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Scientific article
English

Purification of an inositol 1,4,5-trisphosphate-binding calreticulin-containing intracellular compartment of HL-60 cells

Published inBiochemical journal, vol. 281 ( Pt 3), p. 651-656
Publication date1992
Abstract

To investigate the identity of Ins(1,4,5)P3-sensitive intracellular Ca2+ stores in myeloid cells, we have developed a method that yields subcellular fractions highly enriched in Ins(1,4,5)P3 binding. HL-60 cells were disrupted by nitrogen cavitation, and subcellular fractions were obtained by differential centrifugation, followed by Percoll- and sucrose-density-gradient separations. A subcellular fraction enriched 26-fold in Ins(1,4,5)P3-binding sites was obtained. This fraction showed no enrichment in plasma-membrane markers and only a comparatively moderate enrichment (7-fold) in endoplasmic-reticulum markers. The ratio between specific enrichment of Ins(1,4,5)P3 binding and endoplasmic-reticulum markers in the different fractions varied over 50-fold, from less than 0.1 to greater than 5. The purified Ins(1,4,5)P3-binding fraction was enriched to a similar extent (27-fold) in the putative intravesicular Ca(2+)-storage protein calreticulin. Our results favour the concept of a distinct Ins(1,4,5)P3-binding, calreticulin-containing compartment (i.e. the calciosome) in HL-60 cells.

Keywords
  • Calcium-Binding Proteins/ analysis
  • Calreticulin
  • Cell Fractionation
  • Cell Membrane/metabolism
  • Centrifugation, Density Gradient
  • Endoplasmic Reticulum/chemistry/ metabolism
  • Humans
  • Immunoblotting
  • Inositol 1,4,5-Trisphosphate/ metabolism/pharmacology
  • NADPH-Ferrihemoprotein Reductase/analysis
  • Sulfatases/analysis
  • Tumor Cells, Cultured
Citation (ISO format)
VAN DELDEN, Christian et al. Purification of an inositol 1,4,5-trisphosphate-binding calreticulin-containing intracellular compartment of HL-60 cells. In: Biochemical journal, 1992, vol. 281 ( Pt 3), p. 651–656.
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ISSN of the journal0264-6021
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