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.

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.

In vitro synthesis of β-galactosidase by membrane fractions of Escherichia coli

1970 ◽  
Vol 48 (8) ◽  
pp. 893-907 ◽  
Author(s):  
W. Chefurka ◽  
A. Yapo ◽  
B. Nisman
Keyword(s):  
Escherichia Coli ◽  
Actinomycin D ◽  
Heavy Fraction ◽  
Synthetic Activity ◽  
In Vitro Synthesis ◽  
Absolute Requirement ◽  
Discontinuous Sucrose Gradient ◽  
Reconstituted System ◽  
High Concentrations

The induction of β-galactosidase by a membranous fraction P1, prepared by digitonin lysis of spheroplasts of Escherichia coli, was studied in vitro. Electron micrographs of P1 show it to be a heterogeneous mixture of smooth vesicles, rough vesicles, rough vesicles attached to DNA, and ribosomes attached to DNA. P1 was subfractionated by differential centrifugation into an active heavy fraction, P4, and a relatively inactive light fraction, Pm. The P4 fraction consisted mainly of rough vesicles while the Pm fraction consisted mainly of smooth vesicles, but also of some rough vesicles. These vesicles of the Pm fraction were further separated by discontinuous sucrose gradient centrifugation.The induction of β-galactosidase by P1, P4, and Pm fractions was not related to mild contamination by unbroken viable spheroplasts. It was only partially sensitive to DNase and RNase. High concentrations of actinomycin D were required for complete inhibition of activity. This suggests that the transcription and translation components were shielded by the membranes. The synthetic activity of Pm was enhanced by fortification with DNA and/or S30. Only lac-containing DNA was active. The induction of β-galactosidase by this reconstituted system showed an absolute requirement for Pm membranes and for the inducer but only a partial requirement for nucleoside triphosphates. It was completely inhibited by puromycin and chloramphenicol.

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 Nucleic acid enzymology of extremely halophilic bacteria. Halobacterium cutirubrum deoxyribonucleic acid-dependent ribonucleic acid polymerase

1971 ◽  
Vol 121 (4) ◽  
pp. 621-627 ◽  
Author(s):  
B. Gregory Louis ◽  
P. S. Fitt
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Rna Polymerase ◽  
Deoxyribonucleic Acid ◽  
Halophilic Bacteria ◽  
Nucleoside Triphosphate ◽  
Extreme Halophile ◽  
Univalent Cations ◽  
Absolute Requirement ◽  
Nucleoside Triphosphates

1. DNA-dependent RNA polymerase was purified 150-fold from crude extracts of the extreme halophile Halobacterium cutirubrum. 2. The enzyme requires the presence of native DNA and all four nucleoside triphosphates to incorporate 14C-labelled nucleoside triphosphate into an acid-insoluble ribonuclease-sensitive product. 3. It has an absolute requirement for both Mn2+ and Mg2+. 4. The polymerase requires a high salt concentration for stability, but is markedly inhibited by univalent cations. 5. Its molecular weight is very low compared with that of Escherichia coli RNA polymerase.

High-level expression of lipase in Escherichia coli and recovery of active recombinant enzyme through in vitro refolding

2010 ◽  
Vol 70 (1) ◽  
pp. 75-80 ◽  
Author(s):  
Neda Akbari ◽  
Khosro Khajeh ◽  
Sasan Rezaie ◽  
Saeed Mirdamadi ◽  
Mahmoud Shavandi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Enzyme ◽  
High Level Expression ◽  
High Level ◽  
In Vitro Refolding
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