Kenneth M. Blumenthal

A Specific Interaction between the Cardiac Sodium Channel and Site-3 Toxin Anthopleurin B

The polypeptide neurotoxin anthopleurin B (ApB) isolated from the venom of the sea anemone Anthopleura xanthogrammica is one of a family of toxins that bind to the extracellular face of voltage-dependent sodium channels and retard channel inactivation. Because most regions of the sodium channel known to contribute to inactivation are located intracellularly or within the membrane bilayer, identification of the toxin/channel binding site is of obvious interest. Recently, mutation of a glutamic acid residue on the extracellular face of the fourth domain of the rat neuronal sodium channel (rBr2a) was shown to disrupt toxin/channel binding (Rogers, J. C., Qu, Y. S., Tanada, T. N., Scheuer, T., and Catterall, W. A. (1996) J. Biol. Chem. 271, 15950–15962). A negative charge at this position is highly conserved between mammalian sodium channel isoforms. We have constructed mutations of the corresponding residue (Asp-1612) in the rat cardiac channel isoform (rH1) and shown that the lowered affinity occurs primarily through an increase in the toxin/channel dissociation rate k off. Further, we have used thermodynamic mutant cycle analysis to demonstrate a specific interaction between this anionic amino acid and Lys-37 of ApB (ΔΔG = 1.5 kcal/mol), a residue that is conserved among many sea anemone toxins. Reversal of the charge at Asp-1612, as in the mutant D1612R, also affects channel inactivation independent of toxin (−14 mV shift in channel availability). Binding of the toxin to Asp-1612 may therefore contribute both to toxin/channel affinity and to transduction of the effects of the toxin on channel kinetics.

5 Glutamate Dehydrogenases

Publisher Summary This chapter discusses the types of investigations with major emphasis on recent studies that include elucidation of complete or partial amino acid sequences of the enzymes from several sources. L-Glutamate dehydrogenases catalyze the interconversion of α-ketoglutarate and L-glutamic acid. The enzymes are recognized to be important because of the pivotal positions in metabolism occupied by glutamate and α -ketoglutarate and the ability of these compounds to enter into many types of pathways. Glutamate dehydrogenases provide a route for incorporation of nitrogen into organic compounds, and thus a link between carbohydrate and amino acid metabolism. There are at least three types of glutamate dehydrogenases which differ in coenzyme specificity. Those specific for either NAD or NADP and those that can function with both. The successful isolation from some species of modified forms of the enzyme produced by mutant strains, particularly of Neurospora, now permits identification of residues important for maintenance of normal activity.

The complete amino acid sequence of the α-subunit of pea lectin, Pisum sativum

Abstract The complete primary structure of the α-subunit of the lectin from the pea (Pisum sativum) has been determined using a combination of tryptic and staphylococcal protease digestion, purification using Sephadex gel filtration and high-voltage electrophoresis followed by either manual or automated Edman degradation. The molecular weight of the α-subunit from sequence data and gel filtration in guanidine-HCl is close to 5800, which is lower than that determined by sedimentation equilibrium techniques. The sequence reveals considerable homology to concanavalin A and near identity to the α-subunit of the lentil lectin (Lens culenaris). As in the case of the lentil α-subunit, the α-methyl glucose binding site(s) are not present in this region, nor are the S1 and S2 metal ion binding sites as judged by homology consideration, though the residues for the S3 lanthanide binding (Glu 87 and Asp 136) are conserved from the available data on the α- and β-subunits. Preliminary metal exchange experiments on the intact pea lectin indicate some differences in the metal exchange properties of this lectin compared to concanavalin A, and therefore possible ligand variations in this region of the β-subunit.

Identification and characterization of novel sodium channel toxins from the sea anemone Anthopleura xanthogrammica

Six new toxins from the sea anemone Anthopleura xanthogrammica were identified using a molecular biological approach. Five of these novel isoforms resemble the 47 residue type I long polypeptides native to Anthopleura elegantissima, Anthopleura fuscoviridis and Anemonia sulcata, while one appears to be chimera of the two previously identified 49 residue toxins native to A. xanthogrammica. Four of these toxins were expressed in bacteria, purified and characterized by ion flux assays in RT4-B and N1E-115 cell lines expressing the cardiac and neuronal Na channel isoforms, respectively. The novel 47 residue toxin isoforms form a new subclass within the A. xanthogrammica neurotoxin family, although they are related to previously described anemone toxins. One of the three 47 residue toxins characterized, PCR2-10, enhances veratridine-dependent sodium uptake, displaying a K0.5 of 329 nM and 1354 nM in RT4-B and N1E-115 cell lines, respectively. The novel 49 residue toxin, PCR3-7, interacts with the sodium channel with even higher affinity, enhancing sodium uptake with a K0.5 of 47 nM and 108 nM in RT4-B and N1E-115 cells, respectively.

