Biosynthesis of Nitrogenous Phospholipids in Spinach Leaves

1974 ◽  
Vol 52 (6) ◽  
pp. 469-482 ◽  
Author(s):  
M. O. Marshall ◽  
M. Kates
Keyword(s):  
Microsomal Fraction ◽  
Enzyme System ◽  
Supernatant Fraction ◽  
Exchange Reactions ◽  
Methylation Pathway ◽  
Ethanolamine Kinase ◽  
Spinach Leaves

Pathways for biosynthesis of phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylcholine (PC), in spinach leaves have been studied both in vivo (whole leaves and leaf slices) and in vitro (cell-free leaf fractions). Biosynthesis of PS was shown to occur by the action of a particle-bound CDP-diglyceride: serine phosphatidyltransferase, and PE by the action of a PS-decarboxylase localized in the 100 000 × g supernatant fraction. PE was also formed by the operation of the CDP-ethanolamine:diglyceride phosphorylethanolamine transferase, localized in the microsomal fraction. The presence of ethanolamine kinase required for formation of phosphorylethanolamine was demonstrated in vitro, but not the presence of CTP:phosphorylethanolamine cytidyltransferase; however, the latter is presumed present on the basis of in vivo results. Operation of the methylation pathway for biosynthesis of PC was established in vivo, and direct methylation of phosphatidyl-N-methylethanolamine to phosphatidyl-N,N-dimethylethanolamine (PE-diMe) and of PE-diME to PC by S-adenosylmethionine was demonstrated with a particulate enzyme system localized in the microsomal fraction; direct methylation of PE itself could not be shown in this system. PC was also synthesized by the CDP-choline:diglyceride phosphorylcholine transferase system localized in the microsomal fraction. Synthesis of PE and PC by Ca2+-stimulated exchange reactions with ethanolamine and choline, respectively, could be demonstrated, but at low rates. However, no synthesis of PS by exchange reactions with serine could be detected.

DEMETHYLIERUNG VON METHOXYÖSTROGENEN IN VITRO UND IN VIVO

1964 ◽  
Vol 46 (3) ◽  
pp. 361-378 ◽  
Author(s):  
H. Breuer ◽  
Marlene Knuppen ◽  
D. Gross ◽  
C. Mittermayer
Keyword(s):  
Intravenous Injection ◽  
Rat Liver ◽  
Intravenous Administration ◽  
Microsomal Fraction ◽  
Enzyme System ◽  
Average Yield ◽  
Experimental Conditions ◽  
Menten Constant

ABSTRACT The microsomal fraction of rat liver contains an enzyme system which, in the presence of NADPH2 and oxygen, demethylates 2-methoxyoestradiol-17β to 2-hydroxyoestradiol-17β. Under similar experimental conditions, 3-methoxyoestradiol-17β is demethylated to oestradiol-17β. The demethylation of 3-methoxyoestradiol-17β shows an optimum at pH 7.4 and is inhibited by β-diethylaminoethyl diphenylpropylacetate; the type of inhibition seems to be noncompetitive. The Michaelis-Menten constant for 3-methoxyoestradiol-17β was found to be 2.0 × 10−4 m. It appears that demethylation of 2- and 3-methoxyoestrogens is catalysed by a non-specific ether-cleaving enzyme system. After intravenous injection of 20 mg of 3-methoxyoestradiol-17β into 3 patients, the excretion of oestradiol-17β, oestrone and oestriol in the urine showed a marked increase. The average yield of the 3 urinary oestrogens derived from 3-methoxyoestradiol-17β was 2.7% of the dose administered. By using corrections for metabolic and method losses, a demethylation rate of 17.5% for 3-methoxyoestradiol-17β was calculated. The concentrations of oestradiol-17β, oestrone and oestriol in bile also increased after intravenous administration of 3-methoxyoestradiol-17β. The maximum concentrations of oestrone and oestradiol-17β were found immediately after injection, whereas the maximum in the oestriol fraction occurred 4 h later. Oestrone was predominantly excreted in the sulphate fraction, but most of the oestriol was found in the glucuronoside fraction. These results suggest that demethylation of methoxyoestrogens takes place in liver. Some of the biochemical and physiological aspects of demethylation are discussed.

