Ken'ichi Ichihara

Diacylglycerol acyltransferase in maturing safflower seeds: its influences on the fatty acid composition of triacylglycerol and on the rate of triacylglycerol synthesis

Diacylglycerol acyltransferase in a particulate preparation from maturing safflower seeds showed no strict selectivity for acyl-CoA when acyl-CoA substrates were administered as mixtures. This suggested that the fatty acid composition of position 3 in safflower triacyl-sn-glycerol exclusively depends on the acyl-CoA composition in the cell. The specific activity of the acylation was approximately 3 nmol/min per mg protein of the preparation under the optimum assay conditions. This low activity and other data appeared to indicate that the diacylglycerol acyltransferase reaction may be the rate-limiting step in triacylglycerol synthesis in vivo.

Cloning and sequence analysis of a cDNA encoding rice glutaredoxin

A full‐length cDNA clone (RASC8) encoding glutaredoxin (thioltransferase) was isolated from a cDNA library of an aleurone layer prepared from a developing seed of rice (Oryza sativa L.). RASC8, 568bp in length, contained an ATG codon and two possible polyadenylation signals, and encoded 112 amino acid residues. Cys‐Pro‐Phe‐Cys, which is the active site and a highly conserved sequence among thioltransferases, was found in the deduced amino acid sequence. RASC8 was introduced into an expression vector pMALc2 and the translated product possessed thioltransferase activity.

sn-Glycerol-3-phosphate acyltransferase in a particulate fraction from maturing safflower seeds.

The properties of the acyl-CoA:sn-glycerol-3-phosphate O-acyltransferase in a 20,000g particulate fraction from maturing safflower seeds were investigated. The optimum pH of the reaction was 7.2. The apparent Km for glycerophosphate was 0.54 mM. Only monoacylglycerophosphate was accumulated in the particulate fraction under normal conditions. Position 1 of glycerophosphate was exclusively esterified with either palmitoyl-CoA or linoleoyl-CoA as acyl donor, while 2-acylglycerophosphate was the minor product. The specificity and selectivity of the acyltransferase for acyl-CoA were broad and somewhat affected by temperature. The concentration of glycerophosphate did not affect the selectivity. These observations suggested that the fatty acid composition of position 1 of safflower triacylglycerol must primarily depend on the composition of the acyl-CoA pool in the site of synthesis, and that growth temperature and the acyl-CoA selectivity of the glycerophosphate acyltransferase may be rather minor factors regarding regulation of the fatty acid composition of position 1 in triacylglycerol.

Fatty acid composition and lipid synthesis in developing safflower seeds

Abstract Linoleic acid predominated in every lipid class during the whole period of seed development of safflower, while linolenic acid decreased with increasing maturation and it was not detected in mature seeds. Just before the initiation of triacylglycerol accumulation, the fatty acid composition of triacylglycerols changed more rapidly than those of phospholipids and glycolipids. Saturated fatty acids tended to accumulate at the 1- and 3-positions of the glycerol molecule and the more highly unsaturated acids at the 2-position. The fatty acid compositions at the 1- and 3-positions were similar in all cases investigated, but in none of the triacylglycerols was the distribution completely symmetrical. The positional distribution of linolenic acid in triacylglycerols prepared from the immature seeds 2 days after flowering and from the leaves was unusual; in spite of its highest degree of unsaturation, it was preferentially esterified at the 1- and 3-positions. When triacylglycerol was most rapidly accumulated (14–18 days after flowering), the incorporation of acetate-[U- 14 C] into total lipids was also maximum and dienoic fatty acids were the principal acids labelled. Diacylglycerols and compound lipids reached the highest rate of synthesis 15 days after flowering, and then a maximum incorporation into triacylglycerol occurred 18 days after flowering. Incubation temperature affected the synthesis of individual lipid classes. Triacylglycerol was more rapidly synthesized at 32° than at 10°, while diacylglycerols and compound lipids were accumulated under the low-temperature condition. A rise of incubation temperature caused a depression in dienoic acid synthesis.

Some properties of diacylglycerol acyltransferase in a particulate fraction from maturing safflower seeds

Abstract The activity of diacylglycerol acyltransferase of a subcellular particulate fraction from maturing safflower seeds was remarkably stimulated by the addition of 1, 2-diacylglycerols which were previously emulsified in a gelatin solution by sonication. Metal ions were inhibitory to the reaction. Deoxycholate and diisopropyl fluorophosphate were the most effective inhibitors. Sulfhydryl groups seemed to be of limited significance in the enzyme. Both 1, 2-dioleoyl- sn -glycerol and 2, 3-dioleoyl- sn -glycerol were good substrates of diacylglycerol acyltransferase, but the 1, 3-isomer did not serve as an acyl acceptor. The enzyme showed broad specificity for synthetic rac -1, 2-diacylglycerols containing various fatty acids. However, rac -1, 2-diacetylglycerol and rac -1, 2-dibutyrylglycerol, which are soluble in water, were ineffective. The enzyme exhibited no significant specificity for saturated and unsaturated fatty acyl-CoA thioesters as acyl donors. This suggests that the fatty acid composition at the 3-position of the glycerol molecule of safflower triacylglycerols may depend on the composition of the endogenous acyl-CoA pool.

