Studies on the mechanism of lanosterol 14 alpha-demethylation. A requirement for two distinct types of mixed-function-oxidase systems.

Carbon monoxide inhibited the removal of C-32 of dihydrolanosterol (I), but not of its metabolites 5 alpha-lanost-8-ene-3 beta,32-diol (II) and 3 beta-hydroxy-5 alpha-lanost-8-en-32-al (III). It appears therefore that cytochrome P-450 is a component of the enzyme system required to initiate oxidation of the 14 alpha-methyl group, but not of that responsible for the subsequent oxidation steps required for elimination of C-32 as formic acid. Non-radioactive compounds (II) and (III), when added to cell-free systems actively converting dihydrolanosterol into cholesterol, inhibited 14 alpha-demethylation measured by the rate of formation of labelled cholesterol from dihydro[1,7,15,22,26,30-14C]lanosterol or of labelled formic acid from dihydro[32-14C]lanosterol. However, neither compound (II) nor compound (III) accumulated radioactive label under these conditions. These observations could be attributed partly to inhibition of the initial oxidation of the 14 alpha-methyl group by compounds (II) and (III).     

Simplified digitalis-like compounds acting on Na(+), K(+)-ATPase

Digitalis compounds are used in the treatment of congestive heart failure as positive inotropic agents; their action is mainly due to the inhibition of Na(+),K(+)-ATPase. A well-known drawback is their arrhythmogenic potential. Attempts to find safer digitalis-like compounds by means of molecular simplifications of the typical 5beta,14beta-steroidal skeleton, which appeared in the medicinal chemistry literature from 1990 until 2002, are briefly reviewed. Several novel achievements were obtained in order to better understand the requisites of the digitalis binding site on Na(+), K(+)-ATPase. Only minor simplification, such as cleavage of the D ring of the digitalis skeleton, could preserve the desired inotropic activity, while highly simplified digitalis-like compounds failed to give sufficiently high inotropic potency, even in the presence of a powerful pharmacophore, such as the O-aminoalkyloxime group.     

Structure-activity relationships of estrogens. Effects of 14-dehydrogenation and axial methyl groups at C-7, C-9 and C-11

Thirty compounds were evaluated in the rat for uterotropic effects, inhibition of gonadotropin release, and competitive displacement of (3H) estradiol-17 beta from uterine cytosolic preparations. 7 alpha-Methylestradiol-17 beta was 150% as active as estradiol-17 beta as an uterotropic agent. Estradiol-17 beta was the most active inhibitor of gonadotropin release. 11 beta-Methylestradiol-17 beta had 124% of the activity of estradiol-17 beta in displacing (3H) estradiol-17 beta from the "estrogen receptor." The 9 alpha-methyl group considerably decreased the potency of estrogens in any of the three assays. The 14-dehydro modification was advantageous only in the estradiol-17 beta 3-methyl ether series. Uterotropic activities and inhibition of gonadotropin release did not parallel. The best compound for inhibiting gonadotropin release, as compared to uterotropic activity, was estrone. The "estrogen receptor" assay data correlated fairly well with uterotropic assay data, but only for compounds having free 3-hydroxyl groups; even so, some exceptions were noted.     

Fetal lamb 3 beta, 20 alpha-hydroxysteroid oxidoreductase: dual activity at the same active site examined by affinity labeling with 16 alpha-(bromo[2'-14C]acetoxy)progesterone

3 beta,20 alpha-Hydroxysteroid oxidoreductase was purified to homogeneity from fetal lamb erythrocytes. The Mr 35,000 enzyme utilizes NADPH and reduces progesterone to 4-pregnen-20 alpha-ol-3-one [Km = 30.8 microM and Vmax = 0.7 nmol min-1 (nmol of enzyme)-1] and 5 alpha-dihydrotestosterone to 5 alpha-androstane-3 beta, 17 beta-diol [Km = 74 microM and Vmax = 1.3 nmol min-1 (nmol of enzyme)-1]. 5 alpha-Dihydrotestosterone competitively inhibits (Ki = 102 microM) 20 alpha-reductase activity, suggesting that both substrates may be reduced at the same active site. 16 alpha-(Bromoacetoxy)progesterone competitively inhibits 3 beta- and 20 alpha-reductase activities and also causes time-dependent and irreversible losses of both 3 beta-reductase and 20 alpha-reductase activities with the same pseudo-first order kinetic t1/2 value of 75 min. Progesterone and 5 alpha-dihydrotestosterone protect the enzyme against loss of the two reductase activities presumably by competing with the affinity alkylating steroid for the active site of 3 beta,20 alpha-hydroxysteroid oxidoreductase. 16 alpha-(Bromo[2'-14C]acetoxy) progesterone radiolabels the active site of 3 beta,20 alpha-hydroxysteroid oxidoreductase wherein 1 mol of steroid completely inactivates 1 mol of enzyme with complete loss of both reductase activities. Hydrolysis of the 14C-labeled enzyme with 6 N HCl at 110 degrees C and analysis of the amino acid hydrolysate identified predominantly N pi-(carboxy[2'-14C]methyl)histidine [His(pi-CM)].(ABSTRACT TRUNCATED AT 250 WORDS)     

