Related Experiment Video
Updated: Aug 4, 2026
Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays
Published on: July 4, 2018
Purification and characterization of a C5a-inactivating enzyme from human peritoneal fluid
S K Ayesh1, Y Azar, I I Barghouti
1Hematology Unit, Hadassah University Hospital, Mount Scopus, Jerusalem, Israel.
Abstract:
Earlier work has suggested that familial Mediterranean fever, an inherited disorder characterized by sporadic episodes of inflammation involving the pleural and peritoneal cavities and the joints, is caused by the lack of a C5a inactivator normally found in serosal fluid. We have purified this inactivator from ascites fluid and obtained a protein of molecular weight 53 to 56 kD with a specific activity 10,000-fold greater than the crude material. On Western blot, an inhibitory antibody recognized a single antigenic species at the same molecular weight. The enzyme had no activity against denatured bovine serum albumin. With recombinant C5a as substrate, the Km and Vm were 3.4 mumol/L and 52 nmol C5a/min/mg protein, respectively.
Insights
Researchers purified a C5a inactivator protein from ascites fluid, potentially explaining familial Mediterranean fever. This purified protein shows significantly higher activity, offering new insights into the disorder.
Area of Science:
- Biochemistry
- Immunology
- Genetics
Background:
- Familial Mediterranean fever (FMF) is an inherited inflammatory disorder.
- Previous research linked FMF to a deficiency in C5a inactivator in serosal fluid.
Purpose of the Study:
- To purify and characterize the C5a inactivator protein.
- To investigate its role in familial Mediterranean fever.
Main Methods:
- Purification of C5a inactivator from ascites fluid.
- Western blot analysis using an inhibitory antibody.
- Enzyme activity assays with recombinant C5a.
Main Results:
- A C5a inactivator protein (53-56 kD) was purified with 10,000-fold greater specific activity.
- The antibody recognized a single species of the protein.
- Kinetic analysis revealed Km of 3.4 μmol/L and Vm of 52 nmol C5a/min/mg protein.
Conclusions:
- The purified C5a inactivator is a distinct protein species.
- This purified protein's characterization provides a basis for understanding its deficiency in familial Mediterranean fever.
