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Inactivation of secretory phospholipase A2 by ionizing radiation
L J Reynolds1, E S Kempner, L L Hughes
1Department of Chemistry, University of California at San Diego, La Jolla 92093-0601, USA.
Abstract:
The extracellular phospholipase A2s (PLA2) from cobra venom, rattlesnake venom, and porcine pancreas were analyzed by radiation inactivation to determine their functional aggregation states. The analysis was performed in the presence of the protein transferrin at two different concentrations of PLA2: 5 micrograms/ml. The small size of these proteins necessitated the use of high radiation dosages. The catalytic activity of all samples decreased as a single exponential as a function of radiation dosage, to > 97% inactivation. Target size analysis of these curves yielded sizes corresponding to dimers for all three PLA2s, indicating that all three enzymes exist as dimers or larger aggregates under the conditions studied. An analysis of the amount of intact protein remaining by sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed that the loss of protein also followed a dimeric size for all three PLA2s. The loss of protein as a dimer indicates that transfer of radiation energy is occurring between polypeptides.
Insights
Extracellular phospholipase A2s (PLA2) from various sources were studied using radiation inactivation. All analyzed PLA2 enzymes function as dimers, indicating they exist as dimers or larger aggregates.
Area of Science:
- Biochemistry
- Enzymology
- Structural Biology
Background:
- Extracellular phospholipase A2s (PLA2) are enzymes involved in various biological processes.
- Understanding the aggregation state of PLA2 is crucial for comprehending their function.
Purpose of the Study:
- To determine the functional aggregation states of extracellular phospholipase A2s (PLA2).
- To investigate the oligomeric status of PLA2 from cobra venom, rattlesnake venom, and porcine pancreas.
Main Methods:
- Radiation inactivation analysis was employed to assess the functional size of PLA2 enzymes.
- Target size analysis was used to interpret the inactivation curves.
- Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was utilized to analyze protein loss.
Main Results:
- Catalytic activity of all PLA2 samples decreased exponentially with increasing radiation dosage (>97% inactivation).
- Target size analysis indicated that all three PLA2 enzymes exist as dimers.
- Protein loss analysis via SDS-PAGE also suggested a dimeric size for all PLA2s.
Conclusions:
- The study concludes that extracellular phospholipase A2s from cobra venom, rattlesnake venom, and porcine pancreas function as dimers.
- These findings suggest that PLA2 enzymes exist as dimers or larger aggregates under the studied conditions.
- Radiation energy transfer between polypeptides was inferred from the dimeric loss of intact protein.