scholarly journals Rat liver pyruvate carboxylase. Purification, detection and quantification of apo and holo forms by immuno-blotting and by an enzyme-linked immunosorbent assay

1986 ◽  
Vol 236 (2) ◽  
pp. 527-533 ◽  
Author(s):  
F Ahmad ◽  
P M Ahmad ◽  
A Mendez
Keyword(s):  
Rat Liver ◽  
Immunosorbent Assay ◽  
Pyruvate Carboxylase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Rat Liver Mitochondria ◽  
Enzyme Linked Immunosorbent Assay ◽  
Enzyme Preparations ◽  
Detection And Quantification

A simple scheme for the purification of pyruvate carboxylase from rat liver mitochondria is described. It is rapid and provides high-purity pyruvate carboxylase with excellent yield and reproducibility. The final enzyme preparations appear to be homogeneous by the following criteria: elution behaviour on molecular-sizing matrix, SDS/polyacrylamide-gel electrophoresis, Ouchterlony double-diffusion analysis and Western blotting. Detection and quantification of nanogram amounts of pyruvate carboxylase (apo and holo forms) in total tissue homogenates by immuno-blotting and by enzyme-linked immunosorbent assay are described. The data provided suggest that under normal physiological conditions (both in vivo and in vitro) essentially all the pyruvate carboxylase molecules are biotinylated.

Import of the glucocorticoid receptor into rat liver mitochondria in vivo and in vitro

1993 ◽  
Vol 46 (3) ◽  
pp. 401-413 ◽  
Author(s):  
C. Demonacos ◽  
N.C. Tsawdaroglou ◽  
R. Djordjevic-Markovic ◽  
M. Papalopoulou ◽  
V. Galanopoulos ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Rat Liver ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria

scholarly journals Improved Assessment of Plasmodium vivax Response to Antimalarial Drugs by a Colorimetric Double-Site Plasmodium Lactate Dehydrogenase Antigen Capture Enzyme-Linked Immunosorbent Assay

2007 ◽  
Vol 51 (6) ◽  
pp. 2112-2116 ◽  
Author(s):  
Pierre Druilhe ◽  
Philippe Brasseur ◽  
Catherine Blanc ◽  
Michael Makler
Keyword(s):  
Lactate Dehydrogenase ◽  
Plasmodium Vivax ◽  
Immunosorbent Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Response Curves ◽  
In Vitro Susceptibility ◽  
Poor Growth ◽  
Antigen Capture

ABSTRACT The occurrence of Plasmodium vivax resistance to chloroquine has been reported in several countries of Asia and South America. However, the resistance of P. vivax is insufficiently documented for three reasons: it has received far less attention than P. falciparum; in vivo investigations are handicapped by the existence of hypnozoites, which make it difficult to distinguish between recrudescences due to drug failure and relapses due to dormant forms in the liver; and in vitro studies are greatly limited by the poor growth of P. vivax. We report on the adaptation to P. vivax of a colorimetric double-site Plasmodium lactate dehydrogenase antigen capture enzyme-linked immunosorbent assay previously developed for P. falciparum. The assay proved remarkably sensitive, as under optimal conditions it could detect P. vivax parasitemia levels as low as 10−8. The technique, which relies on the detection of protein synthesis by the parasite, yielded steep drug-response curves, leading to the precise determination of the 50% inhibitory concentrations for a high proportion of isolates. Chloroquine-resistant parasites were identified in an area where this phenomenon had been documented by in vivo methods. Thus, the results indicate that the in vitro susceptibility of P. vivax can now be monitored easily and efficiently. The data suggest that the threshold of resistance is similar to that of P. falciparum, i.e., in the range of 100 nM for chloroquine and 15 nM for pyronaridine. However, further studies are required to precisely define the cutoff for resistance and the sensitivity to each drug.

scholarly journals Role of membrane-bound and free polyribosomes in the synthesis of cytochrome c in rat liver

1974 ◽  
Vol 140 (2) ◽  
pp. 157-167 ◽  
Author(s):  
Néstor F. González-Cadavid ◽  
Carmen Sáez De Córdova
Keyword(s):  
Endoplasmic Reticulum ◽  
Cytochrome C ◽  
Rat Liver ◽  
Cell Structure ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mitochondrial Protein ◽  
Sodium Dodecyl ◽  
Membrane Bound

