What role for membranes in determining the higher sodium pump molecular activity of mammals compared to ectotherms?

The major body organs of mammals have sodium pumps that turn over energy (ATP) three to four times faster than those of ectotherms, at the same temperature. To examine if membranes play a role in these differences in molecular activity, membrane cross-over experiments were performed using two representative species, Rattus norvegicus and Bufo marinus. Microsomal membrane of kidney and brain displayed characteristic molecular activity differences (three- to four-fold) between the species. These molecular activity differences could be removed by delipidation. Pre-existing molecular activities and differences could be restored when reconstituted with original membrane. Using the same reconstitution method, species membrane cross-over experiments resulted in toad sodium pumps in rat membrane significantly increasing (≈30–40%) and rat sodium pumps in toad membrane significantly decreasing (≈40%) activities in both kidney and brain. Analysis of membrane composition showed reduced cholesterol content and differences in the fatty acids of phospholipids with higher overall unsaturation in the mammal. The scope for membranes to determine protein performance and its broader implications for metabolism are discussed. Accepted: 17 March 1999     

Cell ATP level of Saccharomyces cerevisiae sensitively responds to culture growth and drug-inflicted variations in membrane integrity and PDR pump activity

Cellular ATP level in Saccharomyces cerevisiae was measured during culture growth of strain US50-18C overproducing all major PDR pumps and its isogenic mutants variously deleted in these pumps. It was found to be inversely proportional to the intensity of cell metabolism during different growth phases and to the activity of PDR pumps, which are thus among major ATP consumers in the cells. The ATP level was increased when membrane integrity was affected by 0.5% butanol, and further increased by compound 23.1, a semisynthetic phenol lipid derivative that acts as inhibitor of Pdr5p and Snq2p pumps. The magnitude of increase in cell ATP caused by inhibition of Pdr5p pump by compound 23.1 and the Pdr5p pump inhibitor FK506 used for comparison reflects the activity and hence the energy demand of the pump. The rise in cell ATP caused by different PDR pump inhibitors can be thus used as an indicator of pump activity and the potency of the inhibitor.     

Characterization of membrane calcium pumps by simultaneous immunoblotting and 32P radiography

Calcium pumps of various plasma membrane, endoplasmic reticulum and sarcoplasmic reticulum preparations were visualized by simultaneous immunoblotting and autoradiography of the 32P-labelled phosphoenzymes. The pump proteins and their fragments produced by a proteolytic pretreatment of the membranes were selectively phosphorylated by [gamma-32P]ATP, separated on an acidic SDS-polyacrylamide gel, blotted onto nitrocellulose and reacted with polyclonal antibodies raised against the purified human erythrocyte and rat skeletal muscle sarcoplasmic reticulum calcium pumps, respectively. The immuno-reaction was detected by peroxidase staining, while the phosphoproteins were shown by autoradiography of the same blot. An antibody against the erythrocyte calcium pump, reacting on the blot with the 140 kDa erythrocyte calcium pump and its 80 kDa proteolytic fragment, did not show a cross-reaction with the calcium pump of similar molecular mass in rat synaptosome membranes or with any of the endoplasmic- or sarcoplasmic-type calcium pumps. An anti-sarcoplasmic reticulum calcium pump antibody cross reacted with several sarcoplasmic and endoplasmic calcium pump proteins and their proteolytic fragments but with none of the plasma membrane pumps. This sensitive double-labelling method can be applied to study structural relationships and molecular alterations in various ion pump proteins.     

Characterization of the plasma membrane Mg2+-ATPase from the yeast, Saccharomyces cerevisiae.

