Bioinformatic and experimental fishing for artemisinin-interacting proteins from human nasopharyngeal cancer cells.

Determining interacting cellular partners of drugs by chemical proteomic techniques is complex and tedious. Most approaches rely on activity-based probe profiling and compound-centric chemical proteomics. The anti-malarial artemisinin also exerts profound anti-cancer activity, but the mechanisms of action are incompletely understood. In the present investigation, we present a novel approach to identify artemisinin-interacting target proteins. Our approach overcomes usual problems in traditional fishing procedures, because the drug was attached to a surface without further chemical modification. The proteins identified effect among others, cell cycle arrest, apoptosis, inhibition of angiogenesis, disruption of cell migration, and modulation of nuclear receptor responsiveness. Furthermore, a bioinformatic approach confirmed experimentally identified proteins and suggested a large number of other interacting proteins. Theoretically predicted interaction partners may serve as a starting point to complete the whole set of proteins binding artemisinin.

Phosphatidylcholine and lysophosphatidylcholine in intestinal mucus of ulcerative colitis patients. A quantitative approach by nanoElectrospray-tandem mass spectrometry.

BACKGROUND: A defective mucus composition represents a key pathogenetic factor for intestinal injury. Phosphatidylcholine (PC) is an essential component contributing to formation of a hydrophobic mucus layer. For evaluation of PC in the pathogenesis of inflammatory bowel disease, the concentration and composition of PC in the rectal mucus of patients with ulcerative colitis was determined. Electrospray ionization (ESI) tandem mass spectrometry (MS/MS) allows quantification of PC species and enables analysis of crude extracts. METHODS: Lipid extracts of material obtained by light scrapings of the intestinal lumen were analysed quantitatively by nanoESI MS/MS with synthetic internal PC and lysophosphatidylcholine (LPC) standards. PC and LPC species from rectoscopically acquired mucus aliquots of patients with ulcerative colitis were compared to Crohn disease and control subjects. RESULTS: Patients with inactive ulcerative colitis showed significantly less PC and LPC (median 346 [IQR: 230-405] pmol total PC/mg dry weight) in rectal mucus compared to Crohn disease (median 126 [IQR: 465-1941] pmol total PC/mg dry weight) and control subjects (median 1285 [IQR: 850-1639] pmol total PC/mg dry weight) (P < 0.05). The molecular species of PC and LPC were not significantly different between the groups. The most abundant species were PC 16:0/18:1; PC 16:0/18:2; PC 18:0/18:1; PC 18:0/18:2; LPC 16:0; and LPC 18:0. CONCLUSION: NanoESI MS/MS is a suitable tool for analysing and quantifying small amounts of PC in human mucus. Patients with ulcerative colitis have significant less PC in their intestinal mucus despite a comparable PC molecular species composition pattern. This suggests that a low amount of protective mucus PC is a characteristic feature in ulcerative colitis and explains an increased susceptibility to luminal contents.

YfiD of Escherichia coli and Y06I of bacteriophage T4 as autonomous glycyl radical cofactors reconstituting the catalytic center of oxygen-fragmented pyruvate formate-lyase.

Reaction of oxygen with the glycyl radical in pyruvate formate-lyase (PFL) leads to cleavage of the polypeptide backbone between N-Calpha of Gly734. A recombinant protein comprising the core of PFL (Ser1-Ser733) is shown here to associate with the YfiD protein (14 kDa) of Escherichia coli and likewise with the homologous T4 encoded Y06I protein, yielding upon reaction with PFL activase a heterooligomeric PFL enzyme that has full catalytic activity (35 U/nmol). Treatment of the activated complexes with oxygen led to cleavage of the 14 kDa proteins into 11 and 3 kDa polypeptides as expected for the localization of the putative glycyl radical at Gly102 (YfiD) or Gly95 (Y06I). For the isolated fragments from Y06I, mass spectrometric analysis (nanoESI-MS) determined a C-terminal serine carboxamide in the 11 kDa fragment, and a N-terminal oxalyl modification in the 3 kDa fragment. We speculate that YfiD in E. coli and other facultative anaerobic bacteria has evolved as a "spare part" for PFL's glycyl radical domain, utilized for rapid recovery of PFL activity (and thus ATP generation) in cells that have experienced oxidative stress.

Protein phosphorylation degree: determination by capillary liquid chromatography and inductively coupled plasma mass spectrometry.

