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Biomedical subjects

A G So

Publications and source records attributed to A G So.

At least 55 records · Page 3Linked to original sources
PubMed1980

Degradation of deoxyribonucleic acid by a 1,10-phenanthroline-copper complex: the role of hydroxyl radicals.

Degradation of deoxyribonucleic acid (DNA) by 1,10-phenanthroline has been shown to require Cu(II), a reducing agent, and O2. Other metal ions do not substitute for Cu(II), and degradation of DNA is inhibited by metal ions that can form stable complexes with 1,10-phenanthroline, such as Co(II), Cd(II), Ni(II), or Zn(II), as well as by chelators that can bind copper, such as triethyltetraamine, neocuproine, or ethylenediaminetetraacetic acid (EDTA). Neocuproine, a specific copper chelator, is more effective than EDTA in inhibiting the breakdown of DNA. The degradation of DNA shows a requirement for a reducing agent which can be satisfied by either ascorbate or a thiol. A free radical generating system, e.g., xanthine oxidase-hypoxanthine, can substitute for the reducing agent. DNA degradation, in the presence of either an organic reducing agent or xanthine oxidase-hypoxanthine, is inhibited by hydroxyl radical scavengers and by catalase, suggesting that hydroxyl radical is the reactive species in DNA degradation and that hydrogen peroxide is an intermediate in hydroxyl radical generation.

Catalase↗
PubMed1980

Purification of deoxyribonucleic acid polymerase delta from calf thymus: partial characterization of physical properties.

Deoxyribonucleic acid (DNA) polymerase delta has been purified 7800-fold from calf thymus, to a specific activity of 28 000 units/mg of protein. Similar to DNA polymerase delta from bone marrow [Byrnes, J.J., Downey, K. M., Black, V. L., & So, A. G. (1976) Biochemistry 15, 2817], the calf thymus enzyme is associated with 3'- to 5'-exonuclease activity. Both DNA polymerase and 3'- to 5'-exonuclease activities copurify on hydroxylapatite, DNA-cellulose, and molecular sieve chromatography. The ratio of exonuclease activity to polymerase activity is approximately 1:12. When the most highly purified fraction is subjected to polyacrylamide gel electrophoresis under nondenaturing conditions, both DNA polymerase and exonuclease activities have the same mobility at several acrylamide gel concentrations. Isoelectric focusing experiments have shown that both activities have the same pI. These data suggest that 3'- to 5'-exonuclease activity is an intrinsic property of DNA polymerase delta. The molecular weight of the enzyme, as estimated from measurements of Stokes radius and sedimentation coefficient, is 152 000.

Animals↗
PubMed1980

Mechanism of inhibition of deoxyribonucleic acid synthesis by 1-beta-D-arabinofuranosyladenosine triphosphate and its potentiation by 6-mercaptopurine ribonucleoside 5'-monophosphate.

The mechanism of inhibition of DNA synthesis by 1-beta-D-arabinofuranosyl-ATP (ara-ATP) and the potentiation of this inhibition by 6-mercaptopurine ribonucleoside 5'-monophosphate (6-MPR-P) have been investigated with mammalian DNA polymerase delty by using poly(dA-dT) as the template. The inhibition of DNA synthesis by ara-ATP correlates with incorporation of ara-AMP into poly(dA-dT). Nearest-neighbor analysis indicates that ara-AMP does not act as an absolute chain terminator but rather that chains with 3'-terminal arabinosyl nucleotides are extended slowly. The inhibition of DNA synthesis by ara-ATP is markedly enhanced by the addition of the nucleotide derivative of 6-mercaptopurine, 6-mercaptopurine ribonucleoside 5'-monophosphate. The increased inhibition of DNA synthesis in the presence of 6-MPR-P is due to increased incorporation of ara-AMP. The mechanism by which 6-MPR-P increases the incorporation of ara-AMP is by selective inhibition of the 3' to 5' exonuclease activity of DNA polymerase, thereby preventing the removal of newly incorporated ara-AMP at 3' termini of DNA chains.

Adenosine Triphosphate↗
PubMed1978

Mechanisms of selective inhibition of 3' to 5' exonuclease activity of Escherichia coli DNA polymerase I by nucleoside 5'-monophosphates.

The 3' to 5' exonuclease activity of Escherichia coli DNA polymerase I can be selectively inhibited by nucleoside 5'-monophosphates, wherease the DNA polymerase activity is not inhibited. The results of kinetic studies show that nucleotides containing a free 3'-hydroxy group and a 5'-phosphoryl group are competitive inhibitors of the 3' to 5' exonuclease. Previous studies by Huberman and Kornberg [Huberman, J., and Kornberg, A. (1970), J. Biol. Chem. 245, 5326] have demonstrated a binding site for nucleoside 5'-monophosphates on DNA polymerase I. The Kdissoc values for nucleoside 5'-monophosphates determined in that study are comparable to the Ki values determined in the present study, suggesting that the specific binding site for nucleoside 5'-monophosphates represents the inhibitor site of the 3' to 5' exonuclease activity. We propose that (1) the binding site for nucleoside 5'-monophosphates on DNA polymerase I may represent the product site of the 3' to 5' exonuclease activity. (2) the primer terminus site for the 3' to 5' exonuclease activity is distinct from the primer terminus site for the polymerase activity, and (3) nucleoside 5'-monophosphates bind at the primer terminus site for the 3' to 5' exonuclease activity.

