Phosphatidylethanolamine synthesis by castor bean endosperm : a base exchange reaction

A base exchange reaction for synthesis of phosphatidylethanolamine by the endoplasmic reticulum of castor bean (Ricinus comminus L. var Hale) endosperm has been examined. The calculated Michaelis-Menten constant of the enzyme for ethanolamine was 5 micromolar and the optimal pH was 7.8 in the presence of 2 millimolar CaCl(2). l-Serine, N-methylethanolamine and N,N-dimethylethanolamine all reduced ethanolamine incorporation, while d-serine and myo-inositol had little effect. These inhibitions of ethanolamine incorporation were found to be noncompetitive and ethanolamine also noncompetitively inhibited l-serine incorporation by exchange. The activity of the ethanolamine base exchange enzyme was affected by several detergents, with the best activity being obtained with the zwitterionic defjtergent 3-3-cholamidopropyl) dimethylammonio-2-hydroxyl-1-propanesulfonate.     

Phosphatidylethanolamine synthesis in castor bean endosperm

Phosphatidylethanolamine synthesis by CDP-ethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) from the endoplasmic reticulum of castor bean (Ricinus communis L. var. Hale) endosperm was characterized. The Michaelis-Menten constant of the enzyme for CDP-ethanolamine was approximately 8.0 micromolar. The pH optimum was 6.5 and a divalent cation was an absolute requirement for activity, with Mg²⁺ giving the greatest stimulation at 3 millimolar. Sulfhydryl reagents variously affected enzyme activity. No discernible differences were detected between the responses of the ethanolaminephosphotransferase and CDP-choline:1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) to a variety of treatments. CDP-choline and CDP-ethanolamine were competitive inhibitors of the ethanolaminephosphotransferase and cholinephosphotransferase reactions, respectively.     

Phosphatidylethanolamine synthesis by castor bean endosperm. Intracellular distribution and characteristics of CTP:ethanolaminephosphate cytidylyltransferase.

The intracellular distribution and catalytic properties of CTP: ethanolaminephosphate cytidylyltransferase from endosperm of castor bean (Ricinus communis L. var. Hale) have been studied. This enzyme was confined to membranes, with about 80% of the activity occurring in mitochondria and the rest in endoplasmic reticulum (ER) following sucrose density gradient centrifugation. The mitochondrial location of this enzyme was supported by further purifying mitochondria on Percoll density gradients. The mitochondrial cytidylyltransferase was detected largely in outer membrane fractions, and lost its activity after trypsin treatment, indicating that the active sites are exposed to the cytoplasm. Both mitochondrial and ER cytidylyltransferase required cations for activity; Mg2+ was preferred over Mn2+ and Ca2+. The pH optima both were 6.5. The apparent Km values for ethanolamine phosphate were 143 and 83 microM and those for CTP were 125 and 1010 microM, respectively, for the mitochondrial and ER activities. The mitochondrial cytidylyltransferase reached a maximal velocity of 3.0 nmol/min/mg protein, whereas ER cytidylyltransferase was 0.424 nmol/min/mg protein. These findings reveal that the majority of the cytidylyltransferase activity in castor bean endosperm is not closely associated with ethanolaminephosphotransferase (predominantly in ER) which catalyzes the subsequent reaction in the synthesis of phosphatidyl-ethanolamine by a nucleotide pathway. The possible roles of these enzymes in phosphatidylethanolamine synthesis in plants are discussed.     

