IMMUNOCHEMICAL STUDIES OF INFECTIOUS-MONONUCLEOSIS .5. ISOLATION AND CHARACTERIZATION OF A GLYCOPROTEIN FROM GOAT ERYTHROCYTE-MEMBRANES

  • 1 January 1976
    • journal article
    • research article
    • Vol. 117  (3) , 722-729
Abstract
A glycoprotein was isolated from goat erythrocyte membranes by extraction with hot 75% ethanol. The glycoprotein was purified by ethanol precipitation, phosphocellulose chromatography gel filtration, ethyl:ether and chloroform:methanol extraction. In aqueous phosphate-buffered saline, pH 7, the glycoprotein was in an aggregated state with a sedimentation coefficient (Sobs) of 21. Addition of 0.5% sodium dodecyl sulfate (SDS) lowered Sobs to 1.5. Electrophoresis of the glycoprotein on polyacrylamide gels containing phosphate-buffered 0.1% SDS gave a single band, staining with periodic acid Schiff (PAS) and Coomassie Blue (CB). The apparent MW, calculated from retardation coefficient, was 25,000. Electrophoresis of the glycoprotein on 1% SDS gels buffered with Tris-acetate (pH 7.4) showed a major band of similar (23,500) apparent MW plus 4 other PAS- and CB-staining bands of lower mobility. 131I-labeled glycoprotein, recovery of bands from gels, sialic acid analysis, heterophile antigen activity and re-electrophoresis showed that these additional bands were aggregated forms of a single or closely related glycoprotein species. The purified glycoprotein contained 50% carbohydrate with molar ratios of sialic acid: galactose: mannose: galactosamine: glucosamine of 3.1:2.1:0.1:1.6:1. The glycoprotein was highly reactive with the Paul-Bunnell heterophile antibody in the sera of patients with infectious mononucleosis, with Limulus polyphemus lectin and weakly reactive with wheat germ agglutinin. These reactivities were destroyed by neuraminidase treatment or by alkaline sodium borohydride. The native glycoprotein did not react with lectins from Canavalia ensiformis, Phaseolus vulgaris, Ricinus communis or Vicia graminea, although it was reactive with the latter 2 after neuraminidase treatment.

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