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W J Ball

Publications and source records attributed to W J Ball.

At least 37 records · Page 2Linked to original sources
PubMed1990

Identification of monoclonal antibody binding domains of Na+,K(+)-ATPase by immunoelectron microscopy.

Treatment of purified preparations of porcine Na+,K(+)-ATPase with phospholipase A2, MgCl2 and NaVO3 leads to the formation of two-dimensional crystals exclusively in a dimeric configuration. Two-dimensional computer-averaged projections of the electron microscopy images of the crystalline enzyme with bound Fab fragments of monoclonal antibody M10-P5-C11 were accomplished using image enhancement software and showed that the antibody fragments caused only a modest increase in the unit cell size, while reducing the extent of asymmetry of the two promoters in each unit cell. The digital imaging also showed that the antibody's epitope on the alpha subunit resides on the 'lobe' or 'hook' region of the intracellular portion of the enzyme. Since functional studies indicate that M10-P5-C11 binds near or between the ATP binding site and the phosphorylation site, this visualized 'lobe' region of alpha may comprise the catalytic site. In addition, the binding of another inhibitory antibody, 9-A5, has been found to prevent crystal formation and the presence of the carbohydrate sugars on the enzyme's beta subunit shown to be required for crystal formation.

Animals↗
PubMed1989

Determination of monoclonal antibody-induced alterations in Na+/K+-ATPase conformations using fluorescein-labeled enzyme.

The fluorescein 5'-isothiocyanate (FITC)-labeled lamb kidney Na+/K+-ATPase has been used to investigate enzyme function and ligand-induced conformational changes. In these studies, we have determined the effects of two monoclonal antibodies, which inhibit Na+/K+-ATPase activity, on the conformational changes undergone by the FITC-labeled enzyme. Monitoring fluorescence intensity changes of FITC-labeled enzyme shows that antibody M10-P5-C11, which inhibits E1 approximately P intermediate formation (Ball, W.J. (1986) Biochemistry 25, 7155-7162), has little effect on the E1 in equilibrium E2 transitions induced by Na+, K+, Mg2+ Pi or Mg2+. ouabain. The M10-P5-C11 epitope, which appears to reside near the ATP-binding site, does not significantly participate in these ligand interactions. In contrast, we find that antibody 9-A5 (Schenk, D.B., Hubert, J.J. and Leffert, H.L. (1984) J. Biol. Chem. 259, 14941-14951) inhibits both the Na+/K+-ATPase and p-nitrophenylphosphatase activity. Its binding produces a 'Na+-like' enhancement in FITC fluorescence, reduces the ability of K+ to induce the E1 in equilibrium E2 transition and converts E2.K+ to an E1 conformation. Mg2+ binding to the enzyme alters both the conformation of this epitope region and its coupling of ligand interactions. In the presence of Mg2+, 9-A5 binding stabilizes an E1.Mg2+ conformation such that K+-, Pi- and ouabain-induced E1----E2 or E1----E2-Pi transitions are inhibited. Oubain and Pi added together overcome this stabilization. These studies indicate that the 9-A5 epitope participates in the E1 in equilibrium E2 conformational transitions, links Na+-K+ interactions and ouabain extracellular binding site effects to both the phosphorylation site and the FITC-binding region. Antibody-binding studies and direct demonstration of 9-A5 inhibition of enzyme phosphorylation by [32P]Pi confirm the results obtained from the fluorescence studies. Antibody 9-A5 has also proven useful in demonstrating the independence of Mg2+ ATP and Mg2+Pi regulation of ouabain binding. In addition, [3H]ouabain and antibody-binding studies demonstrate that FITC-labeling alters the enzyme's responses to Mg2+ as well as ATP regulation.

Animals↗
PubMed1988

Role of the Na+K+-ATPase in the cardiotonic action of cardiac glycosides.

In terms of evolution, the Na+,K+-ATPase is one of the oldest proteins. It is intriguing that this membrane-based enzyme has become biologically consistent with a multifunctional protein. One of the most interesting aspects of the Na+,K+-ATPase is that it contains a specific receptor domain for the oldest plant origin cardiotonic drug and that it is involved in the regulation of myocardial contractility.