Phospholipid synthesis and exchange in castor bean endosperm homogenates

Leaves on a bush of Hyptis emoryi Torr. varied in length from less than 1 cm when development occurred in full sunlight (e.g. 40 Mjoules m⁻²) to over 7 cm when the total daily solar irradiance was less than 3 Mjoules m⁻². The 1-cm sun leaves were 3-fold higher than the 7-cm shade leaves in chlorophyll per unit area, mesophyll thickness, and the internal to external leaf area ratio (A^mes/A). The higher A^mes/A caused a 1.2-cm leaf to have a 3-fold lower CO₂ liquid phase resistance than did a 7.1-cm leaf. Large thin shade leaves captured photosynthetically active radiation effectively (less than 7% passed through), but were not adapted to full sunlight. Specifically, when a 6.9-cm leaf was placed at 910 w m⁻² for 30 min, its temperature exceeded that of the air by nearly 8 C. For the common daytime air temperatures above 30 C for this desert shrub, large shade leaves would have temperatures far in excess of that optimum for photosynthesis for H. emoryi, 29 to 32 C.     

Phosphatidylinositol synthesis by a mn-dependent exchange enzyme in castor bean endosperm

myo-Inositol is incorporated into phosphatidylinositol by an exchange reaction associated with the endoplasmic reticulum fraction isolated from post-germination castor bean endosperm. The reaction requires Mn²⁺, has a pH optimum of 8.0, an apparent K_m for myo-inositol of 26 micromolar, and is stimulated about 15-fold by certain cytidine derivatives. The cytidine derivatives appear to be converted to CMP, which may be the only active stimulator. These optimal exchange reaction conditions, both with and without CMP, differ from those for cytidine-5′ -diphosphodiglyceride: myo-inositol transferase (EC 2.7.8), so the exchange does not appear to be a reversal of the transferase. This conclusion is augmented by the low rates of CDP-diglyceride formation from cytidine derivatives when compared to the high rate of myo-inositol incorporation into phosphatidylinositol in the presence of the same cytidine derivatives and identical reaction conditions.     

Stimulation of phosphatidylethanolamine exchange by castor bean cytosol proteins

Cytosol proteins prepared from castor bean endosperm (4-day-old) seedlings stimulate the exchange of [³H]phosphatidylethanolamine between liposomes and mitochondria. The acceleration of the exchange depends on the quantity of cytosol proteins, the time of incubation, and the respective amounts of liposomes and mitochondria. On a per seedling basis, the active proteins are essentially located in the endosperm, whereas the roots and the cotyledons are less rich in these proteins.     

Cellular distribution of polysome populations of castor bean endosperm

The distribution of polysomes in the cells of the endosperm of very young (48 hours of germination) seedlings of castor bean (Ricinus communis var. Hale) has been examined. Seedlings exposed to 100 micromolar gibberellin A(3) for periods up to 24 hours showed increased numbers of polysomes associated with endomembranes. Cytosol-derived polysomes were generally of a shorter mean length than membrane-derived polysomes. Gibberellin did not lead significantly to increased numbers of cytosol-derived polysomes, nor did it lead to enhancement of any particular size class of polysomes.     

Phosphatidylcholine synthesis in castor bean endosperm : free bases as intermediates

The methylation steps in the biosynthesis of phosphatidylcholine by castor bean (Ricinus communis L.) endosperm have been studied by pulse-chase labeling. Endosperm halves were incubated with [methyl-(14)C]S-adenosyl-l-methionine, [2-(14)C]ethanolamine, [(14)C]ethanolamine phosphate, or [(14)C]serine phosphate. The kinetics of appearance were followed in the free, phospho-, and phosphatidyl-bases. The initial methylation utilized ethanolamine as a substrate to form methylethanolamine, which was then converted to dimethylethanolamine, choline, and phosphomethylethanolamine. Subsequent methylations occurred at the phospho-base and, to a lesser extent, the phosphatidyl-base levels, after which the radioactivity either remained constant or decreased in these compounds and accumulated in phosphatidylcholine. Although the precursors tested did support the synthesis of choline, the kinetics of the labeling make them unlikely to be the major sources of free choline to be utilized for the nucleotide pathway. A model with two pools of choline is proposed, and the implications of these results for the pathways leading to phosphatidylcholine biosynthesis are discussed.     

