Malson N. Lucena

Isolation and functional characterization of proinflammatory acidic phospholipase A2 from Bothrops leucurus snake venom

In the present study, an acidic PLA(2), designated Bl-PLA(2), was isolated from Bothrops leucurus snake venom through two chromatographic steps: ion-exchange on CM-Sepharose and hydrophobic chromatography on Phenyl-Sepharose. Bl-PLA(2) was homogeneous on SDS-PAGE and when submitted to 2D electrophoresis the molecular mass was 15,000Da and pI was 5.4. Its N-terminal sequence revealed a high homology with other Asp49 acidic PLA(2)s from snake venoms. Its specific activity was 159.9U/mg and the indirect hemolytic activity was also higher than that of the crude venom. Bl-PLA(2) induced low myotoxic and edema activities as compared to those of the crude venom. Moreover, the enzyme was able to induce increments in IL-12p40, TNF-α, IL-1β and IL-6 levels and no variation of IL-8 and IL-10 in human PBMC stimulated in vitro, suggesting that Bl-PLA(2) induces proinflammatory cytokine production by human mononuclear cells. Bothrops leucurus venom is still not extensively explored and knowledge of its components will contribute for a better understanding of its action mechanism.

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.

Modulation by K+ Plus NH4+ of microsomal (Na+, K+)-ATPase activity in selected ontogenetic stages of the diadromous river shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae).

We investigate the synergistic stimulation by K+ plus NH4 + of (Na+, K+)-ATPase activity in microsomal preparations of whole zoea I and decapodid III, and in juvenile and adult river shrimp gills. Modulation of (Na+, K+)-ATPase activity is ontogenetic stage-specific, and particularly distinct between juveniles and adults. Although both gill enzymes exhibit two different sites for K+ and NH4 + binding, in the juvenile enzyme, these two sites are equivalent: binding by both ions results in slightly stimulated activity compared to that of a single ionic species. In the adult enzyme, the sites are not equivalent: when one ion occupies its specific binding site, (Na+, K+)-ATPase activity is stimulated synergistically by ≈50% on binding of the complementary ion. Immunolocalization reveals the enzyme to be distributed predominantly throughout the intralamellar septum in the gill lamellae of juveniles and adults. Western blot analyses demonstrate a single immunoreactive band, suggesting a single (Na+, K+)-ATPase α-subunit isoform that is distributed into different density membrane fractions, independently of ontogenetic stage. We propose a model for the modulation by K+ and NH4 + of gill (Na+, K+)-ATPase activity. These findings suggest that the gill enzyme may be regulated by NH4 + during ontogenetic development in M. amazonicum.

A kinetic characterization of the gill V(H+)-ATPase in juvenile and adult Macrobrachium amazonicum, a diadromous palaemonid shrimp

Novel kinetic properties of a microsomal gill V(H(+))-ATPase from juvenile and adult Amazon River shrimp, Macrobrachium amazonicum, are described. While protein expression patterns are markedly different, Western blot analysis reveals a sole immunoreactive band, suggesting a single V(H(+))-ATPase subunit isoform, distributed in membrane fractions of similar density in both ontogenetic stages. Immunofluorescence labeling locates the V(H(+))-ATPase in the apical regions of the lamellar pillar cells in both stages in which mRNA expression of the V(H(+))-ATPase B-subunit is identical. Juvenile (36.6±3.3 nmol Pi min(-1) mg(-1)) and adult (41.6±1.3 nmol Pi min(-1) mg(-1)) V(H(+))-ATPase activities are similar, the apparent affinity for ATP of the adult enzyme (K0.5=0.21±0.02 mmol L(-1)) being 3-fold greater than for juveniles (K0.5=0.61±0.01 mmol L(-1)). The K0.5 for Mg(2+) interaction with the juvenile V(H(+))-ATPase (1.40 ± 0.07 mmol L(-1)) is ≈6-fold greater than for adults (0.26±0.02 mmol L(-1)) while the bafilomycin A1 inhibition constant (KI) is 45.0±2.3 nmol L(-1) and 24.2±1.2 nmol L(-1), for juveniles and adults, respectively. Both stages exhibited residual bafilomycin-insensitive ATPase activity of ≈25 nmol Pi min(-1) mg(-1), suggesting the presence of ATPases other than the V(H(+))-ATPase. These differences may reflect a long-term regulatory mechanism of V(H(+))-ATPase activity, and suggest stage-specific enzyme modulation. This is the first kinetic analysis of V(H(+))-ATPase activity in different ontogenetic stages of a freshwater shrimp and allows better comprehension of the biochemical adaptations underpinning the establishment of palaemonid shrimps in fresh water.

