Cell-free translation of mitochondrial nicotinamide nucleotide transhydrogenase

1985 ◽  
Vol 5 (6) ◽  
pp. 483-490 ◽  
Author(s):  
Elisabeth Carlenor ◽  
Vigg Joste ◽  
B. Dean Nelson ◽  
Jan Rydström
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Liver Mitochondria ◽  
Cytochrome Bc1 Complex ◽  
Free System ◽  
Nicotinamide Nucleotide Transhydrogenase ◽  
Free Translation ◽  
A Cell ◽  
Matrix Fraction

Mammalian nicotinamide nucleotide transhydrogenase is translated as a 5000 daltons larger molecular weight precursor in a cell-free system programmed with rat liver polysomes. The mature rat liver enzyme had the same molecular weight as the purified beef heart enzyme, 115 000 daltons. The precursor was not processed in vitro by liver mitochondria or by a rat liver mitochondrial matrix fraction, nor did it appear to bind to mitochondria. In contrast, pre-FeS protein of the cytochrome bc1 complex was processed in the same samples by both mitochondria and matrix, suggesting an important difference in the processing mechanisms or in the efficiency of processing of the two precursors.

scholarly journals Effects of cytochalasin B on membrane-associated microfilaments in a cell-free system.

1981 ◽  
Vol 91 (2) ◽  
pp. 524-530 ◽  
Author(s):  
M J Phillips ◽  
M Oda ◽  
I M Yousef ◽  
K Funatsu
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
Rat Liver ◽  
Mode Of Action ◽  
Actin Filaments ◽  
Cytochalasin B ◽  
Bile Canaliculus ◽  
Free System ◽  
A Cell

The mode of action of cytochalasin B was examined in vitro using bile canaliculus-enriched plasma membrane fractions isolated from rat liver. The pericanalicular microfilaments, which are mainly actin filaments and which are normally attached to the canalicular membranes, were dissociated from the membranes by cytochalasin B treatment. A microfilamentous network was found in the supernate of the cytochalasin B treatment. A microfilamentous network was found in the supernate of the cytochalasin-treated specimens and a number of polypeptides, of which a polypeptide corresponding in molecular weight to actin was a notable member. These results suggest that actin filaments become detached from the canaliculus membranes by cytochalasin B.

scholarly journals Incorporation of ribonucleic acid in vitro into dense ribonucleoprotein-like materials by isolated rat liver nuclei

1978 ◽  
Vol 174 (2) ◽  
pp. 503-508 ◽  
Author(s):  
I Suzuka
Keyword(s):  
Rat Liver ◽  
Ribonucleic Acid ◽  
Free System ◽  
Isolated Rat Liver ◽  
Cell Free System ◽  
Rat Liver Nuclei ◽  
A Cell ◽  
Liver Nuclei ◽  
Isolated Rat

A cell-free system of isolated rat liver nuclei is described which permits an active incorporation of newly synthesized RNA into ‘dense’ ribonucleoprotein-like materials. The reaction is stimulated with increasing amounts of cytosol protein isolated from rat liver. This indicates that cytosol protein plays an important role in the formation of such material.

scholarly journals Development of a cell-free system to study the membrane assembly of photosynthetic proteins of Rhodobacter capsulatus.

1990 ◽  
Vol 111 (1) ◽  
pp. 87-94 ◽  
Author(s):  
D Troschel ◽  
M Müller
Keyword(s):  
Rhodobacter Capsulatus ◽  
Photosynthetic Apparatus ◽  
Membrane Vesicles ◽  
Translation System ◽  
Intracytoplasmic Membrane ◽  
Free System ◽  
Light Harvesting Complex ◽  
Free Translation ◽  
A Cell

A cell-free translation system from the facultatively photoheterotrophic bacterium Rhodobacter capsulatus is described. Synthesis of two proteins of the bacterium's photosynthetic apparatus (light-harvesting complex B870 alpha and beta) was performed by SP6 polymerase transcription of the subcloned genes, isolation of the mRNA and translation in vitro using a cell-free extract of R. capsulatus cells. The integration of these proteins in vitro into added intracytoplasmic membrane vesicles (ICM) is demonstrated. Without addition of ICM approximately 70% of the synthesized B870 proteins were soluble. If, however, ICM were present during synthesis, the majority of the soluble protein was found to associate with the membranes. The membrane-associated polypeptides could be solubilized only by detergent treatment but could not be extracted by treatment at alkaline pH (Na2CO3), suggesting that the proteins had been firmly inserted into the lipid bilayer. Moreover, the B870 alpha and beta proteins that integrated in vitro into ICM were also found to associate with pigment ligands and to assemble into a native reaction center/B870 complex. The native conformation of this complex isolated from ICM by Triton fractionation was demonstrated by microspectral analysis of the bound pigments.

