Soluble acyl-CoAsn-glycerol 3-phosphate acyltransferases (EC 23.1.15) which are localized in chloroplasts were purified from leaves of Pisum sativum and Spinacia oke.aca and obtained free from interfering activities. The purification raised the specific activities by factors of about 1,000 for pea and 200 for spinach preparations. In pea chloroplasts, acyltransferase activity occurs in two soluble forms with apparent isoelectric points of 6.3 and 6.6. For both forms, the same molecular weight of about 42,000 was determined. The enzyme from spinach chloroplasts showed a slightly higher molecular weight and a lower isoelectric point of 5.2.The enriched enzyme fractions possessed a specificity for glycerol 3-phosphate as acyl acceptor and did not use dihydroxyacetone phosphate. Besides acyl-CoA, acyl-acyl carrier protein also can function as acyl donor. With acyl-CoA as acyl donor, the enzyme shows a high positional specificity, since the predominant product is 1-acylglycerol 3-phosphate.Different acyl-CoAs, when offered separately, were all accepted as substrates, whereas incubations with mixtures of palmitoyl-, stearoyl-, and oleoyl-CoA demonstrated a preference for olec acid. The acyltransferase from spinach displays higher selectivity than does the enzyme from pea and, therefore, may be responsible for the preferred esterification of oleic acid at the C-1 position in chloroplast lipids and the exclusion of palmitic acid from this position as observed during in vivo labeling experiments.When membrane lipids contain different fatty acids, they are usually distributed asymmetrically between positions C-1 and C-2 of the glycerol backbone. In leaf lipids, palmitic acid and unsaturated C18-fatty acids such as linoleic and linolenic acid predominate. In so-called 16:3-plants, in addition to linolenic acid, the lower homologue hexadecatrienoic acid is present and accumulates specifically in monogalactosyl diacylglycerol (13). Positional analyses have shown that this acid is confined to C-2 in this glycolipid, whereas linolenic acid is found in both positions (12). When these analyses were extended to labeled lipids from leaves or chloroplasts of 16:3-plants, common patterns were observed in all chloroplast lipids. After short labeling times, they carried mainly oleate at C-I and palmitate at C-2 (16,22,26,27), which suggests a common diacylglycerol precursor. The only exception in chloroplasts is phosphatidyl choline, which carries palmitate esterified at the C-1 position.The discovery that, apart from other organelles also chloroplasts (3, 15) contain acyl-CoA:glycerol 3-P acyltransferase activity suggested that this enzyme may be involved in the control of fatty acid distribution in chloroplast lipids, since it catalyzes the first acylation step in the de novo synthesis ofcomplex lipids. Therefore, we purified this chloroplast enzyme to investigate its general 'This work was supported by the Deutsche Forschungsgemeinschaft.properties as well as its positional and fatty acid specificity. The results show tha...

De novo-synthesis of glycerolipids in chloroplasts is initiated by a stroma enzyme which catalyzes the formation of lyso-phosphatidic acid from glycerophosphate and acyl-CoA. When these substrates are added to isolated, intact chloroplasts, only glycerophosphate can readily pass through the chloroplast envelope which represents a permeation barrier for acyl-CoA, although higher thioester concentrations destroy this membrane system. At low concentrations of acyl-CoA, which do not impair the envelope, intact chloroplasts metabolize exogenous acyl-CoA in two ways to give free fatty acids and labelled phosphatidyl choline. This indicates that the envelope thioesterase can use exogenous substrates. Isolated, intact chloroplasts fixing radioactive CO2 label free fatty acids and acylglycerols but not galactolipids, since they cannot convert 3-phosphoglycerate into UDP-galactose which in vivo is supplied by the cytoplasm. This cooperation was simulated in vitro by adding all enzymes and cofactors necessary for conversion of 3-phosphoglycerate into UDP-galactose to intact chloro­plasts which then formed labelled monogalactosyl diacylglycerol from labelled CO2. The time required to transfer envelope-made galactolipids from the envelope into thylakoids was studied by incubating intact chloroplasts with radioactive UDP-galactose, subsequent osmotic disruption of organelles with concomitant enzymatic degradation of UDP-galactose followed by separation of envelopes and thylakoids. Only after short times (< 1min) appreciable proportions 920-30%) of radioactive galactolipid export from envelopes into thylakoids.

Optimal reaction conditions were investigated for acylation of sn-[U-(14)C]glycerol 3-phosphate in cell-free systems from leaves of Pisum sativum L. and Spinacia oleracea L. With palmitoyl-CoA as acyl donor the major product formed was monoacyl glycerol phosphate. In pea seedlings enzymatic activity was found to be dependent on the age of shoots going through a maximum 12 days after planting. In such plants the highest enzymatic activity is found in leaves and not in the shoot apex. Also in leaves activity varies according to the age of these organs. In leaves of maximal activity the highest total and specific activity was found in soluble proteins from chloroplasts. In older pea and spinach leaves higher activities were observed in membranes from chloroplasts and microsomes.

No abstract