Fatty Acid Synthetase of Spinacia oleracea Leaves

The molecular organization of fatty acid synthetase system in spinach (Spinacia oleracea L. var. Viroflay) leaves was examined by a procedure similar to that employed for the safflower system (Carthamus tinctorius var. UC-1). The crude extract contained all the component activities (acetyl-CoA:ACP transacylase, malonyl-CoA:ACP transacylase, β-ketoacyl-ACP synthetase, β-ketoacyl-ACP reductase, β-hydroxyacyl-ACP dehydrase, and enoyl-ACP reductase [I]) involved in the synthesis of fatty acids, but enoyl-ACP reductase (II) present in safflower seeds extract could not be detected spectrophotometrically. By polyethylene glycol fractionation followed by several chromatographic procedures, i.e. Sephadex G-200, hydroxyapatite, and blue-agarose, the component enzymes were clearly separated from one another. Properties of β-ketoacyl-ACP reductase, β-hydroxyacyl-ACP dehydrase, and enoyl-ACP reductase (I) from spinach were compared with the same enzymes in safflower seeds and Escherichia coli.     

Cytokinins in tRNA Obtained from Spinacia oleracea L. Leaves and Isolated Chloroplasts

Cytokinin-active ribonucleosides have been isolated from tRNA of whole spinach (Spinacia oleracea L.) leaves and isolated spinach chloroplasts. The tRNA from spinach leaf blades contained: 6-(4-hydroxy-3-methyl-2-butenylamino)-9-β-d-ribofuranosylpurine (cis and trans isomers), 6-(3-methyl-2-butenylamino)-9-β-d-ribofuranosylpurine, and 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9-β-d -ribofuranosylpurine (cis and trans isomers). A method for isolation of large amounts of intact chloroplasts was developed and subsequently used for the isolation of chloroplast tRNA. The chloroplast tRNA contained 6-(3-methyl-2-butenylamino)-9-β-d-ribofuranosylpurine and 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9-β-d -ribofuranosylpurine (the cis isomer only). The structures of these compounds were assigned on the basis of their chromatographic properties and mass spectra of trimethylsilyl derivatives which were identical with those of the corresponding synthetic compounds. The results of this study indicate that ribosylzeatin was present in spinach leaf tRNA, but absent from the purified chloroplast tRNA preparation.     

Fatty Acid Synthesis in Hydrilla Chloroplasts

The rates of carboxylation, photophosphorylation and acetate incorporation have been compared in the intact and broken chloroplasts of Hydrilla verticillata Royle leaves in the presence and absence of certain inhibitors and metabolites. The intact chloroplasts showed low rates of photophosphorylation, high rates of carboxylation, and exhibited normal capacity for fatty acid biosynthesis. In broken chloroplasts a drastic decrease was observed in the rates of carboxylation and acetate incorporation. However, the rate of photophosphorylation was considerably increased. In the presence of light, inhibitors such as iodoacetamide, arsenite and sodium azide decreased the photophosphorylation rate. F-1,6-di-P and PGA stimulated CO₂ fixation rate. In the absence of artificial light, inhibitors such as sodium arsenite, gluconate-6-phosphate, sodium azide and iodoacetamide decreased the rate of CO₂ fixation. CoA, ATP, G-6-P, F-1,6-di-P Stimulated the synthesis of fatty acids. Exogenous supply of ADP. NADH, NADP and NADPH did not stimulate fatty acid biosynthesis probably because these compounds could not gain entry into the chloroplasts. Light was necessary for the in vitro fatty acid biosynthesis.     

De Novo Maltotriose Biosynthesis from the Reducing End by Spinacia oleracea L. Chloroplasts

The distribution of ¹⁴C in the various glucose residues of maltotriose was studied as a function of time of photosynthesis of isolated chloroplasts of spinach (Spinacia oleracea L.) using ¹⁴CO₂. The distribution of label showed that the reducing-end glucose residue was labeled first and the label subsequently distributed to the second and third glucose residues at approximately equal rates.     

