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Cytosolic malic dehydrogenase activity is associated with a putative substrate for the transforming gene product of
Abstract:
A cellular protein of apparent Mr 34,000--36,000 was suggested as a possible physiological substrate for the protein kinase (EC 2.7.1.37) activity associated with the transforming gene product of Rous sarcoma virus. We find this protein to migrate with an apparent Mr of 38,000 in NaDodSO4/polyacrylamide gels. It was not separable from cytosolic malic dehydrogenase activity when purified by chromatography on DEAE-Sephacel, hydroxylapatite, poly(A)-Sepharose, and blue Sepharose, by gel filtration, and by isoelectric focusing. The Mr 38,000 protein as well as cytosolic malic dehydrogenase activity focused with a pI of 7.5. In gel filtration experiments, both displayed an apparent native Mr of 68,000. The male dehydrogenase activity contained in homogeneous preparations of the Mr 38,000 protein had a specific activity of up to 130 units/mg of protein. The recovery of the enzyme was 5--10% of the activity in the extract. Antiserum against the Mr 38,000 protein inactivated the malic dehydrogenase activity associated with the Mr 38,000 protein.
Insights
Researchers identified a 38,000 molecular weight (Mr) protein that is identical to cytosolic malic dehydrogenase. This protein is a potential substrate for Rous sarcoma virus protein kinase activity.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- A cellular protein (34-36 kDa) was previously proposed as a substrate for Rous sarcoma virus protein kinase.
- The transforming gene product of Rous sarcoma virus possesses protein kinase activity.
Purpose of the Study:
- To identify and characterize the cellular protein substrate for Rous sarcoma virus protein kinase.
- To investigate the relationship between the putative substrate and cytosolic malic dehydrogenase.
Main Methods:
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (NaDodSO4/PAGE) to determine molecular weight.
- Multiple chromatography techniques (DEAE-Sephacel, hydroxylapatite, poly(A)-Sepharose, blue Sepharose) for protein purification.
- Gel filtration and isoelectric focusing for protein characterization.
- Enzyme activity assays and antibody inactivation experiments.
Main Results:
- The cellular protein identified has an apparent Mr of 38,000, distinct from the initially suggested range.
- This Mr 38,000 protein was inseparable from cytosolic malic dehydrogenase activity through extensive purification.
- Both the protein and the enzyme activity exhibited a pI of 7.5 and an apparent native Mr of 68,000.
- Homogeneous preparations of the Mr 38,000 protein contained significant malic dehydrogenase specific activity (up to 130 units/mg).
- Antiserum against the Mr 38,000 protein neutralized the associated malic dehydrogenase activity.
Conclusions:
- The cellular protein substrate for Rous sarcoma virus protein kinase is cytosolic malic dehydrogenase.
- The Mr 38,000 protein and cytosolic malic dehydrogenase are identical.
- This finding suggests a potential role for malic dehydrogenase in viral transformation processes.