Toshio Gotoh

An evolutionary tree for invertebrate globin sequences

SummaryA phylogenetic tree was constructed from 245 globin amino acid sequences. Of the six plant globins, five represented the Leguminosae and one the Ulmaceae. Among the invertebrate sequences, 7 represented the phylum Annelida, 13 represented Insecta and Crustacea of the phylum Arthropoda, and 6 represented the phylum Mollusca. Of the vertebrate globins, 4 represented the Agnatha and 209 represented the Gnathostomata. A common alignment was achieved for the 245 sequences using the parsimony principle, and a matrix of minimum mutational distances was constructed. The most parsimonious phylogenetic tree, i.e., the one having the lowest number of nucleotide substitutions that cause amino acid replacements, was obtained employing clustering and branch-swapping algorithms. Based on the available fossil record, the earliest split in the ancestral metazoan lineage was placed at 680 million years before present (Myr BP), the origin of vertebrates was placed at 510 Myr BP, and the separation of the Chondrichthyes and the Osteichthyes was placed at 425 Myr BP. Local “molecular clock” calculations were used to date the branch points on the descending branches of the various lineages within the plant and invertebrate portions of the tree. The tree divided the 245 sequences into five distinct clades that corresponded exactly to the five groups plants, annelids, arthropods, molluscs, and vertebrates. Furthermore, the maximum parsimony tree, in contrast to the unweighted pair group and distance Wagner trees, was consistent with the available fossil record and supported the hypotheses that the primitive hemoglobin of metazoans was monomeric and that the multisubunit extracellular hemoglobins found among the Annelida and the Arthropoda represent independently derived states.

Autoxidation of native oxymyoglobin from bovine heart muscle

A method is described for the preparation of native oxymyoglobin from bovine heart muscle. The aqueous extract is gel filtered on Sephadex G-50 to isolate myoglobin from hemoglobin. Native oxymyoglobin is then separated from metmyoglobin by DEAE-cellulose chromatography. There is a marked effect of temperature on the autoxidation of native oxymyoglobin to metmyoglobin, with Q10 values approximating 5.3 over the pH range of 5–10. The activation energies over this pH range are shown to be almost constant, i.e., 26.5 kcal·mole−1. In contrast to the suggestions in earlier reports, the autoxidation rate of native oxymyoglobin estimated at physiological pH and temperature is quite high with t12 ≤ 1.5 days under air saturation. This suggests the existence of an in vivo system(s) immediately reducing metmyoglobin formed to the ferrous state.

Amino acid sequence of the smallest polypeptide chain containing heme of extracellular hemoglobin from the polychaete tylorrhynchus heterochaetus

Abstract The complete amino acid sequence of 139 residues of the smallest polypeptide chain of extracellular hemoglobin from the polychaete Tylorrhynchus heterochaetus was determined. Mainly owing to the absence of seven residues in the G-helical region, this polypeptide chain is the second shortest, after Chironomus III hemoglobin (136 residues), of all vertebrate and invertebrate globin sequences so far reported. The residues at 42 sites (30%) of the Tylorrhynchus chain are identical with those of Lumbricus extracellular hemoglobin (AIII), which was sequenced very recently (Garlick, R.L. and Riggs, A.F. (1982) J. Biol. Chem. 257, 9005–9015). The C-terminal 20 residues of the Tylorrhynchus chain also showed 35% homology with those of Glycera intracellular hemoglobin, whereas the C-terminal sequence of Tylorrhynchus chain showed little correspondence to that of Lumbricus chain. The molecular mass of the polypeptide chain was determined to be 16327 including the heme group.

Effect of intracellular organic phosphates on the oxygen equilibrium curve of chicken hemoglobin

Abstract The main factor to decrease the oxygen affinity of chicken hemoglobin may be IHP among the organic phosphates in the red blood cell. Only 1 mole of IHP per mole of hemoglobin was sufficient to bring about the lowest oxygen affinity. By the successive addition of ATP to the stripped hemoglobin in more than 1 m ratio, the gradual decrease of oxygen affinity was still observed. Both IHP and ATP gave a biphasic oxygen equilibrium curve on the addition to the stripped hemoglobin in less than 1 m ratio.

HEAVY-METAL INDUCED CHANGES IN NONPROTEINACEOUS THIOL LEVELS AND HEAVY-METAL BINDING PEPTIDE IN TETRASELMIS TETRATHELE (PRASINOPHYCEAE)

The effects of mercury and cadmium on the intracellular level of nonproteinaceous thiols in a unicellular green alga Tetraselmis tetrathele (West) Butcher (Prasinophyceae) were investigated by using a fluorescent dye, 5‐chloromethylfluorescein (5CMF), as a probe for nonproteinaceous thiols. The 5CMF fluorescence was observed in cytoplasm, and the intensity of the fluorescence was decreased by exposure of the cells to HgCl2. Analysis of the fluorescent intensity of 5CMF by flow cytometry made it possible to distinguish cells in three states during the dying process caused by HgCl2: a normal state, a thiol‐depleted state, and a dead state. Depletion of nonproteinaceous thiols began within 30 min, and they were completely depleted at 2 h. Most cells died after 24 h of exposure to more than 3.0 μM HgCl2, whereas exposure up to 1.0 mM CdCl2 did not cause depletion of nonproteinaceous thiols or cell death within 48 h.  HPLC analyses revealed that glutathione was a major nonprotein thiol in T. tetrathele and that it was oxidized by exposing the cells to HgCl2. Phytochelatins, which play a great role in the tolerance to heavy metals of higher plants and many algae, could not be found in T. tetrathele. However, a tripeptide, Arg‐Arg‐Glu, was found to be abundant, and it showed ability to bind Hg2+, suggesting that it functions to scavenge heavy metals as well as thiol molecules.

