Carnitine in Trypanosoma brucei brucei

Abstract

Bloodstream and culture forms of Trypanosoma brucei brucei contain high concentrations of intracellular l-carnitine, determined enzymatically (1.46 ± 0.28 and 4.49 ± 0.34 mM, respectively), a large proportion of which is non-esterified. Uptake of carnitine by bloodstream forms has been shown to take place against a concentration gradient. The K m and V max for carnitine uptake, which was saturable, were 180 μM and 0.242 nmol · min −1 · (mg cell protein) −1. Uptake was inhibited by the sulphydryl reagents p-chloromercuribenzoic acid (PCMB), N-ethylmaleimide (NEM), but not by salicylhydroxamic acid (SHAM), iodoacetic acid (IAA) or fluoride. Over the temperature range 10° – 30°C carnitine uptake possessed a coefficient of 45.4 ± 7.2 kJ · mol −1; between 30 and 40°C this decreased to approximately 7.0 kJ · mol −1. Both bloodstream and culture forms of T. b. brucei have high levels of carnitine-stimulated acetyl-CoA hydrolysis, i.e. carnitine acetyltransferase (CAT) activity. In the absence and presence of 3 mM l-carnitine, these activities were 9.73 ± 3.63 and 117.4 ± 22.4, and 36.13 ± 9.43 and 524.4 ± 99.9 nmol · min −1 · ( mg cell protein) −1, respectively. CAT activity in bloodstream forms was half-maximally stimulated by an l-carnitine concentration of 0.79 mM. Two separate enzyme activities capable of hydrolysing acetyl-CoA have been distinguished based on their sensitivity to freezing and thawing. Acetyl-CoA hydrolysis in the absence of l-carnitine (thioesterase activity) showed an apparent K m for acetyl-CoA of 120 μM and a relatively low value for V max; this activity was very labile. Hydrolysis of acetyl-CoA in the presence of saturating concentrations of l-carnitine (20 mM; ≈ 25 K m) gave an apparent K m of 36 μM and a much higher value for V max; this activity was relatively stable on storage. CAT activity in bloodstream forms was shown to be competitively inhibited by ATP with a K i of 0.46 ± 0.03 mM. Total and free carnitine within the cell were measured for bloodstream and culture forms under various metabolic conditions. Acetyl group pressure decreased the proportion of free carnitine within the cell. It is suggested that the carnitine-CAT system in T. b. brucei has the physiological role of protecting the limited cellular CoA pool from metabolic ‘acetyl pressure’. Attempts to measure the cellular CoA levels in either bloodstream or culture forms were unsuccessful; however, an upper limit of 0.1 and 0.2 nmol CoA · (mg cell protein) −1, respectively can be put on the size of the cellular pool.