The structure of cyanoginosin-LA (previously referred to by us as toxin BE-4) is cyclo(-o-Ala-L-Leuerythro-P-methyl-D-isoAsp-L-Ala-Adda-D-G lu -N-methyldehydroAla) (1 7a), where Adda refers to the novel p-amino acid residue of 3-amino-9-methoxy-2,6,8-trimethyl-1 O-phenyldeca-4,6-dienoic acid (11). The connectivity of Adda is deduced from Hand I3C-n.m.r. data, but the stereochemistry at carbons 2,3,8, and 9 remains unknown. The residue sequence is derived from the mass spectra of cyclic and linear derivatives of the toxin. A new nomenclature for the related toxins of Microcystis aeruginosa is discussed.
The structures of the hepatotoxins of general name cyanoginosins-XY are proposed t o be Cyclo-D-AIa- (1 ) where X and Y represent variable amino acids and Adda is 3-amino-9-methoxy-2,6,8-trimethyl-lO-phenyldeca-4,6dienoic acid (2). The structural stildies on four variant toxins utilised n.m.r. and mass spectral methods analogous t o those recently used to determine the structure of cyanoginosin-LA.
The Macrotyloma axillare plant, belonging to the Leguminosae family, is a perennial climbing or trailing herb 0.2-3.5 m long. The plant is indigenous to South Africa and it occurs in the warm dry northern parts of the Transvaal. It has been introduced into Australia, where the seed are used as animal food. Two protease inhibitors, DE-3 and DE-4, were purified from Macrotyloma axillare seed by gel filtration on Sephadex G-50 followed by ion-exchange chromatography on DEAEcellulose. They each comprise 76 amino acid residues including 14 half-cystine residues. The complete primary structures of the two protease inhibitors have been elucidated and their sequences are 67 % identical. The inhibitor specificities, the sequences, the invariant amino acid residues and the reactive inhibitor sites of protease inhibitors DE-3 and DE-4 resemble the corresponding properties of the Bowman-Birk double-headed protease inhibitor group. The cysteine residues are in similar locations to those in protease inhibitors of known structure so they are presumed to link similarly. In continuation of the studies of protease inhibitors from Leguminosae seed the present paper describes the purification, some of the properties and the complete primary structure of protease inhibitors DE-3 and DE-4 from Macrotyloma axillare seed.
EXPERIMENTAL PROCEDUREMacrotyloma axillare (formerly Dolichos axillaris 1151) seed was supplied by Wright Stephenson Co. (Aust) Pty Ltd, Queensland, Australia. The sources of the trypsin, a-chymotrypsin, thermolysin, subtilisin and the chemical reagents have been described previously [16,17]. The physicochemical methods, the reduction and S-carboxymethylation, the digestion with trypsin, a-chymotrypsin and subtilisin, the fractionation of enzyme digests by chromatography on DEAE-cellulose and paper chromatography and/or high-voltage paper electrophoresis, the amino acid analyses of the protease inhibitors and of the peptides, the sequence determination of reduced and S-carboxymethylated protease inhibitors and peptides by Edman degradation with a Beckman sequencer or manually and the nomenclature of the peptides have been detailed in previous communications [16,17]. The sequence determination of the peptides in the Beckman sequencer was carried out in the presence of Polybrene as described [18,19].Inhibition of proteolytic activities of trypsin and a-chymotrypsin was estimated by Kunitz's casein digestion method [20] as described by Birk [21]. The residual tryptic and a-chymotryptic activities were measured. The specific inhibitor activity was expressed as mg trypsin and a-chymotrypsin inhibited/mg inhibitor.
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