Binding of SSB-protein from Ehrlich ascites carcinoma cells with DNA and polyribonucleotides]

Binding of SSB-protein from Ehrlich ascites tumor to ssDNA from M13 phage leads to its compactization. The structure of the complex at the protein/DNA ratios far from the saturation level looks like "beads-on the string". DNA that was fully saturated with protein forms collapsed globular structure. Binding of the protein to the dsDNA from phage lambda increases its flexibility and decreases the coil dimensions; no "beads-on the string" structure are seen. The protein possess slight destabilizing effect on hairpin helices of M13DNA. Competition studies demonstrate that the binding properties of protein with polyribonucleotide lattices and DNA's decrease in ranking as follows: poly(rG) greater than or equal to poly(rI) greater than or equal to ssDNA greater than dsDNA greater than poly(rA) congruent to approximately poly(rU). Thus SSB-protein from Ehrlich ascites tumor differs significantly from its presumed prokaryotic analogs.     

The role of protein binding with single-stranded DNA (SSB-protein) in DNA replication in Ehrlich ascites carcinoma cells

Possible involvement of the single-strand DNA-binding protein (SSB-protein) in DNA replication in Ehrlich ascite tumour (EAT) cells was studied. There was a direct correlation between the content of SSB-protein in chromatin and the intensity of replicative synthesis of DNA in various preparations of EAT in vitro and in vivo (the computed value of the correlation coefficient was equal to 0.9). It was shown that the addition of exogenous SSB-protein to permeable EAT cells increased the replicative synthesis. It was concluded that although eukaryotic SSB-proteins are not complete analogs of prokaryotic ones, they may participate in DNA replication in eukaryotic cells and, possibly, are intracellular regulators of proliferation.     

Oxygen dependence of nuclear DNA replication in Ehrlich ascites cells

Oxygen was excluded from cultured Ehrlich ascites cells for 5-7 h and then readmitted. During the anaerobic period and for about 1 h following reoxygenation the DNA synthesis of the cells was studied by determining the DNA synthesis rate from [3H] thymidine incorporation data, by evaluation of the thymidine (pulse labelling) index, by DNA fibre autoradiography, and by alkaline sucrose gradients in order to follow the maturation of the daughter chains. The DNA synthesis rate was found to decay to a few percent of the initial value within 5-7 h after deoxygenation. Immediately after reoxygenation it increased to exceed the control level within 0.5-1 h. The only partial process of the genome replication definitely responding to deoxygenation/reoxygenation was the initiation of new replicon units, while progress of the replication forks and maturation of the new daughter chains were not significantly affected. The coordination of replicon initiation within groups or clusters was maintained throughout. The interruption of replication at the level of initiation of clusters upon deoxygenation was interpreted as a regulatory response of the cells to ensure basic viability under unfavourable conditions.     

Isolation of replication forks from growing Ehrlich ascites cells

A procedure is described which permits the large-scale isolation of essentially complete replications forks from the DNA of Ehrlich ascites cells. The whole nuclear DNA is first isolated by a method which involves minimal hydrodynamic shear. The DNA is then degraded by cryolysis, a freeze-thawing procedure, to a size providing the otherwise very labile forked structures with a sufficient resistance against shear forces. Finally, the Y-shaped structures of replicating DNA are separated by nitrocellulose column chromatography. When the newly formed strands of replicating DNA were density-labeled with 5-bromodeoxyuridine the DNA fraction isolated by this procedure banded in isopycnic CsCl gradients at a density expected for Y-shaped molecules with two light-heavy branches and one light-light branch and sedimented significantly faster than the corresponding bulk DNA fraction through neutral sucrose gradients. The forked molecules could be visualized by electron microscopy. The essential step of the procedure is the cryolysis which produces fragments from larger DNA structure essentially at random. When the cryolysis is omitted the forked structures are disrupted within the highly susceptible regions around the branching point.     

DNA ligase from mouse Ehrlich ascites tumor cells

The molecular (Mr = 120,000; s20, w = 5S) and catalytic properties (Km (ATP) = 3 microM; Km (nicked DNA) = 0.2 microM; Km (Mg2+) = 3 mM) of DNA ligase from mouse Ehrlich ascites tumor cells are similar to those of the enzymes from calf thymus and rodent liver. The activity level of DNA ligase from the tumor cells is about 10-fold higher than that from mouse liver. Immunochemical titration of DNA ligase with antibodies against the calf thymus enzyme showed that the higher level of DNA ligase activity in the tumor cells is due to an increase in enzyme quantity and not to elevation of the catalytic efficiency of the enzyme molecule. These results suggest that there is little apparent difference between the qualities of DNA ligases from the tumor cells and normal tissues of rodents and calf.     

DNA methylase: purification from ascites cells and the effect of various DNA substrates on its activity.

DNA methylase has been purified 405-fold from Krebs II ascites cells. The purified enzyme is homogeneous on SDS-poly acrylamide gel electrophoresis (molecular weight about 80,000) and the only product of the reaction with DNA is 5-methyl cytosine. Both native and denatured DNA are methylated by the enzyme; with calf thymus DNA the double stranded form is the better substrate but the enzyme preferentially methylates single stranded E.coli DNA even in "native" preparations. Our results do not support a mechanism whereby the enzyme methylates DNA by binding irreversibly and "walking" along it. By measuring maximum levels of methylation of DNAs from different sources we have estimated the proportion of unmethylated sites present in them. Homologous ascites DNA can be methylated, but only to about 5% of the level of the best substrate, undermethylated mouse L929 cell DNA. DNA isolated from growing cells or tissues is a better substrate than DNA from normal liver or pancreas, or from stationary cells.     

