Spin-labeled derivatives of cardiotonic steroids as tools for characterization of the extracellular entrance to the binding site on Na+ ,K+ -ATPase.

The information obtained from crystallized complexes of the Na+ ,K+ -ATPase with cardiotonic steroids (CTS) is not sufficient to explain differences in the inhibitory properties of CTS such as stereoselectivity of CTS binding or effect of glycosylation on the preference to enzyme isoforms. The uncertainty is related to the spatial organization of the hydrophilic cavity at the entrance of the CTS-binding site. Therefore, there is a need to supplement the crystallographic description with data obtained in aqueous solution, where molecules have significant degree of flexibility. This work addresses the applicability of the electron paramagnetic resonance (EPR) method for the purpose. We have designed and synthesized spin-labeled compounds based on the cinobufagin steroid core. The length of the spacer arms between the steroid core and the nitroxide group determines the position of the reporting group (N-O) confined to the binding site. High affinity to Na+ ,K+ -ATPase is inferred from their ability to inhibit enzymatic activity. The differences between the EPR spectra in the absence and presence of high ouabain concentrations identify the signature peaks originating from the fraction of the spin labels bound within the ouabain site. The degree of perturbations of the EPR spectra depends on the length of the spacer arm. Docking of the compounds into the CTS site suggests which elements of the protein structure might be responsible for interference with the spin label (e.g., steric clashes or immobilization). Thus, the method is suitable for gathering information on the cavity leading to the CTS-binding site in Na+ ,K+ -ATPase in all conformations with high affinity to CTS.

Isolation, crystallization and crystal structure determination of bovine kidney Na(+),K(+)-ATPase.

Na(+),K(+)-ATPase is responsible for the transport of Na(+) and K(+) across the plasma membrane in animal cells, thereby sustaining vital electrochemical gradients that energize channels and secondary transporters. The crystal structure of Na(+),K(+)-ATPase has previously been elucidated using the enzyme from native sources such as porcine kidney and shark rectal gland. Here, the isolation, crystallization and first structure determination of bovine kidney Na(+),K(+)-ATPase in a high-affinity E2-BeF3(-)-ouabain complex with bound magnesium are described. Crystals belonging to the orthorhombic space group C2221 with one molecule in the asymmetric unit exhibited anisotropic diffraction to a resolution of 3.7 Šwith full completeness to a resolution of 4.2 Å. The structure was determined by molecular replacement, revealing unbiased electron-density features for bound BeF3(-), ouabain and Mg(2+) ions.

Multimeric species in equilibrium in detergent-solubilized Na,K-ATPase.

In this work, we find an equilibrium between different Na,K-ATPase (NKA) oligomeric species solubilized in a non-ionic detergent C12E8 by means of Dynamic Light Scattering (DLS), Analytical Ultracentrifugation (AUC), Small Angle X-ray Scattering (SAXS), Spectrophotometry (absorption at 280/350nm) and enzymatic activity assay. The NKA sample after chromatography purification presented seven different populations as identified by AUC, with monomers and tetramers amounting to ∼55% of the total protein mass in solution. These two species constituted less than 40% of the total protein mass after increasing the NKA concentration. Removal of higher-order oligomer/aggregate species from the NKA solution using 220nm-pore filter resulted in an increase of the specific enzymatic activity. Nevertheless, the enzyme forms new large aggregates over an elapsed time of 20h. The results thus point out that C12E8-solubilized NKA is in a dynamic equilibrium of monomers, tetramers and high-order oligomers/subunit aggregates. These latter have low or null activity. High amount of detergent leads to the dissociation of NKA into smaller aggregates with no enzymatic activity.

Purification of Na,K-ATPase from Pig Kidney.

The method of purification of Na,K-ATPase from pig kidney is based on a differential centrifugation and SDS-treatment of a microsomal preparation. The yield is 0.4 mg protein per 1 g tissue with the specific (ouabain-sensitive) activity of 25-28 µmol Pi/min per mg protein and nucleotide binding capacity of 3 nmol/mg. The protein/lipid ratio is 1/1 (mg/mg) with a protein purity of ~80 %.