Intracellular translocation of phosphatidate phosphatase in maturing safflower seeds: a possible mechanism of feedforward control of triacylglycerol synthesis by fatty acids.

Phosphatidate phosphatase activity was found both in the cytosol and in the microsomal membrane of maturing safflower seeds. The combined and relative activities of these two forms varied with seed maturation. During the period of rapid triacylglycerol accumulation in the cell, most of the phosphatidate phosphatase activity was membrane-bound; at the initial and last stages of seed development when triacylglycerol synthesis was at an insignificant level, the majority of the activity was soluble. The potassium salts of palmitic, stearic and oleic acids, which are the fatty acid products of proplastids, caused the translocation of the cytosolic phosphatidate phosphatase to the microsomal membrane, while laurate and linoleate, which are not products of proplastids, showed no effect. Oleoyl-CoA did not convert the soluble form of the enzyme into the membrane-bound form. The translocation induced by oleate was reversible. The cytosolic phosphatidate phosphatase of safflower seeds was not transferred to the microsomal membranes prepared from soybean, a plant species of Leguminosae, and from rapeseed, a species of Cruciferae, but was transferred to that from sunflower, which belongs to the same family as safflower, Compositae. These observations suggest that in maturing oil seeds the rate of fatty acid synthesis in proplastids may regulate the species-specific translocation of phosphatidate phosphatase between the cytosol and the endoplasmic reticulum membrane where triacylglycerol synthesis occurs and that in turn the translocation of this ambiquitous enzyme could control the rate of triacylglycerol synthesis in the cell.

Lipid synthesis and acyl-CoA synthetase in developing rice seeds.

Developing rice seeds rapidly accumulated storage lipids between 5 and 12 d after flowering. The contents of palmitic, oleic, and linoleic acids increased throughout seed development, while the α-linolenic acid content remained low. The activity of acyl-CoA synthetase varied coincidentally during the period of lipid accumulation, and rice seeds had a sufficient capacity to supply acyl-CoA substrates for TAG synthesis. Acyl-CoA synthetase showed a broad specificity for native FA of rice seeds except for stearic acid, and π electrons of a Δ9–Δ11 double bond in the C16−C18 acyl chains were required for its maximal activity.

Triacylglycerol synthesis by subcellular fractions of maturing safflower seeds

Abstract A subcellular particulate fraction (103 g, precipitate) prepared from maturing safflower seeds catalysed triacylglycerol synthesis from oleoyl-CoA.

Lipid biosynthesis in developing perilla seeds

In developing seeds of Perilla frutescens var. crispa, the triacylglycerol fraction was found to accumulate between 15 and 19 days after flowering. Of this, 65% of the total fatty acids was alpha-linolenic acid in the mature seeds, with the latter being esterified in comparable amounts at all positions (sn-1, 2 and 3) of the glycerol residue. It was also demonstrated that, 1-acylglycerol-3-phosphate acyltransferase, which catalyzes esterification at the sn-2 position of the glycerol backbone, showed low activities for alpha-linolenoyl-CoA as substrate. These findings suggest that the diacylglycerol precursor for triacylglycerol synthesis is not directly derived from phosphatidic acid through the glycerol phosphate pathway.

1-Acylglycerophosphocholine O-acyltransferase in maturing safflower seeds

The activity of 1-acylglycerophosphocholine (1-acyl-GPC) O-acyltransferase (EC 2.3.1.23) varied during maturation of safflower (Carthamus tinctorius L.) seeds, and activity per seed was highest in the middle period of seed development when triacylglycerol (TAG) is most rapidly synthesized. The specific activity of acyl transfer in a 20000·g particulate preparation exceeded 500nmol·min-1·(mg protein)-1 and was higher than those of any other enzymes involved in TAG synthesis (K. Ichihara et al., 1993, Plant Cell Physiol. 34, 557–566). This suggested the presence of a large flux of acyl-CoA to phosphatidylcholine in the cell. The reaction was specific to C16 and C18 acyl-CoAs with a double bond at position 9. Lauroyl- and erucoyl-CoA were completely ineffective, while ricinoleoyl- and elaidoyl-CoA were utilized efficiently. The relative order of specificity for native acyl-CoA species was linoleoyl > oleoyl ≫ stearoyl = palmitoyl. When acyl-CoA mixtures were presented, preference for the unsaturated species rather than the saturated species was even more apparent. The enzyme preferentially utilized 1-C16-acyl- and 1-C18-acyl-GPC molecular species, and 1-palmitoyl-, 1-stearoyl-, 1-oleoyl-and 1-linoleoyl-GPC equally served as acyl acceptor. No activity was detected with 1-octanoyl-GPC, and 1-erucoyl-GPC produced little effect. The effectiveness of 1-alkyl-GPC was comparable to that of 1-acyl-GPC. It was thus concluded that the enzyme recognizes the chain lengths of the acyl donor and acceptor, and the double bond at position 9 of the acyl donor.