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Lactate dehydrogenase in rat mitochondria
R B Brandt1, J E Laux, S E Spainhour
1Department of Biochemistry and Molecular Biophysics, Medical College of Virginia/Virginia Commonwealth University, Richmond 23298.
Abstract:
Small but persistent amounts of L-lactate dehydrogenase (LDH) activity were found in mitochondrial preparations isolated from rat heart, kidney, liver, and lymphocytes. Brain mitochondrial preparations were also isolated, but the results were inconclusive. A variety of cytosolic markers were used and it was found that essentially no cytosolic contamination was present except in brain preparations. A bacterial protease was used along with digitonin fractionation to determine localization of the mitochondrial LDH. Approximately 80% of the LDH activity associated with heart and kidney mitochondrial preparations was on the inside compared to about 40% for liver. Lymphocyte mitochondrial LDH activity was about 70% on the inside. Cytosolic LDH-5 preferentially adheres to outer mitochondrial membrane of liver, kidney, and heart. Agarose gel electrophoresis showed LDH isozymes in mitochondria qualitatively similar to that of the corresponding cytosol except in kidney mitochondrial preparations, where a specific electrophoretic band was found which did not correspond to any of the common LDH isozymes.
Insights
Lactate dehydrogenase (LDH) activity exists within mitochondria of rat heart, kidney, liver, and lymphocytes. This mitochondrial LDH, particularly LDH-5, shows varied localization and unique isozymes in kidney mitochondria.
Area of Science:
- Biochemistry
- Cell Biology
- Enzymology
Background:
- L-lactate dehydrogenase (LDH) is primarily known as a cytosolic enzyme.
- Mitochondrial localization of LDH has been reported but remains incompletely characterized.
- Understanding LDH compartmentalization is crucial for metabolic pathway analysis.
Purpose of the Study:
- To investigate the presence and localization of L-lactate dehydrogenase (LDH) activity within isolated mitochondria from various rat tissues.
- To determine the submitochondrial distribution of LDH and identify specific LDH isozymes in mitochondrial fractions.
Main Methods:
- Isolation of mitochondrial preparations from rat heart, kidney, liver, and lymphocytes.
- Assessment of cytosolic contamination using specific marker enzymes.
- Digitonin fractionation and bacterial protease treatment to determine LDH localization.
- Agarose gel electrophoresis for LDH isozyme analysis.
Main Results:
- Persistent LDH activity was detected in mitochondria from heart, kidney, liver, and lymphocytes, with minimal cytosolic contamination.
- Substantial portions of mitochondrial LDH were located internally (80% in heart/kidney, 70% in lymphocytes, 40% in liver).
- Cytosolic LDH-5 was found to associate with the outer mitochondrial membrane in liver, kidney, and heart.
- Kidney mitochondrial preparations exhibited a unique LDH isozyme band not found in corresponding cytosol.
Conclusions:
- Mitochondria possess intrinsic LDH activity across multiple rat tissues.
- LDH exhibits significant intramitochondrial localization, varying by tissue.
- Tissue-specific LDH isozyme patterns exist within mitochondria, notably in the kidney.