Detergent solubilization and hydrophobic chromatography of rat brain phosphatidylinositol kinase.
Rat brain microsomal phosphatidylinositol kinase activity was maximally activated in the presence of either 3 mM sodium deoxycholate, 2% Triton-X-100, or 30-40 mM octylglucoside. Among these detergents, 1% Triton-X-100 was most effective in solubilizing the enzyme, and after treatment with this agent, 100% of the activity was recovered in the high speed supernatant. Octylglucoside solubilized 40% of the enzyme at concentrations below its critical micelle concentration of 25 mM and up to 80% at higher levels. Solubilized phosphatidylinositol kinase failed to absorb to adenosine nucleotide affinity resins. However, when the Triton-X-100 extract was chromatographed on an uncharged hydrophobic resin, consisting of dodecyl chains attached to Sepharose 4B by ether bonds, nearly all the enzyme activity was retained, and from 44-85% could be eluted with 8 mM sodium deoxycholate. Solubilization followed by hydrophobic chromatography resulted in several-fold purification of phosphatidylinositol kinase and may have disrupted interactions of the enzyme with other hydrophobic proteins sufficiently to allow its substantial purification by conventional or affinity chromatography techniques.
D019870:1-Phosphatidylinositol 4-Kinase; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002846:Chromatography, Affinity; D003902:Detergents / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D005960:Glucosides / Q000494:pharmacology; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D017830:Octoxynol; D010716:Phosphatidylinositols / Q000302:isolation & purification / Q000378:metabolism; D010770:Phosphotransferases / Q000302:isolation & purification / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000494:pharmacology; D051381:Rats; D012995:Solubility; D013501:Surface-Active Agents / Q000494:pharmacology*
D003902:Detergents; D005960:Glucosides; D010716:Phosphatidylinositols; D013501:Surface-Active Agents; C018619:octyl-beta-D-glucoside; D011092:Polyethylene Glycols; D017830:Octoxynol; D010770:Phosphotransferases; D019870:1-Phosphatidylinositol 4-Kinase