The inhibitory effect of various fatty acids on aerobic glycolysis in Ehrlich ascites tumour cells

Inhibition of glycolysis in Ehrlich ascites tumour cells by saturated fatty acids, added either in form of potassium salts or incorporated into phosphatidylcholine liposomes, increases with the increasing carbon atom chain length and is independent of the concentration within the range of 0.1 to 1.0 mM. In contrast, the inhibition of glycolysis in the cytosolic fraction from Ehrlich ascites cells depends on the concentration of fatty acids. The content of ATP in Ehrlich ascites cells incubated with fatty acids increases with increasing carbon atom chain length, which leads to a crossing-over in the concentrations of pyruvate and 2-phosphoenolpyruvate. Lowering of the sum of both these metabolites by palmitate and stearate points to the inhibition not only of pyruvate kinase but also of other enzymes of early steps of glycolysis. Fatty acids in intact Ehrlich ascites cells inhibit all three key glycolytic enzymes but added to the cytosolic fraction affect mainly the activity of phosphofructokinase. The inhibition of pyruvate kinase by fatty acids is smaller in the cytosolic fraction from tumour cells than from liver and muscles.     

The binding of SSB-proteins with DNA in chromatin of Ehrlich ascites carcinoma cells]

Using UV-induced cross-linking between proteins and DNA, the contacts between single-stranded DNA-binding proteins (SSB proteins) and chromatin DNA have been demonstrated. Ehrlich ascites tumour DNA was labeled in vivo by inoculation of tumour-bearing mice with 3H-thymidine. The cells were irradiated with the UV light dose of 3000 J/m2, destroyed in a Triton X-100-containing hypotonic medium, and separated by centrifugation into the extrachromatin fraction and chromatin. Chromatin DNA was digested with DNAase 1, and the chromatin proteins were extracted with 2 M NaCl-polyethyleneglycol. SSB proteins from the extrachromatin fraction and chromatin were purified. Only SSB proteins from UV-irradiated cell chromatin appeared to possess a high specific radioactivity which exceeded 7.5-fold that of non-irradiated cells. There were no differences between chromatin SSB proteins in control and irradiated cells as could be evidenced from SDS electrophoresis data. It is assumed that in irradiated cells SSB proteins of DNA-digested chromatin are covalently cross-linked with DNA fragments.     

DNA polymerase in nucleoli isolated from Ehrlich ascites tumor cells

DNA replication was investigated in nucleoli isolated from Ehrlich ascites tumor cells. DNA synthesis was dependent on the presence of the four deoxynucleoside triphosphates and magnesium, but was reduced in the presence of ATP. The pH optimum for DNA replication was 8.5 to 9.0 N-Ethyl-maleimide reduced the reaction significantly. DNA synthesis occurred on nucleolar chromatin and was stimulated by treatment of the nucleoli with a small amount of DNase I. Addition of exogenous DNA to the reaction mixture significantly stimulated [³H]dTMP incorporation.     

Participation of the SSB protein in the repair of the DNA of Ehrlich ascites carcinoma cells following UV irradiation

Synchronous changes were detected in the SSB-protein content of the chromatin and in the rate of repair DNA synthesis at different time intervals after UV-irradiation of Ehrlich ascites tumor cells. The amount of SSB-protein in the extra-chromatin fraction was in an inverse relation to its content in the chromatin, whereas the cumulative SSB-protein content remained invariable. Similar changes in the SSB-protein content of the chromatin and in repair synthesis were also registered after the effect of various doses of UV-light. The increase of the SSB-protein content in the chromatin was not connected with the postirradiation accumulation of single-strand sites in DNA.     

Participation of the SSB protein in the repair of the DNA in Ehrlich ascites carcinoma cells following gamma radiation

Survival of Ehrlich ascites tumor cells, SSB content of the chromatin, and repair DNA synthesis rate were investigated after gamma-irradiation, the rate of repair synthesis was shown to depend on the SSB-protein content of the chromatin. Changes in the amount of SSB-protein in the chromatin were not connected with the postirradiation accumulation of single-strand sites in DNA.     

Purification and characterization of 3-phosphoglycerate kinase from Ehrlich ascites carcinoma cells

3-Phosphoglycerate kinase (3-PGK) has been purified to apparent homogeneity from Ehrlich ascites carcinoma (EAC) cells by (NH4)2SO4 precipitation, gel filtration and ion-exchange chromatography. The enzyme has been partially characterized and compared with the characteristics of this enzyme of other normal and malignant cells. The EAC cell 3-PGK is composed of a single subunit of 47 kDa. It has a broad pH optimum (pH 6.0-7.5) for its enzymatic activity. The apparent Km values of 3-phosphoglycerate (3-PGA) and ATP for 3-PGK have been found out to be 0.25 mM and 0.1 mM respectively. Similar to 3-PGK of other cells, the EAC enzyme requires either Mg2+ or Mn2+ for full activity; the optimum concentrations of Mg2+ and Mn2+ are 0.8 mM and 0.5 mM respectively. When ATP and 3-PGA act as substrates, ADP, the reaction product of 3-PGK-catalyzed reaction has been found to inhibit this enzyme. Kinetic studies were made on the inhibition of ADP in presence of the substrates ATP and 3-PGA. Attempts to hybridize 3-PGK and glyceraldehyde-3-phosphate dehydrogenase of EAC cells by NAD or glutaraldehyde were unsuccessful.