Enzymes of Carbohydrate Metabolism in Four Human Species of Leishmania: A Comparative Survey

600 MACRONUCLEAR AND CYTOPLASMIC R N A

14. Martin RG, Ames BN. 1961. A method for determining Application to protein

15. Moner 1. 1965. RNA svnthesis and cell division in heat-

the sedimentation behaviour of enzymes: mixtures. J . Biol. Chem. 236, 1372-9.

synchronized bopulations of Tltrahymena pyriformis. J. Proto- zool. 12,505-9.

16. Nachtwey DS, Dickinson WJ. 1967. Actinomycin D : Blockage of cell division of synchronized Tetrahymena pyriformis. Exp . Cell Res. 47, 581-95.

17. Nilsson JR. Zeuthen E. 1974. Microscopic studies of heat svnchronized Tetrahvmena bvriformis GL. C . Re- Trav. Lab. Carls- A. , derg 4Q( l ) , 1-18. '

18. Perry RP, Kelley DE. 1968. Persistent synthesis of 5s RNA when production of 28s and 18s ribosomal RNA is in- hibited by low doses of actinomycin D. ]. Cell Physiol. 72, 235-46.

19. ~ , Cheng T, Freed JJ, Greenberg JR, Kelley DE, Tar- tof KD. 1970. Evolution of the transcription unit of ribosomal RNA. Proc. Nut. Acad. Sci. US. 65, 609-16.

20. Rickwood D, Klemperer HG. 1970. Decreased ribonucleic acid synthesis in isolated rat liver nucleic during starvation. Bio- chem. 1. 120, 381-4.

21. Scherbaum OH, Zeuthen E. 1954. Induction of syn- chronous cell division in mass cultures of Tetrahymena pyriformis. Exp. Cell Res. 6, 221-7.

22. Scherrer K, Darnell JE. 1962. Sedimentation character- istics of rapidly labelled RNA from HeLa cells. Biochem. Biophys. Res. Commun. 7, 486-90.

23. Stocco DM, Zimmerman AM. 1975. Adenine nucleotide metabolism in heat-synchronized Tetrahymena. Mol. Cell. Bio- chem. 7 , 187-94.

24. Tavitian A, Uretsky SC, Acs G. 1968. Selective inhibi- tion of ribosomal RNA in mammalian cells. Biochim. Biophys. Acta

1971. Effect of valine deprivation on the biosynthesis of ribosomal and messenger RNA in a mammalian cell line (L1578Y). Eur. J . Biochem. 18, 35-45.

26. Wagner EK, Katz L, Penman S. 1967. The possibility of aggregation of ribosomal RNA during hot phenol-SDS deprotein- ization. Biochem. Biophys. Res. Commun. 28, 152-9.

27. Yoshikawa M, Fukada T, Kawade Y. 1964. Separation of rapidly labelled RNA of animal cells into DNA-type and ribosomal RNA-type components. Biochem. Biophys. Res. Commun.

28. Yuyama S, Zimmerman AM. 1972. RNA synthesis in Tetrahymena : Temperature-pressure studies. Exp. Cell Res. 71,

29. Zeuthen E. 1964. The temperature-induced division synchrony in Tetrahymena, in Zeuthen E, ed., Synchrony in Cell Divi- sion and Growth, Interscience, New York, pp. 99-158.

30. Zimmerman AM. 1969. Effects of hydrostatic pressure on macromolecular svnthesis in svnchronized Tetrahvmena. in Padilla

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J. PROTOZOOL. 23 ( 4 ) , 600-607 ( 1976).

Enzymes of Carbohydrate Metabolism in Four Human Species of Leishmania: A Comparative Survey*

ELMER MARTIN, MICHAEL W. SIMON, FRANK W. SCHAEFER, IIIt and ANTONY J. MUKKADA* Department of Biological Sciences, University of Cincfdnati, Cincinftati, Ohio 45221