The plasma membrane of Saccharomyces cerevisiae has a Mg2+-dependent ATPase which is distinct from the mitochondrial Mg2+-ATPase and at the pH optimum of 5.5 has a Km for ATP of 1.7 mM and a Vmax of 0.42 mumol of ATP hydrolyzed/mg/min. At least three protein components of the crude membrane (Mr = 210,000, 160,000 and 115,000) are labeled with [gamma"32P]ATP at pH 5.5. These phosphoproteins form rapidly in the presence of Mg2+, rapidly turn over the bound phosphate when unlabeled ATP is added, and dephosphorylate after incubation in the presence of hydroxylamine. Vanadate, an inhibitor of the Mg2+-ATPase activity, blocks the phosphorylation of the 210,000- and 115,000-dalton proteins. At pH 7.0, only the 210,000- and 160,000-dalton proteins are phosphorylated. While these three phosphorylated intermediates have not been unambiguously identified as components of the Mg2+-ATPase, the finding of such phosphorylated components in association with that activity implies that this enzyme differs in mechanism from the mitochondrial proton pump and that it is similar in mechanism to the metal ion pumps ((Na+-K+)-ATPase and Ca2+-ATPase) of the mammalian plasma membrane.     

On the functional use of the membrane compartmentalized pool of ATP by the Na+ and Ca++ pumps in human red blood cell ghosts

Previous evidence established that a sequestered form of adenosine triphosphate (ATP pools) resides in the membrane/cytoskeletal complex of red cell porous ghosts. Here, we further characterize the roles these ATP pools can perform in the operation of the membrane''s Na⁺ and Ca²⁺ pumps. The formation of the Na⁺- and Ca²⁺-dependent phosphointermediates of both types of pumps (E_Na-P and E_Ca-P) that conventionally can be labeled with trace amounts of [γ-³P]ATP cannot occur when the pools contain unlabeled ATP, presumably because of dilution of the [γ-³P]ATP in the pool. Running the pumps forward with either Na⁺ or Ca²⁺ removes pool ATP and allows the normal formation of labeled E_Na-P or E_Ca-P, indicating that both types of pumps can share the same pools of ATP. We also show that the halftime for loading the pools with bulk ATP is 10–15 minutes. We observed that when unlabeled “caged ATP” is entrapped in the membrane pools, it is inactive until nascent ATP is photoreleased, thereby blocking the labeled formation of E_Na-P. We also demonstrate that ATP generated by the membrane-bound pyruvate kinase fills the membrane pools. Other results show that pool ATP alone, like bulk ATP, can promote the binding of ouabain to the membrane. In addition, we found that pool ATP alone functions together with bulk Na⁺ (without Mg²⁺) to release prebound ouabain. Curiously, ouabain was found to block bulk ATP from entering the pools. Finally, we show, with red cell inside-outside vesicles, that pool ATP alone supports the uptake of ⁴⁵Ca by the Ca²⁺ pump, analogous to the Na⁺ pump uptake of ²²Na in this circumstance. Although the membrane locus of the ATP pools within the membrane/cytoskeletal complex is unknown, it appears that pool ATP functions as the proximate energy source for the Na⁺ and Ca²⁺ pumps.     

Characterization of a putative Ca2(+)-transporting Ca2(+)-ATPase in the pellicles of Paramecium tetraurelia

In Paramecium, no Ca2(+)-ATPases with the properties of Ca2+ pumps have been identified. Here we report a pellicle associated Ca2(+)-ATPase activity and a corresponding phosphoprotein intermediate characteristic of a pump. The Ca2(+)-ATPase activity requires 3 mM Mg for optimal Ca2+ stimulation (KCa = 90 nM) and is specific for ATP as substrate (Km = 75 microM). Vanadate and calmidazolium inhibit Ca2(+)-stimulated activity with an EC50 of about 2 microM and 0.5 microM, respectively. Likewise, 10 microM trifluoperazine inhibits 80% of Ca2(+)-ATPase activity, but bovine calmodulin fails to stimulate. The Ca2(+)-ATPase is not inhibited by sodium azide (10 mM), oligomycin (10 micrograms/ml) or ouabain (0.2 mM). Incubation of pellicles with [gamma-32P]ATP specifically labels a 133 kDa protein in a Ca2(+)-dependent, hydroxylamine-sensitive manner, and the level of phosphorylation is increased by 100 microM La3+. Phosphorylation of an endoplasmic reticulum-enriched fraction labels a Ca2(+)-dependent protein different from the pellicle protein, being lower in molecular mass and unaffected by La3+. Ca2+ uptake by the alveolar sacs, integral components of the pellicle membrane complex, is poorly coupled to Ca2(+)-stimulated ATP hydrolysis (Ca2+ transported/ATP hydrolysed less than 0.2) and is much less sensitive to vanadate inhibition (EC50 approx. 20 microM) compared to the total Ca2(+)-ATPase activity. Therefore, the majority of the Ca2(+)-ATPase activity is likely to be plasma membrane associated.     

