Allan Keith Stobart

Properties of the glycerol acylating enzymes in microsomal preparations from the developing seeds of safflower (Carthamus tinctorius) and turnip rape (Brassica campestris) and their ability to assemble cocoa-butter type fats

Microsomal membrane preparations from the developing seeds of safflower (Carthamus tinctorius, var. Gila) and turnip-rape (Brassica campestris, var. Bele) catalyzed the assembly of triacylglycerols (triglycerides) from sn-glycerol 3-phosphate and acyl-CoA. The membrane preparations were used to assess the acyl specificity properties of the initial acylating enzymes—glycerol 3-phosphate acyltransferase (GPAT) and 1-acylglycerol 3-phosphate acyltransferase (lysophosphatidic acid acyltransferase, LPAAT)—that are responsible for the fatty acids at positions sn-1 and sn-2 of the sn-triacylglycerol, respectively. In spectrophotometric assays it was possible to evaluate, to some extent, how these enzymes will utilize unusual and foreign fatty acids that are not normally found in these particular plant species. The acylating enzymes from both plants used, to varying extents, a comprehensive range of acyl-CoA donor species and some kinetic properties of the substrates involved are presented. The enzymes from safflower, however, were generally the more selective, whereas the turnip-rape was less particular and could utilize a range of acyl substrates. The enzymes from both plants hardly utilized erucate (C22∶1), and the significance of this is discussed in terms of mechanisms which have evolved in order to exclude certain, perhaps detrimental, fatty acids from structural membrane lipids and dedicate them to storage lipid assembly.The ability of the microsomal preparations, from the developing seeds of both plants, to synthesize cocoabutter type fats was investigated. Microsomal membranes were incubated with glycerol 3-phosphate and equimolar amounts of palmitate, oleate and stearate. Safflower preparations catalyzed the construction of sn-triacylglycerol with largely palmitate, oleate and stearate in positions sn-1, 2 and 3, respectively. The selectivity for acyl species in rape was less pronounced, however, substantial saturated-unsaturated-saturated oils were still produced. The results are discussed in terms of the acyl selectivity properties of the glycerol acylating enzymes. It is evident that given the correct composition of fatty acids, the plant can produce cocoabutter or other exotic fats.

The regulation of the fatty-acid composition of the triacylglycerols in microsomal preparations from avocado mesocarp and the developing cotyledons of safflower

The utilisation of [14C]glycerol 3-phosphate and [14C]linoleoyl-CoA in the synthesis of triacylglycerol has been studied in the microsomal preparations of developing cotyledons of safflower seed. The results confirm that the glycerol backbone, which flows towards triacylglycerol from phosphatidic acid through the Kennedy pathway, can enter phosphatidylcholine from diacylglycerol. The equilibration between diacylglycerol and phosphatidylcholine offers a mechanism for the return of oleate to phosphatidylcholine for desaturation to linoleate. We have established that the oleate entering position 1 of sn-phosphatidylcholine from diacylglycerol is desaturated in situ to linoleate. The results indicate that the diacylglycerol phosphatidylcholine interconvertion coupled to the acyl exchange between acyl-CoA and position 2 of sn-phosphatidylcholine brings about the continuous enrichment of the glycerol backbone with C18-polyunsaturated fatty acids and hence these enzymes are of major importance in regulating the acyl quality of the accumulating triacylglycerols. Microsomal preparations from avocado mesocarp, however, did not have detectable acyl exchange between acyl-CoA and phosphatidylcholine or diacylglycerol phosphatidylcholine interconversion despite the high activity of the enzymes of the Kennedy pathway. A scheme is presented which incorporates many of the observations on triacylglycerol synthesis and provides a working model for the regulation of acyl quality in linoleate-rich vegetable oils.

Safflower microsomes catalyse oil accumulation in vitro: A model system.

Microsomal membrane preparations from the developing cotyledons of safflower (Carthamus tinctorius L.) seed catalyse the formation of triacylglycerol fromsn-glycerol 3-phosphate and linoleoyl-CoA. Conditions of incubation were achieved in which the rate of triacylglycerol synthesis approached activities which were compatible with oil accumulation observed in vivo. Reaction mixtures which contained the microsomes took on a white soup-like appearance as triacylglycerol synthesis proceeded and sufficient oil was produced to form a white fat-pad at the surface after centrifugation. The development of the oil bodies in the microsomal membranes was studied by electron microscopy and showed that lipid droplets were formed in or on the membrane surface and were then released as apparently naked entities into the surrounding medium. The ontogeny of the oil droplet in vitro is discussed in terms of oil-body formation in vivo.

Involvement of acyl exchange between acyl-CoA and phosphatidylcholine in the remodelling of phosphatidylcholine in microsomal preparations of rat lung.

