S. Coderoni

Effect of biotin on phosphorylation, acetylation, methylation of rat liver histones

Biotin deficient rat liver histones showed decreased phosphorylation and methylation, and increased acetylation rates as compared to normal rat liver histones: these alterations may be related to the observed lower stability of the interactions between histones and DNA. The modifications of the metabolic process might be the consequence of an alteration of the synthesis of the enzymes involved in histone phosphorylation, acetylation and methylation mechanisms and are presumably related to a biotin effect upon the synthesis of RNA and proteins.

Small peptides controlling transcription in vitro are bound to chromatin DNA.

Low-molecular-weight peptides are linked to the chromatin DNA of several tissues, from which they can be dissociated by alkaline extraction at pH 9.5. The level of the active peptide fraction ranges between 10 and 35 micrograms/mg DNA. The removal of peptides from DNA causes a relevant amplification of DNA template capacity for prokaryotic and eukaryotic RNA polymerases. Gel filtration on Sephadex G-25 or BioGel P4 shows that the chromatin peptide fraction from purified DNA migrates as a sharp peak with an elution volume corresponding to a molecular weight of about 1000. The chromatin peptides are further purified by Sephadex G-10 and high-performance liquid chromatography. Four active fractions are isolated, one of which shows very high inhibition activity on the RNA synthesis in vitro. The amino acid analysis and the inhibition mechanism of the purified peptides are reported.

Phosphorylation sites for type n ii protein kinase in DNA-topoisomerase i from calf thymus

1. Calf thymus DNA-topoisomerase I has been isolated, in an improved preparation, nearly to SDS-PAGE homogeneity, as a single major protein (100 kDa). 2. In vitro labeling experiments, which employed the purified enzyme [gamma-32P]ATP and N II protein kinase, also showed that the calf thymus topoisomerase I became phosphorylated. 3. Phosphorylation was accompanied by an increase in topoisomerase I activity. 4. Phosphoaminoacid analysis indicated that only serine residues became phosphorylated. 5. Tryptic peptides mapping, by HV electrophoresis, identified five major [32P]peptides. This number is higher than that reported for topoisomerase I from Novikoff hepatoma cells. 6. Separation of each spot, by reverse phase HPLC, resulted in their elution at fractions 1, 2, 3, 4 and 5 with 9, 11, 16, 27 and 28% acetonitrile, respectively. 7. Isolated phosphopeptides will be subjected to sequencing, to DNA-binding and transcription regulation tests; then, it will be speculated whether type N II protein kinase may contribute to the physiological regulation of DNA topoisomerase I activity from calf thymus, as well.

Red cell lysis induced by microorganisms as a case of superoxide- and hydrogen peroxide-dependent hemolysis mediated by oxyhemoglobin

Some bacteria, isolated from the blood of hospitalized patients, have been shown to hemolyze red blood cells through a mechanism which was dependent on the oxygenated state of intracellular hemoglobin, since transformation of hemoglobin into the CO-derivative inhibited the lysis. Hemolysis was also inhibited by superoxide dismutase and catalase, while only catalase prevented the formation of methemoglobin in experiments where isolated oxyhemoglobin was exposed to metabolizing bacteria. Production by bacteria of extracellular superoxide was demonstrated. It is suggested that hemolysis is due to interaction of O-2 and/or H2O2 with intracellular hemoglobin and that some product of such interaction is the lytic agent.

Effect of CPT on the calf thymus Topoisomerase I-mediated DNA breakage-reunion reaction: optimal conditions for the formation and reversal of the CPT trapped Topoisomerase I cleavable complex.

The effects of CPT on the calf thymus Topoisomerase I-mediated DNA breakage-reunion reaction were studied at an enzyme concentration range proper for evidencing, at the same time, both DNA relaxation and DNA cleavage/religation. Some of the requirements and the optimal conditions for the formation and reversal of the CPT-trapped Topoisomerase I-DNA cleavable complex are also characterized. We conclude that:1.Calf thymus (100 kDa) Topoisomerase I requires, for maximal DNA cleavage activity, specific and characteristic reaction conditions.2.CPT does not affect these optimal conditions, but only stabilizes the normal enzyme-DNA intermediate. In this way, the drug lowers the religation process, becoming responsible for the relaxation inhibition.3.The optimum of monovalent salt concentration for cleavable complex formation is found between 30 and 70 mM. These values are lower than those required for the relaxation activity optimum (75–125 mM NaCl).4.The addition of 0.5 M monovalent salt causes reversal of the reaction, and shifts the equlibrium distribution between cleavable intermediate and closed relaxed DNA in the direction of DNA resealing. Therefore, it is suggested that salt affects the cleavage but not the religation reaction.

