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Isolation and characterization of factor I of the bovine complement system

Marilyn MengerDepartment of Microbiology and Immunology, Faculty of Health Sciences, Queen's University, Kingston, ON, Canada K7L 3N6.
Present address is GenVec Incorporated, 12111 Parklawn Dr, Rockville, MD 20852.

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W. Peter AstonDepartment of Microbiology and Immunology, Faculty of Health Sciences, Queen's University, Kingston, ON, Canada K7L 3N6.

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Abstract

Objective—To isolate and characterize factor I of the bovine complement system.

Sample Population—Serum obtained from the blood of beef cattle.

Procedures—Serum samples were fractionated to yield factor I by means of sequential precipitation, ionexchange, and gel-filtration chromatography. The protein was identified throughout the procedure on the basis of its ability to degrade the α'-chain of bovine C3b in the presence of bovine factor H. Electrophoresis in polyacrylamide gels was used to assess the degradation of C3b and determine the molecular weights of factor I and its component polypeptide chains.

Results—Bovine factor I had an apparent molecular weight of 94 kd and consisted of 2 disulfide-bonded polypeptides that had apparent molecular weights of 51 and 42 kd (under reducing conditions). Factor H was required for the factor I cleavage of the α'-chain of bovine C3b into iC3b. A similar cofactor effect was provided by trypsinized bovine erythrocytes or erythrocyte ghosts. Bovine properdin was prepared and shown to be a single polypeptide chain of 58 kd in the reduced form.

Conclusions and Clinical Relevance—Bovine factor I can be purified from serum by a simple 4-step procedure. It is structurally and functionally comparable to factor I of other species, and its purification completes the isolation and characterization of all the soluble components of the bovine alternative complement pathway. (Am J Vet Res 2003;64:989–993)

Abstract

Objective—To isolate and characterize factor I of the bovine complement system.

Sample Population—Serum obtained from the blood of beef cattle.

Procedures—Serum samples were fractionated to yield factor I by means of sequential precipitation, ionexchange, and gel-filtration chromatography. The protein was identified throughout the procedure on the basis of its ability to degrade the α'-chain of bovine C3b in the presence of bovine factor H. Electrophoresis in polyacrylamide gels was used to assess the degradation of C3b and determine the molecular weights of factor I and its component polypeptide chains.

Results—Bovine factor I had an apparent molecular weight of 94 kd and consisted of 2 disulfide-bonded polypeptides that had apparent molecular weights of 51 and 42 kd (under reducing conditions). Factor H was required for the factor I cleavage of the α'-chain of bovine C3b into iC3b. A similar cofactor effect was provided by trypsinized bovine erythrocytes or erythrocyte ghosts. Bovine properdin was prepared and shown to be a single polypeptide chain of 58 kd in the reduced form.

Conclusions and Clinical Relevance—Bovine factor I can be purified from serum by a simple 4-step procedure. It is structurally and functionally comparable to factor I of other species, and its purification completes the isolation and characterization of all the soluble components of the bovine alternative complement pathway. (Am J Vet Res 2003;64:989–993)