Binding Sites↗
PubMed1976

A new mammalian DNA polymerase with 3' to 5' exonuclease activity: DNA polymerase delta.

A new species of DNA polymerase has been purified more than 10 000-fold from the cytoplasm of erythroid hyperplastic bone marrow. This DNA polymerase, in contrast to previously described eukaryotic DNA polymerases, is associated with a very active 3' to 5' exonuclease activity. Similar to the 3' to 5' exonuclease activity associated with prokaryotic DNA polymerases, this enzyme catalyzes the removal of 3'-terminal nucleotides from DNA, as well as a template-dependent conversion of deoxyribonucleoside triphosphates to monophosphates. The exonuclease activity is not separable from the DNA polymerase activity by chromatography on DEAE-Sephadex or hydroxylapatite, and upon sucrose density gradient centrifugation the two activities cosediment at 7 S or at 11 S depending on the ionic strength. Both exonuclease and polymerase activities have identical rates of heat inactivation and both are equally sensitive to hemin and Rifamycin AF/013, inhibitors of DNA synthesis that act by binding to DNA polymerase and causing its dissociation from its template/primer. These results are consistent with the coexistence of two enzyme activities in a single protein.

Bone Marrow↗
PubMed1975

Mechanism of hemin inhibition of erythroid cytoplasmic DNA polymerase.

Hemin, which has an important role in the regulation of hemoglobin synthesis, also regulates the activity of cytoplasmic DNA polymerase from erythroid hyperplastic bone marrow cells and reticulocytes. Hemin inhibits DNA synthesis by binding reversibly to the enzyme. Binding assays demonstrated that hemin prevents association and causes dissociation of the DNA-enzyme complex. This is in contrast to inhibitory compounds that specifically interact with DNA such as ethidium bromide and daunomycin which have little or no effect on the DNA polymerase-template complex. Kinetic analysis reveals that hemin inhibition of DNA synthesis is competitive with respect to template and noncompetitive with respect to substrate. The inhibitory effect of hemin can be reversed by subsequent addition of globin, indicating that the inhibition of DNA synthesis by hemin is not due to irreversible inactivation of the enzyme.

Bone Marrow↗
PubMed1974

Metabolic regulation of cytoplasmic DNA synthesis.

The regulation of cytoplasmic DNA synthesis by the metabolites ATP and citrate has been demonstrated. Other ribonucleoside and deoxyribonucleoside triphosphates as well as alpha,beta-methylene- and beta,gamma-methylene-ATP and alpha,beta-methylene-ADP are able to partially substitute for ATP in stimulating the rate of DNA synthesis with the cytoplasmic DNA polymerase (DNA nucleotidyltransferase, EC 2.7.7.7) from bone marrow. The fact that the methylene analogs of ATP and ADP are effective in stimulating DNA synthesis indicates that the mechanism of stimulation does not involve ATP hydrolysis. The nucleotide activators have been shown by kinetic analysis to affect the V(max) of the enzyme and not the apparent K(m)s for the substrates. The curve that results when the rate of DNA synthesis is plotted as a function of ATP concentration is sigmoidal, suggesting that more than one site on the enzyme interacts with ATP and that these sites are acting cooperatively. The concentration of ATP required for maximal velocity is dependent on the Mn(++) concentration. At pH 7.0 maximal activity is obtained when the molar ratio of ATP to Mn(++) is 1.6:1. When either ATP or Mn(++) is present in relative excess, DNA synthesis is inhibited. The mechanism of ATP activation has been shown to be associated with an alteration in the sedimentation behavior of the DNA polymerase. In the presence of ATP, there is an increase in the fraction of the enzyme that sediments at 8 S with a corresponding decrease in the 11.6S enzyme fraction. Thus, ATP activation corresponds to the dissociation of an 11.6S dimer into 8S monomers. In addition to ATP and other nucleotides, citrate also stimulates DNA synthesis. At present it is not clear whether the stimulatory effects of ATP and citrate are due to their ability to chelate Mn(++), which is inhibitory at high concentrations, or whether an ATP-Mn(++) or citrate-Mn(++) complex is the activator.

Adenosine Diphosphate↗
PubMed1973

Reticulocyte RNA-dependent RNA polymerase.

A cytoplasmic, microsomal bound RNA-dependent RNA polymerase has been purified 2500-fold from rabbit reticulocyte lysates. The synthesis of RNA with the purified enzyme is absolutely dependent on the addition of an RNA template. The best template is hemoglobin messenger RNA, while bacteriophage RNA and poly(A,G) are less active, and DNA is completely inactive as a template. With poly(A,G) as a template, only UTP and CTP are incorporated into polynucleotide chains, indicating that the RNA polymerase is an RNA replicase and not a terminal transferase. With messenger RNA as a template, all four ribonucleoside triphosphates are required for maximal activity. The RNA-dependent RNA polymerase reaction is extremely sensitive to low concentrations of heme, rifamycin AF/013, and ribonuclease and resistant to actinomycin D and DNase. The discovery of RNA-directed RNA synthesis in reticulocytes offers an additional site for control of gene expression in mammalian cells and provides a possible mechanism for amplification of the expression of specific genes.

Adenine Nucleotides↗