Effects of plant lectins on cation-activated brain ATPases.

The effects of concanavalin A (con A) and wheat germ agglutinin (WGA) on cation-activated ATPases in a crude homogenate of rat brain corpus striatum were examined. Con A enhanced the activity of Mg⁺⁺-dependent-, and Ca⁺⁺-activated-ATPases, and inhibited (Na⁺ + K⁺)- ATPase activity. WGA enhanced Mg⁺⁺-dependent ATPase activity, but did not alter the activity of the other two components. The specificity of these interactions was demonstrated by reversal with specific lectin-interacting sugars. The possibility that these effects may be mediated by lectin-binding to physiologic regulatory sites, as well as the possible role of these interactions in the etiopathology of schizophrenia are discussed.     

Characterization of the membrane bound Mg2+-ATPase of rat skeletal muscle

A procedure was developed to isolate a membrane fraction of rat skeletal muscle which contains a highly active Mg²⁺-ATPase (5–25 μmol P_i/mg min). The rate of ATP hydrolysis by the Mg²⁺-ATPase was nonlinear but decayed exponentially (first-order rate constant ≥0.2 s^?1 at 37°C). The rapid decline in the ATPase activity depended on the presence of ATP or its nonhydrolyzable analog 5′-adenylyl imidodiphosphate (AdoPP[NH]P). Once inactivated, removal of ATP from the medium did not immediately restore the original activity. ATP- or AdoPP[NH]P-dependent inactivation could be blocked by concanavalin A, wheat germ agglutinin or rabbit antiserum against the membrane. Additions of these proteins after ATP addition prevented further inactivation but did not restore the original activity. Low concentrations of ionic and nonionic detergents increased the rate of ATP-dependent inactivation. Higher concentrations of detergents, which solubilize the membrane completely, inactivated the Mg²⁺-ATPase. Cross-linking the membrane components with glutaraldehyde prevented ATP-dependent inactivation and decreased the sensitivity of the Mg²⁺-ATPase to detergents. It is proposed that the regulation of the Mg²⁺-ATPase by ATP requires the mobility of proteins within the membrane. Cross-linking the membrane proteins with lectins, antiserum or glutaraldehyde prevents inactivation; increasing the mobility with detergents accelerates ATP-dependent inactivation.     

Effects of wheat germ agglutinin and concanavalin A on parameters of highly purified sodium-potassium adenosine triphosphatases from Squalus acanthias and Electrophorus electricus.

The effects of two lectins, wheat germ agglutinin and concanavalin A, were studied on a variety of parameters of two highly purified (Na+ + K+)-ATPases (ATP phosphohydrolase, EC 3.6.1.3), from the rectal salt gland of Squalus acanthias and from the electroplax of Electrophorus electricus. Both lectins agglutinated the rectal gland enzyme equally, but wheat germ agglutinin inhibited (Na+ + K+)-ATPase activity much more. The electroplax enzyme was only marginally agglutinated and inhibited by the lectins. Neuraminidase treatment of the rectal gland (Na+ + K+)-ATPase had no effect on germ agglutinin inhibition. The inhibition of the rectal gland (Na+ + K+)-ATPase by wheat germ agglutinin could be reversed by N,N'-diacetylchitobiose, which has a high affinity for wheat germ agglutinin. Neither ouabain inhibition nor ouabain binding to the rectal gland enzyme was affected by wheat germ agglutinin. The p-nitrophenylphosphatase activity of the rectal gland enzyme was not inhibited by wheat germ agglutinin. Na+-ATPase activity, which reflects ATP binding and phosphorylation at the substrate site was inhibited by wheat germ agglutinin and this inhibition was reversed by potassium. Evidence is cited (Pennington, J. and Hokin, L.E., in preparation) that the inhibition of the (Na+ + K+)-ATPase by wheat germ agglutinin is due to binding to the glycoprotein subunit.     