Role of calf thymus DNA-Topoisomerase I phosphorylation on relaxation activity expression and on DNA-protein interaction

Calf thymus DNA-Topoisomerase I activity was found to be altered by changing in phosphorylation: it was completely inhibited upon dephosphorylation by alkaline phosphatase, but incubation with N II protein kinase and ATP restored the relaxation activity to a level higher than that observed prior to dephosphorylation. The calf thymus Topoisomerase I-mediated DNA cleavage, induced by camptothecin, also proved to be inhibited by dephosphorylation, which, apparently, stabilizes the initial enzymesubstrate complex. We conclude that:- the native protein is partially phosphorylated,- the phosphorylation involvement is essential for the activity expression and also for DNA-protein interaction,- changes in the degree of phosphorylation might be involved in the regulation of DNA processing; that evokes some properties of chromatinic peptide models, which bind DNA only when phosphorylated and leads to the assumption that they represent the minimum functional substrate for N II protein kinase.

Optimum DNA relaxation reaction conditions for calf thymus DNA-topoisomerase I are determined by specific enzyme features

Reactivity and chemical properties of calf thymus Topoisomerase I have been investigated with respect to enzyme ability to relax supercoiled DNA. The relaxation rate has been analyzed at optimum and relatively high salt concentration. Catalysis is processive at optimum salt concentration and distributive at a higher one; camptothecin decreases the initial rate of reaction in both salt conditions, but more so at the higher one. We conclude that:1.calf thymus Topoisomerase I requires, for its maximum reactivity, specific and characteristic reaction conditions;2.salt concentration affects DNA processing, indeed influencing the initial rate of DNA relaxation and directly reflecting the salt-dependence for the enzyme-duplex DNA binding;3.Topoisomerase I, from various sources, maybe individually responds to alteration of assay parameters such as pH, Mg++ and NaCl concentrations, indicating that individual criteria could be responsible for the catalytic activity optimum.

Effect of CPT on the DNA cleavage/religation reaction mediated by calf thymus Topoisomerase I: evidence of an inhibition of DNA religation. Inhibition of Topoisomerase I-mediated DNA religation by CPT.

The uncoupling of the calf thymus Topoisomerase I-mediated forward DNA cleavage reaction from the religation event by a rapid shift of cleavage temperature either from 37 °C to 0 °C or from 37 °C to 56 °C has been studied and utilized to elucidate the molecular mechanism by which camptothecin, a clinically relevant antineoplastic agent, influences the half reactions of the enzyme. Results of heating and cooling religation-inducing treatments have been compared: both temperature extremes reduce the amount of protein-linked DNA breaks to background levels, thereby affecting cleavage reversal. Camptothecin is found to stabilize the enzyme-DNA intermediate, by inhibition of the Topoisomerase I-mediated rejoining of cleaved DNA, even when the drug is added after formation of the complex. We conclude that:1.Heating and cooling treatments show a pronounced effect on the DNA cleavage-religation equilibrium. The efficacy of cold is more pronounced than that of heat.2.Reversal of the enzyme-DNA intermediate favors the DNA resealing versus the closed relaxed form.3.Camptothecin affects the heat or cold induced religation: in fact in both cases the drug delays the religation step.

Specific regulatory role of phosphorylation of calf thymus DNA-topoisomerase I smaller forms on the relaxational activity expression

Calf thymus Topo I is found to be associated with three active breakdown products, resolved from intact enzyme, which do not appear to be unique to one extraction procedure. They are phosphoproteins, whose enzymatic activity can be modulated through changes in phosphorylation, and which can be phosphorylated ‘in vitro’, by N II protein kinase, in the same five sites as the intact enzyme. Different amounts of 32P incorporated are observed however, in the corresponding sites. We conclude:1.Proteolysis is probably an ‘in vivo’ phenomenon, as the Topo I smaller species are observed, during isolation from the earlier crude fractions, and as a minimum of them is always present, even if precautions are taken to minimize proteolysis;2.a specific regulatory role in the DNA relaxational activity might be played by N II protein kinase phosphorylation, indeed, in the smaller species;3.the different degrees of 32P incorporation, in analogous phosphorylation sites, might represent a different signal for modulating the gene expression.

DNA-binding peptides from rat liver and Novikoff hepatoma cells: quantitative level and possible biochemical differences

DNA isolated from rat liver by intensive deproteinization with chloroform/isoamyl alcohol and phenol contains low molecular weight peptides in a quantity of about 20 μg/mg DNA.These peptides show high specific activity in inhibiting transcription in a reconstituted cell-free system with prokaryotic and eukaryotic RNA polymerase. Their level is markedly decreased in DNA prepared from Novikoff hepatoma cells. Moreover the amino acid analysis and the pattern of analytical separation by high performance liquid chromatography (HPLC) show some biochemical differences between DNA-binding peptides extracted from rat liver and Novikoff hepatoma cells. The possibility that carcinogenesis may involve mechanisms which lead to selective removal of some components of the DNA-binding peptides, is discussed.