Delivery of ion pumps from exogenous membrane-rich sources into mammalian red blood cells.

Using polyethylene glycol-mediated fusion of ATP-ase-enriched (native) microsomes with red blood cells, we have delivered sarcoplasmic reticulum (SR) Ca-ATPase and kidney Na,K-ATPase into the mammalian erythrocyte membrane. Experiments involving delivery of the SR Ca-ATPase into human red cells were first carried out to assess the feasibility of the fusion protocol. Whereas there was little detectable 45Ca2+ uptake into control cells in either the absence or presence of extracellular ATP, a marked time-dependent uptake of 45Ca2+ was observed in the presence of ATP in cells fused with SR Ca-ATPase. Comparison of the kinetics of uptake into microsome-fused cells versus native SR vesicles supports the conclusion of true delivery of pumps into the red cell membrane. Thus, the time to reach steady state was more than two orders of magnitude longer in the (large) cells versus the native SR vesicles. Na,K-ATPase from dog and rat kidney microsomes were fused with red cells of humans, sheep, and dogs. Using dog kidney microsomes fused with dog red cells which are practically devoid of Na,K-ATPase, functional incorporation of sodium pumps was evidenced in ouabain-sensitive Rb+ uptake and Na+ efflux energized by intracellular ATP, as well as in ATP-stimulated Na+ influx and Rb+ efflux from inside-out membrane vesicles prepared from the fusion-treated cells. From analysis of the biphasic kinetics of ouabain-sensitive Na+ efflux under conditions of limited intracellular Na+ concentration, it is concluded that the kidney pumps are incorporated into a relatively small fraction (approximately 15%) of the red cells. This system provides a uniquely useful system for studying the behavior of native sodium pumps in a compartment (red cell) of small surface/volume ratio. The newly incorporated native kidney pumps, while of the same isoform as the endogenous red cell pump, behave differently from the endogenous red cell sodium pump with respect to their very low "uncoupled" Na+/O flux activity.     

Purification of a hexokinase-binding protein from the outer mitochondrial membrane.

Brain hexokinase (ATP:D-hexose-6-phosphotransferase, EC 2.7.1.1) binds selectively to the outer membrane of rat liver mitochondria but not to inner mitochondrial or microsomal membranes nor to the plasma membrane of human erythrocytes. A protein having subunit molecular weight of 31,000, determined by sodium dodecyl sulfate-gel electrophoresis, has been highly purified from the outer mitochondrial membrane by repetitive solubilization with octyl-beta-D-glucopyranoside followed by reconstitution into membranous vesicles when the detergent is removed by dialysis. When incorporated into lipid vesicles, the protein confers the ability to bind brain hexokinase in a Glc-6-P-sensitive manner as is seen with the intact outer mitochondrial membrane. Hexokinase binding ability and the 31,000 subunit molecular weight protein co-sediment during sucrose density gradient centrifugation. Both hexokinase binding ability and the 31,000 subunit molecular weight protein are resistant to protease treatment of the intact outer mitochondrial membrane while other membrane proteins are extensively degraded. It is concluded that this protein, designated the hexokinase-binding protein (HBP), is an integral membrane protein responsible for the selective binding of hexokinase by the outer mitochondrial membrane.     

