Effects of Sodium Chloride and Pancreatic Ribonuclease on the Rat‐Brain Nuclear Particles: The Fate of the Protein Moiety

Abstract

Previous results on RNA-labelled nuclear particles were compatible with a folded ribonucleoprotein strand structure. In the present experiments, particles were labelled with tritiated amino acids or with tritiated amino acids and [32P]orthophosphate in vim; they were prepared by sonication of nuclei.1. Proteins were released progressively from the particles (e = 1.39 g/ml) as shown by the accumulation of material of e = 1.31 -1.35 g/ml after treatment with 0.25 M and 0.4 M NaCl. Concomitantly, high-density ribonucleoproteins were formed.2. Proteins released from the particles at various NaCl concentrations (up to 2 M) sedimented between 0 and 15 S. 30-S aggregates corresponding to "informofers" were never detected. Some proteins remained bound to the RNA even after treatment with 2 M NaCl.3. Large protein aggregates contaminated the smallest particles (up to 50-60 S) as shown by the sedimentation properties of the products of extensive ribonuclease digestion and the density of protein-labelled fractions. 4.About half of the protein released from the particles by pancreatic ribonuclease treatment had a low sedimentation coefficient (0-15 S) after formaldehyde fixation. The other half had higher polydisperse sedimentation coefficient, and possibly resulted from artificial cross-linking. A 30-S protein peak was not detected. 5.The material released from the particles by ribonuclease banded at e = 1.35 g/ml, a higher density than that of free proteins (p = 1.31 g/ml). Treatment with 1 M NaCl after ribonuclease digestion released free proteins, some of which were phosphoproteins (e = 1.31 g/ml) and ribonucleoproteins of higher density. It is suggested that these complexes contain ribonuclease-resistant RNA.These results confirm and complete our previous ones. They are not compatible with the informofer model of the monoparticle since 30-S protein aggregates could not be isolated after salt or ribonuclease treatment. They are consistent with the folded ribonucleoprotein strand model which predicts the sequential release of proteins.In a previous study [1,2] of the action of ribonuclease and salts on nuclear particles containing DNA like RNA we could not find any evidence in favor of the "informofer" model proposed by Samarina et al.[3] and Lukanidin et al. [4,5], according to which, the RNA was localized at the surface of a 30-S protein aggregate, the informofer, stable at high salt concentration. On the contrary, our results were compatible with a folded ribonucleoprotein strand model. However, no direct proof for such a structural organization where only a fraction of the RNA sequences would be external could be furnished.Since our previous experiments [1,2] had only been performed with particles containing labelled RNA, so that the fate of the protein moeity was not followed, the evidence against the informofer was indirect. This paper reports results obtained with particles labelled either on their protein, or on both their RNA and protein moieties. METHODS Labelling of Par tidesIn order to obtain highly labelle...

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