A kinetic study of the soluble 5''-nucleotidase of rat liver.

1. The kinetic properties of the 5'-nucleotidase (EC 3.1.3.5) present in the cytosol of rat liver were investigated in relation to the conversion of adenine nucleotides into uric acid, with particular reference to the stimulation of this process by fructose. The enzyme was assayed by the release of Pi and by a new and more sensitive radiochemical procedure. 2. When IMP was used as substrate, the partially purified enzyme displayed almost hyperbolic kinetics (h = 1.1) with S0.5 = 1.2 mM. Similar kinetics were observed with GMP and other nucleoside 5'-monophosphates, except AMP. 3. Vmax. of the enzyme for AMP was about the same as for IMP, but the kinetics were sigmoidal (h = 1.6) with S 0.5 = 10 mM. 4. The hydrolysis of IMP was inhibited competitively by GMP. IMP, at concentrations up to 0.5 mM, had a paradoxical stimulatory action on the hydrolysis of 2-5 mM-AMP and was inhibitory at higher concentrations. 5. The activity of the enzyme towards AMP and IMP was stimulated by ATP and GTP, and inhibited by Pi. Activators and inhibitor approximately cancelled each others' effects. At pH 7.4, the enzymic activity with 0.2 mM-AMP was undetectable under physiological conditions. 6. It is concluded that, in the liver cell, AMP is not hydrolysed by the soluble 5'-nucleotidase, but that its degradation requires prior deamination to IMP.     

Tyrosine aminotransferase of chicken liver: purification and properties

Tyrosine aminotransferase has been purified from chicken liver to homogeneity by a 5-step procedure. The resultant enzyme preparation has a specific activity (256 units activity/mg protein) comparable to results published for the enzyme purified from rat liver and represented an overall recovery of 35-40%. In terms of structure (native and subunit molecular weights, immunological reactivity, and kinetic parameters) (apparent Michaelis constants for L-tyrosine and 2-oxoglutarate, oxoacid specificity, pH optimum) the purified enzyme from chicken liver exhibits remarkable similarities to tyrosine amino-transferase from rat liver.     

Rabbit liver dCMP phosphohydrolase: a pyrimidine 5'-nucleotidase I-like enzyme in non-erythrocytic cells

A nucleotide phosphomonoesterase activity that preferably hydrolyzed dCMP was detected in rabbit liver and purified approximately 20-fold. The enzyme was similar in the catalytic and molecular properties to pyrimidine 5'-nucleotidase subclass I (P5N-I), which distributed specifically in vertebrate erythrocytes. In addition to liver, the activity was found in rabbit kidney, spleen, heart, intestine, but was not detected in any rat or chicken tissues tested. The rabbit enzyme protein reacted with antibodies against chicken P5N-I. Its pI was estimated to be approximately 5.3, and the enzyme was concluded to consist of single polypeptide of an approximately 38 kDa based on gel filtration and Western blot analysis. The partially purified enzyme preferentially hydrolyzes dCMP, UMP and CMP, K(m) values for these substrates are approximately 0.3 mM, the optimal pH is approximately 7, and the enzyme requires Mg(2+). This nucleotidase may contribute to the regulation of intracellular pyrimidine nucleotides in the rabbit.     

Purification and properties of phosphofructokinase 2/fructose 2,6-bisphosphatase from chicken liver and from pigeon muscle

