Proximity of transmembrane segments M3 and M1 of the alpha subunit of Na+,K+-ATPase revealed by specific oxidative cleavage mediated by a complex of Cu2+ ions and 4,7-diphenyl-1,10-phenanthroline.

This paper describes a novel approach to specific oxidative cleavage of Na(+),K(+)-ATPase, mediated by Cu(2+) ions and a hydrophobic phenanthroline, 4,7-diphenyl-1,10-phenanthroline (DPP), in the presence of ascorbate and H(2)O(2). The cleavage produces two major fragments of the alpha subunit, with apparent molecular masses of 96.5 and 76 kDa, and N-termini near the cytoplasmic entrance of transmembrane segments M1 and M3, respectively, The kinetics indicate that both cleavages are mediated by a single Cu(2+)-DPP complex. We infer that M3 and M1 are in proximity near the cytoplasmic surface. The yields of 96.5 and 76 kDa fragments are not significantly affected by ligands that stabilize different E(1) and E(2) conformations. In E(2)(K) and E(2)P conformations, a minor 5.5 kDa fragment with its N-terminus in M10 is also observed. The 96.5 and 76 kDa fragments are indistinguishable from two fragments near M3 and M1 produced by Fe(2+)-catalyzed cleavage described previously [Goldshleger, R., and Karlish, S. J. D. (1999) J. Biol. Chem. 274, 16213-16221], whereas other Fe(2+)-catalyzed cleavage fragments in the cytoplasmic P and A domains are not observed with the Cu(2+)-DPP complex. These findings provide experimental support for the concept of two separate Fe(2+) sites. A homology model, with Na(+),K(+)-ATPase residues within transmembrane segments and connecting loops substituted into the crystal structure of Ca(2+)-ATPase, shows the proximity between the sequences HFIH in M3 and EVWK in M1, near the cytoplasmic surface. Thus, the model strongly supports the conclusions based on cleavages mediated by the Cu(2+)-DPP complex (or Fe(2+) at site 2). As a corollary, the cleavages provide evidence for similar packing of M1 and M3 of Na(+),K(+)-ATPase and Ca(2+)-ATPase.

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 alpha/beta 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.

Inhibition of amiloride-sensitive Na+ channel by isothiouronium derivatives.

The effects on the amiloride-blockable Na+ channel of a family of recently synthesized isothiouronium derivatives were measured in plasma membrane vesicles from rat distal colon. Some of these derivatives act as high-affinity Na(+)-like antagonists on the Na(+)-K(+)-adenosinetriphosphatase. One of the reagents tested, 1-bromo-2,4,6-tris(isothiouronium methyl)-benzene tribromide (Br-TITU), was found to be a potent blocker of the Na+ channel. At neutral pH, Br-TITU rapidly inhibits the channel mediated 22Na+ uptake, with an inhibition constant of 94 +/- 39 nM. The inhibition observed is specific and reversible. 1,3-Dibromo-2,4,6-tris(isothiouronium methyl)benzene tribromide and Br-TITU derivatives with methyl and phenyl substitutions on the isothiouronium moiety were much less effective blockers. Incubation of cells with Br-TITU at alkaline (but not neutral) pH produces irreversible inactivation of channels, possibly due ot covalent modification of a lysine residue. This inactivation can be attenuated by amiloride but not by Na+. Thus Br-TITU may be a useful reagent in identifying essential residues of the channel protein.

Solubilization of a complex of tryptic fragments of Na,K-ATPase containing occluded Rb ions and bound ouabain.