Capillary liquid chromatography (muLC) interfaced to inductively coupled plasma mass spectrometry (ICPMS) is introduced as a new micromethod to determine the phosphorylation degree in phosphoproteins and phosphopeptides containing cysteine and/or methionine residues. The stoichiometric phosphorus to sulfur (31P to 32S) ratio is experimentally determined by muLC-ICPMS and converted into the degree of phosphorylation using protein/ peptide sequence information. The method is applied to the phosphoproteins beta-casein, beta-casein, and recombinant protein kinase A catalytic subunit and to synthetic phosphopeptides. The accurate data obtained by muLC-ICPMS allow quantitative assessment of the compound-specific discrimination of the electrospray ionization process between nonphosphorylated and phosphorylated proteins and peptides.

Enzymic characterization of epidermis-derived 12-lipoxygenase isoenzymes.

Substrate selectivity and other enzymic characteristics of two epidermis-derived lipoxygenases (LOXs), the epidermis-type (e) (12S)-LOX and (12R)-LOX, were compared with those of the platelet-type (p) (12S)-LOX. In contrast with p(12S)-LOX, e(12S)-LOX and (12R)-LOX exhibited no or very low reactivity towards the customary substrates linoleic acid and arachidonic acid but metabolized the corresponding fatty acid methyl esters, which, in contrast, were not accepted as substrates by p(12S)-LOX. Other esters of arachidonic acid and linoleic acid, including propan-2-yl and cholesterol esters, 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-linoleyl-sn-glycero-3-phosphoethanolamine, and ceramide 1 carrying an omega-linoleic acid ester, were not metabolized by these three LOX isoenzymes. Among various polyunsaturated fatty acids the isomeric eicosatrienoic acids were found to be oxygenated by e(12S)-LOX but not by (12R)-LOX. 4,7,10,13,16,19-Docosahexaenoic acid as a substrate was restricted to p(12S)-LOX. Variations in the pH and the Ca(2+) content of the incubation medium affected the catalytic potential only slightly. Whereas (12R)-LOX activity increased in the presence of Ca(2+) and with an acidic pH, Ca(2+) had no effect on p(12S)-LOX and e(12S)-LOX; an acidic pH decreased the catalytic activity of the latter two. However, the catalytic activity of the epidermis-type isoenzymes, but not of p(12S)-LOX, was found to be markedly increased in the presence of DMSO. Under these conditions, e(12S)-LOX and (12R)-LOX oxygenated 4,7,10,13,16,19-docosahexaenoic acid to 14-hydroxy-4,7,10,12,16,19-docosahexaenoic acid and 13-hydroxy-4,7,10,14,16,19-docosahexaenoic acid respectively. In addition, (9R)-hydroxyoctadeca-10,12-dienoic acid methyl ester was generated from linoleic acid methyl ester by (12R)-LOX. Independently of the substrate, the catalytic activity of e(12S)-LOX and (12R)-LOX was always at most 2% of that of p(12S)-LOX with arachidonic acid as substrate.

Analysis of protein phosphorylation by a combination of elastase digestion and neutral loss tandem mass spectrometry.

Loss of phosphoric acid is the most effective fragmentation reaction of pSer- and pThr-containing phosphopeptides of small size (up to 10-15 residues) in low-energy collision-induced dissociation. Therefore, tandem mass spectrometry with neutral loss scanning was evaluated for its utility to analyze protein phosphorylation using protein kinase A (PKA) catalytic subunit, which is phosphorylated at Thr197 and Ser338, as an example. Analysis of tryptic digests of phosphoproteins by tandem mass spectrometry with scanning for neutral loss of phosphoric acid resulted in spectra with poor signal-to-noise ratio, mainly because of the large size of the phosphopeptides formed (>2 kDa). This unfavorable size was caused by the distribution of tryptic cleavage sites in PKA and by interference of phosphorylation with tryptic cleavage. To generate a set of smaller peptide fragments, digestion was performed using the low-specificity protease elastase. Analysis of the total elastase digest with neutral loss scanning resulted in observation of a set of partially overlapping phosphopeptides with high abundance, providing a complete coverage of PKA phosphorylation sites. The peptide size generated by elastase (0.5-1.5 kDa) is ideally suited for this scan mode, which was found to provide the highest specificity for detection of singly charged phosphopeptides (neutral loss of 98). Identification of the PKA phosphorylation sites was performed by mass spectrometric sequencing of the elastase-derived phosphopeptides, which provided highly informative product ion spectra.