SYNOPSIS. The occurrence and levels of activity of various enzymes of carbohydrate catabolism in culture forms (promastigotes) of 4 human species of Leishmania ( L . brasiliensis, L. donovani, L . mexicana, and L. tropica) were compared. These organisms possess enzymes of the Embden-Meyerhof pathway but lack lactate dehydrogenase. No evidence could be found for the production of lactic acid by growing cultures and lactic acid could not be detected either in cell-free preparations or after incubation of cell-free extracts with pyruvate and NADH under appropriate conditions. All 4 species possess a-glycerophosphate dehydrogenase and a-glycerophosphate phosphatase which together could regenerate NAD, thus compensating for the absence of lactate dehydrogenase. The oxidative and nonoxidative reactions of the hexose monophosphate pathway are present in all 4 species. Cell-free extracts have pyruvate dehydrogenase activity which allows the entry of pyruvate into and its subsequent oxidation through the tricarboxylic acid cycle. All enzymes of this cycle, including a thiamine pyrophosphate dependent a-ketoglutarate dehydrogenase, are present. Both NAD and NADP-linked malate dehydrogenase activities are present. The isocitrate dehydrogenase is NADP specific. There is an active glutamate dehydrogenase which could compete with a-ketoglutarate dehydrogenase for the common substrate (a-ketoglutarate) . Replenishment of C, acids is accomplished by heterotrophic COI fixation catalyzed by pyruvate carboxylase. All 4 species have high levels of NADH oxidase activity. Several enzymes thus far not found in any species of Leishmania have been demonstrated. These are: phosphoglucose isomerase, triose phosphate isomerase, fructose-1, 6-diphosphatase, 3-phosphoglycerate kinase, enolase, a-glycerophosphate dehydrogenase, cu-glycerophosphate phosphatase, pyruvate dehydrogenase complex, citrate synthase, aconitase, a-ketoglutarate dehydrogenase, glutamate dehydrogenase, and NADH oxidase.

Index Key Words: Leishmania brasiliensis; Leishmania donovani; Leishmania mexicana; Leishmania tropica; enzymes of Embden-Meyerhof pathway ; enzymes of hexose monophosphate pathway ; enzymes of tricarboxylic acid cycle ; heterotrophic COS fixation.

number of enzymes normally involved in carbohydrate me- A tabolism have been demonstrated in various species of Leishmania. Chatterjee et al. (13) reported the presence of hexokinase in Leishmania donovani which was later shown to phosphorylate not only glucose but also fructose, mannose, and glucosamine (21 ). Fructose-1, 6-diphosphate aldolase was shown

* This investigation was supported by a grant from the Research

t Present address: Department of Biology, College of Science,

* To whom reprint requests should be addressed.

Council of the University of Cincinnati.

University of Notre Dame, Notre Dame, Indiana 46556.

to occur in L. donovani (22) as well as in Leishmania adleri, Leishmania tarentolae, and Leishmania mexicana (46). Glyc- eraldehyde-3-phosphate dehydrogenase activity was demonstrated in cell-free extracts of L . donovani, L . mexicana, and L . tarentolae ( 3 0 ) . Enzymes of the tricarboxylic acid cycle reported from Leishmania spp. included NADP-linked isocitrate dehydrogenase in L. donovani, L . mexicana, and L . tarentolae (30) ; succinic dehydrogenase in L. donovani (27) and L. brasiliensis ( 11 ) ; NAD-linked malate dehydrogenase in L . tarentolae (35) , Leishmania enrietti, Leishmania tropica, L. donovani, Leish- mania brasiliensis, and L . mexicana (20) and fumarase in L.

Leishmania: CARBOHYDRATE METABOLISM 60 1

donovani ( 15). Glucose-6-phosphate dehydrogenase, ribose-5- phosphate isomerase, transketolase, and transaldolase, which are characteristic of the hexose monophosphate pathway, have been found in L. donovani (23). Another enzyme of the hexose monophosphate pathway, 6-phosphogluconate dehydrogenase, was demonstrated in L. donovani, L . mexicana, and L . tarentolae (30). The presence of heterotrophic C0,-fixing enzymes in L . donovani was inferred from the incorporation of radiolabeled NaHCO, into succinate (14).

The foregoing studies have suggested the operation of the Embden-Meyerhof pathway, the tricarboxylic acid cycle, and the hexose monophosphate pathway in Leishmania. These con- clusions, however, were generalizations based upon partial studies on different species. No single species was thoroughly and systematically studied. Examples are known where different species of the same genus differ in biochemical pathways; generalizations could thus be misleading, e.g. the differences in nitrogen metabolism especially at the level of transaminases between L. donovani and L. enrietti (7) .

MATERIALS AND METHODS Organisms and Growth Conditions

Promastigotes from cultures of L . brasiliensis, L . donovani, L . mexicana, and L . tropica were used in all studies detailed in this report. They were maintained as previously described (41, 56, 62). For routine use in experiments, the cells were cultivated in a liquid medium as previously described (41 ) . Reagents