Animals↗
PubMed1987

Immunochemical studies of (Na+ + K+)-ATPase using site-specific, synthetic peptide directed antibodies.

Rabbit antisera were raised against a series of synthetic peptides corresponding to regions of the alpha subunit of lamb kidney (Na+ + K+)-ATPase which chemical labeling studies and hydropathy plots of the amino-acid sequence suggest are exposed, accessible regions of the enzyme and may comprise the cation selectivity region, the ATP and cardiac glycoside binding sites, and the phosphorylation site. Five of six peptides tested (11-15 residues in length) were immunogenic and the antisera to four peptides recognized the intact, electroblotted (Western blot analysis) alpha subunit. Immunization with peptides conjugated to keyhole limpet haemocyanin (KLH) produced antipeptide antibodies for seven of nine conjugates. Antisera to four peptide conjugates recognized the native enzyme, confirming predictions that these sequence regions are exposed regions of the holoenzyme. In addition, a collection of four polyclonal antisera and five monoclonal antibodies raised to native holoenzyme were tested for their ability to bind to the peptide conjugates. In this way, two NH2-terminal sequence regions (1-12 and 16-30) and the putative ATP-binding site region (496-506) were identified as epitopes of the native enzyme. These results confirm some aspects of the transmembrane folding models proposed by Shull et al. and Kawakami et al. for the membrane-bound (Na+ + K+)-ATPase.

Animals↗
PubMed1987

Immunochemical evidence that the FITC-labeling site on Na+,K+-ATPase is not the ATP binding site.

The covalent labeling of the alpha subunit of lamb kidney Na+,K+-ATPase by fluorescein 5'-isothiocyanate at Lys-501 has generally been assumed to occur at the ATP binding site. We have found that the peptide sequence 496HLLVMKGAPER506 serves as the antigenic determinant for monoclonal antibody M8-P1-A3. This antibody binds to both native and FITC-labeled enzyme and while this epitope undergoes ligand-induced changes these changes are not involved in either enzyme function or the E1 in equilibrium E2 conformational changes monitored by FITC-fluorescence intensity.

Adenosine Triphosphate↗
PubMed1986

Uncoupling of ATP binding to Na+,K+-ATPase from its stimulation of ouabain binding: studies of the inhibition of Na+,K+-ATPase by a monoclonal antibody.

The effects of a monoclonal antibody, prepared against the purified lamb kidney Na+,K+-ATPase, on the enzyme's Na+,K+-dependent ATPase activity were analyzed. This antibody, designated M10-P5-C11, is directed against the catalytic subunit of the "native" holoenzyme. It inhibits greater than 90% of the ATPase activity and acts as a noncompetitive or mixed inhibitor with respect to the ATP, Na+, and K+ dependence of enzyme activity. It inhibits the Na+- and Mg2+ATP-dependent phosphoenzyme intermediate formation. In contrast, it has no effect on K+-dependent p-nitrophenylphosphatase (pNPPase) activity, the interconversion of the phosphoenzyme intermediates, and ADP-sensitive or K+-dependent dephosphorylation. It does not alter ATP binding to the enzyme nor the covalent labeling of the enzyme at the presumed ATP site by fluorescein 5'-isothiocyanate (FITC), but it prevents the ATP-induced stimulation in the rate of cardiac glycoside [3H]ouabain binding to the Na+,K+-ATPase. M10-P5-C11 binding appears to inhibit enzyme function by blocking the transfer of the gamma-phosphoryl of ATP to the phosphorylation site after ATP binding to the enzyme has occurred. In the presence of Mg2+ATP, it also prevents the ATP-induced transmembrane conformational change that enhances cardiac glycoside binding. This uncoupling of ATP binding from its stimulation of ouabain binding and enzyme phosphorylation demonstrates the existence of an enzyme-Mg2+ATP transitional intermediate preceding the formation of the Na+-dependent ADP-sensitive phosphoenzyme intermediate. These results are also consistent with a model of the Na+,K+-ATPase active site being composed of two distinct but interacting regions, the ATP binding site and the phosphorylation site.

