De novo Synthesis and Levels of Cytochrome c and a Biliprotein during Pupal-Adult Development of a Butterfly, Pieris brassicae

1984 ◽  
Vol 39 (9-10) ◽  
pp. 938-947 ◽  
Author(s):  
Hartmut Kayser
Keyword(s):  
Cytochrome C ◽  
Adult Development ◽  
De Novo ◽  
Larval Instar ◽  
Spectroscopic Method ◽  
Pieris Brassicae ◽  
Major Product ◽  
Exchange Chromatography ◽  
De Novo Synthesis ◽  
Large White

Abstract Using a sensitive pH-difference spectroscopic method in combination with a three-column procedure of ion-exchange chromatography (overall yield 94%) the levels of cytochrome c in the large white butterfly, Pieris brassicae, were determined from the last larval instar to the adult insect. In the larva cytochrome concentration reached a maximum at mid instar and sub­sequently decreased to very low values in the pupa. During adult development the level of cytochrome c increased 50-fold in males and 43-fold in females; this sexual difference was expressed only after adult emergence. Biliverdin IXγ which occurs as a specific biliprotein complex was accumulated during the last larval instar and also in young butterflies. De novo synthesis of heme c, biliverdin IXγ and the corresponding apoproteins was demonstrated in newly emerged butterflies by injections of radiolabeled 5-aminolevulinate, lysine, leucine, and succinate, respectively. Cycloheximide inhibited labeling of both apoproteins and of heme c to 90% but that of the bilin to only 25%. This suggests that in cytochrome c but not in the biliprotein formation of the holoprotein depended on a coordinated synthesis of both constituents. In­corporation of 5-aminolevulinate into the biliprotein exceeded that into cytochrome c sevenfold indicating that biliverdin IXγ is the major product of the heme pathway in P. brassicae. The results are discussed in relation to the formation of mitochondria and flight muscles during postembryonic development of insects.

Development-Specific Incorporation of [14C]5-Aminolevulinate and [3H]Leucine into Cytochrome c and Biliprotein in the Butterfly, Pieris brassicae. Correlation with the Ecdysteroid Titer in the Pupa

1984 ◽  
Vol 39 (9-10) ◽  
pp. 948-957 ◽  
Author(s):  
Hartmut Kayser ◽  
Ute Krull-Savage
Keyword(s):  
Cytochrome C ◽  
Time Course ◽  
De Novo ◽  
Larval Instar ◽  
Preceding Paper ◽  
Pupal Stage ◽  
Adult Emergence ◽  
Protein S ◽  
Pieris Brassicae ◽  
Total Soluble Protein

Abstract Incorporation of [14C]5-aminolevulinate and [3H]leucine into cytochrome c, biliprotein and total soluble protein was followed from the last larval instar to the adult stage in Pieris brassicae. The titer of ecdysteroids during the pupal stage was determined with a radioimmunoassay to correlate synthesis of heme products and of protein(s) with adult differentiation. Incorporation of both precursors showed a developmental profile with high synthetic activities in feeding larvae and in pupae after the release of ecdysteroids. Variation of the hormone titer during pupal life differed significantly in males and females. Labeling of cytochrome c by both 14C and 3H was as expected from the variation of its concentration reported in a preceding paper; highest in corporation was around adult emergence. The results demonstrate that i) the accumulation of cytochrome c in the developing adult insect is primarily due to de novo synthesis of both heme and apocytochrome c, performed under coordinate control, and ii) the concentration of 5-aminolevulinate is not rate-limiting in the formation of cytochrome c. Biliverdin IXγ, the major tetrapyrrolic product in this insect, seems to be directly derived from (free) heme and relatively short-lived as deduced from a time-course study. Formation of the bilin, i.e. destruction of heme, increased concomitantly to the initiation of adult differentiation by ecdysteroids in the pupa but later decreased at adult emergence. Synthesis of cytochrome c takes place as a late event during terminal development. Thus, the pathways leading to the two major heme products seem to be differently regulated during development.

Characterization of Monocyte-Associated Factor V

1993 ◽  
Vol 70 (02) ◽  
pp. 273-280 ◽  
Author(s):  
Janos Kappelmayer ◽  
Satya P Kunapuli ◽  
Edward G Wyshock ◽  
Robert W Colman
Keyword(s):  
Monoclonal Antibody ◽  
Light Chain ◽  
Peripheral Blood ◽  
De Novo ◽  
Factor V ◽  
Blood Monocytes ◽  
De Novo Synthesis ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
Peripheral Blood Monocytes

SummaryWe demonstrate that in addition to possessing binding sites for intact factor V (FV), unstimulated peripheral blood monocytes also express activated factor V (FVa) on their surfaces. FVa was identified on the monocyte surface by monoclonal antibody B38 recognizing FVa light chain and by human oligoclonal antibodies H1 (to FVa light chain) and H2 (to FVa heavy chain) using immunofluorescence microscopy and flow cytometry. On Western blots, partially cleaved FV could be identified as a 220 kDa band in lysates of monocytes. In addition to surface expression of FVa, monocytes also contain intracellular FV as detected only after permeabilization by Triton X-100 by monoclonal antibody B10 directed specifically to the Cl domain not present in FVa. We sought to determine whether the presence of FV in peripheral blood monocytes is a result of de novo synthesis.Using in situ hybridization, no FV mRNA could be detected in monocytes, while in parallel control studies, factor V mRNA was detectable in Hep G2 cells and CD18 mRNA in monocytes. In addition, using reverse transcriptase and the polymerase chain reaction, no FV mRNA was detected in mononuclear cells or in U937 cells, but mRNA for factor V was present in Hep G2 cells using the same techniques. These data suggest that FV is present in human monocytes, presumably acquired by binding of plasma FV, and that the presence of this critical coagulation factor is not due to de novo synthesis.