Melittin inhibition of the gastric (H+ + K+) ATPase and photoaffinity labeling with [125I]azidosalicylyl melittin.

Melittin is a 26-amino acid amphipathic polypeptide toxin from bee venom which forms anion-selective ion channels in bilayers and biological membranes under the influence of membrane potential. Melittin has been shown to interact with a number of membrane proteins. We found that melittin inhibited K+-stimulated ATP hydrolysis by the (H+ + K+) ATPase in parietal cell apical membrane vesicles derived from histamine-stimulated rabbit gastric mucosa with a KIapp of 0.5 micron. Melittin also inhibited K+-stimulated p-nitrophenyl hydrolysis activity which is associated with the gastric (H+ + K+) ATPase in a dose-dependent manner with a KIapp of 0.95 micron. ATP-driven, K+-dependent H+ transport was inhibited over this same concentration range, even in the absence of a membrane potential. Melittin did not appear to increase the H+ leak from vesicle with preformed H+ gradients when the H+ pump was arrested by Mg2+ chelation, but all possible membrane perturbation effects were difficult to rule out. However, the data suggest that melittin exerts its inhibitory effect through interaction with the (H+ + K+) ATPase. In order to determine whether direct interactions between the (H+ + K+) ATPase and melittin occurred, a radioactive derivative of melittin, [125I]azidosalicylyl melittin, was prepared and photoreacted with sealed rabbit gastric membranes and highly purified hog gastric membrane containing the (H+ + K+) ATPase. In the purified hog preparation only a 95,000-Da band, the (H+ + K+) ATPase was labeled, while in the rabbit preparation a 95,000-Da band and one other membrane protein of 70,000 Da were labeled with this reagent. Label incorporation into the (H+ + K+) ATPase and the 70,000-Da band was greatly reduced by addition of excess unlabeled melittin, suggesting specificity of the interaction. Label incorporation occurred in the absence of ATP or added salts and was not reduced by SCH28080 (a K+ site inhibitor) suggesting that the melittin binding site was distinct from the luminal K+ site of action of SCH28080.

Structure and action of heteronemertine polypeptide toxins: Inactivation of Cerebratulus lacteus toxin B-IV by tyrosine nitration

Abstract Reaction of Cerebratulus lacteus toxin B-IV with tetranitromethane in the presence of low concentrations of urea results in essentially complete loss of toxicity as measured by a sensitive quantal bioassay. Amino acid analysis and speetrophotometric studies both indicate the primary effect of reaction to be nitration of a single tyrosine residue per molecule of toxin. The nitrated residue has been identified as tyrosine-9 by automated Edman degradation of the modified protein. Since the secondary structure of toxin B-IV is not detectably altered by nitration, it is concluded that tyrosine-9 is directly involved in the interaction of this polypeptide with its axonal receptor, proposed to be involved in the inactivation of voltage-sensitive Na + channels in crustacean nerves.

Membrane damage by Cerebratulus lacteus cytolysin A-III. Effects of monovalent and divalent cations on A-III hemolytic activity

The effects of monovalent and divalent cations on the hemolytic activity of Cerebratulus lacteus toxin A-III were studied. The activity of cytolysin A-III is remarkably increased in isotonic, low ionic strength buffer, the HC50 (the toxin concentration yielding 50% lysis of a 1% suspension of erythrocytes after 45 min at 37 degrees C) being shifted from 2 micrograms per ml in Tris or phosphate-buffered saline to 20-30 ng per ml in sucrose or mannitol buffered with Hepes, corresponding to a 50-100-fold increase in potency. On the contrary, hemolytic activity decreases progressively as the monovalent cation concentration in the medium increases for Na+, K+, or choline salts. The divalent cations Ca2+ and Zn2+ likewise inhibit the cytolysin A-III activity, but more strongly than do the monovalent cations specified above. Zn2+ at a concentration of 0.3 mM totally abolishes both toxin A-III-dependent hemolysis of human erythrocytes and toxin-induced leakage from liposomes. The observation of similar effects in both natural membranes and artificial bilayers suggests an effect of Zn2+ on the toxin A-III-induced membrane lesion, especially since Zn2+ does not alter binding of the cytolysin. The dose-response curve for toxin A-III exhibits positive cooperativity, with a Hill coefficient of 2 to 3. However, analysis of toxin molecular weight by analytical ultracentrifugation reveals no tendency to aggregate at protein concentrations up to 2 mg per ml. These data are consistent with a post-binding aggregational step which may be affected by the ionic strength of the medium.

