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Biomedical subjects

P Reichard

Publications and source records attributed to P Reichard.

At least 109 records · Page 6Linked to original sources
PubMed1987

Regulation of pyrimidine deoxyribonucleotide metabolism by substrate cycles in dCMP deaminase-deficient V79 hamster cells.

A mutant V79 hamster fibroblast cell line lacking the enzyme dCMP deaminase was used to study the regulation of deoxynucleoside triphosphate pools by substrate cycles between pyrimidine deoxyribosides and their 5'-phosphates. Such cycles were suggested earlier to set the rates of cellular import and export of deoxyribosides, thereby influencing pool sizes (V. Bianchi, E. Pontis, and P. Reichard, Proc. Natl. Acad. Sci. USA 83:986-990, 1986). While normal V79 cells derived more than 80% of their dTTP from CDP reduction via deamination of dCMP, the mutant cells had to rely completely on UDP reduction for de novo synthesis of dTTP, which became limiting for DNA synthesis. Because of the allosteric properties of ribonucleotide reductase, CDP reduction was not diminished, leading to a large expansion of the dCTP pool. The increase of this pool was kept in check by a shift in the balance of the deoxycytidine/dCMP cycle towards the deoxynucleoside, leading to massive excretion of deoxycytidine. In contrast, the balance of the deoxyuridine/dUMP cycle was shifted towards the nucleotide, facilitating import of extracellular deoxynucleosides.

Animals↗
PubMed1986

Changes of deoxyribonucleoside triphosphate pools induced by hydroxyurea and their relation to DNA synthesis.

Hydroxyurea inactivates ribonucleotide reductase from mammalian cells and thereby depletes them of the deoxynucleoside triphosphates required for DNA replication. In cultures of exponentially growing 3T6 cells, with 60-70% of the cells in S-phase, 3 mM hydroxyurea rapidly stopped ribonucleotide reduction and DNA synthesis (incorporation of labeled thymidine). The pool of deoxyadenosine triphosphate (dATP) decreased in size primarily, but also the pools of the triphosphates of deoxyguanosine and deoxycytidine (dCTP) were depleted. Paradoxically, the pool of thymidine triphosphate increased. After addition of hydroxyurea this pool was fed by a net influx and phosphorylation of deoxyuridine from the medium and by deamination of intracellular dCTP. An influx of deoxycytidine from the medium contributed to the maintenance of intracellular dCTP. 10 min after addition of hydroxyurea, DNA synthesis appeared to be completely blocked even though the dATP pool was only moderately decreased. As possible explanations for this discrepancy, we discuss compartmentation of pools and/or vulnerability of newly formed DNA strands to nuclease action and pyrophosphorolysis.

Cells, Cultured↗
PubMed1986

Production and characterization of monoclonal antibodies against the two subunits proteins B1 and B2 of Escherichia coli ribonucleotide reductase.

Ribonucleotide reductase from Escherichia coli consists of two nonidentical subunits, named protein B1 (170 000) and protein B2 (87 000). We purified and characterized five monoclonal antibodies against B1 and three against B2 from hybridomas obtained by fusion of spleen cells from immunized mice and the myeloma cell line P3-X63Ag8. All are of the IgG1 class with a high affinity for the antigen with dissociation constants in the nanomolar range. Four of the anti-B1 monoclonals and all three anti-B2 monoclonals neutralize reductase activity while one anti-B1 monoclonal binds tightly to B1 without affecting its activity. Fab fragments prepared from three anti-B1 monoclonals had similar dissociation constants. The anti-B1 monoclonals interacted with separate epitopes while two of the anti-B2 monoclonals appeared to react with the same epitope. In the case of B1, various allosteric states of the protein induced by binding of effectors had no apparent effect on the interaction with monoclonals, nor did their binding prevent subsequent binding of effectors. With B2, binding of monoclonals did not affect the typical electron paramagnetic resonance spectrum of the protein and thus did not involve either the tyrosyl free radical or the iron center of B2. All neutralizing antibodies interfered with the interaction between the two subunits, explaining their effect on enzyme activity, since active ribonucleotide reductase consists of a B1-B2 complex.

Animals↗
PubMed1986

Affinity chromatography on anti-B1 monoclonal gels for purification and characterization of protein B1 from Escherichia coli ribonucleotide reductase.

