The primary structures of ovine a sl -casein variants A, C and D (formerly called Welsh variant) were determined. Separation of variants from whole casein was achieved using a fast and reliable reversed-phase HPLC method. Extended structural characterization of the purified proteins using electrospray mass spectrometry, automated Edman degradation and peptide mapping by means of HPLC-fast atom bombardment-mass spectrometry demonstrated that the mature protein was a mixture of two molecular species that differed in the deletion of residues 141-148 and were therefore 199 and 191 residues long respectively. The 199 residue peptide chain, which accounted for ~ 80% of the entire translated a sl -casein, was as long as its caprine and bovine counterparts, and had a 98 and 89 % degree of identity with those two proteins respectively. Nine serine residues (positions 12, 44, 46, 64 to 68 and 75) were fully phosphorylated in a sl -casein A, whereas Ser 115 and Ser 41 were phosphorylated by ~ 50 and ~ 20% respectively. The differences between the three genetic variants A, C and D were simple silent substitutions, which however involved the degree to which the protein was phosphorylated. Variant C differed from variant A in the substitution Ser 13 -> Pro 13 which determined the loss of the phosphate group on site 12 of the protein chain, SerP 12 ->-Ser 12 . A further substitution, SerP 68 -> Asn 68 caused the disappearance of both phosphate groups in the phosphorylated residues Ser 64 and Ser 66 in variant D; in this last casein variant there was no evidence of phosphorylation at Ser 41 .Following the elucidation of the primary structure of bovine caseins (for a review, see Swaisgood, 1992), attention has now focused on the micellar components of other species. The primary structures of ovine K-and /?-casein have been determined on the mature proteins (Jolles et al. 1974;Richardson & Mercier, 1979) and those of a sl -and a s2 -casein on the cDNA precursors (Boisnard et al. 1991;Mercier et al. 1985). The initial translation products of ovine mammary mRNA coding for caseins are larger molecules containing hydrophobic amino terminal extensions. Comparison of ovine with other a sl -casein nucleotide sequences determined from the cDNA (Mercier et al. * This paper is dedicated to M. Bruno Ribadeau Dumas on the occasion of his retirement from INRA. § Present address: Istituto
Three different phenol oxidases produced by the basidiomycete fungus Pleurotus ostreatus have been isolated and their main structural, enzymatic and physico-chemical properties characterized. Studies have focused on the most abundantly secreted of these proteins, a copper-enzyme specific towards ortho-diphenol substrates. This protein was purified to homogeneity and part of its primary structure determined by direct protein sequencing. The influence of pH, temperature and presence of water-soluble or water-insoluble organic solvents on the activity and stability of the enzyme were also investigated. These data can be used for applying bioreactors to problems of environmental concern such as waste-water treatment.
The structure of the fuscopeptins, bioactive lipodepsipeptides produced in culture by the gramineae pathogen Pseudomonas fuscovaginae, has been determined. The combined use of FAB mass spectrometry, NMR spectroscopy and chemical and enzymatic procedures allowed one to define a peptide moiety corresponding to ZDhb-DPro-LLeu-DAIa-DAIa-DAla-DAIaDVai-Gly-DAIa-DVaI-DAIa-DVaI-ZDhb-DaThr-LAIa-LDabDDab-LPbe with the terminal carboxyl group closing a macrocyclic ring on the hydroxyl group of the allothreonine residue. The N-terminus is in turn acylated by 3-hydroxyoctanoate in fuscopeptin A and 3-hydroxydecanoate in fuscopeptin B. Some preliminary data on the biological activity of fuscopeptins are also reported.
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