Calmodulin-binding proteins in granule and plasma membranes from bovine chromaffin cells.
Calmodulin-binding proteins in chromaffin granule membrane and chromaffin cell plasma membranes have been investigated and compared. Chromaffin granules were purified by centrifugation over a 1.7 M sucrose layer. Plasma membranes were obtained in a highly purified form by differential and isopycnic centrifugation. Enzymatic determinations of 5'-nucleotidase, a generally accepted plasma membrane marker, showed a 40-50-fold enrichment as compared to the cell homogenate. Marker enzyme studies demonstrated only minimal contamination by other subcellular organelles. After solubilization with Triton X-100, calmodulin-binding proteins were isolated from chromaffin granule membranes and plasma membranes by affinity chromatography on a calmodulin/Sepharose 4B column. On two-dimensional polyacrylamide gelelectrophoresis a prominent protein (Mr = 65,000, pI ranging from 5.1 to 6) consisting of multiple spots, was present in the calmodulin-binding fraction from chromaffin granule membranes as well as from plasma membranes. Besides this 65 kDa protein both fractions had at least four groups of proteins in common. Also, proteins typical for either preparation were observed. In the calmodulin-binding protein preparations from chromaffin granule membranes a prominent spot with Mr = 80,000 and a pH ranging from 5.0 to 5.7 was present. This protein was enzymatically and immunologically identified as dopamine-beta-monooxygenase.
De Block
J
J
Department of Medicine, University of Antwerp (UIA), Wilrijk, Belgium.
Petit
K
K
Van Laer
L
L
Dillen
L
L
Roggen
E
E
De Potter
W
W
D000311:Adrenal Glands / Q000648:ultrastructure*; D000818:Animals; D002148:Calmodulin-Binding Proteins / Q000302:isolation & purification*; D002417:Cattle; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry*; D002499:Centrifugation, Density Gradient; D002500:Centrifugation, Isopycnic; D002837:Chromaffin Granules / Q000648:ultrastructure*; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D007425:Intracellular Membranes / Q000737:chemistry*; D007525:Isoelectric Focusing
D002148:Calmodulin-Binding Proteins