Cytoplasmic Ribonucleoprotein Components of the Novikoff Hepatoma

Abstract

Prominent nucleoprotein sedimentation boundaries were demonstrable in cytoplasmic extracts of Novikoff hepatoma. Fractionation of the homogenates by differential centrifugation or a density gradient method revealed that 65 to 75 per cent of the cytoplasmic ribonucleic acid was present in the form of free ribonucleoprotein particles. After purification by differential centrifugation in dilute buffer, the particles contained 37 per cent RNA (ribonucleic acid) very little lipid, and no demonstrable membrane material. Ultracentrifugal boundaries corresponding to those seen in the original extracts were present, the main component having an S20,w of 81 S. Upon exposure to chelating agents, the particles dissociated through an intermediate component with sedimentation rate of 56 S to a final stage in which 46 and 28 S subunits were present in a weight ratio of 21. ATP (adenosine triphosphate) and pyrophosphate were equally effective in causing dissociation. ADP (adenosine diphosphate) was considerably less effective. Treatment of the purified particles with deoxycholate removed one-third of the protein and significantly altered the ultracentrifugal pattern. The particles now dissociated directly to the 46 and 28 S subunit when exposed to chelating agents. Upon electron microscopy, the 81 S particle appeared as an oblate spheroid 24 mu in diameter. The 46 and 28 S subunits also appeared spheroidal.