Daniel M. Tal

Selectivity of Digitalis Glycosides for Isoforms of Human Na,K-ATPase

There are four isoforms of the α subunit (α1–4) and three isoforms of the β subunit (β1–3) of Na,K-ATPase, with distinct tissue-specific distribution and physiological functions. α2 is thought to play a key role in cardiac and smooth muscle contraction and be an important target of cardiac glycosides. An α2-selective cardiac glycoside could provide important insights into physiological and pharmacological properties of α2. The isoform selectivity of a large number of cardiac glycosides has been assessed utilizing α1β1, α2β1, and α3β1 isoforms of human Na,K-ATPase expressed in Pichia pastoris and the purified detergent-soluble isoform proteins. Binding affinities of the digitalis glycosides, digoxin, β-methyl digoxin, and digitoxin show moderate but highly significant selectivity (up to 4-fold) for α2/α3 over α1 (KD α1 > α2 = α3). By contrast, ouabain shows moderate selectivity (≈2.5-fold) for α1 over α2 (KD α1 ≤ α3 < α2). Binding affinities for the three isoforms of digoxigenin, digitoxigenin, and all other aglycones tested are indistinguishable (KD α1 = α3 = α2), showing that the sugar determines isoform selectivity. Selectivity patterns for inhibition of Na,K-ATPase activity of the purified isoform proteins are consistent with binding selectivities, modified somewhat by different affinities of K+ ions for antagonizing cardiac glycoside binding on the three isoforms. The mechanistic insight on the role of the sugars is strongly supported by a recent structure of Na,K-ATPase with bound ouabain, which implies that aglycones of cardiac glycosides cannot discriminate between isoforms. In conclusion, several digitalis glycosides, but not ouabain, are moderately α2-selective. This supports a major role of α2 in cardiac contraction and cardiotonic effects of digitalis glycosides.

Purification of Na+,K+-ATPase Expressed in Pichia pastoris Reveals an Essential Role of Phospholipid-Protein Interactions

Na+,K+-ATPase (porcine α/his10-β) has been expressed in Pichia Pastoris, solubilized in n-dodecyl-β-maltoside and purified to 70–80% purity by nickel-nitrilotriacetic acid chromatography combined with size exclusion chromatography. The recombinant protein is inactive if the purification is done without added phospholipids. The neutral phospholipid, dioleoylphosphatidylcholine, preserves Na+,K+-ATPase activity of protein prepared in a Na+-containing medium, but activity is lost in a K+-containing medium. By contrast, the acid phospholipid, dioleoylphosphatidylserine, preserves activity in either Na+- or K+-containing media. In optimal conditions activity is preserved for about 2 weeks at 0 °C. Both recombinant Na+,K+-ATPase and native pig kidney Na+,K+-ATPase, dissolved in n-dodecyl-β-maltoside, appear to be mainly stable monomers (α/β) as judged by size exclusion chromatography and sedimentation velocity. Na+,K+-ATPase activities at 37 °C of the size exclusion chromatography-purified recombinant and renal Na+,K+-ATPase are comparable but are lower than that of membrane-bound renal Na+,K+-ATPase. The β subunit is expressed in Pichia Pastoris as two lightly glycosylated polypeptides and is quantitatively deglycosylated by endoglycosidase-H at 0 °C, to a single polypeptide. Deglycosylation inactivates Na+,K+-ATPase prepared with dioleoylphosphatidylcholine, whereas dioleoylphosphatidylserine protects after deglycosylation, and Na+,K+-ATPase activity is preserved. This work demonstrates an essential role of phospholipid interactions with Na+,K+-ATPase, including a direct interaction of dioleoylphosphatidylserine, and possibly another interaction of either the neutral or acid phospholipid. Additional lipid effects are likely. A role for the β subunit in stabilizing conformations of Na+,K+-ATPase (or H+,K+-ATPase) with occluded K+ ions can also be inferred. Purified recombinant Na+,K+-ATPase could become an important experimental tool for various purposes, including, hopefully, structural work.

Reactions in dry media: reactions of cholesterol and cholestanes on silica bound ferric chloride : Cholestane—diacholestene rearrangement

Abstract Reaction of cholesterol with silica bound FeCl3 resulted in a mixture of 3β-cholesteryl chloride and dicholesteryl ether. 5-Cholestene and hydroxy- and halogeno-substituted cholestane derivatives gave on heating at 100° with this reagent a 1:1 mixture of 20-epimeric diacholestenes. The 20(R)-isomer gave with meta-chloroperbenzoic acid 20(R)-α-epoxide, while the 20(S)-gave a mixture of 20(S)-α- and 20(S)-β-epoxides. 5α,6β-Dihydrocholestane reacted with the FeCl3/SiO2 under milder conditions (50°) to give 6β-hydroxy-20(R)-diacholestene, which was converted to the 20(R)-diacholestene.

