Effects of diethyl pyrocarbonate on the isolation and recovery of bound polysomes from a particulate fraction of rat liver

Abstract

Listen

Translate article

The conventional treatment of rat liver postmitochondrial supernatant with detergents permits the recovery of a roughly equal mixture of undegraded free and bound polysomes [ 1 ] , whereas similar treatment of the pellet containing mitochondria, lysosomes and the bulk of bound polysomes leads to the recovery of largely degraded polysomes [2,3] in spite of the use of naturally-occurring nuclease inhibitor [4]. This presumably is due to detergent-mediated activation or release of nucleases that are refractory to nuclease inhibitor [5]. Intact bound polysomes can be prepared from purified rough membrane fractions; however, the whole procedure requires about 2 days of centrifugation and polysome recovery is low [l]. Recently, diethyl pyrocarbonate (DEP), a potent inhibitor of nucleases, has been employed for the preparation of undegraded polysomes and RNA from higher plants and bacteria [6-81. The inactivation of pancreatic ribonuclease by DEP in vitro is believed to result from the formation of intraand intermolecular peptide-like bonds which cause conformational changes and yield polymeric proteins [9]. It was, therefore, of interest to determine whether mitochondrial and lysosomal nucleases could be inactivated by DEP while still in their latent state, and thereby permit isolation of undegraded bound polysomes from a crude particulate fraction of rat liver. In this report we provide evidence that the nuclease activities of the more rapidly sedimenting fraction of