Analysis of folding intermediates from the main toxin of Naja naja samarensis containing two blocked cysteines.

The formation of folding intermediates blocked by iodoacetate from the main toxin of Naja naja samarensis was monitored by FPLC and the populations were identified by amino acid analyses. We have determined conditions allowing for the highest yield in the populations with two blocked cysteines. The free cysteines remaining under these conditions were labeled with iodo[14C]acetate and localized by peptide mapping in one of the products isolated by ion-exchange chromatography (NT3III). We have also investigated the effects on the native-like characteristics of such molecules of an incubation at equilibrium with a mixture of cysteine and cystine. We find that many different molecular populations are present during the folding process and that disulfide exchanges allow for the reconstitution of native-like products having open disulfides even under strongly denaturing conditions.

Reoxidation and renaturation studies on the main toxin of Naja naja samarensis.

The main toxin of Naja naja samarensis is a very small and rigid protein (Mr 6850, 8 cysteines). When fully reduced, it regains its native conformation by a mechanism involving a rapid cysteine oxidation and a slower, less temperature-dependent disulfide exchange. In a native-like form of the protein we observed a population whose cysteines were incompletely reoxidized. When intermediates with blocked cysteines were incubated with oxidized and reduced glutathione, the percentage of native-like molecules increased. These findings suggest a multiple folding pathway.

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.

Some properties of porcine carboxypeptidase N.

Some properties of carboxypeptidase N purified from pig serum have been investigated. The amino acid composition resembles that of human carboxypeptidase N, but differs markedly from that of porcine carboxypeptidase B. The enzyme contains a mass fraction of 0.1 carbohydrates. Both ester and peptide substrates are hydrolyzed at the same site. Peptide substrates are hydrolyzed if lysine or arginine is the C-terminal amino acid, and provided that the penultimate amino acid is not proline. Argininic acid and epsilon-aminocaproic acid are good inhibitors of the enzyme.

Study of peptides inhibiting the angiotensin-converting enzyme of human seminal plasma.

Several peptides were investigated for their inhibitory capacity against dipeptidyl carboxypeptidase (angiotensin-converting enzyme) from human seminal fluid. The strongest inhibitor was the nonapeptide SQ 20881. A marked inhibition was also shown by the compounds Phe-Ala-Pro and Boc-Phe-Ala-Pro, which behaved as competitive inhibitors. Among the peptides related to angiotensin, angiotensin III was the strongest inhibitor, followed by angiotensin II and the C-terminal hexapeptide of angiotensin II. The results indicate the dipeptidyl carboxypeptidase of human semen is very similar to pulmonary dipeptidyl carboxypeptidase in its susceptibility to peptide inhibitors. In view of these and other previously reported similarities, it is possible that both enzymes are identical.

Dipeptidyl carboxypeptidase from human seminal plasma.

Dipeptidyl carboxypeptidase (angiotensin I converting enzyme) was purified from human seminal plasma. The apparent relative molecular mass determined by gel filtration on Sephadex G-200 was 330 000. The pI in isoelectric focusing was 4.6--5.0 and the optimum pH 7.7--8.0. The enzyme is activated by chloride. These properties are similar to those reported for the lung enzyme. The specificity is that of a carboxypeptidase releasing dipeptides. A study of different substrates showed the activity to be highest with Z-Leu-Gly-Gly, followed by Z-Phe-His-Leu greater than bradykinin greater than Bz-Gly-Gly-Gly greater than Boc-Phe-Ala-Pro greater than Bz-Gly-His-Leu greater than angiotensin I.

[Structural analysis of lethal snake venom neurotoxins by change transfer]. / Analyse structurale par transfert de charge de neurotoxines létales de venin

Among the two Trp and the unique Tyr present in the amino-acid sequence of the neurotoxin of Naja naja philippinensis, only one Trp is reactive in intermolecular charge transfer experiments, using organic electron acceptors. In the reference toxin-alpha of Naja nigricollis, neither the single Trp nor the Tyr, both invariant and essential for lethality, are available for charge transfer. A minor and very weak complex is obtained, which is assumed to be initiated by an exposed His. Although fully exposed, the additional Trp of the former toxin, adjacent to invariant Trp-28, fails to influence lethality and binding to the cholinergic receptor.

Carboxypeptidase N from pig serum.

Carboxypeptidase N has been purified 865-fold from pig serum. The enzyme has a molecular weight of approximately 315 000. In the presence of dodecylsulfate and mercaptoethanol, it dissociates into three subunits of Mr = 90 000, 50 000, 30 000, respectively. The native enzyme and the subunit of Mr = 90 000 contain carbohydrate; no carbohydrate is found in the subunits of Mr = 50 000 and 30 000. Trypsin transforms carboxypeptidase N into a form having a smaller molecular weight and enhanced activity.