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 homogeneity and its molecular properties were compared with a PtdIns 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 PtdIns 4-kinase. Polyclonal antibodies raised against PtdIns 4-kinase inhibited the enzyme activity in in vitro assays. Analysis of adult rat tissue particulate fractions on immunoblots showed restricted immunoreactivity among PtdIns 4-kinases. However, the immunoreactivity is conserved in lymphoid tissues from mouse to human, suggesting that lymphoid tissue has a distinct PtdIns 4-kinase. Activation of rat splenocytes with Con A showed two fold increase in PtdIns 4-kinase activity. Comparison of PtdIns 4-kinases from spleen and splenic lymphocytes showed identical chromatographic behaviour, molecular mass, immunoreactivity, K(m) values for PtdIns and inhibition by adenosine.
Verghese
M
M
Biotechnology Centre, Indian Institute of Technology, Powai, Mumbai, India.
Fernandis
A Z
AZ
Subrahmanyam
G
G
D019870:1-Phosphatidylinositol 4-Kinase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D000241:Adenosine / Q000494:pharmacology; D000818:Animals; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007120:Immunochemistry; D066298:In Vitro Techniques; D007700:Kinetics; D008214:Lymphocytes / Q000201:enzymology; D051379:Mice; D008970:Molecular Weight; D051381:Rats; D017208:Rats, Wistar; D013154:Spleen / Q000201:enzymology
D004791:Enzyme Inhibitors; D019870:1-Phosphatidylinositol 4-Kinase; D000241:Adenosine