Francois J. Joubert

Isolation and properties of N-acetyllactosamine-specific lectins from nine erythrina species

Abstract Lectins from seeds of nine species of Erythrina have been purified by affinity chromatography on columns of lactose coupled to Sepharose and their properties compared with those of the lectin from Erythrina cristagalli . All lectins are glycoproteins of M , ca 60 000 composed of two identical or nearly identical subunits. They contain between 3–10% carbohydrates comprised of N -acetylglucosamine, mannose, fucose and xylose. The amino acid composition of all Erythrina lectins is very similar. The N -terminal amino acid is valine, with the exception of the lectin from E. flabelliformis in which it is alanine. To the extent tested, identities or near identities have been found in the N -terminal sequences (up to 15 residues in some cases) of the lectins. Hapten inhibition experiments of agglutination have shown that the lectins are specific for N -acetyllactosamine, this disaccharide being 10–30 times more inhibitory than D -galactose and 10–20 times more than N -acetyl- D -galactosamine. All lectins agglutinate human erythrocytes equally well, irrespective of blood type, at minimal concentrations of 5–20 μg/ml. Six of the lectins are also very effective in agglutinating rabbit erythrocytes and are mitogenic for human peripheral blood lymphocytes, whereas three of them are considerably weaker hemagglutinins for rabbit erythrocytes, and two of these are also very weak mitogens. Our results, while demonstrating striking similarities in the molecular properties and sugar specificity of all Erythrina lectins studied, suggest the existence of differences at or close to the carbohydrate-binding site.

Naja mossambica mossambica venom Purification, some properties and the amino acid sequences of three phospholipases A (CM-I, CM-II and CM-III)

Three phospholipases A, CM-I, CM-II and CM-III, were purified from Naja mossambica mossambica venom by gel filtration on Sephadex G-50 followed by ion-exchange chromatography on CM-cellulose. They comprise each 118 amino acid residues and are close-linked by seven intrachain disulphide bridges. The complete primary structure of the three phospholipases A have been elucidated. The sequences and the invariant amino acid residues of CM-I, CM-II and CM-III resemble those of phospholipases A from other snake venoms and also from porcine pancreas. However, the letality (LD50 values) of the three phospholipases A from Naja mossambica mossambica venom, differ among themselves, and are also much higher than the LD100 value encountered for notexin from Notechis scutatus scutatus venom.

Naja melanoleuca (Forest cobra) venom: The amino acid sequence of phospholipase A, fraction DE-III

Reduced and S-carboxymethylated phospholipase A (Fraction DE-III) from Naja melanoleuca venom was digested with trypsin, chymotrypsin and thermolysin. The resulting peptides were purified by ion-exchange chromatography on DEAE-cellulose, gel filtration on Sephadex G-25 or G-50 and chromatography and electrophoresis on paper. The amino acid sequences of the intact enzyme and the pur peptides were determined by the Edman procedure, either through the use of the automatic sequencer or by manual manipulation. The chymotryptic digest provided the necessary overlapping peptides which allowed the alignment of the tryptic peptides into a single chain of 119 amino acids. The amino acid sequence of N. melanoleuca phospholipase A shows a high degree of homology with phospholipases A from Bitis gabonica and also from porcine pancreas.

Snake venom toxins: The amino acid sequences of two toxins from Ophiophagus hannah (King Cobra) venom

Abstract Two toxins were isolated from the venom of Ophiophagus hannah (King Cobra) by ion-exchange chromatography on CM-cellulose and gel filtration on Sephadex G-50. The toxins were homogeneous by free boundary electrophoresis, polyacrylamide electrophoresis and sodium dodecyl sulphate gel electrophoresis, The amino acid sequences of the two toxins were determined by using an automatic sequencer. Both toxins contain 73 amino acid residues and are cross-linked by five disulphide bridges. The amino acid sequences of the Ophiophagus hannah toxins show a high degree of homology with similar toxins from the genera Naja, Dendroaspis and Bungarus.