Endogenous “ouabain-like” activity in bovine plasma

An endogenous inhibitor of the sodium pump has been detected and concentrated 1000-fold from bovine plasma. The steps of purification included deproteinization and extraction with methanol, removal of lipids by coextractions with a lipophilic solvent, desalting and further concentration by adsorption on C18-SepPack cartridges and HPLC fractionation on a weak anionic exchange column. The material isolated displaces 3H-ouabain from brain synaptosomes, inhibits red cell membrane Na,K-ATPase without inhibiting Mg-ATPase or Ca,Mg-ATPase. Deproteinization of plasma by boiling may lead to appearance of non-specific inhibitors. The procedures developed should now permit isolation of sufficient amount of material for further purification and structural characterization.

Hellebrin and its aglycone form hellebrigenin display similar in vitro growth inhibitory effects in cancer cells and binding profiles to the alpha subunits of the Na+/K+-ATPase

BackgroundSurface-expressed Na+/K+-ATPase (NaK) has been suggested to function as a non-canonical cardiotonic steroid-binding receptor that activates multiple signaling cascades, especially in cancer cells. By contrast, the current study establishes a clear correlation between the IC50in vitro growth inhibitory concentration in human cancer cells and the Ki for the inhibition of activity of purified human α1β1 NaK.MethodsThe in vitro growth inhibitory effects of seven cardiac glycosides including five cardenolides (ouabain, digoxin, digitoxin, gitoxin, uzarigenin-rhamnoside, and their respective aglycone forms) and two bufadienolides (gamabufotalin-rhamnoside and hellebrin, and their respective aglycone forms) were determined by means of the MTT colorimetric assay and hellebrigenin-induced cytotoxic effects were visualized by means of quantitative videomicroscopy. The binding affinity of ten of the 14 compounds under study was determined with respect to human α1β1, α2β1 and α3β1 NaK complexes. Lactate releases and oxygen consumption rates were also determined in cancer cells treated with these various cardiac glycosides.ResultsAlthough cardiotonic steroid aglycones usually display weaker binding affinity and in vitro anticancer activity than the corresponding glycoside, the current study demonstrates that the hellebrin / hellebrigenin pair is at odds with respect to this rule. In addition, while some cardiac steroid glycosides (e.g., digoxin), but not the aglycones, display a higher binding affinity for the α2β1 and α3β1 than for the α1β1 complex, both hellebrin and its aglycone hellebrigenin display ~2-fold higher binding affinity for α1β1 than for the α2β1 and α3β1 complexes. Finally, the current study highlights a common feature for all cardiotonic steroids analyzed here, namely a dramatic reduction in the oxygen consumption rate in cardenolide- and bufadienolide-treated cells, reflecting a direct impact on mitochondrial oxidative phosphorylation.ConclusionsAltogether, these data show that the binding affinity of the bufadienolides and cardenolides under study is usually higher for the α2β1 and α3β1 than for the α1β1 NaK complex, excepted for hellebrin and its aglycone form, hellebrigenin, with hellebrigenin being as potent as hellebrin in inhibiting in vitro cancer cell growth.

Structural Organization and Energy Transduction Mechanism of Na+,K+-ATPase Studied with Transition Metal-Catalyzed Oxidative Cleavage

This chapter describes contributions of transition metal-catalyzed oxidative cleavage of Na+,K+-ATPase to our understanding of structure–function relations. In the presence of ascorbate/H2O2, specific cleavages are catalyzed by the bound metal and because more than one peptide bond close to the metal can be cleaved, this technique reveals proximity of the different cleavage positions within the native structure. Specific cleavages are catalyzed by Fe2+ bound at the cytoplasmic surface or by complexes of ATP–Fe2+, which directs the Fe2+ to the normal ATP–Mg2+ site. Fe2+- and ATP–Fe2+-catalyzed cleavages reveal large conformation-dependent changes in interactions between cytoplasmic domains, involving conserved cytoplasmic sequences, and a change of ligation of Mg2+ ions between E1P and E2P, which may be crucial in facilitating hydrolysis of E2P. The pattern of domain interactions in E1 and E2 conformations, and role of Mg2+ ions, may be common to all P-type pumps. Specific cleavages can also be catalyzed by Cu2+ ions, bound at the extracellular surfaces, or a hydrophobic Cu2+-diphenyl phenanthroline (DPP) complex, which directs the Cu2+ to the membrane–water interface. Cu2+- or Cu2+-DPP-catalyzed cleavages are providing information on α/β subunit interactions and spatial organization of transmembrane segments. Transition metal-catalyzed cleavage could be widely used to investigate other P-type pumps and membrane proteins and, especially, ATP binding proteins.

