Comparative studies of Ca2+-dependent proteases (CDP I and CDP II) from Allomyces arbuscula.

Allomyces arbuscula, an aquatic fungus, contains two Ca2+-dependent neutral cysteine proteases (CDP I and CDP II), eluting respectively, at 0.07 and 0.2 M NaCl from DEAE cellulose columns. The purified CDP I has a Mr of 39 kDa whereas CDP II appears as a doublet of 43 and 40 kDa. Both enzymes require free thiol, the same concentration of Ca2+ for half maximal activation, and are inactivated by thiol protease inhibitors. Our results show that despite these similarities the two enzymes are different because affinity-purified CDP II antibodies do not cross-react with CDP I antigen in Western blots. In contrast, there is a strong cross-reaction between the two 43 and 40 kDa CDP II peptides and their respective antibodies. Both enzymes cleave preferentially the carboxy terminus of Arg and to a limited extent Lys on the cleavage site. This primary specificity is governed by the nature of the amino acids in the P2 and P3 positions. In general either Pro or Gly in P2 is required, with preference for Pro and in P3 position, Gly over Val. CDP II has higher catalytic activity than CDP I. The sulfhydryl reagent NEM is a more potent inhibitor of CDP I than CDP II. Although the function of the phosphorylable site(s) is not clear, both CDP I and CDP II contain phosphorylable serine residue(s).

T cell epitopes in experimental autoimmune myasthenia gravis of the rat: strain-specific epitopes and cross-reaction between two distinct segments of the alpha chain of the nicotinic acetylcholine receptor (Torpedo californica).

T cell epitopes on the nicotinic acetylcholine receptor (A ChR) of Torpedo californica were analyzed using T cell lines isolated from Lewis, BN, and (Lewis X BN)F1 rats. All lines selected for reactivity against either native or denatured AChR or for 6 selected synthetic peptides of the AChR alpha chain expressed the CD4 membrane phenotype and recognized their antigen in the context of major histocompatibility complex class II determinants. They were tested in vitro for reactivity with each of these antigens. The results indicate that parental Lewis and BN rat T lymphocytes recognize distinct molecular epitopes on the AChR protein, whereas (Lewis X BN)F1 hybrids respond against both sets of epitopes. Two peptides (P10 and P11) which represent distinct amino acid sequences on the putatively extracellular part of the AChR alpha chain, and which share only 4 common amino acids, two of them contiguous, showed an unexpected cross-reactivity in the Lewis rat. T cells selected for either peptide co-recognize the other peptide in vitro. In addition, these cells are responsive against full length AChR. P11, in particular, appears to be a major epitope for Lewis rats immunized with AChR.

A modified nicotinic acetylcholine receptor lacking the 'ion channel amphipathic helices'.

Antibodies to a synthetic peptide from the 'amphipathic helix' of the alpha-chain of the nicotinic acetylcholine receptor (nAChR) bound both to detergent-solubilised and membrane-bound nAChR, indicating that this region, suggested as a component of the transmembrane ion channel in one model, is not buried in the membrane. Trypsinisation of membranes prior to affinity purification yielded preparations lacking the amphipathic helices of the alpha- and beta-chains and probably also of the gamma- and delta-chains. Such material should allow direct testing, by reconstitution experiments, of the importance of these regions for channel activity.

Antibodies to synthetic peptides as probes of acetylcholine receptor structure.

Peptides containing the C-terminus of the alpha-chain of the nicotinic acetylcholine receptor (nAChR), as deduced from cDNA data, were synthesised and shown to bind to antibodies to denatured nAChR. Conversely, peptide-specific antibodies bound both to native and to denatured nAChR. Binding was exclusively to the alpha-chain. Trypsinization experiments and the use of the unique C-terminal hexapeptide of the alpha-chain demonstrated that the proposed C-terminus does exist on the mature alpha-chain, and that post-translational cleavage can be discounted as an explanation of the discrepancy of the molecular masses of the alpha-chain deduced from SDS gel electrophoresis (40 kDa) and from the DNA sequencing (50 kDa). Cleavage of the alpha-chain in the membrane occurs at two closely linked sites, resulting in the formation of a large fragment (approximately 35 kDa) and the remainder of the chain (approximately 9-10 kDa). No signs of experimental myasthenia gravis were observed in rabbits immunised with C-terminal peptide coupled to carrier protein.

Reconstitution of pure acetylcholine receptor in phospholipid vesicles and comparison with receptor-rich membranes by the use of a potentiometric dye.

