Sterol and triterpene synthesis in the developing and germinating pea seed.

Developing and germinating pea seeds were compared with respect to their capacity to incorporate mevalonate into sterols and triterpenes. The capacity for sterol synthesis is greatest in the least mature fruits and decreases during their development. Label is shown, by gas-liquid chromatography and counting the radioactivity of trapped fractions, to be associated with campesterol, beta-sitosterol and isofucosterol. During early stages of germination sterol synthesis is insignificant. The triterpene fraction becomes heavily labelled during both development and germination. The label is associated almost exclusively with beta-amyrin during germination but with cycloartenol and 24-methylenecycloartanol during development. It is only in the terminal stages of maturation that beta-amyrin becomes significantly labelled. At the same time an unidentified radioactive polar compound appears. The possible significance of the appearance of this polar compound and the regulation of the synthesis of these higher terpenoids is discussed.

Development of the activities of enzymes of the isoprenoid pathway during early stages of pea-seed germination.

The activities of individual enzymes of the isoprenoid pathway from mevalonate kinase to squalene synthetase in homogenates of seeds germinated up to 32h were assayed. Changes in the activity of each enzyme were observed and compared with the activity at the 2h germination stage. Activities of alkaline phosphatase and fructose 1,6-diphosphate aldolase were similarly measured to provide a reference for changes in the general metabolic activity of seeds during imbibition of water. Water uptake reached a plateau after 12h. The reference enzymes almost doubled in activity between 2 and 8h and thereafter their activities steadily declined. All of the enzymes of the isoprenoid pathway increased in activity between 2 and 6h and, thereafter, with the exception of the prenyltransferase, their activities remained relatively constant. With the prenyltransferase activity the initial increase was followed by a short plateau between 6 and 9h and then a second increase to a maximum between 14 and 16h. After 16h the activity declined. The relative activities of the isoprenoid enzymes at 16h of germination were mevalonate kinase>phosphomevalonate kinase>pyrophosphomevalonate decarboxylase approximately isopentenyl pyrophosphate isomerase>squalene synthetase>isopentenyl pyrophosphate/dimethylallyl pyrophosphate prenyltransferase. The finding that the prenyltransferase may be the rate-limiting enzyme in squalene synthesis from mevalonate is discussed in relation to regulation of isoprenoid synthesis during pea-seed germination.

2,3-Oxidosqualene cyclase and cycloartenol-s-adenosylmethionine methyltransferase activities in vivo in the cotyledon and axis tissues of germinating pea seeds.

Axis tissues, root and shoot, of germinating pea seedlings actively synthesize sterol from [2-14C]mevalonate during the first 3 days of germination. In addition to the intermediates of sterol synthesis, cycloartenol and 24-methylenecycloartanol, these tissues also form the triterpene beta-amyrin. The cyclase catalysing the formation of cycloartenol from oxidosqualene is about four times as active as that for beta-amyrin synthesis. 2. Sterol synthesis in the cotyledon is negligible, but cycloartenol and 24-methylenecycloartanol, as well as beta-amyrin, are synthesized there. Oxidosqualene cyclase activity in this tissue is 2.6 times as active for beta-amyrin synthesis as for cycloartenol synthesis. 3. Comparison of the relative amounts of 14C in cycloartenol and 24-methylenecycloartanol in the axis tissues and cotyledons of 3-day-old seedlings point to relatively active cycloartenol-S-adenosylmethionine methyltransferase systems in both axis tissues and a poorly active system in the cotyledon. 4. The role of beta-amyrin synthesis in the germinating pea seedling is discussed.

Characterization of the increased lysophospholipase activity in gibberellic Acid-treated barley aleurone layers.

A lysophospholipase (LPL) activity appears in the aleurone of barley (Hordeum vulgare L. cv Himalaya) half seeds during imbibition on moist agar. Secretion of LPL by half seeds is promoted by GA(3); the increase in secretory rate is almost linear from 10(-10) to 10(-6) molar GA(3). LPL activity is likewise promoted in isolated aleurone layers by GA(3). Its secretion into the incubation medium requires the continued presence of GA(3) and commences after a 10 to 14 hour lag period when 10 millimolar Ca(2+) is present. In the absence of Ca(2+), the lag period remains unchanged but attainment of the maximum secretory rate is delayed. Ca(2+) alone has very little effect either on LPL activity accumulated in the aleurone layer or in the surrounding medium. However, 50 millimolar Ca(2+) together with GA(3) dramatically increase the level of secreted activity and of total (accumulated and secreted) activity.The metabolic inhibitors cycloheximide and actinomycin D inhibit the accumulation of LPL activity in the aleurone and also the secreted activity. Actinomycin D added after the lag period results in a much lower inhibition. The increase in LPL activity in response to GA(3) occurs as a result of de novo synthesis; LPL activity from barley half seeds incubated in 80% D(2)O in the presence of GA(3) undergoes a shift to higher density compared with the activity from similar controls incubated in H(2)O. The characteristics of the GA(3) enhancement of LPL activity are compared specifically with alpha-amylase and generally with other GA(3)-controlled hydrolases.

Uridine diphosphate glucose-sterol glucosyltransferase and nucleoside diphosphatase activities in etiolated pea seedlings.