Expression of Na,K-ATPase and H,K-ATPase Isoforms with the Baculovirus Expression System.

P-type ATPases can be expressed in several cell systems. The baculovirus expressions system uses an insect virus to enter and express proteins in Sf9 insect cells. This expression system is a lytic system in which the cells will die a few days after viral infection. Subsequently, the expressed proteins can be isolated. Insect cells are a perfect system to study P-type ATPases as they have little or no endogenous Na,K-ATPase activity and other ATPase activities can be inhibited easily. Here we describe in detail the expression and isolation of Na,K-ATPase and H,K-ATPase isoforms with the baculovirus expression system.

Colorimetric Assays of Na,K-ATPase.

The Na,K-ATPase is a plasma membrane enzyme that catalyzes active ion transport by the hydrolysis of ATP. Its activity in vivo is determined by many factors, particularly the concentration of intracellular sodium ions. It is the target of the cardiac glycoside class of drugs and of endogenous regulators. Its assay is often an endpoint in the investigation of physiological processes, and it is a promising drug target. As described in this unit, its enzymatic activity can be determined in extracts from tissues by test tube assay using a spectrophotometer or (32)P-ATP. The protocols in this chapter measure inorganic phosphate as the end product of hydrolysis of ATP.

Subunit isoform selectivity in assembly of Na,K-ATPase α-ß heterodimers.

To catalyze ion transport, the Na,K-ATPase must contain one α and one ß subunit. When expressed by transfection in various expression systems, each of the four α subunit isoforms can assemble with each of the three ß subunit isoforms and form an active enzyme, suggesting the absence of selective α-ß isoform assembly. However, it is unknown whether in vivo conditions the α-ß assembly is random or isoform-specific. The α(2)-ß(2) complex was selectively immunoprecipitated by both anti-α(2) and anti-ß(2) antibodies from extracts of mouse brain, which contains cells co-expressing multiple Na,K-ATPase isoforms. Neither α(1)-ß(2) nor α(2)-ß(1) complexes were detected in the immunoprecipitates. Furthermore, in MDCK cells co-expressing α(1), ß(1), and ß(2) isoforms, a greater fraction of the ß(2) subunits was unassembled with α(1) as compared with that of the ß(1) subunits, indicating preferential association of the α(1) isoform with the ß(1) isoform. In addition, the α(1)-ß(2) complex was less resistant to various detergents than the α(1)-ß(1) complex isolated from MDCK cells or the α(2)-ß(2) complex isolated from mouse brain. Therefore, the diversity of the α-ß Na,K-ATPase heterodimers in vivo is determined not only by cell-specific co-expression of particular isoforms, but also by selective association of the α and ß subunit isoforms.

Role of protein kinase C in phospholemman mediated regulation of α2ß1 isozyme of Na⁺/K⁺-ATPase in caveolae of pulmonary artery smooth muscle cells.

We have recently reported that α(2)ß(1) and α(1)ß(1) isozymes of Na(+)/K(+)-ATPase (NKA) are localized in the caveolae whereas only the α(1)ß(1) isozyme of NKA is localized in the non-caveolae fraction of pulmonary artery smooth muscle cell membrane. It is well known that different isoforms of NKA are regulated differentially by PKA and PKC, but the mechanism is not known in the caveolae of pulmonary artery smooth muscle cells. Herein, we examined whether this regulation occurs through phospholemman (PLM) in the caveolae. Our results suggest that PKC mediated phosphorylation of PLM occurs only when it is associated with the α(2) isoform of NKA, whereas phosphorylation of PLM by PKA occurs when it is associated with the α(1) isoform of NKA. To investigate the mechanism of regulation of α(2) isoform of NKA by PKC-mediated phosphorylation of PLM, we have purified PLM from the caveolae and reconstituted into the liposomes. Our result revealed that (i) in the reconstituted liposomes phosphorylated PLM (PKC mediated) stimulate NKA activity, which appears to be due to an increase in the turnover number of the enzyme; (ii) phosphorylated PLM did not change the affinity of the pump for Na(+); and (iii) even after phosphorylation by PKC, PLM still remains associated with the α(2) isoform of NKA.

