Subcellular and Tissue Distribution, Partial Purification, and Sequencing of Calmodulin‐Stimulated Ca2+‐Transporting ATPases from Barley (Hordeum Vulgare L.) and Tobacco (Nicotiana Tabacum)

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Abstract
The subcellular distribution of plasma‐membrane‐type Ca2+‐transporting ATPases was studied in barley leaves (Hordeum vulgare L.). A highly enriched plasma membrane (PM) fraction was analysed for Ca2+ pumps and compared with several inner‐membrane preparations, including the tonoplast, the envelope of the chloroplast, and an endoplasmic reticulum (ER)‐enriched fraction. The enzymes were identified and characterised with regard to the phosphointermediate formation, their nucleotide specificity and their binding to calmodulin. A Ca2+‐transporting ATPase (molecular mass ≈ 130 kDa), which showed high specificity for ATP and high affinity for calmodulin, was localised in the PM. A 116‐kDa Ca2+‐transporting ATPase, probably located in the ER, showed lower nucleotide specificity and weaker affinity for calmodulin. A comparison of the distribution of the pumps in leaves and roots indicated that the ratio of expression of the two enzymes changed in a tissue‐specific manner: the PM pump was dominant in leaves, while the inner‐membrane pump was expressed at a higher level in the roots. For the purification of calmodulin‐binding proteins (Ca2+ pumps), microsomes isolated from tobacco cell cultures were used. Two active Ca2+ pumps were identified, and the one at 116kDa was partially sequenced.