Fat metabolism in higher plants XIX. The biosynthesis of triglycerides by avocado-mesocarp enzymes

Barron, Edward J.; Stumpf, P.K.

doi:10.1016/0006-3002(62)90408-0

Cited by 104 publications

(37 citation statements)

References 24 publications

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“…The system shows a linear response to the amount of CoA added up to the concentration of 1 X 10-4 M. No inhibition of acetate incorporation occurred up to a concentration of 1.5 X 10-4 M, and therefore a concentration of 1.2 X 10-4 M was used routinely in the assay. These results are similar to those reported for the avocado system (1).…”

Section: Resultssupporting

confidence: 91%

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Biosynthesis of Fatty Acids by a Soluble Extract From Developing Soybean Cotyledons

Rinne

1969

Plant Physiol.

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A bstract. Fractionation of developing soybean cotyledons into cellular components demonstrates that most of the activity necessary to incorporate acetate-i-1] C into lipid remains in the supernatant from a 198,000g spin for 1 hr. The system studied is dependent upon ATP, CoA, and CO. Concentrations of ATP greater than 4 X 1-3 M are inhibitory, while 1 X 10-4 M CoA is needed for optimal activity. Avidin inhibition of acetate incorporation into lipid could be reversed by biotin. Studies indicated that NADPH is a better source of reducing power than NADH. The system studied is inhibited by p-chloromercuribenzoic acid and this inhibition can be reversed by an excess of GSH. The system studied shows maximum activity in tris buffer at pH 8.6 or in glycine buffer, pH 9.4. Plants were grown in gravel cuiltul-e (2) tinder a 14 hr photoperiod in the greenihouse. The sovbeanis were removed from the l)ods alld the seed coats.plumule, hypocotyl. and radicle were removed fromll the cotvledons. The cotyledonls were placed in distilled water at room temperatuire unltil grinding. About 10 g of soybean cotyledons were ground with 20 ml of '0.5 M sucrose + 0.01 M MES [2-(N-morpholino)ethanesulfonic acid], pH 6.1. at room temperature. The slurry was passed through 8 layers of cheesecloth and the homogenate was considered the crude enzyme. The crude enzvme was spun at 21,600g for 20 min at 100. The supernatant was then spun at 198,000g for 60 min at 10°. Protein was determined by the biuret method.Assay Method. The reaction was carried out in a 25 ml Erlenmever flask at the temperature and for the time specified. The reaction was stopped with 15 % ethanolic KOH followed by saponification for 3 to 4 hr at 500. After saponification, the 89 www.plantphysiol.org on May 12, 2018 -Published by Downloaded from

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Section: Resultssupporting

confidence: 91%

“…Table V illtustrates the effect of pCMB on the fatty acid synthesizing svstem from soybean cotyledons. The inhibition of acetate incorporation by pCMB and the reversal of the inhibition bv an excess of GSH is in agreement with the accepted idea of sulfhydrvl groups associated with the active sites of fatty acid svnthetase svstems (1,5,11,12).…”

Section: Resultssupporting

confidence: 89%

Biosynthesis of Fatty Acids by a Soluble Extract From Developing Soybean Cotyledons

Rinne

1969

Plant Physiol.

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“…Previous studies in A. thaliana and other members of the Brassicaceae had demonstrated that VLCFAs are biosynthesized by successive condensations of malonyl-CoA with 18:l-CoA or 18:0-CoA in the presence of reductant, whereas 18:l-CoA is desaturated to 18:2 and 18:3 while esterified to PC (Agrawal and Stumpf, 1985;Stymne and Stobart, 1987;Fehling et al, 1990;Taylor et al, 1991Taylor et al, ,1992aKunst et al, 1992b). Then, as in other oilseeds, fatty acyl-CoAs are assembled into glycerolipids via the G-3-P (Kennedy) pathway (Kennedy, 1961;Barron and Stumpf, 1962;Stymne and Stobart, 1987;Kunst et al, 1992b;Taylor et al, 1992c;Taylor and Weber, 1994).…”

Section: Biochemical and Analytical Characterization Of The A S L L Mmentioning

confidence: 99%

Alteration of Seed Fatty Acid Composition by an Ethyl Methanesulfonate-Induced Mutation in Arabidopsis thaliana Affecting Diacylglycerol Acyltransferase Activity

