Arginine kinase of the flagellate protozoa Trypanosoma cruzi. Regulation of its expression and catalytic activity.

In epimastigotes of Trypanosoma cruzi, the etiological agent of Chagas' disease, arginine kinase activity increased continuously during the exponential phase of growth. A correlation between growth rate, enzyme-specific activity and enzyme protein was observed. Arginine kinase-specific activity, expressed as a function of enzyme protein, remains roughly constant up to 18 days of culture. In the whole range of the culture time mRNA levels showed minor changes indicating that the enzyme activity is post-transcriptionally regulated. Arginine kinase could be proposed as a modulator of energetic reserves under starvation stress condition.

Presence of a fatty acid-binding protein and lipid stores in flight muscles of Dipetalogaster maximus (Hemiptera: Reduviidae).

A fatty acid-binding protein (FABP) from the cytosolic fraction of the triatomine Dipetalogaster maximus (Uhler) flight muscles was purified by a procedure based on gel filtration, reverse-phase high performance liquid chromatography, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein has an apparent molecular mass of 14 kDa, and its N-terminus is unblocked. Its N-terminal sequence was obtained by submitting an SDS-PAGE band blotted onto a polyvinylidene difluoride membrane to Edman degradation. The sequence obtained indicates that this FABP belongs to the heart type. This is the first time that a fatty acid-binding protein has been reported for a triatomine. The presence of said FABP, abundant mitochondria, and lipid stores in the flight muscles of D. maximus suggests that beta oxidation of fatty acids is used by the triatomine thoracic muscle as an energy source, and could be related to its dispersal capacity.

Effects of temperature and pH on hexokinase from the flight muscles of Dipetalogaster maximus (Hemiptera: Reduviidae).

Effects of temperature and pH on the catalytic properties of hexokinase (HK, EC 2.7.1.1) from the flight muscles of Dipetalogaster maximus (Uhler) were studied. The enzyme showed a hyperbolic behavior with its two substrates (glucose and ATP). There was no inhibition by glucose. Apparent Km and Vmax increased as pH increased from 7.0 to 8.5. Catalytic efficiency was lowest at pH 7.0. Km, Vmax, and catalytic efficiency were higher at 37 degrees C than at 30 and 20 degrees C. There was marked inhibition by ATP, which was dependent on pH and temperature. Km values for ATP were reduced and catalytic efficiency increased as pH increased. Lowest Vmax was observed at pH 7.0. At this pH there was 87.3% inhibition by ATP, whereas it was only 5.7% at pH 8.5 (at 30 degrees C). Km, Vmax, and catalytic efficiency were higher at 37 degrees C than at 30 and 20 degrees C. The strong inhibition by ATP detected at 20 degrees C (pH 7.6) almost disappeared at 37 degrees C. Therefore, temperature could regulate hexokinase activity by modulating the inhibition produced by ATP. Glucose utilization and ATP production would be promoted when temperature rises from 30 to 37 degrees C. Because insect thoracic muscles increase their temperature over 30 degrees C during flight, this phenomenon elucidates a mechanism enhancing energy supply for muscle activity.

Enzymatic method for branched chain alpha-ketoacid determination: application to rapid analysis of urine and plasma samples from maple syrup urine disease patients.

A new method for the determination of branched-chain alpha-ketoacid concentration using lactate dehydrogenase (E C 1.1.1.27) isozyme C4 (LDH C4) from mouse testes is proposed. The assay is performed on urine and plasma without previous treatment. Alpha-ketoglutarate and pyruvate are determined on the same sample using glutamate dehydrogenase (GDH, EC 1.4.1.2) and lactate dehydrogenase isozyme A4 (LDH5) respectively and subtracted from the total alpha-ketoacid concentration obtained with LDH C4. This value corresponds to the branched chain alpha-ketoacid. Results were linear within the concentration range 8 to 170 mumoles/L. Detection limit was 8 mumoles/L. Analytical recovery was higher than 91%. For microplate assays, recoveries were higher than 84% and the detection limit was 20 mumoles/L. Determinations performed with GDH, LDH A4 and LDH C4 allow differentiation of E3 deficiency from other clinical phenotypes of maple syrup urine disease. The method is simple and fast, and adaptation to microplates would allow screening of newborns.

