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

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 α-subunit with an Mr of ≈ 110 kDa. The α-subunit was immunolocalized to the intralamellar septum of the gill lamellae. The (Na(+), K(+))-ATPase hydrolyzed ATP obeying Michaelis-Menten kinetics with VM = 109.5 ± 3.2 nmol Pi min(-1) mg(-1) and KM = 0.03 ± 0.003 mmol L(-1). Mg(2+) (VM = 109.8 ± 2.1 nmol Pi min(-1 )mg(-1), K0.5 = 0.60 ± 0.03 mmol L(-1)), Na(+) (VM = 117.6 ± 3.5 nmol Pi min(-1 ) mg(-1), K0.5 = 5.36 ± 0.14 mmol L(-1)), K(+) (VM = 112.9 ± 1.4 nmol Pi min(-1 )mg(-1), K0.5 = 1.32 ± 0.08 mmol L(-1)), and NH4 (+) (VM = 200.8 ± 7.1 nmol Pi min(-1 )mg(-1), K0.5 = 2.70 ± 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 (KI = 84.0 ± 2.1 µmol L(-1)) and orthovanadate (KI = 0.157 ± 0.001 µmol L(-1)) inhibited total ATPase activity by ≈ 50 and ≈ 44 %, respectively. Ouabain inhibition increases ≈ 80 % in the presence of NH4 (+) with a threefold lower KI, suggesting that NH4 (+) is likely transported as a K(+) congener.

Low salinity‐induced alterations in epithelial ultrastructure, Na+/K+‐ATPase immunolocalization and enzyme kinetic characteristics in the gills of the thinstripe hermit crab, Clibanarius vittatus (Anomura, Diogenidae)

“…Stimulation of Na + /K + ‐ATPase activity by NH 4 + in C. vittatus was greater than by K + (Table ), and similar to other decapod gill enzymes (França et al., ; Furriel, Masui, McNamara, & Leone, , ; Garçon et al., ; Leone et al., , ; Holliday, ; Masui et al., ; Santos et al., ). NH 4 + affinity in 15‰ S‐acclimated crabs without K + is very similar to that of fresh‐caught (33‰ S) and 45‰ S‐acclimated crabs (Gonçalves et al., ; Lucena et al., ), and crab nerve (Skou, ) and gill tissue (Masui et al., ).…”

“…Gill Na + /K + ‐ATPase affinity for Mg 2+ is not salinity dependent in C. vittatus , being similar in 15‰ S‐acclimated, fresh‐caught (33‰ S), and 45‰ S‐acclimated crabs (Table ). Mg 2+ affinity is similar to C. danae (Masui et al., ), Macrobrachium olfersi (Furriel, McNamara, & Leone, ), M. amazonicum (Leone et al., ; Santos et al., ), Xiphopenaeus kroyeri (Leone et al., ), C. ornatus (Garçon et al., ), and Dilocarcinus pagei (Furriel et al., ), species in which Mg 2+ stimulation is cooperative, suggesting multiple binding sites. The inhibition seen with excess Mg 2+ , likely a result of Mg 2+ binding to a second inhibitory site (Pedemonte & Beaugé, ) corroborates the notion of multiple binding sites.…”

“…The apical pillar cell flanges form a simple epithelium below the fine cuticle, their cell bodies projecting onto an intralamellar septum that divides the lamellar hemolymph space into two flattened compartments, each further subdivided into lacunae by the pillar cell bodies (Freire & McNamara, 1995;. As seen in palaemonid (Boudour-Boucheker, Boulo, Lorin-Nebel, Elguero, & Grousset, 2013Leone et al, 2014;McNamara & Torres, 1999) and penaeid gill lamellae (Leone et al, 2015b), the Na + /K + -ATPase is located unequivocally in the intralamellar septal cells, both in crabs in seawater, and in those acclimated to dilute seawater in which immuno-staining for the -subunit is very intense, owing to augmented membrane area and likely Na + /K + -ATPase gene and protein expression (cf., Lucena et al, 2012). These alterations in pillar cell and septal cell ultrastructure likely underpin the ability of C. vittatus to hyper-regulate its hemolymph F I G U R E 1 0 Effect of ouabain on total ATPase activity in a gill microsomal fraction from C. vittatus acclimated to 15‰ salinity for 10 days.…”

“…Xiphopenaeus kroyeri (Leone et al, 2015b), C. ornatus (Garçon et al, 2007), and Dilocarcinus pagei (Furriel et al, 2010), species in which Mg 2+ stimulation is cooperative, suggesting multiple binding sites. The inhibition seen with excess Mg 2+ , likely a result of Mg 2+ binding to a second inhibitory site (Pedemonte & Beaugé, 1983) corroborates the notion of multiple binding sites.…”

Goniopsis cruentata: caracterização cinética e bioquímica da (Na+, K+)-ATPase do tecido branquial e análise da sequência de RNAm

“…A razão para esta grande variação, se deve ao fato da afinidade da enzima pelo Na + estar relacionada com a isoforma da subunidade α, sendo o tecido onde a mesma é expressa também extremamente importante (SWEADNER, 1989;LEVENSON, 1994;THERIEN et al, 1996;BLANCO e MERCER, 1998;SWEENEY e KLIP, 1998;CRAMBERT et al, 2000;LOPEZ et al, 2002). Tinha-se como premissa que os valores de K0.5 seriam maiores em espécies de água doce, porém, estudos recentes têm mostrado que, independente da salinidade e/ou aclimatação, se tem uma variação constante nos valores de K0.5 (4,0 a 6,5 mmol L -1 ) (LEONE et al, 2015a;GARÇON et al, 2009).…”