The following reagents were obtained from Sigma Chemical Co., (St. Louis, Missouri) : sodium 6-phosphogluconate, /!- mercaptoethanol, coenzyme A (CoA), disodium EDTA, erythrose-4-phosphate, fructose-6-phosphate, glucose-6-phosphate dehydrogenase, glycine, glycylglycine, N-hydroxyethylpiperazine- N-2-ethanesulfonic acid (HEPES ), lactic dehydrogenase (type 11) , NAD, NADH, NADP, NADPH, oxaloacetic acid, phosphoglucose isomerase, ribose-5-phosphate isomerase, pyruvate kinase (types I and 11), sodium ADP, sodium ATP, sodium a-ketoglutarate, sodium fructose-1, 6-diphosphate, sodium phosphoenol- pyruvate, sodium pyruvate, triethanolamine, thiamine pyrophosphate, and Tris. Aldolase, a-glycerophosphate dehydrogenase- triose phosphate isomerase mixture (grade A), and potassium ATP were purchased from Calbiochem, Los Angeles, California. Bovine serum albumin was purchased from Nutritional Bio- chemical Co., Cleveland, Ohio; 3-acetyl pyridine-NAD (AP- NAD) was obtained from General Biochemicals, Chagrin Falls, Ohio. All other chemicals were bought from Fisher Scientific Co., Cincinnati, Ohio.

Enzyme Preparation and Assays Cell-free extracts were prepared as described previously (41 ) .

All spectrophotometric assays were carried out in a Gilford Multisample Absorbance Recorder, Model 2000. Enzyme assays were performed at room temperature and in general, the reaction mixtures were allowed to equilibrate 2-5 min before initiating the reaction. The amount of extract used in individual assays varied and usually corresponded to 100-200 pg protein. Protein determinations were made by the procedure of Lowry et al. (38). Specific activity is expressed as nanomoles substrate converted/ min/mg protein.

All reaction mixtures used for assaying the various enzymes contained cell-free extracts. Except as indicated, enough distilled water was added to bring the sample to a final volume of 3 ml.

Hexokinase (E.C. 2.7.1.2) activity was measured by coupling

it with glucose-6-phosphate dehydrogenase as described by Joshi & Jagnnathan ( 3 1 ) . The reaction mixture contained : Tris-HC1, pH 7.6, 60 pmoles; MgCL, 60 pmoles; EDTA, 0.03 pmoles; NADP, 0.39 pmoles; glucose-6-phosphate dehydrogenase, 0.6 U; ATP, 3 pmoles, and D-glucose, 45 pmoles. The formation of NADPH was followed at 340 nm.

Phosphoglucose Zsomerase (E.C. 5.3.1.9) activity was assayed by the Noltmann method (42). The conversion of fructose-6- phosphate to glucose-6-phosphate was measured by coupling the latter to the glucose-6-phosphate dehydrogenase reaction. The reaction mixture included: Tris-HC1, pH 8.0, 200 pmoles; NADP, 1 pmole; glucose-6-phosphate dehydrogenase, 3 U, and fructose-6-phosphate, 2 pmoles. The production of NADPH was followed at 340 nm.

Phosphofructokinase (E.C. 2.7.1.1 1 ) and pyruvate kinase (E.C. 2.7.1.40) were assayed as described previously (41 ).

Fructose-I, 6-Diphosphatase (E.C. 3.1.3.1 1 ) was assayed by the procedure of Rosen et al. (53). The fructose-6-phosphate formed was converted to glucose-6-phosphate by phosphoglucose isomerase which was coupled to the glucose-6-phosphate dehydrogenase reaction. The reaction mixture contained : glycine buffer, pH 9.5, 120 pmoles; MgCI,, 3 pmoles; NADP, 0.75 pmole; glucose-6-phosphate dehydrogenase, 1.7 U; phosphoglucose isomerase, 2 U; EDTA, 0.6 pmole, and fructose-1, 6- diphosphate, 0.6 pmole. The reduction of NADP was followed at 340 nm.

Fructose-I, 6-Diphosphate Aldolase (E.C. 4.1.2.6) was assayed by the procedure of Rutter et al. (54). The buffer used in this method contained glycylglycine, pH 7.5, 0.1 M, potassium acetate, 0.2 M, and /!-mercaptoethanol, 0.05 M. The reaction mixture contained: buffer, 1.5 ml; NADH, 6 pmoles; a-glycerophosphate dehydrogenase-triose phosphate isomerase mixture, 60 pg, and fructose-1, 6-diphosphate, 4 pmoles. The oxidation of NADH was followed at 340 nm.

Triose Phosphate Zsomerase (E.C. 5.3.1.1 ) was measured according to the procedure of Beisenherz (5 ) . The reaction mixture contained : triethanolamine-HC1, pH 7.9, 100 pmoles; NADH, 0.26 pmole; a-glycerophosphate dehydrogenase, 12 U, and glyceraldehyde-3-phosphate solution, 0.3 ml. The glyceraldehyde phosphate solution used in this assay was prepared from fructose-1, 6-diphosphate through the aldolase reaction, as described by Beisenherz et al. (6 ) . The formation of NAD was followed at 366 nm.

