scholarly journals Uptake of malate dehydrogenase into mitochondria in vitro. Some characteristics of the process

1983 ◽  
Vol 210 (1) ◽  
pp. 207-214 ◽  
Author(s):  
S Passarella ◽  
E Marra ◽  
S Doonan ◽  
E Quagliariello
Keyword(s):  
Enzyme Activity ◽  
Malate Dehydrogenase ◽  
The Other ◽  
Enzyme Treatment ◽  
Carbonyl Cyanide ◽  
Ion Gradient ◽  
Signal Probe ◽  
Energy Dependent ◽  
Mitochondrial Malate Dehydrogenase

1. It was previously shown [Passarella, Marra, Doonan & Quagliariello (1980) Biochem. J. 192, 649-658] that, when mitochondrial malate dehydrogenase from rat liver is incubated with sulphite-loaded mitochondria from the same source, uptake of the enzyme occurs, as judged by a fluorimetric assay of intramitochondrial enzyme activity. Confirmation of sequestration of the enzyme inside the organelles is provided by its proteinase-resistance after uptake. 2. Enzyme uptake into mitochondria is inhibited by enzyme treatment with mersalyl at concentrations that do not affect its catalytic activity. 3. Enzyme uptake is energy-dependent, as shown by inhibition of the process by carbonyl cyanide p-trifluoromethoxyphenylhydrazone and by antimycin. ATP and oligomycin, on the other hand, both stimulate the process, but stimulation by ATP is inhibited by oligomycin. These results suggest that uptake depends on maintenance of transmembrane ion gradient rather than direct ATP involvement. 4. Measurements of delta psi by means of the ‘redistribution signal’ probe safranine suggest no dependence of malate dehydrogenase uptake on membrane potential. 5. Comparison of the effects of the ionophores valinomycin, nonactin, gramicidin and nigericin shows that uptake depends on maintenance of a transmembrane pH gradient.

The properties of acidic compartments in developing schistosomula ofSchistosoma mansoni

Parasitology ◽  
2003 ◽  
Vol 127 (3) ◽  
pp. 253-264 ◽  
Author(s):  
B. H. AL-ADHAMI ◽  
J. THORNHILL ◽  
A. AKHKHA ◽  
M. J. DOENHOFF ◽  
J. R. KUSEL
Keyword(s):  
Enzyme Activity ◽  
Acid Phosphatase ◽  
Histochemical Staining ◽  
Transformation Process ◽  
Percentage Area ◽  
Red Dye ◽  
Energy Dependent ◽  
Acidic Compartments ◽  
Phosphatase Enzyme

A variety of fluorescent probes have been used to study the acidic compartments in cercariae and schistosomula ofSchistosoma mansoni. Freshly transformed schistosomula treated with the LysoTracker Red dye specific for lysosomes showed large acid-containing compartments (0·5–10 μm in size). The uptake of the dye is an energy-dependent process that depends on the metabolic activity of schistosomula. The compartments were quantified individually with respect to area, quantity of fluorescence and the total number/schistosomulum. Under normal conditions these compartments were not found in untreated cercariae, but appeared in cercariae slightly damaged by poly-L-lysine. The formation of these compartments seemed to be related to the development of cercariae into schistosomula as the number of compartments and uptake of fluorescence increased with time after transformation. Also, the method of transformation as well as thein vitroincubation of the parasite affected the percentage area of compartments/schistosomulum. Acid phosphatase enzyme activity was assessed using an endogenous phosphatase probe. Living and fixed schistosomula displayed the presence of enzyme activity in compartments of the same size and distribution as the acid-rich compartments. This was confirmed by histochemical staining showing deposition of enzyme-generated lead at the sites of phosphatase activity. We suggest that the development of acidic compartments is important during the transformation process or as a consequence of damage.

scholarly journals Selective permeability of rat liver mitochondria to purified malate dehydrogenase isoenzymes in vitro

1980 ◽  
Vol 192 (2) ◽  
pp. 649-658 ◽  
Author(s):  
S Passarella ◽  
E Marra ◽  
S Doonan ◽  
E Quagliariello
Keyword(s):  
Malate Dehydrogenase ◽  
Rat Liver ◽  
Liver Mitochondria ◽  
Enzymic Activity ◽  
Rat Liver Mitochondria ◽  
Starch Gel Electrophoresis ◽  
Selective Permeability ◽  
Starch Gel ◽  
Mitochondrial Malate Dehydrogenase

