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Abstract
T200 glycoprotein, a major cell surface component of murine hematopoietic cells, is a phosphorylated transmembrane glycoprotein. Two distinct regions of the molecule can be defined by radiolabeling with a variety of metabolic precursors or by lactoperoxidase-catalyzed iodination, in combination with protease treatments, immunoprecipitation techniques, and peptide "mapping" analysis. A relative protease-resistant domain, which is exposed on the cell surface and contains the antigenic site recognized by a monoclonal anti-T200 antibody known to react with the exterior cell surface, contains most if not all of the mannose-containing oligosaccharide units of the glycoprotein and all of the amino acid residues labeled by lactoperoxidase-catalyzed iodination of intact viable cells. This protease-resistant fragment migrates with an apparent molecular weight of approximately 100,000 in sodium dodecyl sulfate-polyacrylamide gels. The remaining portion of the molecule contains a region, extensively digested by trypsin, which is exposed on the cytoplasmic side of the plasma membrane and contains phosphoserine residues which can be labeled with 32PO4 in vivo. A 125I-labeled tryptic peptide derived from this region of the molecule was obtained if membrane preparations from cells disrupted by nitrogen cavitation were labeled by lactoperoxidase-catalyzed iodination.
Highlights
Thy-1 glycoprotein may not be exposed on the cytoplasmic side of the plasma membrane [3, 16,17,18]
A lZ5I- glycoprotein is presentlyunknown; the restricted labeled tryptic peptide derived from this region of the tissue distribution of this molecule and differencesin its molecule was obtained if membranepreparations from structure on different subpopulations of hematopoietic cells cells disrupted by nitrogen cavitation were labeled by suggest the molecule may have a biological function related lactoperoxidase-catalyzed iodination
16).In eachcase, additional labeled peptides were obtained in tiallyresolved into two species in someexperiments) was experiment.. in which unsealed erythrocyte ghostswere used found in immunoprecipitates of T200 glycoprotein from iodirather than intacctells
Summary
Gens and Ia antigens of murine and human lymphoid cells (IO-14), andmembrane-bound immunoglobulin andThy-1. M PMSF in acetonedepending on the amounot f TPCK-trypsin used While this procedure gave preparations of labeled T200 glycoprotein Proteolysis in the presence of 1mM HgClz was carried out in the same of high purity from iodinated cells or cells labeled with mannose, it way except that 50 p1 of 10 mM disodium ethylenediaminetetraacetic was not satisfactory for the isolation of other preparations of labeled acid (NazEDTA) in 0.09 M NaCl, 0.01 M phosphate buffer (pH 7.2). 0.1% SDS, and 1% Trasylolfor 20 min on ice. The digestion was terminated by adding 10 p1 of 0.2 M iodoacetamide and supernatant of the cell lysate obtained by centrifugation a t 20,000 x S. aureus V8 proteolysis by adding 10 pl of 0.1 M PMSF. The oligosaccharide unitsonthe labeledspecies fromthe mutant and wild type cells
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