Platelets Stimulate Thromboplastin Synthesis in Human Endothelial Cells

1983 ◽  
Vol 49 (02) ◽  
pp. 069-072 ◽  
Author(s):  
U L H Johnsen ◽  
T Lyberg ◽  
K S Galdal ◽  
H Prydz
Keyword(s):  
Endothelial Cells ◽  
De Novo ◽  
Umbilical Vein ◽  
Time Dependent ◽  
De Novo Synthesis ◽  
Human Endothelial Cells ◽  
Tumor Promotor ◽  
Coagulation Assay ◽  
Platelet Aggregates ◽  
Umbilical Vein Endothelial Cells

SummaryHuman umbilical vein endothelial cells in culture synthesize thromboplastin upon stimulation with phytohaemagglutinin (PHA) or the tumor promotor 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The thromboplastin activity is further strongly enhanced in a time dependent reaction by the presence of gel-filtered platelets or platelet aggregates. This effect was demonstrable at platelet concentrations lower than those normally found in plasma, it may thus be of pathophysiological relevance. The thromboplastin activity increased with increasing number of platelets added. Cycloheximide inhibited the increase, suggesting that de novo synthesis of the protein component of thromboplastin, apoprotein III, is necessary.When care was taken to remove monocytes no thromboplastin activity and no apoprotein HI antigen could be demonstrated in suspensions of gel-filtered platelets, platelets aggregated with thrombin or homogenized platelets when studied with a coagulation assay and an antibody neutralization technique.

DE NOVO SYNTHESIS OF STEROIDS BY THE TESTES OF THE HUMAN FOETUS AT MIDGESTATION

1971 ◽  
Vol 68 (1_Supplb) ◽  
pp. S135 ◽  
Author(s):  
R. S. Mathur ◽  
N. Wiqvist ◽  
E. Diczfalusy
Keyword(s):  
De Novo ◽  
De Novo Synthesis ◽  
Human Foetus

In vivo study of the biosynthesis of long-chain fatty acids using deuterated water

1995 ◽  
Vol 269 (2) ◽  
pp. E247-E252 ◽  
Author(s):  
H. O. Ajie ◽  
M. J. Connor ◽  
W. N. Lee ◽  
S. Bassilian ◽  
E. A. Bergner ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
De Novo ◽  
Chain Elongation ◽  
Deuterated Water ◽  
De Novo Synthesis ◽  
Isotopomer Distribution ◽  
Long Chain ◽  
Mass Isotopomer

To determine the contributions of preexisting fatty acid, de novo synthesis, and chain elongation in long-chain fatty acid (LCFA) synthesis, the synthesis of LCFAs, palmitate (16:0), stearate (18:0), arachidate (20:0), behenate (22:0), and lignocerate (24:0), in the epidermis, liver, and spinal cord was determined using deuterated water and mass isotopomer distribution analysis in hairless mice and Sprague-Dawley rats. Animals were given 4% deuterated water for 5 days or 8 wk in their drinking water. Blood was withdrawn at the end of these times for the determination of deuterium enrichment, and the animals were killed to isolate the various tissues for lipid extraction for the determination of the mass isotopomer distributions. The mass isotopomer distributions in LCFA were incompatible with synthesis from a single pool of primer. The synthesis of palmitate, stearate, arachidate, behenate, and lignocerate followed the expected biochemical pathways for the synthesis of LCFAs. On average, three deuterium atoms were incorporated for every addition of an acetyl unit. The isotopomer distribution resulting from chain elongation and de novo synthesis can be described by the linear combination of two binomial distributions. The proportions of preexisting, chain elongation, and de novo-synthesized fatty acids as a percentage of the total fatty acids were determined using multiple linear regression analysis. Fractional synthesis was found to vary, depending on the tissue type and the fatty acid, from 47 to 87%. A substantial fraction (24-40%) of the newly synthesized molecules was derived from chain elongation of unlabeled (recycled) palmitate.

scholarly journals An enzymatic activation of formaldehyde for nucleotide methylation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Charles Bou-Nader ◽  
Frederick W. Stull ◽  
Ludovic Pecqueur ◽  
Philippe Simon ◽  
Vincent Guérineau ◽  
...  
Keyword(s):  
Amino Acids ◽  
Thymidylate Synthase ◽  
Active Site ◽  
De Novo ◽  
Crystallographic Structure ◽  
De Novo Synthesis ◽  
Active Site Residues ◽  
Enzymatic Activation ◽  
Enzyme Cofactors ◽  
Unique Ability

AbstractFolate enzyme cofactors and their derivatives have the unique ability to provide a single carbon unit at different oxidation levels for the de novo synthesis of amino-acids, purines, or thymidylate, an essential DNA nucleotide. How these cofactors mediate methylene transfer is not fully settled yet, particularly with regard to how the methylene is transferred to the methylene acceptor. Here, we uncovered that the bacterial thymidylate synthase ThyX, which relies on both folate and flavin for activity, can also use a formaldehyde-shunt to directly synthesize thymidylate. Combining biochemical, spectroscopic and anaerobic crystallographic analyses, we showed that formaldehyde reacts with the reduced flavin coenzyme to form a carbinolamine intermediate used by ThyX for dUMP methylation. The crystallographic structure of this intermediate reveals how ThyX activates formaldehyde and uses it, with the assistance of active site residues, to methylate dUMP. Our results reveal that carbinolamine species promote methylene transfer and suggest that the use of a CH2O-shunt may be relevant in several other important folate-dependent reactions.

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