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Lipoamide dehydrogenase from Malbranchea pulchella: isolation and characterization
Biochemistry
|October 16, 1979
Summary
Lipoamide dehydrogenase was purified from a thermophilic fungus. The enzyme exhibits high stability and specificity, with properties similar to other sources.
Area of Science:
- Biochemistry
- Enzymology
- Mycology
Background:
- Lipoamide dehydrogenase (EC 1.6.4.3) is a crucial enzyme in metabolic pathways.
- Understanding its properties from diverse sources like thermophilic fungi is important.
Purpose of the Study:
- To isolate and characterize lipoamide dehydrogenase from the thermophilic fungus Malbranchea pulchella var. sulfurea.
- To compare its properties with lipoamide dehydrogenase from other organisms.
Main Methods:
- Enzyme isolation using ammonium sulfate fractionation, affinity chromatography with Procion Brilliant Blue M-R--Sepharose 4B, and hydroxylapatite chromatography.
- Characterization using polyacrylamide gel electrophoresis, molecular weight determination, and substrate specificity assays.
Main Results:
- A single protein band was observed upon electrophoresis, indicating high purity.
- The enzyme is a dimer (102,000 MW) with monomers (51,000 MW) binding one FAD molecule.
- Optimal activity at pH 8.2, specific for dihydrolipoamide and NAD+, with diaphorase activity and thermostability up to 60°C.
Conclusions:
- Lipoamide dehydrogenase from Malbranchea pulchella var. sulfurea was successfully purified.
- The enzyme's properties are comparable to those reported for other sources, suggesting conserved functional characteristics.