Phosphatidylcholine synthesis in castor bean endosperm

Three pathways for phosphatidylcholine synthesis were assayed in castor bean (Ricinus communis var. Hale) endosperm. Phosphatidylethanolamine: S-adenosylmethionine methyl transferase occurred predominantly in the endoplasmic reticulum fraction, but some activity appeared in the mitochondria. Phosphorylcholine glyceride transferase occurred exclusively in the endoplasmic reticulum. The phosphorylcholine glyceride transferase activity was approximately 20-fold greater than the methylation pathway in the endoplasmic reticulum. No exchange activity was found. The Michaelis constant for the methylation was 31 mum for S-adenosylmethionine; phosphatidylethanolamine promoted the reaction slightly while other intermediates stimulated it by about 50%. The pH optimum was 9. Phosphorylcholine glyceride transferase had a Michaelis constant of 9.7 mum for cytidine diphosphate choline but variable results were obtained from diglycerides. The pH optimum was 7.5 and a divalent cation was required, Mg(2+) giving the greatest stimulation.     

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.     

Phosphatidylserine synthesis in castor bean endosperm

Phosphatidylserine synthesis by the endoplasmic reticulum fraction isolated from castor bean (Ricinus communis var. Hale) endosperm was assayed by measuring the incorporation of (14)C-l-serine into chloroform-soluble material. Both phosphatidylserine and phosphatidylethanolamine were identified as products. The incorporation required calcium ions and showed an optimum pH of 7.8 in 2 mm CaCl(2). Phosphatidylethanolamine and CDP-diglyceride stimulated the reaction only about 40 to 50% and primary alcohols had relatively little effect on the incorporation. These and other results suggest the synthesis of phosphatidylserine in this tissue occurs by an exchange reaction but the relative roles of phospholipase D and phosphatidylethanolamine: l-serine phosphatidyltransferase remain to be elucidated.     

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.     

Glutamine synthesis in germinating castor bean endosperm

The pathway of glutamine synthesis in germinating castor beanendosperm was investigated by feeding experiments with (2,3-¹⁴C)succinateand by determining enzyme activities related to pyruvate formationand utilization. ¹⁴C of (2,3-¹⁴C)succinate was rapidly and sequentiallyincorporated into amino acids in the following order: aspartateor alanine, glutamate and glutamine. ¹⁴CO₂ was slowly released,especially during the early hours of incubation. Fluorocitrateinhibited ¹⁴CO₂ release while aminooxyacetate stimulated itslightly. Fluorocitrate inhibited the incorporation of ¹⁴C intoglutamate and glutamine. Aminooxyacetate inhibited ¹⁴C incorporationinto aspartate, alanine, glutamate and glutamine. Glutaminesynthetase activity was detected in a soluble fraction. NAD-malicenzyme activity was detected in mitochondria by sucrose densitygradient centrifugation. Activities of pyruvate decarboxylaseand aldehyde dehydrogenasewere detected. Aldehyde dehydrogenasewas partially purified about 60-fold by ammonium sulfate fractionationand the DEAE-cellulose chromatography. The Km values of theenzyme were 0.71 miu for NAD and 0.43 mM for acetaldehyde. Basedon these results and properties of pyruvate kinase reportedpreviously (9), the metabolism of pyruvate in cytosol and mitochondriawas discussed in connection with glutamine synthesis in germinatingcastor bean endosperm. (Received August 25, 1978; )     

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.     

Accumulation of free ricinoleic Acid in germinating castor bean endosperm

Lipids from the endosperm of germinating castor bean (Ricinus communis var. Hale) were separated by thin layer chromatography and quantitated by gas chromatography. During the later stages of lipid breakdown (4-6 days germination at 30 C), several lipid classes were found in addition to the storage triglycerides, which are triricinoleins for the most part. One was identified as free ricinoleic acid, the proportion of which increased as germination progressed. After 6 days germination, ricinoleic acid comprised more than 30% of the total lipid. The appearance of this fatty acid implies that lipase activity (lipolysis) is not strictly coordinated with beta oxidation in this tissue.     

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.     

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.     