Analysis of protein phosphorylation by capillary liquid chromatography coupled to element mass spectrometry with 31P detection and to electrospray mass spectrometry.

A method for phosphopeptide identification by capillary liquid chromatography (muLC) interfaced alternatively to element mass spectrometry (inductively coupled plasma mass spectrometry, ICPMS) and to electrospray ionization mass spectrometry (ESI-MS) is described. ICPMS is used for 31P detection and ESI-MS provides the corresponding molecular weight information. Alignment of the two separate muLC runs is performed using the baseline distortion at the elution front, which shows up in both muLC-ICPMS and muLC-ESI-MS. Both a quadrupole and a magnetic sector field mass analyzer were used in combination with ICP. The detection limit achieved for the muLC-ICP-HRMS runs is approximately 0.1 pmol of phosphopeptide injected. Without any further precautions, contamination by phosphate-containing compounds at this level was found to be uncritical. The method is demonstrated for the analysis of a complex mixture of synthetic phosphopeptides and a set of tryptic digests of three phosphoproteins. These include beta-casein, activated human MAP kinase ERK1, and protein kinase A catalytic subunit. The tryptic phosphopeptides of these proteins could all be detected and identified by our new strategy. Analysis of three fractions of protein kinase A catalytic subunit with different phosphorylation status gives direct access to the order in which the phosphorylation of the four phosphorylation sites occurs. The two most important aspects of using muLC-ICPMS with 31P detection for phosphopeptide identification are (i) that a high selectivity is achieved and (ii) that the signal intensity is independent of the chemical form of phosphorus and directly proportional to the molar amount of 31P in the muLC eluate. Thus, muLC-ICPMS with 31P detection is introduced as a new, robust, and specific method in phosphoproteomics.

The information encrypted in accurate peptide masses-improved protein identification and assistance in glycopeptide identification and characterization.

Analytically useful information from accurate mass data for peptides with an error of

Evidence for segregation of sphingomyelin and cholesterol during formation of COPI-coated vesicles.

In higher eukaryotes, phospholipid and cholesterol synthesis occurs mainly in the endoplasmic reticulum, whereas sphingomyelin and higher glycosphingolipids are synthesized in the Golgi apparatus. Lipids like cholesterol and sphingomyelin are gradually enriched along the secretory pathway, with their highest concentration at the plasma membrane. How a cell succeeds in maintaining organelle-specific lipid compositions, despite a steady flow of incoming and outgoing transport carriers along the secretory pathway, is not yet clear. Transport and sorting along the secretory pathway of both proteins and most lipids are thought to be mediated by vesicular transport, with coat protein I (COPI) vesicles operating in the early secretory pathway. Although the protein constituents of these transport intermediates are characterized in great detail, much less is known about their lipid content. Using nano-electrospray ionization tandem mass spectrometry for quantitative lipid analysis of COPI-coated vesicles and their parental Golgi membranes, we find only low amounts of sphingomyelin and cholesterol in COPI-coated vesicles compared with their donor Golgi membranes, providing evidence for a significant segregation from COPI vesicles of these lipids. In addition, our data indicate a sorting of individual sphingomyelin molecular species. The possible molecular mechanisms underlying this segregation, as well as implications on COPI function, are discussed.

Radioiodinated N-(2-diethylaminoethyl)benzamide derivatives with high melanoma uptake: structure-affinity relationships, metabolic fate, and intracellular localization.

