Membrane phospholipids associated with nuclei and chromatin: Melting profile, template activity and stability of chromatin

Abstract

A substantial amount of phospholipid, the distribution pattern of which resembled that found in microsomal membranes, was recovered in chromatin prepared from whole rat liver (“straight” chromatin). The amount of phospholipid, which is taken to indicate the presence of membrane fragments, was reduced in chromatin prepared from purified nuclei (control nuclear chromatin) and was extremely reduced if obtained from nuclei pretreated with Triton X100 (Triton nuclear chromatin). Electron microscopy showed that detergent treatment removed the outer nuclear membrane and the resulting chromatin sometimes appeared partially dehydrated. Labelling of phospholipid with 32P, [ 3H]choline and [ 14C]acetate showed that the over-all rate of turnover of total phospholipid in outer nuclear membrane approximated that of microsomal membranes, but those of inner nuclear membrane were about four times more stable. This difference was reflected in labelling kinetics in chromatin preparations which indicated that phospholipids or membrane fragments in chromatin from whole tissue or nuclei untreated with detergent were derived from the endoplasmic reticulum and the outer nuclear envelope and those in Triton nuclear chromatin from only the inner nuclear membrane. The presence of varying amounts of phospholipids was of little consequence to the melting profile or template activity of the three types of fresh chromatin preparations. But storage at 2 °C (with 10 −3 m-NaN 3) had a drastic effect on the stability of straight chromatin and control nuclear chromatin, which resulted in lower protein/DNA ratios, progressive drop in T m and an enhancement of template activity. Triton-treated nuclear chromatin was stable in this respect for over one month, thus suggesting that proteases associated with the endoplasmic reticulum or the outer nuclear envelope contaminate chromatin prepared without detergent treatment of the tissue or nuclei.