The nonionic detergent C12E10 (polyoxyethylene 10-laurylether) has been used to solubilize a complex of tryptic fragments of Na, K-ATPase containing occluded Rb ions and bound ouabain. The aim was to define which fragments are required to maintain Rb occlusion. The experiments utilize "19 kDa membranes" consisting of a 19 kDa and several smaller tryptic fragments (8-11.7 kDa) of the alpha subunit, which include trans-membrane segments M7/M10 and the pairs M1/M2, M3/M4, and M5/M6 [Capasso, J. M., et al (1992) J. Biol. Chem. 267, 1150-1158]. The beta subunit is partially split into a 16 kDa fragment and a glycosylated approximately 50 kDa fragment. Cation occlusion and ouabain binding are intact. After preincubation of "19 kDa membranes" with Rb (5 mM) and then ouabain (10 mM), 90-100% of occluded Rb was solubilized by C12E10 at 0 degrees C. All fragments of the alpha and beta subunits, and also the gamma subunit, were cosolubilized by C12E10, and were observed to sediment together on a sucrose density gradient as a complex containing occluded Rb ions. The soluble complex consists of a monomer containing one copy of each fragment, as indicated by size-exclusion HPLC, as well as estimates of specific Rb occlusion (20.0 +/- 1.2 nmol/mg of protein). In the absence of Rb ions and ouabain, the complex was unstable. Whereas the 19 kDa fragment (M7-M10) and beta subunit remained associated, the smaller fragments, containing M5/M6 and M3/M4 and M1/M2, and the subunit dissociated. Observations on the thermal inactivation of Rb occlusion, and effect of pH and ionic strength, suggest that the soluble complex is stabilized by multiple interactions, both within the lipid bilayer and in hydrophilic domains (e.g., salt bridges).

Novel aromatic isothiouronium derivatives which act as high affinity competitive antagonists of alkali metal cations on Na/K-ATPase.

This paper describes properties of a novel family of aromatic isothiouronium derivatives, which act as Na(+)-like competitive antagonists on renal Na/K-ATPase. The derivatives are reversible competitors of Rb+ and Na+ occlusion. Ki values of the most potent compounds, 1-bromo-2,4,6-tris(methylisothiouronium)benzene (Br-TITU) and 1,3-dibromo-2,4,6-tris(methylisothiouronium)benzene(Br2-TITU ), 0.65 and 0.32 microM, respectively, are 15-30-fold lower than Ki values of the bis-guanidinium derivatives described previously (David, P., Mayan, H., Cohen, H., Tal, D. M., and Karlish, S. J. D. (1992) J. Biol. Chem. 267, 1141-1149), and represent the lowest reported values for cation antagonists. Using fluorescein-labeled Na/K-ATPase, all derivatives have been shown to stabilize the E1 conformation when bound at high affinity sites (i.e. they are sodium-like). In addition, in one condition (10 mM Tris-HCl, pH 8.1), high concentrations of Br-TITU (KD approximately 10 microM) appear to stabilize an E2 conformation. We propose a model which allows for simultaneous binding of the antagonists to high affinity cytoplasmic sites and low affinity sites, which may be at the extracellular surface. Blockage of cation occlusion by the isothiouronium derivatives at the cytoplasmic surface probably occurs at the entrance to the occlusion sites, which is recognized both by Na+ antagonists and by Na+ or K+ ions. Unlike the alkali metal cations, the Na+ antagonists are not occluded or transported (see also Or, E., David, P., Shainskaya, A., Tal, D. M., and Karlish, S. J. D. (1993) J. Biol. Chem. 268, 16929-16937). The isothiouronium derivatives appear to be promising candidates for further development as affinity labels of cation binding domains, for kinetic analysis of isoforms or mutated Na/K pumps, or as probes of other cation transport proteins.

Topology of the alpha-subunit of Na,K-ATPase based on proteolysis. Lability of the topological organization.