Angiotensin II-dependent phosphorylation at Ser11/Ser18 and Ser938 shifts the E2 conformations of rat kidney Na+/K+-ATPase.

Kidney plasma membranes, which contain a single α-1 isoform of Na+/K+-ATPase, simultaneously contain two sub-conformations of E2P, differing in their rate of digoxin release in response to Na+ and ATP. Treating cells with Ang II (angiotensin II) somehow changes the conformation of both, because it differentially inhibits the rate of digoxin release. In the present study we tested whether Ang II regulates release by increasing phosphorylation at Ser11/Ser18 and Ser938. Opossum kidney cells co-expressing the AT1a receptor and either α-1.wild-type, α-1.S11A/S18A or α-1.S938A were treated with or without 10 nM Ang II for 5 min, increasing phosphorylation at the three sites. Na+/K+-ATPase was bound to digoxin-affinity columns in the presence of Na+, ATP and Mg2+. A solution containing 30 mM NaCl and 3 mM ATP eluted ~20% of bound untreated Na+/K+-ATPase (Population #1). Pre-treating cells with Ang II slowed the elution of Population #1 in α-1.wild-type and α-1.S938A, but not α-1.S11A/S18A cells. Another 50% of bound Na+/K+-ATPase (Population #2) was subsequently eluted in two phases by a solution containing 150 mM NaCl and 3 mM ATP. Ang II increased the initial rate and slowed the second phase in α-1.wild-type, but not α-1.S938A, cells. Thus Ang II changes the conformation of two forms of EP2 via differential phosphorylation.

Comparative properties of caveolar and noncaveolar preparations of kidney Na+/K+-ATPase.

To evaluate previously proposed functions of renal caveolar Na(+)/K(+)-ATPase, we modified the standard procedures for the preparation of the purified membrane-bound kidney enzyme, separated the caveolar and noncaveolar pools, and compared their properties. While the subunits of Na(+)/K(+)-ATPase (α,ß,γ) constituted most of the protein content of the noncaveolar pool, the caveolar pool also contained caveolins and major caveolar proteins annexin-2 tetramer and E-cadherin. Ouabain-sensitive Na(+)/K(+)-ATPase activities of the two pools had similar properties and equal molar activities, indicating that the caveolar enzyme retains its ion transport function and does not contain nonpumping enzyme. As minor constituents, both caveolar and noncaveolar pools also contained Src, EGFR, PI3K, and several other proteins known to be involved in stimulous-induced signaling by Na(+)/K(+)-ATPase, indicating that signaling function is not limited to the caveolar pool. Endogenous Src was active in both pools but was not further activated by ouabain, calling into question direct interaction of Src with native Na(+)/K(+)-ATPase. Chemical cross-linking, co-immunoprecipitation, and immunodetection studies showed that in the caveolar pool, caveolin-1 oligomers, annexin-2 tetramers, and oligomers of the α,ß,γ-protomers of Na(+)/K(+)-ATPase form a large multiprotein complex. In conjunction with known roles of E-cadherin and the ß-subunit of Na(+)/K(+)-ATPase in cell adhesion and noted intercellular ß,ß-contacts within the structure of Na(+)/K(+)-ATPase, our findings suggest that interacting caveolar Na(+)/K(+)-ATPases located at renal adherens junctions maintain contact of two adjacent cells, conduct essential ion pumping, and are capable of locus-specific signaling in junctional cells.

Isolation and characterization of BetaM protein encoded by ATP1B4--a unique member of the Na,K-ATPase ß-subunit gene family.