Adenosine Triphosphate↗
PubMed1986

Immunochemical comparison of cardiac glycoside-sensitive (lamb) and -insensitive (rat) kidney (Na+ + K+)-ATPase.

The immunological cross-reactivity of the ouabain-sensitive lamb kidney and the ouabain-insensitive rat kidney (Na+ + K+)-ATPase (EC 3.6.1.37) was examined using polyclonal and monoclonal antibodies. Studies using rabbit antisera prepared against both the lamb kidney and rat kidney holoenzymes showed the existence of substantial antigenic differences as well as similarities between the holoenzymes and the respective denatured alpha and beta subunits of these two enzymes. Quantitation of the extent of cross-reactivity using holoenzyme-directed antibodies showed a 40-60% cross-reactivity. In addition, rabbit antisera monospecific to the purified, denatured alpha and beta subunits of the lamb kidney enzyme showed about a 50% cross-reactivity towards the respective subunit of the rat enzyme. In contrast to the cross-reactivity observed using the polyclonal antibodies, six monoclonal antibodies specific for the alpha subunit of the lamb holoenzyme exhibited no cross-reactivity with the rat holoenzyme. Four of these monoclonal antibodies, however, showed substantial cross-reactivity with rat alpha subunit as resolved by SDS-polyacrylamide gel electrophoresis. A fifth antibody did not bind to the denatured alpha subunit of either the lamb or the rat enzyme. Another monoclonal antibody (M7-PB-E9), which is specific for an epitope previously implicated in the regulation of both ATP and ouabain binding to (Na+ + K+)-ATPase (Ball, W.J., Jr. (1984) Biochemistry 2275-2281) was found to bind to the denatured lamb alpha but not to the rat alpha. This antibody has identified a region of the lamb alpha that has an altered amino acid sequence in the ouabain-insensitive rat enzyme. These immunological studies indicate that there are substantial antigenic differences between the lamb and rat kidney (Na+ + K+)-ATPases. The majority of these antigenic differences appear to be due to variations in the tertiary structures rather than to variations in the primary structures of the alpha subunits.

Animals↗
PubMed1986

Structural studies on H+,K+-ATPase: determination of the NH2-terminal amino acid sequence and immunological cross-reactivity with Na+,K+-ATPase.

The NH2-terminal amino acid sequence of the 100 kilodalton subunit of porcine gastric H+,K+-ATPase has been determined to be YKAENYELYQVELGPGP. Although the NH2-terminal region of this protein is not similar to the same region of the lamb kidney Na+,K+-ATPase catalytic subunit, other regions of these ATPase proteins appear to be homologous. Both monoclonal and polyclonal antibodies raised to lamb kidney Na+,K+-ATPase and its alpha, but not beta, subunit cross-react with the 100 kilodalton protein of H+,K+-ATPase.

Adenosine Triphosphatases↗
PubMed1984

Immunochemical characterization of a functional site of (Na+,K+)-ATPase.

Several hybridoma cell lines secreting antibodies specific to the membrane (Na+,K+)-dependent ATPase from lamb kidney medulla have been isolated by using the methods developed by Kohler and Milstein. One of these antibodies (designated M7-PB- E9 ) has been shown to be directed against a functional epitope or antigenic site of the catalytic (alpha) subunit of the enzyme. Although this antibody was raised to the "native" holoenzyme, it has a higher apparent affinity toward the isolated, delipidated, and inactive alpha subunit than toward the holoenzyme. This antibody shows a 10-fold faster initial rate of binding to the alpha subunit than to the holoenzyme. The antibody dissociation rates from both isolated alpha subunit and holoenzyme are similarly slow, and the binding can be considered a pseudoirreversible reaction. By binding at this site, the antibody, however, acts like a "partial competitive inhibitor" with respect to ATP and acts as an uncompetitive or mixed competitive inhibitor with respect to the Na+ and K+ dependence of ATPase hydrolysis. This antibody also does not alter the cooperativity at either the Na+ or the K+ sites. The antibody causes a partial inhibition of the Na+- and MgATP-dependent phosphoenzyme intermediate formation but has no effect on either ADP in equilibrium ATP exchange or the K+-stimulated dephosphorylation step. In addition, the K+-dependent p-nitrophenylphosphatase activity of the enzyme was not affected. In the presence of Mg2+, the antibody stimulates the rate of cardiac glycoside binding [( 3H]ouabain) to the (Na+,K+)-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Tryptic digest of the alpha subunit of lamb kidney (Na+ + K+)-ATPase.