Enzymatic method for branched chain alpha-ketoacid determination: application to rapid analysis of urine and plasma samples from maple syrup urine disease patients.

A new method for the determination of branched-chain alpha-ketoacid concentration using lactate dehydrogenase (E C 1.1.1.27) isozyme C4 (LDH C4) from mouse testes is proposed. The assay is performed on urine and plasma without previous treatment. Alpha-ketoglutarate and pyruvate are determined on the same sample using glutamate dehydrogenase (GDH, EC 1.4.1.2) and lactate dehydrogenase isozyme A4 (LDH5) respectively and subtracted from the total alpha-ketoacid concentration obtained with LDH C4. This value corresponds to the branched chain alpha-ketoacid. Results were linear within the concentration range 8 to 170 mumoles/L. Detection limit was 8 mumoles/L. Analytical recovery was higher than 91

. For microplate assays, recoveries were higher than 84

and the detection limit was 20 mumoles/L. Determinations performed with GDH, LDH A4 and LDH C4 allow differentiation of E3 deficiency from other clinical phenotypes of maple syrup urine disease. The method is simple and fast, and adaptation to microplates would allow screening of newborns.

Intracellular localization of the testicular and sperm-specific lactate dehydrogenase isozyme C4 in mice.

The proposed dual intracellular distribution of the sperm-specific lactate dehydrogenase (EC 1.1.1.27) isozyme C4 (LDH C4) has been based on indirect evidence. In order to obtain direct evidence on the localization of this LDH isozyme in mice, postembedding immunocytochemistry at ultrastructural level was performed on testes, epididymal spermatozoa, and isolated testicular mitochondria. The immunogold technique was applied to thin sections incubated first in partially purified specific anti-LDH C4 rabbit IgG, and immunoreactive sites were detected with colloidal gold adsorbed to anti-rabbit IgG. In the testis, immunostaining was found in the cytoplasm of spermatocytes and spermatids and in the principal and middle pieces of differentiating spermatozoa. Spermatozoa from epididymis also exhibited heavy labeling of colloidal gold in their middle and principal pieces, but the immunostaining was weak in the special type of mitochondria present in spermatocytes, spermatids, and spermatozoa (sperm-type mitochondria, STM). The isolation of STM produced several morphological changes in comparison with those in situ, including an enhancement of the LDH C4 labeling in the mitochondrial matrix. The other type of mitochondria (non-STM) from spermatocytes and nonspermatogenic cells were not immunostained and served as background control. The results presented here confirm previous findings, gathered by indirect methods, indicating a dual localization of LDH C4 in the cytosol of spermatocytes, spermatids, and spermatozoa, as well as in the matrix of sperm-type mitochondria.

The lactate/pyruvate shuttle in spermatozoa: operation in vitro.

The operation of a shuttle for the transfer of reducing equivalents in the special mitochondria present in the middle piece of spermatozoa (sperm-type mitochondria, STM) was studied in reconstituted systems in vitro with mouse, rat, and rabbit STM. The redox couple lactate/pyruvate and the lactate dehydrogenase isozyme C4 are involved in the shuttle. It is active with rat and rabbit STM, while it does not work with mouse STM, probably because the influx of lactate into the mouse organelles is relatively poor. Ratios of consumption of pyruvate/lactate by STM were 21.6, 1.28, and 1.6 for mouse, rat, and rabbit organelles, respectively. The shuttle is inhibited by 0.6 mM mersalyl, a blocker of lactate transport. The operation of the shuttle would oxidize cytosolic NADH produced during aerobic glycolysis (or fructolysis) in spermatozoa of those species having an efficient lactate carrier in mitochondria.

Effect of L-malate on pyruvate dehydrogenase activity of spermatozoa.