Structure and action of heteronemertine polypeptide toxins: importance of amphipathic helix for activity of Cerebratulus lacteus toxin A-III

The marine heteronemertine Cerebratulus lacteus produces a family of protein cytolysins designated as A-toxins. Limited subtilisin digests of the most abundant homolog, toxin A-III, yield two major products which may be purified by high-performance liquid chromatography. One product is shown to represent residues 1-86 and the other contains the entire toxin sequence (1-95). Both polypeptides are shown to lack internal protease nicks. The 1-95 polypeptide retains full cytolytic activity in comparison to native toxin, whereas 1-86 has an activity that is approximately four times lower. Extensive treatment of A-III with carboxypeptidase Y yields a polypeptide containing residues 1-75 which is totally devoid of hemolytic activity. Residues 63-95 of native A-III have been predicted to form a relatively hydrophobic alpha-helix which is potentially important for activity. The circular dichroism spectrum of 1-95 is in excellent agreement with both experimental and Chou-Fasman-predicted secondary structures of native A-III, while the spectra of 1-86 and 1-75 indicate a loss of helicity quantitatively consistent with the removal of residues 87-95 and 76-95, respectively. Combined with our earlier data on bilayer penetration by N-terminal sequences (K. M. Blumenthal (1982) Biochemistry 21, 4229-4233], the current results indicate a direct involvement of both ends of A-III in lytic activity. The C-terminal region may function by contributing a membrane binding site in the form of an amphipathic helix.

Release of liposomal markers by Cerebratulus toxin A-III

Marker release from liposomes induced by the cytolytic protein Cerebratulus lacteus toxin A-III was studied. No phospholipid specificity was apparent, but the sensitivity of liposomes to A-III varied with the membrane fluidity. With dioleylphosphatidylcholine liposomes, complete release occurred at 10-20 micrograms toxin per ml, depending on marker size. Kinetic experiments showed that release was rapid and exhibited no lag phase. The diameter of the A-III produced membrane lesion must exceed 90 A, as tetrameric Concanavalin A is quantitatively released from A-III treated liposomes.

Role of Electrostatic Interactions in Defining the Potency of Neurotoxin B-IV from Cerebratulus lacteus

Chemical modification implicates arginine residues of the Cerebratulus lacteus neurotoxin B-IV in biological activity. In the present study, we used site-directed mutagenesis to assess the functional contributions of each of these residues. Panels of mutants at each site have been constructed by polymerase chain reaction and recombinant proteins expressed and purified to homogeneity using an Escherichia coli expression system developed in this laboratory. All substitutions for Arg-17 (Gln, Ala, or Lys) yield proteins having undetectable levels of activity, while charge neutralizing replacement of Arg-25 (R25Q) causes a 400-fold reduction in specific toxicity. However, the R25K mutein is almost as active as natural toxin. Circular dichroism spectroscopy indicates that there are no major conformational changes in any of these muteins. These results therefore demonstrate the requirement for a guanidinium group at position 17, and a positive charge at position 25. NMR analyses (Hansen, P. E., Kem, W. R., Bieber, A. L., and Norton, R. S. (1992) Eur. J. Biochem. 210, 231-240) reveal neurotoxin B-IV to contain two antiparallel α-helices, which together include 57% of the sequence. Both Arg-17 and Arg-25 lie on the same face of the N-terminal helix (residues 13-26), as do the carboxyl groups of Glu-13 and Asp-21. However, charge neutralizing mutations of the latter two sites have no effects on biological activity. Arg-34, situated near the N terminus of helix 2 (residues 33-49) is also important for activity, as its replacement by Gln or Ala diminishes activity by 20- and 80-fold, respectively. However, unlike Arg-17 and Arg-25, thermal denaturation experiments suggest that R34Q may be structurally destabilized relative to wild-type B-IV.