Plasmodium falciparum. (solid-support isolation of membrane/polycationic beads). JEAN GRUENBERG AND IRWIN W. SHERMAN. Department of Biology ...
Proc. Nati Acad. Sci. USA Vol. 80, pp. 1087-1091, February 1983
Medical Sciences
Isolation and characterization of the plasma membrane of human erythrocytes infected with the malarial parasite Plasmodium falciparum (solid-support isolation of membrane/polycationic beads)
JEAN GRUENBERG AND IRWIN W. SHERMAN Department of Biology, University of California, Riverside, California 92521
Communicated by George A. Zentmyer, November 22, 1982
was 4% and the parasites were at the trophozoite stage, the cells were labeled metabolically for 12 hr by the addition of 3.7 X 105 becquerels per ml of medium with a mixture of 15 L amino acids. Because no protein synthesis takes place in mature erythrocytes, all of the radioactivity incorporated by infected erythrocytes represents parasite protein synthesis. The absence of leukocytes was checked by examination of Giemsa-stained smears. When 75% of the parasites had matured to multinucleate schizonts (==36-40 hr after synchronization) infected blood cells were collected by centrifugation (1,500 x g, 7 min) and then were separated on a Percoll-Hypaque gradient modified from Vettore et al. (7) (Fig. 1). The top layer of the gradient was recovered, diluted 1:10 with low ionic strength buffer (250 mosM sucrose/50 mosM Na phosphate, pH 7.1), and centrifuged (1,500 x g, 10 min). The cell pellet was washed twice in the same buffer (1,500 X g, 7 min), and then the erythrocytes were immediately processed for isolation of the plasma membranes, as described below. Isolation of the Erythrocyte Plasma Membrane on Polycationic Beads. Polyacrylamide beads coated with polyethylenimine were used as a polycationic solid support (Affi-Gel 731). The procedure was adapted from Jacobson and Branton (8) and Jacobson (9). Beads were hydrated and then were washed twice with 2 M NH4Cl, twice with H20, and twice with the low ionic strength buffer. One-half milliliter of a 50% (vol/vol) suspension of beads was added dropwise to an identical suspension of erythrocytes recovered from four to six gradients. The bare sites on the beads were neutralized immediately with 1 ml of low ionic strength buffer containing dextran sulfate (Mr 5,000) at concentrations which varied from 0.1 to 10.0 mg/ml. (The amount of dextran sulfate corresponds to that added to 0.5 ml of a 50% bead suspension.) The suspension was diluted immediately to 15.0 ml with low ionic strength buffer and the beads were allowed to settle by gravity. The beads were washed twice (gravity sedimentation) in the same buffer, with or without dextran sulfate (0.5 mg/ml). The erythrocyte-covered beads were resuspended either in 1.0 ml of 20 mosM Na phosphate buffer at pH 7.1 or in the same buffer containing uninfected erythrocyte vesicles, vortexed (highest setting) for 15 sec, and centrifuged in a tabletop centrifuge (5 sec). The breakage sequence by vortexing then was repeated. Uninfected erythrocyte vesicles were prepared by osmotic lysis of a 2-ml erythrocyte pellet in 35 ml of 20 mosM Na phosphate buffer, followed by 1 min of vortexing and centrifugation (35,000 x g, 30 min). The membrane pellet was brought to 2 ml by addition of buffer and was sonicated (two times, 1 min each time) by using a probe-type sonicator at 200 W (Braunsonic 1510). After several washings with 15 ml of 20 mosM buffer, the membranes bound to the beads (0.5 ml of a 50% suspension)
ABSTRACT Human erythrocytes infected with the malarial parasite Plasmodiumfalciparum were labeled metabolically with a mixture of 15 radioactive amino acids. When synchronously growing parasites were at the schizont stage of development infected cells were concentrated and purified by using a PercollHypaque gradient. The plasma membrane of the infected erythrocyte, isolated by binding cells to a solid support (Affi-Gel 731, Bio-Rad), was 0.1 mg of dextran sulfate, erythrocytes that were loosely bound to the beads were released, whereas those cells more closely associated with the bead surface were not released, even in the presence of 10.0 mg. The tightly bound cells accounted for -70% of the total number of cells bound in the absence of neutralizing agent, as determined by the amount of Hb (absorption at 410 nm) released during vortexing, as well as the amount of membrane proteins eluted from the beads. The same ratio of tightly bound cells was obtained by monitoring erythrocyte binding to the beads by using either cells infected with metabolically labeled parasites or surface-labeled uninfected cells. Of particular importance was the finding that the binding affinity of uninfected and infected erythrocytes was identical. By contrast, >90% of the binding of radiolabeled parasite debris to the bead surface was blocked by the presence of 0.5 mg or greater amounts of dextran sulfate. Characterization of Infected Erythrocyte Plasma Membranes. When 0.1 mg of dextran sulfate was used, the autoradiographic pattern of parasite proteins recovered from the beads (Fig. 4, lane 3) was identical to the particulate parasite protein pattern (Fig. 4, lane 4), except for slight variations in band in-
Proc. Natl. Acad. Sci. USA 80 (1983)
1089
tensity that resulted from differences in the electrostatic binding capacities of parasite contaminants. The efficiency of the neutralization step was progressively improved by using greater amounts of dextran sulfate-i. e., 0. 1, 1.0, and 10.0 mg. On autoradiograms no increased silver grain density was observed in any particular protein band, although the degree of contamination dropped from 7.0% to 1.2%. By using 10.0 mg of dextran sulfate, contamination of erythrocyte membranes with radiolabeled parasite proteins could be decreased further to 0. 15-0.2% by vortexing beads in the presence of a large excess of normal human erythrocyte cell vesicles. Vesicles competed effectively with parasite debris for binding to the remaining accessible positive charges on the bead surface or possibly binding to sites present on the exposed cytoplasmic face of the erythrocyte membrane. When the latter experiment was repeated with dextran sulfate (0.5 mg/ml) present in the washing solution of intact beadbound cells (as described in Materials and Methods), contamination could be decreased further to
