Fine structure of the band 3 protein in human red cell membranes: Freeze‐fracture studies

Abstract

The major red cell membrane protein, band 3, is a glycoprotein which extends across the membrane from the extracellular space into the cytoplasmic compartment. It is widely held that band 3 is a component of the intramembrane particles (IMP) which can be demonstrated by freeze-fracture electron microscopy. In this study, we find that the outer surface poles of the IMP can be seen by freeze-etching after they are unmasked by proteolysis under conditions which excise the surrounding sialopeptides from the membrane. The poles appear as distinctive projections, 30--50 A in diameter, the "ES particles." The ES particles remain associated with the outer surface of the membrane following cleavage of the band 3 polypeptide by chymotrypsin or pronase. This is consistent with previous biochemical studies which have shown that the 38,000-dalton outer surface segment of band 3 is intercalated in the lipid bilayer. A granulofibrillar component at the inner surface of the membrane is provisionally identified as the 40,000-dalton inner-surface domain of band 3.

Oligomeric structure and the anion transport function of human erythrocyte band 3 protein

“…In the fractured membrane, band 3 remains associated mainly with the inner leaflet [33,149] and thus appears as IMPp (protoplasmic face). Weinstein et al [150] [88] show distinct heterogeneity in particle size and fine structure, suggesting that some particles could represent dimers and others consist of tetramers of band 3.…”

Freeze-fracture studies on barley plastid membranes. III. Location of the light-harvesting chlorophyll-protein

“…The isolation medium contained a higher concentration of divalent cations (Mg *+) to maintain stacking in the mutant (10,41). In order to detect small changes in particle size, shape or density, the technique of rotary shadowing (7,13,26,43) was used and the results are compared with those from conventional uni-directional shadowing.…”

Protein‐dependent lipid lateral phase separation as a mechanism of human erythrocyte ghost resealing

“…To avoid temperatureinduced resealing, proteolytic digestion of unsealed ghosts had to be carried out at low temperature. As reported by Weinstein et al [17], chymotrypsin treatment of unsealed ghosts at 0°C for 1 hr produces extensive digestion of the major membrane proteins (no bands were detectable on SDS gel electrophoresis) but leaves the so-called intramembrane particles morphologically intact. Thus chymotrypsin digested ghosts are a good model to investigate protein-lipid interactions in RBC membrane since, after proteolysis, proteins experiencing polar interactions with lipids are preferentially removed.…”

“…To increase phospholipase activity, erythrocytes were preincubated with 5 mM tetrathionate at 37°C for 3 hr. Proteolytic digestions of intact erythrocytes and unsealed ghosts were performed with 1 mg/ml chymotrypsin or trypsin at 0°C for 1 hr [17].…”