Purification of phosphatidylinositol kinase from bovine brain myelin

Abstract

A membrane-bound phosphatidylinositol (PI) kinase (EC 2.7.1.67) was purified by affinity chromatography from bovine brain myelin. This enzyme activity was solubilized with non-ionic detergent and chromatographed on an anion-exchange column. Further purification was achieved by affinity chromatography on PI covalently coupled to epoxy-activated Sepharose, which was eluted with a combination of PI and detergent. The final step in the purification was by gel filtration on an Ultrogel AcA44 column. This procedure afforded greater than 5500-fold purification of the enzyme from whole brain myelin. The resulting activity exhibited a major silver-stained band on SDS/polyacrylamide-gel electrophoresis with an apparent Mr 45,000. The identity of this band as PI kinase was corroborated by demonstration of enzyme activity in the gel region corresponding to that of the stained protein. The purified enzyme exhibited a non-linear dependence on PI as substrate, with two apparent kinetic components. The lower-affinity component exhibited a Km similar to that observed for the phosphorylation of phosphatidylinositol 4-phosphate by the enzyme.

Insulin increases membrane protein kinase C activity in rat diaphragm

“…Insulin has been reported to activate phospholipase C [29] and increase production of diacylglycerol [30,31]. However, since no insulininduced translocation of protein kinase C was found in our study, this mechanism cannot fully explain the net increase of membrane enzyme activity seen after insulin treatment of the diaphragm.…”

Increased protein synthesis response to insulin in fibroblasts treated with the diacylglycerol kinase inhibitor R59022

“…Such concentrations (1-100/~U/ml) are well below those at which insulin binds to insulin-like growth factor receptors and therefore the effect on DAG synthesis most probably occurs through the insulin receptor itself. Together with earlier findings that insulin alone leads to increased DAG labelling [14,22] and to increased DAG production [23], these results support the hypothesis that insulin receptor activation leads to a stimulation of phospholipase C. The phospholipid substrate from which this DAG arises remains, however, a matter for debate [8,22]. In addition to prolonging the increase in DAG labelling, R59022 treatment of cells also led to a greater response of protein synthesis to insulin and a greater sensitivity to insulin such that the concentration required for half-maximal stimulation was reduced.…”

Identification of the bovine brain Ins(1,4,5)P3 5‐phosphatase after SDS‐polyacrylamide gel electrophoresis

“…Other enzymes, e.g. phosphatidylinositol kinase from bovine brain myelin or from bovine uteri could be extracted after SDSpolyacrylamide gel electrophoresis [13,14]. After electrophoresis, one lane was sliced into 2-mm sections as described in section 2.…”