40.1 mOsm kg(-1) H(2)O). Hemolymph [Na(+)] (323.0 ± 2.5 mmol L(-1)) and [Mg(2+)] (34.6 ± 1.0 mmol L(-1)) are hypo-regulated while [Ca(2+)] (22.5 ± 0.7 mmol L(-1)) is hyper-regulated; [K(+)] is hyper-regulated in fresh-caught crabs (17.4 ± 0.5 mmol L(-1)) but hypo-regulated (6.2 ± 0.7 mmol L(-1)) at 45‰. Protein expression patterns are altered in the 45‰-acclimated crabs, although Western blot analyses reveal just a single immunoreactive band, suggesting a single (Na(+), K(+))-ATPase α-subunit isoform, distributed in different density membrane fractions. A high-affinity (Vm=46.5 ± 3.5 Umg(-1); K(0.5)=7.07 ± 0.01 µmol L(-1)) and a low-affinity ATP binding site (Vm=108.1 ± 2.5 U mg(-1); K(0.5)=0.11 ± 0.3 mmol L(-1)), both obeying cooperative kinetics, were disclosed. Modulation of (Na(+), K(+))-ATPase activity by Mg(2+), K(+) and NH(4)(+) also exhibits site-site interactions, but modulation by Na(+) shows Michaelis-Menten kinetics. (Na(+), K(+))-ATPase activity is synergistically stimulated up to 45% by NH(4)(+) plus K(+). Enzyme catalytic efficiency for variable [K(+)] and fixed [NH(4)(+)] is 10-fold greater than for variable [NH(4)(+)] and fixed [K(+)]. Ouabain inhibited ≈80% of total ATPase activity (K(I)=464.7 ± 23.2 µmol L(-1)), suggesting that ATPases other than (Na(+), K(+))-ATPase are present. While (Na(+), K(+))-ATPase activities are similar in fresh-caught (around 142 nmol Pi min(-1)mg(-1)) and 45‰-acclimated crabs (around 154 nmol Pi min(-1)mg(-1)), ATP affinity decreases 110-fold and Na(+) and K(+) affinities increase 2-3-fold in 45‰-acclimated crabs.

Na+, K+-ATPase activity in gill microsomes from the blue crab, Callinectes danae, acclimated to low salinity: novel perspectives on ammonia excretion.

This investigation provides an extensive characterization of the modulation by ATP, Mg(2+), Na(+), K(+) and NH(4)(+) of a gill microsomal (Na(+),K(+))-ATPase from Callinectes danae acclimated to 15 per thousand salinity. Novel findings are the lack of high-affinity ATP-binding sites and a 10-fold increase in enzyme affinity for K(+) modulated by NH(4)(+), discussed regarding NH(4)(+) excretion in benthic marine crabs. The (Na(+),K(+))-ATPase hydrolyzed ATP at a maximum rate of 298.7+/-16.7 nmol Pi min(-1) mg(-1) and K(0.5)=174.2+/-9.8 mmol L(-1), obeying cooperative kinetics (n(H)=1.2). Stimulation by sodium (V=308.9+/-15.7 nmol Pi min(-1) mg(-1), K(0.5)=7.8+/-0.4 mmol L(-1)), magnesium (299.2+/-14.1 nmol Pi min(-1) mg(-1), K(0.5)=767.3+/-36.1 mmol L(-1)), potassium (300.6+/-15.3 nmol Pi min(-1) mg(-1), K(0.5)=1.6+/-0.08 mmol L(-1)) and ammonium (V=345.1+/-19.0 nmol Pi min(-1) mg(-1), K(0.5)=6.0+/-0.3 mmol L(-1)) ions showed site-site interactions. Ouabain inhibited (Na(+),K(+))-ATPase activity with K(I)=45.1+/-2.5 micromol L(-1), although affinity for the inhibitor increased (K(I)=22.7+/-1.1 micromol L(-1)) in 50 mmol L(-1) NH(4)(+). Inhibition assays using ouabain plus oligomycin or ethacrynic acid suggest mitochondrial F(0)F(1)- and K(+)-ATPase activities, respectively. Ammonium and potassium ions synergistically stimulated specific activity up to 72%, inferring that these ions bind to different sites on the enzyme molecule, each modulating stimulation by the other.

Hemolymph ionic regulation and adjustments in gill (Na+, K+)-ATPase activity during salinity acclimation in the swimming crab Callinectes ornatus (Decapoda, Brachyura).

We evaluate hemolymph osmotic and ionic regulatory abilities and characterize a posterior gill microsomal (Na+, K+)-ATPase from the marine swimming crab, Callinectes ornatus, acclimated to 21 per thousand or 33 per thousand salinity. C. ornatus is isosmotic after acclimation to 21 per thousand but is hyposmotic at 33 per thousand salinity; hemolymph ions do not recover initial levels on acclimation to 21 per thousand salinity but are anisoionic compared to ambient concentrations, revealing modest regulatory ability. NH4+ modulates enzyme affinity for K+, which increases 187-fold in crabs acclimated to 33 per thousand salinity. The (Na+, K+)-ATPase redistributes into membrane fractions of different densities, suggesting that altered membrane composition results from salinity acclimation. ATP was hydrolyzed at maximum rates of 182.6 +/- 7.1 nmol Pi min(-1) mg(-1) (21 per thousand) and 76.2 +/- 3.5 nmol Pi min(-1) mg(-1) (33 per thousand), with little change in KM values (approximately 50 micromol L(-1)). K+ together with NH4+ synergistically stimulated activity to maximum rates of approximately 240 nmol Pi min(-1) mg(-1). KI values for ouabain inhibition (approximately 110 micromol L(-1)) decreased to 44.9 +/- 1.0 micromol L(-1) (21 per thousand) and 28.8 +/- 1.3 micromol L(-1) (33 per thousand) in the presence of both K+ and NH4+. Assays employing various inhibitors suggest the presence of mitochondrial F0F1-, and K+- and V-ATPase activities in the gill microsomes.

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.

Temporal-spatial distribution of the hermit crab Loxopagurus loxochelis (Decapoda: Diogenidae) from Ubatuba Bay, São Paulo State, Brazil.

The present work was conducted to determine the distribution of Loxopagurus loxochelis collected monthly, over a period of one year. in Ubatuba Bay (from September/95 to August/96). A total of 179 specimens were collected (138 males, 30 females and 11 ovigerous females). The greatest depth, predominance of very fine sand and highest mean value of organic matter contents of sediment, in combination with the low temperatures registered in winter (July and August), determined the presence of L. loxochelis in the subarea located at the Ubatuba Bay mouth, exposed to the open sea with high water current energy, important because this position insures that spawned larvae will enter into the oceanic circulation.