Enzyme system of Clostridium stercorarium for hydrolysis of arabinoxylan: reconstitution of the in vivo system from recombinant enzymes

Microbiology ◽  
2004 ◽  
Vol 150 (7) ◽  
pp. 2257-2266 ◽  
Author(s):  
Helmuth Adelsberger ◽  
Christian Hertel ◽  
Erich Glawischnig ◽  
Vladimir V. Zverlov ◽  
Wolfgang H. Schwarz
Keyword(s):  
Extracellular Enzymes ◽  
Enzyme System ◽  
Thermophilic Bacterium ◽  
Recombinant Enzymes ◽  
Type Mode ◽  
Complete Set ◽  
First Time ◽  
Hydrolysis Of

Four extracellular enzymes of the thermophilic bacterium Clostridium stercorarium are involved in the depolymerization of de-esterified arabinoxylan: Xyn11A, Xyn10C, Bxl3B, and Arf51B. They were identified in a collection of eight clones producing enzymes hydrolysing xylan (xynA, xynB, xynC), β-xyloside (bxlA, bxlB, bglZ) and α-arabinofuranoside (arfA, arfB). The modular enzymes Xyn11A and Xyn10C represent the major xylanases in the culture supernatant of C. stercorarium. Both hydrolyse arabinoxylan in an endo-type mode, but differ in the pattern of the oligosaccharides produced. Of the glycosidases, Bxl3B degrades xylobiose and xylooligosaccharides to xylose, and Arf51B is able to release arabinose residues from de-esterified arabinoxylan and from the oligosaccharides generated. The other glycosidases either did not attack or only marginally attacked these oligosaccharides. Significantly more xylanase and xylosidase activity was produced during growth on xylose and xylan. This is believed to be the first time that, in a single thermophilic micro-organism, the complete set of enzymes (as well as the respective genes) to completely hydrolyse de-esterified arabinoxylan to its monomeric sugar constituents, xylose and arabinose, has been identified and the enzymes produced in vivo. The active enzyme system was reconstituted in vitro from recombinant enzymes.

scholarly journals Protein synthesis in chloroplasts. Characteristics and products of protein synthesis in vitro in etioplasts and developing chloroplasts from pea leaves

1975 ◽  
Vol 146 (3) ◽  
pp. 675-685 ◽  
Author(s):  
S G Siddell ◽  
R J Ellis
Keyword(s):  
Energy Source ◽  
Protein Synthesis ◽  
Large Subunit ◽  
Sodium Dodecyl ◽  
Supernatant Fraction ◽  
Pisum Sativum L ◽  
Fraction I ◽  
Plastid Ribosomes

The function of plastid ribosomes in pea (Pisum sativum L.) was investigated by characterizing the products of protein synthesis in vitro in plastids isolated at different stages during the transition from etioplast to chloroplast. Etioplasts and plastids isolated after 24, 48 and 96h of greening in continuous white light, use added ATP to incorporate labelled amino acids into protein. Plastids isolated from greening leaves can also use light as the source of energy for protein synthesis. The labelled polypeptides synthesized in isolated plastids were analysed by electrophoresis in sodium dodecyl sulphate-ureapolyacrylamide gels. Six polypeptides are synthesized in etioplasts with ATP as energy source. Only one of these polypeptides is present in a 150 000g supernatant fraction. This polypeptide has been identified as the large subunit of Fraction I protein (3-phospho-D-glycerate carboxylyase EC 4.1.1.39) by comparing the tryptic ‘map’ of its L-(35S)methionine-labelled peptides with the tryptic ‘map’ of large subunit peptides from Fraction I labelled with L-(35S)methionine in vivo. The same gel pattern of six polypeptides is seen when plastids isolated from greening leaves are incubated with either added ATP or light as the energy source. However, the rates of synthesis of particular polypeptides are different in plastids isolated at different stages of the etioplast to chloroplast transition. The results support the idea that plastid ribosomes synthesize only a small number of proteins, and that the number and molecular weight of these proteins does not alter during the formation of chloroplasts from etioplasts.

