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

Abstract

A Ptdlns 4-kinase from rat spleen particulate fraction was purified to homogeneity and its molecular properties were compared with a Ptdlns 4-kinase from splenic lymphocytes. The enzyme activity was solubilized from spleen particulate

fraction with Triton X-100 and chromatographed sequentially on phosphocellulose, DEAE-sephacel, heparin acrylamide and hydroxyapatite columns. The purified enzyme preparation showed a 55 kDa band on SDS-PAGE with silver staining. Renaturation of the enzyme activity from SDS-PAGE showed that it comigrated with the 55 kDa protein. Characterization of the enzyme showed that it was a type II Ptdlns 4-kinase. Polyclonal antibodies raised against Ptdlns 4-kinase inhibited the enzyme activity in <i>in vitro </i>assays. Analysis of adult rat tissue particulate fractions on immunoblots showed restricted immunoreactivity among Ptdlns 4-kinases. However, the immunoreactivity is conserved in lymphoid tIssues from mouse to human, suggesting that lymphoid tissue has a distinct Ptdlns 4-kinase. Activation of rat splenocytes with Con A showed two fold in crease in Ptdlns 4-kinase activity. Comparison of Ptdlns 4-kinases from spleen and splenic lymphocytes showed

<span style="font-size:12.0pt;font-family:" times="" new="" roman";="" mso-fareast-font-family:"times="" roman";mso-ansi-language:en-in;mso-fareast-language:="" en-in;mso-bidi-language:ar-sa"="" lang="EN-IN">identical chromatographic behaviour, molecular mass, immunoreactivity, <i>K_m </i>values for Ptdlns and inhibition by adenosine.</span>