Affinity gels were prepared from four monoclonal antibodies against the B1 protein of ribonucleotide reductase of Escherichia coli. The gels were used to purify protein B1 and also to study some of its properties. Gels from the nonneutralizing monoclonal anti-B1-k bound as much as 2 mg of B1/mL and were employed to prepare essentially pure B1 protein in a single step from extracts of wild-type E. coli and strains overproducing the subunit. However, B1 prepared from wild-type extracts had a lowered specific activity, suggesting some denaturation during elution of the protein from the column. Addition of the allosteric effector dATP during affinity chromatography changed the chromatographic pattern. Some protein B2, the second subunit of the reductase, remained in all cases bound to the gels together with B1. The gel prepared from anti-B1-c retained two additional proteins. In other experiments involving binding of proteolytic fragments of B1 to various antibodies, we also found a striking effect of dATP, suggesting that dATP made protein B1 less accessible to proteolysis. In these experiments fragments around 15K still had the ability to bind monoclonals, making possible more detailed investigations of the structural contacts between B1 and the monoclonals.

Antibodies, Monoclonal↗
PubMed1986

A nitrocellulose filter binding assay for ribonucleotide reductase.

Protein B1, one of the two nonidentical subunits of Escherichia coli ribonucleotide reductase, contains two classes of binding sites for nucleoside triphosphates. One class (h-sites), with a high affinity for dATP (KD = 30 nM) regulates the substrate specificity, while the other (l-sites), with a lower affinity for dATP, regulates overall activity. These classes were defined from experiments involving equilibrium dialysis. Here we describe a sensitive alternative method to measure nucleotide binding to ribonucleotide reductases that gave the same results as equilibrium dialysis. The method involves the protein-specific binding of radioactive nucleotides to nitrocellulose filters. We believe that this method will be useful in binding studies with pure reductases from sources other than E. coli, for the characterization of mutants with changed allosteric properties, and as an assay during purification of reductases containing an h-site for dATP.

Binding Sites↗
PubMed1986

Interrelations between substrate cycles and de novo synthesis of pyrimidine deoxyribonucleoside triphosphates in 3T6 cells.

Degradation of pyrimidine deoxyribonucleoside triphosphates plays a major role in the regulation of their pool sizes in 3T6 cells. During normal growth, these cells excrete deoxyribonucleosides (mostly deoxyuridine) into the medium. When DNA strand elongation is inhibited, de novo synthesis of dCTP and dTTP continues, followed by degradation of the deoxyribonucleotides. We now demonstrate that inhibition of de novo synthesis with hydroxyurea stops degradation of deoxyribonucleotides. We now demonstrate that inhibition of de novo synthesis with hydroxyurea stops degradation of deoxyribonucleotides and leads to an influx of deoxyuridine from the medium. This effect appears to be caused by a large drop in the size of the intracellular dUMP pool. We propose that substrate cycles, involving phosphorylation of deoxyribonucleosides by kinases and dephosphorylation of deoxyribonucleoside 5'-phosphates by a nucleotidase, participate in the regulation of the size of pyrimidine deoxyribonucleoside triphosphate pools by directing the flow of deoxyribonucleosides across the cell membrane. While kinases are regulated mainly by allosteric effects, the activity of the nucleotidase appears to be regulated by substrate concentration.

Animals↗
PubMed1986

Superoxide dismutase participates in the enzymatic formation of the tyrosine radical of ribonucleotide reductase from Escherichia coli.

One of the two nonidentical subunits of Escherichia coli ribonucleotide reductase, protein B2, contains in its active form two antiferromagnetically coupled Fe(III) ions and an organic free radical that arises by the one-electron oxidation of tyrosine-122 of the polypeptide chain. Protein B2 lacking the tyrosine radical but with the iron center intact (called protein B2/HU because it is produced by treatment with hydroxyurea) is enzymatically inactive. Previously, it was found that a crude extract from E. coli transforms B2/HU into B2 in the presence of dithiothreitol, Mg2+, and oxygen. On purification of the enzyme system, we now find that radical introduction requires three separate proteins as well as NADPH and FMN. One of the proteins is superoxide dismutase. We hypothesize that the overall reaction involves a reduction of the iron center followed by the oxidation of iron and tyrosine-122. Superoxide dismutase may then be involved in the second step to protect an oxidation-sensitive intermediate. Alternatively, the enzyme might be directly involved in the oxidation step.

Enzyme Activation↗
PubMed1985

Evidence for the involvement of substrate cycles in the regulation of deoxyribonucleoside triphosphate pools in 3T6 cells.

