Isolation and Characterization of the Vascular-specific 22-kDa Zn-binding Protein

Abstract

A 22-kDa Zn-binding protein (ZBP) was isolated from the phloem tissue and evacuated xylem sap of `Valencia' sweet orange [Citrus sinensis (L.) Osbeck] on rough lemon [C. jambhiri (L.)], as well as Valencia on Rangpur lime [Citrus limonia Osbeck]. Phloem and xylem Zn was associated with the 22 kDa ZBP. The Mr value of this ZBP was estimated to be 19,500 by size exclusion chromatography and 22,800 by SDS-PAGE. This protein was isolated with an isoelectric point of 7.5. Ion exchange chromatography demonstrated that 22-kDa ZBP was highly anionic, requiring 0.43 M NaCl for elution from QAE Sepharose. The 22-kDa ZBP appears unique to citrus, having no cross reaction with protein from several tissues from a range of plant species. Accumulation decreased under Zn-deficient conditions, was enhanced by osmotic stress, and the protein completely disappeared with wounding. Amino acid composition demonstrated that the protein was rich in aspartate, and glutamate; and contained 6 cysteine, and 4 histidine residues. These amino acids may be involved in metal binding. N-terminal amino acid sequencing demonstrated that the 22-kDa ZBP had identity with sporamin A&B precursors, Kunitz-type trypsin inhibitors, and miraculin. It is suggested that the genes that encode these proteins are derived from a common ancestral gene.