Separate beta subunits are derivatized with 14C and 3H when the bovine heart mitochondrial F1-ATPase is doubly labeled with 7-chloro-4-nitro[14C]benzofurazan and 5'-p-fluorosulfonylbenzoyl[3H]inosine.

Tyrosine residues 311 and 345 of the beta subunit of the bovine heart mitochondrial F1-ATPase (MF1) are present on the same peptide when the enzyme is fragmented with cyanogen bromide. Maximal inactivation of MF1 with 7-chloro-4-nitro[14C]benzofurazan [( 14C]Nbf-Cl) derivatizes tyrosine-311 in a single beta subunit. Cyanogen bromide digests of MF1 containing the [14C]Nbf-O-derivative of tyrosine-beta 311 were submitted to reversed-phase HPLC, with and without prior reduction of the nitro group on the incorporated reagent with dithionite. The retention time of the radioactive cyanogen bromide peptide was shifted substantially by reduction. When a cyanogen bromide digest of MF1 inactivated with 5'-p-fluorosulfonylbenzoyl[3H]inosine [( 3H]FSBI), which proceeds with derivatization of tyrosine-345 in a single beta subunit, was submitted to HPLC under the same conditions, the fragment labeled with 3H eluted with the same retention time as the [14C]Nbf-O-derivative before reduction. Doubly labeled enzyme was prepared by first derivatizing Tyr-beta 311 with [14C]Nbf-Cl and then derivatizing tyrosine-beta 345 with [3H]FSBI with and without reducing the [14C]Nbf-O-derivative of tyrosine-beta 311 with dithionite before modification with [3H]FSBI. The doubly labeled enzyme preparations were digested with cyanogen bromide and submitted to HPLC. The 14C and 3H in the cyanogen bromide digest prepared from doubly labeled enzyme not submitted to reduction eluted together. In contrast, the 14C and 3H in the digest prepared from doubly labeled enzyme which had been reduced eluted separately. From these results it is concluded that different beta subunits are derivatized when MF1 is doubly labeled with [14C]Nbf-Cl and [3H]FSBI.     

14CO2 fixation in incubated rat diaphragms

The time course (0-60 min) of label incorporation from NaH14 CO3 into citric-acid-cycle intermediates and amino acids was investigated in incubations of isolated rat diaphragms. On the basis of these results, 14CO2 exchange by isocitrate dehydrogenase and 14CO2 fixation by propionyl-CoA carboxylation and pyruvate carboxylation could be estimated. Apparent rates amounted to about 30-40, 2, and 35 nmol/min per g of muscle, respectively. About 90 percent of C4-carbon compounds originating from 14CO2 fixation were subsequently removed by decarboxylation. 2-Cyano-4-hydroxycinnamate, an inhibitor of mitochondrial pyruvate transport, effectively reduced 14CO2 production from [1-14C]pyruvate but did not affect incorporation of radioactive label from NaH14CO3. In cell-free muscle extracts, 14CO2 fixation was demonstrable under assay conditions suitable for NADP -dependent 'malic' enzyme(s). Addition of hydroxymalonate, an inhibitor of the latter enzyme(s), significantly reduced 14CO2 incorporation. The results provide evidence for a continuous cytosolic replenishment and mitochondrial depletion of citric-acid-cycle carbon skeletons in resting skeletal muscle tissue. The functional role of malic (iso)enzyme activities in these processes is discussed.     