et al. 1995

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In characterizing the enzymes involved in the formation of very long-chain fatty acids (VLCFAs) i n the Brassicaceae, we have generated a series of mutants of Arabidopsis thaliana that have reduced VLCFA content. Here we report the characterization of a seed lipid mutant, AS1 1, which, in comparison to wild type (WT), has reduced levels of 20:l and 18:l and accumulates 18:3 as the major fatty acid in triacylglycerols. Proportions of 18:2 remain similar to WT. Cenetic analyses indicate that the fatty acid phenotype is caused by a semidominant mutation in a single nuclear gene, designated TAC1, located on chromosome 2. Biochemical analyses have shown that the AS11 phenotype is not due to a deficiency in the capacity t o elongate 18:l or to an increase in the relative A1 5 or A1 2 desaturase activities. Indeed, the ratio of desaturase/elongase activities measured in vitro is virtually identical in developing WT and AS1 1 seed homogenates. Rather, the fatty acid phenotype of AS1 1 is the result of reduced diacylglycerol acyltransferase activity throughout development, such that triacylglycerol biosynthesis is reduced. This leads to a reduction in 20:l biosynthesis during seed development, leaving more 18:l available for desaturation. Thus, we have demonstrated that changes to triacylglycerol biosynthesis can result i n dramatic changes i n fatty acid composition and, in particular, i n the accumulation of VLCFAs i n seed storage lipids.The fatty acyl composition of seed TAGs determines their physical and chemical properties and, thus, their use in edible oil or industrial applications. TAG composition depends on the interaction of several different groups of enzymes in the lipid biosynthesis pathway. The enzymes of the fatty acid synthase complex in the plastids of developing seeds are responsible for the biosynthesis of fatty acids up to and including oleic acid. Modifying enzymes, such as the extraplastidic A12 and A15 desaturases, elongases, hydroxylases, and epoxidases, yield polyunsaturated, very long-chain, hydroxy-, and epoxy-fatty acids, respectively. Acyltransferases insert specific acyl moieties onto the glyc-

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“…Therefore, care must be taken to ensure that the hydrolysis ofphosphatidate is not due to acid phosphatase. (1). Acid phosphatase, which is intrinsically Mg2+-independent and nonspecific, also may be responsible for the formation of monoacylglycerol from lysophosphatidate in maturing oil seeds.…”

Section: Specificity For Water-soluble Phosphate Estersmentioning

confidence: 99%

“…Phosphatidate phosphatase in maturing oil seeds has been studied to a very minor extent (29) despite pioneering work by Barron and Stumpf (1), who have suggested operation of the phosphatidate phosphatase reaction in avocado mesocarp. It has so far been reported that phosphatidate phosphatase in maturing safflower seeds requires Mg2+ (10) and that the enzyme activity in groundnut varies with seed maturation (30).…”

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confidence: 99%

Microsomal Phosphatidate Phosphatase in Maturing Safflower Seeds

Ichihara¹,

Norikura²,

Fujii³

1989

Plant Physiol.

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An assay system comprising sodium phosphatidate, phosphatidylcholine, and bovine serum albumin has been developed for the reproducible determination of phosphatidate phosphatase activity in maturing seeds of safflower (Carthamus tinctorius L.). The activity was detected in both membrane and soluble fractions, and the microsomal phosphatidate phosphatase was characterized. The optimum pH for Pi release was 6.7, and the activity depended on the concentration of Mg2 . Phosphatidylcholine and bovine serum albumin stimulated the phosphatase reaction. This phosphatase was highly specific for phosphatidate; lysophosphatidate, and water-soluble phosphate esters did not serve as substrate. The specific activity was approximately 20 nanomoles per minute per milligram of protein, which was close to that of glycerol-phosphate acyltransferase and higher than that of diacylglycerol acyltransferase. Furthermore, the activity per seed was enough to account for the rate of triacylglycerol accumulation in vivo. The step of diacylglycerol formation by phosphatidate phosphatase does not appear to be rate-limiting for triacylglycerol synthesis during seed maturation.Phosphatidate phosphatase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) is a key enzyme of glycerolipid biosynthesis, and catalyzes the hydrolysis of phosphatidate to 1,2-diacylglycerol and Pi. The diacylglycerol product is not only the direct precursor of triacylglycerol but also a substrate for the synthesis of membrane glycerolipids. Although this enzyme was first demonstrated in plants by Kates (19) In mammalian tissues, the phosphatidate phosphatase reaction is considered to be the rate-limiting step of triacylglycerol synthesis (2). We will discuss whether this is also the case for maturing safflower seeds.To assay the activity of phosphatidate phosphatase with good reproducibility, we have developed an effective phosphatidate substrate, the preparative procedure for which is also described. MATERIALS AND METHODS Plant Material and Preparation of MicrosomesMaturing seeds of safflower (Carthamus tinctorius L.) were harvested 14 to 15 d after flowering when '4C-labeled acetate was most rapidly incorporated into diacylglycerol (15), and cotyledons were homogenized in 10 mm Tris-HCl (pH 7.0) containing 0.4 M sorbitol. The homogenate was centrifuged at 5000g for 10 min, and then the supernatant obtained was centrifuged at 100,000g for 1 h. The precipitate was suspended in the same buffer and stored at -20°C. Protein was determined by the method of Lowry et al. (21)

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Fat metabolism in higher plants XIX. The biosynthesis of triglycerides by avocado-mesocarp enzymes

Cited by 104 publications

References 24 publications

Biosynthesis of Fatty Acids by a Soluble Extract From Developing Soybean Cotyledons

Biosynthesis of Fatty Acids by a Soluble Extract From Developing Soybean Cotyledons

Alteration of Seed Fatty Acid Composition by an Ethyl Methanesulfonate-Induced Mutation in Arabidopsis thaliana Affecting Diacylglycerol Acyltransferase Activity

Microsomal Phosphatidate Phosphatase in Maturing Safflower Seeds

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