Topology of the alpha-subunit of Na,K-ATPase has been analyzed utilizing proteolytic digestion. Evidence is presented for a model with 10 transmembrane segments and lability of the C-terminal domain (M7-M10). Using reconstituted proteoliposomes, inside-out oriented pumps were digested with trypsin at the cytoplasmic surface. Evidence was obtained for the M7/M8 pair and cytoplasmic splits between M8 and M9 and between M9 and M10. Because an extracellular split between M9 and M10 was also observed, using right-side-out oriented renal microsomes, we propose that the M9/M10 pair either is destabilized by cytoplasmic digestion or is intrinsically mobile. Using renal microsomes, extracellular digestion of the alpha-subunit by trypsin, chymotrypsin, or an endogenous protease has been observed, after incubation at 55 or at 45 degrees C with beta-mercaptoethanol (beta-ME) and n-butanol. Both perturbations inactivate enzyme activity. Rb ions protect against inactivation and digestion. At 45 degrees C, with beta-ME and n-butanol, trypsin and chymotrypsin cut between M7 and M8 and between M9 and M10, consistent with the 10-segment model. At 55 degrees C, the topological organization is altered, the M8/M9 connecting loop is exposed at the extracellular surface, and an additional split between M8 and M9 is observed. Extracellular digestion of the alpha-subunit is associated with digestion of the beta-subunit near the first extracellular S-S bridge. Rb ions protect the beta-subunit. Exposure to proteases of extracellular domains of both subunits appears to be caused by disruption of subunit interactions.

Effects of competitive sodium-like antagonists on Na,K-ATPase suggest that cation occlusion from the cytoplasmic surface occurs in two steps.

Information on cation occlusion sites of renal NA,K-ATPase has been obtained by comparing the ability of competitive Na-like antagonists (David, P., Mayan, H., Cohen, H., Tal, D. M., and Karlish, S. J. D. (1992) J. Biol. Chem. 267, 1141-1149) with that of transported alkali metal cations to protect against covalent modification and structural perturbations of the protein. Sodium antagonists include p- or m-xylylenebisguanidium, guanidinium ions, and ethylenediamine. Experiments with proteoliposomes reconstituted with Na,K-ATPase demonstrate that p-xylylenebisguanidium has pronounced selectivity for the cytoplasmic surface. Tryptic digestion of Na,K-ATPase leading to "19-kDa membranes," a specifically truncated enzyme with intact cation occlusion sites, requires the presence of alkali metal cations. Sodium antagonists do not protect 19-kDa membranes against further digestion, and occlusion is destroyed. Incubation of 19-kDa membranes at 37 degrees C, in the absence of occluded ions, leads rapidly to loss of ability to occlude rubidium ions. Rubidium, sodium, or other alkali metal cations protect fully, whereas sodium antagonists do not protect against this thermal inactivation. Like the alkali metal cations, sodium antagonists protect Na,K-ATPase and, somewhat less effectively, 19-kDa membranes against inactivation by the carboxyl reagent N,N'-dicyclohexylcarbodiimide. Cation occlusion from the cytoplasmic surface is suggested to occur in two steps. In an initial recognition, either transported cations or sodium antagonists interact with carboxyl groups. The second step is selective for transported cations and involves occlusion of cations (either potassium or sodium ions) and a conformational change to a compact structure, which is resistant to proteolysis and thermal inactivation. Sodium antagonists are sterically hindered from becoming occluded and block Na,K-ATPase activity. Implications for the structural basis of cation specificity of the Na/K pump are discussed.

Separation of membrane-embedded tryptic peptides of Na,K-ATPase by size-exclusion chromatography.

Several attempts to separate hydrophobic tryptic and cyanogen bromide-digested short peptides from Na,K-ATPase, using HPLC and different acid-organic solvent gradients, failed because of the insolubility of the peptides in the initial or final solvents of the gradients used for elution. Therefore, we opted to use a detergent-containing mobile phase. For sodium dodecyl sulphate-solubilized tryptic peptides of M(r) 7 x 10(3)-100 x 10(3), elution on a TSK-G3000SW size-exclusion column successfully separates families of peptides with a resolution of M(r) 5 x 10(3)-10 x 10(3). Peptides in these size ranges can then be resolved completely by tricine-sodium dodecyl sulphate gel electrophoresis, and identified by microsequencing after transfer to polyvinylidene difluoride paper. For separation of smaller peptides a Biosep-SEC-S2000 column, eluted at slow flow-rates, was evaluated. Use of ammonium chloride buffer allows sensitive detection at 214 nm. The separated fractions are resolved and identified on 16.5% tricine gels. Reasonable resolution has been obtained with defined cyanogen bromide fragments of myoglobin. Resolution of small tryptic and cyanogen bromide fragments of Na,K-ATPase is less successful, but the experiments suggest ways of improving the resolution of peptides in the range M(r) 2 x 10(3)-10 x 10(3).