Phosphofructokinase 2 and fructose 2,6-bisphosphatase extracted from either chicken liver or pigeon muscle co-purified up to homogeneity. The two homogeneous proteins were found to be dimers of relative molecular mass (Mr) close to 110,000 with subunits of Mr 54,000 for the chicken liver enzyme and 53,000 for the pigeon muscle enzyme. The latter also contained a minor constituent of Mr 54,000. Incubation of the chicken liver enzyme with the catalytic subunit of cyclic-AMP-dependent protein kinase in the presence of [gamma-32P]ATP resulted in the incorporation of about 0.8 mol phosphate/mol enzyme. Under similar conditions, the pigeon muscle enzyme was phosphorylated to an extent of only 0.05 mol phosphate/mol enzyme and all the incorporated phosphate was found in the minor 54,000-Mr constituent. The maximal activity of the native avian liver phosphofructokinase 2 was little affected by changes of pH between 6 and 10. Its phosphorylation by cyclic-AMP-dependent protein kinase resulted in a more than 90% inactivation at pH values below 7.5 and in no or little change in activity at pH 10. Intermediary values of inactivation were observed at pH values between 8 and 10. Muscle phosphofructokinase 2 had little activity at pH below 7 and was maximally active at pH 10. Its partial phosphorylation resulted in a further 25% decrease of its already low activity measured at pH 7.1 and in a negligible inactivation at pH 8.5. Phosphoenolpyruvate and citrate inhibited phosphofructokinase 2 from both origins non-competitively. The muscle enzyme and the phosphorylated liver enzyme displayed much more affinity for these inhibitors than the native liver enzyme. Fructose 2,6-bisphosphatase from both sources had about the same specific activity but only the chicken liver enzyme was activated about twofold upon incubation with ATP and cyclic-AMP-dependent protein kinase. All enzyme forms were inhibited by fructose 6-phosphate and this inhibition was released by inorganic phosphate and by glycerol 3-phosphate. Both liver and muscle fructose 2,6-bisphosphatases formed a 32P-labeled enzyme intermediate when incubated in the presence of fructose 2,6-[2-32P]bisphosphate.     

Inhibitory effect of pH5 enzyme from non-lactating bovine mammary gland on various stages of protein synthesis in the rat liver amino acid-incorporating system

1. pH5 enzyme from non-lactating bovine mammary gland was found to contain potent inhibitors of protein synthesis in the rat liver cell-free system. These inhibitors affect (a) formation of aminoacyl-tRNA where tRNA represents transfer RNA, (b) transfer of labelled amino acids from rat liver amino[(14)C]acyl-tRNA to protein in rat liver polyribosomes, and (c) incorporation of (14)C-labelled amino acids into peptide by rat liver polyribosomes supplemented with rat liver pH5 enzyme. 2. Increasing amounts of pH5 enzyme from bovine mammary gland progressively inhibited the incorporation of labelled amino acids into protein by a complete incorporating system from rat liver. Approx. 80% inhibition was observed at a concentration of 2mg. of protein of pH5 enzyme from bovine mammary gland. The inhibitory effect of the bovine pH5 enzyme fraction could not be overcome by the addition of increasing amounts of rat liver pH5 enzyme. 3. Fractionation of bovine pH5 enzyme with ammonium sulphate into four fractions showed that all the fractions inhibited the incorporation of (14)C-labelled amino acids in the rat liver system, but to varying extents. The highest inhibition observed (90%) was exhibited by the 60%-saturated-ammonium sulphate fraction. 4. Heat treatment of bovine pH5 enzyme at various temperatures caused only a partial loss of its inhibitory effect on labelled amino acid incorporation by the rat liver system. Treatment at 105 degrees for 5min. resulted in the bovine pH5 enzyme fraction losing 30% of its inhibitory activity. 5. pH5 enzyme from bovine mammary gland strongly inhibited the charging of rat liver tRNA in the presence of its own pH5 enzymes. 6. The transfer of labelled amino acids from rat liver amino[(14)C]acyl-tRNA to protein in a system containing rat liver polyribosomes and pH5 enzyme was almost completely inhibited by bovine pH5 enzyme at a concentration of 2mg. of protein of the enzyme fraction. 7. One of the inhibitors of various stages of protein synthesis in rat liver present in bovine pH5 enzyme was identified as an active ribonuclease, and the second inhibitor present was shown to be tRNA.     

Partial purification, properties and regulation of inosine 5''phosphate dehydrogenase in normal and malignant rat tissues.

IMP dehydrogenase (EC 1.2.1.14) was purified 180-fold from rat liver and from the transplantable rat hepatoma 3924A. The enzymes from the two sources were apparently identical; they exhibited hyperbolic saturation kinetics and an ordered, sequential mechanism, and were subject to inhibition by a number of purine nucleotides. Km values for the substrates, IMP and NAD+, were 12 and 24 micrometer respectively. IMP dehydrogenase activity in a spectrum of rat hepatomas was increased, relative to normal liver, by 2.5--13-fold; these increases correlated with tumour growth rate. Activity in two rat kidney tumours was increased 3-fold relative to that in normal renal cortex; control of activity of this enzyme is apparently altered in neoplastic cells. After partial hepatectomy, IMP dehydrogenase activity began to rise 6 h after operation, reaching a peak of 580% of normal activity by 18 h. Activity in neonatal liver, however, was only slightly higher than that in the adult. Organ-distribution studies showed highest enzyme activities in spleen and thymus. In livers of rats starved for 3 days, where all enzymes, except those involved in gluconeogenesis, showed decreased activity IMP dehydrogenase activity was increased; this change was accompanied by a rise in hepatic GTP concentrations. It is concluded that IMP dehydrogenase is a key enzyme in the regulation of GTP production, and thus involved in regulation of nucleic acid biosynthesis. The increased activity of IMP dehydrogenase in liver of starved rats may be related to the requirements for GTP for gluconeogenesis.     