Presence of L-malate (from 0.5 to 4 mM concentrations) in the medium produces a marked increment of pyruvate consumption by the special type of mitochondria found in the middle piece of spermatozoa (sperm-type mitochondria, STM). Pyruvate uptake by liver mitochondria is not increased by malate. A comparative study on pyruvate dehydrogenase (PDH) of STM and liver mitochondria from mouse, rat, and rabbit showed that 2 mM L-malate does not modify significantly the activity of liver PDH, while it increases markedly that of spermatozoal PDH in the three species. The differential sensitivity to L-malate appears to be a peculiar regulatory property of the PDH complex in the gametes, which contains at least one component (E1, pyruvate decarboxylase, EC 1.2.4.1) known to be a sperm-specific isozyme.

Effect of gossypol on trypomastigotes and amastigotes of Trypanosoma cruzi.

Bloodstream Trypanosoma cruzi trypomastigotes isolated from infected mice undergo reduction of motility and structural damages after 5 to 45 min exposure to gossypol at concentrations ranging from 5 to 50 microM. When 1% serum albumin is added to the incubation medium, no alterations of parasites are observed, even with 100 microM gossypol. Intracellular T. cruzi amastigotes in infected Vero cell cultures exposed to 5 microM gossypol for 2 h do not show changes. Incubation with 5 microM gossypol for 48 h produces complete disruption of host cells; however, the amastigotes they contain show only minor alterations. The observations indicate that, in protein-rich media, gossypol is complexed into associations which have no activity on the different forms of the T. cruzi biological cycle.

Properties of the branched-chain 2-hydroxy acid/2-oxo acid shuttle in mouse spermatozoa.

Operation of the branched-chain 2-hydroxy acid/2-oxo acid shuttle for the transfer of reducing equivalents in mitochondria of mouse spermatozoa was studied in vitro in reconstituted systems. Results show that the branched-chain 2-oxo acids within the mitochondria are offered several metabolic pathways. (a) Decarboxylation: mouse sperm mitochondria possess high branched-chain 2-oxo acid decarboxylase activity. (b) Recycling to the cytosol by using a transport system which can be inhibited by alpha-cyano-3-hydroxycinnamate and pH 6.8. (c) Transamination to the corresponding amino acids: experiments presented indicate that leucine formed from 4-methyl-2-oxopentanoate may pass to the external phase, re-initiating the cycle. These two last possibilities would allow autocatalytic operation of the shuttle. The branched-chain 2-hydroxy acids apparently do not utilize the monocarboxylate carrier to penetrate the mitochondria.

In vitro inhibition by gossypol of oxidoreductases from human tissues.

The effect of gossypol, a polyphenolic compound with antifertility action on human males, has been investigated on the following oxidoreductases purified from human tissues: lactate dehydrogenase (EC 1.1.1.27) isozymes 1 or B4 from heart, 5 or A4 from liver and X or C4 from spermatozoa; malate dehydrogenase (EC 1.1.1.37) mitochondrial and "soluble" isozymes from heart and NADP-glutamate dehydrogenase (EC 1.4.1.4) from liver. Gossypol proved to be a powerful inhibitor of the six enzymes studied. For all of them, inhibition was of the competitive type with respect to the coenzyme and non-competitive in relation to substrate. The lowest ki values were shown for lactate dehydrogenase isozyme 1 or B4 and for the two isozymes of malate dehydrogenase. Results did not show selectivity of gossypol for the sperm-specific isozyme X or C4 of lactate dehydrogenase.

Inhibition by gossypol of oxidoreductases from Trypanosoma cruzi.

The effects of gossypol, a polyphenolic compound isolated from the cotton plant upon six oxidoreductases from cultured epimastigotes of Typanosoma cruzi were studied. Gossypol was a powerful inhibitor of the alpha-hydroxyacid and malate dehydrogenases, NAD-linked enzymes, and of glutamate dehydrogenase, malic enzyme and glucose-6-phosphate dehydrogenase, NADP-dependent enzymes. The drug did not have an effect on succinate dehydrogenase, a flavoprotein. The Ki values with respect to substrate were 0.73, 0.3 and 3.5 microM for alpha-hydroxyacid, malate and glutamate dehydrogenases, respectively, and 1.1, 0.19 and 7.8 microM with respect to the coenzyme. Inhibition was noncompetitive with respect to substrate and uncompetitive in relation to the coenzyme.