Redox Sensitive Polysaccharide Based Nanoparticles for Improved Cancer Treatment: A Comprehensive Review

2018 ◽  
Vol 24 (28) ◽  
pp. 3303-3319 ◽  
Author(s):  
Erfaneh Ghassami ◽  
Jaleh Varshosaz ◽  
Somayeh Taymouri
Keyword(s):  
Drug Delivery ◽  
Cancer Treatment ◽  
Drug Delivery Systems ◽  
Chemical Properties ◽  
Delivery Systems ◽  
Exchange Reactions ◽  
Triggered Release ◽  
Redox Responsive

Background: Among the numerous bio-responsive polymeric drug delivery systems developed recently, redox-triggered release of molecular payloads have gained great deal of attention, especially in the field of anticancer drug delivery. In most cases, these systems rely on disulfide bonds located either in the matrix crosslinks, or in auxiliary chains to achieve stimuli-responsive drug release. These bonds keep their stability in extracellular environments, yet, rapidly break by thiol–disulfide exchange reactions in the cytosol, due to the presence of greater levels of glutathione. Polysaccharides are macromolecules with low cost, natural abundance, biocompatibility, biodegradability, appropriate physical and chemical properties, and presence of numerous functional groups which facilitate chemical or physical cross-linking. Methods: With regards to the remarkable advantages of polysaccharides, in the current study, various polysaccharide-based redox-responsive drug delivery systems are reviewed. In most cases the in vitro/in vivo effects of the developed system were also evaluated. Results: Considering the hypoxic and reducing nature of the tumor microenvironment, with several folds higher glutathione levels than the systemic tissues, redox-sensitive polymeric systems could be implemented for tumorspecific drug delivery and the results of the previous researches in this field indicated satisfactory achievements. Conclusion: According to the reviewed papers, the efficiency of diverse redox-responsive polysaccharide-based nanoparticles with therapeutic payloads in cancer chemotherapy could be concluded. Nevertheless, more comprehensive studies are required to understand the exact intracellular and systemic fate of these nano-carriers, as well as their clinical efficacy for cancer treatment.

scholarly journals Stimulation in vitro of vitamin B12-dependent methionine synthase by polyamines

1996 ◽  
Vol 316 (2) ◽  
pp. 661-665 ◽  
Author(s):  
Susan H. KENYON ◽  
Anna NICOLAOU ◽  
Tamara AST ◽  
William A. GIBBONS
Keyword(s):  
Enzyme Activity ◽  
Vitamin B12 ◽  
Cell Signalling ◽  
Methionine Synthase ◽  
Polyamine Metabolism ◽  
Physiological Concentration ◽  
Methylation Pathway ◽  
Important Enzyme

Vitamin B12-dependent methionine synthase is an important enzyme for sulphur amino acid, folate polyamine metabolism, S-adenosylmethionine metabolism and also in the methylation pathway of DNA, RNA, proteins and lipids. Consequently, studies aiming at exploring the control and regulation of methionine synthase are of particular interest. Here we report the modulation of enzyme activity in vitro by polyamines. Although putrescine, cadaverine, spermine and spermidine all stimulated enzyme activity, the last two were the most potent, causing increases in enzyme activity up to 400%. The EC50 for spermine was determined as 8 μM and for spermidine 40 μM. The physiological concentration for spermine has been reported to be 15–19 μM. Spermine was found to increase both the Km and the Vmax with respect to methyltetrahydrofolate for the enzyme. These data support the hypothesis that spermine and spermidine are feedback regulators of methionine synthase both in vivo and in vitro and are consistent with the polyamines' regulating cell signalling pathways.

Conversion of Flavanone to Flavone, Dihydroflavonol and Flavonol with an Enzyme System from Cell Cultures of Parsley

1981 ◽  
Vol 36 (9-10) ◽  
pp. 742-750 ◽  
Author(s):  
L. Britsch ◽  
W. Heller ◽  
H. Grisebach
Keyword(s):  
Microsomal Fraction ◽  
Specific Activity ◽  
Enzyme System ◽  
High Specific Activity ◽  
Cell Suspension Cultures ◽  
Soluble Enzyme ◽  
Enzyme Preparations ◽  
Malonyl Coa ◽  
In Vitro Biosynthesis

Abstract Soluble enzyme preparations from irradiated cell suspension cultures of parsley (Petroselinum hortense Hoffm.) catalyse the conversion of flavanone to flavone, dihydroflavonol and flavonol. These reactions require 2-oxoglutarate, Fe2+ and ascorbate as cofactors. In the presence of these cofactors conversion of dihydroflavonol to flavonol was also observed. With this system in vitro biosynthesis of radioactive flavone, dihydroflavonol and flavonol from [2-14C]malonyl-CoA and 4-coumaroyl-CoA in good yield and with high specific activity is possible.We postulate that synthesis of flavone and flavonol from flavanone proceeds via 2-hydroxy-and 2,3-dihydroxyflavanone, respectively, with subsequent dehydration.The microsomal fraction of the parsley cells contains an NADPH-dependent flavanone 3'-hydroxylase.

