Takehiko Ochiai

Zinc as modulator of oxygenation function and stabilizer of quaternary structure in earthworm hemoglobin

Blood of the earthworm Pheretima hilgendorfi contains 10.7 mM Ca, 2.0 mM Mg, 75.5 mM Na, 5.9 mM K, 0.9 mM Zn and 0.3 mM Mn. Some of these cations cannot be removed completely from the blood by dialysis, and 36 atoms of Ca, 1-3 atoms of Mg and 1-2 atoms of Zn per 164 atoms of Fe were retained in purified preparation of the hemoglobin (Hb). At physiological pH, oxygen affinity and the Hill coefficient at half saturation (n1/2 value) of the Hb increased in the presence of 100 mM of Ca, Mg or Na. These effects were in the order of Ca > Mg > Na. At physiological concentration, however, the effect of each of these three cations on oxygenation was rather small. On the other hand, Zn gave a remarkable effect at less than 1 mM. This suggests a possibility that Zn acts as a primal modulator for the oxygenation function of the Hb in vivo. Oxygenation data at various pH values in the presence of each cation strongly suggest that Zn binds to a site different from those for the other three cations. Zn at a concentration of only 1 mM protected considerably the dissociation of the whole molecule to smaller components at alkaline pH and Zn thus contributes to the stabilization of the quaternary structure of the Hb.

Molecular cloning of the cDNA for the major hemoglobin component from paramecium caudatum

Nucleotide sequence of the cDNA for the major hemoglobin component of Paramecium caudatum was determined. An oligonucleotide was synthesized on the basis of the amino acid sequence, and the Paramecium cDNA library constructed in phage lambda gt11 was screened with it. Three positive clones, of which insert sizes were 0.4, 0.6, and 0.9 kbp, were obtained. Sequence analysis made clear that the 0.4-kbp cDNA retains a full length of the nucleotides encoding 116 amino acid residues, and that in the coding region it contains four TAA codons which are known to encode glutamine.

Effects of magnesium and calcium on the oxygenation reaction of erythrocruorin from the marine polychaete Arenicola marina and the terrestrial oligochaete Lumbricus terrestris.

Abstract Oxygenation function of annelid erythrocruorin (Er) is affected by Mg and Ca concentration in the blood. Four classes of responces may be encountered in different species: 1) Mg=Ca (equal effects), 2) Mg>Ca, 3) Mg<Ca and 4) no effect. In the marine polychaete Arenicola marina, at physiological pH and 20°C, Mg and Ca exerted almost equivalent effects in increasing oxygen affinity in the range of 1–200 mM. As measured from the slope of Δlog P1/2 /Δlog [Cation] the effect of Mg was larger than that of Ca at the physiological concentration of respective ion (55 mM Mg; 10 mM Ca). The n1/2 value was similar in the presence of both cations (pH 7.0) or higher for Mg (pH 7.6). In the terrestrial oligochaete Lumbricus terrestris, at the same condition, Ca was more effective than Mg, in raising oxygen affinity at both pHs, also at the physiological concentration (2–4 mM Mg; 8 mM Ca), and n1/2 was similar for Mg and Ca (pH 7.0) or higher for Ca (pH 7.6). The Bohr factor, -[Δlog P1/2 /ΔpH], is maintained its maximum value within the span of the physiological concentration of Mg in Arenicola. In Lumbricus, Ca can contribute to the increase of the Bohr factor at the physiological concentration, but Mg cannot contribute to it. These results reveal that Arenicola and Lumbricus belong to classes 1) and 3), respectively, and that the oxygenation function of both Ers may be controlled by effective utilization of the more dominant of the divalent cations Mg and Ca.

Leech Extracellular Hemoglobin: Two Globin Strains That are Akin to Vertebrate Hemoglobin α and β Chains

Abstract Leech (Whitmania edentula, Haemadipsa zeylanica var. japonica and Erpobdella lineata) extracellular hemoglobins are basically composed of three constituent subunits, a dimer (D1 and D2 chains) and two monomers (M1 and M2 chains). We isolated these four chains from respective species by a combination of reversed-phase chromatography on a Resource RPC column and gel-filtration on a Superdex 75 column. The apparent molecular masses of the four globin chains were estimated by SDS-PAGE analysis to be 13 kDa (M1), 16 kDa (M2; 19 kDa in its reduced form) and about 27 kDa for the dimer subunit (13 kDa for D1;15 kDa for D2), regardless of the source. The amino (N)-terminal segments (21–30 residues) from twelve globin chains of the above three species were determined and aligned. It was found that the twelve sequences could be separated into two distinct globin groups A and B. This finding supports the original idea of “two globin strains in annelid hemoglobin”, which was proposed without any evidence for leech hemoglobins. Comparing the sequences in the three classes of Annelida, Hirudinea, Oligochaeta and Polychaeta, we found two invariant amino acids, Cys and Trp, which are interposed by eleven amino acid residues. Furthermore, the globin chains belonging to strain A were readily discernible as they had three more invariants, Ser-13, Asp-16 and Trp-28, while the globin chains of strain B had two more invariants, Lys-12 and Arg-27. Consequently, we propose that each of the three classes of Annelida have two distinct groups of globin chains that are akin to vertebrate hemoglobin α and β chains.