Conversion of 5-aminolaevulinate into haem by homogenates of human liver. Comparison with rat and chick-embryo liver homogenates.

To assess whether the synthesis of haem can be studied in small amounts of human liver, we measured kinetics of the conversion of 5-aminolaevulinate into haem and haem precursors in homogenates of human livers. We used methods previously developed in our laboratory for studies of rat and chick-embryo livers [Healey, Bonkowsky, Sinclair & Sinclair (1981) Biochem. J. 198, 595-604]. The maximal rate at which homogenates of human livers converted 5-aminolaevulinate into protoporphyrin was only 26% of that for rat, and 58% of that for chick embryo. In the absence of added Fe2+, homogenates of fresh human liver resembled those of chick embryos in that protoporphyrin and haem accumulated in similar amounts, whereas fresh rat liver homogenate accumulated about twice as much haem as protoporphyrin. However, when Fe2+ (0.25 mM) was added to human liver homogenates, mainly haem accumulated, indicating that the supply of reduced iron limited the activity of haem synthase, the final enzyme in the haem-biosynthesis pathway. Addition of the potent iron chelator desferrioxamine after 30 min of incubation with 5-amino[14C]laevulinate stopped further haem synthesis without affecting synthesis of protoporphyrin. Thus the prelabelled haem was stable after addition of desferrioxamine. Since the conversion of 5-amino[14C]laevulinate into haem and protoporphyrin was carried out at pH 7.4, whereas the pH optimum for rat or bovine hepatic 5-aminolaevulinate dehydratase is about 6.3, we determined kinetic parameters of the human hepatic dehydrase at both pH values. The Vmax was the same at both pH values, whereas the Km was slightly higher at the lower pH. Our results indicate that the synthesis of porphyrins and haem from 5-aminolaevulinate can be studied with the small amounts of human liver obtainable by percutaneous needle biopsy. We discuss the implications of our results in relation to use of rat or chick-embryo livers as experimental models for the biochemical features of human acute porphyria.     

Diadenosine tetraphosphate activates cytosol 5'-nucleotidase

The rate of hydrolysis of IMP (0.5 mM) by cytosol 5'-nucleotidase from Artemia embryos was increased up to 7-fold by concentrations of around 10 microM diadenosine tetraphosphate (Ap4A). Half maximal activation of the enzyme was accomplished with 5 microM Ap4A. The Km (S 0.5) values of the nucleotidase for IMP, GMP, AMP, XMP and CMP decreased about 10 fold in the presence of 10 microM Ap4A. Maximum velocity of the enzyme was not affected by Ap4A. ATP had been previously described as an activator of the enzyme. However, comparatively with Ap4A, concentrations of ATP two orders of magnitude higher are needed to elicit similar effects on the enzyme. Preliminary results indicate that Ap4A is also an activator of the cytosol 5'-nucleotidase from rat liver.     

An improved procedure for purifying 5'-nucleotidase from various sources. Evidence for tissue and species differences in their molecular mass and affinity for F-actin

5'-Nucleotidase from chicken gizzard smooth muscle has been extracted, using a sulfobetaine derivate of cholic acid, and purified to homogeneity by employing three chromatographic steps. It is shown that the purification scheme can be applied to 5'-nucleotidase from other sources, such as rat liver. On sodium dodecyl sulfate polyacrylamide gels, stained with silver nitrate, the purified enzyme from chicken gizzard shows a single polypeptide band with an apparent molecular mass of 79 kDa. The enzyme purified from rat liver exhibits a molecular mass of 73 kDa in agreement with published data [Bailyes, E.M., Soos, M., Jackson, P., Newby, A. C., Siddle, K. & Luzio, J.P. (1984) Biochem. J. 221, 369-377). Gel filtration, using non-denaturating detergent solutions, indicates that the native enzyme may exist as a homodimer (152 kDa) or homotetramer (310 kDa). Antibodies raised against the enzyme purified from chicken gizzard bind only 5'-nucleotidase, solubilized from chicken muscular sources, when immobilized, but not from chicken or rat liver. The existence of tissue specific variants of 5'-nucleotidase is therefore postulated and it appears that these particular isoforms can also be classified in membranous and secretory forms of 5'-nucleotidase. They also differ in their mode of interaction with actin. The AMPase activity of the membranous (= muscular) isoform is inhibited to a considerably higher percentage by F-actin than the enzyme isolated from rat liver.     