Glyceraldehyde-3-Phosphate Dehydrogenase (E.C. 1.2.1.12) was assayed according to the method of Krebs (36). The reaction mixture was made up of: pyrophosphate, pH 8.5, 60 pmoles; cysteine, 12 pmoles; NAD, 0.76 pmole; sodium arsenate, 20 pmoles, and glyceraldehyde-3-phosphate solution (6) , 0.2 ml. The formation of NADH was measured a t 340 nm.

Phosphoglycerate Kinase (E.C. 2.7.2.3) was measured according to the procedure of Bucher ( 9 ) . The reaction mixture included: potassium-sodium phosphate, pH 6.9, 150 &moles; ADP, 1 pmole; MgSO,, 15 pmoles; glycine, 400 pmoles; glyceraldehyde-3-phosphate dehydrogenase, 5 U; NAD, 1.2 pmoles, and glyceraldehyde-3-phosphate solution (6 ) , 0.25 ml. The formation of NADH was followed at 366 nm.

Enolase (E.C. 4.2.1.11) was assayed according to the method of Bucher (10). The reaction mixture contained: carbonate buffer, pH 7.4, 150 pmoles; glycine, 81 pmoles; MgCI,, 8 pmoles, and ~-~-2-phosphoglycerate, 10 pmoles. The reaction was followed by measuring the formation of phosphoenolypyruvate at 240 nm.

a-Glycerophosphate Dehydrogenase (E.C. 1.1.1.8) was assayed according to the method of Beisenherz et al. (6 ) . The reaction

602 Leishmania: CARBOHYDRATE METABOLISM

mixture contained: triethanolamine-HC1, pH 7.5, 150 pmoles; EDTA, 5.3 pmoles; NADH, 0.4 pmole, and dihydroxyacetone phosphate solution (6 ) , 0.3 ml. The formation of NAD was measured at 340 nm.

a-Glycerophosphate Phosphatase (E.C. 3.1.3.b) was assayed according to the method of Rao & Vaidyanathan (50). The reaction mixture was made up of: sodium-acetate-acetic acid, pH 5.7, 75 pmoles; MgCI,, 20 pmoles; a-glycerophosphate, 6 pmoles; extract, and H,O to make a final volume of 1.5 ml. The reaction mixture was incubated at 30 C for 30 min and then quenched by the addition of 1 ml 70% (V/V) perchloric acid. After removal of the precipitate by centrifugation, the supernate was tested for inorganic phosphate by the method of Fiske & SubbaRow (19).

Lactate Dehydrogenase (E.C. 1.1.1.27) was assayed in both directions, viz. pyruvate to lactate and lactate to pyruvate. Lac- tate formation from pyruvate was measured by the Kornberg (33 ) method. The reaction mixture contained: phosphate buffer, pH 7.4, 100 pmoles; NADH, 0.2 pmole, and sodium pyruvate, 1 pmole. The formation of NAD was followed at 340 nm. Formation of pyruvate from lactate was measured by the Stockland & San Clemente procedure (65). The reaction mixture included: Tris-HCI, pH 7.5, 69.5 pmoles; KCN, 12.6 pmoles; NAD, 3 pmoles, and D-L-sodium lactate, 37.5 pmoles. The formation of NADH was followed at 340 nm.

Lactate dehydrogenase was also assayed by determining lactate formation. A reaction mixture containing 250 pmoles phosphate buffer, pH 7.4; 0.2 pmole NADH; 1.5 pmole sodium pyruvate and extract in a final volume of 3 ml was incubated for 30 min at 30 C in a shaking water bath. At the end of 30 min, the reaction was terminated by adding an equal volume of 10% (w/v) trichloroacetic acid. After removing the precipitate by centrifugation, the supernate was analyzed for lactic acid by the method of Barker & Summerson (3).

Pyruvate Dehydrogenase Complex, consisting of pyruvate dehydrogenase ( E.C. 1.2.4.1 ) , dihydrolipoamide transacetylase (E.C. 2.3.1.12), and dihydrolipoamide dehydrogenase (E.C. 1.6.4.3), was assayed according to the procedure of Schwartz & Reed (58). The reaction mixture included: Tricine, pH 7.8, 200 pmoles; NAD, 10 pmoles; thiamine pyrophosphate, 1 pmole; MgCI,, 3 pmoles; CoA, 1 pmole, and sodium pyruvate, 15 pmoles. The formation of NADH was followed at 340 nm.

Glucose-6-Phosphate Dehydrogenase (E.C. 1.1.1.49) was assayed according to the method of Loehr & Waller (37). The reaction mixture contained: triethanolamine-HCI, pH 7.5, 90 pmoles; EDTA, 12.9 pmoles; NADP, 1.5 pmoles, and glucose- 6-phosphate, 2 pmoles. The reduction of NADP was followed at 340 nm.