Superoxide Anion-Generating Activities of Macrophages as Studied by Using Cytochalasin E and Lectins as Synergistic Stimulants for Superoxide Release

Treatment of macrophages with cytochalasin E in combination with a lectin was found to stimulate the generation of superoxide anions (O₂^-) very efficiently. The macrophages stimulated with concanavalin A, phytohemagglutinin or wheat germ agglutinin released superoxide, but cells pretreated with cytochalasin E released much greater amounts of superoxide, without notable lag time, upon stimulation with the lectin. Wheat germ agglutinin was found to be the most efficient stimulant among the lectins tested. Superoxide generation in guinea pig macrophages was shown to be dependent largely on cytoplasmic glucose metabolism and to some extent on mitochondrial respiration, since the superoxide release was largely but not totally inhibited by 2-deoxyglucose and to a lesser extent by antimycin A or KCN. The method presented is sensitive and allows rapid assay of the superoxide-generating activity with only 1–5 × 10⁵ macrophages for a single determination. In application of this technique, elevation of the superoxide-generating activity was shown with macrophages elicited by chemical inflammation or those obtained from mice after treatment with tubercle bacilli.     

Nature of the lectin-induced activation of plasma membrane Mg2+ATPase.

The Mg2+ATPase activity of liver plasma membranes decreases markedly with increasing temperature above 30 degrees. This negative temperature dependency is counteracted by the binding of wheat germ agglutinin, concanavalin A, or Ricinus communis agglutinin (at concentrations greater than or equal 0.5 mg/ml) to membranes prior to assay of the enzyme. With one of these lectins bound, the enzyme has a single energy of activation between 20 degrees and 45 degrees. The binding of dimeric succinyl concanavalin A, soybean agglutinin, fucose-binding lectin from Lotus tetragonolobus, or the leucoagglutinin from Phaseolus vulgaris does not alter the temperature dependency of the enzyme. The latter two lectins, however, do prevent the concanavalin A-induced activation of the enzyme at 37 degrees. At saturating substrate concentrations, the enzyme is not inhibited by any of the lectins tested over a wide range of concentrations. Cytochalasin B and colchicine separately or in combination have little influence on the lectin-induced enhancement of enzyme activity. Chlorpromazine and vinblastine sulfate each partially prevent the activation and in combination do so completely. Treatment of the membranes with the detergent Lubrol-PX or phospholipase A prevents activation of the enzyme by concanavalin A. The results are consistent with a restriction by the lectin of an environment which is normally too disordered for maximal enzyme activity above 30 degrees.     

Ordering of bulk membrane lipid or protein promotes activity of plasma membrane Mg2+ATPase

Treatment of liver plasma membranes with phospholipase A2 or high doses of concanavalin A enhances the activity of Mg2+ATPase assayed at temperatures greater than 30 degrees C. The effects of the two treatments are not additive. Both the removal of phospholipids and binding of the lectin increase the degree of polarization of fluorescence of the lipid-soluble fluorophores, diphenylhexatriene and beta-parinaric acid, suggesting that decreased lipid fluidity may activate Mg2+-ATPase. In fact modification of lipid fluidity by reconstitution of phospholipase-treated membranes with phosphatidylcholines of defined fatty acid composition or by addition of cis-vaccenic acid showed a strong inverse correlation between Mg2+ATPase activity and lipid fluidity as monitored by fluorescence polarization. However, despite the ability of concanavalin A to nonspecifically order membrane lipid, its effect on Mg2+ATPase is apparently not mediated in this manner because other enzyme-activating lectins such as Ricinus communis agglutinin and wheat germ agglutinin are without effect on lipid fluidity. The facts that lectins of lower valency than tetravalent native concanavalin A such as divalent succinyl concanavalin A are far less effective in activating the enzyme and that paraformaldehyde treatment also activates suggests that cross-linking of membrane proteins is responsible. Hence, the diminution in activity of this membrane enzyme due to the disordering effect of heat in the physiological temperature range can be counteracted by isothermally increasing the order of either membrane lipid or protein.     

