ISOLATION OF CHROMAFFIN CELL THAPSIGARGIN-SENSITIVE CA2-MEDULLA( STORE IN LIGHT MICROSOMES FROM BOVINE ADRENAL)

1. A subcellular fractionation procedure for bovine adrenal glands was designed with the aim to study the biochemical properties of Ca2+ sto res in chromaffin cells. 2. The thapsigargin-sensitive compartment of Ca2+ stores was found to be highly enriched in a light microsomal frac tion (LMF) on a 15-30% linear sucrose gradient, and was found to be es sentially devoid of contamination by plasma, mitochondrial or secretor y granule membranes. 3. A Ca2+-pumping ATPase was identified in this L MF as a 97 kDa protein forming an acid-stable, Ca2+-dependent, thapsig argin-sensitive phosphorylated intermediate upon incubation with [gamm a-P-32]ATP, suggesting this protein to represent a SERCA-3 isoform of Ca2+ ATPases. 4. A major 162 kDa protein, previously demonstrated in t he isolated chromaffin cells, was enriched in the LMF, distributing on sucrose gradients in parallel with the thapsigargin-sensitive Ca2+ up take. 5. LMF appears to represent a part of the thapsigargin-sensitive Ca2+ store of chromaffin cells, and should be useful for further stud ies of the store properties at the subcellular and molecular level.