Effect of IC14, an anti-CD14 antibody, on plasma and cell-associated chemokines during human endotoxemia

To determine the role of CD14 in lipopolysaccharide (LPS)-induced release of chemokines, 16 humans were injected with LPS (4 ng/kg) preceded (-2 h) by intravenous IC14, an anti-human CD14 monoclonal antibody, or placebo. LPS elicited increases in interleukin (IL)-8 concentrations in plasma and in lysates of red blood cell (RBC), polymorphonuclear cell and mononuclear cell fractions, which were all reduced by IC14. LPS also induced rises in the plasma and RBC levels of monocyte chemoattractant protein (MCP)-1, which were diminished by IC14. Macrophage inflammatory protein (MIP)-1alpha and MIP-1beta, chemokines that in contrast to IL-8 and MCP-1 can not bind to the Duffy antigen receptor for chemokines on RBCs, were only detected in plasma. IC14 attenuated the LPS-induced release of MIP-1beta, but not of MIP-1alpha. IL-8 and MCP-1, but not MIP-1alpha and MIP-1b, circulate in RBC-associated form during endotoxemia. LPS-induced chemokine release is, in part, mediated by an interaction with CD14.     

Inhibitors of sterol synthesis. Chemical synthesis, structure, and biological activities of (25R)-3 beta,26-dihydroxy-5 alpha-cholest-8(14)-en-15-one, a metabolite of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one

3 beta-Hydroxy-5 alpha-cholest-8(14)-en-15-one (I) is a potent inhibitor of sterol synthesis with significant hypocholesterolemic activity. (25R)-3 beta,26-Dihydroxy-5 alpha-cholest-8(14)-en-15-one (II) has been shown to be a major metabolite of I after incubation with rat liver mitochondria. Described herein is the chemical synthesis of II from diosgenin. As part of this synthesis, improved conditions are described for the conversion of diosgenin to (25R)-26-hydroxycholesterol. Benzoylation of the latter compound gave (25R)-cholest-5-ene-3 beta,26-diol 3 beta,26-dibenzoate which, upon allylic bromination followed by dehydrobromination, gave (25R)-cholesta-5,7-diene-3 beta,26-diol 3 beta,26-dibenzoate. Hydrogenation-isomerization of the delta 5.7-3 beta,26-dibenzoate to (25R)-5 alpha-cholest-8(14)-ene-3 beta,26-diol 3 beta,26-bis(cyclohexanecarboxylate) followed by controlled oxidation with CrO3-dimethylpyrazole gave (25R)-3 beta,26-dihydroxy-5 alpha-cholest-8(14)-en-15-one 3 beta,26-bis(cyclohexanecarboxylate). Acid hydrolysis of the delta 8(14)-15-ketosteryl diester gave II. 13C NMR assignments are given for all synthetic intermediates and several major reaction byproducts. The structure of II was unequivocally established by X-ray crystal analysis. II was found to be highly active in the suppression of the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase in cultured mammalian cells and to inhibit oleoyl coenzyme A-dependent esterification of cholesterol in jejunal microsomes.     

Affinity labeling of the digitalis receptor with p-nitrophenyltriazene-ouabain, a highly specific alkylating agent

Three derivatives of ouabain have been synthesized which alkylate the digitalis receptor. These derivatives were formed through reductive amination of p-nitrophenyltriazene (NPT) ethylenediamine to the periodate-oxidized rhamnose moiety of ouabain. The non-covalent binding of the ouabain derivatives (NPT-ouabain, designated I, II, and III) was followed (i) by their ability to inhibit the activity of sodium- and potassium-activated ATPase ((Na+,K+)-ATPase) purified from the electric organ of Electrophorus electricus, (ii) by the binding of [3H]NPT-ouabain I to the enzyme, and (iii) by the inhibition of [3H]ouabain binding with unlabeled NPT-ouabain I. Covalent modification of the digitalis site of (Na+,K+)-ATPase occurs after long periods of time. At pH 7.5 (25 degrees C) the best alkylating derivative, NPT-ouabain I, gives maximum covalent labeling after 6 h. Only the large polypeptide chain (Mr = 93,000) of the purified enzyme is specifically labeled with [3H]NPT-ouabain I while the glycoprotein chain (Mr = 47,000) is not significantly labeled. Labeling of a microsomal fraction of the electric organ with [3H]NPT-ouabain I gave the same type of gel pattern as that observed with the purified enzyme. [3H]NPT-ouabain I was also used to label the digitalis receptor in highly purified axonal membranes and in cardiac membranes prepared from embryonic chick heart. Although the (Na+,K+)-ATPase in both types of membranes has a low affinity for ouabain, [3H]NPT-ouabain I proved to be a very efficient affinity label for the digitalis receptor. In the complex mixture of polypeptides found in these membrane preparations, only a single polypeptide chain having a Mr = 93,000 is specifically labeled by [3H]NPT-ouabain I.     

