Soluble and Membrane Lectin‐binding Glycoproteins of the Chromaffin Granule

Abstract

Abstract— Fluorescein isothiocyanate‐labelled lectins were used to identify lectin‐binding glycoproteins of the chromaffin granule after electrophoresis of the membrane and soluble granule proteins on sodium dodecyl sulphate polyacrylamide slab gels. The glycoprotein nature of all lectin‐binding bands was confirmed by staining the gels for carbohydrates, and the specificity of the lectin‐binding was demonstrated by hapten sugar inhibition of binding. In samples of granule membrane proteins reduced with dithiothreitol 10 concanavalin A (Con A), 5 wheat germ agglutinin, 8 Ricinus communis agglutinin‐60, and 7 Ricinus communis agglutinin‐120 (RCA‐120) binding glycoproteins were identified. Molecular weights of these glycoproteins varied from 20,000 to 200,000 daltons. All but two of the Con A‐binding bands and one of the RCA‐120 binding bands appeared to react with more than one lectin, suggesting possible carbohydrate heterogeneity in these membrane glycoproteins. The band identified as dopamine β‐hydroxylase reacted most intensely with all four lectin tested, and in the soluble core material this enzyme was the sole significant lectin binding glycoprotein.

Glycoproteins of the Chromaffin Granule Membrane: Separation by Two‐Dimensional Electrophoresis and Identification by Lectin Binding

“…Are all the lectin-binding bands really glycoproteins? For a number of reasons we believe that they are: first, as observed by Cahill and Morris (1979), the binding of each lectin was specifically abolished by the hapten sugar; second, although many components bound more than one lectin, most lectins showed considerable selectivity; third, binding was very sensitive to the procedure used for iodinating the lectins, often not being observed if the standard chloramine-T procedure was used, or the protecting hapten sugar omitted ; fourth, neuraminidase treatment of the membranes greatly increased the binding of several lectins, notably DBA, SBA, and UEA.…”

“…H u b e r et al (1979) used t h e periodate-Schiff method of staining electrophoretograms and affinity chromatography on lectin columns to isolate detergent-solubilized glycoproteins; by this means they detected five glycosylated components. Cahill and Morris (1979) used fluorescein-conjugated lectins to detect glycoproteins, after separation on sodium dodecyl sulphate (SDS)-polyacrylamide slab gels, and by this means detected about 12 components. Abbs and Phillips (1980) used both chemical and lectin labelling, and their results are in good agreement with those of Cahill and Morris (1979).…”

“…Cahill and Morris (1979) used fluorescein-conjugated lectins to detect glycoproteins, after separation on sodium dodecyl sulphate (SDS)-polyacrylamide slab gels, and by this means detected about 12 components. Abbs and Phillips (1980) used both chemical and lectin labelling, and their results are in good agreement with those of Cahill and Morris (1979). T h e studies of Roda e t al.…”

“…2; "indicates a protease-sensitive band. Identities refer to (1) Abbs and Phillips (1980), who used numbers for Coomassie Blue-staining bands, and letters for glycoproteins; (2) Huber et al (1979); (3) Cahill and Morris (1979). Strengths of labelling by different lectins are on the scale + (detectable) to + + + + (very strong).…”

Dopamine β‐Hydroxylase and Other Glycoproteins from the Soluble Content and the Membranes of Adrenal Chromaffin Granules: Isolation and Carbohydrate Analysis

“…Furthermore, it has not yet been established whether the two forms of the enzyme, i.e., the soluble and the membranebound one, have the same sugar composition (see Skotland and Ljones, 1979). In addition to this enzyme the membranes of chromaffin granules contain several other glycoproteins, as shown by electrophoretic studies employing periodic acid-Schiff (PAS) stain o r lectin binding (Eagles et al, 1975;Zinder et al, 1978;Cahill and Morris, 1979;Huber et al, 1979;Abbs and Phillips, 1980;Roda et al, 1980). These glycoproteins have different affinities for two lectins, a s demonstrated by affinity chromatography with Concanavalin A (Con A) a n d wheat germ agglutinin (Huber et al, 1979).…”