Chemical modification of Glu-953 of the alpha chain of Na+,K(+)-ATPase associated with inactivation of cation occlusion.

We have investigated the role, number, and identity of glutamate (or aspartate) residues involved in cation occlusion on Na+, K(+)-ATPase, using the carboxyl reagent N,N'-dicyclohexylcarbodiimide (DCCD). Extensive use is made of selectively trypsinized Na+,K(+)-ATPase--the so-called "19-kDa membranes"--containing a 19-kDa COOH-terminal, smaller (8-11 kDa) membrane-embedded fragments of the alpha chain, and a largely intact beta chain; these membranes have normal Rb+ and Na+ occlusion capacities. The 19-kDa peptide and a smaller (approximately 9 kDa) unidentified peptide(s) are labeled by [14C]DCCD in a Rb(+)-protectable fashion. Rb(+)-protected [14C]DCCD incorporation into the "19 kDa membranes" and into native Na+,K(+)-ATPase is linearly correlated with inactivation of Rb+ occlusion. Similar linear correlations are observed when Rb(+)-protected [14C]DCCD incorporation is measured by examination of labeling of 19-kDa peptide purified from "19-kDa membranes" or of alpha chain purified from native enzyme. Stoichiometries, estimated by extrapolation, are as follows: (for "19-kDa membranes") close to one DCCD per Rb+ site and one DCCD per 19-kDa peptide; and (for native enzyme) close to two DCCD per phosphoenzyme and two DCCD per alpha chain. We suggest that each of two K+ (or Na+) sites contains a carboxyl group, one located in the 19-kDa peptide and one elsewhere in the alpha chain. After cyanogen bromide digestion of purified, labeled alpha chain, or of 19-kDa peptide, a labeled fragment of apparent M(r) approximately 4 kDa was detected and was identified as that with NH2-terminal Lys-943. Rb(+)-protected [14C]DCCD incorporation was associated almost exclusively with Glu-953. We suggest that the cation occlusion "cage" consists of ligating groups donated by different trans-membrane segments and includes two carboxyl groups such as Glu-953 (and perhaps Glu-327) as well as neutral groups, in two K+ (or Na+) sites, but only neutral groups in the third Na+ site.

Identification of the cation binding domain of Na/K-ATPase.

This paper summarises results and conclusions from experiments with renal Na/K-ATPase, utilising proteolytic digestion to define minimal peptide structures involved in cation occlusion and chemical modification with dicyclohexylcarbodiimide (DCCD) to investigate the role of carboxyl groups and location of K (Rb) and/or Na binding residues. Extensive digestion with trypsin or non-selective proteases in the presence of Na or Rb and absence of divalent cations reveals an essential C-terminal 19Kd fragment of the alpha chain (N-terminal asn 830) and indicates that occlusion sites of Na or K ions must reside within transmembrane segments. The bulk of the beta chain is not involved. Kinetics of inactivation of Rb or Na occlusion and covalent labelling with DCCD indicate that each of two Rb(K) or Na sites contains a carboxyl group. The third Na site may contain only neutral ligating groups. One carboxyl group is located on the 19Kd fragment and the other on tryptic fragment of about 9Kd. When cyanogen bromide was used to digest labelled alpha chain, glu 953 was found to be labelled in a Rb-protectable fashion. In tryptic "19Kd-membranes", fragments containing all putative transmembrane segments of the alpha chain have been identified (i.e. 19, 10.9, 8.7 and 8.0 Kda respectively). The cation occlusion "cage" is apparently composed of ligating groups from different trans-membrane segments, including segments of the 19Kd fragment. Construction of models is hampered by the fact that the number of the transmembrane segments is still uncertain, particularly in the crucial C-terminal domain. Alternative ways of arranging the tryptic fragments across the membrane are discussed.