Fat Metabolism in Higher Plants: XLV. Some Factors Regulating Fatty Acid Synthesis by Isolated Spinach Chloroplasts

In the biosynthesis of fatty acids from 1-¹⁴C-acetate by intact spinach chloroplasts, ATP and Triton X-100 exert opposing effects on the conversion of palmitic acid to stearic acid; thus, ATP decreases the conversion and Triton X-100 increases the conversion. Changes in the availability of photosynthetically generated reduced nicotinamide adenine dinucleotide phosphate apparently does not markedly affect the C₁₆-C₁₈ ratio. Various H₂O₂-generating systems, such as viologen dyes, inhibit oleate synthesis from acetate and cause stearate to accumulate. Catalase partially reverses the effect of these days.     

Salinity and in vitro Ageing Effects on Primary Photosynthetic Processes of Thylakoids Isolated from Pisum sativum and Spinacia oleracea

The effects of in vitro ageing and salinity of the reactionmedium on the primary photochemistry of photosystem II and thepattern of energy distribution within the photochemical apparatusof thylakoids isolated from Pisum sativum and Spinacia oleraceaare described. Analyses of the low temperature induction curvesof fluorescence emission at 695 and 735 nm and of low temperatureabsorption and fluorescence emission spectra were used to examinethese processes. In vitro ageing over short periods reducedthe photochemical activity and changed the energy distributionwithin thylakoids of P. sativum, but had little effect on thylakoidsof S. oleracea. A synergistic effect of in vitro ageing andsalinity of the reaction medium was observed for P. sativumthylakoids. Ageing effects could be minimized by addition of100 mM NaCl to the resuspension medium. Changes in NaCl concentrationin the reaction medium produced large and similar changes inthe primary photochemical functioning of thylakoids from P.sativum and S. oleracea, which could be attributed mainly tothe cation species, Na^$; however, experiments using mannitolto produce osmotic stress indicated some small osmotically inducedchanges in photofunction of the thylakoids. Optimal primaryphotochemical activity of photosystem II, for both species,was observed with 200 mM NaCl. Cation-induced regulation ofexciton distribution appears to be facilitated by controllingthe degree of energy coupling between the light-harvesting chlorophylla/b complex and the two photosystems, I and II, and not by regulationof coupling between photosystems I and II.     

Amino Acid Biosynthesis by Isolated Chloroplasts during Photosynthesis

The pool sizes of the common amino acids in purified intact chloroplasts from Vicia faba L. were measured (nanomoles per milligram chlorophyll). The three amino acids present in the highest concentrations were glutamate, aspartate, and threonine. Alanine, serine, and glycine were each present at levels between 15 and 20 nanomoles per milligram chlorophyll and 13 other amino acids were detectable at levels below 10.     

Localization of Filipin-Sterol Complexes in the Membranes of Beta vulgaris Roots and Spinacia oleracea Chloroplasts

Filipin was used as a cytochemical probe for membrane sterols in the root storage tissue of the red beet Beta vulgaris L. and the chloroplasts of Spinacia oleracea L. In unfixed beet tissue, filipin lysed the cells. Freeze-fracture replicas revealed that the filipin-sterol complexes were tightly aggregated in the plasma membrane, while in thin section the complexes corrugated the plasma membrane. If the cells were fixed with glutaraldehyde prior to the filipin treatment, the cell structure was preserved. Filipin-induced lesions were dispersed or clustered loosely in the plasma membrane. A few filipin-sterol complexes were observed in the tonoplast. In spinach chloroplasts, filipin-sterol complexes were limited to the outer membrane of the envelope and were not found in the inner membrane of the envelope or in the lamellar membranes. If the filipin-sterol complexes accurately mapped the distribution of membrane sterols, then sterol was located predominantly in the plasma membrane of the red beet and in the outer membrane of the chloroplast envelope. Furthermore, the sterol may be heterogenously distributed laterally in both these membranes.     

Effects of Hydroxamic Acids Isolated from Gramineae on Adenosine 5'-triphosphate Synthesis in Chloroplasts

Two hydroxamic acids isolated from maize extracts, 2,4-dihydroxy-7-methoxy-1,4-(2H)-benzoxazin-3(4H)-one (DIMBOA) and the 2-O-beta-d-glucopyranoside of DIMBOA, inhibit photophosphorylation by spinach chloroplasts. Both cyclic and noncyclic photophosphorylations were inhibited to the same extent. The concentrations producing 50% inhibition for DIMBOA and its glucoside were about 1 and 4 millimolar, respectively. These compounds inhibit coupled electron transport but do not affect basal or uncoupled electron transport. Both acids inhibit the ATPase activities of membrane-bound coupling factor 1 (CF(1)) and of purified CF(1). On the basis of these results, it is concluded that DIMBOA and its glucoside act as energy transfer inhibitors of photophosphorylation.     