ATP1B4 genes represent a rare instance of the orthologous gene co-option that radically changed functions of encoded BetaM proteins during vertebrate evolution. In lower vertebrates, this protein is a ß-subunit of Na,K-ATPase located in the cell membrane. In placental mammals, BetaM completely lost its ancestral role and through acquisition of two extended Glu-rich clusters into the N-terminal domain gained entirely new properties as a muscle-specific protein of the inner nuclear membrane possessing the ability to regulate gene expression. Strict temporal regulation of BetaM expression, which is the highest in late fetal and early postnatal myocytes, indicates that it plays an essential role in perinatal development. Here we report the first structural characterization of the native eutherian BetaM protein. It should be noted that, in contrast to structurally related Na,K-ATPase ß-subunits, the polypeptide chain of BetaM is highly sensitive to endogenous proteases that greatly complicated its isolation. Nevertheless, using a complex of protease inhibitors, a sample of authentic BetaM was isolated from pig neonatal skeletal muscle by a combination of ion-exchange and lectin-affinity chromatography followed by SDS-PAGE. Results of the analysis of the BetaM tryptic digest using MALDI-TOF and ESI-MS/MS mass spectrometry have demonstrated that native BetaM in neonatal skeletal muscle is a product of alternative splice mRNA variant B and comprised of 351 amino acid residues. Isolated BetaM protein was also characterized by SELDI-TOF mass spectrometry before and after deglycosylation. This allowed us to determine that the carbohydrate moiety of BetaM has molecular mass 5.9kDa and consists of short high-mannose type N-glycans. The results of direct analysis of the purified native eutherian BetaM protein provide first insights into structural properties underlying its entirely new evolutionarily acquired functions.

Isolation and cloning of the K+-independent, ouabain-insensitive Na+-ATPase.

Primary Na+ transport has been essentially attributed to Na+/K+ pump. However, there are functional and biochemical evidences that suggest the existence of a K+-independent, ouabain-insensitive Na+ pump, associated to a Na+-ATPase with similar characteristics, located at basolateral plasma membrane of epithelial cells. Herein, membrane protein complex associated with this Na+-ATPase was identified. Basolateral membranes from guinea-pig enterocytes were solubilized with polyoxyethylene-9-lauryl ether and Na+-ATPase was purified by concanavalin A affinity and ion exchange chromatographies. Purified enzyme preserves its native biochemical characteristics: Mg2+ dependence, specific Na+ stimulation, K+ independence, ouabain insensitivity and inhibition by furosemide (IC50: 0.5 mM) and vanadate (IC50: 9.1 µM). IgY antibodies against purified Na+-ATPase did not recognize Na+/K+-ATPase and vice versa. Analysis of purified Na+-ATPase by SDS-PAGE and 2D-electrophoresis showed that is constituted by two subunits: 90 (α) and 50 (ß) kDa. Tandem mass spectrometry of α-subunit identified three peptides, also present in most Na+/K+-ATPase isoforms, which were used to design primers for cloning both ATPases by PCR from guinea-pig intestinal epithelial cells. A cDNA fragment of 1148 bp (atna) was cloned, in addition to Na+/K+-ATPase α1-isoform cDNA (1283 bp). In MDCK cells, which constitutively express Na+-ATPase, silencing of atna mRNA specifically suppressed Na+-ATPase α-subunit and ouabain-insensitive Na+-ATPase activity, demonstrating that atna transcript is linked to this enzyme. Guinea-pig atna mRNA sequence (2787 bp) was completed using RLM-RACE. It encodes a protein of 811 amino acids (88.9 kDa) with the nine structural motifs of P-type ATPases. It has 64% identity and 72% homology with guinea-pig Na+/K+-ATPase α1-isoform. These structural and biochemical evidences identify the K+-independent, ouabain-insensitive Na+-ATPase as a unique P-type ATPase.

Neutral endopeptidase neprilysin is copurified with Na,K-ATPase from rabbit outer medulla and hydrolyzes its α-subunit.