Small-angle X-ray scattering studies of giant haemoglobin from the annelid Tylorrhynchus heterochaetus

Abstract The extracellular haemoglobin of the polychaete Tylorrhynchus heterochaetus was studied in solution by small-angle X-ray scattering. The following molecular parameters were determined: radius of gyration 10.8±0.2 nm and a maximum intraparticular distance of 29.5±0.5 nm. Models which fit well the experimental data and reflect also the biochemical structure especially the known number of polypeptide chains are presented.

Wheat germ agglutinin-reactive chains of giant hemoglobin from the polychaete Perinereis aibuhitensis.

Wheat germ agglutinin-reactive chains of multisubunit extracellular hemoglobin from the polychaete Perinereis aibuhitensis were identified to clarify the carbohydrate gluing which is the carbohydrate-dependent supramolecular architecture of the hemoglobin (Ebina S. et al. (1995) Proc. Natl. Acad. Sci. USA 92, 7367-7371). Electron microscope micrographs of Perinereis hemoglobin showed a characteristic shape of two-tiered hexagonal rings whose diameter and height were determined to be 29.4 +/- 1.7 nm and 20.0 +/- 1.8 nm, respectively. Four types of globins and two types of linkers were isolated from the giant hemoglobin by reverse-phase chromatography and SDS-PAGE. These constituents showed similar NH2-terminal sequences as those previously reported for corresponding chains of Tylorrhynchus hemoglobin (Suzuki T. and Gotoh T. (1986) J. Biol. Chem. 261, 9257-9267; Suzuki T. et al. (1990) J. Biol. Chem. 265, 12168-12177). Thus, each globin of Perinereis hemoglobin was identified in terms of amino acid sequence homology and designated using names common to Tylorrhynchus hemoglobin, namely, a, A, b, and B. The linkers were stained by horseradish peroxidase (HRP)-lectins and PAS staining kits, indicating the presence of carbohydrate oligomers. Lectin staining was also significantly positive to globins a and A, which belong to strain A, but negative to globins b and B, which belong to strain B. Results showed that linkers and globins of strain A had a site in a carbohydrate oligomer to which wheat germ agglutinin (WGA) could bind. On the other hand, an alignment between known amino acid sequences of annelid globins and linkers and the sequences of lectins revealed that only the domain of the cysteine-rich motif in linkers has a homology with WGA-type lectins. The results of this study clarify the structuring mechanism of a supramolecule by lectin-like binding, called carbohydrate gluing.

Separation of constituent polypeptide chains containing heme from extracellular hemoglobin of the polychaete Perinereis brevicirris (Grube)_

Abstract 1. 1 Perinereis cyanomethemoglobin was reduced with 1 mM DTT and applied to a column of Sephadex G-100 equilibrated with 5 mM phosphate buffer (pH 7.2) containing 1 mM DTT. The polypeptides eluted in the position corresponding to a mol. wt of about 18,000. Each retained heme without any appreciable loss. 2. 2. These polypeptides were separated into six fractions on a column of DEAE-Sepharose CL-6B equilibrated with 5 mM phosphate buffer (pH 7.2) containing 1 mM DTT. Each polypeptide showed a clear absorbance maximum in the Soret region. 3. 3. The amino acid compositions and NH2-terminal sequences of these polypeptides were determined. 4. 4. The procedure developed for separation of constituent polypeptides with attached heme was applicable to other annelid extracellular hemoglobins; i.e. those of Tylorrhynchus and Neanthes. 5. 5. In contrast to previous suggestions based on heme analysis, these results strongly suggest that each constituent polypeptide chain of polychaete hemoglobins contains heme.

Autoxidation of extracellular oxyhaemoglobin from the polychaete Perinereis brevicirris (Grube)

Abstract 1. 1. SDS-polyacrylamide gel electrophoresis showed that the Perineis haemoglobin consisted of three subunits with mol. wt of 12,000 (subunit 1), 40,000 (subunit 2) and 54,000 (subunit 3) in a molar ratio of 1:4:3. 2. 2. At alkaline pH, the oxyhaemoglobin dissociates into subunits. Therefore, the two haem-containing, subunits 1 and 2, could be separated by gel filtration on a Sephadex G-75 column. 3. 3. The autoxidation rates of oxyhaemoglobin and the oxy-forms of the haem-containing subunits to met-forms were measured in the pH range of 5.4–11.6 and the pH dependencies of their autoxidation rates were compared with that of vertebrate oxymyoglobin. 4. 4. The autoxidation rates of subunits 1 and 2 were approx 500 and 30 times, respectively, that of the intact haemoglobin at pH 7.5. 5. 5. The activation energy of the autoxidation reaction of the whole oxyhaemoglobin was calculated to be 30.5 kcal/mol at pH 7.2.

Nomenclature of the Major Constituent Chains Common to Annelid Extracellular Hemoglobins

We propose a new nomenclature for the similar constituent chains in the Hbs of the polychaete Tylorrhynchus heterochaetus and the oligochaete Lumbricus terrestris, and suggest that, since these two species are far apart in the phylum Annelida, the proposed nomenclature may be extended to other extracellular annelid Hbs, as well (1).