Purification and characterization of a type II phosphatidylinositol 4-kinase from rat spleen and comparison with a PtdIns 4-kinase from lymphocytes

A PtdIns 4-kinase from rat spleen particulate fraction was purified to homo geneity and its molecular properties were compared with a PtdIns 4-kinase f rom splenic lymphocytes. The enzyme activity was solubilized from spleen pa rticulate fraction with Triton X-100 and chromatographed sequentially on ph osphocellulose, DEAE-sephacel, heparin acrylamide and hydroxyapatite column s. The purified enzyme preparation showed a 55 kDa band on SDS-PAGE with si lver staining. Renaturation of the enzyme activity from SDS-PAGE showed tha t it comigrated with the 55 kDa protein. Characterization of the enzyme sho wed that it was a type II PtdIns 4-kinase. Polyclonal antibodies raised aga inst PtdIns 4-kinase inhibited the enzyme activity in in vitro assays. Anal ysis of adult rat tissue particulate fractions on immunoblots showed restri cted immunoreactivity among PtdIns 4-kinases. However, the immunoreactivity is conserved in lymphoid tissues from mouse to human, suggesting that lymp hoid tissue has a distinct PtdIns 4-kinase. Activation of rat splenocytes w ith Con A showed two fold increase in PtdIns 4-kinase activity. Comparison of PtdIns 4-kinases from spleen and splenic lymphocytes showed identical ch romatographic behaviour, molecular mass, immunoreactivity, K-m values for P tdIns and inhibition by adenosine.