Morgan, Sarah Jane; (1991) Purification and characterisation of phosphatidylinositol 3-kinase from bovine brain. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Phosphatidylinosilol 3-kinase (Ptdlns 3-kinase) activity is circumstantially correlated with mitogenic responses and transformation, however little is known about the enzyme. The identification and structural analysis of this enzyme have been the principal objectives of the work described here. Ptdlns 3-kinase was purified from bovine brain cytosol. Initially small scale preparations were used to develop the purification, these were then scaled up linearly to allow purification of sufficient material for structural analysis. The purification developed yielded a heterodimeric complex containing proteins of 85 and 110 kDa, which co-migrated with activity over the final purification steps. The net purification of Ptdlns 3-kinase was 650 fold. The purified material was free from Ptdlns 4-kinase contamination. The purified enzyme was characterised with respect to substrates and potential activators and has a Km for ATP of 67 μM, for pure sonicated Ptdlns of 34 μM and an apparent Ka for Mg2+ of 6.9 mM. Mg2+ is preferred over Mn2+ and the activity is Ca2+ independent. The purified enzyme is capable of utilising Ptdlns, PtdIns(4)P and PtdIns(4,5)P2 as substrates, the ratios of phosphorylation of these three lipids were similar with substrates presented as pure sonicated phospholipids. A large scale purification of Ptdlns 3-kinase was developed to allow gel purification and peptide sequence analysis of both the 85 kDa and 110 kDa proteins. Tryptic peptide sequencing of the 85 kDa protein revealed that the protein contained a src homology 3 (SH3) domain. This was subsequently confirmed by sequence analysis of a cDNA clone which encoded p85. The predicted sequence of p85α and the related p85β (for which no tryptic peptides were obtained) show the presence of an SH3 domain, two SH2 domains and a bcr-related sequence. Recombinant expressed p85 α and p85β associate with and are phosphorylated by both platelet-derived growth factor receptor and Polyoma middle T antigen;pp60c-src complexes in vitro. This is consistent with the reported associations of Ptdlns 3-kinase in vivo, as discussed.

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