Hiromichi Okuda

Mechanism of the phosphorylation of thymidine by the culture filtrate of Clostridium perfringens and rat liver extract

Abstract 1. 1. A factor from the culture filtrate of Clostridium perfringens (Factor CP) involved in thymidine phosphorylation utilizing ATP as phosphate donor in the presence of a factor from liver extract (Factor L) was purified approximately 9000-fold. 2. 2. The reaction product of Factor CP and Factor L was identified as thymidine 5′-monophosphate. 3. 3. Factor L was found to be identical to the ATP hydrolyzing enzyme in its behavior on a DEAE-cellulose column, on zone electrophoresis and in its inhibition by NaF. 4. 4. Factor CP catalyzed thymidine phosphorylation in the presence of AMP as phosphate donor. 5. 5. In the thymidine phosphorylation the ATP hydrolyzing enzyme and nucleoside phosphotransferase acted in cooperation. 6. 6. A nucleoside phosphotransferase was purified approximately 50-fold from Cl. perfringens . The purified enzyme gave one peak on acrylamide gel electrophoresis. 7. 7. Its optimum pH was at about 7.0 in Tris-HCl buffer. 8. 8. Its K m values for thymidine and AMP were 1.43 · 10 −4 M and 1.57 · 10 −4 M, respectively. 9. 9. Various nucleoside monophosphates and nucleoside diphosphates acted as phosphate donor. 10. 10. Deoxycytidine, deoxyuridine, thymidine and cytidine were better acceptors than uridine.

Alternative methods of thymidine phosphorylation in different organisms

Abstract Thymidine was found to be phosphorylated to thymidine 5′-monophosphate using ATP as phosphate donor in germinating potatoes and Tetrahymena pyriformis as well as in regenerating rat liver and tumor tissues. The properties of the phosphorylating enzymes from these sources were studied. The results obtained suggest that germinating potatoes and T. pyriformis may not contain thymidine kinase (ATP : thymidine 5′-monophosphotransferase, EC 2.7.1.21). In germinating potatoes and T. pyriformis , thymidine was phosphorylated by the combined actions of nucleoside phosphotransferase and the ATP hydrolyzing enzyme. The nucleoside phosphotransferase and ATP hydrolyzing enzyme from germinating potatoes were separated by DEAE-Sephadex A-50 column chromatography, but these enzymes from T. pyriformis could not be separated under the same conditions. When an extract of T. pyriformis was treated with trypsin, both the thymidine phosphorylating activities using ATP as phosphate donor and ATP hydrolyzing activities disappeared, while the nucleoside phosphotransferase activity remained. These results suggest that thymidine can be phosphorylated in two ways: one is by the action of thymidine kinase (in mammalian tissues and Escherichia coli ) and the other is by the combined actions of nucleoside phosphotransferase and the ATP hydrolyzing enzyme (in potatoes and T. pyriformis ).

Studies on a reciprocal relationship between nucleoside kinases and 5′-nucleotidase

Abstract The activities of thymidine kinase (EC 2.7.1.21), deoxycytidine kinase, uridine kinase (EC 2.7.1.48) and 5′-nucleotidase (EC 3.1.3.5) for dTMP, dCMP and UMP were investigated during liver regeneration. A reciprocal relationship was found between the activity of thymidine kinase and 5′-nucleotidase activity for dTMP during liver regeneration. This relationship was also found in various normal and tumor tissues. Similar relationships were also found between uridine kinase and 5′-nucleotidase for UMP and between deoxycytidine kinase and 5′-nucleotidase for dCMP during liver regeneration. Treatment of animals with actinomycin D inhibited the elevation of thymidine kinase activity during liver regeneration, but had no effect on that of uridine kinase. This treatment also inhibited the decreases of 5′-nucleotidase activity for dTMP and UMP during liver regeneration. The thymidine kinase and uridine kinase activities of regenerating liver were inhibited by addition of the microsomal fraction from normal liver. The microsomal fraction from regenerating liver was less inhibitory. The microsomal fraction contained various phosphatases, such as 5′-nucleotidase and ATPase, and it seems probable that the inhibotory effect of this fraction on the kinases may result from the actions of these phosphatases. It was found that the activities of the phosphatases in the microsomal fraction of regenerating liver were much lower than those of normal liver.

Activation of thymidine kinase of adult rat liver by the culture filtrate of Clostridium perfringens.

Abstract 1. When adult rat-liver extract was treated with the culture filtrate of Clostridium perfringens, marked stimulation of the thymidine kinase activity was observed. The extract did not stimulate thymidine kinase of either regenerating liver or Yoshida sarcoma. 2. The activity of the culture filtrate depended on the age of the culture, being maximal after 82 h growth. 3. The activator in the culture filtrate was heat-labile and nondialyzable and was inactivated by pronase. 4. This activator was purified approx. 500-fold by a procedure involving (NH4)2SO4 fractionation, acetone treatment and refractionation with (NH4)2SO4.

Studies on aminopeptidases in rat liver and plasma

Abstract It was found that aminopeptidases (EC 3.4.1.2) were easily solubilized from rat liver by bromelain (EC 3.4.4.24) treatment. The solubilized form of the enzymes was subject to TEAE-cellulose column chromatography resulting in separation of five types of aminopeptidases. Rat plasma also contained some aminopeptidases, which were separated by gel filtration on a Sephadex G-200 column or TEAE-cellulose column chromatography. These enzymes showed different substrate specificities toward l -leucine amide and l -leucyl-β-naphthylamide. It was found that these enzymes increased in the plasma after CCl 4 injection or ligation of the common bile duct.

Preparation of adrenaline-sensitive lipid micelles from rat epididymal adipose tissue

Abstract Lipid micelles were prepared from rat epididymal adipose tissue. The micelles were mainly composed of triglyceride with small amounts of protein, sugar, phospholipid, choresterol and choresterol ester. Adrenaline stimulated lipolysis in intact micelles, but not when they were homogenized.

Studies of the clinical application of serum leucine aminopeptidase (LAP) activity determined with leucinamide as substrate

SummaryA new method was presented for the determination of leucine aminopeptidase (LAP). The principle of the method consisted in the measurement of ammonia liberated by the action of LAP with direct colorimetry. Leucine naphthylamide has been widely used as substrate for the determination of LAP (Nap-method), but leucineamide was used (NH3-method) in this study, and the enzyme activities determined by the both methods were compared in various diseases.Serum LAP activity in acute hepatitis was much higher in NH3-method than in Nap-method, but the activity in obstructive jaundice was much higher in the latter than in the former.It was demonstrated that the serum of normal rats and CC14 treated rats contained isozymes (LAP-I and II) which showed the different substrate specificities toward leucinamide and leucine naphthylamide. LAP-I activity was much higher, but LAP-II activity was much lower in the NH3-method than in the Napmethod. LAP-I activity was remarkably elevated by the NH3-method, but not by the Nap-method in the serum of CC14 treated rats.The results suggested that leucinamide was preferable substrate for the measurements of activities of serum LAP in the clinical examinations.

Purification of the factor involved in thymidine phosphorylation in adult rat liver

Abstract Adult rat liver contains a factor involved in thymidine phosphorylation which is readily activated by the culture filtrate of Clostridium perfringens. The enzyme was purified approx. 110-fold by a procedure involving pH treatment, ammonium sulfate fractionation and DEAE-cellulose column chromatography.