Keizo Tsushima

5'-nucleotidase of chicken liver.

1. 1.|5′-Nucleotidase (5′-ribonucleotide phosphophydrolase, EC 3.1.3.5) was partially purified from chicken liver. This is the first time it has been possible to obtain 5′-nucleotidase from the hepatic tissue of uricotelic animals and it was found to be kinetically distinct from 5′-nucleotidases obtained from other sources. 2. 2.|5′-Mononucleotides having a keto group at position 6 in the purine base are the most active substrates of this enzyme. 5′-IMP is the most active substrate among the 5′-nucleotides tested, and it is about 10 times more active than 5′-AMP. 3. 3.|This enzyme has an optimum pH at 6.5 and requires divalent metal ions. In the absence of divalent metal ions, the enzyme is almost inactive. 4. 4.|Inosino, guanosine p-chloromercuribenzoate (PCMB) and NaF inhibit this enzyme. Of these inhibitors, PCMB was found to be the most potent. 5. 5.|The general properties of the enzyme are described, and its possible metabolic function is discussed.

Kinetic properties of cytosol 5'-nucleotidase from chicken liver.

Abstract A highly purified preparation of cytosol 5′-nucleotidase (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) from chicken liver was effectively activated by ATP and inhibited by P i . When AMP was used as substrate, the enzyme displayed sigmoidal kinetics. When either IMP, GMP, UMP or CMP was used as substrate, the substrate saturation curve was slightly sigmoidal or hyperbolic. In the presence of 10 mM ATP, the substrate saturation curves for all substrates tested were hyperbolic, whereas P i increased the sigmoidicity of the saturation curves. The enzyme had much higher affinities for IMP and GMP than for AMP, UMP and CMP. ATP decreased the s 0.5 , whereas P i increased the s 0.5 for all the substrates tested. Inhibition by P i was removed by ATP, and activation by ATP was removed by P i . V of the enzyme was determined to be in the same order of the magnitude for all the substrates tested. The hydrolysis of GMP was inhibited competitively by the other 5′-nucleotides. AMP at low concentration had a stimulatory effect on the hydrolysis of GMP. The enzyme activity was absolutely dependent on the presence of bivalent cations. Increasing concentration of MgCl 2 increased the V and affinity of the enzyme for AMP and vice versa. ATP protected the enzyme against the inactivation by heat or trypsin digestion. It seems likely that this enzyme is an allosteric protein regulated by various ligands through conformational changes.

AMP deaminase from baker's yeast. Purification and some regulatory properties

AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) was found in extract of bakers yeast (Saccharomyces cerevisiae), and was purified to electrophoretic homogeneity using phosphocellulose adsorption chromatography and affinity elution by ATP. The enzyme shows cooperative binding of AMP (Hill coefficient, nH, 1.7) with an s0.5 value of 2.6 mM in the absence or presence of alkali metals. ATP acts as a positive effector, lowering nH to 1.0 and s0.5 to 0.02 mM. P1 inhibits the enzyme in an allosteric manner: s0.5 and nH values increase with increase in Pi concentration. In the physiological range of adenylate energy charge in yeast cells (0.5 to 0.9), the AMP deaminase activity increases sharply with decreasing energy charge, and the decrease in the size of adenylate pool causes a marked decrease in the rate of the deaminase reaction. AMP deaminase may act as a part of the system that protects against wide excursions of energy charge and adenylate pool size in yeast cells. These suggestions, based on the properties of the enzyme observed in vitro, are consistent with the results of experiments on bakers yeast in vivo reported by other workers.