Identification of Na+/K+-ATPase inhibitors in bovine plasma as fatty acids and hydrocarbons.

A preparative purification of endogenous inhibitors of the Na+/K+-ATPase has been carried out from bovine blood. Dried plasma was deproteinized, hexane-extracted and desalted, followed by further purification through a series of reverse-phase HPLC fractionations. Fractions active in inhibiting Na+/K+-ATPase activity and displacing ouabain were collected and purified further. By comparison with ouabain, the final extract was found to have a steeper concentration-effect curve in the inhibition of Na+/K+-ATPase. In displacement of [3H]ouabain, the extract had again a steeper concentration-effect curve than does ouabain, and in addition it enhanced ouabain binding at high dilutions. These properties are indicative of nonspecific interactions with the Na+/K+-ATPase. The active fraction was identified by TLC, HPLC, NMR, GLC and GC-MS, to be a mixture of three unesterified fatty acids, mainly oleic acid (72% of the total) and three saturated hydrocarbons. The assignment of structures was corroborated by comparison with authentic samples.

Ozonolysis of olfefins and acetylenes adsorbed on silica gel

Abstract Ozonation of phenyl ethylenes adsorbed on untreated silica gel results in aromatic aldehydes and on dried silica gel in ozonides and aldehydes. On the other hand ozonation of alkyl ethylenes on both types of silica gel results in a mixture of ozonides or polymeric peroxides similar to that obtained in aprotic solvents.

6,7-Dihydroxy-6,7-dihydrocanrenone isomers: improved synthesis and proton NMR study.

The synthesis in improved yields of one 6,7-epoxide and three 6,7-dihydroxycanrenone derivatives is described. Canrenone was the starting material for all derivatives and was obtained by acid-catalyzed lactonization of potassium canrenoate. The epoxidation of canrenone to 6 alpha, 7 alpha-epoxycanrenone by m-chloroperbenzoic acid was improved by addition of a free radical inhibitor. This epoxide was efficiently cleaved to 6 beta, 7 alpha-dihydroxy-6,7-dihydrocanrenone by perchloric acid in a dioxane-water mixture; 6 beta, 7 beta- and 6 alpha, 7 alpha-dihydrocanrenone were obtained by OsO4 oxidation of canrenone in either-pyridine and subsequent reduction of the osmates by hydrogen sulfide. The stereochemistry of the products obtained from the reaction of osmium tetroxide with the 6,7-double bond of steroidal 4,6-dien-3-ones has been a controversial issue for some time. A detailed proton-NMR study of the three diol derivatives unequivocally confirmed the proposed stereochemical structure.

Selective assembly of Na,K-ATPase α2β2 heterodimers in the heart: distinct functional properties and isoform-selective inhibitors.

The Na,K-ATPase α2 subunit plays a key role in cardiac muscle contraction by regulating intracellular Ca2+, whereas α1 has a more conventional role of maintaining ion homeostasis. The β subunit differentially regulates maturation, trafficking, and activity of α-β heterodimers. It is not known whether the distinct role of α2 in the heart is related to selective assembly with a particular one of the three β isoforms. We show here by immunofluorescence and co-immunoprecipitation that α2 is preferentially expressed with β2 in T-tubules of cardiac myocytes, forming α2β2 heterodimers. We have expressed human α1β1, α2β1, α2β2, and α2β3 in Pichia pastoris, purified the complexes, and compared their functional properties. α2β2 and α2β3 differ significantly from both α2β1 and α1β1 in having a higher K0.5K+ and lower K0.5Na+ for activating Na,K-ATPase. These features are the result of a large reduction in binding affinity for extracellular K+ and shift of the E1P-E2P conformational equilibrium toward E1P. A screen of perhydro-1,4-oxazepine derivatives of digoxin identified several derivatives (e.g. cyclobutyl) with strongly increased selectivity for inhibition of α2β2 and α2β3 over α1β1 (range 22–33-fold). Molecular modeling suggests a possible basis for isoform selectivity. The preferential assembly, specific T-tubular localization, and low K+ affinity of α2β2 could allow an acute response to raised ambient K+ concentrations in physiological conditions and explain the importance of α2β2 for cardiac muscle contractility. The high sensitivity of α2β2 to digoxin derivatives explains beneficial effects of cardiac glycosides for treatment of heart failure and potential of α2β2-selective digoxin derivatives for reducing cardiotoxicity.