Studies on ribonucleic acid metabolism using nuclear columns. Release of rapidly labeled RNA from rat liver nuclei.
A method is described to study the effect of successively changing incubation conditions on the release of rapidly labeled RNA from isolated nuclei. Nuclear columns containing immobilized rat liver nuclei isolated after in vivo application of labeled orotic acid are perfused with different non-radioactive media. Within the course of one perfusion, the rate of RNA release can be repeatedly altered by variation of temperature, acidity and concentrations of nucleoside triphosphates, complexing agents, sodium chloride and manganese chloride. RNA release can be started and stopped, indicating that the reaction does not result from damage to nuclei. During 60 min perfusion the same product, labeled ribonucleoprotein (sigma = 1.43 g/cm3 in CsCl), is released. High release rates depend on the ratio of nucleoside triphosphate to divalent cation concentration, not on the concentration of the agents per se. Ribonucleoside and deoxyribonucleoside triphosphates exert the same effect as ATP. The SH reagents iodoacetamide and iodoacetate only slightly affect the ATP-induced reaction. In contrast, p-chloromercuribenzoate, after an initial stimulation, causes inhibition of RNA release.
Studies on ribonucleic acid metabolism using nuclear columns. Release of rapidly labeled RNA from rat liver nuclei. A method is described to study the effect of successively changing incubation conditions on the release of rapidly labeled RNA from isolated nuclei. Nuclear columns containing immobilized rat liver nuclei isolated after in vivo application of labeled orotic acid are perfused with different non-radioactive media. Within the course of one perfusion, the rate of RNA release can be repeatedly altered by variation of temperature, acidity and concentrations of nucleoside triphosphates, complexing agents, sodium chloride and manganese chloride. RNA release can be started and stopped, indicating that the reaction does not result from damage to nuclei. During 60 min perfusion the same product, labeled ribonucleoprotein (sigma = 1.43 g/cm3 in CsCl), is released. High release rates depend on the ratio of nucleoside triphosphate to divalent cation concentration, not on the concentration of the agents per se. Ribonucleoside and deoxyribonucleoside triphosphates exert the same effect as ATP. The SH reagents iodoacetamide and iodoacetate only slightly affect the ATP-induced reaction. In contrast, p-chloromercuribenzoate, after an initial stimulation, causes inhibition of RNA release.