Regulation of surfactant phospholipid secretion from isolated rat alveolar type II cells by lectins

The major surfactant-associated protein is a potent inhibitor of surfactant phospholipid secretion from isolated type II cells. Since the major surfactant-associated protein contains a carboxy terminal polypeptide domain which is homologous to the lectin-like liver mannose-binding protein, we tested whether lectins inhibit surfactant phospholipid secretion from rat alveolar type II cells. Concanavalin A, wheat germ agglutinin and Maclura pomifera agglutinin were potent inhibitors of surfactant phospholipid secretion. When adenosine 5'-triphosphate (ATP) was utilized as a secretagogue, the IC50 values for inhibition of surfactant phospholipid secretion were 5.10(-7) (wheat germ agglutinin), 1.10(-6) (concanavalin A) and 2.5.10(-5) M (M. pomifera agglutinin). Similar results were obtained when 12-O-tetradecanoylphorbol 13-acetate was utilized as a secretagogue: IC50 values of 1.10(-6) M for concanavalin A and wheat germ agglutinin and 2.5.10(-5) M for M. pomifera agglutinin. Hapten sugars were utilized to antagonize the inhibitory effect of the lectins. N-Acetyl-D-glucosamine significantly reversed inhibition of phospholipid secretion by wheat germ agglutinin in a dose-dependent fashion and methyl alpha-D-mannoside significantly reversed inhibition of phospholipid secretion by concanavalin A. N-Acetyl-D-galactosamine had no significant effect on inhibition of secretion produced by any of the lectins. The inhibitory effect of the lectins did not appear to be due to cytotoxicity since lactate dehydrogenase was not released above control levels and the inhibition of the surfactant phospholipid secretion by wheat germ agglutinin could be reversed after treatment of cells with wheat germ agglutinin by washing the lectin from the cells followed by treatment of the cells with ATP. These studies demonstrate a direct inhibitory effect of plant lectins on phospholipid secretion from type II cells in vitro.     

Interactions of lectins with plasma membrane glycoproteins of the Ehrlich ascites carcinoma cell.

Several aspects of the interaction of various lectins with the surface of Ehrlich ascites carcinoma cells are described. The order of agglutinating activity for various lectins is Ricinus communis greater than wheat germ greater than or equal to concanavalin A greater than or equal to soybean greater than Limulus polyphemus. No agglutination was noted for Ulex europaeus. Using 125I-labeled lectins it was determined that there are 1.6 and 7 times as many Ricinus communis lectin binding sites for concanavalin A and soybean lectins. Sodium deoxycholate-solubilized plasma membrane material was subjected to lectin affinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The lectin receptors of the plasma membrane appeared to be heterogeneous and some qualitative differences could be discerned among the electrophoretically analyzed material, which bound to and was specifically eluted from the various lectin affinity columns. The characteristics of elution of bound material from individual lectin columns indicated secondary hydrophobic interactions between concanavalin A or wheat germ agglutinin and their respective lectin receptor molecules.     