1. The mitochondrial malate dehydrogenase from rat liver has been purified to a state of homogeneity as judged by starch-gel electrophoresis and the cytoplasmic isoenzyme has been obtained in a partically purified state. 2. Inhibition of the isoenzymes by sulphite has been studied. 3. In mitochondria loaded with sulphite, the catalytic activity of the (partially inhibited) internal malate dehydrogenase has been measured by addition of oxaloacetate to the suspension medium and observation of the consequent decrease in fluorescence of NADH. 4. Addition of mitochondrial malate dehydrogenase to suspensions of mitochondria loaded with sulphite resulted in an increase in the level of intramitochondrial enzymic activity as measured by the above technique. Addition of the cytoplasmic isoenzyme did not result in such an increase. 5. These results show that mitochondria in suspension are permeable to the mitochondrial malate dehydrogenase but not to the cytoplasmic isoenzyme. 6. This conclusion has been confirmed by direct measurement of a decrease of enzyme activity in solution and an increase inside the mitochondria after incubation of organelles in solutions containing mitochondrial malate dehydrogenase. No such effect was observed with the cytoplasmic isoenzyme. 7. Some features of the permeation process have been studied.

scholarly journals Refolding and recognition of mitochondrial malate dehydrogenase by Escherichia coli chaperonins cpn 60 (groEL) and cpn10 (groES)

1994 ◽  
Vol 302 (2) ◽  
pp. 405-410 ◽  
Author(s):  
J P Hutchinson ◽  
T S el-Thaher ◽  
A D Miller
Keyword(s):  
Escherichia Coli ◽  
Secondary Structure ◽  
Malate Dehydrogenase ◽  
Binding Assay ◽  
Specific Binding ◽  
Guanidinium Chloride ◽  
Mitochondrial Malate Dehydrogenase ◽  
In Vitro Refolding ◽  
Cpn 60

In vitro refolding of pig mitochondrial malate dehydrogenase is investigated in the presence of Escherichia coli chaperonins cpn60 (groEL) and cpn10 (groES). When the enzyme is initially denatured with 3 M guanidinium chloride, chaperonin-assisted refolding is 100% efficient. C.d. spectroscopy reveals that malate dehydrogenase is almost unfolded in 3 M guanidinium chloride, suggesting that a state with little or no residual secondary structure is the optimal ‘substrate’ for chaperonin-assisted refolding. Malate dehydrogenase denatured to more highly structured states proves to refold less efficiently with chaperonin assistance. The enzyme is shown not to aggregate under the refolding conditions, so that losses in refolding efficiency result from irreversible misfolding. Evidence is advanced to suggest that the chaperonins are unable to rescue irreversibly misfolded malate dehydrogenase. A novel use is made of 100 K Centricon concentrators to study the binding of [14C]acetyl-labelled malate dehydrogenase to groEL by an ultrafiltration binding assay. Analysis of the data by Scatchard plot shows that acetyl-malate dehydrogenase, which has previously been extensively unfolded with guanidinium chloride, binds to groEL at a specific binding site(s). At saturation, one acetyl-malate dehydrogenase homodimer (two polypeptides) is shown to bind to each groEL homooligomer with a binding constant of approx. 10 nM.

The effect of five fasciolicides on malate dehydrogenase activity and mortality of Fasciola gigantica, Fasciolopsis buski and Paramphistomum explanatum

1981 ◽  
Vol 55 (2) ◽  
pp. 115-122 ◽  
Author(s):  
A. J. Probert ◽  
R. K. Sharma ◽  
K. Singh ◽  
R. Saxena
Keyword(s):  
Enzyme Activity ◽  
Dehydrogenase Activity ◽  
Malate Dehydrogenase ◽  
Fasciola Gigantica ◽  
High Concentrations ◽  
Fasciolopsis Buski ◽  
Reduced Activity ◽  
Inhibited Enzyme ◽  
Malate Oxidation

ABSTRACTThe effect of oxyclozanide, hexachlorophene, nitroxynil, rafoxanide and diamphenethide on malate dehydrogenase activity of homogenates of Fasciola gigantica, Fasciolopsis buski and Paramphistomum explanatum was investigated. The ratio of oxaloacetate reduction to malate oxidation in homogenates of Fasciola gigantica, Fasciolopsis buski and P. explanatum was 4·5:1, 3·6:1 and 5·2:1 respectively. Oxyclozanide and rafoxanide at 10−3 M inhibited enzyme activity by 100% in homogenates from all three species while hexachlorophene at 10−3M also caused 100% inhibition in homogenates from Fasciola gigantica and P. explanatum but only 65% of malate oxidation in Fasciolopsis buski homogenates. Nitroxynil at 10−3M produced 60% inhibition in F. buski homogenates yet had little effect at this concentration on preparations from the other species. Little inhibition was seen with diamphenethide, even at high concentrations. Rapid death of Fasicola gigantica and P. explanatum resulted in vitro when 10−3M oxyclozanide, hexachlorophene, nitroxynil or rafoxanide, were added to the incubation medium. Fasciolopsis buski was killed by 10−3M oxyclozanide but at this concentration the remaining compounds only caused reduced activity. Assay of malate dehydrogenase following drug treatment in vitro failed to show any appreciable reduction in enzyme activity in Fasciola gigantica and P. explanatum but oxyclozanide and hexachlorophene produced inhibition in Fasciolopsis buski. The mode of action of these compounds is discussed.

scholarly journals Plasma clearance and endocytosis of mitochondrial malate dehydrogenase in the rat