Ultrastructure of reconstituted rat liver microsomal membranes and cytochrome b5- or P-450-containing proteoliposomes

Thin sectioning and freeze-fracture electron microscopy have been used to show that it is possible to obtain topologically closed vesicles by means of reconstitution of rat liver microsomal membrane "ghosts." The reconstitution by 15 hr dialysis resulted in the formation of vesicles with intramembrane particles (IMP) while after 40 hr dialysis no IMP were observed in the membranes. The protein/lipid ratio and functional activity of NADPH- and NADH-linked enzyme systems were similar in both cases. Cytochrome P-450 (LM2) was incorporated into liposomes of different composition (protein: lipid ratio--1:200). IMP were observed only when the incorporation of cytochrome P-450 was performed in the presence of detergent Emulgen 913 as specific additive to the initial protein-lipid-sodium cholate mixture or in the course of incubation of proteoliposomal suspensions at 37 degrees C. After the incorporation of cytochrome b5 into azolectin liposomes vesicular membranes contain IMP if the incorporated membrane protein: lipid ratio is at least 1:50. Pronase-induced splitting off of a 11 kDa heme-containing fragment of cytochrome b5 did not affect IMP content. The conditions of IMP formation in reconstituted membranes and in microsomal ghosts are discussed.     

High Km soluble 5'-nucleotidase from human placenta. Properties and allosteric regulation by IMP and ATP

A human placental soluble "high Km" 5'-nucleotidase has been separated from "low Km" 5'-nucleotidase and nonspecific phosphatase by AMP-Sepharose affinity chromatography. The enzyme was purified 8000-fold to a specific activity of 25.6 mumol/min/mg. The subunit molecular mass is 53 kDa, and the native molecular mass is 210 kDa, suggesting a tetrameric structure. Soluble high Km 5'-nucleotidase is most active with IMP and GMP and their deoxy derivatives. IMP is hydrolyzed 15 times faster than AMP. The enzyme has a virtually absolute requirement for magnesium ions and is regulated by them. Purine nucleoside 5'-triphosphates strongly activate the enzyme with the potency order dATP greater than ATP greater than GTP. 2,3-Diphosphoglycerate activates the enzyme as potently as ATP. Three millimolar ATP decreased the Km for IMP from 0.33 to 0.09 mM and increased the Vmax 12-fold. ATP activation was modified by the IMP concentration. At 20 microM IMP the ATP-dependent activation curve was sigmoidal, while at 2 mM IMP it was hyperbolic. The A0.5 values for ATP were 2.26 and 0.70 mM, and the relative maximal velocities were 32.9 and 126.0 nmol/min, respectively. Inorganic phosphate shifts the hyperbolic substrate velocity relationship for IMP to a sigmoidal one. With physiological concentrations of cofactors (3 mM ATP, 1-4 mM Pi, 150 mM KCl) at pH 7.4, the enzyme is 25-35 times more active toward 100 microM IMP than 100 microM AMP. These data show that: (a) soluble human placental high Km 5'-nucleotidase coexists in human placenta with the low Km enzyme; (b) under physiological conditions the enzyme favors the hydrolysis of IMP and is critically regulated by IMP, ATP, and Pi levels; and (c) kinetic properties of ATP and IMP are each modified by the other compound suggesting complex interaction of the associated binding sites.     

