African trypanosomes have thiol‐dependent proteolytic activity that resembles some of the cathepsin‐like activity found in mammalian lysosomes [Lonsdale‐Eccles, J. D. & Mpimbaza, G. W. N. (1986) Eur. J. Biochem. 155, 469–473]. Here we show that this activity is found in lysosome‐like organelles which we have isolated (density = 1.082 g/cm3 in Percoll) from bloodstream forms of Trypanosoma brucei brucei. They are approximately 250 nm in diameter, are bounded by a single limiting membrane, and contain acid phosphatase. The predominant proteolytic and peptidolytic activity of these organelles has a pH optimum about 6.0, exhibits latency, and has the characteristics of mammalian cathepsin L (and possibly cathepsin H) with respect to its hydrolysis of small fluorogenic peptidyl substrates such as benzyloxycarbonyl‐phenylalanyl‐arginyl‐7‐amido‐4‐methylcoumarin. This substrate appears to be a good marker for trypanosomal lysosomes. The cathepsin‐L‐like activity is inhibited by the thiol‐protease inhibitors, E‐64, cystatin, leupeptin and mercurial compounds.The proteolytic activity of the lysosome‐like fraction is observed as a single band of activity with an approximate molecular mass of 27 kDa when measured after electrophoresis in the fibrinogen‐containing sodium dodecyl sulphate/polyacrylamide gels. The addition of mammalian serum to this purified fraction, or to whole trypansosome homogenates, results in the appearance of additional bands of activity, with a concomitant increase in the total observed proteolytic activity. The serum of some species of animal (e.g. goat and guinea pig) appear to lack the ability to generate this new and increased activity, while rat, rabbit, human and bovine sera exhibit varying capacities to generate the new activity, the cow being the most effective. The apparent molecular masses of the new bands of activity are different for each mammalian species, suggesting that the activator is a species‐specific molecule or class of molecules.We also show that Trypansosoma brucei contains soluble peptidolytic activity with an alkaline pH optimum. It is inhibited by the serine‐protease inhibitor diisopropylfluorophosphate, but not by inhibitors such as phenylmethylsulphonyl fluoride, α1‐antitrypsin, or aprotinin. Nor is it inhibited by the thiol‐protease‐specific inhibitors E‐64 or cystatin, although it is susceptible to inhibition by tosyllysylchloromethane, leupeptin, HgCl2 and p‐chloromercuribenzoate. This enzymic activity has a preference for arginyl residues in the primary binding site (the P1 position), as also does the activity from the lysosomes. However, the subsite specificity of the soluble enzymic activity is distinct from that of the lysosomes. The soluble enzyme(s) prefers small neutral molecules in the P2 and P3 subsites whereas the lysosomal enzyme(s) prefer hydrophobic molecules in these positions.

Lysates of different life‐cycle stages of Trypanosoma congolense, Trypanosoma vivax and Trypanosoma brucei were analysed for endopeptidase activity, using reaction conditions which permitted a distinction to be made between lysosomal and non‐lysosomal activity [Lonsdale‐Eccles, J. D. & Grab, D. J. (1987) Eur. J. Biochem. 169, 467–475]. Hydrolysis of Z‐Arg‐Arg‐NHMec (Z = benzyloxycarbonyl, NHMec = 7‐amino‐4‐methylcoumaryl) and Z‐Gly‐Gly‐Arg‐NHMec occurred predominantly at alkaline pH and was observed in lysates of both insect and mammalian infective forms of T. brucei and T. congolense. Compared to their other life‐cycle stages, procyclic forms of T. brucei and epimastigote forms of T. congolense exhibited enhanced hydrolysis of these substrates. Low levels of hydrolysis of Z‐Arg‐Arg‐NHMec were observed in the bloodstream and epimastigote forms of T. vivax. The hydrolysis of Z‐Gly‐Gly‐Arg‐NHMec in each of the life‐cycle stages of T. vivax was generally below detectable levels.In lysates of T. congolense, proteolytic and Z‐Phe‐Arg‐NHMec‐hydrolytic activity in bloodstream forms > metacyclic > epimastigote > procyclic forms. In T. vivax Z‐Phe‐Arg‐NHMec‐hydrolytic activity differed slightly according to the origin of the parasite but, in general, followed the same pattern (i.e. bloodstream forms > epimastigote forms, with metacyclic forms usually intermediate between these two). In T. brucei, Z‐Phe‐Arg‐NHMec‐hydrolytic activity in bloodstream forms > procyclic forms. Upon differentiation of the long, slender bloodstream forms into short, stumpy forms the Z‐Phe‐Arg‐NHMec‐hydrolytic activity was elevated even further. Thus, during their life cycle, each of these African trypanosomes exhibits complex changes of endopeptidase activity, suggestive of an induction of lysosomal activity between the insect and mammalian forms.

A gene, TvHT1, encoding a glucose transporter protein, has been cloned from the haemoflagellate protozoon, Trypanosoma vivax, which has an active Kreb's cycle in the mammalian stage. The deduced polypeptide is similar in amino acid sequence to other kinetoplastid hexose transporters from Trypanosoma brucei (THT1 and THT2), Trypanosoma cruzi (TcrHT1) and Leishmania (Pro-1). The similarity is higher with THT2 (expressed in T. brucei insect forms) than with the other isoforms. The kientic properties of glucose uptake in Chinese Hamster Ovary (CHO) cells expressing TvHT1 and in trypanosomes show a saturable transport mechanism typical of a facilitated crrier system, with a similar affinity for d-glucose as that of the T. brucei bloodstream from carrier, THT1 (Km=0.548 + 0.01 mM, V max = 4.26+0.12 nmol min-1. mg protein-1 in CHO cells and Km=0.585+0.068 mM, V max = 88.5+6.2 nmol min-1. in T. vivax). The specificity of the TvHT1 protein for various D-glucose analogues, as judged by inhibition of 2-deoxy D-arabinose-hexose transport, shows properties that are intermediate between those ofTHT1 on the one hand and TcrHT1 and THT2 on the other. As with the hexose transporters in the other members of Kinetoplastida, the TvHT1-encoded system differs from erythrocyte-type glucose transport by its moderte sensitivity to cytochalasin B and its capacity to transport fructose.