6-Phosphogluconate Dehydrogenase (E.C. 1.1.1.44) was assayed by the procedure of Marks (40). The reaction mixture included : glycylglycine, pH 7.5, 125 ymoles; MgCl,, 20 pmoles; NADP, 0.25 pmole, and 6-phosphogluconate, 5 pnoles. The formation of NADPH was followed at 340 nm.

Transaldolase (E.C. 2.2.1.2) was assayed by the method of Tchola & Horecker (66). The glyceraldehyde-3-phosphate formed from fructose-6-phosphate and erythrose-4-phosphate was converted to dihydroxyacetone phosphate by triose phosphate isomerase. The dihydroxyacetone phosphate was then reduced to a-glycerophosphate by coupling with a-glycerophosphate dehydrogenase. The reaction mixture contained: triethanolamine- HCl, pH 7.5, 47 pmoles; EDTA, 6.4 pmoles; a-glycerophosphate dehydrogenase-triose phosphate isomerase mixture, 100 pg; NADH, 0.5 pmole; CaCl,, 0.7 pmole; fructose-6-phosphate, 14

-

pmoles, and erythrose-4-phosphate, 1 ,pmole. The oxidation of NADW was followed at 340 nm.

Transketolase (E.C. 2.2.1.1) was assayed according to the procedure of De La Haba & Racker ( 16). The transketolase reaction generates glyceraldehyde-3-phosphate and sedoheptu- lose-7-phosphate from a mixture of ribulose-5-phosphate and ribose-5-phosphate. When coupled to triose phosphate isomerase and a-glycerophosphate dehydrogenase, glyceraldehyde-3-phosphate is eventually reduced to a-glycerophosphate with the simultaneous oxidation of NADH which is measured at 340 nm. The substrates (mixture of ribose-5-phosphate and ribulose- 5-phosphate) were prepared by treating ribose-5-phosphate with ribose-5-phosphate isomerase. The reaction mixture consisted of: glycylglycine, pH 7.6, 250 pmoles; NAD, 0.3 pmole; a- glycerophosphate dehydrogenase-triose phosphate isomerase mixture, 100 pg; thiamine pyrophosphate, 50 pg; MgCl,, 15 pmoles; ribose-5-phosphate, 13.5 pmoles, and ribose-5-phosphate isomerase, 0.8 U.

Ribulose-5-Phosphate-3-Epime~ase (E.C. 5.1.3.1 ) was assayed according to the method of Racker (49). The xylulose-5- phosphate formed from ribulose-5-phosphate in this reaction was used in a transketolase reaction to yield glyceraldehyde-3- phosphate which was then linked to triose phosphate isomerase and a-glycerophosphate dehydrogenase. Ribulose-5-phosphate was produced in the reaction mixture by adding ribose-5-phosphate and ribose-5-phosphate isomerase. Since crude extracts already contained high levels of transketolase, this enzyme was not added. The assay mixture contained: glycylglycine, pH 7.5, 150 pmoles; MgCl,, 10 pmoles; NADH, 0.75 pmole; triose phosphate isomerase-a-glycerophosphate dehydrogenase, 100 pg; ribose-5-phosphate, 13.5 pmoles, and ribose-5-phosphate isomerase, 0.8 U. The formation of NAD was monitored at 340 nm.

Ribose-5-Phosphate Isomerase (E.C. 5.3.1.6) was assayed according to the method of Eldan & Blum (18). The formation of triose phosphate from ribose-5-phosphate was measured in the presence of transketolase and ribulose-5-phosphate-3-epimerase both of which were present in the crude extract and, therefore, were not added to the reaction mixture. The reaction mixture contained: glycylglycine, pH 7.5, 150 pmoles; MgCl,, 10 ymoles; NADH, 0.75 pmole; tnose phosphate isomerase-a- glycerophosphate dehydrogenase, 100 pg, and ribose-5-phosphate, 3 pmoles. The formation of NAD was followed at 340 nm.

Citrate Synthase (E.C. 4.1.3.7) was assayed according to the method of Srere (63). The reaction mixture contained: Tris- HCI, pH 8.0, 100 pmoles; 5-5’-dithiobis-( 2-nitrobenzoate), 0.1 pmole; oxaloacetate, 0.5 pmole, and acetyl CoA, 0.3 pmole. Acetyl CoA was prepared fresh from CoA following the procedure of Simon & Shemin (61). The formation of the mer- captan ion was followed at 412 nm.

Aconitase (E.C. 4.2.1.3) was assayed according to the method of Racker (48). The reaction mixture consisted of: phosphate buffer, pH 7.4, 140 ymoles, and sodium citrate, 87 pmoles. The formation of aconitate was monitored at 240 nm.

