Some kinetic properties of pyruvate kinase from Trypanosoma brucei: influence of pH and fructose-1,6-diphosphate.

The influence of pH on the activity of purified pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from Trypanosoma brucei has been studied. The Km for the coenzyme ADP is pH-dependent and shows the involvement of a dissociable group on the free enzyme with a pKa of 6.5-6.7. The cooperative interaction of the multiple phosphoenolpyruvate (PEP) binding sites is independent of pH in the range of 5.7-7.8. Variation of the Vmax value with pH indicates the presence of a dissociated group (pKa 6.2-6.3) and of an undissociated group (pKa 7.5-7.6) in the enzyme-substrate complex. A doubly dissociated phosphate group on PEP is shown to be essential by the effects of pH on the S0.5 value for this substrate, as is an undissociated enzyme group with a pKa in the range 6.7-7.0. It is shown that PEP and frucotse-1,6-diphosphate (FDP) act entirely in conjunction in allosterically activating the enzyme, FDP, the heterotropic effector, decreases the interaction between PEP binding sites at low concentration, and decreases the S0.5 value for PEP at higher concentration. A model for the interaction of the enzyme with its substrates is discussed.

Studies on glycerol kinase and its role in ATP synthesis in Trypanosoma brucei.

Glycerol kinase of Trypanosoma brucei has been shown to be capable of catalysing sn-glycerol-3-phosphate dependent ADP phosphorylation for ATP generation. The rate of this reaction (Vr) is sufficient to account for the observed rate of glycerol production from anaerobic glucose metabolism by intact cells and to account for net ATP synthesis. Glycerol kinase has been purified by preparing a post-nuclear, particulate fraction and solubilizing the enzyme with 0.5% (w/v) Triton X-100. This treatment results in a 3.5-fold increase in total activity, demonstrating the latent nature of particulate glycerol kinase, and an overall 10-fold increase in specific activity in the soluble fraction. The ratio of the velocities of the forward (Vf) reverse (Vr) reactions of this enzyme is altered from 21 to 170 upon solubilization. The Michaelis constants for the solubilized enzyme are KmADP = 0.12 +/- 0.04 mM, KmG-3-P = 5.12 +/- 1.47 mM, Kmglycerol = 0.12 +/- 0.05 and KmATP = 0.19 +/- 0.04 mM. Endogenous hexokinase acts as an ATP trap favouring ATP synthesis sn-glycerol-3-phosphate and ADP. This can be demonstrated in reconstituted systems using trypanosome glycerol kinase and varying hexokinase activities. Mass action inhibition of ATP synthesis by glycerol is more marked with lower hexokinase activities. High glycerol kinase activity (> 0.5 mumol/min/mg protein) has been found in the T. brucei complex of trypanosomes that produce glycerol anaerobically whereas only low activities (less than or equal to 0.03 mumol/min/mg protein) are present in Trypanosoma cruzi, Trypanosoma lewisi and Crithidia fasciculata, organisms that do not produce glycerol. Trypanosoma congolense has a glycerol kinase activity of 0.17 mumol/min/mg protein and shows poorer ATP synthesis from anaerobic glucose metabolism than organisms of the T. brucei complex.

Trypanosoma brucei: the effect of glycerol on the anaerobic metabolism of glucose.

Studies measuring the glycolytic intermediate and adenine nucleotide concentrations in Trypanosoma brucei metabolising glucose either aerobically or under conditions where glycerol-3-phosphate oxidase is inactive have shown the following: 1. Inhibition with 0.5 mM salicylhydroxamic acid (SHAM) accurately simulates anaerobic conditions in T. brucei; 2. On inhibition of respiring cells with 0.5 mM SHAM, the concentrations of most glycolytic intermediates decrease; they decrease further as the concentration of glycerol, an end product, increases. Only the concentration of sn-glycerol-3-phosphate is increased. This increase depends upon the method of preparation but is independent of time and glycerol concentration. 3. Glycerol formation from sn-glycerol-3-phosphate is coupled to the phosphorylation of another compound. The results of these studies are consistent with this compound being ADP; 4, The degree of inhibition of the anaerobic metabolism of glucose exerted by glycerol varies with the sn-glycerol-3-phosphate concentration, implying that the effect of glycerol is at the site of sn-glycerol-3-phosphate: ADP transphosphorylation.

An improved method for the estimation of suramin in plasma and trypanosome samples.

An improved method for the chemical estimation of suramin is described in which the aromatic amines released from the drug by acid hydrolysis are diazotised and then coupled to N-(1-naphthyl)-ethylenediamine to form a pink coloured product (EmM545nm267 /+- 5) Provided certain precautions are followed, the assay method is highly reproducible (+/- 2%) and sufficiently sensitive to measure 2.5 nmol; suramin in mixtures with plasma (0.5 ml) or trypanosomes (200 mg wet wt.).

Uptake of the trypanocidal drug suramin by bloodstream forms of Trypanosoma brucei and its effect on respiration and growth rate in vivo.

After a single intravenous injection of suramin the rate of removal of the drug from the plasma into other tissue compartments of the rat is independent of initial concentration. The data can be fitted to the sum of two exponential functions, consistent with a two-compartment, open model system. Trypanosomes take up only small amounts of suramin in vivo and do not actively concentrate the drug within the cell. Uptake is apparently by a non-saturable process that decreases with time and is dependent on the amount of suramin already taken up. Once within the cell, suramin progressively inhibits respiration and glycolysis, such that, for a given exposure in vivo, inhibition of oxygen consumption is proportional to the total amount of suramin absorbed. It can be calculated that only a fraction (4--9%) of this total is required to inhibit respiration to the extent found in broken cell preparations. The combined inhibition of two key enzymes in glycolysis--the sn-glycerol-3-phosphate oxidase (EC unassigned) and the glycerol-3-phosphate dehydrogenase (NAD+) (sn-glycerol-3-phosphate: NAD+ 2-oxidoreductase, EC 1.1.1.8)--are sufficient to account for the differential inhibition of glucose and oxygen consumption and of pyruvate production, together with the small, but significant, production of glycerol. Even at the highest dose of suramin tolerated by the rat, trypanosomes continue to increase exponentially in the bloodstream for at least 6 h. The mean doubling time is increased from 4.6 h to a maximum of about 12.5 h in rats treated with doses of suramin in the range 25--150 mg/kg. In the light of these and other findings, it is concluded that part of the trypanocidal action of suramin results from the inhibition of ATP production by glycolysis.