Phosphatidylcholine biosynthesis in castor bean endosperm : purification and properties of cytidine 5'-triphosphate:choline-phosphate cytidylyltransferase

Cytidine 5′-triphosphate:choline-phosphate cytidylyltransferase (EC 2.7.7.15) has been purified to near homogeneity (3350-fold) from castor bean (Ricinus communis L. var Hale) endosperm. The steps of purification included a differential solubilization of this enzyme with n-octyl β-d-glucopyranoside (OGP) and column chromatography on sequential DEAE-sepharose, sepharose-6B, and second DEAE-sepharose columns. The uses of appropriate concentrations of the detergent, OGP, in each step were crucial to obtain the highly purified enzyme. The purified enzyme gave a single protein band on nondenaturing polyacrylamide gel electrophoresis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed one major protein band of 40 kilodaltons. Gel filtration chromatography indicated that native cytidylyltransferase was approximately 155 kilodaltons, suggesting that it exists naturally as a tetramer. The purified enzyme used methylethanolamine-phosphate as a substrate but not ethanolamine-phosphate and dimethylethanolamine-phosphate. ATP and other nucleotides tested showed little effect on the purified enzyme. The purified enzyme activity was stimulated by both phospholipids extracted from castor bean endosperm and phosphatidylcholineoleate vesicles.     

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.     

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.     

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.     

Organelle membranes from germinating castor bean endosperm

Summary Endoplasmic reticulum, mitochondria, and glyoxysomes were obtained from germinating castor bean endosperm,Ricinus communis, by sucrose gradient centrifugation. When each of the three organelle preparations was diluted in 150 mM KCl and centrifuged, all of the component membrane material, measured as phospholipid, was sedimented. Also, the respective membrane enzymes, phosphorylcholine-glyceride transferase, cytochrome c oxidase and alkaline lipase were recovered. The endoplasmic reticulum retained most (60%) of its protein. The mitochondria lost almost no protein while the glyoxysomes lost much of their soluble contents.The isolated endoplasmic reticulum was in the form of vesicles, 0.02 to 1 m, lacking bound ribosomes. The size, 0.5 to 0.8 m, and the structure of the mitochondria were unchanged by the purification procedure. The mitochondria were contracted, whereas the glyoxysomes were distended. The diameter of the glyoxysomes remained 0.4 to 1.5 m, but they lost much of their internal matrix. The small amount of matrix that survived was not especially associated with the membrane. The glyoxysome membrane was about the same thickness as that of the endoplasmic reticulum, 70 Å.     

Pyruvate dehydrogenase complex from germinating castor bean endosperm

Subcellular organelles from castor bean (Ricinus communis) endosperm were isolated on discontinuous sucrose gradients from germinating seeds which were 1 to 7 days postimbibition. Marker enzyme activities of the organelles were measured (fumarase, catalase, and triose phosphate isomerase) and the homogeneity of the organelle fractions was examined by electron microscopy. Pyruvate dehydrogenase complex activity was measured only in the mitochondrial fraction and attempts to activate or release the enzyme from the proplastid were not successful. A pathway is proposed for the most efficient use of endosperm carbon for de novo fatty acid biosynthesis that does not require the presence of the pyruvate dehydrogenase complex in the proplastid to provide acetyl-coenzymeA.     

ATPase in lipid body membranes of castor bean endosperm

Lipid body membranes purified from castor seed endosperm of dry seeds and 4 d old seedlings were found to have an ATPase activity associated with them. This was confirmed by equilibrium density centrifugation of the membranes using acid lipase as a marker enzyme. The specific activity ranged from 45 to 200 nanomoles per milligram protein per minute. The pH optimum was 9.0 but at pH 7.5 nearly 40% of the maximum activity was retained. The apparent K_m for Mg-ATP was 0.5 millimolar. A divalent cation was required for activity and Mg²⁺ was the most effective. Other nucleoside triphosphates were also hydrolyzed but there was no hydrolysis of pyrophosphate or p-nitrophenylphosphate. The ATPase was not inhibited by oligomycin, vanadate, dicyclohexylcarbodiimide, or molybdate but was inhibited by sodium azide. Washing the membranes with increasing concentrations of NaCl removed up to 60% of the ATPase activity but none was removed by 3 millimolar ethylene-diaminetetraacetate.