Several radioiodinated N-(dialkylaminoalkyl)benzamides have been used for planar scintigraphy and single-photon emission computed tomography (SPECT) of melanoma metastases. In a quest for improved melanoma uptake and tissue selectivity, structure-activity studies for N-(2-diethylaminoethyl)benzamides with variation of phenyl substituents were performed using C57Bl/6 mice bearing B16 melanoma. Compounds 2 (4-amino-5-bromo-N-(2-diethylaminoethyl)-3-[(131)I]iodo-2-methoxybenz amide) and 6 (4-acetamido-N-(2-diethylaminoethyl)-5-[(131)I]iodo-2-methoxybenzamid e) showed at 6 h post iv injection, for example, melanoma uptake of 16.6 and 23.2% ID/g, respectively (mean values, n = 3). Uptake was 3-5 times higher (P < 0.01) than observed with benzamides known from the literature and was probably facilitated by the relatively slow urinary excretion of 2 or 6. In contrast, analogues lacking either the MeO, Ac, AcNH, or Br substituents exhibited reduced tumor uptake and high urinary excretion of radioactivity in various benzamide metabolites. Uptake of radioiodinated benzamides in B16 melanoma is not mediated by a specific mechanism such as sigma-receptor binding. 2 and 6 exhibited similar melanoma uptake values but quite different sigma(1)-receptor affinities of K(i) = 0.278 +/- 0.018 and 5.19 +/- 0.40 microM, respectively. Uptake studies with IMBA (N-(2-diethylaminoethyl)-3-[(131)I]iodo-4-methoxybenzamide) or BZA (N-(2-diethylaminoethyl)-4-[(131)I]iodobenzamide) showed that with increasing dose of unlabeled compound the measured uptake of label was unchanged (IMBA) or even enhanced (BZA) while receptor binding of label decreased. Differential and equilibrium density-gradient centrifugation revealed that most of the radioactivity from labeled IMBA was associated with fractions containing melanin granules. Thus, structure-activity studies indicate that blood clearance rates and metabolic stability are the main determinants for benzamide uptake in melanoma. The high uptake and slow clearance of 6 offer considerable potential for melanoma imaging in patients, and this compound may also prove to be useful for radionuclide therapy.

Enhanced urinary excretion of cysteinyl leukotrienes in patients with acute alcohol intoxication.

BACKGROUND & AIMS: Leukotrienes are proinflammatory mediators. Ethanol inhibits the catabolism of both cysteinyl leukotrienes (leukotriene E(4) [LTE(4)] and N-acetyl-LTE(4)) and leukotriene B(4) (LTB(4)) in hepatocytes. We examined the metabolic derangement of leukotriene inactivation by ethanol in humans in vivo. METHODS: LTE(4), N-acetyl-LTE(4), LTB(4), and 20-hydroxy-LTB(4) were quantified in urine samples from 16 patients with acute alcohol intoxication (mean blood ethanol, 75 mmol/L). In 9 healthy volunteers, urinary LTE(4) was determined before and after ethanol consumption (mean blood ethanol, 14 mmol/L). RESULTS: The excretion of LTE(4) during alcohol intoxication was 286 compared with 36 nmol/mol creatinine in healthy subjects (P < 0.01); the corresponding values for N-acetyl-LTE(4) were 101 and 11 nmol/mol creatinine, respectively (P < 0.001). This excretion of cysteinyl leukotrienes decreased when the blood ethanol concentration returned to normal. LTB(4) and 20-hydroxy-LTB(4) were detectable only in patients with excessive blood ethanol concentrations (mean, 95 mmol/L). In healthy volunteers, LTE(4) excretion increased 3-5 hours after ethanol consumption (mean peak concentration of 1.5 nmol/L compared with 0.5 nmol/L for basal values; P < 0.005). CONCLUSIONS: Ethanol at high concentration induces increased leukotriene excretion into urine. These changes are consistent with inhibition of leukotriene catabolism and inactivation induced by ethanol, as well as with a higher leukotriene formation caused by ethanol-induced endotoxemia.

Analysis of isoaspartate in peptides by electrospray tandem mass spectrometry.

In view of the significance of Asn deamidation and Asp isomerization to isoAsp at certain sites for protein aging and turnover, it was desirable to challenge the extreme analytical power of electrospray tandem mass spectrometry (ESI-MS/MS) for the possibility of a site-specific detection of this posttranslational modification. For this purpose, synthetic L-Asp/L-isoAsp containing oligopeptide pairs were investigated by ESI-MS/MS and low-energy collision-induced dissociation (CID). Replacement of L-Asp by L-isoAsp resulted in the same kind of shifts for all 15 peptide pairs investigated: (1) the b/y intensity ratio of complementary b and y ions generated by cleavage of the (L-Asp/L-isoAsp)-X bond and of the X-(L-Asp/L-isoAsp) bond was decreased, and (2) the Asp immonium ion abundance at m/z 88 was also decreased. It is proposed that the isoAsp structure hampers the accepted mechanism of b-ion formation on both its N- and C-terminal side. The b/y ion intensity ratio and the relative immonium ion intensity vary considerably, depending on the peptide sequence, but the corresponding values are reproducible when recorded on the same instrument under identical instrumental settings. Thus, once the reference product ion spectra have been documented for a pair of synthetic peptides containing either L-Asp or L-isoAsp, these identify one or the other form. Characterization and relative quantification of L-Asp/L-isoAsp peptide mixtures are also possible as demonstrated for two sequences for which isoAsp formation has been described, namely myrG-D/isoD-AAAAK (deamidated peptide 1-7 of protein kinase A catalytic subunit) and VQ-D/isoD-GLR (deamidated peptide 41-46 of human procollagen alpha 1). Thus, the analytical procedures described may be helpful for the identification of suspected Asn deamidation and Asp isomerization sites in proteolytic digests of proteins.