Mammalian and avian liver phosphoenolpyruvate carboxykinase. Alternate substrates and inhibition by analogues of oxaloacetate

Phosphoenolpyruvate carboxykinase from chicken liver mitochondria and rat liver cytosol catalyzes the phosphorylation of alpha-substituted carboxylic acids such as glycolate, thioglycolate, and DL-beta-chlorolactate in reactions with absolute requirements for divalent cation activators. 31P NMR analysis of the reaction products indicates that phosphorylation occurs at the alpha-position to generate the corresponding O- or S-bridged phosphate monoesters. In addition, the enzymes catalyze the bicarbonate-dependent phosphorylation of hydroxylamine. The chicken liver enzyme also catalyze the bicarbonate-dependent phosphorylation of hydroxylamine. The chicken liver enzyme also catalyzes the bicarbonate-dependent phosphorylation of fluoride ion. The kappa cat values for these substrates are 20-1000-fold slower than the kappa cat for oxaloacetate. Pyruvate and beta-hydroxypyruvate are not phosphorylated, since the enzyme does not catalyze the enolization of these compounds. Oxalate, a structural analogue of the enolate of pyruvate, is a competitive inhibitor of phosphoenolpyruvate carboxykinase (Ki of 5 microM) in the direction of phosphoenolpyruvate formation. Oxalate is also an inhibitor of the chicken liver enzyme in the direction of oxaloacetate formation and in the decarboxylation of oxaloacetate. The chicken liver enzyme is inhibited by beta-sulfopyruvate, an isoelectronic analogue of oxaloacetate. The extensive homologies between the reactions catalyzed by phosphoenolpyruvate carboxykinase and pyruvate kinase suggest that the divalent cation activators in these reactions may have similar functions. The substrate specificity indicates that phosphoenolpyruvate carboxykinase decarboxylates oxaloacetate to form the enolate of pyruvate which is then phosphorylated by MgGTP on the enzyme.     

pH dependence of kinetic parameters for oxalacetate decarboxylation and pyruvate reduction reactions catalyzed by malic enzyme

Both chicken liver NADP-malic enzyme and Ascaris suum NAD-malic enzyme catalyze the metal-dependent decarboxylation of oxalacetate. Both enzymes catalyze the reaction either in the presence or in the absence of dinucleotide. The presence of dinucleotide increases the affinity of oxalacetate for the chicken liver NADP-malic enzyme, but this information could not be obtained in the case of A. suum NAD-malic enzyme because of the low affinity of free enzyme for NAD. The kinetic mechanism for oxalacetate decarboxylation by the chicken liver NADP-malic enzyme is equilibrium ordered at pH values below 5.0 with NADP adding to enzyme first. The Ki for NADP increases by a factor of 10 per pH unit below pH 5.0. An enzyme residue is required protonated for oxalacetate decarboxylation (by both enzymes) and pyruvate reduction (by the NAD-malic enzyme), but the beta-carboxyl of oxalacetate must be unprotonated for reaction (by both enzymes). The pK of the enzyme residue of the chicken liver NADP-malic enzyme decreases from a value of 6.4 in the absence of NADP to about 5.5 with Mg2+ and 4.8 with Mn2+ in the presence of NADP. The pK value of the enzyme residue required protonated for either oxalacetate decarboxylation or pyruvate reduction for the A. suum NAD-malic enzyme is about 5.5-6.0. Although oxalacetate binds equally well to protonated and unprotonated forms of the NADP-enzyme, the NAD-enzyme requires that oxalacetate or pyruvate selectively bind to the protonated form of the enzyme. Both enzymes prefer Mn2+ over Mg2+ for oxalacetate decarboxylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rat thyroid phosphofructokinase. Comparison of the regulatory and molecular properties with those of rat muscle enzyme.

The kinetic and molecular properties of rat thyroid phosphofructokinase (specific activity 134 units/mg) were compared with those of rat muscle phosphofructokinase (specific activity 135 units/mg). Thyroid and muscle phosphofructokinase showed similar sedimentation patterns in sucrose density gradients; their affinity for DEAE-cellulose was similar but not identical. A comparison of the kinetic properties revealed differences in the pH optima. Striking differences in the kinetic properties were shown below pH 7.4; the thyroid enzyme was less inhibited by ATP or citrate and more sensitive to activation by cyclic 3':5'-AMP than the muscle enzyme. A study of the effects of some cyclic as well as linear mononucleotides, such as cyclic AMP, cyclic IMP, cyclic GMP, cyclic CMP, cyclic UMP, 5'-AMP, and 3'-AMP on thyroid phosphofructokinase showed that at concentrations as low as 1 micrometer only cyclic AMP and cyclic IMP were able to activate thyroid enzyme in the presence of low fructose-6-P and high ATP concentrations.     

