Leishmanial Excreted Factor: Protein-Bound and Free Forms from Promastigote Cultures of Leishmania tropica and Leishmania donovani

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Abstract
Leishmania spp. growing in culture produce an immunologically active substance called excreted factor (EF) which precipitates antibodies raised against intact cells and may be the conditioning agent for parasite infection of host macrophages. An improved method for isolation of the material is described, based on Sephadex column chromatography of growth medium which was boiled at pH 5.0. This procedure allows detection of differences among the EF molecules of different species, and overcomes previous shortcomings by monitoring of immunological activity throughout. Analysis of the products of this procedure revealed that EF from L. tropica and L. donovani share a common carrier protein identified as rabbit serum albumin and are chemically similar. Growth medium from L. tropica boiled at acidic pH contains primarily an EF-albumin complex of 75,000 MW. Treatment growth medium from L. donovani contains the albumin complex and a smaller molecule (less than 27,000 MW) that is not associated with rabbit protein. This material accounts for nearly 20% of the EF of 1 L. donovani strain but constitutes only a minute fraction of L. tropica EF. Treatment of the EF-albumin complex with trichloroacetic acid [TCA] separates the molecule into 2 major subunits, 1 having a MW of about 61,000 (without anti-Leishmania activity) and the other having a MW of about 18,000 (with no anti-rabbit activity). The protein-free EF of L. tropica differs from that released by TCA extraction in that it can precipitate antisera of non-homologous serotypes, whereas the albumin complex and the TCA-treated EF fragment can not. EF from both species display pH-dependent affinity for certain lectins.