Novel side chain modified Delta8(14)-15-ketosterols

Synthesis of five novel Delta8(14)-15-ketosterols comprising modified side chains starting from ergosterol is described. Ergosteryl acetate was converted into (22E)-3beta-acetoxy-5alpha-ergosta-8(14),22-dien-15-one through three stages in 32% overall yield; further transformations of the product obtained led to (22E)-3beta-hydroxy-5alpha-ergosta-8(14),22-dien-15-one, (22S,23S)-3beta-hydroxy-22,23-oxido-5alpha-ergost-8(14)-en-15-one, (22R,23R)-3beta-hydroxy-22,23-oxido-5alpha-ergost-8(14)-en-15-one, (22R,23R)-5alpha-ergost-8(14)-en-15-on-3beta,22,23-triol and (22R,23R)-3beta-hydroxy-22,23-isopropylidenedioxy-5alpha-ergost-8(14)-en-15-one. New Delta8(14)-15-ketosterols were evaluated for their cytotoxicity and effects on sterol biosynthesis in human hepatoma Hep G2 cells in comparison with known 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one. Among the compounds tested, (22R,23R)-3beta-hydroxy-22,23-oxido-5alpha-ergost-8(14)-en-15-one was found to be the most potent inhibitor of sterol biosynthesis (IC(50)=0.6+/-0.2microM), whereas (22R,23R)-5alpha-ergost-8(14)-en-15-on-3beta,22,23-triol exhibited the highest cytotoxicity (TC(50)=12+/-3microM at a 24h incubation).     

Inhibition of red cell Na, K-ATPase in digitalized patients

Na⁺, K⁺-ATPase activities of the red cells obtained from 75 patients for whom serum digoxin determinations had been ordered are compared with the enzyme activities of the 34 blood samples known not to have been exposed to digitalis. Partial inhibition of the enzyme in a substantial number of samples obtained from patients is observed. These results, in conjunction with previous observations on changes in red cell electrolytes of the digitalized subjects, provide strong support for the assumption that the inhibition of red cell Na⁺, K⁺-ATPase may occur in the course of therapy with digitalis.     

Enzymatic synthesis of aliphatic beta-lactosides as mimic units of glycosphingolipids by use of Trichoderma reesei cellulase

Aliphatic beta-lactosides were directly synthesized by beta-lactosyl transfer reaction from p-nitrophenyl beta-lactoside (Lac beta-pNP) to various 1-alkanols (n = 2-12), utilizing commercially available cellulase preparation of Trichoderma reesei C1. With ethanol acceptor, the enzyme induced ethyl beta-lactoside (1) in 18% yield based on the donor added in aqueous buffer system. When 1-octanol and dodecanol were acceptors, octyl beta-lactoside (2) and dodecyl beta-lactoside (3) were also obtained as transfer products, respectively. In both cases, the addition of sodium cholate as detergent to the reaction system ensured a sufficient solubility of these acceptors and resulted in a remarkable increase of the desired compounds (5-13% yields based on the donor added). Furthermore, the enzyme catalyzed the N-acetyllactosaminyl transfer reaction from p-nitrophenyl beta-N-acetyllactosaminide (LacNAc beta-pNP) not only to 1-alkanol, but also to the OH-4 position of Man and Glc to produce the trisaccharides, Gal beta1-4GlcNAc beta1-4Man (4) and Gal beta1-4GlcNAc beta1-4Glc (5), respectively. The enzyme activities transferring lactosyl and N-acetyllactosaminyl groups were not separated by chromatographies using DEAE-Sepharose Fast Flow and Sephadex 75 pg columns, indicating that the two reactions were catalyzed by a single enzyme. It was specified that a single enzyme works both transglycosylations, based on the substrate competition assay on hydrolysis.     

3 beta, 17 beta-hydroxysteroid dehydrogenase of Pseudomonas testosteroni. Kinetic evidence for the bifunctional activity at a common catalytic site

3 beta, 17 beta-Hydroxysteroid dehydrogenase (3 beta 17 beta HSDH) is an NAD-dependent dehydrogenase which has a double specificity for the 3- and 17-positions on the steroid skeleton. When dehydroepiandrosterone (DHEA) is used as steroid substrate, and the assay coupled with ketosteroid-isomerase, the two reactions occur alternately and each reaction on the 3-position produces a chromophoric molecule. These two reactions can follow one another without dissociation of the coenzyme from the enzyme binding site. This is confirmed by competition experiments with another dehydrogenase.     