Synthesis of Proteins by Isolated Euglena gracilis Chloroplasts

Intact Euglena gracilis chloroplasts, which had been purified on gradients of silica sol, incorporated [³⁵S]methionine or [³H]leucine into soluble and membrane-bound products, using light as the only source of energy. The chloroplasts were osmotically shocked, fractionated on discontinuous gradients of sucrose, and the products of protein synthesis of the different fractions characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The soluble fraction resolved into three zones of radioactivity, the major one corresponding to the large subunit or ribulose diphosphate carboxylase. The thylakoid membrane fraction contained nine labeled polypeptides, the two most prominent in the region of 31 and 42 kilodaltons. The envelope fraction contained a major radioactive peak of about 48 kilodaltons and four other minor peaks. The patterns of protein synthesis by isolated Euglena chloroplasts are broadly similar to those observed with chloroplasts of spinach and pea.     

Acetate Binding of Spinach Chloroplasts as a Facet of Fatty Acid Synthesis

A particulate fraction of spinach chloroplasts is the major site of binding when either acetate or acetyl-CoA is used as substrate. The acetate is linked covalently, and the binding is inhibited by reagents which react with sulfhydryl groups. The amount of acetate bound is lowered by both citrate and oxaloacetate; however, the binding is not reversed by oxaloacetate. Reversal of binding is also not brought about by the addition of unlabeled acetyl-CoA. If cofactors for fatty acid synthesis and cold acetyl-CoA are added, the binding of labeled acetate is reversed. Acyl carrier protein from E. coli increases the binding of labeled acetate.     

Fatty Acid Synthesis in Leucoplasts Isolated from Developing Seeds of Brassica campestris

Fatty acid synthesis from Na (1-¹⁴C) acetate in leucoplasts isolated from developing seeds of Brassica compestris was found to be maximum when leucoplasts were supplied with 0.8 mM acetate, 20 mM NaHCO₃, 8 mM ATP, 8 mM MgCl₂, 4 mM MnCl₂, 0.6 mM CoA, 1 mM NADH, 1 mM NADPH and 0.2 M sorbitol and incubated at 30°C for 2 h. The rate of fatty acid synthesis was highest at pH 8.5 In presence of 0.4 M Bistris-propane buffer and linear for upto 4 h at 30°C with 80–110 μg plastid protein. Sorbitol was an essential requirement as it prevented the rupturing of leucoplasts by osmosis. ATP and divalent cations were almost absolute requirements, whereas nucleotides, CoA and bicarbonate improved the rate of fatty acid synthesis by two to ten folds. Mg²⁺ and NADH were the preferred cation and nucleotide, respectively. High concentration of dithiothreltol inhibited the incorporation of (¹⁴C) acetate Into fatty acids. The system developed as above could be used for in vitro studies.     

Purification and properties of cysteine synthase from Spinacia oleracea

Cysteine synthase was purified 3200-fold from Spinacia oleracea leaves. The purified enzyme has an apparent M, of 60 000 ± 2000 and can be dissociated into identical subunits of M, 32 000 ± 2000. The subunits contain one molecule of pyridoxal 5′-phosphate. The K_m value is 2.9 mM for O-acetyl-L-serine and 22 μM for sulphide. Cysteine synthase from S. oleracea catalysed the formation of β-(pyrazol-1-yl)-L-alanine, and β-(3-amino-1,2,4-triazol-1-yl)-L-alanine, and significant differences were found between this enzyme and β-substituted alanine synthases and cysteine synthase from other sources. Amino acid composition of the purified enzyme was also determined.     