Kinetic studies with cytosol and mitochondrial phosphoenolpyruvate carboxykinases

1. Measurements of Michaelis constants for oxaloacetate in the reaction catalysed by liver phosphoenolpyruvate carboxykinase give values much lower than previously reported. With Mg(2+) as bivalent cation, the Michaelis constant was approx. 2.5x10(-5)m whether the enzyme used was the mitochondrial phosphoenolpyruvate carboxykinase purified from sheep liver or chicken liver or the cytosol enzyme purified from rat liver or sheep liver. 2. When Mn(2+) replaced Mg(2+) in the reaction a lower Michaelis constant of 9x10(-6)m was found, but only with the mitochondrial enzymes. 3. With all enzymes malate at high concentration was a competitive inhibitor with respect to oxaloacetate when Mn(2+) was the added cation. With Mg(2+) the inhibition by malate was competitive with the mitochondrial enzymes and non-competitive with the cytosol enzymes.     

Inhibition of glycine N-methyltransferase by 5-methyltetrahydrofolate pentaglutamate

Glycine N-methyltransferase (EC 2.1.1.20) catalyzes the methylation of glycine by S-adenosylmethionine to form sarcosine and S-adenosylhomocysteine. The enzyme was previously shown to be abundant in both the liver and pancreas of the rat, to consist of four identical monomers, and to contain tightly bound folate polyglutamates in vivo. We now report that the inhibition of glycine N-methyltransferase by (6S)-5-CH(3)-H(4)PteGlu(5) is noncompetitive with regard to both S-adenosylmethionine and glycine. The enzyme exhibits strong positive cooperativity with respect to S-adenosylmethionine. Cooperativity increases with increasing concentrations of 5-CH(3)-H(4)PteGlu(5) and is greater at physiological pH than at pH 9.0, the pH optimum. Under the same conditions, cooperativity is much greater for the pancreatic form of the enzyme. The V(max) for the liver form of the enzyme is approximately twice that of the pancreatic enzyme, while K(m) values for each substrate are similar in the liver and pancreatic enzymes. For the liver enzyme, at pH 7.0 half-maximal inhibition is seen at a concentration of about 0.2 microM (6S)-5-CH(3)-H(4)PteGlu(5), while at pH 9.0 this value is increased to about 1 microM. For the liver form of the enzyme, 50% inhibition with respect to S-adenosylmethionine at pH 7.4 occurs at about 0.27 microM. The dissociation constant, K(s), obtained from binding data at pH 7.4 is 0.095. About 1 mol of (6S)-5-CH(3)-H(4)PteGlu(5) was bound per tetramer at pH 7.0, and 1.6 mol were bound at pH 9.0. The degree of binding and inhibition were closely parallel at each pH. At equal concentrations of (6R,6S)- and (6S)-5-CH(3)-H(4)PteGlu(5), the natural (6S) form was about twice as inhibitory. These studies indicate that glycine N-methyltransferase is a highly allosteric enzyme, which is consistent with its role as a regulator of methyl group metabolism in both the liver and the pancreas.     

Carboxylesterases (EC 3.1.1). The molecular sizes of chicken and pig liver carboxylesterases.

The molecular size of pig liver carboxylesterase has been investigated under a variety of conditions of pH and ionic strength. From equilibrium and velocity sedimentation at pH 4.0 and pH 7.5, and from chromatography on Sephadex G-200,we conclude that the monomeric molecular weight is similar to 65,000 daltons and that the enzyme associates to form trimers. Association equilibrium constants for the monomer-trimer system were estimated to be 0.02 1-2 g-2 at pH 4 (concentration-dependent molecular weight data) and 2 times 10-5 1-2g-2 at pH 7.5 (frontal gel chromatographic results). These studies were aided by comparisons of the properties of the pig liver enzyme with those of chicken liver carboxylesterase, which is shown to exhibit the velocity and equilibrium sedimentation characteristics of a homogeneous protein with molecular weight similar to 65,000. Studies of pig and chicken liver carboxylesterases in 6 M guanidinium chloride, 0.1 M in beta-mercaptoethanol, support the proposition that the monomeric species of these enzymes have molecular weights of similar to 65,000. On polyacrylamide gel electrophoresis in SDS, there is no evidence for a major species of molecular weight less than similar to 65,000 for the pig enzyme, but ca. 50 percent of the chicken esterase is dissociated into two species of molecular weight similar to 30,000.     