Pool sizes of deoxyribonucleoside triphosphates (dNTPs) in cultured cells are tightly regulated by i.al., the allosteric control of ribonucleotide reductase. We now determine the in situ activity of this enzyme from the turnover of the deoxycytidine triphosphate (dCTP) pool in rapidly growing 3T6 mouse fibroblasts, as well as in cells whose DNA replication was inhibited by aphidicolin or amethopterin, by following under steady state conditions the path of isotope from [5-3H]cytidine into nucleotides, DNA, and deoxynucleosides excreted into the medium. In normal cells as much as 28% of the dCDP synthesized was excreted as deoxynucleoside (mostly deoxyuridine), leading to an accumulation of deoxyuridine in the medium. Inhibition with amethopterin slightly increased ribonucleotide reductase activity, while aphidicolin halved the activity of this enzyme (and thymidylate synthase). In both instances all dCDP synthesized was degraded and excreted as nucleosides. This continued synthesis and turnover in the absence of DNA synthesis is in contrast to the earlier found inhibition of dCTP (and dTTP) turnover when hydroxyurea, an inhibitor of ribonucleotide reductase, was used to block DNA synthesis. To explain our results, we propose that substrate cycles between deoxyribonucleosides and their monophosphates, involving the activities of kinases and phosphatases, participate in the regulation of pool sizes. Within the cycles, a block of the reductase activates net phosphorylation, while inhibition of DNA polymerase stimulates degradation.

Animals↗
PubMed1985

Azidocytidine is a specific inhibitor of deoxyribonucleotide synthesis in 3T6 cells.

2'-Azidocytidine is a specific inhibitor of DNA synthesis in cultured 3T6 mouse fibroblasts. Earlier work (Akerblom, L., Pontis, E., and Reichard, P. (1982) 257, 6776-6782) indicated that the nucleoside, after phosphorylation, acted by inhibiting both ribonucleotide reduction and DNA strand elongation. We now demonstrate that the effect on strand elongation was due to a contamination of azidocytidine with less than 0.3% of arabinosyl cytosine. Pure azidocytidine inhibits specifically ribonucleotide reductase and its effects on DNA synthesis are secondary to this inhibition. The results with azidocytidine concerning the size and turnover of deoxyribonucleoside triphosphate pools parallel those of hydroxyurea and are distinct from those of arabinosyl cytosine. Together with hydroxyurea, azidocytidine is a useful compound in studies aiming at a specific block of the production of deoxynucleoside triphosphates in intact cells. Comparisons of the effects of azidocytidine and arabinosyl cytosine complement earlier studies with hydroxyurea and aphidicolin concerning inter-relations between dNTP synthesis and DNA replication.

Animals↗
PubMed1985

Relations between synthesis of deoxyribonucleotides and DNA replication in 3T6 fibroblasts.

In exponentially growing 3T6 cells, the synthesis of deoxythymidine triphosphate (dTTP) is balanced by its utilization for DNA replication, with a turnover of the dTTP pool of around 5 min. We now investigate the effects of two inhibitors of DNA synthesis (aphidicolin and hydroxyurea) on the synthesis and degradation of pyrimidine deoxynucleoside triphosphates (dNTPs). Complete inhibition of DNA replication with aphidicolin did not decrease the turnover of pyrimidine dNTP pools labeled from the corresponding [3H]deoxynucleosides, only partially inhibited the in situ activity of thymidylate synthetase and resulted in excretion into the medium of thymidine derived from breakdown of dTTP synthesized de novo. These data demonstrate continued synthesis of dTTP in the absence of DNA replication. In contrast, hydroxyurea decreased the turnover of pyrimidine dNTP pools 5-50-fold. Hydroxyurea is an inhibitor of ribonucleotide reductase and stops DNA synthesis by depleting cells of purine dNTPs but not pyrimidine dNTPs. Our results suggest that degradation of dNTPs is turned off by an unknown mechanism when de novo synthesis is blocked.

Animals↗
PubMed1985

Ribonucleotide reductase and deoxyribonucleotide pools.

Ribonucleotide reductase plays a crucial role in the maintenance of dNTP pools. A molecular model for the allosteric regulation of the enzyme is substantiated by the effects of nucleosides or inhibitors on pool sizes in intact cells as well as by manifestations of mutations of the reductase and other enzymes involved in deoxynucleoside metabolism. Studies of the dynamics of dNTP pools in 3T6 cells suggest compartmentation of dCTP pools, with perferential DNA synthesis from a pool labeled from cytidine. In contrast, no kinetic evidence for more than one dTTP pool was obtained. Our experiments also suggest that all dTTP formed in growing 3T6 cells is used up for DNA synthesis.

