STUDIES ON THE RIBONUCLEOPROTEIN PARTICLES

III. SOME PHYSICOCHEICAL CHARACTERISTICS AND THE DEGRADATION WITH RIBONUCLEASE AND TRYPSIN OF THE MICROSOMAL RIBONUCLEOPROTEIN PARTICLES

Abstract

Some physicochemical characteristics of the ribonucleprotein particles isolated from the rat liver microsomes with sodium deoxycholate were examined, and the particles were digested with ribonuclease and with trypsin to estimate the internal structure of the particles.
1. The isoelectric point of the particles was estimated to be pH 3.2-3.6 by turbidimetry. When pH of solution was kept between pH 2-9, these particles were stable and did not dissociate into the component macro-molecules. Whereas below pH 2, considerable protein was released from the particles.
2. About 80-85 per cent of ribonucleic acid of the particles was easily removed by the incubation at 37° for 20 minutes with pancreatic ribonuclease. The removal of nucleic acid resulted in the turbidity increase of the solution probably due to the mutual aggregateon of the particles.
3. Trypsin digested about 30-50 per cent protein of the particles at 37° within 20 minutes. When the ribonucleoprotein particles previously labeled with C¹⁴-leucine was digested with trypsin, the radioactivity per unit weight of protein decresed remarkably with time.
4. Ultracentrifugal studies demonstrated that the core nucleic acid, which was resistant th ribonuclease, was sedimented by centrifugation at 105, 000×g. for 120 minutes. Therefore the core nucleic acid is probably not a small polymucleotide as suggested by Novikoff, but a macromolecular one, and is either covered by protein as in the case of tobacco mosaic virus or is firmly combined with basic protein as in the case of nucleic acid-protamin complex.
Trypsin destructed the particles into smaller fragments, which were not sedimented by the same centrifugal forces.
5. The protein part of the ribonucleoprotein obtained by the incubation with ribonuclease was hardly soluble to Miller-Golder's buffer of pH 2-9 and its maximum turbidity was at pH 5-6. This protein combines easily with yeast ribonucleic acid, and the combination of the both resulted in the turbidity increase in acidic region (pH 2-5) and the turbidity decrease in neutral region (pH 6-8).
5. From these findings there appears no doubt that a considerable part of ribonucleic acid exist on the surface of the particles to give them the negative charge, which is probably the most important factor for the stability of the particles. Further it was estimated that there existed the stable nucleoprotein in the core of the particles, of which metabolic activity was far less active than the surface.
The author wishes to express his thanks to Prof. K. Sasagawa and to Prof. A. Inouye for their encouragement.