Purification and characterization of gill (Na+, K+)-ATPase in the freshwater crayfish Orconectes limosus rafinesque

Kosiol, Barbara; Bigalke, T; Graszynski, Kai

doi:10.1016/0305-0491(88)90279-9

Cited by 7 publications

(5 citation statements)

References 26 publications

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“…ATPase activities were measured according to Onishi et al ('76) as liberated inorganic phosphate in the homogenate. Measurement of Na + /K + -ATPase activity was conducted similar to Kosiol et al ('88), incubating in each assay 9 μL of the homogenate for 30 min with 21 μL of a reaction buffer containing NaCl (100 mmol L −1 ), KCl (20 mmol L −1 ), MgCl 2 (4 mmol L −1 ), HEPES (50 mmol L −1 ), TRIS (9 mmol L −1 ) and Na 2 ATP (3 mmol L −1 ) adjusted to a pH of 7.5 with TRIS. Na + /K + -ATPase activity was defined as the difference between the activities obtained with and without 5 mmol L −1 ouabain.…”

Section: Methodsmentioning

confidence: 99%

Strong alkalinization in the anterior midgut of larval yellow fever mosquitoes (Aedes aegypti): involvement of luminal Na⁺/K⁺‐ATPase

et al. 2008

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Recently, Na + /K + -ATPase has been detected in the luminal membrane of the anterior midgut of larval yellow fever mosquitoes (Aedes aegypti) with immunohistochemical techniques. In this study, the possible involvement of this ATPase in strong alkalinization was investigated on the level of whole larvae, isolated and perfused midgut preparations and on the molecular level of the Na + /K + -ATPase protein. Ouabain (5 mM) did not inhibit the capability of intact larval mosquitoes to alkalinize their anterior midgut. Also in isolated and perfused midgut preparations the perfusion of the lumen with ouabain (5 mM) did not result in a significant change of the transepithelial voltage or the capacity of luminal alkalinization. Na + /K + -ATPase activity was completely abolished when KCl was substituted with choline chloride, suggesting that the enzyme cannot act as an ATP-driven Na + /H + -exchanger. Altogether the results of the present investigation indicate that apical Na + /K + -ATPase is not of direct importance for strong luminal alkalinization in the anterior midgut of larval yellow fever mosquitoes.Strong luminal alkalinization of up to pH values of 12 has been observed in midgut regions of many larvae of endopterygote insects, including members of the orders Coleoptera, Diptera, Trichoptera and Lepidoptera (for references see Clark, '99). Two tissues have been studied especially intensively with regard to their mechanisms and capacities of alkalinization during the past 20 years: the midgut of the tobacco hornworm (Manduca sexta) and the anterior midgut of larval yellow fever mosquitoes (Aedes aegypti). For the lepidopteran larvae, a transport model has been established in which luminal V-type H + -pumps (V-ATPases) energize luminal, electrogenic K + /2H + -exchangers, resulting in active K + secretion and strong luminal alkalinization (for a review see Wieczorek et al., '99). Unfortunately, this mechanism can so far not be tested with the isolated epithelium, because it loses the capacity for strong luminal alkalinization after isolation, whereas active K + secretion continues in vitro at a high rate (Clark et al., '98). Strong luminal alkalinization in the anterior midgut of larval yellow fever mosquitoes appears to significantly differ from lepidopterans, including the methodological aspect that it can readily be observed with isolated and perfused midgut preparations (Onken et al., 2008). In larval A. aegypti, the V-ATPase is not localized in the luminal membrane of the anterior midgut, but instead in the basolateral membrane (Zhuang et al., '99). The epithelium of larval yellow fever mosquitoes generates a lumen negative transepithelial voltage (Clark et

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Section: Methodsmentioning

confidence: 99%

Strong alkalinization in the anterior midgut of larval yellow fever mosquitoes (Aedes aegypti): involvement of luminal Na⁺/K⁺‐ATPase

et al. 2008

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“…SDS activation and measurement of Na + /K + -ATPase activity were conducted according to Kosiol et al (1988). In each assay, 10-20 g of membrane protein was used.…”

Section: Atpase Assaysmentioning

confidence: 99%

“…The reaction was initiated with 3 mmol l Ϫ1 Tris-ATP. After 15 min, the amount of liberated phosphate was measured as above (Kosiol et al 1988). V-ATPase activity was defined as the difference between the activity obtained with and without 1 mol l Ϫ1 bafilomycin A1 (Bowman et al 1988).…”

