Antonio Orengo

The turnover of deoxyuridine triphosphate during the HeLa cell cycle

Abstract The synthesis and breakdown of deoxyuridine triphosphate (dUTP) was studied to determine whether a dUTP pool is present at any stage of the HeLa cell cycle. Although cell extracts were found to be capable of phosphorylating dUMP to dUTP, only minimal quantities of intracellular dUMP, dUDP or dUTP could be detected. When thymidylate synthetase was blocked with FUdR the dUMP pool increased but no substantial increase in dUDP or dUTP was seen. A powerful and specific dUTP nucleotidohydrolase (dUTPase, EC3.6.1.23) which hydrolyses dUTP to dUMP and PPi was detected. The activity of this enzyme as well as that of the dUTP synthesizing enzymes was low in G1, rose through S and G2 and reached a maximum just prior to cell division. Pulsing experiments with [5-3H]UdR and [14C]TdR suggest that the size of the dUTP pool is 1% of the dTTP pool.

The preparation of an immunoglobulin-amyloglucosidase conjugate and its quantitation by an enzyme-cycling assay☆

Abstract The production and characterization of covalent amyloglucosidase-antibody conjugates using anti-human serum albumin immunoglobulin G are described. The conjugation procedure is based on the periodate oxidation of carbohydrate moieties that are covalently linked to the enzyme, followed by Schiffs base formation with amino residues on IgG. An ultrasensitive enzyme cycling assay for glucose, the product of maltose cleavage by amyloglucosidase, was developed in order to increase the sensitivity of detecting the enzyme-antibody conjugate. The cycling assay, which allows the accurate measurement of glucose in the picomole range, involves an enzymatic conversion of glucose to glucose-6-phosphate and then isomerization to fructose-6-phosphate. A futile cycle between fructose-6-phosphate and fructose-1,6-diphosphate results in accumulation of adenosine diphosphate at a rate proportional to the original glucose concentration. The rate was monitored by a spectrophotometric system involving pyruvate kinase, phospho(enol)pyruvate, lactate dehydrogenase, and diphosphopyridine nucleotide.

The relationship of cAMP levels and the rate of pool labelling of cAMP and related bases, nucleosides and nucleotides to the HeLa cell cycle

Abstract Cellular cAMP levels as well as the rate of pool labelling of cAMP and related bases, nucleosides and nucleotides were determined in synchronized cultures of HeLa cells after pulse-labelling with [14C]adenine. The cAMP levels were found to be maximal in G 1 and minimal in G 2 and mitosis, as previously reported by others. The rate of labelling of the cAMP pools, however, was found to be maximal in G 2 and decreased to a minimum in G 1. This suggests that the rate of cAMP synthesis is highest when pool level is lowest and vice versa. A comparison of cAMP levels and the rate of 5′AMP pool labelling throughout the HeLa cell cycle indicated an inverse relationship. Such a relationship emphasizes the role of the cyclic 3′,5′-phosphodiesterase activity during the cell cycle. The kinetics of pool labelling of IMP, ATP, and hypoxanthine throughout the cell cycle suggested that the adenylate energy charge fluctuated as a function of the cell cycle. The apparent activation of the adenylate cyclase during G 2 and mitosis as reflected by the increased rate of cAMP pool labelling suggests that the super phosphorylation of H 1 histone during G 2-mitotic transition may be mediated by cAMP-dependent phosphokinases.

Activation of rat liver pyrimidine nucleoside monophosphate kinase

The activity of the pyrimidine nucleoside monophosphate kinase (ATP:dCMP phosphotransferase, EC 2.7.4.14) from rat liver is dependent upon the presence of sulfhydryl-reducing agents. Addition to the inactive enzyme of 2-mercaptoethanol (5 mM), a reagent specific for cleavage of disulfide bonds, effects a reduction in molecular weight from approx. 53 000 to 17 000, measured by molecular sieve chromatography. This low molecular weight form is partially active in the presence of 2-mercaptoethanol (f mM). In absence of 2-mercaptoethanol, the low molecular weight form is inactive. Higher concentrations of 2-mercaptoethanol (50 mM) fully reactivate the CMP(ATP) kinase activity followed by dCMP(ATP) and CMP(dCTP) kinase activities in a sequential manner, without further change in moelcular weight. Alkylation by iodoacetamide of the enzyme at different stages of reactivation in dithiothreitol suggests an ordered appearance of the various enzyme activities. Furthermore, iodoacetamide inactivates the fully active enzyme. Thioredoxin was found to activate the enzyme in a manner similar to 2-mercaptoethanol and dithiothreitol. These results are consistent with the interpretation that the mechanism of activation of the enzyme involves cleavage of inter- and intramolecular disulfide bonds.

