A mixture of iturines extracted from Bacillus subtilis gave, on column chromatography, iturine A, iturine B, and iturine C. Iturine A has the entire antifungal activity. It is a mixture of two homologous peptidolipids C48,H74N12O14 and C49H76N12O14 (mp 177 degrees C, [alpha]D-1.7 degrees in methanol (c 0.05 g/mL); mol wt 1042 and 1056). The lipid moiety is a mixture of 3-amino-12-methyltridecanoic acid and 3-amino-12-methyltetradecanoic acid. The peptide moiety contains 7 mol of amino acids: D-Asn2, L-Asn, L-Gln, L-Pro, L-Ser, and D-Tyr. A cyclic structure for iturine A with the serine residue linked to the fatty amino acids through a peptide bond has been domonstrated. By mild HCl hydrolysis, lipid-soluble and water-soluble peptides were obtained. They were analyzed by chemical methods and by mass spectrometry. Permethylated and perdeuteriomethylated derivatives of iturine A were also subjected to mass spectrometric analysis. Both chemical analysis and mass spectrometry led to the cyclic structure I for iturine A.

Although a-granules are usually amorphous, some species of teleost fish have agranules with the morphology of rhombic dodecahedra, closely resembling the crystals formed in uitro (Lange, 1973). These granules when dried for study in the electron microscope have a repeat dimension of 4.26nm (42.6A) (R. H. Lange & C. Klein, unpublished work), which as expected is slightly less than the repeat dimension [4.71 nm (47.1 A)] in pig glucagon granules containing solvent. Thus these granules almost certainly have a structure similar to that of the crystals studied by X-ray analysis and probably contain glucagon trimers.Histochemical staining techniques have been carried out on the amorphous granules (Bussolati et al., 1971). Xanthydrol, a reagent specific for tryptophan residues, produces an unusual blue-grey colour in a-granules, as distinct from the normal violet colour of other structures containing tryptophan. It is noteworthy that glucagon crystals also are stained this unusual colour, suggesting that the environment of the tryptophan residues is the same in both. Thus it seems likely that the crystal trimer also exists in the amorphous granules. The formation of crystals is sensitive to ionic strength, pH, temperature, and the presence of counter-ions and organic substances. Unfavourable combinations of these factors easily cause amorphous precipitation of trimers in the test tube, especially at neutral pH, and it is not surprising that the granules are often amorphous in mammalian a-cells, even though conditions may be conducive to crystallization in the teleost .The glucagon-receptor complex is destabilized at lower temperatures and by the presence of urea (Rodbell et al., 1971). These results suggest that the complex is formed through hydrophobic interactions. It is interesting to speculate that these arise from those hydrophobic side chains brought together in the helical conformer. Glucagon forms a helical structure in the presence of cetyltrimethyl bromide, lysophosphatidylcholine, and other glucagon molecules, which can provide a hydrophobic environment. Fragments which are unable to form helices (residues 1-21, 1-23, 20-29 and 22-29) in these systems do not compete with glucagon at the receptor and have weak or no glucagon activity. Thus it is possible that the helical form found in the crystals binds to the receptor through the two hydrophobic patches. The N-terminal residues may initially remain flexible. Subsequent binding appears to be important in eliciting the biological response, but may not give a proportional further stabilization of the hormone-receptor complex (Rodbell et al., 1971).

The sporulation-related gamma-D-glutamyl-(L)meso-diaminopimelic-acid-hydrolysing peptidase I of Bacillus sphaericus NCTC 9602 has been analysed by proton-induced X-ray emission. It contains 1 equivalent Zn2+ per mol of protein. As derived from gene cloning and sequencing, the B. sphaericus Zn peptidase I is a two-module protein. A 100-amino-acid-residue N-terminal domain consisting of two tandem segments of similar sequences, is fused to a 296-amino-acid-residue C-terminal catalytic domain. The catalytic domain belongs to the Zn carboxypeptidase A family, the closest match being observed with the Streptomyces griseus carboxypeptidase [Narahashi (1990) J. Biochem. 107, 879-886] and with the family prototype, bovine carboxypeptidase A. The catalytic domain of the B. sphaericus peptidase I possesses, distributed along the amino-acid sequence, peptide segments, a triad His162-Glu165-His307 and a dyad Tyr347-Glu366 that are equivalent to secondary structures, the zinc-binding triad His69-Glu72-His196 and the catalytic dyad Tyr248-Glu270 of bovine carboxypeptidase A respectively. The N-terminal repeats of the B. sphaericus peptidase I have similarity with the C-terminal repeats of the Enterococcus hirae muramidase 2, the Streptococcus (now Enterococcus) faecalis autolysin and the Bacillus phi PZA and phi 29 lysozymes, to which a role in the recognition of a particular moiety of the bacterial cell envelope has been tentatively assigned. Detergents enhance considerably the specific activity of the B. sphaericus peptidase I.

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