Use of streptavidin to detect biotin-containing proteins in plants.

A procedure to detect biotinyl proteins after fractionation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was developed. Proteins were immobilized on nitrocellulose and biotin-containing proteins were detected by probing with 125I-streptavidin. Using this procedure a small survey of biotinyl protein in plants was undertaken. In total four biotin-containing proteins were detected in higher plants of molecular weights 62,000, 50,000, 34,000, and 31,000. These biotinyl proteins were not ubiquitous in the plants surveyed. In the cyanobacterium Anabeana variabilis, a single biotin-containing protein of 21,000 Da was detected. In isolated spinach chloroplasts, the two biotinyl proteins detected were soluble. The results are discussed in relation to acetyl-CoA carboxylase.

Characterization and solubilization of an acyl chain elongation system in microsomes of leek epidermal cells.

Microsomes prepared from leek epidermal tissue readily elongate stearoyl-CoA to very long chain fatty acid with malonyl-CoA as the C2 unit. In the absence of stearoyl-CoA, but in the presence of ATP, microsomes elongate endogenous free fatty acids. Endogenous CoA is the source of CoA. Palmitoyl, stearoyl, and higher saturated acyl-CoAs are readily elongated by the microsomal system but oleoyl-CoA is ineffective; however, the higher monounsaturated acyl-CoAs can be elongated. Since the very long chain fatty acids of the leek epidermis are all saturated, it would appear that the reaction controlling the nature of the final acyl product is the inactivity of oleoyl-CoA as a substrate. There is no evidence that acyl carrier protein participates in the elongation reactions. Evidence is also presented suggesting that (a) there may be two elongation systems, one responsible for the conversion of stearoyl-CoA to arachidonyl-CoA and the second involved in the conversion of arachidonyl-CoA to very long chain fatty acids, and that (b) the elongation activities may be associated with a large polypeptide.

Studies of the delta 12 desaturase of Carthamus tinctorius L.

The delta 12 desaturase of developing safflower seeds responsible for the conversion of an oleoyl moiety to the linoleoyl moiety of phospholipids was further characterized. The protein concentration of the microsomal preparation, the oleoyl-CoA concentration (the primary substrate), short incubation periods, and the addition of lysophospholipids must be controlled to obtain optimal desaturation. No evidence could be obtained to implicate cytochrome b5 as the intermediate electron carrier. Attempts to solubilize the desaturase with a variety of detergents and chaotropic reagents were not successful. Brief exposure of the microsomal preparation to trypsin resulted in rapid loss of activity. The overall evidence would suggest that the delta 12 desaturase requires a reductant (NADPH), a NADPH:electron carrier reductase, an electron carrier, a specific desaturase, and an acyltransferase with oleoyl-CoA as the substrate to acylate lysophospholipid to the active oleoyl phospholipids (presumably phosphatidylcholine or phosphatidylethanolamine). The complexity of this system suggests that purification of the components and a reassembling of the purified components will be difficult.

Subcellular distribution of acetyl-coenzyme A carboxylase in mesophyll cells of barley and sorghum leaves.

The subcellular distribution of acetyl-CoA carboxylase [acetyl-CoA-carbon dioxide ligase (ADP-forming), EC 6.4.1.2] was determined in mesophyll protoplasts isolation from barley, a C3 plant, and sorghum, a C4 plant. In both species, all of the mesophyll acetyl-CoA carboxylase was demonstrated to be chloroplastic. In barley leaves and mesophyll protoplasts, a single biotinyl protein of 60,000 Da was identified by a modified Western-blotting procedure. The subcellular distribution of this biotinyl protein was identical to that found for acetyl-CoA carboxylase. These results are discussed in relation to the compartmentation of reactions requiring malonyl-CoA as a substrate.

The effect of hypolipidemic drugs on plant lipid metabolism.

