Nuclear Membranes and Plasma Membranes from Hen Erythrocytes: I. ISOLATION, CHARACTERIZATION, AND COMPARISON

Abstract

The bird erythrocyte provides a cell system which comprises only two sorts of membranes, namely the plasma membrane and the nuclear envelope. As calculated morphometrically, both of these membranes are present in an almost 1:1 membrane surface ratio. Contamination with other types of membranes is a priori excluded in this cell. Purified fractions of plasma membranes and nuclear envelope membranes were isolated by using nondetergent methods, in which high speed rotating knife homogenization is combined with differential and gradient centrifugation steps. Nuclear membranes were separated from other nuclear constituents after high salt extraction of nucleoproteins and sonic oscillation, with or without an additional digestion with DNase. Purity and structural integrity of the fractions are shown in electron micrographs. The buoyant densities of nuclear membranes (p422 = 1.20) and plasma membranes (p422 = 1.14) are different. The gross compositions of intact cells and the nuclei, nuclear membrane, and plasma membrane fractions, with respect to lipids, phospholipids, cholesterol, protein, RNA, and DNA, are given, as well as corresponding recoveries. The nuclear membrane is distinct from the plasma membrane as shown in a much lower cholesterol-phospholipid ratio, in a higher content in protein, and a certain amount of DNA remaining firmly attached to it. In general, the properties of the bird erythrocyte plasma membrane agree with those known from the non-nucleated erythrocytes. Among the enzymes examined, the plasma membrane showed ATPase activity, including a ouabain-sensitive one. On the other hand, the nuclear membrane fraction was almost devoid of ATPase activity, but did show the activity of a typical endoplasmic reticulum type enzyme, the NADH-cytochrome c reductase. While the nuclear fraction demonstrated a strong enrichment for NAD+ pyrophosphorylase, this activity was not found in the plasma membranes or in the nuclear membranes, which indicates the low degree of nucleoplasmic protein contamination present in the nuclear membrane fraction. The differences between the only two membrane systems of this dead end differentiated cell type are discussed in context with the present concepts on the cytomembrane → plasma membrane polarization and diversity.