Glycerol phosphate acylation

The initial acylation at the 1-position of glycerol 3-phosphate is catalysed by glycerol 3-phosphate acyl-transferase-1, abbreviated to GPAT-1. This enzyme is specific for a saturated fatty acid (in the acyl form). 

Fatty acyl CoA-glycerol-3-phosphate acyl transferase... 

In human adipose tissue, palmitoyl-CoA is usually used in the first glycerol-3-phosphate acylation reaction. The next two acyl residues are normally unsaturated fatty acids oleic acid and, less commonly, linoleic acid. Triglyceride biosynthesis is stimulated by insulin, most likely via its activation of lipoprotein lipase and its activity in moving glucose into the cells. 

The biological effects of IPG include tissue-specific regulation of lipolysis, lipogen-esis, glycolytic flux, protein synthesis and/or phosphorylation, DNA and RNA synthesis and also long-term actions such as cellular prohferation. IPG-P activates glycerol-3-phosphate acyl transferase [17], but almost aU the other insulin-mimetic activities reported were tested for IPG-A prepared in vitro by hydrolysis of highly purified GPI [1, 7, 8, 75, 76]. 

Fig. 5. Pathway depicting how flux through phosphatidylcholine (product of reaction 3) can promote acyl group diversity in plant triacylglycerols. Production of 18 2 (boxed) at the sn-2 position and its transfer to TG is used as a sample modification. Other fatty acid alterations may be substituted. Enzymes 1, glycerol-3-phosphate acyl-CoA acyltransferase and lysophosphatidic acid acyl-CoA acyltransferase 2, phosphatidic acid phosphatase 3, diacylglyceroliCDP-aminoalcohol aminoalcoholphosphotransferase 4, 18 l-desaturase or other fatty acid modifying enzyme 5, phosphlipid diacylglycerol acyltransferase 6, diacylglycerol acyltransferase 7, acyl-CoA phosphatidylcholine acyltransferase or phospholipase plus acyl-CoA synthetase.

The first stage is the acylation, in the presence of glycerol-3-phosphate acyl-transferase, of the free alcohol groups of the glycerol-3-phosphate by two molecules of fattyacyl-CoA to yield a phosphatidic acid ... 

Currently available cultivars of high erucic acid rape (HEAR) have a theoretical maximum of 66% erucic acid (22 1, A13) in their seed oil due to the specificity of the membrane-bound l-acyl-572-glycerol-3-phosphate acyl transferase (LPA-AT) enzyme. In HEAR the LPA-AT does not incorporate erucic acid at the sn-2 position of triacylglycerols (TAG) but preferentially incorporates oleic acid (18 1, A9), even if this is only a minor component of the total fatty acid pool (1). However, some plant species, Q.g Limnanthes, can utilise erucoyl-CoA as a substrate and effectively incorporate erucic acid at the sn-2 position (2, 3). 

The acyl pattern of the final mixture of triacylglycerols is characteristic for each seed oil. The specific acyl chain targeting depends on both the selectivity of the various acyltransferases (AT 1, acyl-CoA sn-glycerol-3-phosphate acyl-transferase, E.C. 2.3.1.15 AT 2, acyl-CoA l-acyl-j -glycero-3-phosphate acyl-transferase, E.C.2.3.1.51 ATS, acyl-CoA l,2-diacyl-5n-glycerol acyltransferase, E.C. 2.3.1.20) of the Kennedy pathway and the compartmentation of triacylglycerol biosynthesis and/or acyl chain modification [6, 8],... 

Glycerol 3-phosphate acyl transferase (G3P-AT) and 1-acylglycerol 3-phosphate acyl transferase (LPA-AT), which catalyse the first two steps in course of de novo biosynthesis of glycerolipids, are known enzymic activities not only of plastids and microsomes (Frentzen 1986) but also of mitochondria from higher plants (Douce 1971, Vick and Beevers 1977, Sparace and Moore 1979). In mitochondria of castor bean endosperm the two enzymic activities were localized in both the inner and outer membrane (Sparace and Moore 1979), but little is known about the enzymic properties. 

Frentzen, M., Heinz, E., McKeon, T.A., Stumpf, P.K. 1983 Specificities and selectivities of glycerol-3-phosphate acyl transferase and monoacylglycerol-3-phosphate acyl-transferase from pea and spinach chloroplasts. Eur. J. Biochem. 129, 629-636. 

