RESUMO
Analysis of the properties of the DNA polymerase (pol) system as a function of fundamental factors of the assay environment allowed a rather accurate estimation of its dependence on the HeLa cell cycle. For pol alpha, the temperature and pH optima were 38.1 degrees C and 8.0, respectively; for pol beta, these optima were 36.2 degrees C and pH 7.4. Pol gamma showed a pH optimum at 7.7. Optimum activity for both the alpha and beta enzymes was observed at 60 mM Tris. The maximal activity at 36.2 degrees C and pH 7.4 was associated with resistance to N-ethylmaleimide (MalNEt), whereas that at 38.1 degrees C and pH 8.0 was sensitive to MalNEt. Incorporation of [3H]dTTP was maximal after 1 hr of incubation for the former activity and after 4 hr, for the latter. In extracts from cells in early S phase, the pol activity decreased after 1 hr of incubation, was MalNEt-resistant, and was characterized by temperature and pH optima at 36.2 degrees C and 7.4, respectively. In extracts of late S-phase cells, the pol-catalyzed incorporation of [3H]dTTP continued after 4 hr of incubation, was MalNEt-sensitive, and was characterized by temperature and pH optima at 38.1 degrees C and 8.0, respectively. Thus, a pol beta-type activity appeared in early S phase, whereas a pol alpha-type activity appeared in late S. During the G1, M, and G2 phases, a background level of pol activity was observed that showed intermediate kinetic properties.
Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Células HeLa/enzimologia , Ciclo Celular , DNA Polimerase I/metabolismo , DNA Polimerase II/metabolismo , DNA Polimerase III/metabolismo , Etilmaleimida/farmacologia , Células HeLa/citologia , Humanos , Concentração de Íons de Hidrogênio , Inibidores da Síntese de Ácido Nucleico , TemperaturaAssuntos
Abrina/antagonistas & inibidores , Concanavalina A/farmacologia , Leucemia Experimental/tratamento farmacológico , Proteínas de Plantas/antagonistas & inibidores , Ricina/antagonistas & inibidores , Abrina/metabolismo , Animais , Fracionamento Celular , Linhagem Celular , Vírus da Leucemia Murina de Friend , Células HeLa/efeitos dos fármacos , Humanos , Camundongos , Proteínas de Neoplasias/biossíntese , Ricina/metabolismoAssuntos
Abrina/toxicidade , Concanavalina A/farmacologia , Proteínas de Plantas/toxicidade , Ricina/toxicidade , Abrina/antagonistas & inibidores , Abrina/metabolismo , Animais , Linhagem Celular , Interações Medicamentosas , Vírus da Leucemia Murina de Friend , Células HeLa/efeitos dos fármacos , Células HeLa/metabolismo , Humanos , Leucemia Eritroblástica Aguda/metabolismo , Leucemia Experimental/metabolismo , Camundongos , Ricina/antagonistas & inibidores , Ricina/metabolismoRESUMO
The glycoproteins ricin and abrin intoxicate cells by inhibiting protein synthesis. Pretreatment of HeLa cells with cholera toxin partially protects them from ricin and abrin activity. The involvement in this phenomenon of the various effects of cholera toxin, namely, redistribution of membrane receptors elicited from protomer B and increasing cyclic AMP concentrations induced by protomer A, were studied. Substances able to enhance cyclic AMP concentrations do not affect ricin and abrin activity, while protomer B alone protects cells. In addition, the effects of several lectins on ricin or abrin toxicity were examined. Almost complete prevention of ricin or abrin activity was obtained using concanavalin A (Con A) and wheat germ agglutinin (WGA). Conversely, neither succinyl Con A nor Ulex europeus agglutinin (UEA) affected the cellular response. Both protomer B of cholera toxin and Con A did not alter the binding of ricin or abrin; they seem to protect cells by altering membrane structure.
