Purification of a Heat-Stable Activator for Phospholipase C-γ1 from Bovine Brain Cytosol and its Identification as Microtubule-Associated Protein Tau

다국어 초록 (Multilingual Abstract)

During the purification of phospholipase C (PLC)-rl which was over-expressed in HeLa cells transfected with a recombinant vaccinia virus carrying a complete cDNA sequence for PLC-γ₁ we noticed the presence of a heat-stable activator in crude cytosolic extract of HeLa cells which markedly stimulated phosphatidylinositol (PI) hydrolysis, when reconstituted with purified PLC-γ₁. Moreover, this putative factor was also found to be present in bovine brain cytosol. Subsequently, based on its ability to stimulate PI hydrolysis of PLC-γ₁ as an assay, this activation factor was purified to homogeneity from bovine brain cytosol. It was purified by heat treatment, trichloroacetic acid precipitation, and successive chromatographic steps on DEAE-5PW, phenyl-5PW, and heparin-5PW HPLC columns. The purified protein, as seen on SDS-PAGE, consisted of 4 or 5 closely spaced bands of apparent molecular weight between 48 and 62 kDa. However, on gel filtration chromatography on TSK-G3000SW HPLC, the estimated molecular weight was revealed to be approximately 350 kDa.
The purified activator was identified as a microtubule-associated protein tau by electroelution from an SDS-polyacrylamide gel, partial peptide mapping by Staphylococcal V_(8) protease, and amino acid sequencing of cyanogen bromide cleaved peptides. The identity of the activator was re-confirmed by an immunoblotring using a specific anti-lau monoclonal antibody. In addition, reconstituting PLC-γ₁ with tau protein purified by an alternative method described elsewhere^ resulted in the same magnitude of activation.
Tau protein mediated activation of PLC isozymes was calcium dependent. Isozyme specific activation of PLCs in 0.1% deoxycholate substrate appeared to be preferential toward PI hydrolysis. The magnitude of activation of PLC isozymes in PI substrate were PLC-γ₁ > PLC-r2 > PLC-δ₁ > PLC-β₁ in decreasing order. Approximately 200 nM concentration of tau-protein produced 15.5-, 5.4-, 4.2-, and 1.6-fold increase in activity of these enzymes, respectively, in the presence of 1 mM free calcium ion.