The amino terminus of PKA catalytic subunit--a site for introduction of posttranslational heterogeneities by deamidation: D-Asp2 and D-isoAsp2 containing isozymes.

Conserved deamidation of PKA catalytic subunit isozymes Calpha and Cbeta--more than 25% at Asn2 in vivo in both cases--has been shown to yield Asp2- and isoAsp2-containing isozymes (Jedrzejewski PT, Girod A, Tholey A, König N, Thullner S, Kinzel V, Bossemeyer D, 1998, Protein Sci 7:457-469). Isoaspartate formation in proteins in vivo is indicative of succinimide intermediates involved in both the initial deamidation reaction as well as the "repair" of isoAsp to Asp by the action of protein L-isoaspartyl (D-aspartyl) O-methyl transferase (PIMT). L-Succinimide is prone to racemization to D-succinimide, which may hydrolyze to D-isoAsp- and D-Asp-containing diastereomers with, respectively, no and poor substrate character for PIMT. To analyze native PKA catalytic subunit from cardiac muscle for these isomers the N-terminal tryptic peptides (T1) of the enzyme were analyzed following procedures refined specifically with a set of corresponding synthetic peptides. The methods combined high resolution high-performance liquid chromatography and a new mass spectrometric procedure for the discrimination between Asp- and isoAsp-residues in peptides (Lehmann et al., 2000). The results demonstrate the occurrence of D-isoAsp- and D-Asp-containing T1 fragments in addition to the L-isomers. The small amount of the L-isoAsp isomer, representing only part of the D-isoAsp isomer, and the relatively large amounts of the L-Asp and D-Asp isomers argues for an effective action of PIMT present in cardiac tissue.

Five-membered ring formation in unimolecular reactions of peptides: a key structural element controlling low-energy collision-induced dissociation of peptides.

Unimolecular fragmentation reactions of peptides in low-energy collision-induced dissociation are reviewed in the mechanistic context of five-membered ring formation. This structure of intermediates or of fragment ions is recognized as a key element that governs unimolecular peptide fragmentation within the structural framework determined by the peptide backbone and its side-chains. A collection of collision-induced dissociation reactions is presented covering (i) b-ion formation, (ii) the fragmentation of N-terminally acylated peptides, (iii) neutral loss of the C-terminal amino acid in alkali or silver cationized peptides, (iv) the fragmentation of isoAsp-containing peptides and (v) the fragmentation of negatively charged Asp- or Glu-containing peptides. It appears that for all possible nucleophile-electrophile interactions leading to a five-membered ring structure an associated unimolecular peptide fragmentation reaction can be observed.

Determination of cholesterol at the low picomole level by nano-electrospray ionization tandem mass spectrometry.

A mass spectrometric method for the quantification of free cholesterol in cells and subcellular membranes is presented. The method is based on a simple one-step chemical derivatization of cholesterol to cholesterol-3-sulfate by a sulfur trioxide-pyridine complex. Quantification is performed by nano-electrospray ionization tandem mass spectrometry (nanoESI-MS/MS) using a stable isotope labeled internal standard. The determination of free cholesterol is demonstrated in about 250 cells of a Chinese hamster ovary (CHO) cell line. With this method a molar ratio of free cholesterol to total phospholipids of 0.34 mol/mol in CHO cells was determined. In a subcellular membrane fraction enriched in Golgi membranes, a molar ratio of free cholesterol to total phospholipids of 0.57 mol/mol was determined. The method should be of value for quantification of other sterols as demonstrated for ergosterol and stigmasterol.

Electrospray tandem mass spectrometric studies of phosphopeptides and phosphopeptide analogues.