Development of mouse embryos in media containing lectins

Lectins known to stimulate mitosis in cultured cells were evaluated for effects on development of mouse embryos in vitro. Two-cell mouse embryos were cultured in one of the following treatments: Whitten's medium as the control medium; Whitten's medium with 1, 10 or 100 mug/ml concanavalin A; Whitten's medium with 1, 10 or 100 mug/ml leucoagglutinin; Whitten's medium with 1, 10 or 100 mug/ml phytohemagglutinin; Whitten's medium with 1, 10 or 100 mug/ml pokeweed-mitogen; and Whitten's medium with 1, 10 or 100 mug/ml wheat germ agglutinin. Development to the morula stage was blocked in media with 100 mug/ml concanavalin A and 10 and 100 mug/ml wheat germ agglutinin, whereas blastocyst formation was blocked in all pokeweed-mitogen supplemented media. Embryos incubated in 10 and 100 mug/ml wheat germ agglutinin underwent premature cavitation or vacuolation at 24 to 48 h of culture. More embryos formed blastocysts in media with 1 and 100 mug/ml phytohemagglutinin and 10 mug/ml leucoagglutinin than in Whitten's medium (P<0.05). The percentage of embryos hatching was greatest in 1 mug/ml phytohemagglutinin (P<0.05), but it was the same in Whitten's medium, 1 mug/ml concanavalin A and 1 mug/ml leucoagglutinin (P>0.05). Cell division was not stimulated by the lectins; however, it was significantly suppressed in media with 10 and 100 mug/ml concanavalin A, 100 mug/ml phytohemagglutinin, 1, 10 and 100 mug/ml pokeweed-mitogen, and 10 and 100 mug/ml wheat germ agglutinin. Solubility of the zona pellucida in sodium isothicyanate (NaSCN) was reduced in 100 mug/ml phytohemagglutinin, 100 mug/ml leucoagglutinin and 1 mug/ml wheat germ agglutinin media (P<0.05) when compared to Whitten's medium and may have accounted for the reduced hatching observed in these treatments. Development of isolated blastomeres into blastocysts was reduced in media with 1 mug/ml wheat germ agglutinin, 1 mug/ml concanavalin A, and 10 and 100 mug/ml leucoagglutinin (P<0.05) but was similar in media with 1 mug/ml leucoagglutinin and 1, 10 and 100 mug/ml phytohemagglutinin when compared to Whitten's medium (P>0.05). The extent of embryo development in media with lectins depended upon the degree of cytotoxicity and potential biochemical modifications induced in the zona pellucida. Greatest embryo development took place in medium with 1 mug/ml phytohemagglutinin; however, the mechanism was not that of stimulation of cell division or a change in zona pellucida solubility.     

Studies on lectin-induced agglutination of Drosophila embryonic cell lines

The agglutination responses of three Drosophila cell lines to concanavalin A and wheat germ agglutinin have been examined. Although the cell lines were originally derived from late embryonic stages of the Ore-R strain of Drosophila melanogaster, they show quantitative differences in lectin-induced agglutination. Line 1 cells were least agglutinable with both lectins. All three cell lines reached maximum agglutination with concanavalin A concentrations at 25 μg/ml, but the agglutination response to wheat germ agglutinin was biphasic such that an initial rapid increase in agglutination with concentrations up to 25 μg/ml was followed by slower agglutination above this concentration. Cells of lines 1 and 2 from ten-day old cultures exhibited greater lectin-induced agglutination than cells from three-day old cultures. Age-dependent differences were not found for line 3 cells which gave maximum agglutination responses in both young and old cultures. Cell agglutination by concanavalin A was almost completely inhibited by pretreatment of the lectin with methyl-α-d-mannopyranoside, but preincubation of wheat germ agglutinin with N-acetyl-d-glucosamine caused only partial blockage. Lectin-induced agglutination was not reversible by treatment with the monosaccharide inhibitors. These observations have been discussed with reference to the origin of the three cell lines and their cell surface properties.     

Lectin affinity chromatography of cell surface proteins of novikoff tumor cells

Novikoff hepatocellular carcinoma cells were radioiodinated by a cell surface-specific method using lactoperoxid ase/¹²⁵I. The iodinated proteins were solubilized in 0.5% Nonidet P-40 and subjected to affinity chromatography on Sepharose-conjugated lectins (Ricinus communis agglutinins I or II, soybean agglutinin, concanavalin A, or wheat germ agglutinin) and analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Almost all the iodinated proteins bound to one or more of the Sepharose-conjugated lectins, presumptive evidence that these peptides are glycosylated. Lectin affinity chromatography resolved defined subsets of iodinated glycoproteins and suggested that certain glycoproteins could be fractionated on the basis of heterogeneity of their heterosaccharide moieties. Incubation of the iodinated cells with neuraminidase resulted in increased binding of iodinated proteins to Sepharose-conjugated Ricinus communis agglutinins I and II and soybean agglutinin and decreased binding to Sepharose-conjugated wheat germ agglutinin. Binding of iodinated proteins to concanavalin A was unaffected by neuraminidase treatment of the cells. These studies demonstrate the utility of lectins for the multicomponent analysis of plasma membrane proteins.     