Subcellular distribution of gluconeogenetic enzymes in germinating castor bean endosperm

The intracellular distribution of enzymes capable of catalyzing the reactions from oxaloacetate to sucrose in germinating castor bean endosperm has been studied by sucrose density gradient centrifugation. One set of glycolytic enzyme activities was detected in the plastids and another in the cytosol. The percentages of their activities in the plastids were less than 10% of total activities except for aldolase and fructose diphosphatase. The activities of several of the enzymes present in the plastids seem to be too low to account for the in vivo rate of gluconeogenesis whereas those in the cytosol are quite adequate. Furthermore, phosphoenolypyruvate carboxykinase, sucrose phosphate synthetase, and sucrose synthetase, which catalyze the first and final steps in the conversion of oxaloacetate to sucrose, were found only in the cytosol. It is deduced that in germinating castor bean endosperm the complete conversion of oxaloacetate to sucrose and CO₂ occurs in the cytosol. The plastids contain some enzymes of the pentose phosphate pathway, pyruvate dehydrogenase and fatty acid synthetase in addition to the set of glycolytic enzymes. This suggests that the role of the plastid in the endosperm of germinating castor bean is the production of fatty acids from sugar phosphates, as it is known to be in the endosperm during seed development.     

Fatty Acid synthesis in endosperm of young castor bean seedlings

Enzyme assays on organelles isolated from the endosperm of germinating castor bean (Ricinus communis) by sucrose density gradient centrifugation showed that fatty acid synthesis from [¹⁴C]malonyl-CoA was localized exclusively in the plastids. The optimum pH was 7.7 and the products was mainly free palmitic and oleic acids. Both NADH and NADPH were required as reductants for maximum activity. Acetyl-CoA, and acyl-carrier protein from Escherichia coli increased the rate of fatty acid synthesis, while low O₂ levels suppressed synthesis. In the absence of NADPH or at low O₂ concentration, stearic acid became a major product at the expense of oleic acid. Fatty acid synthesis activity was highest during the first 3 days of germination, preceding the maximum development of mitochondria and glyoxysomes. It is proposed that the plastids are the source of fatty acids incorporated into the membranes of developing organelles.     

Heterogeneous distribution of glycosyltransferases in the endoplasmic reticulum of castor bean endosperm

Endoplasmic reticulum membranes stripped of attached ribosomes were isolated from homogenates of germinating castor bean (Ricinus communis L.) endosperm by sucrose density gradient centrifugation. The isolated endoplasmic reticulum fraction was further separated into two major membrane subfractions by centrifugation on a flotation gradient. Both subfractions appeared to be derived from the endoplasmic reticulum inasmuch as they share several enzymic markers including cholinephosphotransferase, NADH-cytochrome c reductase, and glycoprotein fucosyl-transferase and phase separation of membrane polypeptides using Triton X-114 revealed a striking similarity in both their hydrophilic and hydrophobic protein components. The endoplasmic reticulum membrane subfractions contain glycoproteins which were readily labeled by incubating intact endosperm tissue with radioactive sugars prior to fractionation.

Castor bean endosperm endoplasmic reticulum apparently exhibits a degree of enzymic heterogeneity, however, since the enzymes responsible for the synthesis of dolicholpyrophosphate N-acetylglucosamine and dolicholmonophosphate mannose together with their incorporation into the oligosaccharide-lipid precursor of protein N-glycosylation were largely recovered in a single endoplasmic reticulum subfraction.

     

Phosphatidylinositol synthesis in castor bean endosperm: cytidine diphosphate diglyceride:inositol transferase

CDP-diglyceride:inositol transferase in endoplasmic reticulum fractions from castor bean (Ricinus communis) endosperm was partially characterized. The enzyme had a pH optimum of 8.5 and required Mn²⁺ for activity. Maximal activity was at 1.5 millimolar MnCl₂. A K_m of 0.30 mM was calculated for myo-inositol and 1.35 millimolar was estimated for CDP-dipalmitoylglyceride. Concentrations of CDP-dipalmitoylglyceride above 1.2 millimolar inhibited the enzyme. A deoxycholate concentration of 0.1% (w/v) stimulated the reaction slightly while Triton X-100 inhibited at all concentrations tested. Some incorporation of myo-inositol into phosphatidylinositol occurred in the absence of CDP-diglyceride.     