1981 ◽  
Vol 200 (1) ◽  
pp. 115-121 ◽  
Author(s):  
M K Bijsterbosch ◽  
A M Duursma ◽  
J M W Bouma ◽  
M Gruber ◽  
P Nieuwenhuis
Keyword(s):  
Bone Marrow ◽  
Enzyme Activity ◽  
Malate Dehydrogenase ◽  
Plasma Clearance ◽  
Reticuloendothelial System ◽  
First Order ◽  
First Order Kinetics ◽  
Long Run ◽  
Mitochondrial Malate Dehydrogenase ◽  
Adsorptive Endocytosis

1. Pig mitochondrial malate dehydrogenase was labelled with 125I and intravenously injected into rats. Enzyme activity and radioactivity were cleared from plasma identically, with first-order kinetics, with a half-life of only 7 min. 2. Radioactivity accumulated in liver, spleen, bone (marrow) and kidneys, reaching maxima of 3 1, 4, 6 and 9% of the injected dose respectively, at 10 min after injection. 3. Our data allow us to calculate that in the long run 59, 5, 11 and 13% of the injected dose is taken up and subsequently broken down by liver, spleen, bone and kidneys respectively. 4. Differential fractionation of liver showed that the acid-precipitable radioactivity was mainly present in the lysosomal and microsomal fractions, suggesting that the endocytosed protein is transported via endosomes to lysosomes, where it is degraded. 5. Radioautography of liver and spleen suggested that the labelled protein was taken up by macrophages of the reticuloendothelial system. 6. Mitochondrial malate dehydrogenase is probably internalized in liver, spleen and bone marrow by adsorptive endocytosis, since uptake of the enzyme of these tissues is saturable.

scholarly journals An in silico and in vitro pipeline for the rapid screening of helicase modulators

2020 ◽  
Vol 25 (1) ◽  
pp. e927
Author(s):  
Eleni Papakonstantinou ◽  
Flora Bacopoulou ◽  
Vasileios Megalooikonomou ◽  
Aspasia Efthimiadou ◽  
Dimitrios Vlachakis
Keyword(s):  
Enzyme Activity ◽  
High Throughput Screening ◽  
Screening Method ◽  
The Other ◽  
Rapid Screening ◽  
Helicase Activity ◽  
Luminescence Signal ◽  
Coated Plate ◽  
Biotin Molecule

To evaluate the potency of potential helicase modulators, we developed an assay of helicase enzyme activity. Using a DNA or RNA biotin labelled oligonucleotide and after the addition of a recombinant helicase, the nucleic acid unwinds, causing the emission of luminescence, which is quantified with a particular antibody. In our assay, one of the DNA oligos was biotinylated, while the other was labelled with digoxygenin (DIG), both in their 5’ termini. The biotin molecule immobilises the DNA duplex on a neutravidin-coated plate and the helicase activity is measured through the unwinding of DNA, due to ATP activation. The subsequent release of DIG-labelled oligos results in a luminescence signal measured with a chemiluminescence antibody. Our goal was to provide a high throughput screening method for potential helicase inhibitors. The method described in this paper has been demonstrated to be fast, easy and reproducible and doesn’t use radiochemicals.

scholarly journals Escherichia coli chaperonins cpn60 (groEL) and cpn10 (groES) do not catalyse the refolding of mitochondrial malate dehydrogenase

1993 ◽  
Vol 291 (1) ◽  
pp. 139-144 ◽  
Author(s):  
A D Miller ◽  
K Maghlaoui ◽  
G Albanese ◽  
D A Kleinjan ◽  
C Smith
Keyword(s):  
Escherichia Coli ◽  
Kinetic Analysis ◽  
Malate Dehydrogenase ◽  
Molar Excess ◽  
Absolute Requirement ◽  
Nucleoside Triphosphates ◽  
Atp Analogues ◽  
Mitochondrial Malate Dehydrogenase ◽  
In Vitro Refolding

In vitro refolding of pig mitochondrial malate dehydrogenase is investigated in the presence and absence of Escherichia coli chaperonins cpn60 (groEL) and cpn10 (groES). The refolded yields of active malate dehydrogenase are increased almost 3-fold in the presence of groEL, groES, Mg2+/ATP and K+ ions. Chaperonin-assisted refolding of malate dehydrogenase does not have an absolute requirement for K+ ions but Mg2+/ATP is obligatory. When ATP is replaced by other nucleoside triphosphates, or by non-hydrolysable ATP analogues, assisted refolding is prevented. Optimal chaperonin-assisted refolding requires both groEL and groES homo-oligomers in molar excess over malate dehydrogenase. Kinetic analysis shows that the chaperonins do not catalyse the refolding of malate dehydrogenase but increase the flux of unfolded enzyme through the productive refolding pathway without altering and/or accelerating that pathway. Although not acting as refolding catalysts, the chaperonins are able to assist at least six consecutive cycles of malate dehydrogenase refolding.

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