Masazumi Takeshita

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.

Agglutination of leukemic cells and daunomycin entrapped erythrocytes with lectin in vitro and in vivo

Wheat germ agglutinin (WGA) agglutinated the L1210 leukemic cells and daunomycin entrapped erythrocytes in vitro. Comparing with control preparations, the greatest increase in survival was obtained in vivo when the daunomycin entrapped erythrocytes and WGA were given to BDF1 mice bearing L1210 cells.

Mechanism of tumor and liver concentration of 67Ga: 67Ga binding substances in tumor tissues and liver

Tumor-bearing animals were administered with 67Ga citrate and tumor homogenates, from which nuclear fraction was removed, and mitochondrial fraction of the host livers were digested with protease (pronase P). After digestion, the supernatants of the reaction mixtures were applied to a Sephadex G-100 column. Resultant eluates were analyzed for radioactivity, protein, uronic acid and sialic acids. Three peaks of radioactivity were obtained by gel filtration. The first peak eluted in the void volume contained a species whose molecular weight exceeded 40,000. The second peak consisted of substances with molecular weights of 9400-40,000. Radioactivity in the third peak was from liberated gallium-67. 67Ga in the second peak was bound to acid mucopolysaccharide and/or the sulfated carbohydrate chain of sulfated glycoprotein. It was thought that 67Ga in the first peak might be bound to some acid mucopolysaccharides. Considering the results of cellulose acetate electrophoresis, 67Ga in the second peak seemed to be bound to acid mucopolysaccharide which contained no uronic acids, and/or to the sulfated carbohydrate chain of sulfated glycoprotein. It was concluded that 67Ga was bound to the acid mucopolysaccharides and/or the sulfated carbohydrate chain of sulfated glycoprotein in tumor tissues and liver lysosomes.

Affinity of 167Tm-citrate for tumor and liver tissue

AbstractStrong affinity of 167Tm-citrate for tumor tissue was reconfirmed by using Ehrlich tumor. Excellent tumor imaging was obtained with 167Tm-citrate because of its strong tumor affinity and because of the suitable physical charateristics of 167Tm.A large amount of 167Tm had accumulated in the connective tissue which contained inflammatory tissue, quite large amounts were found in areas containing viable and necrotic tumor tissue, and small amounts were present in viable tumor tissue. 167Tm was not seen in necrotic tumor tissue.It was concluded that lysosomes did not play a major role in the tumor concentration of 167Tm, but played an important role in the liver concentration of this nuclide. In the case of hepatoma AH109A, it was presumd that lysosomes played a considerably important role in the tumor concentration of 167Tm, hepatoma AH109A possessing some residual features of the liver.n167Tm was bound to acid mucopolysaccharides and transposed by the acid mucopolysaccharides in the tumor tissues and liver. The acid mucopolysaccharides to which 167Tm were bound in tumor and liver, were heparan sulfate, chondroitin sulfate (or keratosulfate) and heparin (or keratosulfate).

Mechanism of tumor and liver concentration of 111In and 169Yb: 111In and 169Yb binding substances in tumor tissues and liver

Tumor-bearing animals were injected with 111In- and 169Yb-citrate. Tumor homogenates, from which the nuclear fraction was removed, and the mitochondrial fractions of the host livers were digested with pronase P. After digestion, the supernatants of the reaction mixtures were applied to Sephadex G-100 columns. The resultant eluates were analyzed for radioactivity, protein, uronic acid, and sialic acids. Three peaks of radioactivity were obtained by gel filtration. The first peak, eluted in the void volume, contained a species whose molecular weight exceeded 40000. The second peak consisted of substances with molecular weights of 9400–40000. Radioactivity in the third peak was liberated 111In and 169Yb. These two nuclides in the second peak were bound to acid mucopolysaccharide and/or the sulfated carbohydrate chain of sulfated glycoprotein. It was thought that the nuclides in the first peak might be bound to some acid mucopolysaccharides.The second peak nuclides seemed to be bound to acid mucopolysaccharide that contained no uronic acids, and/or to the sulfated carbohydrate chain of sulfated glycoprotein. It was concluded that they were bound to the acid mucopolysaccharides and/or the sulfated carbohydrate chain of sulfated glycoprotein in tumor tissues and liver lysosomes.

Effect of lipid on protoheme ferro-lyase.

1. n1. 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. n n2. n2. 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. n n3. n3. 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. n n4. n4. Detergency and charge of phospholipids were discussed in relation to the mechanism of protoheme ferro-lyase.

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.

Subcellular distribution of gallium-67 in tumor and liver

Abstract Subcellular distribution of 67 Ga was quantitatively determined to evaluate the role of the lysosome in accumulation of 67 Ga in malignant tumor tissue and the liver using three different tumor models and the host liver. In Yoshida sarcoma and Ehrlich tumor, most of the radioactivity of 67 Ga was localized in the supernatant fraction, and only a small amount of radioactivity was localized in the mitochondrial fraction, which contains lysosomes. In the liver, however, most of the radioactivity was concentrated in the mitochondrial fraction. The radioactivity of this fraction increased with time after the administration of 67 Ga and reached approximately 50% of total radioactivity within 24 h. In the case of hepatoma AH 109A, radioactivity of the mitochondrial fraction increased with time after administration, and about 30%, of total radioactivity was concentrated in this fraction after 24 h. It is concluded that lysosome does not play a major role in the tumor concentration of 67 Ga, although it may play an important role in the liver concentration of 67 Ga. In the case of hepatoma AH109A, it is presumed that lysosome plays a considerably important role in the tumor concentration of 67 Ga, hepatoma AH 109A possessing some residual features of the liver.

Subcellular distribution of 111In and 169Yb in tumor and liver

Subcellular distribution of 111In and 169Yb was quantitatively determined to evaluate the role of the lysosome in accumulation of these nuclides in malignant tumor tissue and in the liver using three different tumor models and the host liver. In Yoshida sarcoma and Ehrlich tumor, most of the radioactivity of these nuclides was localized in the supernatant fraction, and only a small amount of radioactivity was localized in the mitochondrial fraction, which contains lysosomes. In the liver, most of the radioactivity was concentrated in the mitochondrial fraction. The radioactivity of this fraction increased with time after the administration of these nuclides and reached approximately 50% of the total radioactivity within 24 h. In the case of hepatoma AH109A, radioactivity of the mitochondrial fraction increased with time after administration, and about 30% of the total radioactivity was concentrated in this fraction after 24 h. It is concluded that the lysosome does not play a major role in the tumor concentration of these nuclides, although it may play an important role in their liver concentration. In the case of hepatoma AH109A, it is presumed that lysosome plays a considerably important role in the tumor concentration of these nuclides, hepatoma AH109A possessing some residual features of the liver.