Concanavalin A and wheat germ agglutinin receptor activity of sialoglycopeptides isolated from the surface of Novikoff hepatoma cells

Novikoff ascites hepatoma cells were highly agglutinable by the plant lectins concanavalin A and wheat germ agglutinin. Treatment of the intact cells with papain released from the cell surface a glycopeptide fraction which possessed concanavalin A and wheat germ agglutinin receptor activity, as judged by its ability to inhibit lectin-induced hemagglutination. A component of the cell-surface glycopeptide fraction, excluded from Sephadex G-50, possessed lectin receptor activities reflecting the cytoagglutination properties of the intact cells from which it was derived. Further resolution of this component by pronase digestion, gel filtration, and ion-exchange chromatography resulted in the isolation of sialoglycopeptides which exhibited potent and specific concanavalin A receptor activity.     

An application of ellipsometry: Assessment of polysaccharide and glycoprotein interaction with lectin at a liquid/solid interface

Lectins were specifically adsorbed from solution onto metallized glass slides coated with polysacchride, glycopeptide and glycoprotein films. The degree of interaction was determined by measuring the thickness of the bound lectin layer with an ellipsometer after washing and drying the slide. The binding of concanavalin A (tetrameric) and succinyl concanavalin A (dimeric) to a yeast mannan film was studied as a function of lectin concentration, temperature, rinsing time and the extent of stirring of the slide. The maximum thickness of bound concanavalin A and succinyl concanavalin A was 11 and 3.8 nm, respectively. The method permitted the measurement of the association constants for both lectins (1.0 · 10⁷ M^?1 for concanavalin A, 2 · 10⁶ M^?1 for succinyl concanavalin A) and the detection of 0.6 pmol concanavalin A. The same sensitivity was observed with anti-mannan antibodies. The binding of both lectins was shown to be specific using sugar haptens. When compared with methyl α-D-mannoside, the affinity of concanavalin A for D-mannose and D-glucose was 14 and 3%, respectively. A film of mucin glycopeptide (universal adsorbent) interacted similarly with concanavalin A, Ricinus communis I, soya bean and wheat germ lectins. However, films of glycoproteins such as fetuin, ceruloplasmin and Aspergillus niger β-D-galactosidase interacted to different degrees with these lectins. The relative affinity of wheat germ agglutinin for $\text{N-}\text{acetyl}\text{-}\text{D}\text{-}\text{glucosamine}$ and for chitin-derived oligosaccharides was also determined. When films of sialoglyproteins were treated with neuraminidase, the thickness of the bound peanut agglutinin layer increased. Although this method cannot determine quantitatively the sugar composition of the film, it permits rapid estimation of the interaction of lectins with polysaccharides and glycoproteins, usingg little material.     

Effects of morphine tolerance and dependence on Mg++ dependent ATPase activity of synaptic vesicles.