Acetylcholine receptor, isolated in Triton X-100 on a cobra alpha-neurotoxin affinity column was incorporated into unilamellar phospholipid vesicles by a detergent depletion method using Amberlite XAD-2. Vesicles of an average diameter of 25 nm were formed, as verified by freeze-fracture electron microscopy and gel filtration. 85 to 95% of the alpha-bungarotoxin binding sites of the reconstituted acetylcholine receptor were oriented towards the outside of the vesicles. In the reconstituted receptor one molecule of residual Triton X-100 per 2.5 alpha-bungarotoxin binding sites on the receptor molecule could be assessed. The reconstituted protein was not accessible to papain digestion, whereas the pure acetylcholine receptor, solubilized by Triton X-100 was split into smaller polypeptides under the same condition. Reconstituted acetylcholine receptor and receptor-rich membranes did not exhibit the same behavior as measured by use of a potentiometric dye. This is interpreted as an irreversible alteration of at least 95% of the receptors purified in the presence of Triton X-100. Furthermore, it could be shown that the fluorescence intensity changes induced by carbamylcholine in receptor-rich membranes did not reflect ion fluxes, but conformational changes of the protein or a displacement of the dye from the protein.

Specific binding to isolated acetylcholine receptor of a synthetic peptide duplicating the sequence of the presumed active center of a lethal toxin from snake venom.

To verify the existence of a lethal "active center" in snake venom neurotoxins and to assess its delineation within their polypeptide sequences, a tritriacontapeptide matching residues 16-48 of the natural "major" toxin of Naja naja philippinensis (Hauert, J., Maire, M., Sussmann, A., and Bargetzi, J. P. (1974) Int. J. Pept. Protein Research 6, 201-222) has been synthesized by solid-phase technology (Juillerat, M. A., and Bargetzi, J. P. (1980), results presented at the 16th European Peptide Symposium, Helsingor, Denmark, September, (1980). After deblocking, cyclization by reoxidation, and purification, one of the resulting peptides exhibiting the correct chemical and physical characteristics was found to be highly "active" in binding isolated, purified, and standardized acetylcholine receptor protein. A new assay procedure had been developed using 3H-labeled alpha-bungarotoxin as nonreversible back-titrant. It has the advantage of measuring only specific binding of an unknown ligand competing for the same receptor protein. The observed KD was 2.2 x 10(-7) M, a value attesting to a higher affinity than acetylcholine itself, 2.5 x 10(-6) M, as well as curare and the small organic cholinergic ligands, albeit somewhat lower than the affinity of the parent native toxin, as expected from differences in molecular size.

Role of glycerol permeation in the bloodstream form of Trypanosoma brucei.

Under aerobic conditions, we have determined glycerol uptake in the long slender (LS) bloodstream form of Trypanosoma (Trypanozoon) brucei brucei by studying glycerophosphate accumulation in the parasites. The coupled enzyme theory applies to the permeation-phosphorylation sequence. Glycerol passage through the plasma membrane is asymmetric, the efflux process being favored over the influx process. No free diffusion of glycerol can be detected even under conditions under which free glycerol accumulates within the cells; most probably, glycerol permeation is mediated by a specific transport system. In the absence of respiratory activities, glycerol is known to be an end-product of T. brucei glycolysis; its production from glycerophosphate should allow ATP synthesis. The observed efflux of free glycerol following intracellular accumulation of glycerophosphate confirms the hypothesis that glycerol production occurs through reversal of glycerol kinase activity. We conclude that in vivo the role of the carrier-mediated asymmetric permeation process is to prevent inhibition of the reversal of the glycerol kinase-mediated reaction by removing free glycerol.

Characterization of iodinated derivatives of alpha-bungarotoxin.

The iodination of alpha-bungarotoxin and the reactivity of iodinated derivatives towards nicotinic acetylcholine receptor are described. 125I2- and 125I-alpha-bungarotoxin can be resolved, but the latter was not separated from unreacted alpha-bungarotoxin. A study of the reactivities of the various forms of the toxin towards nicotinic acetylcholine receptor indicated that di-iodination had modified its reactivity. The 125I2-form bound with a slower rate constant than alpha-bungarotoxin to the receptor. 125I-alpha-bungarotoxin showed no modification of reactivity towards the receptor. Apart from the A280, two methods for calibrating 125I-alpha-bungarotoxin are described. They may be employed in the presence of other proteins. The first of these is an immunological assay using the complex formed between toxin and antitoxin antibodies. The second is a dilution assay, where competition between iodinated and noniodinated toxins for binding sites on nicotinic acetylcholine receptor is exploited.