Guanidinium derivatives act as high affinity antagonists of Na+ ions in occlusion sites of Na+,K(+)-ATPase.

We have screened various alkyl- and arylguanidinium derivatives as possible competitors of Na+ or Rb+ for the cation sites on renal Na+,K(+)-ATPase. Alkyl-monoguanidinium or alkylbisguanidinium (BisG) compounds (chain lengths of C3 to C10) competitively inhibit the occlusion of Rb+ and Na+ with an order of affinities C10 greater than C8 greater than C6 greater than C4 greater than C3. BisG compounds are approximately twice as effective as the equivalent alkylmonoguanidinium compounds. In media of high ionic strength, affinities of tens of micromolar are observed, e.g. 26 microM for BisG 8. m-(mXBG)- and p-xylylenebisguanidinium were synthesized and were found to compete with Rb+ or Na+ with intrinsic affinities of 7.7 and 8.2 microM, respectively. The hydrophobicity rather than the degree of proximity of the guanidinium groups in all BisG compounds appears to determine the binding affinity. A systematic search has been made of conditions in occlusion assays for which the inhibitor affinities are highest. When the pH is raised from 7.0 to 8.5, a 5-fold increase in affinity is observed, suggesting that the guanidinium derivatives compete with protons at sites of pKa approximately 7.5. Replacing Tris-HCl with choline chloride-containing media raised apparent affinities approximately 2-fold. All guanidinium derivatives stabilize the E1 conformation of fluorescein-labeled Na+,K(+)-ATPase, acting as competitive Na+ analogues. In media containing only 1 mM Tris-HCl, pH 8.55, very high affinities were observed for binding to the fluorescein-labeled enzyme (e.g. 0.08 microM for mXBG). In very low ionic strength medium, the inhibition was still competitive with Rb+ ions. However, there was also evidence for nonspecific adsorption to the membranes. The following findings show that mXBG, a typical guanidinium derivative, behaves as a Na(+)-like antagonist. (a) It inhibits Na+,K(+)-ATPase activity, competing strongly with Na+ but only weakly with K+ ions. (b) It inhibits phosphorylation from ATP, competing with Na+ ions. (c) Like Na+ ions, it blocks phosphorylation from inorganic phosphate. Based on these results, we propose that the guanidinium group binds to a relatively wide vestibule at the cytoplasmic surface; but, unlike Na+ or K+ ions, it cannot pass into a narrower region of the cation transport path within the membrane. Therefore, it blocks the occlusion and active transport of cations. In the future, high affinity guanidinium derivatives may serve the purpose of locating cation-binding domains of the pump protein after being converted to reactive affinity or photoaffinity covalent labels.

Extensive digestion of Na+,K(+)-ATPase by specific and nonspecific proteases with preservation of cation occlusion sites.