Preparations of Na,K-ATPase from outer medulla of rabbit kidney purified in accordance with the method of P. L. Jorgensen were shown to contain as admixture a protease that moves with α-subunit (~100 kDa) as a single protein band during one-dimensional SDS-PAGE. The electro-elution of proteins of this band from polyacrylamide gel results in the appearance of two protein fragments (~67 and 55 kDa) that are stained with polyclonal antibodies against Na,K-ATPase α-subunit. Liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis showed that the neutral membrane-bound endopeptidase neprilysin is located in one protein band together with the Na,K-ATPase α-subunit. Addition of thiorphan, a specific inhibitor of neutral endopeptidase, eliminates proteolysis of the α-subunit. The data demonstrate that Na,K-ATPase α-subunit may be a natural target for neprilysin.

Glyceraldehyde-3-phosphate dehydrogenase as a surface associated antigen on human breast cancer cell lines MACL-1 and MGSO-3.

Breast cancer is a major health burden, responsible for >10% of all cases of cancer worldwide. Advances in breast cancer diagnosis and treatment have contributed to an improved rate of survival, although mortality rates remains significantly high. The establishment of breast cancer cell lines is an important model for understanding biological processes involved in this disease and for identifying potential therapeutic targets. The novel human breast cancer cell lines, MACL-1 and MGSO-3, were used in this study to identify possible surface antigens by antibodies directed against two commercial breast cancer cell lines MCF-7 and MDA-MB-231. We purified a 37 kDa antigen by affinity chromatography from MDA-MB-231, and its N-terminal amino acid sequence was homologous to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Therefore, immunohistochemical experiments, using specific monoclonal antibodies, evidenced a co-localization of GAPDH and Na+/K+-ATPase on the surface of commercially available and recently established breast cancer cell lines. It is of note that Na+/K+-ATPase was used as a plasma membrane marker. This finding opens new perspectives for breast cancer diagnosis and treatment since GAPDH could be used as a biomarker or as a potential therapeutic target in breast cancer.

Extrusion of Na,K-ATPase and transferrin receptor with lipid raft-associated proteins in different populations of exosomes during reticulocyte maturation in dogs.

The present study characterizes canine reticulocyte exosomes. Exosomes are small membrane vesicles involved in membrane remodeling that are released from reticulocytes during the final maturation step of red blood cells. The vesicles collected from reticulocyte culture supernatants by differential centrifugation contained major exosomal proteins including heat shock protein cognate 70 (Hsc70) and transferrin receptors (TfR), consistent with the definition of the exosome. In addition, the Na,K-ATPase alpha-subunit and stomatin, a lipid raft-associated protein, were extruded by the exosome pathway, possibly leading to the absence of these proteins in erythrocytes, while the major protein constituents of erythrocyte membranes, spectrin and band 3 were retained in reticulocytes and not expelled into exosomes. The Na,K-ATPase alpha-subunit, as well as TfR and about half of the stomatin contained in exosomes, was obtained in a detergent-soluble fraction that was distinct from the lipid raft microdomain. Moreover, Na,K-ATPase and a portion of stomatin were distributed differently to Hsc70, TfR, stomatin, and ganglioside GM1 in vesicles separated with sucrose density gradient centrifugation. These results demonstrate that a heterogeneous group of exosomes participates in the loss of Na,K-ATPase and membrane remodeling during reticulocyte maturation in dogs.

Identification, purification and partial characterization of a 70kDa inhibitor protein of Na(+)/K(+)-ATPase from cytosol of pulmonary artery smooth muscle.

