Determination of a putative phosphate-containing peptide in calreticulin.
Calreticulin is an abundant endo/sarcoplasmic reticulum (ER/SR) protein that may carry out multiple functions inside cells. Except for calreticulin, all of the major ER/SR Ca2+-binding proteins are substrates for protein kinase CK2 in vitro, which led us to hypothesize that native calreticulin might exist in the phosphorylated form. To investigate this possibility, we purified calreticulin from cardiac microsomes and verified its identity by immunoblot analysis and sequencing of tryptic peptides. Purified calreticulin, like cardiac calsequestrin, contained endogenous phosphate as determined by a Malachite green assay for phosphate. Previous analyses of cardiac calsequestrin have localized phosphate to a single tryptic peptide containing serine phosphate on sites phosphorylated by protein kinase CK2. Using a similar procedure, we analyzed calreticulin tryptic peptides with Malachite green, localizing phosphate binding to a single calreticulin peptide 367LKEEEEDKK. As this peptide contains no phosphorylatable residues, our results suggest that calreticulin may tightly bind phosphate or a phosphate-containing molecule at this site.
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Program in Molecular and Cellular Cardiology, Wayne State University School of Medicine, 421 East Canfield Avenue, Detroit, Michigan 48201, USA.
D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification; D037282:Calreticulin; D002851:Chromatography, High Pressure Liquid; D004285:Dogs; D006352:Heart Ventricles / Q000737:chemistry; D008862:Microsomes, Liver / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry*; D010710:Phosphates / Q000032:analysis*; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000302:isolation & purification; D012394:Rosaniline Dyes; D012519:Sarcoplasmic Reticulum / Q000737:chemistry; D017421:Sequence Analysis; D014357:Trypsin
D002135:Calcium-Binding Proteins; D037282:Calreticulin; D010446:Peptide Fragments; D010710:Phosphates; D012261:Ribonucleoproteins; D012394:Rosaniline Dyes; C005095:malachite green; D014357:Trypsin