1‐Acyl‐sn‐glycerol‐3‐phosphate acyltransferase in maturing safflower seeds and its contribution to the non‐random fatty acid distribution of triacylglycerol

Abstract

A 20000 x g particulate preparation isolated from maturing safflower seeds catalyzed the acylation of 1 -acylsn-glycerol 3-phosphate with acyl-CoA to form phosphatidate. The specific activity of the reaction exceeded 200 nmol min-' mg protein-'. Although this preparation was also capable of catalyzing the acylation of snglycerol 3-phosphate with acyl-CoA, the hydrolysis of phosphatidate, and the acylation of 1,2-diacylglycerol, phosphatidate was the only major product when the preparation was incubated with 1-acyl-glycerol-3-P and acylCoA.The enzyme responsible for this phosphatidate synthesis, 1-acyl-glycerol-3-P acyltransferase, showed a strict acyl-CoA specificity. The relative order of specificity for acyl-CoA was linoleoyl = oleoyl > palmitoleoyl > elaidoyl > cis-vaccenoyl > stearoyl = palmitoyl. This observation strongly suggests that the fatty acid composition of position 2 in phosphatidate synthesized in vivo primarily depends on both the acyl-CoA specificity of the 1 -acyl-glycerol-3-P acyltransferase and the fatty acid composition of the acyl-CoA pool in the cell. Thus, the absence of saturated fatty acids at position 2 of safflower triacylglycerol may be explained in terms of the acylCoA specificity of the 1-acyl-glycerol-3-P acyltransferase. The fatty acid moiety esterified at position 1 of glycerol-3-P also affected the effectiveness of the reaction. The 1-acyl-glycerol-3-P acyltransferase utilized 1 -acyl-glycerol-3-P molecular species in the following order of effectiveness : linoleoyl = oleoyl > palmitoyl. With a rise in incubation temperature, the initial rates of acylation with unsaturated acyl-CoA species increased more rapidly than those for saturated acyl-CoA species. A similar tendency was observed for saturated and unsaturated acyl acceptors. These data suggest that affinity of the acyltransferase for substrates may vary in response to changes in temperature, and that 1 -acyl-glycerol-3-P acyltransferase may be involved in the alteration of the individual fatty acid compositions at positions 1 and 2 of glycerolipids in tissues grown at different temperatures. Based on these findings, further metabolism of 1 -acyl-glycerol-3-P acyltransferase products could be the major factor determining the non-random distribution of fatty acids in safflower triacylglycerol.The stereospecific analysis of fatty acid distribution in triacylglycerol, the first method for which had been developed by Brockerhoff in 1965 [l], revealed that the positional distribution of fatty acids in naturally occurring plant triacylglycerols is not random, and that each position of the glycerol backbone has a characteristic fatty acid pattern [2, 31. In general, positions 1 and 3 were esterified with both saturated and unsaturated fatty acids, while position 2 was exclusively occupied by unsaturated fatty acids.In plant tissues, triacylglycerol is synthesized via the glycerol-3-P pathway [3 -51, which has originally been established with animal tissues [6] : glycerol-3-P + 1 -acyl-glycerol-3-P + phosphatidate + 1,2-diacylglyc...

Microsomal Phosphatidate Phosphatase in Maturing Safflower Seeds

“…The enzyme activity was stable at 30C for at least 10 min, but was thoroughly inactivated at 550C. Thus, the enzyme appeared to be more thermolabile than the acyltransferases (14,16). The soluble phosphatidate phosphatase was similar to the microsomal enzyme in thermolability (K Ichihara, unpublished data).…”

“…Since the substrate was an emulsion of phosphatidate/ phosphatidylcholine/BSA and hence phosphatidate might not directly available to the microsomal enzyme, the soluble activity might be overestimated. Unlike phosphatidate phosphatase, all of the three acyltransferases involved in the glycerol-phosphate pathway (29) were membrane bound in safflower seeds (14,16,17). This difference in subcellular distribution between phosphatidate phosphatase and the acyltransferases is interesting.…”

“…Phosphatidate phosphatases from a variety ofanimal tissues and yeast are stimulated or inhibited by Mg2" (2,12 system of safflower seeds, the acyltransferase reactions are inhibited or stimulated to a minor extent by Mg2+ (14,16).…”

“…Its fatty acid composition was 36.4% palmitic, 10.4% stearic, 35.0% oleic, 16.0% linoleic, and 2.2% arachidonic acid (by GLC). The egg yolk phosphatidylcholine (1 g) was dissolved in 20 mL diethyl ether containing 3 mg 2,6-di-tert-butyl-p-cresol, an antioxidant.…”

“…Lysophosphatidic acid (ammonium salt) was prepared from egg yolk phosphatidylcholine by the procedure described earlier ( 16 …”