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Human spleen dihydroorotate dehydrogenase: properties and partial purification
Biochemical Medicine
|August 1, 1985
Summary
Human spleen dihydroorotate dehydrogenase, an enzyme linked to the respiratory chain, was purified and characterized. Its product, orotate, was found to be a potent inhibitor, offering insights into metabolic regulation.
Area of Science:
- Biochemistry
- Enzymology
- Mitochondrial research
Background:
- Human spleen dihydroorotate dehydrogenase (DHODH) is a key enzyme in pyrimidine biosynthesis.
- This enzyme is localized to the mitochondrial membrane and linked to the respiratory chain via ubiquinone.
Purpose of the Study:
- To characterize the human spleen dihydroorotate dehydrogenase.
- To investigate its association with the mitochondrial respiratory chain.
- To understand its inhibition by reaction products and other molecules.
Main Methods:
- Enzyme purification using acetone powder, Triton X-100 extraction, and polyacrylamide gel electrophoresis.
- Activity assays with artificial electron acceptors like ubiquinone 50 and PMS.
- Characterization of kinetic properties, pH optimum, and molecular weight.
Main Results:
- Orotate, the reaction product, was identified as a potent inhibitor of DHODH.
- No significant inhibition was observed with other natural pyrimidines or purines.
- Purification altered the enzyme's pH optimum and kinetic characteristics.
- Two molecular weight species (88,000 and 98,000 Da) were consistently observed.
Conclusions:
- Human spleen DHODH properties are broadly similar to the rat liver enzyme.
- Differences exist in the enzyme's respiratory chain linkage and purified characteristics.
- Orotate inhibition suggests a feedback mechanism in pyrimidine synthesis.