When [ 1 -14C]palmitic acid was incubated with young pea leaves or solutions of their acetonedried powders, 2-hydroxy [l-14C]palmitic acid accumulated. This metabolite has been isolated as its methyl ester. When diluted with unlabelled synthetic methyl D-2-hydroxypalmitate to known specific activity, and recrystallised, the crystals and mother liquor residues retained the same specific activity. When diluted with unlabelled synthetic methyl L-2-hydroxypalmitate to known specific activity and recrystallised, the crystals were almost non-radioactive. The radioactive metabolite therefore has the D-COnfigUratiOn.Unlabelled 2-hydroxypalmitic acid isolated from pea leaves was shown to have the n-configuration by direct measurement.
ration of acetone-dried powder has been described previously [1,2]. This was stirred in about forty times its weight of cold 0.2M phosphate buffer (pH 7.0) for 1 h and briefly centrifuged to remove most of the insoluble material. Homogenates were made by coarsely chopping the leaves into about five times their weight of cold 0.2 M phosphate buffer (pH7.0) and homogenising with an Ultra Turrax tissue grinder (Hudes Merchandising, Leeke St., London) for about 45 sec a t a temperature below 10". Homogenates and acetone-dried powder solutions were used immediately after preparation.The Radiochemical Centre (Amersham) provided [1-14C]palmitic acid (specific activity, 44 mC/mmole).
Preparation of ~-[ 2 -~H ] 1 6 :0 and ~-[ 2 -~H ] 1 6 : 0The preparation of D-and L-2-hydroxypalmitic acid, essentially by the method of Horn and Pretorius [5], has been described previously [3]. Each enantiomer was converted into its tosyl derivative with retention of configuration by treatment with p-toluenesulphonyl chloride in pyridine. Each tosylate was refluxed in anhydrous tetrahydrofuran for 20 h with lithium boron [3H]hydride, when hydrogenolysis with inversion of configuration occurs [6,7]. The hexadecanol formed was oxidised t o the acid with chromium trioxide in acetic acid; D-16h:O thus gave ~-[2-~H]16 : 0, which was purified by thin-layer and gas-liquid chromatography. Similarly L-16 h : 0 yielded ~-[ 2 -~H ] 1 6 : 0 (Fig.2).
METHODSSubstrates were prepared by mixing [1-l4C]16: 0 and the [2-3H]16:0 in benzene solution, aliquots of which were assayed for 14C and 3H simultaneously in a Packard Tri Carb spectrometer. The 3H :14C ratio
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