Isocitrate Dehydrogenase was assayed for both NAD and NADP-dependent activity according to the procedure of Siebert (60). For the NADP enzyme (E.C. 1.1.1.42) the assay system contained: Tris-HC1, pH 7.4, 100 pmoles; EDTA, 1 pmole; MnS04, 4 pmoles; NADP, 0.25 pmole, and sodium isocitrate, 5 pmoles. The reduction of NADP was recorded at 340 nm. The assay system for the NAD enzyme (E.C. 1.1.1.41) was the same except that it contained 2.5 pmoles of NAD instead of NADP.

a-Ketoglutarate Dehydrogenase (E.C. 1.2.4.2) was assayed by several procedures (32, 51, 52). The Kaufman procedure (32) employed the following reaction mixture: phosphate

Leishmania : CARBOHYDRATE METABOLISM 603

buffer, pH 7.4, 100 pmoles; L-cysteine, 8.3 pmoles; NAD, 0.27 pmole; CoA, 0.13 pmole, and sodium a-ketoglutarate, 25 pmoles. The formation of NADH was monitored at 340 nm. For the procedure of Reeves et al. (52) the reaction mixture was Tris- HCI, pH 8.5, 500 pmoles; L-cysteine, 7.8 pmoles; CoA, 0.26 pmole; sodium a-ketoglutarate, 25 pmoles, and AP-NAD, 6 pmoles. The reduction of AP-NAD was followed at 365 nm. The reaction mixture used in the Reed & Mukherjee method (51) was: Tris-HCI, pH 8.5, 170 pmoles; thiamine pyrophosphate, 1 pmole; sodium a-ketoglutarate, 10 pmoles; NAD, 6 pmoles; CoA, 3 pmoles, and L-cysteine, 10 pmoles. The formation of NADH was monitored at 340 nm.

Succinate Dehydrogenase (E.C. 1.3.99.1 ) was assayed according to the procedure of Bonner (8 ) . The reaction mixture contained: phosphate buffer, pH 7.2, 300 pmoles; KCN, 30 pmoles; potassium ferricyanide, 3 pmoles, and sodium succinate, 40 pmoles. The reduction of potassium ferricyanide was followed at 400 nm.

Fumarase (E.C. 4.2.1.2) was assayed according to the method of Racker (48). The reaction mixture contained: potassium phosphate buffer, pH 7.4, 100 pmoles, and potassium L-malate, 51 pmoles. The formation of fumarate was followed at 240 nm.

NAD-dependent Malate Dehydrogenase (E.C. 1.1.1.37) was assayed according to the method of Ochoa (43). The reaction mixture contained : glycylglycine, pH 7.4, 75 pmoles; NADH, 0.12 pmole, and oxaloacetate, 0.76 pmole. The oxidation of NADH was measured at 340 nm.

NADP-dependent Malate Dehydrogenase (E.C. 1.1.1.40) was assayed according to the method of Ochoa (44). The reaction mixture contained: glycylglycine, pH 7.4, 75 pmoles; MnCI,, 3 pmoles; NADP, 0.14 pmole, and potassium-L-malate, 1.5 pmoles. The formation of NADPH was followed at 340 nm.

Pyruvate Carboxylase (E.C. 6.4.1.1) was assayed by the method of Scrutton (59). The reaction mixture consisted of: HEPES, pH 7.8, 300 pmoles; ATP, 9.9 pmoles; NaHCO,, 60 pmoles; MgCI,, 20.1 prnoles; NADH, 0.48 pmole; acetyl CoA, 0.26 pmole; NAD-dependent rnalate dehydrogenase, 15 U, and sodium pyruvate 30 pmoles. NADH oxidation was monitored at 340 nm.

Glutamate Dehydrogenase (E.C. 1.4.1.3) was assayed according to the procedure of Schmidt (57). The reaction mixture contained : triethanolamine-HCI, pH 8.0, 100 pmoles; EDTA, 7.8 pmoles; ammonium acetate, 900 pmoles, NADH, 0.5 pmole, and sodium a-ketoglutarate, 6 pmoles. The formation of NAD was followed at 366 nm.

N A D H Oxidase (E.C. 1.6.4.3) was measured according to the procedure of Mahler (39). The reaction mixture contained: Tris-HCI, pH 7.5, 60 pmoles; 2,6-dichlorophenol-indophenol, 0.12 pmole; NADH, 0.6 pmole, and KCN, 1 pmole. The reduction of 2,6-dichlorophenol-indophenol was followed at 600 nm.

RESULTS

Enzymes of the Embden-Meyerhof Pathway

It is evident from the data presented in Table 1 that cell-free extracts of Leishmania spp. contain all enzymes of the Embden- Meyerhof pathway.