Phorbol 12-myristate 13-acetate down-regulates Na,K-ATPase independent of its protein kinase C site: decrease in basolateral cell surface area.

The effect of protein kinase C (PKC) stimulation on the pump current (Ip) generated by the Na,K-ATPase was measured in A6 epithelia apically permeabilized with amphotericin B. Phorbol 12-myristate 13-acetate (PMA) produced a decrease in Ip carried by sodium pumps containing the endogenous Xenopus laevis or transfected Bufo marinus alpha 1 subunits (approximately 30% reduction within 25 min, maximum after 40 min) independent of the PKC phosphorylation site (T15A/S16A). In addition to this major effect of PMA, which was independent of the intracellular sodium concentration and was prevented by the PKC inhibitor bisindolylmaleimide GF 109203X (BIM), another BIM-resistant, PKC site-independent decrease was observed when the Ip was measured at low sodium concentrations (total reduction approximately 50% at 5 mM sodium). Using ouabain binding and cell surface biotinylation, stimulation of PKC was shown to reduce surface Na,K-ATPase by 14 to 20% within 25 min. The same treatment stimulated fluid phase endocytosis sevenfold and decreased by 16.5% the basolateral cell surface area measured by transepithelial capacitance measurements. In conclusion, PKC stimulation produces a decrease in sodium pump function which can be attributed, to a large extent, to a withdrawal of sodium pumps from the basolateral cell surface independent of their PKC site. This reduction of the number of sodium pumps is parallel to a decrease in basolateral membrane area.     

Purified fusion enzyme between rat cytochrome P4501A1 and yeast NADPH-cytochrome P450 oxidoreductase

A genetically engineered fusion enzyme between rat P4501A1 and yeast P450 reductase in the microsomal fraction of the recombinant yeast AH22/pAFCR1 was purified. The purified enzyme showed a typical CO-difference spectrum of P4501A1 and a single band with an apparent molecular weight of 125,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis. This agreed with the molecular weight of 131,202 calculated from the amino acid sequence. The purified enzyme showed both 7-ethoxycoumarin o-deethylase activity and horse heart cytochrome c reductase activity in the presence of NADPH. The 7-ethoxycoumarin o-deethylase activity depended on the species of lipid used for the reconstitution of the purified fusion enzyme although the purified enzyme showed the activity without reconstitution. The purified fusion enzyme had the Km value of 26 microM for 7-ethoxycoumarin and the maximal turnover rate of 29 mol product/min/mol enzyme at 30 degrees C.     

Inhibition of the coated vesicle proton pump and labeling of a 17,000-dalton polypeptide by N,N'-dicyclohexylcarbodiimide

N,N'-Dicyclohexylcarbodiimide (DCCD) inhibits 100% of proton transport and 80-85% of (Mg2+)-ATPase activity in clathrin-coated vesicles. Half-maximum inhibition of proton transport is observed at 10 microM DCCD after 30 min. Although treatment of the coated vesicle (H+)-ATPase with DCCD has no effect on ATP hydrolysis in the detergent-solubilized state, sensitivity of proton transport and ATPase activity to DCCD is restored following reconstitution into phospholipid vesicles. In addition, treatment of the detergent-solubilized enzyme with DCCD followed by reconstitution gives a preparation that is blocked in both proton transport and ATP hydrolysis. These results suggest that although the coated vesicle (H+)-ATPase can react with DCCD in either a membrane-bound or detergent-solubilized state, inhibition of ATPase activity is only manifested when the pump is present in sealed membrane vesicles. To identify the subunit responsible for inhibition of the coated vesicle (H+)-ATPase by DCCD, we have labeled the partially purified enzyme with [14C]DCCD. A single polypeptide of molecular weight 17,000 is labeled. The extremely hydrophobic nature of this polypeptide is indicated by its extraction with chloroform:methanol. The 17,000-dalton protein can be labeled to a maximum stoichiometry of 0.99 mol of DCCD/mol of protein with 100% inhibition of proton transport occurring at a stoichiometry of 0.15-0.20 mol of DCCD/mol of protein. Amino acid analysis of the chloroform:methanol extracted 17,000-dalton polypeptide reveals a high percentage of nonpolar amino acids. The similarity in properties of this protein and the DCCD-binding subunit of the coupling factor (H+)-ATPases suggests that the 17,000-dalton polypeptide may function as part of a proton channel in the coated vesicle proton pump.     