Changes in 5′-nucleotidase activity in chick liver during development and dietary treatment

Abstract 1. 1. To elucidate the physiological roles of hepatic 5′-nucleotidase (EC 3. I .3.5), changes in IMP-hydrolysing activity in chick liver extracts during dietary adaptation were investigated. 2. 2. IMP-hydrolysing activity in chick liver extracts increased 2-fold within one day when the casein content of the diet was increased from 5 to 75%. 3. 3. Increased IMP-hydrolysing activity was Mg 2+ -dependent and has a pH optimum at about 6.5. 4. 4. A correlation between IMP-hydrolysing activity in chick liver extracts and serum uric acid concentration was observed. 5. 5. In rat liver extracts, no dependency of IMP-hydrolysing activity upon the casein content of the diet was observed. 6. 6. Within two days after hatching, a 2-fold increase in IMP-hydrolysing acitivity in chick liver extracts was observed compared with the activity immediately before hatching. 7. 7. These results suggest that the 5′-nucleotidase, which has been described by us previously 1 , would play a role in the excretory metabolism of α-amino nitrogen of amino acid in a uricotelic animal.

5′-Nucleotidase of chicken liver: A comparison of soluble 5′-nucleotidase activities in chicken and rat liver

Abstract 1. 1. Most of the IMP-hydrolyzing activity in the homogenate of chicken liver was recovered in the cell sap. This activity, which has a pH optimum of around 6-5 and is Mg 2+ dependent, is due to the 5′-nucleotidase (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) previously described (Itoh, Mitsui & Tsushima, 1967). 2. 2. The activity of this enzyme in chicken liver was much higher than in rat liver.

Purine nucleoside phosphorylase of chicken liver

Abstract Purine nucleoside phosphorylase (purine nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) has been purified 125-fold from the homogenate of chicken livers and some of the properties of the purified enzyme have been studied. This enzyme had a pH optimum at around 6.o. At high substrate levels of inosine the reaction rate was increased, suggesting that substrate activation of the enzyme had occurred. The enzyme activity was completely lost after 48 h at −20°. The inhibition by nucleotides and SO 4 2− is a characteristic of this enzyme and has not been previously reported.

The role of polyamines in the regulation of AMP deaminase isozymes

The differential effects of polyamines on the activity of AMP deaminase isozyme A (from rat muscle) and isozyme B (from rat liver) are reported. Polyamines activate isozyme B but inhibit isozyme A.

Molecular properties and a nonidentical trimeric structure of purine nucleoside phosphorylase from chicken liver

Some molecular properties of crystalline purine nucleoside phosphorylase (purine nucleoside: orthophosphate ribosyltransferase, EC 2.4.2.1) from chicken liver were investigated and discussed. The molecular weight of the native enzyme was determined to be 89 000 by gel filtration and sedimentation coefficient, and 90 000 by sedimentation equilibrium, respectively. The enzyme was assumed to be a trimer consisting of one large subunit and two identical small subunits. The molecular weights of two different sized subunits were determined to be 32 000 and 28 000 by sodium dodecyl sulfate gel electrophoresis, and 30 000 and 27 000 by 6 M guanidine hydrochloride gel filtration. The amino acid composition was determined and the partial specific volume was estimated to be 0.735 ml/g.

Polyamines as activators of AMP nucleosidase fromAzotobacter vinelandii

Polyamines at physiological concentrations activate AMP nucleosidase fromAzotobacter vinelandii. Biological significance of the activation is discussed in relation to the control of adenylate energy charge and the purine nucleotide synthesis in prokaryotes.

Studies on chicken liver xanthine dehydrogenase with reference to the problem of non-equivalence of FAD moieties

1. Reduction of chicken liver xanthine dehydrogenase (xanthine: NAD+ oxidoreductase, EC 1.2.1.37) by xanthine under anaerobic condition proceeded in two phases. This biphasicity may be due to functional and non-functional enzymes in the enzyme preparation. 2. Cyanolysis of a persulfide group of chicken liver enzyme resulted in an inactivation of the enzyme. The non-functional enzyme in the standard enzyme preparation was found to lack persulfide groups at the active sites. 3. The remaining NADH-Methylene Blue oxidoreductase activity, after KI treatment of the xanthine-reduced enzyme of a high flavin activity ratio, is not at the level of 50% of the initial activity, differing from the report suggesting non-equivalence of FAD chromophores. 4. The findings in the present report indicate that FAD chromophores of chicken liver enzyme are essentially equivalent.