Lactate dehydrogenase activity could not be demonstrated by assays involving the reactions leading from pyruvate to lactate and those proceeding in the opposite direction, i.e. from lactate to pyruvate, using NAD and its acetylpyridine derivative, AP- NAD. Attempts to demonstrate a flavin-linked dehydrogenase, as in bacteria such as Pseudomonas natriegens [= Beneckea natriegens, (4) ] (68) also failed. Negative results from these

TABLE 1. Enzymes of the Embden-Meyerhof pathway.

Specific activity*

Enzyme L. brasi- L. L. L.

liensis donovani mexicana tropica

Hexokinase PhPsphoglucose

isomerase Phosphofructokinase Fructose- 1,6-di-

phosphatase Fructose-1,6-di-

phosphate aldolase Triose phosphate

isomerase Glyceraldehyde-3-

phosphate dehydrogenase

3-Phosphoglycerate kinase

Enolase Pyruvate kinase Lactate dehydrogenase L-a-glycerophosphate

dehydrogenase Glycerophosphate

phosphatase

250.1

414.5 60.0

3.6

214.5

186.7

9.7

6.4 47.0 14.5 NDt

16.9

31.7

306.8

217.3 7.2

13.5

79.3

67.9

2.9

4.6 34.2 54.3 ND

16.9

45.0

66.7

256.5 9.9

3.2

47.3

57.1

1.8

2.8 36.9 75.0 ND

7.5

16.0

537.2

172.7 33.2

8.5

191.7

35.6

3.1

3.1 16.6

342.6 ND

5.8

23.0

* Nanomoles of substrate converted/min/mg protein. t Not detected.

spectrophotometric assays were supported by the lack of lactic acid formation when cell-free extracts were incubated with pyruvate along with the standard reagents of the lactate dehydrogenase assay. Deproteinized samples of extracts of cells at different times during growth also failed to show any lactic acid. To substantiate these findings further, the possibility of lactic acid being excreted into the medium during growth was examined. Three species of Leishmania have been previously reported to accumulate lactic acid in the medium (12). The un- inoculated medium we used contained 480-498 pg/ml lactic acid. No increase of lactic acid, however, was noted in the medium after growth of the promastigotes. The absence of lactate dehydrogenase is compensated by the presence of L-a-glycerophosphate dehydrogenase and a-glycerophosphate phosphatase (for details, see Discussion). Fructose-l,6-diphosphatase, necessary for the reversal of the Embden-Meyerhof pathway also was demonstrated.

Enzymes of the Hexose Monophosphate Pathway

Enzymes of both oxidative (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) and nonoxidative

TABLE 2. Enzymes of the hexose monophosphate pathway.

Specific activity* L. brasi- L. L. L.

Enzyme liensis donovani mexicana trojica

Glucose-6-phosphate

6-Phosphogluconate

Ribulose-5-phos-

Ribose-5-uhosuhate

dehydrogenase 69.3 31.5 106.3 75.5

dehydrogenase 19.6 3.9 43.5 4.8

phate-3-epimerase 22.5 9.4 6.0 4.2 _ - - - - a ~~.._.. ~

isomerase Transaldolase Transketolase

5.7 3.5 3.6 6.8 817.0 324.3 136.3 417.5

7.2 8.3 12.4 6.0

* See note to Table 1.


TABLE 3. Activity o f pyruvate dehydrogenase complex.

Specific Organism activity*

L. brasiliensis L. donovani L. mexicana L. tropica

2.8 2.2 1.4 7.7

* See note to Table 1.

( ribulose-5-phosphate-3-epimerase, ribose-5-phosphate isomerase, transaldolase and transketolase) reactions of the hexose monophosphate pathway were found in Leishmania (Table 2).

Of the enzymes studied, glucose-6-phosphate dehydrogenase was quite active, and oxidation rate of glucose-6-phosphate by the dehydrogenase remains linear for at least 10-12 min under the assay conditions employed in this study. On the other hand, activities of transketolase and 6-phosphogluconate dehydrogenase declined very rapidly.

Oxidation of Pyruvate The pyruvate dehydrogenase complex which converts pyruvate

to acetyl CoA was found in cell-free extracts (Table 3). This complex ensures further metabolism of pyruvate through the tricarboxylic acid cycle. Only the overall activity of pyruvate dehydrogenase was demonstrated by the assay employed; no efforts were made to identify the components of this multi- enzyme complex. Addition of various concentrations of lipoic acid and FAD, cofactors of the enzyme complex, had no effect on enzymic activity.