AIMS: We sought to identify, purify and partially characterize a protein inhibitor of Na(+)/K(+)-ATPase in cytosol of pulmonary artery smooth muscle. MAIN METHODS: (i) By spectrophotometric assay, we identified an inhibitor of Na(+)/K(+)-ATPase in cytosolic fraction of pulmonary artery smooth muscle; (ii) the inhibitor was purified by a combination of ammonium sulfate precipitation, diethylaminoethyl (DEAE) cellulose chromatography, hydroxyapatite chromatography and gel filtration chromatography; (iii) additionally, we have also purified Na(+)/K(+)-ATPase alpha(2)beta(1) and alpha(1)beta(1) isozymes for determining some characteristics of the inhibitor. KEY FINDINGS: We identified a novel endogenous protein inhibitor of Na(+)/K(+)-ATPase having an apparent mol mass of approximately 70kDa in the cytosolic fraction of the smooth muscle. The IC(50) value of the inhibitor towards the enzyme was determined to be in the nanomolar range. Important characteristics of the inhibitor are as follows: (i) it showed different affinities toward the alpha(2)beta(1) and alpha(1)beta(1) isozymes of the Na(+)/K(+)-ATPase; (ii) it interacted reversibly to the E(1) site of the enzyme; (iii) the inhibitor blocked the phosphorylated intermediate formation; and (iv) it competitively inhibited the enzyme with respect to ATP. CD studies indicated that the inhibitor causes an alteration of the conformation of the enzyme. The inhibition study also suggested that the DHPC solubilized Na(+)/K(+)-ATPase exists as (alphabeta)(2) diprotomer. SIGNIFICANCE: The inhibitor binds to the Na(+)/K(+)-ATPase at a site different from the ouabain binding site. The novelty of the inhibitor is that it acts in an isoform specific manner on the enzyme, where alpha(2) is more sensitive than alpha(1).

Solubilization and partial characterization of ouabain-insensitive Na(+)-ATPase from basolateral plasma membranes of the intestinal epithelial cells.

It has been proposed that intestinal sodium transport is mediated by two different active mechanisms: the ouabain-sensitive Na+/K(+)-ATPase and ouabain-insensitive Na(+)-ATPase. In order to determine the optimum conditions to solubilize the membrane-bound Na(+)-ATPase of enterocyte, basolateral plasma membranes were solubilized using different amounts of octyl glucoside (O.G), Tween 20, octaethylene glycol monododecyl ether (C12E8), and polyoxyethylene 9-lauryl ether (C12E9). Solubilized fractions were assayed for protein concentration and ATPase activity and characterized by electrophoresis analysis. Optimal solubilization of Na(+)-ATPase was obtained after mixing of 1 mg of basolateral plasma membrane with 1.5 mg of C12E9. Under these conditions, C12E9 solubilized over 60% membrane protein and Na(+)- and Na+/K(+)-ATPases activities were recovered over 80% in the soluble fraction without inactivation. In addition, when 25% glycerol and 2 mM ATP were added, the solubilized Na(+)-ATPase was stable after 3 days at 4 degrees C. The C12E9-solubilized Na(+)-ATPase presented the following kinetic characteristics: 1) is only stimulated by the Na+ salt, 2) K0.5 for Na+ = 4.62 +/- 0.06 mM, 3) is similarly stimulated by the Na+ salt of different anions, 4) optimal pH = 7.0, 5) inhibited by furosemide (IC50 = 0.52 +/- 0.10 nm). These kinetic properties of the solubilized Na(+)-ATPase were similar to those described to the native membrane-bound enzyme. This work reports for the first time, solubilization and characterization of a fully active and stable Na(+)-ATPase from basolateral plasma membranes of enterocyte using C12E9.

Epigallocatechin-3-gallate is an inhibitor of Na+, K(+)-ATPase by favoring the E1 conformation.

