Chitinases in trypanosomatidae and in Trypanosoma brucei brucei: Reply

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In their letter to Parasitology Todaj, Arnold, Gooday and Chappell report the observation that foetal calf serum (FCS) has chitinolytic activity. They therefore assume that the chitinolytic activity recorded in our study of various trypanosomatids 2 includes the activity of the FCS that was added to the growth medium [Dulbeco's modified Eagle's medium with high glucose content (DMEMHG), supplemented with 10% FCS and antibiotics]. In this study, using high pressure liquid phase chromatography, we have shown that only one d ~stinct chitinase, with an estimated weight of I I 0 kDa, was present. Chitinase activity was not present in other fractions, including that of 47 kDa, which is the molecular weight of chitinase in bovine calf serum 3. The possibility of the involvement of FCS chitinase was also excluded by additional cont-ols (Y. Schlein and R.L. Jacobson, unpublished). We have incubated the culture medium for the period in which growing cul:ures reached the stationary phase (five days at 28°C) and, using our standard procedure, we have found no chitinolytic activity in culture overlayer protein precipitates (fraction II). Therefore it is unlikely that the activity of the FCS chitinase was measured in our tests. On the basis of the above reservation and negative results in an experiment with T. b. brucei, Arnold and co-workers presume that Tryponosoma spp do not produce chitinase (the culture medium for the T. b. brucei was SDM79 supplemented with 10% heat- inactivated FCS). We have grown procyclic T. b. brucei in a different medium (Schneider's Drosophila medium, supplemented with 10% FCS and 10 mg ml-~ haemin) and found a considerable chitinase activity of 5. I enzyme units per ~g protein (one unit of activity is defined as the amount of enzyme that generates 1.0 pmol of the substrate p-nitrophenyl j3-D-N,N'-diacetychitobiose in one hour under standard assay conditions2). According to other experiments (Y. Schlein and R.L. Jacobson, unpublished), the secretion of chitinase by trypanosomatids is determined by the culture medium composition. The activity of enzyme preparations of Crithidia fasciculata made from cultures grown in DMEMHG supplemented with 10% FCS was 13.5 enzyme units per [~g protein; when the same medium was supplemented with 20 mg ml-~ haemin, the activity of the preparations decreased to 0.35 enzyme units per I~g protein. Furthermore, when the C. fasciculata were grown in brain-heart infusion (BHI) supplemented with 10% FCS, the activity of the preparations was 0.6 enzyme units per ~g protein. Enzyme preparations of Sauroleishmania agamae (LRC-L409) had no chitinase activity if the parasites were grown in BHI with 10% FCS, but they had 7.7 enzyme units per I~g protein activity following growth in DMEMHG supplemented with 10% FCS. Evidently some Trypanosoma spp secrete chitinase, and the lack of enzyme reported q can probably be attributed to the growth medium of the parasites.