The effect of hypolipidemic drugs, WY14643 and DH990, on plant lipid metabolism has been studied. The total incorporation of [14C]acetate into lipids was inhibited by addition of both drugs to aged potato (Solanum tuberosum) tuber discs, spinach (Spinacia oleracea) leaves, and spinach chloroplasts, while the incorporation in Chlorella vulgaris cells was affected only by DH990. Moreover, DH990 inhibited the incorporation of 14C-labeled fatty acids into phosphatidylcholine and phosphatidylethanolamine of potato discs, and decreased the incorporation into phosphatidylglycerol of Chlorella cells. DH990 inhibited the formation of polyunsaturated fatty acids in potato discs, Chlorella cells, and spinach leaves, whereas WY14643 had no effect on the formation of these fatty acids. Stearoyl-ACP desaturase from safflower (Carthamus tinctorius) seeds was very sensitive to both drugs, especially DH990, which completely blocked the activity at 2 mM levels. When safflower lysophospholipid acyltransferases were solubilized by detergent treatment, only DH990 inhibited the incorporation of [14C]oleoyl-CoA into lysophosphatidylcholine or lysophosphatidylethanolamine. Both drugs inhibited fatty acid synthesis from [14C]malonyl-CoA in the microsomal fraction from safflower seeds, but only DH990 inhibited FAS activity in the soluble fraction; both drugs inhibited severely the formation of stearic acid. Both acetyl-CoA carboxylase and acetyl-CoA synthetase were sensitive to both drugs.

Tissue distribution of acetyl-coenzyme a carboxylase in leaves.

Acetyl-CoA carboxylase [acetyl-CoA-carbon dioxide ligase (ADP forming), EC 6.4.1.2] is a biotin-containing enzyme catalyzing the formation of malonyl-CoA. The tissue distribution of this enzyme was determined for leaves of C(3)- and C(4)-plants. The mesophyll tissues of the C(3)-plants Pisum sativum and Allium porrum contained 90% of the leaf acetyl-CoA carboxylase activity, with the epidermal tissues containing the remainder. Western blotting of proteins fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, using (125)I-streptavidin as a probe, revealed biotinyl proteins of molecular weights 62,000, 51,000, and 32,000 in P. sativum and 62,000, 34,000, and 32,000 in A. porrum.In the C(4)-plant sorghum, epidermal protoplasts, mesophyll protoplasts and strands of bundle sheath cells contained 35, 47, and 17%, respectively, of the total leaf acetyl-CoA carboxylase activity. In Zea mays leaves the respective figures were 10% for epidermal protoplasts, 56% for mesophyll protoplasts, and 32% for bundle sheath strands. Biotinyl proteins of molecular weights 62,000 and 51,000 were identified in leaves of sorghum and Z. mays.The results are discussed with respect to each tissue's requirements for malonyl-CoA for various metabolic pathways.

In Vitro Fatty Acid Synthesis and Complex Lipid Metabolism in the Cyanobacterium, Anabaena Variabilis: II. Acyl Transfer and Complex Lipid Formation.

In vitro fatty acid transfer to form complex lipids was observed in crude cell extracts of Anabaena variabilis using [1-(14)C]palmitoyl-acyl carrier protein, [1-(14)C]stearoyl-acyl carrier protein, and [1-(14)C]oleoyl-acyl carrier protein substrates. The data indicated that there was a rapid transfer of the fatty acids into the complex lipids. The greatest amount of radioactivity was observed in the monogalactosyl diacylglycerol fractions and there appeared to be a preference for the transfer of stearate over palmitate. The exogenously added lysophospholipids, (lysophosphatidylglycerol, lysophosphatidylcholine) and 2-monopalmitin acted as acceptors in acyl transfer. Addition of the hypolipidemic drug, WY14643, inhibited the fast acyl transfer reaction and showed that the first product of acyl transfer was diglyceride followed by monogalactosyl diacylglycerol. Thioesters of Coenzyme A do not seem to be involved in these reactions.

Biosynthesis of very long chain fatty acids in microsomes from epidermal cells of Allium porrum L.

The elongation system present in leek epidermal cells functions to synthesize very long chain fatty acids which, in turn, are the precursors to alkanes. The elongation system is microsomal, employs only saturated acyl components of the endogenous lipid pool as acceptors, utilizes malonyl-CoA as the C2 donor, has an absolute requirement for ATP, and is markedly inhibited by acetyl-ACP. Only saturated acyl-CoAs are readily elongated to very long chain fatty acids by malonyl-CoA in the absence of ATP. ACP is not required by the microsomal system.

In Vitro Fatty Acid Synthesis and Complex Lipid Metabolism in the Cyanobacterium Anabaena variabilis: I. Some Characteristics of Fatty Acid Synthesis.

