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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)

Full access
in American Journal of Veterinary Research

Abstract

Objective

To isolate and characterize the eighth component of the complement system (C8) in cattle.

Sample Population

Fresh plasma obtained from beef cattle.

Procedures

Plasma samples were fractionated, using sequential precipitation and ion-exchange and gel-filtration chromatography, to yield C8. The protein was identified throughout the procedure on the basis of its hemolytic function. Electrophoresis in polyacrylamide gels was used to determine molecular weight and composition of polypeptide chains. Reconstitution of classical and alternative complement pathways was used to characterize the hemolytic function of bovine C8.

Results

The bovine C8 protein consisted of a disulfide-bonded α-γ heterodimer that was noncovalently associated with a β chain. Apparent molecular weight of the α, β, and γ chains under reducing conditions were 66, 61, and 23 kd, respectively. In the classical pathway of activation, bovine C8 and the ninth component of the complement system (C9) had species incompatibility with human C8 and C9 on sheep erythrocyte target cells.

Conclusions

A simple 4-step fractionation procedure provided good yield of bovine C8 from plasma. The isolated protein was structurally comparable to C8 from other species. Purified bovine C8 may be useful in functional hemolytic assays to investigate the roles of complement-mediated lysis in the pathogenesis of inflammatory diseases and the killing of susceptible microorganisms. (Am J Vet Res 1999;60:1474–1477)

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in American Journal of Veterinary Research