Acetyl coenzyme A carboxylase in species of Triticum of different ploidy.

The cellular amounts and cellular activities of acetyl CoA carboxylase (ACC; EC 6.4.1.2.) were determined in the first leaves of diploid, tetraploid and hexaploid species of Triticum (wheat). Per leaf the ACC activities were very similar in T. monococcum (2 χ), T. dicoccum (4 χ) and T. aestivum (6 χ). The ACC activity per chloroplast also showed little variation between species of different ploidy but since chloroplast number increases with ploidy, the ACC activities and ACC amounts per cell also increased with ploidy. These cellular increases in ACC amounts associated with increases in gene dosage were highly co-ordinated in the diploids T. monococcum and T. tauschii and their respective autotetraploids so the specific activity of ACC was highly conserved in these plants. The relevance of these findings to attempts to genetically manipulate lipid biosynthesis in chloroplasts is discussed.

Acetyl-CoA-carboxylase activity in normally developing wheat leaves.

In order to investigate the role of acetyl CoA carboxylase (ACC) in the regulation of fatty-acid biosynthesis in chloroplasts, the activities and relative amounts of the enzyme have been measured in the tissue of wheat (Triticum aestivum L.) leaves undergoing development and cellular differentiation. The total activity in the first leaves of 5- to 7-d-old plants was similar but decreased to less than half in 9-d-old plants. The activity of ACC in the cells of the first leaf of 7-d-old plants doubled when cell age increased from 24 to 48 h, remained relatively constant for a further 24 h and then declined. The amount of ACC in cells increased 15-fold during the first 36 h of cell enlargement. Cells more than 36 h old contained about two-thirds the maximum amount of ACC found in younger cells. The most rapid phase of fatty-acyl accumulation in lipids was in cells aged between 60 and 84 h. Tenfold changes in the activity of ACC were observed when the assay conditions with respect to ATP, ADP, Mg(2+) and pH were changed to correspond to the physiological conditions in chloroplasts during light/dark transitions. This observation and the magnitude of the changes in the optimum activity and amount of ACC in leaf cells undergoing development are consistent with a role for ACC in the regulation of the flow of carbon from acetyl CoA to fatty acids in chloroplasts.

Purification and characterization of maize leaf acetyl-coenzyme A carboxylase.

Maize leaf acetyl-CoA carboxylase was purified from whole tissue homogenates by precipitation with polyethylene glycol and ammonium sulfate, and gel filtration. Recoveries were approximately 5% with 100-fold increases in specific activity. The molecular weight of the native enzyme is estimated at 500,000 from the elution volume of a calibrated Ultrogel AcA 22 column. Electrophoresis in polyacrylamide gel containing 1% sodium dodecyl sulfate revealed a single subunit of Mr 60,000-61,000. Investigation of the kinetic properties of the purified enzyme indicates that Mg X ATP is the active substrate, with free ATP inhibiting and Mg2+ activating the enzyme. Km's for acetyl-CoA and HCO3- are about 0.1 and 2 mM, respectively. ADP inhibition is competitive with respect to ATP, but uncompetitive with respect to acetyl-CoA. The observed responses of purified acetyl-CoA carboxylase to changes in pH, and in concentrations of Mg2+, ATP, and ADP, and the reported changes in the chloroplastic concentrations of these effectors during light-dark transitions of chloroplasts are consistent with increased acetyl-CoA carboxylase activity upon illumination of chloroplasts.

Desaturation of Oleic and Linoleic Acids by Leaves of Dark- and Light-grown Maize Seedlings.

Oleate and linoleate desaturation in leaves of maize seedlings was largely independent of previous light treatment of the seedlings; there was no evidence of light-induced desaturase activities. These results are in sharp contrast to those observed with developing cucumber cotyledons in which pronounced increase in desaturation occurs after exposure of tissue to light. The rates of desaturation of oleate were about four times those of linoleate in both etiolated and 16-hour greened maize leaves. In both etiolated and greened tissues, about two-thirds of the label from oleate was esterified after 4 hours, half of which was in phosphatidylcholine. Phosphatidylcholine and diglyceride contained large proportions of [(14)C]linoleate formed from [(14)C]oleate but not [(14)C]linolenate. In monogalactolipid, about two-thirds of the labeled fatty acids were linolenate. In vivo desaturase activity was present in tissue of widely different levels of differentiation and chlorophyll content obtained from light-grown maize seedlings.

Influence of elevated levels of linoleic acid on the thermal properties of bovine milk fat.

The thermal properties of bovine milk fat containing 15.5% linoleic acid have been compared with those of milk fat containing a normal level (1.8%) of linoleic acid in order to examine the influence of altered triglyceride (TG) composition on their physical characteristics. The total TGs of 18:2-rich milk fat melted over the range -38 to 30 C compared with the range -33 to 34 C for control milk fat. Polymorphism exhibited by the high mol wt TGs of control milk fat was absent in the same fraction of 18:2-rich milk fat. Similarly, the complex melting thermogram of the low mol wt TGs of control milk fat and its obvious polymorphic behavior contrasted with the single broad melting peak of the low mol wt TGs of 18:2-rich milk fat. This solid miscibility in the 18:2-rich milk fat could be a consequence of the lower proportion of saturated TGs or the presence of high proportions of diene and triene TGs containing 18:2 instead of monoene and diene TGs containing 18:1.

