Acta biochimica et biophysica; Academiae Scientiarum Hungaricae最新文献

A method is described which allows studying specific cation transport pathways of the cell membranes by converting ion fluxes into volume changes. Lipophilic weak electrolytes, such as propionate, rapidly penetrate the cell membranes in their undissociated acid from but not as negatively charged ions. When human red cells are incubated in isoosmotic K-propionate media an intracellular acidification occurs with a limited propionate uptake and volume increase (corresponding to the buffering capacity of the cytoplasm). If both protons and alkali cations are rendered permeable, a rapid salt influx and volume increase is observed. The latter can be quantitatively followed by electronic sizing methods. A detailed characterization of the system is provided through studies with ionophores, inhibitors of the red cell anion exchange system and drugs which activate or inhibit the Ca2+-induced K+ transport. It is demonstrated that in K-propionate media the permeability of the Ca2+-induced K+ pathway can be directly estimated. The method is suitable to observe population (all-or-none) responses in the activation of the K+ pathway under certain experimental conditions. The application of the method in the search for cation-proton exchanger systems is discussed and its use for demonstration purposes by producing selective lysis of red cells is described.

Human tonsillar lymphocytes incorporate about 7-10 times more [3H]-deoxy-thymidine than [3H]-deoxycytidine at the same extracellular nucleoside concentration, and both incorporations could be inhibited by 10(-5) M arabinosyl-cytosine to 99%. On the other hand, the crude extract of these cells had about 10 times more deoxycytidine kinase than deoxythymidine kinase specific activity, as it was reported previously as well. Therefore, the differences in the labelling of these cells by [3H]-deoxycytidine and [3H]-thymidine cannot be simple due to the different levels of phosphorylating enzymes in the cells. The deoxycytidine kinase of human tonsillar lymphocytes was purified 38.6-fold by DEAE-Sephadex chromatography, and some kinetic and regulatory properties of the purified enzyme were investigated. Deoxycytidine kinase was eluted as a single peak from DEAE-Sephadex column and also from Sephadex G-100 column indicating a molecular weight of about 60 000; no isoenzymes could be detected. During the purification procedure an increase of the enzyme activity was observed suggesting the inhibition of the enzyme in the cells. An apparent Km of 13 microM for deoxycytidine and Ki of 55 microM for arabinosyl-cytosine were found for the tonsillar deoxycytidine kinase. The enzyme was strongly inhibited by dCTP, but not by the other deoxyribonucleoside triphosphates.

We have investigated the interaction of mammalian cells with liposome encapsulated DNA. Tissue cultured mammalian cells were exposed to large, unilamellar phosphatidyl serine liposomes containing DNA molecules from different animal cells or prokaryotic organisms. The liposomes bind rapidly to the surface and are taken up by the cells and significant proportion of the encapsulated DNA is transported to the nuclei. Transient expression of the foreign genetic material could be detected in high percentage of the treated cells for a few days. During this period of time foreign DNA is present in both free and integrated form, however, the free form soon disappears. Stable transformant cell colonies--with continuous expression of new gene(s)--were isolated under selective pressure with a frequency of approx. 10(-5).

Chick thymus glucocorticoid receptor activation was followed in the presence of heparin and/or plasmin. Heparin, at a concentration of 9 microM, accelerated the rate of activation at 25 degrees C without influencing significantly the maximum activated fraction of the 3H-triamcinolone acetonide-receptor complex. On the contrary, 0.7 microM plasmin added prior to incubation at 25 degrees C (activation) of the complex blocked DNA cellulose binding. Thrombin did not influence receptor activation. Added to the activated complex, plasmin resulted in a rapid deactivation, i.e. an irreversible loss of DNA binding capacity. Plasmin and heparin appeared to exert their effects independent of each other in spite of the fact that they are known to interact in the concentration range in question.

The ATP release from "caged" ATP by laser flash was determined at different temperatures and pH values. We measured the time resolved absorbance changes of the coloured intermediate, which is involved in the photolytic process. The linearity of the Arrhenius curves suppose only one process and the calculated activation enthalpies (around 55 kJ/mole) did not change markedly at the pH values of 6.0, 7.0 and 8.0

A 10.3 kilobase EcoRI fragment of Novikoff hepatoma rat ascites tumor ribosomal gene was cloned in pBR322 vector. The cloned fragment contained part of the 18S RNA gene, and about 8 kilobases of the 5' spacer region. A restriction map was constructed by cleavage of the fragment with BamHI, HindIII, KpnI, PstI, SstI and XhoI enzymes. A putative transcription initiation site for the 45S pre-ribosomal RNA was localized by P1 nuclease protection mapping at a distance of about 130 base pairs 5' to the HindIII site on the restriction map. Both the restriction map and the position of the initiation site of transcription were almost identical to those of the corresponding rat ribosomal DNA fragments.

A comparative study of the actions of antibiotics primycin and gramicidin on tracer exchange of red blood cells at steady state salt distribution was carried out. The equilibrium radioactive ion distribution was not achieved at definite concentrations of either antibiotics. A development of Adam's statistical mechanical model for cooperative transitions in membranes are presented to explain the results obtained.