Ryoiti Shukuya

Quaternary structure of Limnodrilus haemoglobin.

Structure of the haemoglobin from Limnodrilus gotoi (molecular weight, 3.01 x 10(6)) was studied electron microscopically and chemically. The molecule appears to be a regular hexagonal cylinder having the dimensions 220 A x 160 A, and to consist of 12 identical submultiples arranged six and six into two hexagonal discs laid one above the other. The protein contains 108 haems per molecule and 12 seryl, 12 threonyl, 35 glutamyl and 45 valyl residues to give a total number of 104 residues per molecule as N-terminal amino acids. These results lead us to a conclusion that the minimum molecular weight either per haem or per N-terminal residue is approximately 28,000 and the whole molecule consists of about 108 subunits or peptide chains, each containing single haem. Further it is presumed that submultiple is a nonamer, composed of nine subunits.

A simple non-enzymatic method to regenerate oxyhemoglobin from methemoglobin

Abstract Methemoglobin can be reduced to hemoglobin by NADH in the presence of catalytic amount of phenazine-methosulfate. A simple non-enzymatic method to obtain oxyhemoglobin from methohemoglobin was established using NADH-PMS system. Regenerated hemoglobin obtained by this method revealed normal oxygen equilibria in the presence of 2,3-diphosphoglycerate.

Subunits of limnodrilus erythrocruorin

Abstract The dissociation of the erythrocruorin of the oligochaete Limnodrilus gotoi was investigated using polyacrylamide gel electrophoresis at neutral pH. In the presence of 0.1% SDS, the erythrocruorin dissociated into five subunits possessing molecular weights of 13,000 (1), 20,000 (2), 23,000 (3), 25,000 (4) and 47,000 (5). In the presence of SDS and mercaptoethanol, the erythrocruorin dissociated into two subunits, whose molecular weights were 13,000 (I) and 28,000 (II). Subunit I accounts for 70–80% of the whole molecule. SDS electrophoresis of the isolated subunits 1 through 5 in the presence of mercaptoethanol showed that subunit I was derived from both subunits 1 and 5, while subunit II was derived from subunits 2–4. These results suggest that Limnodrilus erythrocruorin consists of at least five polypeptide chains: two chains of 13,000 and three chains of 28,000.

Purification and properties of serum albumin from Rana catesbeiana

Abstract Serum albumin was purified from the plasma of the bullfrog, Rana catesbeiana , by a combination of Sephadex G-200 and DEAE-cellulose column chromatography, and isoelectric focusing fractionation. The purified albumin was judged to be homogeneous by several criteria: acrylamide disc and cellulose acetate electrophoresis, immunological methods and ultracentrifugation. Serum albumin is a single polypeptide chain of molecular weight 67 000 and s 20, w 4.6, and has an isoelectric point of 5.6 at 24°C. When purified in the absence of mercaptoethanol, the albumin polymerized and the addition of the reagent caused dissociation to the monomer. The band of the albumin-bromophenol blue complex moved faster than the free albumin in acrylamide disc electrophoresis. The above-mentioned characteristics of serum albumin from R. catesbeiana are homologous to those of the other species.

The effect of organic phosphates on the allosteric property of Rana catesbeiana hemoglobins

Abstract Difference in the oxygen affinity between tadpole and frog hemoglobins of Rana catesbeiana was investigated in relation to the interaction of organic phosphates with the hemoglobin. The oxygen affinity of both hemoglobins increased remarkably by the stripping procedure. The addition of DPG, ATP and IHP which were proved to be present in the erythrocytes, to the stripped hemoglobins resulted in notable decrease of the oxygen affinity, except that IHP showed little effect on the frog hemoglobin. Affinity constants of organic phosphates for the hemoglobins were obtained from the oxygen equilibrium data. IHP was found to have the highest affinity for tadpole hemoglobin, while DPG was the highest for frog hemoglobin.

The effects of organic phosphates on the oxygen equilibria of two distinct hemoglobins of the eel, Anguilla japonica.

Summary The effects of organic phosphates, such as adenosine triphosphate, 2,3-diphosphoglycerate and inositol hexaphosphate (phytic acid), on the oxygen equilibria of two major hemoglobins isolated from eel erythrocytes and freed of phosphate were investigated. One of these hemoglobins (E 1 ) was found to be greatly affected on its oxygen affinity and pH dependency by added organic phosphates, while the other (E 2 ) was almost insensitive to these phosphates. Results were described under considerations of the possible role of organic phosphates in regulating the function of eel hemoglobins in situ .

Isolation and properties of γ chain from human fetal hemoglobin

Abstract 1. 1. Dissociation of human fetal hemoglobin (FII fraction) into its constituent subunits was investigated electrophoretically. 2. 2. A simple method of isolating the γ chain from fetal hemoglobin is described. The procedure is a slight modification of the method of Bucci and Fronticelli 1 for adult hemoglobin, consisting of p-chloromercuribenzoate treatment at pH 4.7 and fractionation by CM-cellulose column chromatography. 3. 3. One of the three fractions obtained by this method was identified as the γ chain by means of electrophoretic behavior, ultraviolet absorption spectra, alkali resistance and amino-terminal amino acid analysis. The yield of γ chain was 40–60%.

Cytochrome c oxidase from the liver of bullfrog, Rana catesbeina and change in its turnover rate during metamorphosis

1. Cytochrome c oxidase was purified from the liver mitochondria of bullfrog (Rana catesbeiana). The heme a content of the purified enzyme was 13.5 nmol per mg protein. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the enzyme protein was composed of nine polypeptide subunits having molecular weights of 42000, 27000, 25000, 20000, 15000, 13000, 8600, 5400 and 3600. The purified enzyme from the adult frog was immunological identified with that from the tadpole. 2. The ratio of synthesis and degradation of cytochrome c oxidase were 5.2- and 2.0-times higher at metamorphic climax than at premetamorphic stage, respectively. The amount of the enzyme in the liver was highest at metamorphic climax.

The fastest migrating component of lamprey serum in zone electrophoresis.

Abstract 1. 1. The fastest migrating fraction to anode of serum from lamprey, Entosphenus japonicus, was composed mainly of a single glycoprotein “the fastest migrating component”. 2. 2. The fastest migrating component was purified and the physicochemical properties were studied. 3. 3. From results of cellulose acetate and acrylamide disc electrophoreses, ultracentrifugation and immunological experiments, the purified sample was homogeneous. 4. 4. The molecular weight was estimated to be approx 160,000 by disc gel electrophoresis in the presence of sodium dodecyl sulfate. An approximate molecular weight of 300,000 or more was obtained by Sephadex G-200 gel filtration, suggesting that the fastest migrating component was composed of two identical subunits. 5. 5. The sedimentation coefficient (S2 0, w0) was 12.2 S and the isoelectric point was 4.5. Results of amino acid and carbohydrate analyses are presented.

Seasonal variation in hepatic phosphoenolpyruvate carboxykinase activity of bullfrog, Rana catesbeiana.

Abstract 1. 1. Seasonal variations in phosphoenolpyruvate carboxykinase activity in the bullfrog, Rana catesbeiana , were detected. 2. 2. The cytosolic activity of adult frog river is about five to six times higher in the summer than in the winter. On the other hand, the activity located in the mitochondrial fraction remains nearly constant throughout the year.