The trypanocidal Cape buffalo serum protein is xanthine oxidase

Abstract

Plasma and serum from Cape buffalo (Syncerus caffer) kill bloodstream stages of all species of African trypanosomes in vitro. The trypanocidal serum component was isolated by sequential chromatography on hydroxylapatite, protein A-G, Mono Q, and Superose 12. The purified trypanocidal protein had a molecular mass of 150 kDa, and activity correlated with the presence of a 146-kDa polypeptide detected upon reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid sequences of three peptide fragments of the 146-kDa reduced polypeptide, ligand affinity and immunoaffinity chromatography of the native protein, and sensitivity to pharmacological inhibitors, identified the trypanocidal material as xanthine oxidase (EC 1.1.3.22). Trypanocidal activity resulted in the inhibition of trypanosome glycolysis and was due to H 2 O 2 produced during catabolism of extracellular xanthine and hypoxanthine by the purine catabolic enzyme.

Laminar xanthine oxidase, superoxide dismutase and catalase activities in the prodromal stage of black‐walnut induced equine laminitis

“…Hydrogen peroxide (H 2 O 2 ), generated during catabolism of xanthine by XO, was quantified using a coupled horseradish peroxidase (HRP)-dependent dye-reduction assay as previously described (Muranjan et al 1997). Specificity was established by inclusion of allopurinol (0.5 mmol/l) (A-8003) 1 , an inhibitor of XO (Massey et al 1970), or oxonic acid (10 mmol/l) (O-9376) 1 , an inhibitor of uricase (Kang et al 2002).…”

“…Under aerobic conditions, with xanthine present at a nonlimiting concentration, XO yields O 2 -and H 2 O 2 in a ratio of 2:1 and under this condition the addition of a nonlimiting concentration of SOD to the reaction results in a doubling in the output of H 2 O 2 by catalysing the following reaction: . To ascertain whether XO-positive tissue extracts also contained SOD, a nonlimiting amount of the enzyme (SOD; S-2515, 255 u/ml) 1 was included in the XO assay buffer and output of H 2 O 2 monitored by HRP-coupled dye reduction as described by Muranjan et al (1997).…”

Biology and Diseases of Ruminants: Sheep, Goats, and Cattle

“…The more common in the United States include Angus (also called Aberdeen-Angus), Hereford (both polled and horned), and Simmental (Briggs and Briggs, 1980;Schmidt et al, 1988). Breeds indigenous to other continents, such as the Cape Buffalo, have been found to have unique innate immune characteristics that protect them from endemic trypanosomiasis (Muranjan et al, 1997).…”

Infection-Associated Decline of Cape Buffalo Blood Catalase Augments Serum Trypanocidal Activity

“…Paradoxically, the trypanocidal activity becomes evident only as serum is diluted (28). The trypanocidal serum component is xanthine oxidase (4,21) (21), and trypanocidal activity is due to H 2 O 2 generated by reduction of O 2 during oxidation of hypoxanthine and xanthine to uric acid (11,21). The infection-induced change in Cape buffalo blood that leads to efficient control of trypanosome parasitemia might therefore involve an increase in the amount of H 2 O 2 produced in blood, an increase in the life span of this oxygen radical, or both.…”

“…Here we examine the impact of modifying the concentration of xanthine oxidase, purine substrate, and H 2 O 2 catabolic activity on expression of trypanocidal activity of high concentrations of serum from naive Cape buffalo in vitro, identify the condition that allows expression of the latent oxidative capacity of serum, and show that this condition is established in trypanosome-infected Cape buffalo coincident with control of the sole parasitemic wave. immunoglobulin (Ig) was isolated from immune serum by affinity chromatography on bovine liver catalase conjugated to cyanogen bromide-activated Sepharose 4B (5 mg of catalase/ml of gel; Pharmacia) as previously described (21); trace activity against xanthine oxidase was removed by affinity chromatography on xanthine oxidase that had been isolated from the aqueous phase of fresh cow milk by using a mouse monoclonal antibody as described previously (4) and immobilized on Sepharose 4B. Catalase-specific Ig was conjugated to cyanogen bromide-activated Sepharose 4b (3 mg of Ig/ml of gel), and catalase was removed from serum by immunoaffinity chromatography as previously described for xanthine oxidase (21).…”