The Mr approximately equal to 100 000 alpha subunit was prepared from highly purified lamb kidney (Na+ + K+)-ATPase. Its N-terminal sequence is Gly-Arg-Asx-Lys-Tyr-Glu. The alpha subunit was S-carboxymethylated, succinylated, and cleaved at its 40 arginine residues with trypsin. Four major, well-differentiated peptide fractions (A to D) were obtained by chromatography of the digest on a Sephadex G-50 column. Fraction A eluted at the void volume of the column and contained aggregated, very hydrophobic peptides, possibly from regions of alpha that are buried within the membrane lipid bilayer in the native enzyme. Fractions B to D, which together accounted for about 75% of the total protein, contained water-soluble peptides. To test the feasibility of using antibodies to identify and purify specific peptides of alpha subunit, studies were carried out using antibodies to native (Na+ + K+)-ATPase. Carboxymethylation and succinylation did not significantly decrease total antibody binding to alpha subunit, although the affinity of the anti-(Na+ + K+)-ATPase antibodies for alpha subunit was reduced by about 50%. The tryptic peptides of alpha subunit also retain significant immunochemical reactivity. Fractions A, B and C (but not D) of the digest all bind antibodies. To characterize further the tryptic digest, 16 peptides from fraction D were isolated and sequence studies on these were carried out.

Amino Acid Sequence↗
PubMed1983

Studies of the antigenic properties of the catalytic and glycoprotein subunits of Na+,K+-ATPase.

Antibodies were raised against isolated, delipidated catalytic [alpha] and glycoprotein [beta] subunits of the Na+,K+-dependent ATPase purified from lamb kidney medulla. The specificity of each antiserum was confirmed by agar double-diffusion precipitation, immunoelectrophoresis, and polyacrylamide gel electrophoresis. A solid phase adsorption assay was also employed to determine antibody binding titers and to further test the specificity of these antisera. Antibodies raised to the alpha subunit had a strong reactivity and similar titer values for both the holoenzyme and the alpha subunit and a low-affinity cross-reactivity with the beta subunit. In contrast, beta-subunit-directed antibodies had little reactivity or binding with the holoenzyme and a low-affinity cross-reactivity with the alpha subunit. Competition binding studies revealed that about 80% of the alpha-subunit-specific antibodies bound to the holoenzyme, indicating that similar sets of antigenic sites are exposed in the lipid-embedded holoenzyme complex and in the isolated alpha subunit. Competition binding studies also suggest that the subunit cross-reactivities of the antisera may not result from simple contamination of the respective antigens, but that there may be partial homologies of some antigenic sites. In addition, the beta-directed antibodies had no effect on Na+,K+-ATPase activity, while the alpha-directed antibodies were effective inhibitors of activity. This indicates that at least some functionally important antigenic sites of the alpha subunit may be unaltered by its isolation and delipidation.

Animals↗
PubMed1982

Isolation and characterization of monoclonal antibodies to (Na+ + K+)-ATPase.

