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.     

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 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

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.     

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; )     

Phosphatidic Acid synthesis in castor bean endosperm

Enzyme assays on organelles isolated from the endosperm of castor bean (Ricinus communis var. Hale) by sucrose density gradient centrifugation showed that palmitoyl-CoA:sn-glycerol 3-phosphate acyltransferase (EC 2.3.1.15) was localized in the membranes of the endoplasmic reticulum. Mn(2+) was required for activity, but Ca(2+) and Mg(2+) could substitute for Mn(2+) at higher concentrations. The apparent Km was 170 mum for sn-glycerol 3-phosphate and approximately 8 mum for palmitoyl-CoA. The optimum pH range was 7 to 7.5 and the principal reaction product was diacyl-sn-glycerol 3-phosphate (phosphatidic acid). Monoacyl-sn-glycerol 3-phosphate (lysophosphatidic acid) was not released as a free intermediate in the reaction. The maximum activity of the enzyme occurred immediately after imbibition, preceding the development of mitochondria and glyoxysomes.     

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.     

Proteinases and enzyme stability in crude extracts of castor bean endosperm

The stability of catalase, fumarase, and isocitrate lyase from deliberately broken organelles in crude extracts from endosperm tissue of castor bean seedlings has been examined. These enzymes are relatively stable at 2 C in extracts from endosperm of 2-day seedlings, but rapid losses of activity occur in extracts from older seedlings. These losses are shown to be brought about by the thiol-proteinase present in the extracts. The inclusion of 35% glycerol prevented the loss of catalase, fumarase, and isocitrate lysase activity, and various inhibitors of proteinases afforded limited protection. The most striking protectant was leupeptin, an inhibitor of serine and thiol-proteinases. Leupeptin completely inhibited the loss of activity of the three enzymes in crude extracts and improved yields when included in the grinding medium.     

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.     

Hydrolases in vacuoles from castor bean endosperm

Vacuoles were prepared from endosperm tissue of 4-day-old castor bean seedlings (Ricinus communis var. Hale) and purified on a stepped sucrose gradient. It was shown by assays of marker enzymes that there was only trace contamination of the final preparation by other organelles (mitochondria, glyoxysomes, nuclei, spherosomes, and plastids) and by cytoplasmic components. Hydrolytic enzymes (acid protease, carboxypeptidase, phosphodiesterase, RNAase, phytase and β-glucosidase) were present in the isolated vacuoles in amounts indicating a primarily vacuolar localization in vivo. The vacuoles also contained storage protein and high concentrations of sucrose. The over-all results indicate that the vacuoles from castor bean endosperm are the site of hydrolysis of the constituents of the protein bodies and are a temporary storage compartment for the sucrose produced from fat and protein reserves.     

Phosphatidylethanolamine synthesis by castor bean endosperm : membrane bilayer distribution of phosphatidylethanolamine synthesized by the ethanolaminephosphotransferase and ethanolamine exchange reactions

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 CaCl₂. 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.     

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.     

Characterization of glyoxysomes from castor bean endosperm

Electron micrographs are presented which establish the identity of the components of the 3 major bands observed after sucrose density centrifugation of the crude particulate fraction from the endosperm of germinating castor bean seedlings. These are: mitochondria (density 1.19 g/cc), proplastids (density 1.23 g/cc) and glyoxysomes (density 1.25 g/cc). Further evidence is provided on the enzymatic composition of the glyoxysomes. Essentially all of the particulate malate synthetase, isocitrate lyase, catalase, and glycolic oxidase is present in these organelles. The distribution of glyoxysomal enzymes on sucrose density gradients is contrasted with that of the strictly mitochondrial enzymes fumarase, NADH oxidase, and succinoxidase. Malate dehydrogenase and citrate synthetase are present in both organelles. The functional role of glyoxysomes and their relationship to cytosomes from other tissues is discussed.     

Membrane lipid metabolism in germinating castor bean endosperm

Castor bean (Ricinus communis L. var. Hale) endosperms, excised after 2 days germination at 30 C, were incubated 5 min to 8 hr with ¹⁴C-acetate and ³H-glycerol. Homogenates were fractionated by sucrose gradient centrifugation. Organelles found to be active in lipid synthesis were the lipid bodies and the endoplasmic reticulum. The products of incorporation in the lipid bodies were ³H-diglycerides containing ¹⁴C-fatty acids of more than 20 carbons. In contrast, the endoplasmic reticulum produced ³H-phospholipids as well as ³H-diglycerides rich in ¹⁴C-linoleate. The phospholipids synthesized and their acyl contents were of the types known to be the major components of organelle membranes in this tissue. Phospholipids and diglycerides containing ¹⁴C and ³H were found in the glyoxysomes and mitochondria subsequent to their appearance in the endoplasmic reticulum. The results show that germinating castor bean endosperm synthesizes membrane lipids de novo from acetate rather than reutilizing stored lipid components directly. It is also apparent that the endoplasmic reticulum is responsible for several steps in membrane lipid production.     

Properties of a soluble ATPase from castor bean endosperm mitochondria

An ATPase was extracted and purified from castor bean endospermmitochondria. The enzyme is stable at 60°C only in the presenceof ATP in the incubation medium. It is less stable at 0°Cthan at 30°C but is stabilized by ammonium sulfate or glycerol.Activity is dependent on the presence of Mg⁺⁺, and in the presenceof Mg⁺⁺ is enhanced by 2,4-dinitrophenol, but is not inhibitedby oligomycin. The enzyme hydrolyzes ITP in addition to ATP,but ITPase activity is hardly enhanced by 2,4-dinitrophenol.This preparation has many properties in common with the ATPase(coupling factor 1) from beef heart mitochondria. (Received November 8, 1969; )     

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.