Interactions of the complex secretory vesicle binding protein chromobindin A with nucleotides.
Chromobindin A is a large, multisubunit protein that binds to chromaffin granule membranes in a Ca2+- and ATP-regulated manner. Ca2+ stimulates binding to the membrane, whereas ATP, in the the absence of Ca2+, is required for release of the protein from the membrane. We now report that spectral and HPLC data indicate that nucleotides are associated with the native chromobindin A complex and that the protein can bind two molecules of [3H]ATP in vitro. Chromobindin A also appears to be a novel nucleotide triphosphatase. ATPase activity was detected in fractions containing chromobindin A isolated by affinity chromatography, gel filtration, or ion exchange chromatography. Kinetic studies indicated that the Vmax is 44 nmol of Pi/mg/min and the Km is 0.115 mM, whereas the nonhydrolyzable ATP analog 5'-adenylylimidodiphosphate acts as a competitive inhibitor of this reaction with a Ki of 0.08 mM. The activity was found to be sensitive to protease treatment or to preincubation at 65 degrees C and was inhibited by Ca2+ or low pH. The ATPase activity was not inhibited by N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide, vanadate, oligomycin, or azide.
Martin
W H
WH
Department of Pharmacology, University of Virginia School of Medicine, Charlottesville 22908.
Creutz
C E
CE
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D017302:Annexins; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002837:Chromaffin Granules / Q000378:metabolism; D002845:Chromatography; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D006868:Hydrolysis; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008566:Membranes / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism*
D017302:Annexins; D002352:Carrier Proteins; D008565:Membrane Proteins; D009711:Nucleotides; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases