Isolation of chromaffin cell thapsigargin-sensitive Ca2+ store in light microsomes from bovine adrenal medulla.
1. A subcellular fractionation procedure for bovine adrenal glands was designed with the aim to study the biochemical properties of Ca2+ stores in chromaffin cells. 2. The thapsigargin-sensitive compartment of Ca2+ stores was found to be highly enriched in a light microsomal fraction (LMF) on a 15-30% linear sucrose gradient, and was found to be essentially devoid of contamination by plasma, mitochondrial or secretory granule membranes. 3. A Ca(2+)-pumping ATPase was identified in this LMF as a 97 kDa protein forming an acid-stable, Ca(2+)-dependent, thapsigargin-sensitive phosphorylated intermediate upon incubation with [gamma-32P]ATP, suggesting this protein to represent a SERCA-3 isoform of Ca2+ ATPases. 4. A major 162 kDa protein, previously demonstrated in the isolated chromaffin cells, was enriched in the LMF, distributing on sucrose gradients in parallel with the thapsigargin-sensitive Ca2+ uptake. 5. LMF appears to represent a part of the thapsigargin-sensitive Ca2+ store of chromaffin cells, and should be useful for further studies of the store properties at the subcellular and molecular level.
Mathiasen
D
D
Department of Medical Physiology, Panum Institute, University of Copenhagen, Denmark.
Røssum
L M
LM
Robinson
I M
IM
Burgoyne
R D
RD
East
J M
JM
Møller
M
M
Rasmussen
H N
HN
Treiman
M
M
D000313:Adrenal Medulla / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D008854:Microscopy, Electron; D008861:Microsomes / Q000378:metabolism*; D010766:Phosphorylation; D013729:Terpenes / Q000494:pharmacology*; D019284:Thapsigargin
D013729:Terpenes; D019284:Thapsigargin; D000252:Calcium-Transporting ATPases; D002118:Calcium