External Labeling Research Articles

External labeling of galactose in surface membrane glycoproteins of the intact myelin sheath.

The molecular organization of surface galactose residues in glycoproteins of the intact myelin sheath was investigated using the enzymatic membrane probe, galactose oxidase. Rat spinal cords treated under physiological conditions with this nonpermanent probe were labeled specifically in galactose residues by reduction with tritiated sodium borohydride. The enzymatically modified proteins from isolated myelin were analyzed electrophoretically and their specific radioactivities determined. Results indicated tritium label associated with a surprising variety of high molecular weight proteins. The most extensively labeled peak corresponded to the major myelin glycoprotein as indicated by the coincidence of tritium label with that of [14C]fucose used as an internal marker for the glycoproteins. The radioactivity associated with this protein was 1.1 to 2.7 times higher after treatment with galactose oxidase when compared to reduction in the absence of the enzyme and 1.4 to 4.8 times higher when oxidized and reduced after prior treatment with neuraminidase. The results suggest a complex heterogeneity of minor glycoproteins associated with isolated myelin. It is concluded that from this complexity of glycoproteins, a major glycoprotein is at least partially localized on the external surface of either the intact myelin sheath or the closely associated oligodendroglial plasma membrane. Such a localization of this glycoprotein and the probable localization of the other glycoproteins enhances their potential role in specific interactions in the process of mpyelination or myelin maintenance.

Structure of the mouse mammary tumor virus: polypeptides and glycoproteins.

The polypeptide and glycoprotein compositions of the mouse mammary tumor virus virion from primary monolayer cultures of BALB/cfC3H mouse mammary tumor cells were studied by polyacrylamide gel electrophoresis by using internal and external labeling and Coomassie blue and periodic acid Schiff (PAS) staining. Twelve polypeptides were reproducibly resolved by the combined methods. Five major polypeptides were demonstrable with estimated molecular weights of 52,000, 36,000, 28,000, 14,000, and 10,000. Seven minor polypeptides were also consistently detected and had estimated molecular weights of 70,000, 60,000, 46,000, 38,000, 30,000, 22,000, and 17,000. Carbohydrate was associated with five of these polypeptides as measured by PAS stain or [(3)H] glucosamine labeling, or both. These glycoproteins had estimated molecular weights of 70,000, 60,000, 52,000, 36,000 and 10,000. The majority of the PAS stain and glucosamine was found in the 52,000 and 36,000 dalton peaks.

External Labeling of Cell Surface Galactose and Galactosamine in Glycolipid and Glycoprotein of Human Erythrocytes

Abstract Treatment of erythrocytes with galactose oxidase (EC 1.1.3.9) followed by reduction with tritiated sodium borohydride (NaB3H4) at pH 7.4 allowed labeling of galactosyl and N-acetylgalactosaminyl residues on external surfaces of cells with tritium (3H). Labeling patterns and specific activities of galactose and galactosamine in glycolipids and glycoproteins were determined after separation with gel electrophoresis and thin layer chromatography. The labeling patterns of normal adult cells differed greatly from fetal cells, and were significantly altered when cell surfaces were modified by proteases and neuraminidase. The results of analysis indicated that (a) carbohydrate moieties of two glycolipids (globoside and ceramide trihexoside) and at least three glycoproteins (molecular weight 9.5, 8.2, and 6.4 x 104) were exposed to external environment, but not ceramide dihexoside, ceramide monohexoside, or other glycoproteins with higher molecular weights; (b) specific activities of galactosamine in glycolipid and of galactose in glycoprotein increased after protease treatment, although total activity of glycoprotein did not change; (c) labeling of glycoprotein was greatly enhanced by neuraminidase treatment, while that of glycolipid was enhanced to a lesser degree; (d) the relative exposures of glycoprotein and glycolipid differed greatly between normal and fetal erythrocyte surfaces. Glycoproteins of fetal cells had a very low label as compared to glycolipid.