Identification and characterization of glycoproteins after extraction of bovine chromaffin-granule membranes with lithium di-iodosalicylate. Purification of glycoprotein II from the soluble fraction.
Chromaffin-granule membranes were separated into insoluble and soluble fractions after extraction with lithium di-iodosalicylate (LDIS). These fractions were characterized by one- and two-dimensional gel electrophoresis, and glycoproteins were detected after electroblotting with peroxidase-labelled concanavalin A and wheat-germ agglutinin (WGA). The LDIS-insoluble fraction contained components identified as glycoproteins III, H, J and K (carboxypeptidase H). Microsequence analysis indicated that component J is an N-terminally extended form of glycoprotein K. A major glycoprotein, GpII (Mr 80,000-100,000), present in the LDIS-soluble fraction was purified by affinity chromatography on WGA-Sepharose. This was characterized by one- and two-dimensional gel electrophoresis with Coomassie Blue staining, by amino acid analysis and automated N-terminal sequence analysis. Extraction of chromaffin-granule membranes with LDIS is a simple and rapid procedure that facilitates studies concerned with the structure and function of membrane glycoproteins from these and other secretory granules.
Department of Biochemistry, University of Auckland, New Zealand.
D L
DL
Christie
D J
DJ
Palmer
D000313:Adrenal Medulla / Q000032:analysis*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002837:Chromaffin Granules / Q000032:analysis*; D002838:Chromaffin System / Q000032:analysis*; D015180:Electrophoresis, Gel, Two-Dimensional; D007463:Iodobenzoates; D008562:Membrane Glycoproteins / Q000032:analysis / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D011975:Receptors, Mitogen / Q000032:analysis; D012459:Salicylates; D012995:Solubility
D000596:Amino Acids; D007463:Iodobenzoates; D008562:Membrane Glycoproteins; D011975:Receptors, Mitogen; D012459:Salicylates; C020891:3,5-diiodosalicylic acid