Phosphatidylcholine synthesis in castor bean endosperm: characteristics and reversibility of the choline kinase reaction

Choline kinase (EC 2.7.1.32) was measured in concentrated 100,000gav supernatants from castor bean endosperm (Ricinus communis L. var. Hale). Initial velocity analysis, along with competitive inhibitor (hemicholinium-3) and product inhibition (ADPMg2+) studies suggested that the forward reaction followed a sequentially ordered mechanism with ATPMg2+ binding to the enzyme first, followed by choline and then activation of the ternary complex by free Mg2+. The kinetic constants of the forward reaction are reported. A reverse reaction was measured which had a pH optimum of 6.5 and produced 1 mol of ATP for every mole of choline phosphate. The estimated maximum possible Keq at 7.25 was 5 X 10(-3) which suggested that this reaction is highly reversible in this tissue. The possible physiological significance of this is discussed.     

Glyoxysomal membrane proteins are present in the endoplasmic reticulum of castor bean endosperm

The membrane proteins of glyoxysomes and ER from germinating castor bean endosperm were analyzed by a two-dimensional system: the proteins were first separated by reversed-phase high performance liquid chromatography and subsequently by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Several proteins were common to glyoxysomal and ER membranes, including those at 93, 88, 75, 72, 57, 46, and 32 kDa. Each of these common components was resistant to sodium carbonate extraction and eluted from the reversed phase column at the same acetonitrile concentration. Two proteins, 23 and 33 kDa, were unique to the ER while 30 and 24 kDa characterized the glyoxysomal membranes.     

Phosphatidylglycerol synthesis in castor bean endosperm: kinetics, requirements, and intracellular localization

The synthesis of phosphatidylglycerol in castor bean (Ricinus communis var. Hale) endosperm tissue was found to be located in both the endoplasmic reticulum and mitochondrial fractions separated on sucrose density gradients. The enzyme of both fractions attained maximum activity at 5 mm Mn(2+), 0.075% Triton X-100, and pH 7.3. The addition of dithiothreitol produced little effect, but sulfhydryl inhibitors reduced activity in both systems. Cytidine diphosphate-diglyceride exhibited an apparent Michaelis constant for the endoplasmic reticulum enzyme of 2.8 mum and for the mitochondrial enzyme of 2.0 mum; the maximum reaction rate was achieved at about 20 mum. For the second substrate, glycerol-phosphate, the apparent Michaelis constant for both fractions was about 50 mum and maximum velocity was reached at 400 mum. The specific activity of the mitochondrial enzyme was generally twice that of the endoplasmic reticulum.     

Phosphatidylcholine synthesis in castor bean endosperm: the localization and control of CTP: choline-phosphate cytidylyltransferase activity

The reaction catalyzed by CTP:choline-phosphate cytidylyltransferase (EC 2.7.7.15) has been postulated to be a control reaction in the synthesis of phosphatidylcholine (PtdCho) in many animal tissues and some plants. In 3-day-old castor bean (Ricinus communis L. var. Hale) endosperm the majority of cytidylyltransferase activity resided in a 12,000gav 10-min pellet. Following density-gradient fractionation, 60 to 70% of the enzyme activity was associated with the endoplasmic reticulum (ER) fraction, with the remainder in the particulate fraction being in an unidentified membrane band (band A), less than occurred in the soluble fractions. The properties and kinetics of the forward and reverse reactions are described. About 40% of the total ER activity could be solubilized by treatment of the fraction with 0.32 M KCl, which resulted in a threefold increase in the specific activity of the enzyme. The Michaelis constants of the solubilized enzyme were similar to those of the ER activity. The activity of the solubilized enzyme was stimulated 35% by addition of phosphatidylglycerol or phosphatidylinositol to the assay. Addition of a number of other phospholipids to the incubation medium caused only a small change in activity (+/- 10%) but the enzyme could be stimulated up to 60% by the addition of 0.01-1 mM sodium oleate. A combination of 0.25 mM PtdCho with oleate in the assay resulted in additional stimulation at all concentrations of oleate. Oleate had no effect on the ER activity. These results are discussed in relation to the regulation of cytidylyltransferase activity in plants.     

