A calcium-binding protein with similarity to serum albumin localized to the ER-Golgi network and cell walls of spinach (Spinacia oleracea)

Using polyclonal antibodies raised against human serum albumin (HSA), a 70- kDa microsomal protein with an isoelectric point of approximately 6.5 was d etected in spinach (Spinacia oleracea L.). The protein was purified by sele ctive ammonium sulfate precipitation and anion exchange HPLC. The protein s hared 100%, identity with the first 15 amino acids at the NH, terminus of H SA, including the X-X-H amino acid region, which was identified in HSA as b eing responsible for binding of copper, zinc, indole derivatives and calciu m. Blue staining of the protein with the cationic carbocyanine dye 'Stains- all' and Ca-45 overlay following SDS-PAGE also suggest that the 70-kDa plan t protein binds calcium. The protein reacted positively with carbohydrate s pecific thymol stain, and the carbohydrates associated with the protein wer e identified by gas chromatography-mass spectrometry (GC-MS) as galactose a nd galacturonic acid. The 70-kDa plant protein was present in the detergent -poor phase following Triton X-114 extraction of the microsomal proteins. C ell fractionation using continuous sucrose gradients showed that the protei n is present in membrane fractions with high activity of endoplasmic reticu lum (ER) and Golgi marker enzymes. Using nitrocellulose tissue prints probe d with anti-HSA antibodies, we demonstrated that the protein is present in the apoplastic space of petioles, suggesting that the protein is secreted t o the apoplast of cortex cells in plants. Localization and binding properti es suggest that the plant protein identified in the present study may parti cipate in secretion processes, possibly involved with the transport of prec ursors required for cell-wall synthesis.