In vitro fatty acid synthesis was examined in crude cell extracts, soluble fractions, and 80% (NH(4))(2)SO(4) fractions from Anabaena variabilis M3. Fatty acid synthesis was absolutely dependent upon acyl carrier protein and required NADPH and NADH. Moreover, fatty acid synthesis and elongation occurred in the cytoplasm of the cell. The major fatty acid products were palmitic acid (16:0) and stearic acid (18:0). Of considerable interest, both stearoyl-acyl carrier protein and stearoyl-coenzyme A desaturases were not detected in any of the fractions from A. variabilis. The similarities and differences in fatty acid synthesis between A. variabilis and higher plant tissues are discussed with respect to the endosymbiotic theory of chloroplast evolution.

The effect of hypolipidemic drugs WY14643 and DH990, and lysophospholipids on the metabolism of oleate in plants.

The effects of the addition of hypolipidemic drugs and 1-acylglycerolipids on the metabolism of oleate in plants have been studied in vivo and in vitro. Using aged potato slices with [14C]oleate as a precursor, it was found that these drugs markedly inhibited both the incorporation into complex lipids and the desaturation of oleate to linoleate. Moreover, in vitro experiments, carried out with microsomes prepared from developing safflower seeds and [14C]oleate or [14C]oleoyl-CoA as precursors, confirmed the inhibitory effect of the drugs on oleate desaturation, and showed that while WY14643 mainly affected oleoyl thiokinase activity, DH990 exerted its strongest effect on the formation of PL, indicating that the mode of action of these two drugs in safflower microsomes is essentially different. Addition of LPC or LPE stimulated the incorporation of radiolabeled precursor into PC and PE, respectively, as well as the desaturation of oleate to linoleate when [14C]oleoyl-CoA was the precursor. The evidence obtained suggests that oleoyl-PE, as well as oleoyl-PC, should be considered as a possible substrate for oleate desaturation in plants.

The Metabolism of the Germinating Oil Palm (Elaeis guineensis) Seedling : In Vivo Studies.

The metabolism of (14)C-labeled fatty acids and triacylglycerols was followed in intact germinating oil palm seedlings as well as in tissue slices. In the germinating seedling, the shoot contained a normal pattern of membrane fatty acids (mainly C(16), C(18:1), C(18:2)) but the kernel contained about 68% C(12) and C(14) fatty acids. Haustorium fatty acids were intermediate between the two. [(14)C]Acetate was actively metabolized by shoot and haustorium slices but not so actively by the kernel. Approximately 9% to 17% was converted to water-soluble substances, 4% to 6% to CO(2), and 0.5% to 5.9% to lipids. The fatty acids synthesized in the shoot and haustorium were mainly C(16), C(18), and C(18:1) fatty acids but in the kernel about 18% to 32% of the (14)C-fatty acids were C(12) fatty acids.[(14)C]Lauric acid was absorbed and metabolized by haustorium slices and by the haustorium in intact seedlings; it was partly esterified to triacylglycerols and also converted to water-soluble substances and insoluble tissue material. In contrast, tri-[(14)C]laurin was absorbed but not metabolized. The haustorium also absorbed other fatty acids but the longer chain (C(16) and C(18)) fatty acids were not esterified or metabolized further. Preincubation of the haustorium with plant hormones or in the presence of kernel tissue did not alter its inactivity towards tri-[(14)C]laurin.When tri-[(14)C]laurin or [(14)C]lauric acid were injected into the seed or the shoot, there was no movement or radioactivity to other parts of the seedling. When injected into the shoot, but not into the seed, tri-[(14)C] laurin was hydrolyzed and partly metabolized to water-soluble substances.

Some Enzymic Activities in the Germinating Oil Palm (Elaeis guineensis) Seedling.

In germinating oil palm (Elaeis guineensis var D x P) seedling, an active lipase was present in the shoot but absent from both the kernel and the haustorium. It has an optimum pH of 6.2 and a smaller peak at pH 8.6. The shoot lipase was active against a number of mono-, di-, and triacylglycerols as well as the endogenous lipids present in the shoot, haustorium, and kernel. Activity against related substrates were in the order: trilaurin > dilaurin > monolaurin but monopalmitin > dipalmitin > tripalmitin. The level of the enzyme in the seedling was highest at a relatively early stage of growth (18-21 days) and also higher in dark-grown seedlings. Glyoxylate bypass enzymes (malate synthetase and isocitrate lyase), glutamate-oxaloacetate transaminase, phosphoenolpyruvate carboxykinase and lauroyl-coenzyme A oxidase were located in the haustorium. The levels of the enzymes paralleled seedling development and were slightly higher in light-grown seedlings. Fatty acyl-coenzyme A synthetase activity was very low and was found in both the shoot and haustorium.