scholarly journals Inhibition of the biosynthesis of sterols by phenyl and phenolic compounds in rat liver

1973 ◽  
Vol 134 (3) ◽  
pp. 737-743 ◽  
Author(s):  
S. Ranganathan ◽  
T. Ramasarma
Keyword(s):  
Phenolic Compounds ◽  
Organic Acids ◽  
Aromatic Ring ◽  
Rat Liver ◽  
Carboxyl Group ◽  
Free System ◽  
A Cell ◽  
High Concentrations

The presence of mitochondria increased the incorporation of [2-14C]mevalonate into sterols in a cell-free system from rat liver. Various phenyl and phenolic compounds inhibited the incorporation of mevalonate when added in vitro. p-Hydroxycinnamate, a metabolite of tyrosine, was the most powerful inhibitor among the compounds tested. Catechol, resorcinol and quinol were inhibitory at high concentrations. Organic acids lacking an aromatic ring were not inhibitory. Two hypocholesterolaemic drugs, Clofibrate (α-p-chlorophenoxyisobutyrate) and Clofenapate [α,4-(p-chlorophenyl)phenoxyisobutyrate], which are known to affect some step before the formation of mevalonate in the biosynthesis of cholesterol in vivo, showed inhibition at a step beyond the formation of mevalonate in vitro. The presence of the aromatic ring and the carboxyl group in a molecule appears to be necessary for the inhibition.

scholarly journals Cross-talk between IRAK-4 and the NADPH oxidase1

2007 ◽  
Vol 403 (3) ◽  
pp. 451-461 ◽  
Author(s):  
Sandrine Pacquelet ◽  
Jennifer L. Johnson ◽  
Beverly A. Ellis ◽  
Agnieszka A. Brzezinska ◽  
William S. Lane ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nadph Oxidase ◽  
P38 Mapk ◽  
Interleukin 1 ◽  
Mitogen Activated Protein Kinase ◽  
Free System ◽  
Mitogen Activated Protein ◽  
A Cell ◽  
Tlr4 Activation

Exposure of neutrophils to LPS (lipopolysaccharide) triggers their oxidative response. However, the relationship between the signalling downstream of TLR4 (Toll-like receptor 4) after LPS stimulation and the activation of the oxidase remains elusive. Phosphorylation of the cytosolic factor p47phox is essential for activation of the NADPH oxidase. In the present study, we examined the hypothesis that IRAK-4 (interleukin-1 receptor-associated kinase-4), the main regulatory kinase downstream of TLR4 activation, regulates the NADPH oxidase through phosphorylation of p47phox. We show that p47phox is a substrate for IRAK-4. Unlike PKC (protein kinase C), IRAK-4 phosphorylates p47phox not only at serine residues, but also at threonine residues. Target residues were identified by tandem MS, revealing a novel threonine-rich regulatory domain. We also show that p47phox is phosphorylated in granulocytes in response to LPS stimulation. LPS-dependent phosphorylation of p47phox was enhanced by the inhibition of p38 MAPK (mitogen-activated protein kinase), confirming that the kinase operates upstream of p38 MAPK. IRAK-4-phosphorylated p47phox activated the NADPH oxidase in a cell-free system, and IRAK-4 overexpression increased NADPH oxidase activity in response to LPS. We have shown that endogenous IRAK-4 interacts with p47phox and they co-localize at the plasma membrane after LPS stimulation, using immunoprecipitation assays and immunofluorescence microscopy respectively. IRAK-4 was activated in neutrophils in response to LPS stimulation. We found that Thr133, Ser288 and Thr356, targets for IRAK-4 phosphorylation in vitro, are also phosphorylated in endogenous p47phox after LPS stimulation. We conclude that IRAK-4 phosphorylates p47phox and regulates NADPH oxidase activation after LPS stimulation.

scholarly journals Trafficking of lipids from the endoplasmic reticulum to the Golgi apparatus in a cell-free system from rat liver

1991 ◽  
Vol 266 (7) ◽  
pp. 4322-4328 ◽  
Author(s):  
P Moreau ◽  
M Rodriguez ◽  
C Cassagne ◽  
D M Morré ◽  
D J Morré
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Rat Liver ◽  
Free System ◽  
Cell Free System ◽  
A Cell
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