ARACHIDONATE-CONTAINING TRIACYLGLYCEROLS - BIOSYNTHESIS AND A LIPOLYTIC MECHANISM FOR THE RELEASE AND TRANSFER OF ARACHIDONATE TO PHOSPHOLIPIDS IN HL-60 CELLS

When HL-60 cells are incubated in media containing 10 muM [H-3]arachid onic acid the label is immediately incorporated into both triacylglyce rols and phospholipids. About one-half of the cellular tritium was ass ociated with triacylglycerols after 2 h of incubation and this [H-3]ar achidonate was then transferred to phospholipids as soon as the labele d cells were placed in arachidonate-free media. A technique was devise d to analyze the stereospecific distribution of [H-3]arachidonate at t he three sn-positions of glycerol in order to identify the mechanism(s ) responsible for the biosynthesis of the labeled triacylglycerols. [H -3]Arachidonate was found to be distributed in nearly equal amounts am ong all three glycerol positions of the triacylglycerols. In addition, analysis of intact triacylglycerols containing [H-3]arachidonate reve aled that 24% of the tritium eluted from reverse-phase HPLC with triar achidonoylglycerol. Both of these findings would be expected if a sign ificant portion of the arachidonate-containing triacylglycerols were s ynthesized de novo. Homogenates prepared from [H-3]arachidonate prelab eled HL-60 cells were capable of hydrolyzing the endogenous [H-3]arach idonate-containing triacylglycerols to produce mainly free fatty acids and smaller amounts of monoacylglycerols. The relatively small amount of monoacyl- and diacylglycerols produced by the lipolytic activity o f the homogenates indicated that [H-3]arachidonate was hydrolyzed from all three sn-positions of the [H-3]triacylglycerols. This lipase acti vity had a pH optimum of 4.5 and was associated to a greater extent wi th the soluble fraction than in the total membrane fraction. Although it is not known whether this lipolytic activity is the same as that ex pressed in the intact cells, the activity of the cell-free triacylglyc erol lipase was of sufficient magnitude to have easily accounted for t he decrease in [H-3]triacylglycerols that was observed after transfer of the intact HL-60 cells (prelabeled with [H-3]arachidonate) to fresh media. The data suggest that transfer of arachidonate from triacylgly cerols to phospholipids probably occurs through an acyltransferase uti lizing a lysophospholipid and arachidonoyl-CoA.