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.

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 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.

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

scholarly journals Separate physiological roles of specific and non-specific DNA binding of HU protein in Escherichia coli

2021 ◽  
Author(s):  
Sankar Adhya ◽  
Subhash Verma
Keyword(s):  
Escherichia Coli ◽  
Dna Binding ◽  
Protein Interactions ◽  
Specific Binding ◽  
Bacterial Genome ◽  
Dna Structure ◽  
Binding Modes ◽  
Chromosomal Dna

Conserved in bacteria, the histone-like protein HU is crucial for genome organization and expression of many genes. It binds DNA regardless of the sequence and exhibits two binding affinities in vitro, low-affinity to any B-DNA (non-specific) and high-affinity to DNA with distortions like kinks and cruciforms (structure-specific), but the physiological relevance of the two binding modes needed further investigation. We validated and defined the three conserved lysine residues, K3, K18, and K83, in Escherichia coli HU as critical amino acid residues for both non-specific and structure-specific binding and the conserved proline residue P63 additionally for only the structure-specific binding. By mutating these residues in vivo, we showed that two DNA binding modes of HU play separate physiological roles. The DNA structure-specific binding, occurring at specific sites in the E. coli genome, promotes higher-order DNA structure formation, regulating the expression of many genes, including those involved in chromosome maintenance and segregation. The non-specific binding participates in numerous associations of HU with the chromosomal DNA, dictating chromosome structure and organization. Our findings underscore the importance of DNA structure in transcription regulation and promiscuous DNA-protein interactions in a dynamic organization of a bacterial genome.

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