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

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Title Purification and characterization of a type II phosphatidylinositol 4-kinase from rat spleen and comparison with a PtdIns 4-kinase from lymphocytes
 
Creator VERGHESE, M
FERNANDIS, AZ
SUBRAHMANYAM, G
 
Subject t-cell activation
antigen receptor
signal-transduction
a431 cells
phosphoinositide kinases
tyrosine phosphorylation
protein-kinase
membranes
stimulation
turnover
 
Description 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.
 
Publisher NATL INST SCIENCE COMMUNICATION
 
Date 2011-08-19T06:22:36Z
2011-12-26T12:56:06Z
2011-12-27T05:44:16Z
2011-08-19T06:22:36Z
2011-12-26T12:56:06Z
2011-12-27T05:44:16Z
1999
 
Type Article
 
Identifier INDIAN JOURNAL OF BIOCHEMISTRY & BIOPHYSICS, 36(1), 1-9
0301-1208
http://dspace.library.iitb.ac.in/xmlui/handle/10054/10255
http://hdl.handle.net/10054/10255
 
Language en