Phenylalanine Analogues: Potent Inhibitors of Phenylalanine Ammonia-Lyase Are Weak Inhibitors of Phenylalanine-tRNA Synthetases

1994 ◽  
Vol 49 (11-12) ◽  
pp. 781-790 ◽  
Author(s):  
Gerhard Leubner Metzger ◽  
Nikolaus Amrhein
Keyword(s):  
Wheat Germ ◽  
Phenylalanine Ammonia Lyase ◽  
Inhibitory Effect ◽  
Baker’S Yeast ◽  
Baker's Yeast ◽  
Exchange Reactions ◽  
Trna Synthetases ◽  
Different Sources

(1-Amino-2-phenylethyl)phosphonic acid (APEP), (1-amino-2-phenylethyl)phosphonous acid (APEPi), α-aminooxy-β-phenylpropionic acid (AOPP) and several other phenylalanine analogues are potent inhibitors of (S)-phenylalanine ammonia-lyase (PAL) in vitro and in vivo. The ability of these compounds to inhibit (S)-phenylalanine-tRNA synthetases (PRSs) from wheat germ, soybean, and baker’s yeast has been investigated and compared to the inhibition of PAL. APEP and APEPi were found to inhibit the tRNAphe-aminoacylation reactions catalyzed by the three PRSs studied in vitro in a competitive manner with respect to (5)-phenylalanine. (R)-APEP inhibits the PRSs with apparent Ki values of 144 μᴍ for wheat germ (app. Km for (S)-phe 5.2 μᴍ) , 130 μᴍ for soybean (app. Km for (S)-phe 0.9 μᴍ) , and 1096 μᴍ for baker’s yeast (app. Km for (S)-phe 5.5 μᴍ ) . The apparent Ki values for (R)-APEPi are 315 μᴍ , 160 μᴍ , and 117 μᴍ , respectively. APEP and APEPi inhibit the ATPpyrophosphate exchange reactions catalyzed by the PRSs from wheat germ and baker’s yeast, but they are not activated and do not serve as substrates in these reactions. AOPP has no affinity to any of the three PRSs, whereas it is a potent inhibitor of PAL. In light of our in vitro results with PRSs from different sources it appears unlikely that the PAL inhibitors we have studied have any significant inhibitory effect on this essential step in protein synthesis in vivo.

Distribution and Properties of Na+, K+-ATPase in Human Post-Mortem Kidney and the Effects of Diuretics

1972 ◽  
Vol 42 (3) ◽  
pp. 265-275 ◽  
Author(s):  
B. R. Tulloch ◽  
K. Gibson ◽  
P. Harris
Keyword(s):  
Adenosine Triphosphatase ◽  
Microsomal Fraction ◽  
Enzyme System ◽  
Post Mortem ◽  
Inhibit Activity ◽  
Adenosine Triphosphatase Activity ◽  
Diuretic Drugs ◽  
High Concentrations ◽  
Activity Dependent

1. Adenosine triphosphatase activity dependent on Mg2+ and activated by Na+ and K+ has been found in a microsomal fraction of homogenates of human postmortem kidneys. 2. Various characteristics of the enzyme system are described. 3. Activity is greater in the medulla than in the cortex. 4. Diuretic drugs inhibit activity in vitro only at high concentrations.

scholarly journals Decreased liver cytochrome P-450 in rats caused by norethindrone or ethynyloestradiol

1977 ◽  
Vol 166 (1) ◽  
pp. 57-64 ◽  
Author(s):  
I N H White ◽  
U Muller-Eberhard
Keyword(s):  
Marked Effect ◽  
Enzyme System ◽  
Time Dependent ◽  
Female Rats ◽  
Liver Cytochrome ◽  
Mixed Function Oxidases ◽  
Green Pigments ◽  
Cytochrome P 450