Synergistic stimulation by potassium and ammonium of K(+)-phosphatase activity in gill microsomes from the crab Callinectes ornatus acclimated to low salinity: novel property of a primordial pump.

We provide an extensive characterization of the modulation by p-nitrophenylphosphate, Mg²⁺, Na⁺, K(+), Rb⁺, NH(4)(+) and pH of gill microsomal K⁺-phosphatase activity in the posterior gills of Callinectes ornatus acclimated to low salinity (21‰). The synergistic stimulation by K⁺ and NH(4)(+) of the K⁺-phosphatase activity is a novel finding, and may constitute a species-specific feature of K(+)/NH(4)(+) interplay that regulates crustacean gill (Na⁺, K⁺)-ATPase activity. p-Nitrophenylphosphate was hydrolyzed at a maximum rate (V) of 69.2 ± 2.8nmolPimin⁻¹mg⁻¹ with K(0.5)=2.3 ± 0.1mmolL(-1), obeying cooperative kinetics (n(H)=1.7). Stimulation by Mg²⁺ (V=70.1 ± 3.0nmolPimin⁻¹mg⁻¹, K(0.5)=0.88 ± 0.04mmolL⁻¹), K⁺ (V=69.6 ± 2.7nmolPimin⁻¹mg⁻¹, K(0.5)=1.60 ± 0.07mmolL⁻¹) and NH(4)(+) (V=90.8 ± 4.0nmolPimin⁻¹mg⁻¹, K(0.5)=9.2 ± 0.3mmol L⁻¹) all displayed site-site interaction kinetics. In the presence of NH(4)(+), enzyme affinity for K⁺ unexpectedly increased by 7-fold, while affinity for NH(4)(+) was 28-fold greater in the presence than absence of K⁺. Ouabain partially inhibited K⁺-phosphatase activity (K(I)=320 ± 14.0μmolL⁻¹), more effectively when NH(4)(+) was present (K(I)=240 ± 12.0μmolL⁻¹). We propose a model for the synergistic stimulation by K⁺ and NH(4)(+) of the K⁺-phosphatase activity of the (Na⁺, K⁺)-ATPase from C. ornatus posterior gill tissue.

Na+,K+-ATPase Activity in the Posterior Gills of the Blue Crab, Callinectes ornatus (Decapoda, Brachyura): Modulation of ATP Hydrolysis by the Biogenic Amines Spermidine and Spermine

We investigated the effect of the exogenous polyamines spermine, spermidine and putrescine on modulation by ATP, K+, Na+, NH4+ and Mg2+ and on inhibition by ouabain of posterior gill microsomal Na+,K+-ATPase activity in the blue crab, Callinectes ornatus, acclimated to a dilute medium (21‰ salinity). This is the first kinetic demonstration of competition between spermine and spermidine for the cation sites of a crustacean Na+,K+-ATPase. Polyamine inhibition is enhanced at low cation concentrations: spermidine almost completely inhibited total ATPase activity, while spermine inhibition attained 58%; putrescine had a negligible effect on Na+,K+-ATPase activity. Spermine and spermidine affected both V and K for ATP hydrolysis but did not affect ouabain-insensitive ATPase activity. ATP hydrolysis in the absence of spermine and spermidine obeyed Michaelis–Menten behavior, in contrast to the cooperative kinetics seen for both polyamines. Modulation of V and K by K+, Na+, NH4+ and Mg2+ varied considerably in the presence of spermine and spermidine. These findings suggest that polyamine inhibition of Na+,K+-ATPase activity may be of physiological relevance to crustaceans that occupy habitats of variable salinity.

Gill Ion Transport ATPases and Ammonia Excretion in Aquatic Crustaceans

Crustaceans inhabit diverse biotopes, often subject to alterations that constitute a severe challenge to their homeostatic mechanisms. These challenges have driven the evolution of biochemical and physiological processes that have enabled their survival in such niches. Ion-transporting enzymes like the (Na+, K+)-ATPase and V(H+)-ATPase present in the gill epithelia underpin the ion regulatory abilities of these highly diversified organisms. The present chapter examines the structure and function of these two gill ATPases that also participate actively in ammonia excretion. We summarize current knowledge on their role in osmotic and ionic regulation and associated with ontogenetic changes. We analyze the effects of polyamines on (Na+, K+)-ATPase activity and phosphoenzyme formation, aiming to provide insights into the biochemical bases of physiological homeostasis in crustaceans. We examine future perspectives that should provide a better understanding of the role of gill ATPases in active ammonia excretion.