This paper extends our recent report that renal Na+,K(+)-ATPase is digested by trypsin in the absence of Ca2+ and presence of Rb+ ions to a stable 19-kDa fragment and smaller membrane-embedded fragments of the alpha chain and essentially intact beta chain. These are referred to as "19-kDa membranes." Occlusion of both Rb+ (K+) or Na+ ions is preserved, but ATP-dependent functions are lost (Karlish, S. J. D., Goldshleger, R., and Stein, W. D. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4566-4570). We now show that extensive digestion with nonselective fungal proteases (Pronase and proteinase K) alone, in combination, or after tryptic digestion can remove up to 70% of membrane protein without destroying Rb+ occlusion. In the most heavily digested membranes, the 19-kDa fragment or a slightly shorter 18.5-kDa fragment and smaller fragments of the alpha chain remain, whereas the beta chain is largely digested, leaving smaller membrane-embedded fragments (13-15 kDa). For either trypsin or Pronase digestion, preservation of Rb+ occlusion and the specific fragmentation pattern is observed only in the absence of divalent metal ions (Mg2+ or Ca2+) and presence of either Rb+ or Na+ or congener ions. Tryptic digestion at pH 7.0 can split the beta chain into two fragments of approximately 50 and 16 kDa joined by an S-S bridge. The 16-kDa fragment is protected against further digestion by the presence of Rb+ ions, but probably is not directly involved in occluding cations. Tryptic 19-kDa membranes show a clear and reproducible fragmentation pattern in which all predicted membrane segments are identifiable. Families of fragments from 19-kDa membranes, including seven peptides of 7.6-11.7 kDa, have been separated by size-exclusion high performance liquid chromatography, concentrated, and resolved on 16.5% Tricine gels. N-terminal sequences of the different fragments have been determined after transfer to polyvinylidene difluoride paper. The most interesting findings are as follows. (a) Whereas the 19-kDa tryptic fragment begins at Asn831 as reported previously, the 18.5-kDa Pronase fragment begins at Thr834. (b) Fragments in tryptic 19-kDa membranes of 7.6-11.7 kDa begin at Asp68, Ile263, and Gln737, respectively. These include all putative transmembrane segments other than those in the 19-kDa fragment. (c) A Pronase fragment of 7.8 kDa begins at Thr834, i.e. apparently the 19-kDa fragment has been partially cut, without loss of Rb+ occlusion. (d) Tryptic 16- and approximately 50-kDa fragments of the beta chain begin at Ala5 and Gly143, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

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.

Inactivation of Rb+ and Na+ occlusion on (Na+,K+)-ATPase by modification of carboxyl groups.

This paper demonstrates and characterizes inactivation by N,N'-dicyclohexylcarbodiimide (DCCD) of Rb+ and Na+ occlusion in pig kidney (Na+,K+)-ATPase. Rb+ and Na+ occlusion dependent on oligomycin are measured with a manual assay. Parallel measurement of phosphorylation (by Pi plus ouabain) and Na+ or Rb+ occlusion lead to stoichiometries of 3 Na+ or 2 Rb+ per pump molecule. Inactivation of cation occlusion by DCCD shows the following features: (a) Rb+ and Na+ occlusion are inactivated with identical rates and (b) DCCD concentration dependence shows first-order kinetics and also proportionality to the ratio of DCCD to protein, (c) Rb+ and Na+ occlusion are equally protected from DCCD, by Rb+ ions with high affinity (or Na+ ions with lower affinity), (d) inactivation is only slightly pH-dependent between 6 and 8.5 but (e) is significantly accelerated by several hydrophobic amines while a water-soluble nucleophile, glycine ethyl ester has no effect, and (f) inactivation is exactly correlated with inactivation of (Na+,K+)-ATPase activity of ATP-dependent Na+/K+ exchange in reconstituted vesicles and with the magnitude of E1Na+----E2(Rb+) conformational transitions measured with fluorescence probes. The simplest hypothesis to explain the results is that DCCD modifies one (or a small number of) critical carboxyl residues in a non-aqueous cation binding domain and so blocks occlusion of 2 Rb+ or 3 Na+ ions. The results suggest further that Na+ and K+(Rb+) bind to the same sites and are transported sequentially on the same trans-membrane segments. A second effect of the DCCD treatment is a 4-8-fold shift of the conformational equilibrium E2(Rb+)----E1Rb+ toward E1Rb+. This is detected by (a) reduction in apparent Rb+ affinity for Rb+ occlusion or Rb+/Rb+ exchange in vesicles and (b) direct demonstration of an increased rate of E2(K+)----E1Na+ and decreased rate of E1Na+----E2(K+). This effect is not protected against by Rb+ ions and probably reflects modification of a second group of residues. Modification of (Na+,K+)-ATPase by carbodiimides is complex. Depending on the nature of the carbodiimide (water- or lipid-soluble), ratio of carbodiimide to protein, and perhaps source of the enzyme, inactivation might result either from modification of critical carboxyls, as suggested by this work, or from internal cross-linking as proposed by Pedemonte, C. H. and Kaplan, J. H. ((1986) J. Biol. Chem. 261, 3632-3639).

