ABSTRACT
The present report is dealing with the identification, in various unrelated proteins, of protein fragments sharing local sequence and structure similarities with the chymosin-sensitive linkage surrounding the Phe-Met/Ile bond of kappa-caseins. In all these proteins, this linkage is observed within an exposed beta-strand-like structure, as also predicted for kappa-caseins. The structure of one of these fragments, included in glutamine synthetase, particularly superimposes well with the conformation observed for a chymosin inhibitor (CP-113972) within the complex it forms with chymosin and can be similarly accommodated by specificity pockets within the enzyme substrate binding cleft. The effect of the enzyme activity of chymosin was thus tested on glutamine synthetase. Chymosin cut the latter at the Phe-Met linkage, suggesting that this system may locally resemble the kappa-casein/chymosin complex.
Subject(s)
Caseins/chemistry , Chymosin/chemistry , Animals , Butyrates/chemistry , Caseins/antagonists & inhibitors , Cattle , Cysteine/chemistry , Glutamate-Ammonia Ligase/chemistry , Humans , Molecular Sequence Data , Molecular Structure , Protease Inhibitors/chemistry , Protein Conformation , Substrate SpecificityABSTRACT
On the basis of a partial N-terminal sequence, Jollès and Jollès previously proposed that the lysozyme from the starfish Asterias rubens represents a new form of lysozyme, called type i (invertebrate) lysozyme. Indeed, it differed from both the types c (chicken) and g (goose) known in other animals, as well as from plant and phage lysozymes. Recently, several proteins belonging to the same family have been isolated from protostomes. Here we report the complete mature protein sequence and cDNA sequence of the lysozyme from Asterias. These sequences vindicate the previously proposed homology between the starfish, a deuterostome, and protostome lysozymes. In addition, we present a structural analysis that allows us to postulate upon the function of several conserved residues.
Subject(s)
Anti-Infective Agents/chemistry , Muramidase/chemistry , Starfish/enzymology , Amino Acid Sequence , Animals , Anti-Infective Agents/isolation & purification , Base Sequence , Chickens , Cloning, Molecular , Conserved Sequence , DNA, Complementary , Geese , Molecular Sequence Data , Muramidase/isolation & purification , Protein Conformation , Protein Structure, Secondary , Sequence Homology, Amino AcidABSTRACT
One-electron oxidation of six different c-type lysozymes from hen egg white, turkey egg white, human milk, horse milk, camel stomach and tortoise was studied by gamma- and pulse-radiolysis. In the first step, one tryptophan side chain is oxidized to indolyl free radical, which is produced quantitatively. As shown already, the indolyl radical subsequently oxidizes a tyrosine side chain to the phenoxy radical in an intramolecular reaction. However this reaction is not total and its stoichiometry depends on the protein. Rate constants also vary between proteins, from 120 x s(-1) to 1000 x s(-1) at pH 7.0 and room temperature [extremes are hen and turkey egg white (120 x s(-1)) and human milk (1000 x s(-1))]. In hen and turkey egg white lysozymes we show that another reactive site is the Asn103-Gly104 peptidic bond, which gets broken radiolytically. Tryptic digestion followed by HPLC separation and identification of the peptides was performed for nonirradiated and irradiated hen lysozyme. Fluorescence spectra of the peptides indicate that Trp108 and/or 111 remain oxidized and that Tyr20 and 53 give bityrosine. Tyr23 appears not to be involved in the process. Thus new features of long-range intramolecular electron transfer in proteins appear: it is only partial and other groups are involved which are silent in pulse radiolysis.
Subject(s)
Muramidase/chemistry , Tryptophan/chemistry , Tyrosine/analogs & derivatives , Tyrosine/chemistry , Animals , Asparagine/chemistry , Binding Sites , Dimerization , Free Radicals/chemistry , Glycine/chemistry , Humans , Kinetics , Muramidase/metabolism , Peptide Fragments/analysis , Peptide Fragments/chemistry , Pulse Radiolysis , Spectrometry, Fluorescence , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Trypsin/metabolism , Tryptophan/metabolism , Tyrosine/metabolismABSTRACT
We isolated and sequenced the cDNAs coding for lysozymes of six bivalve species. Alignment and phylogenetic analysis showed that, together with recently described bivalve lysozymes, the leech destabilase, and a number of putative proteins from extensive genomic and cDNA analyses, they belong to the invertebrate type of lysozymes (i type), first described by Jollès and Jollès (1975). We determined the genomic structure of the gene encoding the lysozyme of Mytilus edulis, the common mussel. We provide evidence that the central exon of this gene is homologous to the second exon of the chicken lysozyme gene, belonging to the c type. We propose that the origin of this domain can be traced back in evolution to the origin of bilaterian animals. Phylogenetic analysis suggests that i-type proteins form a monophyletic family.
