Yoshimasa Yoneyama

A single nucleotide base transition is the basis of the common human glucose-6-phosphate dehydrogenase variant A (+).

The X-chromosome-linked glucose-6-phosphate dehydrogenase (G6PD) A(+) is a common variant found in about 20% of blacks. The amino acid substitution of Asp in the variant G6PD A(+) for Asn in the normal G6PD B(+) was previously found (A. Yoshida, 1967, Proc. Natl. Acad. Sci. USA 57: 835), but the exact substitution position has not been identified. By screening a DNA library prepared from genomic DNA of a G6PD A(+) male subject, we obtained a genomic clone that contained the mutation site. Characterization of the clone revealed that AT----GC transition occurred in the variant A(+) gene, thus producing the amino acid substitution Asn----Asp at the 142nd position from the NH2 terminus of the enzyme. The nucleotide change created an additional FokI cleavage site in the variant A(+) gene; thus, the FokI fragment type of the variant subjects differed from that of normal B(+) subjects in Southern blot hybridization analysis.

NADPH-flavin reductase in human erythrocytes and the reduction of methemoglobin through flavin by the enzyme

Abstract A NADPH-dehydrogenase of human erythrocytes was exhaustively purified to a homogeneous protein judging from the electrophoresis on a polyacrylamide gel in the presence of sodium dodecyl sulfate. Studies on the specificity for the electron acceptor of this enzyme suggest that flavins serve as the natural and direct electron acceptor. The enzyme showed a broad specificty for flavins and the Michaelis constants for flavins were estimated to be 5 × 10 −5 M for both FMN and riboflavin. Rapid reduction of methemoglobin by the enzyme in the presence of flavin was demonstrated, and the reduction was explained by the reduction of flavin by the enzyme, and subsequent non-enzymatic reduction of methemoglobin by the reduced flavin. The therapeutic significance of flavins was discussed with reference to the flavin reductase activity in hereditary methemoglobinemia.

Mechanism of o-aminophenol metabolism in human erythrocytes

o‐Aminophenol was found to be rapidly metabolized to a brown compound in the presence of purified human oxy‐ and methemoglobin, coupled with the oxidation and reduction of these hemoglobins by o‐amino‐phenol. The final product of o‐aminophenol was identified as 2‐aminophenoxazine‐3‐one, by using spectrophotometry and HPLC. The metabolism of o‐aminophenol was also observed in human erythrocytes. The production rates of 2‐aminophenoxazine‐3‐one in the cells were very fast, but these were strongly decreased by bubbling carbon monoxide into the cell suspension when intracellular hemoglobin was in the ferrous state. The production of 2‐aminophenoxazine‐3‐one from o‐aminophenol in the cells was completely suppressed by cyanide and azide when intracellular hemoglobin was in the ferric state. These results suggest that oxy‐ and methemoglobin are involved in metabolism of o‐aminophenol to 2‐aminophenoxazine‐3‐one in human erythrocytes.

δ-Aminolevulinic acid synthetase of Rhodopseudomonas spheroides: Purification and properties of the enzyme

Abstract δ-Aminolevulinic acid (ALA) synthetase was purified from Rhodopseudomonas spheroides by treatment with calcium phosphate, ammonium sulfate and column chromatography. Protoporphyrin IX and Mg-protoporphyrin inhibited the enzyme activity apparently at a concentration of 10−7 m and 10−6 m , respectively, but protoporphyrin IX to a lesser extent. Fifty percent inhibition by these inhibitors was observed at a concentration of 4.0 × 10−7 m hemin, 2.2 × 10−6 m Mg-protoporphyrin and 3 × 10− m protoporphyrin, respectively. Unnatural hemins (dimethylprotohemin IX, mesohemin IX and deuterohemin IX) also inhibited activity to an extent comparable to that produced by natural hemin. Hemoglobin and myoglobin was also slightly inhibitory, but cyt. c and cyt. a, not. ALA synthetase activity was inhibited by sulfhydryl reagents such as PCMB, HgCl2 and NEM and by some metal ions such as Cu2+, Zn2+, Fe2+ and Fe3+ ions. Almost all of these inhibitions were partially reversed by further incubation of the treated enzyme with 2–5 m m 2-mercaptoethanol at room temperature. Molecular weight of the purified ALA synthetase was estimated to be 61,000 by the gel filtration method.

Purification and molecular properties of two cadmium-binding glycoproteins from the hepatopancreas of a whelk, Buccinum tenuissimum

