Subcellular and tissue distribution, partial purification, and sequencing of calmodulin-stimulated Ca2+-transporting ATPases from barley (Hordeum vulgare L.) and tobacco (Nicotiana tabacum)

Eur J Biochem. 1997 Feb 15;244(1):31-8. doi: 10.1111/j.1432-1033.1997.00031.x.

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 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Amino Acid Sequence
  • Binding Sites
  • Calcium-Transporting ATPases / chemistry
  • Calcium-Transporting ATPases / isolation & purification*
  • Calcium-Transporting ATPases / metabolism*
  • Calmodulin / metabolism
  • Calmodulin / physiology*
  • Calmodulin-Binding Proteins / isolation & purification
  • Cell Membrane / enzymology
  • Enzyme Activation
  • Guanosine Triphosphate / metabolism
  • Hordeum / enzymology*
  • Intracellular Membranes / enzymology
  • Microsomes
  • Plant Leaves / enzymology
  • Plant Roots / enzymology
  • Plants, Toxic*
  • Subcellular Fractions / enzymology
  • Tobacco / enzymology*

Substances

  • Calmodulin
  • Calmodulin-Binding Proteins
  • Guanosine Triphosphate
  • Adenosine Triphosphate
  • Calcium-Transporting ATPases