Orientation of electron transport activities in the membrane of intact glyoxysomes isolated from castor bean endosperm

Intact glyoxysomes were isolated from castor bean endosperm on isometric Percoll gradients. The matrix enzyme, malate dehydrogenase, was 80% latent in the intact glyoxysomes. NADH:ferricyanide and NADH:cytochrome c reductase activities were measured in intact and deliberately broken organelles. The latencies of these redox activities were found to be about half the malate dehydrogenase latency. Incubation of intact organelles with trypsin eliminated NADH:cytochrome c reductase activity, but did not affect NADH:ferricyanide reductase activity. NADH oxidase and transhydrogenase activities were negligible in isolated glyoxysomes. Mersalyl and Cibacron blue 3GA were potent inhibitors of NADH:cytochrome c reductase. Quinacrine, Ca²⁺ and Mg²⁺ stimulated NADH:cytochrome c reductase activity in intact glyoxysomes. The data suggest that some electron donor sites are on the matrix side and some electron acceptor sites are on the cytosolic side of the membrane.     

Formation of lipid-linked mono- and oligosaccharides from GDP-mannose by castor bean endosperm homogenates

A crude organelle preparation from germinating castor bean endosperm catalysed the incorporation of mannose from GDP[¹⁴C]mannose into acid-labile mannolipids. Solubility and chromatographic properties have identified the most rapidly synthesized products as mannosyl-phosphoryl-polyisoprenol, while the more polar lipid formed was shown to contain oligosaccharide. Little radioactivity from GDP[¹⁴C]mannose accumulated in insoluble product in the cell-free system, but supplying GDP[¹⁴C]mannose to intact endosperm tissue has shown that the major incorporation product in vivo is glycoprotein. This product was readily solubilized by either pronase or sodium dodecyl sulphate treatment suggesting it was membrane bound glycoprotein. Incorporation of mannose into mannosyl-phosphoryl-polyisoprenol during the cell-free assay was stimulated by the addition of dolichol monophosphate. This enzymic activity was optimal at pH 7.5 and in the presence of 10 mM Mg²⁺. The K_m for GDP-mannose was estimated to be 5×10^-7 M. Cellular mannosyl transferase activity changed markedly during early post-germinative growth; from being absent in the dry seed, enzyme activity increased to peak between the second and third days of growth and subsequently declined.Abbreviations TCA trichloroacetic acid - SDS sodium dodecyl sulphate     

Partial purification and characterization of CTP:cholinephosphate cytidylyltransferase from castor bean endosperm

CTP: cholinephosphate cytidylyltransferase (EC 2.7.7.15) has been purified approximately 600-fold from postgermination endosperm of castor bean. The enzyme was solubilized with n-octyl beta-D-glucopyranoside and then subjected to ion exchange and gel filtration chromatography. The Km's of the purified enzymatic activity were 0.37 and 1.1 mM for CTP and choline phosphate, respectively. Magnesium was required for activity. The purified cytidylyltransferase activity was inhibited by both phosphate and ATP. The extent of ATP inhibition was dependent on preincubation time, temperature, and Mg2+ and Ca2+ concentrations. The possible regulation of cytidylyltransferase in castor bean endosperm by protein phosphorylation is discussed.