Effect of temperature upon catalytic properties of alpha-hydroxyacid dehydrogenase from Trypanosoma cruzi.

A comparative study of the effect of temperature (10, 20, 30 and 37 degrees C) upon Km and V of alpha-hydroxyacid dehydrogenase (HADH), isozyme I and II, from Trypanosoma cruzi, a parasite whose life cycle comprises stages in an insect vector, and of another enzyme with analogous substrate specificity, the lactate dehydrogenase, isozyme X (LDH X) from mouse, a homeotherm, is presented. The Km for alpha-ketoisocaproate of HADH is markedly reduced as temperature decreases. This effect can compensate the reduction in thermal energy and produce stabilization of the reaction rate. This compensation does not occur with mouse LDH X. The activation energy for both HADH isozymes is about half the value determined for mouse LDH X. Results indicate that HADH from T. cruzi is able to adjust instantaneously to thermal changes of the environment, behaving as other enzymes of terrestrial poikilothermic animals.

Catalytic properties of the sperm-specific lactate dehydrogenase (LDH X or C4) from different species.

A comparative study of catalytic properties of the sperm-specific lactate dehydrogenase (EC 1.1.1.27) isozyme X or C4 from a variety of animals (boar, bull, goat, Guinea pig, man, mouse, pigeon, rabbit, and rat) is presented. Optimum concentration and Km values for pyruvate, inhibition by substrate, and activity against analog substrates (alpha-ketoacids with linear and branched chains from 4 to 6 carbon atoms) for isozyme X of different species showed significant differences. The observed properties are correlated with available evidence on the metabolic role of the enzyme.

Inhibitory action of gossypol on enzymes and growth of Trypanosoma cruzi.

Gossypol, a phenolic compound isolated from the cotton plant, is a powerful inhibitor of nicotinamide adenine dinucleotide-linked enzymes (alpha-hydroxyacid dehydrogenase and malate dehydrogenase) of Trypanosoma cruzi, the parasite that causes Chagas' disease. Parasites at the epimastigote stage that were incubated for 5 minutes with 100 micromolar gossypol were completely immobilized. Concentrations of gossypol as low as 0.01 micromolar markedly reduced the growth rate of T. cruzi in culture.

Properties of alpha-hydroxyacid dehydrogenase isozymes from Trypanosoma cruzi.

Whole cell extracts of culture epimastigotes of Trypanosoma cruzi (Tulahuén strain) have alpha-hydroxyacid dehydrogenase activity which catalyzes the NAD-linked reaction alpha-ketoacid in equilibrium with alpha-hydroxyacid, with a variety of substrates. Two molecular forms of the enzyme have been separated by means of gel electrophoresis. These isozymes were partially purified by DEAE-cellulose chromatography and ammonium sulfate precipitation. Molecular weights were estimated and some catalytic properties were determined with purified isozymes. The faster migrating fraction (isozyme I) has a molecular weight of 85 500 and showed significant activity against linear 3-5 carbon chain substrates. The lowest Km value was obtained for pyruvate. Isozyme II (MW 60 500) utilizes linear and branched chain substrates with 4-6 carbon atoms. Its highest activity and lowest Km value were recorded with alpha-keto-isocarproate as substrate.

Substrate specificity of the lactate dehydrogenase isoenzyme C4 from human spermatozoa and a possible selective assay.

The activity of lactate dehydrogenase (EC 1.1.1.27) in normal human sperm lysates and in human heart and liver homogenates was determined by using a variety of 2-oxoacids as substrates. Sperm preparations were active with pyruvate, 2-oxobutanoate, 2-oxopentanoate and 2-oxohexanoate, while heart and liver extracts utilized only pyruvate and 2-oxobutanoate. Selective staining after gel electrophoresis indicated that the fraction corresponding to lactate dehydrogenase C4, the sperm-specific isoenzyme, was responsible for the utilization of substrates with a linear chain of 3 to 6 carbon atoms. The use of 5 mM 2-oxohexanoate allowed the selective determination of isoenzyme C4 in preparations containing different lactate dehydrogenase molecular forms.