Isolation and characterization of a plasma membrane calcium pump from Dictyostelium discoideum.

During the aggregation and differentiation of amoebae of Dictyostelium discoideum, changes in free cytosolic Ca2+ appear to regulate a number of physiological processes. To understand the mechanisms regulating free intracellular Ca2+ in this organism, we have isolated and characterized an ATP/Mg2+-dependent, high-affinity Ca2+ pump. When homogenates of 2 h starved cells were fractionated on Percoll/KCl gradients, one peak of high-affinity Ca2+-pumping activity was detected. This activity was resolved from enzyme markers of the mitochondrion and the rough endoplasmic reticulum but it cosedimented with the plasma membrane marker, alkaline phosphatase. Further studies suggested that the pump was associated with 'inside-out' plasma membrane vesicles. Like plasma membrane Ca2+-transport ATPases from other systems, this isolated Ca2+ pump: (1) was Mg2+-dependent, (2) displayed a high specificity for ATP as an energy source, (3) exhibited a high affinity for free Ca2+ with a Km of 0.3 microM, and (4) was very sensitive to inhibition by vanadate (IC50 2 microM) but was unaffected by mitochondrial inhibitors, ouabain and Ca2+-channel blockers. Unlike plasma membrane Ca2+ pumps from most other systems, this enzyme appeared not to be regulated by calmodulin. During development, non-mitochondrial, vanadate-sensitive, high-affinity Ca2+-pumping activity in crude lysates remained relatively constant for at least 15 h. These observations suggest that this plasma membrane Ca2+ pump probably functions in Dictyostelium to maintain Ca2+ homeostasis by extruding free cytosolic Ca2+ from the cells.     

Characterization of sodium transport in Acholeplasma laidlawii B cells and in lipid vesicles containing purified A. laidlawii (Na+-Mg2+)-ATPase by using nuclear magnetic resonance spectroscopy and 22Na tracer techniques.

The active transport of sodium ions in live Acholeplasma laidlawii B cells and in lipid vesicles containing the (Na+-Mg2+)-ATPase from the plasma membrane of this microorganism was studied by 23Na nuclear magnetic resonance spectroscopic and 22Na tracer techniques, respectively. In live A. laidlawii B cells, the transport of sodium was an active process in which metabolic energy was harnessed for the extrusion of sodium ions against a concentration gradient. The process was inhibited by low temperatures and by the formation of gel state lipid in the plasma membrane of this organism. In reconstituted proteoliposomes containing the purified (Na+-Mg2+)-ATPase, the hydrolysis of ATP was accompanied by the transport of sodium ions into the lipid vesicles, and the transport process was impaired by reagents known to inhibit ATPase activity. At the normal growth temperature (37 degrees C), this transport process required a maximum of 1 mol of ATP per mol of sodium ion transported. Together, these results provide direct experimental evidence that the (Na+-Mg2+)-ATPase of the Acholeplasma laidlawii B membrane is the cation pump which maintains the low levels of intracellular sodium characteristic of this microorganism.     