The effect of morphine on ATPase of synaptic plasma membranes (SPM) and synaptic vesicles isolated from the mouse brain was studied. The activity of synaptic vesicle Mg⁺⁺-dependent ATPase from mice rendered morphine tolerant and dependent by pellet implantation was 40% higher than that from placebo implanted mice. However, the activities of Mg⁺⁺-dependent ATPase and Na⁺, K⁺ activated ATPase of SPM of tolerant and nontolerant mice were not significantly different. The activity of synaptic vesicular Mg⁺⁺-dependet ATPase was dependent on the concentration of Mg⁺⁺ but not of Ca⁺⁺; maximum activity was obtained with 2 mM MgCl₂. On the other hand, Mg⁺⁺-dependent ATPase activity of SPM was dependent on both Mg⁺⁺ and Ca⁺⁺, activity being maximum using 2 mM MgCl₂ and 10^?5 M CaCl₂. It is suggested that this stimulation of ATPase activity may alter synaptic transmission and may thus be involved in some aspects of morphine tolerance and dependence.     

Glycoprotein characteristics of the sodium channel saxitoxin-binding component from mammalian sarcolemma

The saxitoxin-binding component of the excitable membrane sodium channel exhibits glycoprotein characteristics as evidenced by its specific interaction with various agarose-immobilized lectins. The detergent-solubilized saxitoxin-binding component interacts quantitatively with immobilized wheat germ agglutinin and concanavalin A and fractionally with immobilized Lens culinaris hemagglutinin and Ricinus communis agglutinin. These lectins preferentially bind $\text{N-}\text{acetylglucosamine}$ and sialic acid (wheat germ agglutinin), mannose (concanavalin A and Lens cunilaris and galactose (Ricinus communis). Removal of terminal sialic acid residues by neuraminidase markedly decreases binding to immobilized wheat germ agglutinin but uncovers sites capable of interacting with lectins specific for galactose and $\text{N-}\text{acetylgalactosamine}$. $\text{β-N-}\text{acetylglucosaminidase}$, an exoglycosidase has no effect on the binding of the channel protein to wheat germ agglutinin. Similarly, phospholipase C has no effect on binding of the solubilized toxin binding component to this lectin. Neither wheat germ agglutinin nor concanavalin A free in solution alters the number of toxin binding sites or their affinity for toxin. The sodium channel saxitoxin-binding component appears to be a glycoprotein containing terminal sialic acid residues and internal mannose, galactose, $\text{N-}\text{acetylglucosamine}$, and $\text{N-}\text{acetylgalactosamine}$ residues. The toxin binding site is spatially separated from the binding sites for the lectins studied. The effect of these sugar moieties must be considered when evaluating the biophysical parameters of the sodium channel.     

Trypanosoma brucei brucei, T. brucei gambiense, and T. brucei rhodesiense: common glycoproteins and glycoprotein oligosaccharide heterogeneity identified by lectin affinity blotting and endoglycosidase H treatment

Whole cell extracts of 10 clones of bloodstream forms of African trypanosomes representing two strains of Trypanosoma brucei gambiense, one strain of T. b. rhodesiense and one strain of T. b. brucei were fractionated on sodium dodecyl sulfate-polyacrylamide gels, electrophoretically transferred to nitrocellulose paper, and probed with horseradish peroxidase conjugated lectins to detect glycoproteins. Variant specific glycoproteins of all 10 clones bound peroxidase labeled concanavalin A, but peroxidase labeled wheat germ agglutinin bound to the variant specific glycoproteins of only 3 of the 10 clones examined. In addition, 22 other glycoproteins expressed in common by all clones bound peroxidase labeled concanavalin A; 19 common glycoproteins bound peroxidase labeled wheat germ agglutinin. Lectin binding to transferred glycoproteins was specifically inhibited by appropriate monosaccharides, alpha-methyl mannoside for concanavalin A and N-acetyl glucosamine for wheat germ agglutinin. Prior incubation of blots in endo-beta-N-acetylglucosaminidase H eliminated binding of peroxidase-labeled concanavalin A to most of the 22 common glycoproteins. Two glycoproteins, designated Gp 81 and Gp 110, were the major Endoglycosidase H resistant components. Endoglycosidase H treatment also reduced binding of peroxidase labeled concanavalin A to the variant specific glycoproteins of 7 clones. The variant specific glycoproteins from the 3 clones that bound peroxidase labeled concanavalin A following enzyme treatment were those that bound peroxidase labeled wheat germ agglutinin. These results show that African trypanosomes express a greater number of glycoproteins than has been reported previously and that only a limited number of these glycoproteins bear Endoglycosidase H resistant oligosaccharides.     