Microsomal membrane preparations from rat lung catalyse the incorporation of radioactive linolenic acid from [14C]linolenoyl-CoA into position 2 of sn-phosphatidylcholine. The incorporation was stimulated by bovine serum albumin and free CoA. Free fatty acids in the incubation mixtures were not utilised in the incorporation into complex lipids. Fatty acids were transferred to the acyl-CoA pool during the incorporation of linolenic acid into phosphatidylcholine. An increase in lysophosphatidylcholine occurred in incubations containing both bovine serum albumin and free CoA and in the absence of acyl-CoA. The results were consistent with an acyl-CoA: lysophosphatidylcholine acyltransferase operating in both a forwards and backwards direction and thus catalysing the acyl exchange between acyl-CoA and position 2 of sn-phosphatidylcholine. In incubations with mixed species of acyl-CoAs, palmitic acid was the major fatty acid substrate transferred to phosphatidylcholine in acyl exchange, whereas this acid was completely selected against in the acylation of added lysophosphatidylcholine. The selectivity for palmitoyl-CoA was particularly enhanced when the mixed acyl-CoA substrate was presented to the microsomes in molar concentrations equivalent to the molar ratios of the fatty acids in position 2 of sn-phosphatidylcholine. During acyl exchange, the predominant fatty acid transferred to phosphatidylcholine from acyl-CoA was palmitic acid, whereas arachidonic acid was particularly selected for in the reverse reaction from phosphatidylcholine to acyl-CoA. A hypothesis is presented to explain the differential selectivity for acyl species between the forward and backward reactions of the acyltransferase that is based upon different affinities of the enzyme for substrates at high and low concentrations of acyl donor. Acyl exchange between acyl-CoA and phosphatidylcholine offers, therefore, a possible mechanism for the acyl-remodelling of phosphatidylcholine for the production of lung surfactant.

Oil synthesis in vitro in microsomal membranes from developing cotyledons of Linum usitatissimum L.

Microsomal preparations from developing linseed (Linum usitatissimum L.) cotyledons catalyzed i) acyl exchange between acyl-CoA and position 2 of sn-phosphatidylcholine, ii) acylation of sn-glycerol 3-phosphate to yield phosphatidic acid, and iii) the utilisation of phosphatidic acid in the production of diacylglycerol and triacylglycerol. Selectivity studies for C18 acyl species of acyl-CoA indicated a bias for the channelling of oleate to phosphatidylcholine for, presumably, its desaturation, and the utilisation of the polyunsaturated fatty-acid products in the acyl-CoA pool for phosphatidic acid and subsequent triacylglycerol synthesis. The microsomal preparations were capable of returning glycerol backbone with associated acyl components to phosphatidylcholine from diacylglycerol where it may be further enriched with polyunsaturated C18 acids by desaturation. The acyl quality in linolenate-rich oilseeds appears to be under similar control to that found in linoleate-rich species.

The synthesis of linoleate and phosphatidic acid and its relationship to oil production in the microsomes of developing seeds of safflower (Carthamus tinctorius, L. var. Gila)

Abstract Microsomes from the developing cotyledons of safflower seeds (var. Gila) were incubated, under desaturating conditions, with [1- 14 C]oleoyl-CoA in the presence of glycerol 3-phosphate. The mass and specific radioactivity of oleate and linoleate in acyl-CoA, phosphatidylcholine and phosphatidic acid showed that (i) the oleate in oleoyl-CoA was selectively transferred to position 2 of phosphatidylcholine by acyl exchange where it underwent desaturation to linoleate; (ii) the newly formed linoleate was returned by further acyl exchange, to the acyl-CoA pool from which it was selectively utilised in the acylation of glycerol 3-phosphate to yield phosphatidic acid, a precursor of triacylglycerol. The results confirm that acyl exchange between acyl-CoA and phosphatidylcholine plays a major role in regulating the quality of C18 unsaturated fatty acids in the acyl-CoA pool for oil synthesis.

Desaturation of oleate-[14C] in leaves of barley

Abstract Etiolated barley leaves when exposed to light desaturate oleate-[ 14 C] to linoleate. The production of substantial amounts of radioactive linolenate was found only in very young, tightly rolled leaves. In oleate-[ 14 C] pulse experiments, radioactive linolenate first appeared in phosphatidylcholine (PC) and only after a lag period did it begin to accumulate in monogalactosyldiacylglycerol (MGDG). The results indicate that in young, immature barley leaves linolenate is synthesized from oleate on the parent lipid, PC, and is then transferred to MGDG.

Phosphatidylcholine and its relationship to triacylglycerol biosynthesis in oil-tissues

Abstract During maturation of safflower cotyledons a marked change occurred in the pattern of [ 14 C] glycerol (G-OH) incorporation into the complex lipids. In the early stages of development, the G-OH-backbone of phosphatidylcholine (PC) with associated acyl-groups, is rapidly turned-over and channelled into the accumulating storage oils (triacylglycerol, TAG). In almost mature cotyledons, [ 14 C]G-OH accumulated in diacylglycerol (DAG). In contrast, the germinating seed cotyledons of marrow, despite active incorporation of [ 14 C]G-OH into TAG, principally synthesized PC which did not participate in oil synthesis. The role of the PC-DAG interconversion in generating polyunsaturated fatty acids, and thus affecting acyl-quality, is discussed.

8 – Triacylglycerol Biosynthesis

Publisher Summary This chapter discusses triacylglycerol biosynthesis. The outstanding problems in oil biosynthesis are numerous and offer many exciting challenges that are still of a sufficiently diverse nature to satisfy the interests and demands of most biochemists. A particularly major contribution will be the purification of those proteins that are present in the endoplasmic reticulum of the developing seed and that catalyze the synthesis of triacylglycerol from glycerol phosphate and regulate the acyl quality of the final oil. The solubilization and purification of membrane-bound enzymes is an active area of research and still has many technical problems to overcome. The microsomal membrane from the developing oil seed offers a fundamental system that, because of its specialized nature, is most abundant in those enzymes associated with lipid metabolism. In the longer term, success in purifying the enzymes of triacylglycerol synthesis leads to the preparation of antibodies, mRNA, cDNA, and the ultimate production of cloned genes.