Roles of lipids and proteins in the calcium(2+)-phosphate-induced aggregation of cytoskeleton-free erythrocyte vesicle membranes

Abstract

The roles of lipids and proteins in Ca2+-PO4-induced membrane aggregation were investigated. Cytoskeleton-free vesicles derived from intact human and rabbit erythrocytes (HEves and REves, respectively) were employed as a model system. The HEves and REves have a simplified membrane protein composition [band 3 proteins and glycoproteins PAS-1, -2, and -3 (HEves)] and normal lipid composition. Optimal experimental conditions for pH, [PO4], and [CaCl2] were determined for quantitatively examining the dynamics and extent of HEves and REves aggregation, measured turbidimetrically. The aggregation process was found to be quite sensitive to small changes in pH and [PO4] and much less sensitive to [CaCl2]. The roles of membrane proteins in vesicle aggregation were examined by selectively modifying the proteins enzymatically. The roles of lipids were studied by using sonicated lipid vesicles [small unilamellar vesicles (SUVs)] made from Dodge ghost lipid extracts. Enzymatic treatment with trypsin, chymotrypsin, or Pronase had no effect on either the rates or the extent of vesicle aggregation (2-min incubation period). Neuraminidase treatment reduced both factors by approximately 20%. SUVs aggregated with Ca2+-PO4 in a way which depended on the PO4/lipid ratio. Together the results suggest the following: (1) PO4 is associated with the vesicle surface, involving the membrane lipids; (2) the vesicle + PO4 incubation time component of the PO4 effect is eliminated by enzymatically modifying the vesicle membrane proteins; (3) qualitative, rather than quantitative, properties of sialic acid containing molecules affect vesicle aggregation; and (4) with the exception of the incubation time effect, membrane proteins seem neither to promote nor to inhibit Ca2+-PO4-induced HEves or REves aggregation.

A possible role of cholesterol in membrane adhesion

“…Calcium phosphate induced vesicle aggregation was monitored turbidimetrically. For a detailed discussion concerning the turbidity assay, its limitations, and the optimization of the experimental conditions for studying the aggregation of these vesicles, see Leonards & Ohki (1984). In brief, an aliquot (see figures) of the stock erythrocyte or lipid vesicles was suspended in 0.1 M NaCl and 2 mM phosphate at pH 7.6.…”

“…It should be noted that the experimental conditions employed in the present study were specifically chosen to avoid membrane fusion (Leonards & Ohki, 1984). One of the advantages of using calcium phosphate is that turbidity changes due to vesicle aggregation are fully reversible (OD400 returns to 0) by acidifying the medium with acetic acid, while those due to membrane fusion are not (Zakai et al, 1976).…”

“…In order to investigate further the molecular processes involved in these membrane aggregation and fusion events, we have produced a simple model membrane from erythrocytes, specifically cytoskeleton-free erythrocyte membrane vesicles (Leonards & Ohki, 1983), and studied the aggregation and fusion of these vesicles. It was observed that these vesicles aggregated in the presence of calcium phosphate and that aggregation was not affected by modifying various protein residues of the membrane surface with enzyme treatments (Leonards & Ohki, 1984). This suggested to us that membrane lipids are responsible for this aggregation phenomenon.…”