Purification of an inositol 1,4,5-trisphosphate-binding calreticulin-containing intracellular compartment of HL-60 cells.
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.
Van Delden
C
C
Division of Infectious Diseases, Geneva University Hospital, Switzerland.
Favre
C
C
Spät
A
A
Cerny
E
E
Krause
K H
KH
Lew
D P
DP
D002135:Calcium-Binding Proteins / Q000032:analysis*; D037282:Calreticulin; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism* / Q000494:pharmacology; D009251:NADPH-Ferrihemoprotein Reductase / Q000032:analysis; D013429:Sulfatases / Q000032:analysis; D014407:Tumor Cells, Cultured
D002135:Calcium-Binding Proteins; D037282:Calreticulin; D015544:Inositol 1,4,5-Trisphosphate; D009251:NADPH-Ferrihemoprotein Reductase; D013429:Sulfatases