A set of synthetic phosphopeptides and phosphopeptide analogues was studied by tandem nano-electrospray mass spectrometry. The influence of the collision offset and of the charge state of the molecular ion on phosphate-specific fragmentation processes was investigated in detail. H--D exchange experiments and structural considerations support a six-centered transition being present in the neutral loss of H3PO4 from serine, threonine and homoserine phosphopeptides, where the C-alpha hydrogen of serine or threonine or the C-beta hydrogen of homoserine is transferred to the protonated phosphate group. Neutral loss of H3PO4 at moderate collision offset potential represents a very abundant fragmentation process for serine, threonine and homoserine phosphopeptides. The most specific feature for discrimination of these phosphopeptides from tyrosine phosphopeptides is the m/z 79:97 ratio in the negative ion product spectra, which is consistently elevated in tyrosine phosphopeptides as compared with serine, threonine and homoserine phosphopeptides. The fragment ions of methylphosphono- and H-phosphonopeptides can be explained by the same mechanisms as are applicable to phosphopeptides.

Lipid patches in membrane protein oligomers: crystal structure of the bacteriorhodopsin-lipid complex.

Heterogenous nucleation on small molecule crystals causes a monoclinic crystal form of bacteriorhodopsin (BR) in which trimers of this membrane protein pack differently than in native purple membranes. Analysis of single crystals by nano-electrospray ionization-mass spectrometry demonstrated a preservation of the purple membrane lipid composition in these BR crystals. The 2.9-A x-ray structure shows a lipid-mediated stabilization of BR trimers where the glycolipid S-TGA-1 binds into the central compartment of BR trimers. The BR trimer/lipid complex provides an example of local membrane thinning as the lipid head-group boundary of the central lipid patch is shifted by 5 A toward the membrane center. Nonbiased electron density maps reveal structural differences to previously reported BR structures, especially for the cytosolic EF loop and the proton exit pathway. The terminal proton release complex now comprises an E194-E204 dyad as a diffuse proton buffer.

Multidrug resistance protein-mediated transport of chlorambucil and melphalan conjugated to glutathione.

The human multidrug resistance protein (MRP1) confers resistance of cells to a number of different cytostatic drugs and functions as an export pump for glutathione S-conjugates, glucuronides and other amphiphilic anions. The present study details for the first time MRP1-mediated ATP-dependent transport of various glutathione S-conjugates of the bifunctional alkylating agents chlorambucil and melphalan. In membrane vesicles prepared from cells expressing recombinant MRP1, the conjugates were transported at rates in the following order: monoglutathionyl chlorambucil > bisglutathionyl chlorambucil > monohydroxy monoglutathionyl chlorambucil and monoglutathionyl melphalan > monohydroxy monoglutathionyl melphalan. In addition, we show that membranes from chlorambucil-resistant GST-alpha-overexpressing CHO cells as well as from their parental cells express the hamster homologue of MRP1. With both CHO cell membrane preparations, we observed ATP-dependent transport of monoglutathionyl chlorambucil and of leukotriene C4, a glutathione S-conjugate and high-affinity substrate of MRP1. The transport rates measured in the resistant cells were only two- to three-fold higher than those measured in the control cells. These results together with cytotoxicity assays comparing MRP1-overexpressing cell pairs with the CHO cell pair indicate that, although MRP1-mediated transport is active, it may not be the rate-limiting step in chlorambucil resistance in these cell lines.

Bovine adrenals contain, in addition to ouabain, a second inhibitor of the sodium pump.

In the search for endogenous cardiac glycosides, two different inhibitors of the sodium pump have been isolated from bovine adrenals. Inhibitor A with a molecular mass of 600 Da and a UV maximum at 250 nm was purified from 16 kg of bovine adrenals. The pure substance (<1 ng) inhibited the sodium pump of human red blood cells with an affinity similar to that of ouabain, yet it cross-reacted with antibodies against the bufadienolide proscillaridin A but not against the cardenolide ouabain. Inhibitor A was slightly more hydrophilic than ouabain on RP-C18 high pressure liquid chromatography. Hence, it showed properties similar to the proscillaridin A immunoreactivity (Sich, B., Kirch, U., Tepel, M., Zideck, W., and Schoner, W. (1996) Hypertension 27, 1073-1078) that increased in humans with systolic blood pressure and pulse pressure. Inhibitor B of the sodium pump with a molecular mass of 584 Da was purified 10(6)-fold from 20 kg of bovine adrenals. It cross-reacted with antibodies against ouabain but not with antibodies against proscillaridin A and inhibited the sodium pump of human and rat red blood cells with the same affinity as ouabain. All other properties, such as the retention time in a C18-reversed phase chromatography, molecular mass determination by electrospray mass spectrometry and fragmentation pattern, and UV and 1H NMR spectroscopic data, were identical to ouabain. Hence, sodium pump inhibitor B from bovine adrenals is the cardenolide ouabain.