Allosteric Regulation↗
PubMed1985

On the regulation of deoxyribonucleotide synthesis.

The results reviewed here demonstrate the feasibility of measuring the turnover of pyrimidine dNTPs in cultured cells and of relating such data to rates of DNA synthesis. Growing 3T6 cells appear to contain two kinetically distinct pools of dCTP. One pool is preferentially labeled by reduction of CDP and used for DNA synthesis, the second pool is preferentially labeled by phosphorylation of deoxycytidine. Within the time resolution of our experiments dTTP does not show similar compartmentation. We have also found that pyrimidine dNTPs continue to be synthesized, albeit at reduced rate, when DNA replication is blocked.

Animals↗
PubMed1983

Ribonucleotides are not channeled into DNA in permeabilized mammalian cells.

Reddy and Pardee earlier reported that permeabilized CHEF/18 cells (J. Biol. Chem 257, 12526) or soluble extracts from such cells (Cell 32, 443) under the proper conditions preferentially incorporated a labeled ribonucleotide (CDP) into DNA without dilution by an excess of non-labeled dCTP, suggesting channeling of ribonucleotides into DNA by a multiprotein complex. Here we present results suggesting that in these experiments CDP was incorporated into RNA and not into DNA.

Animals↗
PubMed1983

Dynamics of pyrimidine deoxynucleoside triphosphate pools in relationship to DNA synthesis in 3T6 mouse fibroblasts.

The 3H-labeled nucleosides cytidine, deoxycytidine, and thymidine are rapidly incorporated into DNA via dCTP or dTTP pools. Between 30 and 60 min after addition of tracer amounts of a labeled nucleoside to the medium of rapidly growing 3T6 cells, dNTP pools attained a constant specific activity resulting from a steady-state equilibrium between incorporation of nucleoside, de novo synthesis, and linear incorporation of isotope into DNA. Removal of labeled deoxycytidine or thymidine depleted the dNTP pools of isotope within a few minutes and incorporation into DNA stopped. When de novo synthesis of dTTP was blocked with amethopterin, the intracellular dTTP pool rapidly reached the specific activity of thymidine of the medium and isotope incorporation into DNA then measured absolute rates of DNA synthesis. In experiments with and without amethopterin, we found no kinetic evidence for the existence of more than one dTTP pool and the decay of the pool suggested that all dTTP served as precursor of DNA. In contrast, experiments with deoxycytidine and cytidine suggested the presence of separate dCTP pools with preferential DNA synthesis from the pool labeled from cytidine.

Animals↗
PubMed1983

Enzymic modification of a tyrosine residue to a stable free radical in ribonucleotide reductase.

Protein B2, a subunit of ribonucleotide reductase from Escherichia coli, contains in its active form a tyrosyl free radical as part of the polypeptide chain and a dimeric iron center that stabilizes the radical. The enzyme depends on this radical for its catalytic activity. Treatment with hydroxyurea scavenges the radical without disturbing the iron center and, thereby, results in an inactive form of the subunit, B2/HU. A second inactive form, apoB2, lacking both the radical and the iron center, is obtained by treatment of B2 with 8-hydroxyquinoline. Here we describe an enzyme activity in extracts from E. coli that transforms the catalytically inactive B2/HU form into the active B2 subunit by regeneration of the tyrosyl radical. This reaction requires the presence of oxygen, dithiothreitol, and Mg2+ and does not proceed through apoB2. Under anaerobic conditions, we obtained evidence for a second activity in the bacterial extract that destroys the free radical and transforms B2 into B2/HU. We suggest that this novel type of protein modification is functionally related to the synthesis of deoxyribonucleotides and DNA.

Apoproteins↗
PubMed1983

Is glucose intolerance after pancreatitis related to pancreatic tissue damage?

Endoscopic retrograde pancreatography (ERP) was performed in 48 patients who had been hospitalized for pancreatitis. The findings were related to results of oral glucose tolerance tests. Patients with gross changes at ERP tended to have latent or manifest diabetes. In patients with latent or manifest diabetes, the increase in C-peptide after oral glucose was lower than in healthy subjects, while insulin sensitivity, estimated with euglycemic insulin clamp technique, was within the same range as in healthy subjects. It is concluded that gross changes of the pancreatic ducts after pancreatitis are often accompanied by widespread tissue damage leading to deficient B-cell function and decreased glucose tolerance.

Adult↗