Subcellular and tissue distribution, partial purification, and sequencing of calmodulin-stimulated Ca2+-transporting ATPases from barley (Hordeum vulgare L.) and tobacco (Nicotiana tabacum).
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 approximately 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 116 kDa was partially sequenced.
Dainese
P
P
Department of Biology, Swiss Federal Institute of Technology, Zürich, Switzerland.
James
P
P
Baldan
B
B
Carafoli
E
E
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism / Q000502:physiology*; D002148:Calmodulin-Binding Proteins / Q000302:isolation & purification; D002462:Cell Membrane / Q000201:enzymology; D004789:Enzyme Activation; D006160:Guanosine Triphosphate / Q000378:metabolism; D001467:Hordeum / Q000201:enzymology*; D007425:Intracellular Membranes / Q000201:enzymology; D008861:Microsomes; D018515:Plant Leaves / Q000201:enzymology; D018517:Plant Roots / Q000201:enzymology; D010947:Plants, Toxic; D013347:Subcellular Fractions / Q000201:enzymology; D014026:Nicotiana / Q000201:enzymology*
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases