EVIDENCE THAT SPINACH LEAVES EXPRESS CALRETICULIN BUT NOT CALSEQUESTRIN

The presence of either calreticulin (CR) or calsequestrin (CS)-like pr oteins in spinach (Spinacia oleracea L.) leaves has been previously de scribed. Here we report the purification from spinach leaves of two hi ghly acidic (isoelectric point 5.2) Ca2+-binding proteins of 56 and 54 kD by means of DEAF-cellulose chromatography followed by phenyl-Sepha rose chromatography in the presence of Zn2+ (i.e. under experimental c onditions that allowed the purification of CR from human liver). On th e other hand, we failed to identify any protein sharing with animal CS the ability to bind to phenyl-Sepharose in the absence of Ca2+. Based on the N-terminal amino acid sequence, the 56- and 54-kD spinach Ca2-binding proteins were identified as two distinct isoforms of CR. Ther efore, we conclude that CR, and not CS, is expressed in spinach leaves . The 56-kD spinach CR isoform was found to be glycosylated, as judged by ligand blot techniques with concanavalin A and affinity chromatogr aphy with concanavalin A-Sepharose. Furthermore, the 56-kD CR was foun d to differ from rabbit liver CR in amino acid sequence, peptide mappi ng after partial digestion with Staphylococcus aureus V8 protease, pH- dependent shift of electrophoretic mobility, and immunological cross-r eactivity with an antiserum raised to spinach CR, indicating a low deg ree of structural homology with animal CRs.