1. 19-Nor-17alpha-pregna-1,3,5(10)-trien-20-yne-3,17-diol (ethynyloestradiol) or 17beta-hydroxy-19-nor-17alpha-pregn-4-en-20-yn-3-one (norethindrone) but not 17alpha-ethyl-17beta-hydroxy-19-norandrost-4-en-3-one (norethandrolone) caused a time-dependent loss of cytochrome P-450 when incubated in vitro with rat liver microsomal fractions and NADPH-generating systems. 2. The enzyme system catalysing the norethindrone-mediated loss of cytochrome P-450 had many characteristics of the microsomal mixed-function oxidases. It required NADPH and air, and was inhibited by Co. However, it was unaffected by 1 mM-compound SKF 525A. 3. In microsomal fractions from phenobarbitone-pretreated rats the norethindrone-mediated loss of cytochrome P-450 was increased relative to controls. The norethindrone-mediated cytochrome P-450 loss was less pronounced when the animals were pretreated with 3beta-hydroxy-pregn-5-en-2-one 16alpha-carbonitrile (pregnenolone 16alpha-carbonitrile). Pretreatment with 3-methylcholanthrene rendered the animals resistant to the norethindrone effect. 4. Administration in vivo [100mg/kg, intraperitoneally] of norethindrone or ethinyl oestradiol also produced a time-dependent loss of liver cytochrome P-450. Norethandrolone had a similar, though much less-marked, effect. All three steroids lead to an induction of 5-aminolaevulinate synthase and an accumulation of porphyrins in the liver. 5. The loss of cytochrome P-450 and the accumulation of porphyrins in the liver 2 h after the administration of norethindrone to female rats was similar to that seen in males. 6. Rats pretreated with phenobarbitone and given norethindrone or ethynyloestradiol (100mg/kg, intraperitoneally) formed green pigments in their livers. These had characteristics similar to the green pigments produced in the livers of rats after the administration of 2-allyl-2-isopropylacetamide. No green pigments could be extracted from the livers of control rats or those given norethandrolone, oestradiol or progesterone.

scholarly journals ALTERATIONS IN BIOCHEMICAL COMPOSITION AND RIBONUCLEIC ACID METABOLISM INDUCED IN RAT LIVER BY CORTISONE

1956 ◽  
Vol 2 (3) ◽  
pp. 331-350 ◽  
Author(s):  
Charles Upton Lowe ◽  
Royden N. Rand
Keyword(s):  
Rat Liver ◽  
Biochemical Composition ◽  
Microsomal Fraction ◽  
Acid Metabolism ◽  
Chemical Constituents ◽  
Differential Centrifugation ◽  
Sucrose Solutions ◽  
The Relationship

An investigation of the effect of cortisone administration upon the chemical composition of intracellular particulates of rat liver has been made. Livers were homogenized in 0.25 M sucrose solutions and submitted to differential centrifugation. Five fractions were prepared: mitochondria (Mit), microsomes (Mi), ultracentrifugable (U), non-sedimentable (S), and nuclear (Nuc). Measurement was made of total and polymerized RNA, nitrogen, lipide P, and uptake of P32 by the RNA of each fraction. The following observations were made:— Cortisone administration caused a fall in concentration in all measured constituents except glycogen. On a per liver basis, however, total liver RNA was unchanged in amount; nitrogen content of Mi fell and that of S increased; the lipide P of Mit and Mi also decreased. The biochemical composition of a statistical mitochondrion was significantly altered; in contrast, the microsomal fraction decreased in amount, but the relationship between the chemical constituents was unchanged. When polymerized RNA was sought by a process involving precipitation from ethanol at 20°C., none was found in the Mit of cortisone livers and the amount in Mi was much less than found in the normal. When, however, precipitation was conducted at 4°C., yields of polymerized RNA in all fractions after cortisone were equal to or greater than those found in the normal. Furthermore, incubation of mixtures of homogenates from normal and cortisone livers resulted in loss of warm precipitable RNA. These data strongly suggest the presence of an enzyme in cortisone livers which upon incubation with normal livers made preparation of polymerized RNA virtually impossible by use of the warm method. This agent, thought to operate in vivo and in vitro, was not present in significant amounts in normal livers, since incubation in this instance had no effect upon the amount of polymerized RNA. Mit from cortisone livers obtained by the cold technique had a significantly decreased rate of incorporation of P32 even though the yield of RNA from this fraction was increased. To reconcile these observations, it was proposed that under the influence of cortisone a variant of normal RNA is synthesized or normal RNA is converted to this variant. This "new" RNA has new solubility properties, a new rate of incorporation of P32, and conceivably it cannot act as a template for normal protein synthesis.

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