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.     

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.     

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.     

Mixed function oxidases from germinating castor bean endosperm

Homogenates from germinating castor bean endosperm were fractionated by sucrose density gradient centrifugation and examined for mixed function oxidase activity. Activity of cinnamic acid 4-hydroxylase and p-chloro-N-methylaniline N-demethylase was highest in the endoplasmic reticulum fraction. Activity of both enzymes is dependent on NADPH and on molecular oxygen; both activities are inhibited by carbon monoxide. When challenged with a number of potential inhibitors the enzymes responded in ways fairly typical of mixed function oxidases from other plants and animals. The N-demethylase appears to be specific for N-methylarylamines. In the absence of NADPH, cumene hydroperoxide is able to support N-demethylation. The mechanistic significance of this activity is discussed.     

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.     

Purification of pyruvate kinase from germinating castor bean endosperm

Cytosolic pyruvate kinase from endosperm of germinating castor beans (Ricinus communis L.; cv Hale) has been purified 3100-fold to apparent homogeneity and a final specific activity of 203 micromole pyruvate produced/minute per milligram protein. Purification steps included: heat treatment, polyethylene glycol fractionation, Q-Sepharose, ADP-agarose, Mono-Q and Phenyl Superose chromatography. Nondenaturing polyacrylamide gel electrophoresis of the final sample resulted in a single protein staining band which co-migrated with pyruvate kinase activity. Two protein staining bands of 57 and 56 kilodaltons were observed following SDS polyacrylamide gel electrophoresis of the final preparation. The native molecular mass was found to be about 240 kilodaltons. This enzyme appears to be a tetramer composed of two different subunits. The presence of dithioerythritol (2 millimolar) was required for optimal activity of the purified enzyme.     

Lipid composition of organelles from germinating castor bean endosperm

Glyoxysome, endoplasmic reticulum, mitochondria, and proplastid fractions were isolated from endosperm of castor beans (Ricinus communis) germinated for 5 days at 30 C. Samples from sucrose density gradients were diluted with 0.15 m KCI and the membranes pelleted. Lipid extracts of these membranes were analyzed for phosphoglyceride, acyl lipid, and sterol content. The endoplasmic reticulum contains 1.24 mumol of phosphoglyceride per mg of protein; the mitochondria, 0.65 mumol/mg; and the glyoxysome membranes, 0.55 mumol/mg. Phosphatidyl choline and phosphatidyl ethanolamine are the most abundant lipids in all membranes studied, accounting for 70% or more of the lipid phosphorus and 50% or more of the fatty acid. Glyoxysome membranes and endoplasmic reticulum also contain phosphatidyl inositol (respectively, 9 and 17% of the lipid phosphorus) and free fatty acids (13% of the total fatty acid in each). Compared with other organelles, mitochondrial membranes have more phosphatidyl ethanolamine relative to phosphatidyl choline and are characterized by the presence of cardiolipin, in which 80% of the fatty acid is linoleate. The relative amounts of linoleate, palmitate, oleate, stearate, and linolenate in each of the phosphotoglycerides are constant regardless of the membrane source. Stimasgasterol and beta-sitosterol are present in the membranes (1-9 nmol each/mg protein).The data provide further evidence that glyoxysome membranes are derived from the endoplasmic reticulum but at the same time indicate some differentiation.     

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.     

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.     

Hydration dependent polysome-monosome interconversion in the germinating castor bean endosperm

Castor bean seeds germinated for 40 br under a condition ofunlimited water availability were placed for 1 to 5 days ina condition such as to interrupt any further water uptake. Thefollowing phenomena were observed: a) the rate of synthesisof glucose-6-phosphate, phosphogluconate and NADP isocitratedehydrogenases progressively decreased, then completely stopped;b) polysomes disappeared while monosomes correspondingly increased;c) the endogenous capacity of isolated ribosomes to incorporateamino acid into proteins fell to zero, while in the presenceof poly-U phenylalanine incorporation remained practically constant.When the seeds were placed again in contact with water, theenzyme activities began to increase again; polyribosomes werereformed both from old ribosomes preexisting in the treatedendosperms and from new synthesized ribosomes; the endogenouscapacity of the ribosomes to perform protein synthesis (on anRNA basis) regained their original levels. These data suggest that some factor affecting mRNA or its interactionwith ribosomes, rather than a modification of ribosomal structureis responsible for the stoppage of protein synthesis causedby interrupting water uptake. (Received July 15, 1968; )     

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.     

Subcellular localization of glycolytic key enzymes in germinating castor bean endosperm

Phosphofructokinase and pyruvate kinase activities in castorbean endosperm increased during germination. Subcellular localizationof pyruvate kinase and phosphofructokinase in germinating endospermtissues was studied by differential and sucrose density gradientcentrifugation techniques. Eighty five percent or more of thepyruvate kinase and phosphofructokinase activities were locatedin cytosol. The remaining activities were mainly detected inproplastids. (Received June 30, 1977; )     

Developmental changes in endosperm of germinating castor bean independent of embryonic axis

Dry castor bean (Ricinus communis) seeds were cut transversely into halves and the half without the embryonic axis was placed in moist vermiculite at 30 C for 5 days. The development of the endosperm in the half-bean was found to be qualitatively similar to that in the whole seedling in the appearance of various enzymes of gluconeogenesis, the accumulation of glucose and sucrose as the end products of fat utilization, and the development of subcellular structure. It is concluded that during germination of castor bean, the embryonic axis does not directly control the developmental changes in the endosperm.     

Transfer of phospholipids from microsomes to mitochondria in germinating castor bean endosperm

¹⁴C-labeled microsomes were prepared by feeding [1-¹⁴ C]acetateto endosperm tissues from 4-day-old seedlings of castor beanseeds and incubated with unlabeled mitochondria from the sametissues. The loss of ¹⁴C-lipids from the microsomes was accompaniedby an increase of ¹⁴C-lipids in the mitochondria. The additionof 105,000?g supernatant and also pH 5.1-treated supernatant,both of which had been prepared from castor bean endospermsat the same stage, markedly enhanced the lipid transfer frommicrosomes to mitochondria. The activity in this fraction wasprecipitated by ammonium sulfate and lost with trypsin or heattreatment. The transfer of lipids was limited to phospholipids.Thus, it is concluded that in castor bean endosperms, phospholipidsare transferred from the endoplasmic reticulum to the mitochondriaby a phospholipid-exchange protein contained in the cytosol. (Received August 8, 1977; )     

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.     

Control of the citric acid cycle in mitochondria from germinating castor bean endosperm

Only a part of the citric acid cycle seems to be functional in the endosperm of germinating castor bean seeds. Mitochondria isolated from the endosperm can oxidize all of the citric acid cycle substrates. This was investigated further by studying the enzymic activities of isolated mitochondria during germination. Whilst all enzymic activities increase during germination there is an imbalance in the absolute levels of activities, with very low activities of those enzymes involved in converting pyruvate to succinate. It is suggested that the enzymic activity represents a coarse control of the cycle 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.