Electrogenic transport of sodium ions in cytoplasmic and extracellular ion access channels of Na+,K+-ATPase probed by admittance measurement technique

Electrogenic movements of sodium ions in cytoplasmic and extracellular access channel of the Na⁺,K⁺-ATPase have been studied by the admittance measurement technique which allows the detection of small changes of the membrane capacitance and conductance induced by phosphorylation of the ion pump. The measurements were carried out on a model system consisting of a bilayer lipid membrane, to which membrane fragments with ion pumps were adsorbed that contain the ion pumps in high density. Small changes of the membrane capacitance and conductance were induced by a fast release of ATP from caged ATP. The effect was measured at various frequencies and in solutions with different Na⁺ concentrations. The experimentally observed frequency dependences were explained using a theoretical model assuming that Na⁺ movement through the cytoplasmic access channel occurs in one step and through the extracellular access channel, in two steps. The phosphorylation of the protein by ATP leads to a block of the cytoplasmic access channel and an opening the extracellular access channel. The disappearance of electrogenic Na⁺ movements on the cytoplasmic side produces a negative change of capacitance and conductance, while the emergence of extracellular Na⁺ movements generates a positive change. Fitting the experimental dependences of capacitance and conductance by theoretical curves allowed the determination equilibrium and kinetic parameters of sodium transport in the access channels. The text was submitted by the authors in English.     

Characterization of native and reconstituted hydrogen ion pumping adenosinetriphosphatase of chromaffin granules

The ATP-dependent H+ pump from adrenal chromaffin granules is, like the platelet-dense granule H+ pump, essentially insensitive to the mitochondrial ATPase inhibitors sodium azide, efrapeptin, and oligomycin and also insensitive to vanadate and ouabain, agents that inhibit the Na+,K+-ATPase. The chromaffin granule H+ pump is, however, sensitive to low concentrations of NEM (N-ethylmaleimide) and Nbd-Cl (7-chloro-4-nitro-2,1,3-benzoxadiazole). These transport ATPases may thus belong to a new class of ATP-dependent ion pumps distinct from F1F0-and phosphoenzyme-type ATPases. Comparisons of ATP hydrolysis with ATP-dependent serotonin transport suggest that approximately 80% of the ATPase activity in purified chromaffin granule membranes is coupled to H+ pumping. Most of the remaining ATPase activity is due to contaminating mitochondrial ATPase and Na+,K+-ATPase. When extracted with cholate and octyl glucoside, the H+ pump is solubilized in a monodisperse form that retains NEM-sensitive ATPase activity. When reconstituted into proteoliposomes with crude brain phospholipid, the extracted enzyme recovers ATP-dependent H+ pumping, which shows the same inhibitor sensitivity and nucleotide dependence as the native pump. These data demonstrate that the predominant ATP hydrolase of chromaffin granule membrane is also responsible for ATP-driven amine transport and granule acidification in both native and reconstituted membranes.     

Influence of dietary linseed oil and sunflower seed oil on some mechanical and metabolic parameters of isolated working rat hearts

The role played by membrane lipid environment on cardiac function remains poorly defined. The polyunsaturated fatty acid profile of myocardial phospholipids could be of utmost importance in the regulation of key-enzyme activities. This study was undertaken to determine whether selective incorporation of n-6 or n-3 fatty acids in membrane phospholipids might influence cardiac mechanical performances and metabolism. For 8 wk, male weaning Wistar rats were fed a semi-purified diet containing either 10% sunflower seed oil (72% C18:2 n-6) or 10% linseed oil (54% C18:3 n-3) as the sole source of lipids. The hearts were then removed and perfused according to working mode with a Krebs-Henseleit buffer containing glucose (11 mM) and insulin (10 Ul/l). Cardiac rate, coronary and aortic flows and ejection fraction were monitored after 30 min of perfusion. Myocardial metabolism was estimated by evaluating the intracellular fate of 1-14C palmitate. Sunflower seed oil and linseed oil feeding did not modify either coronary or aortic flow, which suggests that cardiac mechanical work was not affected by the diets. Conversely, cardiac rate was significantly decreased (-18%; P less than 0.01) when rats were fed the n-3 polyunsaturated fatty acid rich diet. Radioanalysis of the myocardial metabolism suggested that replacing n-6 polyunsaturated fatty acids by n-3 polyunsaturated fatty acids: i) did not alter palmitate uptake; ii) prolonged palmitate incorporation into cardiac triglycerides; iii) reduced beta-oxidation of palmitic acid. These results support the assumption that dietary fatty acids, particularly n-6 and n-3 fatty acids, play an important role in the regulation of cardiac mechanical and metabolic activity.     

