Substoichiometric amounts of the molecular chaperones GroEL and GroES prevent thermal denaturation and aggregation of mammalian mitochondrial malate dehydrogenase in vitro

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.

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

Biochemical Journal ◽

10.1042/bj3020405 ◽

1994 ◽

Vol 302 (2) ◽

pp. 405-410 ◽

Cited By ~ 20

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.

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

Biochemical Journal ◽

10.1042/bj2100207 ◽

1983 ◽

Vol 210 (1) ◽

pp. 207-214 ◽

Cited By ~ 13

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.

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

Biochemical Journal ◽

10.1042/bj2910139 ◽

1993 ◽

Vol 291 (1) ◽

pp. 139-144 ◽

Cited By ~ 45

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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