Snake venom toxins The isolation and purification of three cytotoxin homologues from the venom of the forest cobra (Naja melanoleuca) and the complete amino acid sequence of toxin VII1

Three proteins (VII1–VII3) have been isolated from the venom of Naja melanoleuca (Forest cobra). The complete amino acid sequence of VII1 has been established. The sequence of VII1 and the amino acid composition and other data for VII2 and VII3 indicate that the three proteins are homologoes of the cyto- or cardiotoxins which have been isolated from the venoms of other members of the Elapidae sub-family. Protein VII1 showed the intravenous toxicity level typical of cytotoxin type proteins, while VII2 and VII3 were considerably less toxic. The sequence of the first 34 residues, out of the total of 60, was determined using an automatic sequenator. The remaining 26 residues were aligned with the aid of tryptic and chymotryptic peptides and thermolysin produced sub-fragments of one tryptic peptide. It was found that VII1 consisted of a pair of isoproteins having leucine and isoleucine NH2-terminal residues.

Studies on puff adder (Bitis arietans) venom—I. Purification and properties of protease A

Abstract The proteases from puff adder venom were separated by gradient elution from CM-cellulose, and protease A was further purified by Sephadex G-75 gel filtration. It was found to have a molecular weight of 21,400 and it showed a tendency to associate particularly at higher concentrations. The amino-acid composition and physical properties of protease A are reported. It had no esterase activity towards ATEE or TAME and could not be inhibited by DFP, TPCK, TLCK or PMA. The enzyme requires the presence of calcium to prevent autolysis.

Naja melanoleuca (Forest cobra) venom: Purification and some properties of phospholipases A

Three phospholipases A (Fractions DE-I, DE-II and DE-III) were purified from Naja melanoleuca (Forest cobra) venom by a combination of gel filtration on Sephadex G-50 and chromatography on DEAE-cellulosemthe purified phospholipases A were homogeneous by various physicochemical criteria. Whereas Fraction DE-I contains 118 amino acid residues, Fractions DE-II and DE-III comprise 119 residues. The three enzymes are cross-linked by seven disulphide bridges, have asparagine as N-terminal amino acid and the C-terminal is glutamic acid or glutamine. The molecular weights of the three phospholipases A from sedimentation analysis at pH 2.1, also by the sodium dodecylsulphate-gel method and calculated from the amino acid composition, were close to 13 000. Studies of circular dichroism in the spectral region between 195 to 305 nm showed that the three phospholipases A contain similar helical contents but revealed conformational differences between their side-chain chromophores.

Trypsin isoinhibitors from Momordica repens seeds

Abstract Four trypsin isoinhibitors (CM-1 to CM-4) were purified from Momordica repens seeds by gel filtration on Sephadex G-50 followed by ion exchange chr

Purification and properties of the proteinase inhibitors from Erythrina caffra (coast erythrina) seed

1. Four proteinase inhibitors (DE-1 to DE-4) were purified from Erythrina caffra seed by gel filtration on Sephadex G-50 followed by ion-exchange chromatography involving DEAE-cellulose and DEAE-sepharose. 2. They comprise 164-166 amino acid residues (mol. wt 18,100) including 4 half-cystine residues and resemble the Kunitz-type proteinase inhibitors. 3. The N-terminal primary structure of DE-3 revealed also homology with those of the Kunitz-type inhibitors. For DE-1, DE-2 and DE-4 no free N-terminal amino acid was found. 4. DE-1 contains a potent inhibitor for both porcine trypsin and bovine alpha-chymotrypsin. Whereas DE-2 inhibits alpha-chymotrypsin strongly and has practically no action on trypsin, DE-3 inhibits both trypsin and alpha-chymotrypsin strongly. DE-4 is a potent inhibitor for trypsin but it binds alpha-chymotrypsin only weakly.

The complete primary structures of three cytotoxins (CM-6, CM-7 and CM-7A) from Naja naja kaouthia (Siamese cobra) snake venom

Abstract Toxins CM-6, CM-7 and CM-7A were purified from Siamese cobra ( N. n. kaouthia ) venom. They contain 60 amino acids including eight half-cystines and are devoid of glutamic acid or glutamine, histidine and tryptophan. On elucidation of the primary structures, the sequences and invariant amino acids of toxin CM-6, CM-7 and CM-7A appear to resemble those of a group of cytotoxins, which are also devoid of the same four amino acids. In toxin CM-6, CM-7 and CM-7A the ten structurally invariant amino acid residues of the neurotoxins and the cytotoxins are conserved.