Activation and solubilization by Triton X-100 of membrane-bound phospholipase D of rat brain.
In the present study, we examined the ability of detergents to stimulate and solubilize phospholipase D (PLD) of a particulate fraction of rat brain. PLD activity was assayed by measuring the [3H]choline produced from the exogenous substrate dipalmitoyl phosphatidyl[3H]choline (dipalmitoyl [3H]PC). In the absence of detergents, PLD activity was not detectable. Of the detergents examined, Triton X-100 was found to markedly enhance PLD activity, whereas other detergents including sodium deoxycholate, sodium cholate, CHAPS and Lubrol-PX caused only a small, if any increase in activity. Enhancement by Triton X-100 was maximal at 0.1-0.2% (w/v) and decreased at higher concentrations. The optimal pH was 7.1-7.3. Both Ca2+ and Mg2+ inhibited enzyme activity stimulated by Triton X-100 in a concentration-dependent manner. Triton X-100 effectively solubilized PLD from the particulate fraction of rat brain; more than 70% of the activity of the particulate fraction was extracted by 0.5-1.0% (w/v) Triton X-100. Furthermore, when the PLD activities in brains of three different species (rat, rabbit and bovine) were measured under optimal conditions, the activities were found to differ greatly. PLD activity was highest in rabbit brain, followed by rat and bovine brains; the activity in bovine brain was extremely low compared to the activities in rat and rabbit brains. We conclude that Triton X-100 is potentially useful for the purification of PLD and that rabbit and rat brains are the preferred sources.
Kanoh
H
H
Department of Biochemistry, Gifu University School of Medicine, Japan.
Kanaho
Y
Y
Nozawa
Y
Y
D015060:1,2-Dipalmitoylphosphatidylcholine / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology*; D002794:Choline / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects*; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D017830:Octoxynol; D010739:Phospholipase D / Q000302:isolation & purification / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000494:pharmacology*; D011817:Rabbits; D051381:Rats; D012995:Solubility
D003902:Detergents; D015060:1,2-Dipalmitoylphosphatidylcholine; D011092:Polyethylene Glycols; D017830:Octoxynol; D010739:Phospholipase D; D008274:Magnesium; D002794:Choline; D002118:Calcium