A 67-KDA PLASMA-MEMBRANE-BOUND CA2-STIMULATED PROTEIN-KINASE ACTIVE IN SINK TISSUE OF HIGHER-PLANTS()

A novel 67-kDa protein kinase (p67(cdpk)) was identified in the micros omal membrane fraction of apple (Malus domestica Borkh. cv. Braeburn) suspension cultures and subsequently found to be active in sink tissue s. Microsomal proteins were blotted onto Nylon or polyvinylidenedifluo ride membranes, and p67(cdpk) assayed by insitu-labelling renatured pr oteins with [gamma-P-32]ATP; thin-layer electrophoresis/thin-layer chr omatography of acid hydrolysates of the P-32-labelled protein band ind icated that serine and threonine, but not tyrosine residues were phosp horylated. A detailed analysis of the ion-dependency of p67(cdpk) reve aled that it was a Ca2+-stimulated, Mg2+-dependent protein kinase. How ever, p67(cdpk) was ten times more active in the presence of 10 mM Mn2 +, and these assay conditions were used routinely to increase the sens itivity of the assay. Activity of p67(cdpk) was found at high levels i n the plasma membrane, and solubilisation experiments with a number of detergents suggested that p67(cdpk) is a, integral membrane protein. A homologous protein kinase with similar biochemical properties was al so present in cell-suspension cultures of pear and maize. In maize (Ze a mays L.) plants, sink tissues, such as young expanding leaves of bot h 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 lea ves and coleoptiles the kinase activity was highest in expanding tissu e and decreased as the cells expanded. When etiolated maize plants wer e exposed to light, the activity of p67(cdpk) was reduced to backgroun d levels after 8 h. Although p67(cdpk) has biochemical properties simi lar to those of other plant calcium-dependent protein kinases, this is the first identification of a membrane-bound calcium-dependent protei n kinase which is specifically active in sink tissues.