M. Paparelli

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.

Electron microscopical evidence of the evolution of corynebacteria-like microorganisms within human erythrocytes

The corynebacteria-like microorganisms evoluting in the haemocultures take origin from electron dense granular bodies carried within the erythrocytes.

Intracellular distribution of biotin-14COOH in rat liver

Biotin clearance, its distribution in liver and liver fractions after intravenous administration of 5 μCi/100 g body weight (21.55 μg) of biotin-14COOH in normal and biotin-deficient rats are reported. In the biotin deficient animal there is a more rapid disappearance of the labeled biotin from the blood stream. Biotin-14COOH incorporation in the liver of the deficient rat is more rapid and larger than the incorporation in normal rat liver. Almost all the biotin recovered from liver homogenate is found in the mitochondria and in the pH 5.2 cytosol fraction; whereas in the microsomes only a very small amount is present. The intracellular distribution of biotin is in agreement with its known metabolic roles.

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.

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.

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.

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.

Effects of biotin deficiency on serum proteins and plasma amino acids

In biotin-deficient rats, a decrease of total proteins, attributable to a decrease of albumin anda 1-globulin fractions, a decrease of the pre-β-lipoproteins and an increase of the α-lipoproteins, was observed, together with a rise of total amino acids. Such a situation may be related to the influence of biotin on the synthesis of RNA and proteins.