Four stable hybridoma cell lines secreting antibodies specific to the membrane (Na+ + K+)-dependent ATPase isolated from lamb kidney medulla have been produced by fusing mouse myeloma cells with spleen cells from immunized mice. These cell lines produce IgG gamma 1 heavy chain and kappa light chain antibodies which are directed against the catalytic or alpha-subunit of the (Na+ + K+)-ATPase enzyme. Binding studies, using antibodies that were produced by growing hybridomas in vivo and purified by affinity column chromatography, suggest a somewhat higher affinity of these antibodies for the isolated alpha-subunit than for the 'native' holoenzyme. In addition, these monoclonal antibodies show no reactivity with either the glycoprotein (beta) subunit of the lamb enzyme nor the (Na+ + K+)-ATPase from rat kidney, an ouabain-insensitive organ. Cotitration binding experiments have shown that the antibodies from two cell lines originally isolated independently from the same culture plate well population of fused cells bind to the same determinant site and are probably the same antibody. Cotitration and competition binding studies with two other antibodies have revealed two additional distinct antibody binding sites which appear to have little overlap with the first site. One of the three different antibodies isolated caused a partial inhibition of the (Na+ + K+)-ATPase activity. This antibody appears to be directed against a specific functionally important site of the alpha-subunit and is a competitive inhibitor of ATP binding. Under optimum conditions of ATPase activity, this inhibitory effect is not altered by the presence of the other two antibodies.

Animals↗
PubMed1981

Effect of quinidine on the digoxin receptor in vitro.

To investigate the basis for a clinically important digitalis-quinidine interaction that is characterized by increases in serums digoxin concentrations when quinidine is administered to digoxin-treated patients, we have studied in vitro the interaction of quinidine with the digoxin receptor. Evidence has been obtained that quinidine is capable of decreasing the affinity for digoxin of cardiac glycoside receptor sites on purified Na,K-ATPase and on intact human erythrocyte membranes. As others have shown, quinidine is capable of inhibiting Na,K-ATPase activity, and evidence has been obtained in the current study that, while quinidine can reduce the affinity of the enzyme for digoxin, it is also capable of acting together with digoxin in inhibiting enzyme activity to a degree greater than the inhibitory effect of digoxin alone. The concentrations of digoxin and quinidine used in this study were considerably greater than their therapeutic serum concentrations. Nevertheless, these observations are consistent with the hypothesis that the increases in serum digoxin concentrations and the decreases in volumes of digoxin distribution observed clinically when quinidine is administered to digoxin-treated patients may reflect, at least in part, a decrease in the affinity of tissue receptors for digoxin. The possibility must also be considered that enhanced cardiac effects of digoxin may occur clinically as the result of an augmentation, by quinidine, of digoxin effects, which more than compensates for the modest reduction in digoxin binding.

Biological Transport, Active↗
PubMed1976

Biochemical changes in preneoplastic rodent intestines.

Two enzymes were examined as potential indicators of early precancerous changes. Ornithine decarboxylase, an enzyme normally associated with rapid cell division, is low in the rapidly dividing, cancer-susceptible colon. The level of this enzyme was also very high in the nondividing cells of the small intestines. Administration of an intestinal carcinogen, dimethylhydrazine, led to a large increase in colonic ornithine decarboxylase but did not affect the enzyme in liver. A liver carcinogen, acetylaminofluorene, induced manyfold increases in ornithine decarboxylase of the liver but not of the colon. Studies of thymidine kinase of the gut showed that this enzyme changed quantitatively and qualitatively throughout the life of the animal, from fetal rat to newborn and adult. The tumor enzyme has many fetal-like properties. Long-term treatment with dimethylhydrazine led to changes in thymidine kinase reminiscent of the fetal enzyme. Short-term treatment caused sharp increases in the thymidine kinase of nondividing cells of the jejunum and the proximal end of the colon; similar changes in the distal end of the colon were slower in appearing and less pronounced.

2-Acetylaminofluorene↗
PubMed1976

Changes in ornithine decarboxylase activity in rat intestines during aging.

The ornithine decarboxylase (ODC) activity of rat intestines, liver, and brain was found to vary dramatically as animals develop and age. Unusually high activity was present in the small intestines of adult animals. The ODC activity of the small intestines approximated that of fetal tissues and of regenerating rat liver. Putrescine, spermine, and spermidine levels of fetal and adult animal tissues were determined. In all tissues but the stomach mucosa, high putrescine levels correlated with high IDC activity. However, the total polyamine concentrations of the stomach and colon could not be correlated with ODC levels, and no simple relationship between polyamine levels of ODC levels and cellular proliferation in the gut was found.

Aging↗