Acyl-(acyl-carrier protein) glycerol-3-phosphate acyl transferase (glycero-P acyltransferasc) in higher-plant chloroplasts transfers the acyl group from acyl-(acyl-carrier protein) to the C-1 position of glycerol 3-phosphate to synthesize 1-acylglycerol 3-phosphate. Since this reaction is the first step of glycerolipid synthesis in the chloroplasts, it is of special interest to study this enzyme. 

The present work deals with purification and immunological properties of pea chloroplast acyl-ACP glycerol-3-phosphate acyl transferase. 

The Acyl-CoA or Acyl-ACP glycerol-3-phosphate acyl transferase was purified (Douady and Dubacq 1987). Homogeneity was achieved by FPLC on a MonoQ column (fig.lA).The activity estimated with Acyl-CoA indicated that the purification factor was about 15000 (Table 1). 

Bertrams, M.and Heinz, E. (1981) Positional specificity and fatty acid selectivity of purified sn-glycerol 3-phosphate acyl transferases from chloroplasts. Plant Physiol. 68, 653-657. 

Douady, D. and Dubacq, J.P. (1987) Purification of acylCoA glycerol-3-phosphate acyl transferase from pea leaves. Biochim. Biophys. Acta 921,... 

Frentzen, M , Nishida, I. and Murata, N. (1987) Properties of the plastidial acyl-(Acyl-Carrier-Protein) glycerol-3-phosphate acyl transferase from the chilling-sensitive plant squash (Cucurbita moschata). Plant Cell Physiol. 28, 1195-1201. 

Molecular species of PG, which are entirely synthesized in the plastid, always contain a "saturated" (16 0 or 16 1 ) fatty acid esterified at the sn-2 position of glycerol-3-phosphate. Therefore, the only variability in molecular species of PG of any significance to the chilling phenomenon occurs at the sn-1 position. These observations have focussed attention on the role of sn-glycerol-3-phosphate acyl-ACP acyltransferase (E.C. 2.3.1.15 henceforth, "ATI") in determining the relative proportions of saturated and unsaturated fatty acids which are esterified at the sn-1 position of molecular species of PG. 

The pattern of total synthesis is closely reflected by the acyl-CoA fraction (data not shown). However, when compared with total fatty acid synthesis, PA, DG and PG are always relatively enriched in saturated fatty acids (Table 2). Although we have not actually performed molecular species analysis on the products of the chloroplast incubations, most, but not all, of this enrichment for saturated fatty acids can be explained by the apparent absolute specificity of the plastid 1-acyl-sn-glycerol-3-phosphate acyl-ACPacyltransferase (henceforth,... 

Two forms of ACP have been purified from spinach and barley leaf, but, only one ACP Isoform predominates In spinach seed tissue (2, 3). Oleic acid Is the major product of fatty acid synthesis by Isolated chloroplasts (4). The regulation of plant lipid metabolism Is believed to Involve the export of oleic acid from Its site of synthesis (plastld) to sites of complex lipid metabolism (eg. ER). This pathway requires the Initial release of oleic acid from ACP by oleoyl-ACP thloesterase (5). In addition, the oleoyl moiety may also enter glycerollpld synthesis within the plastld through the action of acyl-ACP acyl-transferase (6). The preferred substrate for both the thloesterase and glycerol—3-phosphate acyl transferase reactions Is 18 1-ACP (6,7). 

The occurrence of two forms of ACP raises the question of their metabolic function. Because 18 1—ACP Is the substrate for two competing reactions, ACP Isoforms might facilitate acyl chain distribution within the cell. He have analyzed the reactivity and kinetics of oleoyl-ACP thloesterase and oleoyl-ACP glycerol-3—phosphate acyl transferase with oleoyl-ACP-I and oleoyl-ACP-II. Our results suggest that ACP Isoforms may partially regulate the flow of oleic acid from Its site of de novo synthesis In higher plants. 

The enzymatic synthesis of [ C]18 1-ACP Isoforms was according to a published procedure (9), and was purified through the octyl sepharose step. Oleoyl-ACP thloesterase was partially purified from spinach tissue by methods which Involved acid precipitation and either Ion-exchange or affinity chromatography (7,8). Glycerol-3-phosphate acyl transferase was purified by ammonium sulfate fractionation, DEAE and... 

The glycerol—3—phosphate acyl transferase reaction preferred oleoyl-ACP-11 over oleoyl-ACP-1 at all substrate concentrations tested (Figure lA). This Is In direct contrast to the competing thloesterase reaction shown In Figure IB. The Km for, oleoyl—ACP-1 by the acyl transferase was 0.75 uM while that for oleoyl-ACF-11 was 0.16 yM. 

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