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

Abstract: Chromogranin A and two other proteins (A1 and A2) of the soluble proteins of bovine chromaffin granules were isolated by extraction from polyacrylamide gels after electrophoresis. The carbohydrate content of these proteins was 5%, with galactose, N‐acetylgalactosamine, and sialic acid as the main sugars. Membranes of chromaffin granules were solubilized with sodium dodecyl sulphate (SDS) and three glycoproteins were isolated by sequential affinity chromatography on Concanavalin A (Con A) and wheat germ lectin (WGL) Sepharose columns. Two glycoproteins, designated GP II and III, were found to have a high carbohydrate content of about 30%. Mannose, galactose, N‐acetylgalactosamine, and sialic acid were the main sugars. In addition membrane‐bound dopamine β‐hydroxylase was isolated by this procedure. No significant differences between the carbohydrate composition of the membrane‐bound and the soluble enzyme were revealed. It was shown that all four subunits of dopamine β‐hydroxylase possess carbohydrate chains with an affinity for Con A. The isolation methods established in this study will be useful for immunological studies on these glycoproteins.

[1]  R. U. Margolis,et al.  Glycoproteins and proteoglycans of the chromaffin granule matrix. , 1982, The Journal of biological chemistry.

[2]  D. L. Wong,et al.  Regulation of dopamine beta-hydroxylase synthesis and degradation ascorbic acid stabilization of the enzyme against tryptic proteolysis. , 1981, The Journal of biological chemistry.

[3]  E. Westhead,et al.  Th molecular organization of adrenal chromaffin granules , 1980, Neuroscience.

[4]  S. Udenfriend,et al.  Structure of two adrenal polypeptides containing multiple enkephalin sequences. , 1980, Archives of biochemistry and biophysics.

[5]  I. Hanbauer,et al.  Secretion of enkephalin-like peptides from canine adrenal gland following splanchnic nerve stimulation , 1980, Neuropeptides.

[6]  O. H. Viveros,et al.  Synthesis of enkephalins by adrenal medullary chromaffin cells: reserpine increases incorporation of radiolabeled amino acids. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[7]  J. Phillips,et al.  Organisation of the proteins of the chromaffin granule membrane. , 1980, Biochimica et biophysica acta.

[8]  R. Fischer‐Colbrie,et al.  ISOLATION, SUGAR COMPOSITION AND IMMUNOLOGICAL CHARACTERISATION OF GLYCOPROTEINS OF CHROMAFFIN GRANULES , 1980 .

[9]  T. Flatmark,et al.  Subcellular distribution of ascorbate in bovine adrenal medulla. Evidence for accumulation in chromaffin granules against a concentration gradient. , 1980, Biochimica et biophysica acta.

[10]  E. Bock,et al.  Immunochemically identical hydrophilic and amphiphilic forms of the bovine adrenomedullary dopamine beta-hydroxylase. , 1979, The Biochemical journal.

[11]  T. Flatmark,et al.  On the amphiphilic and hydrophilic forms of dopamine β‐mono‐oxygenase in bovine adrenal medulla , 1979, Journal of neurochemistry.

[12]  S. Morris,et al.  SOLUBLE AND MEMBRANE LECTIN‐BINDING GLYCOPROTEINS OF THE CHROMAFFIN GRANULE , 1979, Journal of neurochemistry.

[13]  H. Plattner,et al.  CHARACTERIZATION AND TOPOGRAPHY OF THE GLYCOPROTEINS OF ADRENAL CHROMAFFIN GRANULES , 1979, Journal of neurochemistry.

[14]  G. Avvakumov,et al.  Analysis of methyl glycosides as their trimethylsilyl ethers: on-columns Re-N-acetylation and improved gas— liquid chromtographic separation , 1978 .

[15]  R. U. Margolis,et al.  Composition and biogenesis of complex carbohydrates of ox adrenal chromaffin granules , 1977, Neuroscience.

[16]  R. Lotan,et al.  Activities of lectins and their immobilized derivatives in detergent solutions. Implications on the use of lectin affinity chromatography for the purification of membrane glycoproteins. , 1977, Biochemistry.

[17]  K. Helle,et al.  Resistance of Dopamine-β-Hydroxylase to proteolysis during tryptic digestion of the adrenomedullary chromogranins , 1977 .

[18]  H. Winkler.  The composition of adrenal chromaffin granules: An assessment of controversial results , 1976, Neuroscience.

[19]  H. Hörtnagl.  Membranes of the adrenal medulla: A comparison of membranes of chromaffin granules with those of the endoplasmic reticulum , 1976, Neuroscience.

[20]  T. Flatmark,et al.  Purification and Characterization of Dopamine β ‐Hydroxylase from Bovine Adrenal Medulla , 1976 .

[21]  L. Johnson,et al.  The distribution of Concanavalin A receptor sites on the membrane of chromaffin granules. , 1975, Journal of cell science.

[22]  P. Mandel,et al.  Bovine adrenal medullary chromogranin A: studies on the structure and further evidence for identity with dopamine-beta-hydroxylase subunit. , 1975, Biochimica et biophysica acta.

[23]  R. E. Stephens.  High-resolution preparative SDS-polyacrylamide gel electrophoresis: fluorescent visualization and electrophoretic elution-concentration of protein bands. , 1975, Analytical biochemistry.

[24]  S. Udenfriend,et al.  The interaction of dopamine-beta-hydroxylase with concanavalin A and its use in enzyme purification. , 1974, Biochemical and biophysical research communications.

[25]  H. Lochs,et al.  Immunological studies on the acidic chromogranins and on dopamine β hydroxylase (EC 1.14.2.1) of bovine chromaffin granules , 1974, Journal of neurochemistry.

[26]  A. D. Smith,et al.  Adrenal chromaffin granules: isolation and disassembly. , 1974, Methods in enzymology.

[27]  E. F. Wallace,et al.  Dopamine-β-Hydroxylase: A Tetrameric Glycoprotein , 1973 .

[28]  F. Schneider.  Isolation of and production of antibody to cow chromaffin granule membrane. , 1972 .

[29]  H. Lochs,et al.  Membrane proteins of chromaffin granules, dopamine -hydroxylase, a major constituent. , 1972, The Biochemical journal.

[30]  F. H. Schnideer.  Isolation of and production of antibody to cow chromaffin granule membrane. , 1972, Biochemical Pharmacology.

[31]  J. Clamp,et al.  An assessment of methanolysis and other factors used in the analysis of carbohydrate-containing materials. , 1971, The Biochemical journal.

[32]  D. Wallach,et al.  Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. , 1971, Biochemistry.

[33]  K. Helle,et al.  Chromogranin: The soluble and membrane-bound lipoprotein of the chromaffin granule , 1969 .

[34]  A. D. Smith,et al.  Some properties of soluble proteins from chromaffin granules of different species. , 1968, Biochemical pharmacology.

[35]  A. D. Smith,et al.  Purification and properties of an acidic protein from chromaffin granules of bovine adrenal medulla. , 1967, The Biochemical journal.

[36]  A. D. Smith,et al.  A simple method for the isolation of adrenal chromaffin granules on a large scale. , 1967, The Biochemical journal.

[37]  N. Hillarp,et al.  The Isolation of n Granular Fraction from the Suprarenal Medulla, Containing the Symyathomimetic Catechol Amines1 , 1953 .

[38]  O. H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.