Four catechins, epigallocatechin-3-gallate, epigallocatechin, epicatechin-3-gallate, and epicatechin, inhibited activity of the Na(+),K(+)-ATPase. The two galloyl-type catechins were more potent inhibitors, with IC(50) values of about 1 microM, than were the other two catechins. Inhibition by epigallocatechin-3-gallate was noncompetitive with respect to ATP. Epigallocatechin-3-gallate reduced the affinity of vanadate, shifted the equilibrium of E1P and E2P toward E(1)P, and reduced the rate of the E1P to E2P transition. Epigallocatechin-3-gallate potently inhibited membrane-embedded P-type ATPases (gastric H+, K(+)-ATPase and sarcoplasmic reticulum Ca(2+)-ATPase) as well as the Na(+),K(+)-ATPase, whereas soluble ATPases (bacterial F(1)-ATPase and myosin ATPase) were weakly inhibited. Solubilization of the Na(+),K(+)-ATPase with a nonionic detergent reduced sensitivity to epigallocatechin-3-gallate with an elevation of IC50 to 10 microM. These results suggest that epigallocatechin-3-gallate exerts its inhibitory effect through interaction with plasma membrane phospholipid.

UNBS1450 from Calotropis procera as a regulator of signaling pathways involved in proliferation and cell death.

Despite significant progress in oncology therapeutics in the last decades, the urge to discover and to develop new, alternative or synergistic anti-cancer agents still remains. For centuries it has been known that the coarse shrub Calotropis procera is a very promising source of ascaricidal, schizonticidal, anti-bacterial, anthelmintic, insecticidal, anti-inflammatory, anti-diarrhoeal, larvicidal and cytotoxic chemicals. Different compounds like norditerpenic esters, organic carbonates, the cysteine protease procerain, alkaloids, flavonoids, sterols as well as numerous types of cardenolides have provided this plant for centuries with scientists' interest. The chemical class of cardenolides and their related bioactivity evaluation and structure-activity relationship (SAR) studies pointed out their therapeutic utility and led to the discovery of promising drug candidates. Recently the cardiotonic steroid UNBS1450 01 (derived from 2-oxovoruscharin 02) from C. procera was shown to additionally exert an anti-cancer activity. UNBS1450 01 has been proven to be a potent sodium pump inhibitor, showing anti-proliferative and cell death-inducing activities. This anti-cancer potential of UNBS1450 01 is achieved by disorganization of the actin cytoskeleton after binding to the sodium pump at the cellular membrane, by inducing autophagy-related cell death, by repressing NF-kappaB activation as well as by down-regulating c-Myc in cancer cells. We aim to review pharmacologically important chemical extracts from C. procera and focus more specifically on the anti-cancer activities of UNBS1450 01.

Angiotensin II stimulates elution of Na-K-ATPase from a digoxin-affinity column by increasing the kinetic response to ligands that trigger the decay of E2-P.

We earlier observed that treating rat proximal tubules with concentrations of angiotensin II (ANG II) that directly stimulate Na-K-ATPase activity changed how Na-K-ATPase subsequently eluted from an ouabain-affinity column. In this study we tested whether ANG II increases the rate of elution in response to ligands that trigger the decay of E(2)-P, which implies a change in functional properties of Na-K-ATPase, or by decreasing the amount subsequently eluted with SDS, which suggests a change in how Na-K-ATPase interacts with other proteins. We utilized a new digoxin-affinity column and novel lines of opossum kidney (OK) cells that coexpress the rat AT(1a) receptor and either the wild-type rat alpha(1)-isoform of Na-K-ATPase or a truncation mutant missing the first 32 amino acids of its NH(2) terminus. We characterized how rat kidney microsomes bind to and elute from the digoxin-affinity column and demonstrated that they are heterogeneous in the rate at which they release digoxin in response to ligands that trigger the decay of E(2)-P. Incubating OK cells with ANG II stimulated the ensuing elution of wild-type rat alpha(1)-subunit by increasing the kinetic response to ligands that cause a decay of E(2)-P without affecting the amount later eluted with SDS. In contrast, ANG II had no effect on the kinetic response of the truncation mutant but decreased the amount eluted with SDS. These data suggest that ANG II regulates both the kinetic properties of Na-K-ATPase and its interaction with other proteins by a mechanism(s) involving its NH(2) terminus.