Section: Atpase Assaysmentioning

confidence: 99%

A V-ATPase DRIVES ACTIVE, ELECTROGENIC AND Na+-INDEPENDENT Cl−ABSORPTION ACROSS THE GILLS OF ERIOCHEIR SINENSIS

Onken¹,

Putzenlechner²

1995

Journal of Experimental Biology

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Using biochemical and electrophysiological techniques, we have examined the proposal that an H+-ATPase is involved in Cl- uptake across the gills of the Chinese crab Eriocheir sinensis. Bafilomycin A1 (1 µmol l-1), a specific inhibitor of V-ATPases, was used to investigate the importance of this H+-translocating enzyme in Cl- transport across the gill. In homogenates of ion-transporting posterior gills, we found the activity of a bafilomycin-sensitive V-ATPase to be markedly higher than in the anterior gills, which are not involved in ion transport. A similar distribution was found for the Na+/K+- and the mitochondrial F1Fo-ATPase. After differential and density centrifugation, the specific activity of the V-ATPase was enriched by a factor of 5. Neither Na+/K+- and F1Fo-ATPase activities nor acid phosphatase activity copurified with the bafilomycin-sensitive ATPase activity, indicating that at least the major portion of V-ATPase activity is not of basolateral, mitochondrial or lysosomal origin. In fluorescence studies, using Acridine Orange or Oxonol V as dyes, membrane vesicles displayed ATP-dependent proton transport and membrane potential generation, which were markedly reduced in the presence of bafilomycin. In addition to these biochemical studies, we mounted split lamellae of posterior gills in an Ussing-type chamber and measured the negative short-circuit current (Isc), which was shown to reflect active, electrogenic, Na+-independent and ouabain-insensitive Cl- absorption. After the addition of 1 µmol l-1 bafilomycin to the external bath, this Isc was reduced to about 5060 % of its original value. Concomitantly, the conductance of the preparation decreased by about 13 %. From these results, we conclude that an apical V-ATPase drives electrogenic Cl- uptake across the posterior gills of the Chinese crab.

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“…Activity of CA was completely inhibited in all fractions with 10 -6 tool/1 acetazolamide. Na~-/K~--ATP'ase activity, localized in the basolateral membrane of epithelial gill cells (Towle & Kays, 1986), was determined measuring the liberated inorganic phosphate according to Kosiol et al (1988). Succinate dehydrogenase (SDH) activity, a marker for mitochondrial contamination, was measured according to the methods used by Clarke & Porteous (1964) and Green & Narahara (1980).…”

mentioning

confidence: 99%

The carbonic anhydrase of the Chinese crabEriocheir sinensis: Effects of adaption from tap to salt water

Olsowski,

Putzenlechner,

Böttcher

et al. 1995

Helgolander Meeresunters

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In the present investigation we studied the carbonic anhydrase (CA) in various tissues of Chinese crab Eriocheir sinensis which were acclimated to different salinities (0, 10, 20, 30 ~/~). We found only negligible CA activity in haemolymph, heart, hypodermis, antennat gland, leg muscle and digestive gland, irrespective of the acclimation medium. However, high amounts of CA activity were found in the gills. In the case of the posterior gills, a strong dependence on the acclimatization of the animals was demonstrated; the highest activities were found in those adapted to tap water. To investigate the cellular distribution of the CA in the posterior gills, the additional enzyme activities were measured in all fractions of a differential centrifugation of the gill homogenate: Na+/K +-ATP'ase (a marker for the plasmamembrane); lactate dehydrogenase (LDH; as marker for the cytosot); and succinate dehydrogenase (SDH; as marker for mitochondria). Independent of the acclimation salinity (0 or 36 %,, salinity), we found about 70 % of CA associated with the highest level of the Na+/K+-ATP'ase in the second 100000 g pellet (membrane fraction), while only 15 % were found in the cytosolic fractions (associated with highest levels of LDH). We conclude that the carbonic anhydrase of posterior gills of the Chinese crab is mainly membrane-bound. Furthermore, the activity of CA shows a strong dependence on the salinity of the water in which the crabs were kept.

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Purification and characterization of gill (Na+, K+)-ATPase in the freshwater crayfish Orconectes limosus rafinesque

Cited by 7 publications

References 26 publications

Strong alkalinization in the anterior midgut of larval yellow fever mosquitoes (Aedes aegypti): involvement of luminal Na⁺/K⁺‐ATPase

Strong alkalinization in the anterior midgut of larval yellow fever mosquitoes (Aedes aegypti): involvement of luminal Na⁺/K⁺‐ATPase

A V-ATPase DRIVES ACTIVE, ELECTROGENIC AND Na+-INDEPENDENT Cl−ABSORPTION ACROSS THE GILLS OF ERIOCHEIR SINENSIS

The carbonic anhydrase of the Chinese crabEriocheir sinensis: Effects of adaption from tap to salt water

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Purification and characterization of gill (Na+, K+)-ATPase in the freshwater crayfish Orconectes limosus rafinesque

Cited by 7 publications

References 26 publications

Strong alkalinization in the anterior midgut of larval yellow fever mosquitoes (Aedes aegypti): involvement of luminal Na+/K+‐ATPase

Strong alkalinization in the anterior midgut of larval yellow fever mosquitoes (Aedes aegypti): involvement of luminal Na+/K+‐ATPase

A V-ATPase DRIVES ACTIVE, ELECTROGENIC AND Na+-INDEPENDENT Cl−ABSORPTION ACROSS THE GILLS OF ERIOCHEIR SINENSIS

The carbonic anhydrase of the Chinese crabEriocheir sinensis: Effects of adaption from tap to salt water

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Strong alkalinization in the anterior midgut of larval yellow fever mosquitoes (Aedes aegypti): involvement of luminal Na⁺/K⁺‐ATPase

Strong alkalinization in the anterior midgut of larval yellow fever mosquitoes (Aedes aegypti): involvement of luminal Na⁺/K⁺‐ATPase