Gill (Na+, K+)-ATPase from the Amazon River shrimp, Macrobrachium amazonicum (Decapoda, Palaemonidae): effect of exogenous biogenic amines on enzyme activity in juveniles and adults

Despite the biological and economic importance of the freshwater shrimp Macrobrachium amazonicum, a role for biogenic amines in the osmoregulatory capability of this diadromous species remains unstudied. This investigation provides an extensive kinetic characterization of the effects of the exogenous polyamines spermine, spermidine, and putrescine on (Na+, K+)-ATPase activity in gill microsomal preparations from juvenile and adult shrimps at varying concentrations of ATP, Mg2+, Na+, and K+, and on inhibition by ouabain. (Na+, K+)-ATPase activity in both juvenile and adult shrimps is inhibited by spermidine (60–95%) and putrescine (40–70%) while spermine has a negligible inhibitory effect (<10%). Putrescine affects the kinetics of ATP hydrolysis and enzyme affinity (KM) for ATP in both enzymes, while spermidine alters specific activity only. Spermidine increases Mg2+ affinity of the adult (Na+, K+)-ATPase while putrescine decreases Mg2+ affinity in both enzymes. Spermidine decreases Na+ affinity by ≈50% in both enzymes, while putrescine increases affinity of the juvenile enzyme twofold. These biogenic amines cause slight changes in KM values in both enzymes. These findings suggest that the effect of biogenic amines on gill (Na+, K+)-ATPase activity and kinetics may be species and stage specific.

Kinetic characterization of the gill (Na+, K+)-ATPase in a hololimnetic population of the diadromous Amazon River shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae)

We provide a kinetic characterization of (Na+, K+)-ATPase activity in a posterior gill microsomal fraction from a hololimnetic population of the diadromous Amazon River shrimp Macrobrachium amazonicum. Sucrose density gradient centrifugation reveals two distinct membrane fractions showing considerable (Na+, K+)ATP-ase activity, but also containing other microsomal ATPases. Only a single immune-reactive (Na+, K+)-ATPase with Mr of ≈110 kDa is present that hydrolyzes ATP with VM = 130.3 ± 4.8 nmol Pi min-1 mg protein-1 and K0.5 = 0.065 ± 0.00162 mmol L-1, exhibiting site-site interactions. Stimulation by Na+ (VM = 127.5 ± 5.3 nmol Pi min-1 mg protein-1, K0.5 = 5.3 ± 0.42 mmol L-1), Mg2+ (VM = 130.6 ± 6.8 nmol Pi min-1 mg protein-1, K0.5 = 0.33 ± 0.042 mmol L-1), K+ (VM = 126.7 ± 7.7 nmol Pi min-1 mg protein-1, K0.5 = 0.65 ± 0.0079 mmol L-1) and NH4+ (VM = 134.5 ± 8.6 nmol Pi min-1 mg protein-1, K0.5 = 1.28 ± 0.44 mmol L-1) also obeys cooperative kinetics. Ouabain (KI = 0.18 ± 0.058 mmol L-1) inhibits total ATPase activity by ≈70%. This study reveals considerable differences in the kinetic characteristics of the gill (Na+, K+)-ATPase in a hololimnetic population that appear to result from the adaptation of diadromous Macrobrachium amazonicum populations to different limnic habitats.

BIO011 Kinetic Characterization of a (Na+,K+)-ATPase Activity in the Freshwater Shrimp Zoeae I Macrobrachium rosenbergii

Macrobrachium rosenbergii is the species of freshwater shrimp more used in the commercial aquaculture. This freshwater prawn, also known as the giant Malaysian prawn, is native to the tropical Indo-Pacific region and has been introduced in several countries due to its economic interest. M. rosenbergii belongs to the family Palaemonidae which include the brackish and freshwater shrimps, where most species which comprise this family require brackish water to complete the early stages of their life cycle. The newly hatched larvae must reach brackish water with salinities of 10 to 14 parts per thousand (ppt) within two days or they will not survive. At this stage, they feed on zooplankton, worms and the larvae of other aquatic organisms and to reach the post-larval stage, the larvae undergo about 11 molts in approximately 35 days. During larval development many changes occur both in structure and physiology of the shrimp. The ability to survive in different salinities, until its complete establishment in freshwater, is dependent on enzymes that control and regulate the metabolism of the animal. Various enzymes are responsible for active ion transport in crustacean although their importance in osmoregulatory mechanisms differs among species. The (Na+,K+)-ATPase and the V(H+)-ATPase are the two most important enzymes.