Lipid Synthesis and Ultrastructure of Isolated Barley Chloroplasts

The cell organelle contents of chloroplast preparations made from barley leaves with salt and sucrose isolation media at pH 6 and 8 were determined and compared with the acetate incorporating activity of these preparations. A chloroplast preparation obtained with 0.5 m sucrose at pH 8 gave the highest number of intact chloroplasts (with envelope and stroma), the lowest number of contaminating mitochondria, and the highest activity in light dependent acetate incorporation into lipids. In the preparations observed, the light induced lipid synthesizing capacity correlates well with the percentage of intact chloroplasts. It is suggested that the intact chloroplasts are responsible for the light induced lipid synthesis of the preparations and that the synthesizing enzymes are localized in the chloroplast stroma. Acetate is mainly incorporated into palmitic and oleic acids. The low yield of intact chloroplasts and of light induced lipid synthesis in preparations isolated at pH 6 seem to result from the action of galactolipid lipase(s).     

菠菜种子胰蛋白酶抑制剂对K-562细胞增殖的抑制及促凋亡作用研究

应用MTT法分析了菠菜种子胰蛋白酶抑制剂SOTI对人慢性髓原性白血病K-562细胞生长的影响。结果显示SOTI能够抑制K-562细胞的增殖,其抑制细胞增殖的作用呈现明显的剂量-效应关系;进一步观察了不同浓度的SOTI处理K-562细胞所导致的形态学变化和诱导凋亡作用,证实SOTI具有明显的体外抗癌活性,SO—TI的抗癌活性与其诱导凋亡有关。     

Effects of Azide on Photophosphorylation in Isolated Spinach Chloroplasts

The influence of sodium azide on open-chain and flavine mononucleotide mediated cyclic photophosphorylation in isolated spinach chloroplasts was investigated under anaerobic conditions. Open chain phosphorylation was completely inhibited with DCMU both in the presence and absence of sodium azide in the experimental medium. Flavine mononucleotide mediated photophosphorylation was only slightly inhibited by DCMU in the absence of sodium azide but inhibited in two steps by increasing amounts of DCMU when sodium azide was present in the medium. The first step can be explained as being mainly an effect of DCMU on an open chain electron transport, with water and H₂O₂ as electron donors and with flavine mononucleotide — kept in an oxidized state by sodium azide — as the electron acceptor. The second step, as well as the comparatively insensitivity to DCMU in the absence of sodium azide, depends on cyclic photophosphorylation mediated by flavine mononucleotide.     

Positional Specificity and Fatty Acid Selectivity of Purified sn-Glycerol 3-Phosphate Acyltransferases from Chloroplasts

Soluble acyl-CoA:sn-glycerol 3-phosphate acyltransferases (EC 2.3.1.15) which are localized in chloroplasts were purified from leaves of Pisum sativum and Spinacia oleracea and obtained free from interfering activities. The purification raised the specific activities by factors of about 1,000 for pea and 200 for spinach preparations. In pea chloroplasts, acyltransferase activity occurs in two soluble forms with apparent isoelectric points of 6.3 and 6.6. For both forms, the same molecular weight of about 42,000 was determined. The enzyme from spinach chloroplasts showed a slightly higher molecular weight and a lower isoelectric point of 5.2.     

Synthesis and Absolute Configuration of a Cytotoxic Fatty Acid Isolated from the Mushroom,Hericium erinaceum

The naturally occurring (–)-enantiomer of a fatty acid isolated from the mushroom (Hericium erinaceum) was synthesized from (R)-(–)-benzyl glycidyl ether in 8 steps. A comparison of the specific rotation of the synthetic sample with that of the natural compound established the absolute configuration of the latter.     

Histochemical Study of Photoperiodic and Low Temperature Effects on Floral Induction of Spinacia oleracea

Shoot apices of Spinacia oleracea plants have been induced toflower either by: (a) subjecting leaves to 24 h long day, or(b) exposure to a short photoperiod but displaced by 8 h (displacedshort day) in the usual 24 h short-day cycle, or (c) exposureto low temperature (5 °C) during the dark period of thenormal short day. A quantitative cytochemical assay of pentosephosphate pathway activity during floral induction indicatesan approximate doubling of the rate of activity when comparedto that of vegetative apices (short day) (21 °C). Exposure to either low temperature, or a displaced short photoperiodstimulates pentose phosphate pathway activity in the shoot apexin a manner similar to that seen by long-day induction. Thischange in metabolic activity is accompanied by changes in theshape of the shoot apex which resembles that seen at an earlystage during floral induction. Spinacia oleracea, pentose phosphate pathway, shoot apex, glucose-6-phosphate dehydrogenase, floral induction, chilling, displaced short day