Membrane homoeostasis and multidrug resistance in yeast

The development of MDR (multidrug resistance) in yeast is due to a number of mechanisms. The most documented mechanism is enhanced extrusion of drugs mediated by efflux pump proteins belonging to either the ABC (ATP-binding cassette) superfamily or MFS (major facilitator superfamily). These drug-efflux pump proteins are localized on the plasma membrane, and the milieu therein affects their proper functioning. Several recent studies demonstrate that fluctuations in membrane lipid composition affect the localization and proper functioning of the MDR efflux pump proteins. Interestingly, the efflux pumps of the ABC superfamily are particularly susceptible to imbalances in membrane-raft lipid constituents. This review focuses on the importance of the membrane environment in functioning of the drug-efflux pumps and explores a correlation between MDR and membrane lipid homoeostasis.     

Sodium pump molecular activity and membrane lipid composition in two disparate ectotherms,and comparison with endotherms

Previous research has shown that the lower sodium pump molecular activity observed in tissues of ectotherms compared to endotherms, is largely related to the lower levels of polyunsaturates and higher levels of monounsaturates found in the cell membranes of ectotherms. Marine-based ectotherms, however, have very polyunsaturated membranes, and in the current study, we measured molecular activity and membrane lipid composition in tissues of two disparate ectothermic species, the octopus (Octopus vulgaris) and the bearded dragon lizard (Pogona vitticeps), to determine whether the high level of membrane polyunsaturation generally observed in marine-based ectotherms is associated with an increased sodium pump molecular activity relative to other ectotherms. Phospholipids from all tissues of the octopus were highly polyunsaturated and contained high concentrations of the omega-3 polyunsaturate, docosahexaenoic acid (22:6 (n–3)). In contrast, phospholipids from bearded dragon tissues contained higher proportions of monounsaturates and lower proportions of polyunsaturates. Sodium pump molecular activity was only moderately elevated in tissues of the octopus compared to the bearded dragon, despite the much greater level of polyunsaturation in octopus membranes. When the current data were combined with data for the ectothermic cane toad, a significant (P=0.003) correlation was observed between sodium pump molecular activity and the content of 22:6 (n–3) in the surrounding membrane. These results are discussed in relation to recent work which shows a similar relationship in endotherms.     

Pump currents generated by the purified Na+K+-ATPase from kidney on black lipid membranes.

The transport activity of purified Na+K+-ATPase was investigated by measuring the electrical pump current induced on black lipid membranes. Discs containing purified Na+K+-ATPase from pig kidney were attached to planar lipid bilayers in a sandwich-like structure. After the addition of only microM concentrations of an inactive photolabile ATP derivative [P3-1-(2-nitro)phenylethyladenosine 5'-triphosphate, caged ATP] ATP was released after illumination with u.v.-light, which led to a transient current in the system. The transient photoresponse indicates that the discs and the underlying membrane are capacitatively coupled. Stationary pump currents were obtained after the addition of the H+, Na+ exchanging agent monensin together with valinomycin to the membrane system, which increased the permeability of the black lipid membrane for the pumped ions. In the absence of ADP and Pi the half saturation for the maximal photoeffect was obtained at 6.5 microM released ATP. The addition of ADP decreased the pump activity. Pump activity was obtained only in the presence of Mg2+ together with Na+ and Na+ and K+. No pump current was obtained in the presence of Mg2+ together with K+. The electrical response was blocked completely by the Na+K+-ATPase-specific inhibitors vanadate and ouabain. No pump currents were observed with a chemically modified protein, which was labelled on the ATP binding site with fluoresceine isothiocyanate. The method described offers the possibility of investigating by direct electrical measurements ion transport of Na+K+-ATPase with a large variety of different parameters.