Abstract
Endothelial cell CD36 (glycoprotein IV) has been purified from bovine heart tissue by detergent partitioning and immunoaffinity chromatography. Bovine CD36 differs from human CD36 in its apparent mass (85 versus 88 kDa), primary structure, and immunological cross-reactivity. Of the 18 N-terminal residues identified, 17 conformed to the human CD36 sequence. Mouse monoclonal antibodies E-1 and 8A6 defined bovine- and human-specific epitopes, respectively. Because human CD36 has been identified as a receptor for erythrocytes infected with the malaria parasite Plasmodium falciparum, we examined the ability of bovine CD36 to bind infected erythrocytes. Bovine CD36, unlike human CD36, did not bind infected erythrocytes, suggesting that human CD36-specific structural features are responsible for recognition of the infected erythrocyte ligand.
Highlights
CD36 differs from human CD36 in its apparent mass (85 uersus 88 kDa), primary structure, and immunological cross-reactivity
The human sequence reveals two putative transmembrane regions, one at either end of the protein. Beyond this nothing is known about the structure of CD36 or the mechanisms by which it binds IRBC, collagen, and thrombospondin
Whereas human CD36 has been purified from platelet membrane preparations, purification of this protein from a tissue such as heart tissue in which CD36 constitutes a very small percentage of the total protein would be expected to be more difficult
Summary
Materials-Aprotinin, 2,2’-azinobis(3-ethylbenzthiazolinesulfonic acid), a-_lvcine, SDS, Tris. bovine serum albumin, phenylmethylsulfonyl fluoride, Triton X-114, Triton X-100, nitro blue tetrazolium, amino-n-caproic acid. and SDS-PAGE molecular weight standards were purchased from Sigma. Centrifugation of the extract resulted in a clear supernatant or aqueous phase and a more dense detergent phase layered above an insoluble pellet. 114 was added to the isolated aqueous phase to a final concentration of l%, and the partitioning was repeated This “wash” of the original aqueous phase yielded a second detergent phase and a small pellet of insoluble material. The supernatant was centrifuged at 100,000 X g for 1 h to obtain the final platelet membrane preparation. Infected blood of 75% parasitemia mature asexual parasites was obtained by sedimentation through gelatin (Jensen, 1978) and incubated on plastic Petri dishes coated with lo-r1 spots of either bovine or human CD36 diluted in PBS. When Triton X-114 was present, all samples and standards were adjusted to a final concentration of 0.1% SDS prior to the addition of the bicinchoninic acid reagent
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