The role of holotrichs in the metabolism of dietary linoleic acid in the rumen.

The uptake and metabolism of linoleic acid by rumen holotrichs (mainly Isotricha prostoma and I. intestinalis) has been examined in in vitro infusion experiments. Maximum absorption and metabolism of [1-14C]linoleate by 2 . 10(6) Isotricha suspended in 100 ml buffer was obtained using an infusion rate of 1.6 mg linoleate/h. After 90 min, 84% of the added substrate was recovered within the cells, mainly as free fatty acid or phospholipid. There was a rapid incorporation of radioactivity into phospholipid, mainly phosphatidylcholine, at the commencement of linoleate infusion but no further incorporation after about 40 min. The presence of bacteria during incubations, in approximately the same Isotricha/bacteria ratio as found in the rumen, reduced the uptake of linoleate and the accumulation of free fatty acid by holotrichs but the incorporation into phospholipid remained similar to that obtained in the absence of bacteria. Very little biohydrogenation of linoleic acid occurred in incubations with holotrichs alone. Bacterial suspensions converted linoleic acid to mainly trans monoene and a small amount of stearic acid, but in incubations containing both bacteria and holotrichs, both stearic acid and trans monoene were major products. Using the latter mixed culture, about 20% of the added [1-14C]linoleic acid was present in holotrich phospholipid of which 62% remained as octadecadienoic acid. The Isotricha population was 3 . 10(3)--2 . 10(4)/ml rumen fluid and it contributed about 23% of the linoleic acid in the rumen of a cow on a hay diet.

Triglyceride composition of bovine milk fat with elevated levels of linoleic acid.

The effect of increasing the linoleic acid (18:2) content of milk fat on the composition and structure of the triglycerides (TG) was investigated. Protected sunflower seed supplement was added to the diet of a cow grazing on pasture, and the structure and composition of the milk fat compared with the milk fat from its monozygous twin which had been fed a control diet. The relative proportions of TG fractions of high, medium, and low molecular weight in the milk fat with elevated levels of 18:2 (15.5% 18:2) were 43.0, 19.5, and 37.5 moles %, respectively, compared with 36.1, 19.7, and 44.2 moles %, respectively in the milk fat from the cow fed the control diet. Separation of these three TG fractions of each milk fat into TG classes with different levels of unsaturation showed that the milk fat with elevated levels of 18:2 contained higher proportions of diene, triene, and tetraene TG and correspondingly lower proportions of saturated and, to a lesser extent, monoene TG. The saturated and monoene TG from the two milk fats had similar fatty acid compositions. However, the diene TG of the 18:2-rich milk fat included high proportions of the combination of 18:2 with two saturated fatty acids (FA) which are minor constituents of normal milk fats. Likewise, the triene TG reflected the presence of 18:2 in combination with 18:1 and a saturated FA.

Lipid biosynthesis in green leaves of developing maize.

Successive leaf sections from the base to the tip of rapidly developing leaves of 7-day-old maize (Zea mays var. Kelvedon Glory), grown in the light, utilized acetate for fatty acid biosynthesis in a very divergent manner. Basal regions of the leaf containing proplastids synthesized insignificant proportions of unsaturated fatty acids and appreciable proportions of fatty acids with 20 or more carbon atoms. An increase in the light intensity during incubations with acetate-1-(14)C resulted in very little enhancement of either total or polyunsaturated fatty acid biosynthesis in this tissue.When the distal leaf sections, containing mesophyll chloroplasts with well developed grana and bundle sheath chloroplasts without grana, were incubated with acetate at 150 ft-c and 30 C, approximately 30% of the newly synthesized fatty acids were unsaturated (mainly 18: 1 and 18: 2). At 2800 ft-c and 20 C, 60% of the fatty acids were unsaturated and the total synthesis of fatty acids increased 4-fold. No detectable amount of fatty acids with 20 or more carbon atoms were synthesized in this morphologically mature tissue, and the proportions of newly synthesized fatty acids more closely resembled the endogenous fatty acids in the immature tissue.Only 4% of the newly synthesized fatty acids were 18: 3 but most of this was incorporated into monogalactolipid. In the distal sections, 20 to 25% of the newly synthesized fatty acids in monogalactolipid were 18: 3 compared with the endogenous proportions of 85%. The differences in the composition of the newly synthesized fatty acids and the endogenous fatty acids appear to be related very largely to the low rate of 18: 3 biosynthesis from 18: 1 and 18: 2. Phosphatidyl choline and phosphatidyl ethanolamine, with lower proportions of 18: 3, contained radioactive fatty acids which resembled the endogenous composition more closely.Phosphatidyl choline was quantitatively the most important acyl lipid synthesized under both light conditions. In addition, there was considerable stimulation of acetate incorporation into phosphatidyl glycerol and monogalactolipid, especially in the morphologically most mature regions of the tissue at the higher light intensity.