Separation of cyclic AMP from adenine and hypoxanthine, their nucleosides and nucleotides by high voltage paper electrophoresis

A simple and rapid technique which permits the separation of cyclic AMP, adenine, adenosine, hypoxanthine, inosine, 5′-AMP, IMP, ADP, and ATP by the use of unidirectional high-voltage paper electrophoresis has been described. The separation of these compounds based on their charge difference utilizes the following properties: (1) the protonation of the NH2 group of the adenine, (2) the primary and secondary ionization of the phosphate group of the nucleotides, and (3) the formation of the chelated oxyderivative of boron with the two cis (OH) groups of the ribose moieties of nucleosides and nucleotides.

Deoxyuridine triphosphate nucleotidohydrolase of HeLa cells.

Abstract 1. 1. A kinetically pure preparation of dUTPase was prepared from HeLa cells. 2. 2. dUTPase is a 62,500 dalton pyrophosphatase. The Km of dUTPase for dUTP is 1.22 × 10−5 M± 0.19. In the presence of Mg2+ the Km is 0.47 × 10−5M. 3. 3. dUTPase is highly specific for dUTP and will not hydrolyze any naturally occurring deoxynucleoside triphosphate nor UTP. 4. 4. dUTPase is activated by Mg2+ and K+ and inhibited by EDTA, Cu2+ and sodium polyphosphate.

Exploitable molecular mechanisms in hibernation—IL: Aromatic circular dichroism of hamster and rat liver diphosphofructose phosphatase

Abstract 1. 1. The aromatic circular dichroism of diphosphofructose phosphatase of the albino rat, golden Syrian hamster and New Zealand rabbit were used to compare their conformations. 2. 2. The conformational transitions incited by Na + , K + , pH, and spontaneous denaturation were compared and studied. 3. 3. From a comparative analysis of the tertiary structure of the phosphatases, as it is revealed by circular dichroism, it was concluded that the strong interactions of tyrosyl residues with non-polar micro-environments confer to the hamster enzyme a degree of rigidity which was found unmatched in the isofunctional protein of the rat.

Human liver fructose 1,6-bisphosphatase (EC 3.1.3.11). purification and properties

Abstract 1. 1. Human fructose 1,6-bisphosphatase (EC 3.1.3.11) is composed of subunits of 44,000 daltons ±9% and exists predominantly as a dimer in 0.005 M Tris-glycine buffer, pH 8.3. 2. 2. The human enzyme is more sensitive to 5′-AMP inhibition (I (0.5) 3.2 × 10 −5 M) than that of 2 other ominivorous species, i.e. the albino rat (2.3 × 10 −4 M) and the Syrian hamster (3.6 × 10 −4 M). 3. 3. The circular dichroism spectrum reveals highly oriented thyrosine and phenylalanine residues and an alpha helix content of 23%. 4. 4. As all the phosphatases previously studied, the enzyme has a low specific activity at physiological pHs.

A rapid procedure for separation and purification of purine and pyrimidine nucleoside monophosphate kinases

1. 1. The partial purification of pyrimidine nucleoside monophosphate kinase, adenylate kinase and guanylate kinase has been applied directly to cytosol preparations of rat liver, erythrocytes, Novikoff hepatoma, mouse adenocarcinoma and human liver. 2. 2. Solutions of dCTP or CTP (1.0 mM) appear to be the most efficient eluents of pyrimidine nucleoside monophosphate kinase and erythrocyte adenylate kinase from cellulose phosphate column. 3. 3. Liver and hepatoma adenylate kinase require ATP for elution. 4. 4. The erythrocyte adenylate kinase is inhibited by pHMB (Ki 0.0087 mM) and NEM (Ki 0.0611 mM) and has a low affinity for AMP (Km 0.717 mM) in contrast to the liver adenylate kinase which is not inhibited by NEM and pHMB and has a higher affinity for AMP (Km 0.0951 mM).

On the activation of human and rat fructose 1,6-bisphosphatase

Abstract 1. 1. Human and rat liver Fructose 1,6-bisphosphatase have low specific activities at physiological pHs. 2. 2. Cysteine, threonine, serine and glycine activate the enzyme present in cytosols. A small amount (5%) of the enzyme is bound to microsomes. This fraction does not require any activator for maximal activity. 3. 3. A factor that is capable of activating the cytosol enzyme has been isolated and partially purified from liver microsomes and co-elutes with the microsomal-bound enzyme. A similar factor has been isolated from the cytosol fraction of 2 hepatomas.