Characterization of a multicatalytic proteinase complex (20S proteasome) from Trypanosoma brucei brucei.

African trypanosomes are tsetse-transmitted protozoan parasites that cause sleeping sickness in humans and 'Nagana' in animals. A high relative molecular mass multicatalytic proteinase complex (MCP) was purified and biochemically characterized from the cytosolic fraction of Trypanosoma brucei brucei. The isolation procedure consisted of fractionation of the lysate by high speed centrifugation, chromatography on Q-sepharose molecular sieve filtration on Sephacryl S-300, chromatography on HA-Ultrogel and glycerol density gradient centrifugation (10-40%). The final enzyme preparation yielded a single protein band corresponding to a relative molecular mass of 630 kDa on a non-denaturing polyacrylamide gel. The enzyme hydrolyses a wide range of peptide substrates characteristic of chymotrypsin-like, trypsin-like, peptidylglutamylpeptide-hydrolysing activities determined by fluorogenic peptides, Z-Gly-Gly-Leu-NHMec, Z-Arg-Arg-NHMec and Z-Leu-Leu-Glu-beta NA, respectively. The enzyme was found to have a wide variation in pH optimal activity profile, with optimum activity against Z-Gly-Gly-Leu-NHMec at 7.8, Z-Arg-Arg-NHMec at pH 10.5 and Z-Leu-Leu-Glu-beta NA at pH 8.0, showing that the different activities are distinct. The enzyme hydrolysed oxidized proteins. In addition, the chymotryptic and trypsin-like activities were susceptible to inhibition by peptide aldehyde inhibitors with variable inhibition effects. The study demonstrates the presence of a non-lysosomal proteasome pathway of intracellular protein degradation in the bloodstream form of T. b. brucei. Further, the ability of the enzyme to hydrolyse most oxidized proteins, and the high immunogenicity exhibited suggests a possible involvement of the enzyme in pathogenesis of the disease.

Thyroid status and adenosine triphosphatase activity in experimental Trypanosoma congolense infection in rabbits.

The effect of trypanosome infection on the plasma levels and ratios of tri-iodothyronine (T3) and thyroxine (T4) as well as the activity of mitochondrial adenosine triphosphatase (ATPase) were investigated. Three groups of sexually mature white New Zealand rabbits were used. Group 1 consisted of the normal non-infected rabbits, group 2 were experimentally infected with Trypanosoma congolense and group 3 were infected but given replacement doses of thyroxine. The infected animals (group 2) showed a rapid decline in both T3 and T4 but an increase in the T3/T4 ratio indicating differential production or clearance rates between the two hormones. The mitochondrial ATPase activity was found to be depressed in the infected group whereas there was no significant difference in the ATPase activity between the non-infected (group 1) and infected-treated animals (group 2). It is postulated that trypanosome induced hypothyroid status may play a role in the impairment of mitochondrial ATPase activity, a key enzyme in energy metabolism.

The effect of L-thyroxine on the anaemia response in Trypanosoma congolense infected rabbits.

The development of anaemia is a major pathological manifestation in chronic trypanosomosis. The anaemia in African trypanosomosis coincides with a marked decrease in plasma concentration of both thyroxine (T4) and 3,5,3' triiodothyronine (T3). To evaluate the effect of trypanosome-induced hypothyroidism on the development of anaemia, sexually mature white New Zealand rabbits were used. Three groups were set up, each of ten rabbits: one group was infected with Trypanosoma congolense; the second group was infected but given replacement doses of thyroxine (treated); the third group was not infected. Small volumes of blood were collected for the determination of parasitaemia and packed cell volume (PCV). The concentrations of T3 and T4 were measured in plasma by radioimmunoassay. The decrease in PCV correlated closely (y = -0.38x + 15.2; r = 0.82, P = 0.001) with the intensity and duration of parasitaemia. The critical PCV value was 0.15 11-1 with a peak parasitaemia of approximately 5 x 10(6) trypanosomes ml-1 of blood. There was a significant correlation between the plasma T3 and PCV (y = 0.049x + 0.57; r = 0.66, P = 0.020). There was also a good positive correlation between T4 and PCV (y = 14.5 + 3.03; r = 0.95, P < 0.001) in the infected untreated group. The PCV levels were significantly different among the three groups of animals (P < 0.05). The infected-treated animals sustained longer periods of infection than the infected and untreated ones. The sustained physiological level of bioactive thyroid hormones T3 and T4 significantly arrested the decline in PCV as the disease progressed.(ABSTRACT TRUNCATED AT 250 WORDS)

Altered 3,5,3'-triiodothyronine thyroxine ratio in experimentally induced kwashiorkor and obesity in rats.

The plasma triiodothyronine (T3) and thyroxine (T4) ratios have been evaluated in kwashiorkor and diet-induced obese weaned rats. The concentrations of T3 and T4 were determined in plasma by radio-immunoassay. A significant decrease in T3 level in the order kwashiorkor < obese < control was observed. However T4 concentration was more elevated (P < 0.01) in the obese than the normal controls, while more significantly depressed (P < 0.001) in the kwashiorkor than in control animals. The T3/T4 ratio decreased in the order obese < kwashiorkor < control. It was concluded from these studies that kwashiorkor and diet-induced obesity not only interfere with the absolute concentration of the thyroid hormones but also alter the T3/T4 ratio. The altered T3 and T4 ratio perhaps contributes to the maintenance of the isoenergetic state rather than to the promotion of negative or positive energy balance in kwashiorkor and obese subjects respectively.

Respiratory activity of isolated liver Mitochondria following Trypanosoma congolense infection in rabbits: the role of thyroxine.

1. The effect of trypanosome infection on rabbit liver mitochondrial oxidative phosphorylation was investigated, with and without thyroxine replacement. 2. State 3 respiration, respiratory control ratio (RCR) and ADP/O ratio were significantly reduced in mitochondria from trypanosome-infected animals whereas there was no change in state 4 respiration. 3. State 3 respiration, RCR and ADP/O ratio were not significantly altered in trypanosome-infected animals given thyroxine replacement therapy. 4. Trypanosome infection leads to impairment of mitochondrial integrity, apparently through lowered thyroxine levels. Replacement of thyroxine therefore sustains optimal mitochondrial respiratory activity.