PLANT PHYSIOLOGY , Vol 109, Issue 3 983-990, Copyright © 1995 by American Society of Plant Biologists
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BIOCHEMISTRY AND ENZYMOLOGY |
Evidence that Spinach Leaves Express Calreticulin but Not Calsequestrin
L. Navazio, B. Baldan, P. Dainese, P. James, E. Damiani, A. Margreth and P. Mariani
Dipartimento di Biologia, Universita di Padova, Via Trieste 75, 35121 Padova, Italy (L.N., B.B., P.M.)
The presence of either calreticulin (CR) or calsequestrin (CS)-like
proteins in spinach (Spinacia oleracea L.) leaves has been previously
described. Here we report the purification from spinach leaves of two
highly acidic (isoelectric point 5.2) Ca2+-binding proteins of 56 and 54 kD
by means of DEAE-cellulose chromatography followed by phenyl-Sepharose
chromatography in the presence of Zn2+ (i.e. under experimental conditions
that allowed the purification of CR from human liver). On the 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. Therefore, 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 chromatography with concanavalin A-Sepharose.
Furthermore, the 56-kD CR was found to differ from rabbit liver CR in amino
acid sequence, peptide mapping after partial digestion with Staphylococcus
aureus V8 protease, pH-dependent shift of electrophoretic mobility, and
immunological cross-reactivity with an antiserum raised to spinach CR,
indicating a low degree of structural homology with animal CRs.
