A 67-kDa plasma-membrane-bound Ca2+-stimulated protein kinase active in sink tissue of higher plants |
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Authors: | Laurence D P Barker Matthew D Templeton Ian B Ferguson |
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Institution: | (1) Horticulture and Food Research Institute of New Zealand, Mt. Albert Research Centre, Private Bag 92-169, Auckland, New Zealand, NZ;(2) School of Biological Sciences, University of Auckland, Auckland, New Zealand, NZ |
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Abstract: | A novel 67-kDa protein kinase (p67
cdpk
) was identified in the microsomal membrane fraction of apple (Malus domestica Borkh. cv. Braeburn) suspension cultures and subsequently found to be active in sink tissues. Microsomal proteins were blotted
onto Nylon or polyvinylidenedifluoride membranes, and p67
cdpk
assayed by in situ-labelling renatured proteins with γ-32P]ATP; thin-layer electrophoresis/thin-layer chromatography of acid hydrolysates of the 32P-labelled protein band indicated that serine and threonine, but not tyrosine residues were phosphorylated. A detailed analysis
of the ion-dependency of p67
cdpk
revealed that it was a Ca2+-stimulated, Mg2+-dependent protein kinase. However, p67
cdpk
was ten times more active in the presence of 10 mM Mn2+, and these assay conditions were used routinely to increase the sensitivity of the assay. Activity of p67
cdpk
was found at high levels in the plasma membrane, and solubilisation experiments with a number of detergents suggested that
p67
cdpk
is an integral membrane protein. A homologous protein kinase with similar biochemical properties was also present in cell-suspension
cultures of pear and maize. In maize (Zea mays L.) plants, sink tissues, such as young expanding leaves of both light-grown and etiolated plants, mature etiolated tissue
and roots all had high levels of p67
cdpk
activity. However, mature light-grown (source) tissues had barely detectable levels. In etiolated maize leaves and coleoptiles
the kinase activity was highest in expanding tissue and decreased as the cells expanded. When etiolated maize plants were
exposed to light, the activity of p67
cdpk
was reduced to background levels after 8 h. Although p67
cdpk
has biochemical properties similar to those of other plant calcium-dependent protein kinases, this is the first identification
of a membrane-bound calcium-dependent protein kinase which is specifically active in sink tissues.
Received: 14 July 1997 / Accepted: 25 September 1997 |
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Keywords: | : Calcium-dependent protein kinase Malus (protein kinase) Plasma membrane Sink tissue Zea (protein kinase) |
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