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Localization of L-lactate dehydrogenase in mitochondria
Abstract:
Relatively small but persistent amounts of L-lactate dehydrogenase (LDH) activity were found in mitochondrial preparations isolated from liver of the rat. Using a variety of cytosolic markers, it was found that essentially no cytosolic contamination was present. Respiratory velocities and respiratory control with L-lactate were somewhat lower than with glutamate, but equal or superior to those with pyruvate. Agarose gel electrophoresis showed LDH isoenzymes in mitochondria similar to that in corresponding cytosol. Subtilisin BPN', a bacterial protease, was incubated with intact mitochondria and enzyme activities were measured. Following mitochondrial disruption, the proteolytic treatment was repeated. Digitonin was also used in the fractionation of mitochondria. These techniques helped to determine the location of the LDH in the mitochondria as being mainly in the outer membrane and periplasmic space.
Insights
Lactate dehydrogenase (LDH) activity was detected in rat liver mitochondria, with isoenzymes similar to the cytosol. Further analysis localized this enzyme primarily to the outer mitochondrial membrane and periplasmic space.
Area of Science:
- Biochemistry
- Cell Biology
- Mitochondrial Research
Background:
- Mitochondria are crucial organelles for cellular respiration and metabolism.
- L-lactate dehydrogenase (LDH) is typically known as a cytosolic enzyme involved in glycolysis.
- The presence and localization of LDH within mitochondria remain areas of investigation.
Purpose of the Study:
- To investigate the presence and quantify L-lactate dehydrogenase (LDH) activity in isolated rat liver mitochondria.
- To determine the precise subcellular localization of LDH within these mitochondria.
- To characterize the functional implications of mitochondrial LDH activity.
Main Methods:
- Isolation of rat liver mitochondria and assessment of cytosolic contamination using specific markers.
- Measurement of respiratory activities and respiratory control ratios using L-lactate, glutamate, and pyruvate as substrates.
- Analysis of LDH isoenzyme profiles via agarose gel electrophoresis.
- Subcellular localization studies using protease (Subtilisin BPN') treatment on intact and disrupted mitochondria, and digitonin fractionation.
Main Results:
- Persistent, albeit small, amounts of L-lactate dehydrogenase (LDH) activity were identified in highly purified rat liver mitochondrial preparations.
- LDH isoenzymes found in mitochondria exhibited profiles similar to those in the corresponding cytosol.
- Respiratory studies indicated that mitochondrial LDH activity with L-lactate was functional, with respiratory control ratios comparable or superior to pyruvate.
- Proteolytic treatment and digitonin fractionation experiments localized LDH predominantly to the outer mitochondrial membrane and periplasmic space.
Conclusions:
- L-lactate dehydrogenase (LDH) is present in rat liver mitochondria, primarily associated with the outer membrane and periplasmic space.
- Mitochondrial LDH exhibits enzymatic activity and influences mitochondrial respiration.
- These findings suggest a potential role for mitochondrial LDH beyond its traditional cytosolic function in cellular metabolism.