Related Experiment Videos
Chemical cross-linking reveals a dimeric structure for CTP:phosphocholine cytidylyltransferase
1Department of Chemistry, Simon Fraser University, British Columbia, Canada.
The Journal of Biological Chemistry
|May 25, 1989
Summary
Rat liver CTP:phosphocholine cytidylyltransferase (CTP:PC-CT) is a dimer of two 42-kDa subunits when bound to membranes or micelles. This enzyme dimer reversibly self-aggregates into higher-order structures when detergent or membrane binding is absent.
Area of Science:
- Biochemistry
- Enzymology
- Protein chemistry
Background:
- CTP:phosphocholine cytidylyltransferase (CTP:PC-CT) is a key enzyme in phosphatidylcholine biosynthesis.
- Understanding the quaternary structure of CTP:PC-CT is crucial for elucidating its regulatory mechanisms.
Purpose of the Study:
- To determine the native oligomeric state and subunit interactions of purified rat liver CTP:PC-CT.
- To investigate the role of membrane/micelle binding in the enzyme's structure and activity.
Main Methods:
- Purification of CTP:PC-CT from rat liver.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and non-reducing conditions.
- Chemical cross-linking with thiol-cleavable reagents (DTSSP, DSS) and glutaraldehyde.
- Second-dimension electrophoresis to analyze cross-linked products.
- Enzyme activity assays.
- DEAE-Sepharose chromatography to remove detergent.
Main Results:
- Purified CTP:PC-CT consists of a predominant 42-kDa subunit, which can form disulfide-linked dimers (84-kDa) under non-reducing conditions.
- Thiol-cleavable cross-linking reagents converted the 42-kDa subunit to a dimer, indicating a dimeric structure when bound to Triton X-100 micelles or membrane vesicles.
- Cross-linking with glutaraldehyde also generated dimers in the presence of micelles/vesicles.
- Detergent removal led to the formation of hexamers and higher aggregates.
- The dimeric form was restored upon re-addition of Triton X-100 or vesicles.
- Cross-linking inactivated the enzyme, with partial reactivation upon disulfide reduction.
Conclusions:
- Purified, native CTP:PC-CT exists as a dimer of two non-covalently linked 42-kDa subunits when associated with detergent micelles or membrane vesicles.
- In the absence of membrane or micelle association, CTP:PC-CT dimers undergo reversible self-aggregation.
- Membrane/micelle binding stabilizes the dimeric form and is essential for enzyme activity.