The enzymatic conversion of cis-(14C)phytofluene, trans-(14C)phytofluene, and trans-zeta-(14C)carotene to more unsaturated acyclic, monocyclic, and dicyclic carotenes by a cell-free preparation of red tomato fruits

This paper reports the conversion of cis-[¹⁴C]phytofluene to trans-[¹⁴C|phytofluene and the conversion of the latter compound to trans-ζ-[¹⁴C]carotene by a soluble enzyme system obtained from the plastids of red tomato fruits. Each of these radioactive compounds was also converted to labeled neurosporene, lycopenc, α-carotene, and β-carotene by the same enzyme system. The incorporation of each substrate into more unsaturated carotenes was carried out under nitrogen at pH 7.5–8.2 (borate buffer), at 25 °C in the dark.Proof of the formation of the above carotenes from each of the three radioactive substrates was demonstrated by cochromatography with authentic nonradioactive carotenes on an alumina chromatographic column. A close correspondence between radioactivity and light absorbance for each carotene was observed. Confirmation of these conversions was achieved by cochromatography with authentic samples on thinlayer plates. Final proof for the formation of the acyclic and cyclic carotenes from the above radioactive substrates was obtained by gas-liquid chromatography of the hydrogenated products. Coincidence between mass and radioactivity was observed.Maximum conversion of cis- and trans-phytofluenes to more unsaturated carotenes by the red tomato fruit enzyme system appears to be dependent upon the presence of NADP⁺, FAD, and Tween 80. The formation of the carotenes is also increased in the presence of Mg²⁺ or Mn²⁺ ions.     

The enzymatic conversion of cis-(14C)phytofluene, trans-(14C)phytofluene, and trans-zeta-(14C)carotene to poly-cis-acyclic carotenes by a cell-free preparation of tangerine tomato fruit plastids

The conversion of cis-[¹⁴C]phytofluene to trans-[¹⁴C]phytofluene and the conversion of the latter to trans-ζ-[¹⁴C]carotene by a soluble enzyme system obtained from plastids of tangerine tomato fruits is reported. Each of these compounds is also converted to cis-ζ-carotene, proneurosporene, prolycopene, neurosporene, lycopene, and γ- and β-carotenes. [¹⁴C]Prolycopene was also incubated with the above enzyme system. No conversion of this compound to trans-lycopene or cyclic carotenes was observed. Proof for the formation of the above carotenes from each of the substrates mentioned above was obtained by cochromatography with authentic samples on an alumina column. A close correspondence between radioactivity and light absorbance of each carotene was observed. Further proof for the formation of acyclic and cyclic carotenes from the above radioactive substrates was obtained by gas-liquid chromatography of the hydrogenated products. Coincidence between mass and radioactivity was observed in each case.     

A modified procedure for the preparation of UDP-beta-L-(U-14C)rhamnose.

This paper describes the synthesis of UDP-L-(U-14C)rhamnose from UDP-D-(U-14C)glucose and NADPH using an enzyme preparation of Nicotiana tabacum var. Xanthi. A procedure to separate UDP-l-rhamnose from the other compounds in the reaction mixture is described. Optimal separation was achieved in ethanol 95%-1 M ammonium acetate (pH 3.8) (7:3, v/v) at 30 degrees C.     

Metabolism of [14C]glucose by regenerating spheroplasts of Candida albicans

Spheroplasts of Candida albicans were regenerated in [14C]glucose and buffered magnesium sulphate (0.1 M-Tris/HCl; 0.5 M-MgSO4, pH 7.2) at 35 degrees C. Uptake of glucose by spheroplasts was faster than that by intact yeast cells. After 6 h, 65% of the glucose taken up by the yeast appeared as CO2 and 30% was incorporated into the cellular material. With spheroplasts, 55% of the glucose taken up was expired as CO2, 25% was excreted into the medium as other metabolites and 20% was incorporated into the cells. The regenerating spheroplasts excreted 14C-labelled carbohydrates into the medium which were fractionated on a Sephadex G-15 column. Acid hydrolysis of the low molecular-weight fraction yielded the following sugars: mannose (75.7%), fucose (3.8%), arabinose (3%), galactose (2.1%) and an unidentified monosaccharide (14%). Spheroplasts did not incorporate mannoprotein into the regenerated wall. The wall carbohydrate from regenerated spheroplasts was fractionated on the basis of solubility in sodium hydroxide. The alkali-insoluble fraction was analysed by sequential enzyme hydrolysis; 40% of the incorporated counts were associated with beta (1----3)-linked glucan and 50% with a mixed glucan comprising beta (1----3)- and beta (1----6)-linkages and chitin.     