Do canrenone and 6,7-dihydroxylated derivatives compete with ouabain at the same site on Na,K-ATPase?

Canrenone is the major metabolic product of the synthetic steroids spironolactone and K+-canrenoate, used in antihypertensive therapy. Canrenone can competitively displace [3H]ouabain from Na,K-ATPase [Na+- and K+-activated, Mg2+-dependent adenosine triphosphatase (E.C.3.6.1.3)] and partially inhibit enzymatic activity. These features have led to a hypothesis that competition between canrenone and endogenous digitalis-like materials, suggested to be involved in etiology of essential hypertension, could underly the antihypertensive effect of canrenone. Surprisingly, three derivatives of canrenone (6 beta,7 alpha-,6 beta,7 beta-, and 6 alpha,7 alpha-dihydroxy-6,7-dihydrocanrenone) reportedly occur in normal human and rat urine. This paper characterizes the interactions with partially purified renal Na,K-ATPase of canrenone, the three 6,7-dihydroxylated derivatives, and one epoxide intermediate, synthesized from K+-canrenoate. Canrenone and all the 6,7-substituted derivatives partially inhibited Na,K-ATPase activity (39-45%), with approximately the same apparent affinity, 100-200 microM. Canrenone displaced [3H]ouabain in an apparently competitive fashion (Ki = 100-300 microM) but none of the tested derivatives significantly displaced ouabain even at very high concentrations. The ability to differentiate the ATPase inhibition and [3H]ouabain displacement by modification of the 6,7-double bond indicates that inhibition of ATPase activity does not occur from within the ouabain binding site. We suggest that neither canrenone nor the 6,7-derivatives bind to the ouabain site, but rather interact with it 'allosterically.' Our findings do not support the proposed mechanisms for the antihypertensive action of canrenone.

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.

Bile acids. LXXII. High-performance liquid chromatographic analysis of bile acid coenzyme A derivatives.

The mobilities of coenzyme A and coenzyme A derivatives of cholate, chenodeoxycholate, deoxycholate, lithocholate, and their 5 alpha analogs were studied in reversed-phase high-performance liquid chromatography. With a C18 Radial-PAK A cartridge (10-micron particles) and a solvent mixture of 2-propanol/10 mM phosphate buffer (pH 7.0, 140:360), separation of the chenodeoxycholyl and deoxycholyl coenzyme A derivatives was not observed. An increase in ionic strength of the buffer to 50 mM afforded separation, which was markedly augmented with a C18 Radial-PAK A cartridge with 5-micron particles. Lowering the pH of the buffer to 5.5 did not materially change the separations regardless of the ionic strength. Quantitation was carried out to a lower level of 8.5 X 10(-12) mol.

Bile acids. LXVIII. Allylic and benzylic photo-chemical oxidation of steroids.

To provide 7-oxocholesterol derivatives in yields superior to those obtained by chemical oxidation, the preparation of steroidal allylic or benzylic ketones was studied. Air-induced oxidation was investigated with a highly selective low energy UV lamp in the presence of mercuric bromide. With this procedure cholesterol acetate, 5-cholestene-3 beta, 27-diol diacetate, 24(R)-24-methyl-5-cholesten-2 beta-yl acetate, 24(R)-24-ethyl-5-cholesten-28-yl acetate and 24(R)-24-ethyl-(22E)-cholesta-5,22-dien-3 beta-yl acetate were oxidized to the allylic keto-derivative in good yields; estradiol-17 beta diacetate was similarly converted to the 6-oxo-product in improved yield. This method can be very useful in the synthesis of 7-oxocholesteryl acetate and its analogs and 6-oxo-estratrienes.