The functional distinction of membrane-bound and free polyribosomes for the synthesis of exportable and non-exportable proteins respectively is not so strict as was initially thought, and it was therefore decided to investigate their relative contribution to the elaboration of an internal protein integrated into a cell structure. Cytochrome c was chosen as an example of a soluble mitochondrial protein, and the incorporation of [14C]leucine and δ-amino[14C]laevulinate into the molecule was studied by using different ribosomal preparations from regenerating rat liver. A new procedure was devised for the purification of cytochrome c, based on ion-exchange chromatography combined with sodium dodecyl sulphate–polyacrylamide-gel electrophoresis. In spite of cytochrome c being a non-exportable protein, the membrane-bound polyribosomes were at least as active as the free ribosomes in the synthesis in vitro of the apoprotein and the haem moiety. The detergent-treated ribosomes could also effect the synthesis of cytochrome c, although at a lower rate. Since in liver more than two-thirds of the ribosomes are bound to the endoplasmic-reticulum membranes, it is considered that in vivo they are responsible for the synthesis of most of the cytochrome c content of the cell. This suggests that in secretory tissues the endoplasmic reticulum plays a predominant role in mitochondrial biogenesis, although free ribosomes may participate in the partial turnover of some parts of the organelle. The hypothesis on the functional specialization of the different kinds of ribosomes was therefore modified to account for their parallel intervention in the synthesis of proteins associated with membranous structures.

scholarly journals Selective permeability of rat liver mitochondria to purified aspartate aminotransferases in vitro

1977 ◽  
Vol 164 (3) ◽  
pp. 685-691 ◽  
Author(s):  
E Marra ◽  
S Doonan ◽  
C Saccone ◽  
E Quagliariello
Keyword(s):  
Rat Liver ◽  
Aspartate Aminotransferase ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Transferase Activity ◽  
Selective Permeability ◽  
The Matrix ◽  
Mitochondrial Aspartate Aminotransferase

1. A method was devised to allow determination of intramitochondrial aspartate amino-transferase activity in suspensions of intact mitochondria. 2. Addition of purified rat liver mitochondrial aspartate aminotransferase to suspensions of rat liver mitochondria caused an apparent increase in the intramitochondrial enzyme activity. No increase was observed when the mitochondria were preincubated with the purified cytoplasmic isoenzyme. 3. These results suggest that mitochondrial aspartate aminotransferase, but not the cytoplasmic isoenzyme, is able to pass from solution into the matrix of intact rat liver mitochondria in vitro. 4. This system may provide a model for studies of the little-understood processes by which cytoplasmically synthesized components are incorporated into mitochondria in vivo.

scholarly journals A New Cell Enzyme-Linked Immunosorbent Assay Demonstrates Gamma Interferon Suppression by Beta Interferon in Multiple Sclerosis

1999 ◽  
Vol 6 (3) ◽  
pp. 415-419 ◽  
Author(s):  
Moiz Bakhiet ◽  
Volkan Özenci ◽  
Carin Withagen ◽  
Maha Mustafa ◽  
Sten Fredrikson ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
Immunosorbent Assay ◽  
Gamma Interferon ◽  
T Cell Response ◽  
Unknown Etiology ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cell Enzyme ◽  
Ifn Γ

ABSTRACT Multiple sclerosis (MS) is a demyelinating disorder of the central nervous system of unknown etiology. Immune mechanisms involving the proinflammatory cytokine gamma interferon (IFN-γ) are believed to play an important role in the pathogenesis of MS. IFN-β-1b has been introduced as a treatment for MS and was found to reduce the number and severity of clinical exacerbations. To examine the influence of IFN-β-1b on myelin basic protein (MBP)-specific and phytohemagglutinin-induced IFN-γ production, we developed a cell-released capturing enzyme-linked immunosorbent assay (CRC-ELISA), which rapidly measures spontaneous and antigen- or mitogen-induced cellular IFN-γ production. CRC-ELISA documented a significant MBP-specific T-cell response in the blood of untreated MS patients, as assessed by IFN-γ production. This response was suppressed in MS patients treated with IFN-β-1b. The present work confirms in vivo the in vitro suppressive effects of IFN-β-1b on IFN-γ production in MS. Moreover, it provides a powerful new technique for detection of cytokines.

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