Alkaline phosphatase in HeLa cells. Stimulation by phospholipase A2 and lysophosphatidylcholine.
Treatment of homogenates and plasma membrane preparations from HeLa cells with phospholipase A2 (EC 3.1.1.4) caused a 50% increase in activity of membrane-associated alkaline phosphatase. Lysophosphatidylcholine, dispersed in 0.15 M KCl, affected alkaline phosphatase in a similar fashion by releasing the enzyme from particulate fractions into the incubation medium and by elevating its specific activity. Higher concentrations of lysophosphatidylcholine solubilized additional protein from particulate fractions but did not further increase the specific activity of the released alkaline phosphatase. Particulate fractions from HeLa cells were exposed to the effects of liposomes prepared from lysophosphatidylcholine and cholesterol. The ratio of particulate protein/lysophosphatidylcholine (by weight) required for optimal activation of alkaline phosphatase was one. Kinetic studies indicated that phospholipase A2 and lysophosphatidylcholine enhanced the apparent V of the enzyme but did not significantly alter its apparent Km. The increased release of alkaline phosphatase from the particulate matrix by lysophosphatidylcholine was confirmed by disc electrophoresis. The release of the enzyme by either phospholipase A2 or by lysophosphatidylcholine appeared to be followed by the formation of micelles that contained lysophosphatidylcholine. The new complexes had relatively less cholesterol and more lysophosphatidylcholine than the native membranes. The possibility that lysophosphatidylcholine formed a lipoprotein complex with the solubilized alkaline phosphatase was indicated by a break point in the Arrhenius plot which was evident only in the lysophosphatidylcholine-solubilized enzyme but could not be demonstrated in alkaline phosphatase that had been released with 0.15 M KCl alone.
D000469:Alkaline Phosphatase / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D003840:Deoxycholic Acid / Q000494:pharmacology; D006367:HeLa Cells / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D008244:Lysophosphatidylcholines / Q000494:pharmacology*; D010740:Phospholipases / Q000494:pharmacology*; D011092:Polyethylene Glycols / Q000494:pharmacology; D013347:Subcellular Fractions / Q000201:enzymology; D013696:Temperature; D013816:Thermodynamics
D008244:Lysophosphatidylcholines; D003840:Deoxycholic Acid; D011092:Polyethylene Glycols; D010740:Phospholipases; D000469:Alkaline Phosphatase