Characterization of the Carbohydrate Chain on the Substance P Receptor in the Rat Brain Cortex: Effect of Lectins on [3H]Substance P Binding

The characteristics of the carbohydrate chain on the rat cerebral cortical substance P (SP) receptor were studied. We examined the effects of pretreatment with three lectins (concanavalin A, wheat germ agglutinin, lens culinaris agglutinin) on the [3H]SP binding activities. Each lectin can bind to the specific carbohydrate chain. Among these lectins, only concanavalin A inhibited specific [3H]SP binding by reducing the affinity of the binding sites. The inhibitory action of concanavalin A was dose-dependent and diminished by the addition of alpha-methyl-D-mannoside. The present results suggest that the rat cortical SP receptor has either a biantennary complex-type or a high mannose-type of carbohydrate chain, and that the carbohydrate chain is implicated in the SP binding activity of the SP receptor system.     

Interactions of lectins with plasma membrane glycoproteins of the Ehrlich ascites carcinoma cell

Several aspects of the interaction of various lectins with the surface of Ehrlich ascites carcinoma cells are described. The order of agglutinating activity for various various lectins is Ricinuscommunis > wheat germ concanavalin A soybean >Limuluspolyphemus. No agglutination was noted for Ulex europaeus. Using ¹²⁵I-labeled lectins it was determined that there are 1.6 and 7 times as many Ricinus communis lectin binding sites as sites for concanavalin A and soybean lectins. Sodium deoxy-cholate-solubilized plasma membrane material was subjected to lectin affinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The lectin receptors of the plasma membrane appeared to be heterogeneous and some qualitative differences could be discerned among the electrophoretically analyzed material, which bound to and was specifically eluted from the various lectin affinity colums. The characteristics of elution of bound material from individual lectin columns indicated secondary hydrophobic interactions between concanavalin A or wheat germ agglutinin and their respective lectin receptor molecules.     

Interactions of lectins with (Na+ + K+)-ATPase of eel electric organ

Interaction of lectins with a detergent-solubilized ATPase from eel electric organ was studied. Concanavalin A, which binds to α-mannosides, altered the rate of enzyme migration in agar and inhibited the formation of an antigen-antibody precipitate; other lectins had no such effects. Concanavalin A similar amounts partially inhibited $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$; this inhibition was reversible by α-methylglucoside. There was no corresponding effect of concanavalin A on the potassium $\text{p-}\text{nitrophenyl-phosphatase}$. Concanavalin A also did not interfere with ouabain binding. Thus, concanavalin A binds to an antigenic region also involved in Na⁺ and/or ATP binding, but does not interact with a K⁺ site.     

Effect of the diazonium salt of sulfanilic acid on human erythrocyte ATPase activity

External treatment of human erythrocytes with the diazonium salt of sulfanilic acid does not inhibit the Mg²⁺-dependent ATPase but does markedly inhibit the Ca²⁺-stimulated ATPase activity. Inhibition of the (Na⁺ + K⁺)-dependent activity is dependent upon the concentration of diazonium salt used. Treatment of membrane fragments does not irreversibly inhibit the (Na⁺ + K⁺)-dependent ATPase even though the diazonium salt binds covalently to membrane components. However, the Mg²⁺-dependent and Ca²⁺-stimulated ATPase activities are irreversibly inhibited. ATP and Mg-ATP will completely protect the (Na⁺ + K⁺)-dependent ATPase when present during treatment of membrane fragments with the diazonium salt, but only Mg-ATP will protect the Mg²⁺-dependent ATPase from inhibition. The Ca²⁺-stimulated ATPase activity is not protected.