A novel cytotoxin (intermedilysin) specific for human cells was identified as a cytolytic factor of Streptococcus intermedius UNS46 isolated from a human liver abscess. Intermedilysin caused human cell death with membrane blebs. Intermedilysin was purified from UNS46 culture medium by means of gel filtration and hydrophobic chromatography. The purified toxin was resolved into major and minor bands of 54 and 53 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These proteins reacted with an antibody against intermedilysin. Five internal peptide fragments of intermedilysin were sequenced and found to have 42 to 71% homology with the thiol-activated cytotoxin pneumolysin. However, the action of intermedilysin differed from that of thiol-activated cytotoxins, especially in terms of a lack of activation by dithiothreitol and resistance to treatments with N-ethylmaleimide and 5,5-dithio-bis-(2-nitrobenzoic acid), although cholesterol inhibited the toxin activity. Intermedilysin was potently hemolytic on human erythrocytes but was 100-fold less effective on chimpanzee and cynomolgus monkey erythrocytes. Intermedilysin was not hemolytic in nine other animal species tested. Since human erythrocytes treated with trypsin were far less sensitive to intermedilysin than were the intact cells, a cell membrane protein(s) may participate in the intermedilysin action. These data demonstrated that intermedilysin is distinguishable from all known bacterial cytolysins.

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The extracellular hemoglobin (Hb) of the earthworm, Lumbricus terrestris, has four major kinds of globin chains: a, b, c, and d, present in equimolar proportions, and additional non-heme, non-globin scaffolding chains called linkers that are required for the calcium-dependent assembly of the full-sized molecule. The amino acid sequences of all four of the globin chains and one of the linkers (L1) have previously been determined. The amino acid sequences via cDNA of each of the three remaining linkers, L2, L3, and L4, have been determined so that the sequences of all constituent polypeptides of the hemoglobin are now known. Each linker has a highly conserved cysteine-rich segment of approximately 40 residues that is homologous with the seven ligand-binding repeats of the human low-density lipoprotein receptor (LDLR). Analysis of linker L1 shows that the connectivity of the three disulfide bonds is exactly the same as in the LDLR ligand-binding repeats. The presence of a calcium-binding site comprising one glutamyl and three aspartyl residues in both the LDLR repeats and in the linkers supports the suggestion that calcium is required for the folding and disulfide connectivity of the linkers as in the LDLR repeats. Linker L2 is markedly heterogeneous and contains unusual glycine-rich sequences near the NH2-terminus and a polar zipper-like sequence with imperfect repeats of Asp-Asp-His at the carboxyl terminus. Similar Asp-Asp-His repeats have been found in a protein homologous to superoxide dismutase in the hemolymph of certain mussels. These repeats may function as metal-binding sites.

The penultimate tyrosine in the hemoglobin subunit is considered to be one of the most important residues for the normal structure and function of hemoglobin. To elucidate the functional and structural role of the penultimate residue in the alpha-subunit, we prepared new artificial mutants; Hb Y140 alpha Q, in which Tyr-140 alpha is replaced by a nonaromatic residue, Gln, and Hb Y140 alpha F, which loses its hydrogen bond to Val-93 alpha by the substitution of Phe for Tyr. HB Y140 alpha Q exhibited a markedly increased oxygen affinity and almost completely diminished cooperativity, whereas Hb Y140 alpha F showed similar but less extensively impaired function, indicating that the aromatic residue at the penultimate position in the alpha-subunit contributes to the stabilization of the T-quaternary structure as does the corresponding residue in the beta-subunit. However, the deoxygenated forms of these mutants bear significant T-state character in their spectroscopic properties observed at high protein concentrations. The tetramer-dimer equilibrium data of the mutants suggested that a significant part of the functional alterations observed for dilute solution appears to result from partial dissociation into alpha beta dimers rather than direct destabilization of the T-quaternary structure in the deoxygenated form. Therefore, we can conclude that the penultimate tyrosine in the alpha-chain plays a key role not only in the stabilization of the T-state but also in the subunit assembly.(ABSTRACT TRUNCATED AT 250 WORDS)

To elucidate phylogenetic relationships among amniotes and the evolution of alpha globins, hemoglobins were analyzed from the Komodo dragon (Komodo monitor lizard) Varanus komodoensis, the world's largest extant lizard, inhabiting Komodo Islands, Indonesia. Four unique globin chains (alpha A, alpha D, beta B, and beta C) were isolated in an equal molar ratio by high performance liquid chromatography from the hemolysate. The amino acid sequences of two alpha chains were determined. The alpha D chain has a glutamine at E7 as does an alpha chain of a snake, Liophis miliaris, but the alpha A chain has a histidine at E7 like the majority of hemoglobins. Phylogenetic analyses of 19 globins including two alpha chains of Komodo dragon and ones from representative amniotes showed the following results: (1) The a chains of squamates (snakes and lizards), which have a glutamine at E7, are clustered with the embryonic alpha globin family, which typically includes the alpha D chain from birds; (2) birds form a sister group with other reptiles but not with mammals; (3) the genes for embryonic and adult types of alpha globins were possibly produced by duplication of the ancestral alpha gene before ancestral amniotes diverged, indicating that each of the present amniotes might carry descendants of the two types of alpha globin genes; (4) squamates first split off from the ancestor of other reptiles and birds.