Functional mosaicism of the lymphocyte plasma membrane. Characterization of membrane subfractions obtained by affinity chromatography on concanavalin A-Sepharose

Abstract

Microsomal membranes (consisting largely of plasma membrane) or purified plasma membranes from calf thymocytes were fractionated by affinity chromatography on concanavalin A-Sepharose. Two fractions were obtained: one, designated MF1, eluted freely from the affinity adsorbent containing two thirds of the membrane protein. A second fraction, designated MF2, containing one third of the membrane protein, adhered to concanavalin A-Sepharose and was recovered after mechanical dissociation. Both fractions were homogeneous as assessed by rechromatography. The following control experiments suggested that the fractions were derived from different areas of an individual cell. (1) Fractionation required the binding of membranes to (Sepharose-bound) concanavalin A. (2) The non-retarded fraction MF1 exhibited more binding sites for concanavalin A, suggesting that both fractions were right side-out. (3) Intact thymocytes could not be fractionated on concanavalin A-Sepharose, which made it likely that the membrane subfractions did not originate from different cells. This was supported by an identical fractionation of plasma membranes isolated from a homogeneous T-lymphocyte tumor line. (4) Several membrane-bound enzymes (γ-glutamyltransferase, adenylate cyclase, Mg 2+-ATPase) exhibited similar specific activities in both subfractions as well as in the unseparated membrane. In addition to previous findings that the cholesterol/phospholipid ratio was identical (Brunner, G., Ferber, E. and Resch, K. (1976) Differentiation 5, 161–164), this suggests that MF1 and MF2 consisted predominantly of identical membranes, i.e. the plasma membrane. The affinity for concanavalin A was 2.5 times higher in MF2 as compared to MF1. Both fractions were distinct in plasma membrane-bound enzymes: ( K + + Na + )-ATPase exhibited high specific activity in MF2 compared to MF1 or the unseparated membrane. Similarly, acyl-CoA:lysophosphatidylcholine acyltransferase was alos enriched in MF2. Alkaline p- nitrophenyl phosphatase showed also higher activity in this fraction. The data suggest a close association of receptors with high affinity for concanavalin A and certain membrane-bound enzymes. As activation of lymphocytes was initiated when 14–24% of the available binding sites interacted with concanavalin A, the data suggest a functional mosaicism in the vicinity of activating receptors.