Evidence for the involvement of cytosolic 5'-nucleotidase (cN-II) in the synthesis of guanine nucleotides from xanthosine

In this paper, we show that in vitro xanthosine does not enter any of the pathways known to salvage the other three main natural purine nucleosides: guanosine; inosine; and adenosine. In rat brain extracts and in intact LoVo cells, xanthosine is salvaged to XMP via the phosphotransferase activity of cytosolic 5'-nucleotidase. IMP is the preferred phosphate donor (IMP + xanthosine --> XMP + inosine). XMP is not further phosphorylated. However, in the presence of glutamine, it is readily converted to guanyl compounds. Thus, phosphorylation of xanthosine by cytosolic 5'-nucleotidase circumvents the activity of IMP dehydrogenase, a rate-limiting enzyme, catalyzing the NAD(+)-dependent conversion of IMP to XMP at the branch point of de novo nucleotide synthesis, thus leading to the generation of guanine nucleotides. Mycophenolic acid, an inhibitor of IMP dehydrogenase, inhibits the guanyl compound synthesis via the IMP dehydrogenase pathway but has no effect on the cytosolic 5'-nucleotidase pathway of guanine nucleotides synthesis. We propose that the latter pathway might contribute to the reversal of the in vitro antiproliferative effect exerted by IMP dehydrogenase inhibitors routinely seen with repletion of the guanine nucleotide pools.     

The regulation of rat liver xanthine oxidase. Involvement of thiol groups in the conversion of the enzyme activity from dehydrogenase (type D) into oxidase (type O) and purification of the enzyme

1. The ;xanthine oxidase' activity of rat liver supernatant, most of which behaves as an NAD(+)-dependent dehydrogenase (type D) can be rapidly converted into an oxidase (type O) by thiol reagents such as tetraethylthiuram disulphide, copper sulphate, 5,5'-dithiobis-(2-nitrobenzoic acid), N-ethylmaleimide and p-hydroxymercuribenzoate. Treatment with copper sulphate, if prolonged, leads to almost complete inactivation of the enzyme. The effect of these reagents is prevented by dithioerythritol, and in all cases but that of N-ethylmaleimide is reversed by the same thiol. 2. Dithioerythritol prevents and reverses the conversion of xanthine oxidase from type D into type O brought about by storage of rat liver supernatant at -20 degrees C, preincubation under anaerobic conditions, treatment with carbon or with diethyl ether, and reverses, but does not prevent, the conversion obtained by preincubation of the whole liver homogenate. 3. Conversion of the enzyme from type D into type O is effected by preincubation of rat liver supernatant with the sedimentable fraction from rat liver but not from chick or pigeon liver. The xanthine dehydrogenase activity of chick liver supernatant is not changed into an oxidase by preincubation with the sedimentable fraction from rat liver. 4. The enzyme activity of rat liver supernatant is converted from type D into type O during purification of the enzyme: the purified enzyme can be reconverted into type D by dithioerythritol. 5. The enzyme appears as an oxidase in the supernatant of rat heart, intestine, spleen, pancreas, lung and kidney. The enzyme of all organs but intestine can be converted into a dehydrogenase by dithioerythritol.     

Chicken erythrocyte pyrimidine 5'-nucleotidase: purification and characterization of the subclass I enzyme

Nucleotidase activities resembling subclass I and subclass II of human pyrimidine 5'-nucleotidases (P5N) were detected in chicken red blood cells (RBCs). In chicken RBCs from untreated controls, the activity of the subclass II enzyme was about one third of that of subclass I enzyme, whereas that ratio was approximately 5:1 in rat or human RBCs. The subclass I activity in chicken RBCs was increased 5- to 6-fold upon erythropoietic induction by phenylhydrazine administration, but the subclass II activity did not increase under these conditions. The subclass I enzyme was purified to near homogeneity. Its molecular mass was about 35 kDa as estimated by gel filtration and SDS-polyacrylamide gel electrophoresis. Its N-terminal 12 amino acids, PEFQKKTVHIKD, were also determined. The catalytic properties of the subclass I enzyme were very similar to those of the human enzyme with regard to substrate (preferential hydrolysis of CMP, dCMP, UMP), Km values, optimum pH, and metal ion requirements. Antibodies against chicken P5N subclass I were raised in rats. The chicken P5N-I as well as the rat P5N-I proteins could be detected by antibodies in Western blot analyses, but not the P5N-II proteins. These findings indicate that P5N subclass I may have an important function in chicken erythropoiesis.