Tissue Localization of Active Na,K-ATPase Isoenzymes by Determination of their Profile of Inhibition with Ouabain,Digoxin, Digitoxigenin and LND 796, A new Aminosteroid Cardiotonic

Abstract

Recently, Na, K-ATPase isoforms with differential affinities for digitalis have been identified that may contribute to different toxicity profiles. Our objectives were to localize them and to define tissue receptor patterns by examining the effect of different glycosides on the Na, K-ATPase activity. The digitalis derivatives used exhibit variation in lipophilicity and rate of enzyme inhibition. Membrane fractions enriched in Na, K-ATPase were prepared from canine heart, brain, aorta and peripheral nerves. The inhibition of enzyme activities indicates a pattern of differential sensitivities with IC₅₀ values starting from 3 nM in heart and 30 nM in brain. Therefore, high and low affinity active forms of the Na, K-ATPase enzyme coexist in these tissues. The data also suggest the existence of two Na, K-ATPase isoforms in aorta and peripheral nerves as identified by the action of digitoxigenin and LND 796 where the predominant expression is that of a high affinity form. The comparison of the patterns of digitalis sensitivities in these different tissues, suggests a more complex molecular interaction than that which can be explained by the presence of only two forms.     

Cleavage of CD14 and LBP by a protease from<Emphasis Type="Italic"> Prevotella intermedia</Emphasis>

Periodontitis is an inflammatory disease caused by subgingival microorganisms and their components, such as lipopolysaccharide (LPS). Responses of the host to LPS are mediated by CD14 and LPS-binding protein (LBP). In this study, it was determined that proteases from a periodontal pathogen, Prevotella intermedia, cleave CD14 and LBP, and thereby modulate the virulence of LPS. Culture supernatants from two strains of P. intermedia (ATCC 25611 and 25261) cleaved CD14 and LBP in a concentration-dependent manner. Zymographic and molecular mass analysis revealed the presence of a membrane-associated, 170-kDa, monomeric protease. Class-specific inhibitors and stimulators demonstrated that this enzyme is a metal-requiring, thiol-activated, cysteine protease. The protease was stable over a wide range of temperatures (4-56 degrees C) and pH values (4.5-8.5). This enzyme also decreased the expression of interleukin-1beta (IL-1beta)-specific mRNA in the LPS-activated macrophage-like cell lines U937 and THP-1 in a concentration-dependent manner, indicating that it also cleaves membrane-associated CD14. Furthermore, addition of soluble CD14 abrogated protease-mediated inhibition of IL-1 mRNA expression induced by LPS. The observations suggest that proteolysis of CD14 and LBP by P. intermedia protease might modulate the virulence of LPS at sites of periodontal infections.     

Monoclonal antibodies that distinguish between two related digitalis glycosides, ouabain and digoxin.

The exogenous digitalis glycosides, ouabain and digoxin, have been widely used in humans to treat congestive heart failure and cardiac arrhythmias. Several reports have also pointed to the existence of endogenous ouabain- and digoxin-like compounds, but their precise roles in mammalian physiology and various disorders of the circulation are not clear. In an attempt to produce specific Abs for the purification and identification of endogenous ouabain-like compounds, somatic cell fusion was used to produce mAbs specific for ouabain. Our attempts to produce ouabain-specific mAbs were unsuccessful when ouabain was coupled to exogenous proteins such as bovine gamma-globulins, BSA, and human serum albumin. However, when ouabain was coupled to an Ab of A/J mice origin and the same strain of mouse was used for immunization with ouabain-Ab conjugate, three Abs (1-10, 5A12, and 7-1) specific for ouabain were obtained. In assays of fluorescence quenching and saturation equilibrium with tritiated ouabain, Ab 1-10 exhibited 200 nM affinity for ouabain. These three mAbs are distinguished from existing Abs to ouabain and digoxin by their specificity for ouabain and lack of cross-reactivity with digoxin. Specificity studies showed that the loss of cross-reactivity was correlated with the presence of a hydroxyl group at either position 12beta (digoxin) or 16beta (gitoxin) of the steroid ring. These Abs can be used to develop assays for detection and characterization of ouabain-like molecules in vivo.