Inositol 1,4,5-trisphosphate binding sites copurify with the putative Ca-storage protein calreticulin in rat liver.
Rat liver was homogenized and subjected to differential centrifugation. When the low speed nuclear pellet was processed on a Percoll gradient, plasma membrane markers and Ins(1,4,5)P3 binding activity purified together. The high speed (microsomal) fraction was subfractionated by sucrose density gradient centrifugation, resulting in 10-fold enrichment of [32P]-Ins(1,4,5)P3 binding. In the sucrose density gradient fractions there was an inverse relationship between the enrichment of plasma membrane markers and Ins(1,4,5)P3 binding sites. Endoplasmic reticulum markers showed a moderate enrichment in the fractions displaying high Ins(1,4,5)P3 binding activity. Calcium binding proteins in the homogenate and in the microsomal subfractions were separated by SDS/PAGE. A 60 kD protein, stained metachromatically with Stains-All was identified as calreticulin with immunoblotting. Its enrichment pattern was similar to that of Ins(1,4,5)P3 binding sites, indicating the co-existence of these two elements of Ca(2+)-metabolism in the same intracellular compartment in the liver.
Department of Physiology, Semmelweis University of Medicine, Budapest, Hungary.
P
P
Enyedi
G
G
Szabadkai
K H
KH
Krause
D P
DP
Lew
A
A
Spät
D000818:Animals; D015415:Biomarkers; D015220:Calcium Channels; D002135:Calcium-Binding Proteins / Q000302:isolation & purification*; D037282:Calreticulin; D002451:Cell Compartmentation; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000737:chemistry; D005260:Female; D015151:Immunoblotting; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008099:Liver / Q000737:chemistry* / Q000648:ultrastructure; D008862:Microsomes, Liver / Q000737:chemistry; D008970:Molecular Weight; D051381:Rats; D017208:Rats, Wistar; D011956:Receptors, Cell Surface / Q000302:isolation & purification*; D018160:Receptors, Cytoplasmic and Nuclear; D012261:Ribonucleoproteins / Q000302:isolation & purification*; D013347:Subcellular Fractions / Q000737:chemistry*
D015415:Biomarkers; D015220:Calcium Channels; D002135:Calcium-Binding Proteins; D037282:Calreticulin; D053496:Inositol 1,4,5-Trisphosphate Receptors; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D012261:Ribonucleoproteins; D015544:Inositol 1,4,5-Trisphosphate