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Isolation of chromaffin cell thapsigargin-sensitive Ca2+ store in light microsomes from bovine adrenal medulla

1993; Elsevier BV; Volume: 25; Issue: 5 Linguagem: Inglês

10.1016/0020-711x(93)90348-i

1878-6014

David P. Mathiasen, L.M. Røssum, Iain M. Robinson, Robert D. Burgoyne, J. Malcolm East, Mads P. Møller, Hanne N. Rasmussen, Marek Treiman,

Heat shock proteins research

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.