Two cytoplasmic cadmium-binding glycoproteins from the hepatopancreas of a whelk, Buccinum tenuissimum, were purified to homogeneity by Sephadex G-75 gel filtration and two DEAE-Sephadex A-25 chromatographies in the presence of 2-mercaptoethanol. These two cadmium-binding glycoproteins, termed FIIA and FIIB, each showed a single band after electrophoresis on 10% polyacrylamide gel at pH 8.9. The molecular weight of FIIA was estimated as 8000 and that of FIIB as 13000 by SDS-polyacrylamide gel electrophoresis. Based on molecular weights of 6500 and 11900 for the protein moiety of FIIA and FIIB, respectively, the total number of amino acid residues was 52 in the former and 94 in the latter. Three and two cysteine residues in FIIA and FIIB, respectively, were titratable with p-chloromercuribenzoate. FIIB also contained two more half-cystine residues. The sugar contents of FIIA and FIIB were about 20.5% and 8.7% by weight, respectively, consisting of galactose, mannose, fucose and amino sugar. The purified glycoproteins FIIA and FIIB contained about 0.6% and 1.0% cadmium by weight, respectively, and both showed strong metal-binding capacity, especially for cadmium, copper and mercury. The apparent cadmium dissociation constants for FIIA and FIIB after treatment with 2-mercaptoethanol were 7.3 X 10(-6) and 9.1 X 10(-7) M, respectively. Cadmium contents at saturation were nearly 6 and 8 gatom per mole for FIIA and FIIB, respectively.

Effect of lipid on protoheme ferro-lyase.

1. 1. Protoheme ferroy-lyase (EC 4.99.1.1), extracted from chicken erythrocyte stroma with sodium cholate, showed similar properties to those of duck erythrocyte stroma which were reported previously. 2. 2. The preparation extracted with cholate showed high enzyme activity and contained lipids, while that extracted with 0.4 M KCl contained little lipid and showed little enzyme activity. Crude lipids from acetone-dried powder of chicken erythrocyte stimulated heme synthesis when they were added to the 0.4 M KCl-extracted preparation. Crude lipids from acetone-treated egg yolk cake also stimulated heme synthesis. When the lipids were previously digested with Naja naja phospholipase A (EC 3.1.1.4), the stimulating effect was increased in the presence of cholate and decreased in the absence of cholate. 3. 3. The effects of purified lipids on 0.4 M KCl-extracted preparation were as follows. Acidic phospholipids, phosphatidylethanolamine and lysophospholipids were effective, while choline-containing lipids were ineffective. Palmitate was effective, whereas tripalmitin was ineffective. In the presence of cholate, choline-containing lipids were effective, acidic lipids and phosphatidylethanolamine were slightly effective or neutral, and lyso-lipids were neutral. The effects of crude lipids could be explained by those of pure phospholipids. Sonication of lipid gave stronger stimulation than manual shaking. 4. 4. Detergency and charge of phospholipids were discussed in relation to the mechanism of protoheme ferro-lyase.

Isolation of human erythrocyte membranes in glucose solution

A method is described for the preparation or removal of erythrocyte membranes from hemolysates by a glucose solution. The procedure is simple and rapid, requiring centrifugation at 8000g for 2 min. The preparation has microscopic shape and two-dimensional peptide patterns similar to those of the membrane isolated by conventional procedures (10,000g for 20 min). The present procedure is suitable for dealing with a bulky preparation or for removal of erythrocyte membranes from large volumes of hemolysates to purify enzymes and proteins of soluble or membrane fractions.

Tryptophan fluorescence of human hemoglobin. I. Significant change of fluorescence intensity and lifetimes in the T − R transition

Abstract The fluorescence spectra and fluorescence lifetimes due to tryptophan residues in HbA, Hb Chesapeake, NES-des-Arg Hb and Hb Kempsey were determined at room temperature. The fluorescence intensity and apparent fluorescence lifetimes decrease when the deoxy or T structure in HbA changes to the oxy or R structure, while no significant difference was observed in Hb Kempsey. The difference of fluorescence behavior was ascribed to the quaternary conformational transition of T- and R-states.

Acceleration of methaemoglobin reduction by riboflavin in human erythrocytes.

The effect of riboflavin on nitrite treated erythrocytes from normal subjects and patients with hereditary methaemoglobinaemia due to the deficiency of NADH‐cytochrome b5 reductase was studied in the presence of glucose, 2‐deoxy‐d‐glucose or lactate. When glucose or 2‐deoxy‐d‐glucose was used as a substrate for these erythrocytes, the rate of methaemoglobin reduction in these cells was accelerated more than two‐fold in the presence of riboflavin. The acceleration was dependent on the concentration of riboflavin and was suppressed by the addition of atebrin. The stimulative effect of riboflavin was, however, not observed when lactate was used in place of glucose or 2‐deoxy‐d‐glucose. On the basis of these results, the acceleration of methaemoglobin reduction by riboflavin was considered to be due to the activation of NADPH‐flavin reductase (Yubisui et al, 1977) in erythrocytes by the reagent. The availability of riboflavin for patients with methaemoglobinaemia due to the deficiency of NADH‐cytochrome b5 reductase and for those with toxic methaemoglobinaemia is discussed in relation to methaemoglobin reducing systems in erythrocytes.

Effect of inositol hexaphosphate on hemoglobin oxidation by nitrite and ferricyanide

Abstract In the presence of inositol hexaphosphate (IHP), the rate of hemoglobin oxidation by nitrite was much inhibited; however, that of the hemoglobin oxidation by ferricyanide was much accelerated. The difference in the reaction mode was discussed in relation to the interaction of hemoglobin with IHP. The dissociation constant of IHP to oxyhemoglobin was estimated from the rate of the hemoglobin oxidation by ferricyanide in different concentrations of IHP under oxygen saturated conditions.