1. UDP-glucose-sterol glucosyltransferase and nucleoside diphosphatases were isolated in a particulate fraction from 7-day-old etiolated pea seedlings. The glucosyltransferase and UDPase (uridine diphosphatase) are stimulated by Ca2+ cation, less so by Mg2+ cation, and inhibited by Zn2+. 2. Each activity has a pH optimum near 8. 3. The glucosyltransferase is specific for UDP-glucose as the glucosyl donor and is inhibited by UDP. Partial recovery from UDP inhibition is effected by preincubation of the enzyme. 4. Freeze-thaw treatment and subsequent sucrose-density-gradient centrifugation of the particulate fraction shows the glucosyltransferase to be widely distributed among cell fractions but to be most active in particles with a density of 1.15 g/ml. UDPase is most active in particulate material with a density of over 1.18 g/ml but an activity peak also appears at 1.15 g/ml. Of several nucleoside diphosphatase activities, UDPase activity is most enhanced by the freeze-thaw and sucrose-density-gradient-fractionation procedures. 5. Detergent treatment with 0.1% sodium deoxycholate allows the partial solubilization of the glucosyltransferase and UDPase. The two activities are similarly distributed between pellet and supernatant after high-speed centrifugation for two different time intervals. 6. A role for UDPase in the functioning of glucosylation reactions is discussed.

Albumin stimulates the release of lysophosphatidylcholine from cultured rat hepatocytes.

The effect of albumin on the release of [3H]lysophosphatidylcholine from cultured rat hepatocytes prelabelled with [Me-3H]choline was studied. In the absence of serum and albumin from the medium, the cells released essentially no [3H]lysophosphatidylcholine. Albumin stimulated this process dramatically, and it reached a plateau at 2 mg/ml. After an initial lag of 30 min, the release of [3H]lysophosphatidylcholine was linear for at least 4 h. At low concentrations, albumin slightly stimulated [3H]phosphatidylcholine release. The albumin had no measurable effect on the metabolism of cellular [3H]phosphatidylcholine, [3H]lysophosphatidylcholine or [3H]glycerophosphocholine. In addition, albumin did not alter the release of 3H-labelled water-soluble compounds, including [3H]glycerophosphocholine, into the medium. The possibility that the [3H]lysophosphatidylcholine was arising from catabolism of [3H]phosphatidylcholine in the medium by secreted enzymes was excluded. The effect on [3H]lysophosphatidylcholine secretion was also observed when the cells were incubated with alpha-cyclodextrin, a cyclic polysaccharide that has the ability to bind lysophosphatidylcholine. The albumin-released lysophosphatidylcholine was enriched in unsaturated fatty acids. Alteration of the fatty acid composition of cellular phosphatidylcholine gave rise to parallel changes in phosphatidylcholine and lysophosphatidylcholine in the medium. It is concluded that phosphatidylcholine is constantly being degraded in the rat hepatocyte to lysophosphatidylcholine which is released into the medium only when a suitable acceptor is present.

Comparison of albumin-mediated release of lysophosphatidylcholine and lysophosphatidylethanolamine from cultured rat hepatocytes.

We have investigated the albumin-stimulated release from cultured rat hepatocytes of lysophosphatidylcholine derived from methylation of phosphatidylethanolamine and of lysophosphatidylethanolamine. In the absence [corrected] of albumin, neither lysophosphatidylethanolamine nor lysophosphatidylcholine was released into the culture medium. Albumin stimulated the accumulation of both phospholipids in the medium. After 2 h, 14.1 nmol of lysophosphatidylcholine and 2.0 nmol of lysophosphatidylethanolamine per 3 x 10(6) cells had accumulated in the medium. The rate of release of [3H]ethanolamine-labelled lysophosphatidylethanolamine was rapid in the first 2 h and then was decreased, whereas there was a 1 h lag in the release of [3H]ethanolamine-labelled lysophosphatidylcholine. This apparent lag probably reflected the time necessary for the synthesis of phosphatidylcholine from phosphatidylethanolamine in the cells. Albumin caused a decrease in labelled cellular lysophosphatidylethanolamine and lysophosphatidylcholine which only partially accounted for the accumulation of the labelled phospholipids in the medium. Albumin also stimulated the release of labelled phosphatidylethanolamine (almost 3-fold) and phosphatidylcholine (2-fold) into the medium. There was no detectable change in the labelling of the cellular pools of these phospholipids, most likely owing to the large amounts in the cells compared with the medium. The labelled lysophospholipids did not arise from catabolism of the parent phospholipid in the medium. Analysis of the fatty acids of the secreted lysophospholipids showed a preferential release of unsaturated fatty acyl species of lysophosphatidylcholine, whereas lysophosphatidylethanolamine contained similar amounts of saturated and unsaturated fatty acids.

Lysophosphatidylcholine metabolism and lipoprotein secretion by cultured rat hepatocytes deficient in choline.

The metabolism of lysophosphatidylcholine was studied in cultured rat hepatocytes deficient in choline and methionine. Even though the cells were defective in phosphatidylcholine biosynthesis, the albumin-stimulated release of lysophosphatidylcholine (1.9 nmol/h per mg of cellular protein) was similar to that in hepatocytes supplemented with choline. Albumin also stimulated (1.4-fold) the release of phosphatidylcholine from the deficient cells. The extra phosphatidylcholine and lysophosphatidylcholine in the medium were largely recovered in the albumin fraction (density greater than 1.18 g/ml), suggesting that albumin released these lipids from hepatocytes because of binding to this protein. The secretion of glycerophosphocholine was decreased by about 40% by the addition of albumin. When choline-deficient hepatocytes were supplemented with lysophosphatidylcholine, it was transported into the cells and mainly acylated to form phosphatidylcholine, which increased in mass by 30-35% in the first 4 h of incubation. Lysophosphatidylcholine was shown to be as effective as choline in restoring the secretion of very-low-density lipoproteins to normal amounts, as judged by the secretion of triacylglycerol, phosphatidylcholine and the apolipoproteins associated with very-low-density lipoproteins. Thus phosphatidylcholine synthesis via reacylation of lysophosphatidylcholine, via the CDP-choline pathway or via methylation of phosphatidylethanolamine, will satisfy the requirements for secretion of very-low-density lipoprotein from hepatocytes.