Enzymes of Tricarboxylic Acid Cycle All enzymes of the tricarboxylic acid cycle have been identi-

fied in Leishmania (Table 4 ) . Isocitrate dehydrogenase is NADP-linked; no NAD-linked activity was detected. High levels of NAD-linked malate dehydrogenase activity were recognized in all species but by comparison the levels of succinate dehydrogenase and a-ketoglutarate dehydrogenase were low. The activity of a-ketoglutarate dehydrogenase was particularly difficult to demonstrate. No activity was detected by the procedures of Kaufman (32) and Reeves et al. (52), using both NAD and AP-NAD. The latter compound is less susceptible

TABLE 4. Enzymes of the tricarboxylic acid cycle.

Specific activity" L. brasi- L. L. L.

Enzyme liensis donovani mexicana tropica

Citrate synthase 0.29 0.05 0.11 0.04 Aconitase 41.5 38.8 23.3 55.9 NAD-linked isocitrate

dehydrogenase NDt ND ND ND NADP-linked isoci-

a-Ketoglutarate dehydrogenase 0.8 1.5 1.6 1.5

Succinic dehydro-

Fumarase 44.4 37.3 44.9 32.3 NAD-linked malate

dehydrogenase 132.0 238.0 61.8 372.3 NADP-linked malate

dehydrogenase 202.6 44.5 130.6 28.4

trate dehydrogenase 28.5 115.2 80.1 35.3

genase 2.8 1.6 0.7 1.2

*t See notes to Table 1.

TABLE 5 . Glutamate dehydrogenase activity.

Specific activity* Organism

L. brasiliensis 18.5 L. donovani 21.7 L. mexicana 12.9 L. tropica 21.8

See note to Table 1.

to NADH oxidase and thus has an advantage over NAD. The enzyme was, however, demonstrated when the extracts were assayed according to the procedure of Reed & Mukherjee (51) in which thiamine pyrophosphate (TPP) is used in the reaction mixture. I t was found that TPP is an in vitro activator of a-ketoglutarate dehydrogenase from L. tropica. Optimum results were attained in the presence of 0.3 mM TPP, and it was demonstrated that the a-ketoglutarate dehydrogenase in Leish- mania spp. has an absolute requirement for TPP.

Glutamate Dehydrogenase

An active glutamate dehydrogenase is present in all 4 species of Leishmania studied (Table 5). This enzyme reduces a-ketoglutarate to glutamic acid as follows:

a-ketoglutarate + NH, + NADH glutamic acid + NAD.

Thus, glutamate dehydrogenase shares a common substrate with a-ketoglutarate dehydrogenase. The enzyme in Leishmania is NAD-specific; its activity could not be demonstrated with NADP. In this attribute, it differs from the corresponding enzymes from the liver fluke, Fasciola hepatica (47), and from livers of van- ous other animals (17) in which both NAD- and NADP-linked activities have been found.

Enzymes of Heterotrophic CO, Fixation

Since C4 acids of the tricarboxylic acid cycle are drawn off as biosynthetic precursors, most heterotrophs have one or more mechanisms for replenishing them. This is frequently accomplished by fixing CO, into either pyruvate or phosphoenolpyru- vate (PEP). Cell-free extracts of Leishmania spp. possess pyruvate carboxylase (Table 6), which catalyzes the formation of oxaloacetate by fixing COP into pyruvate. The NADP-specific malic enzyme capable of generating malate by carboxylating pyruvate could not be demonstrated in any of the species.

N A D H Oxidase

As is true of most aerobic organisms, Leishmania spp. possess an active NADH oxidase (Table 7). Although this enzyme is not directly concerned with carbohydrate metabolism, deter- mination of its activity was relevant since it frequently inter- feres in the assay of other NADH-dependent enzymes. Appro-

TABLE 6. Enzymes of heterotrophic CO, fixation.

Specific activity* L. brasi- L. L. L.

Enzyme liensis donovani mexicana tropica

Pyruvate carboxylase 1.3 2.8 3.7 2.2 Malic enzyme NDt ND ND ND

*t See notes to Table 1.

Leishmania: CARBOHYDRATE METABOLISM 605

TABLE 7 . NADH oxidase activity.

Specific Organism activity*

L. brasiliensis L. donovani L. mexicana L. tropica

289.9 351.3 331.3 272.8

See note to Table 1 .

priate corrections were made to avoid exaggerated values and false positive results.

DISCUSSION

As mentioned earlier, the occurrence of the Embden-Meyerhof pathway, the hexose monophosphate shunt and the tricarboxylic acid cycle, is generally assumed in most leishmania1 parasites (7, 21). No single species, however, was systematically examined and shown to bear out these assumptions. Results from our comparative study of 4 species of Leishmania support these generalizations. Except for the variations in the specific activities of individual enzymes in different species, no significant differences were noted among them with respect to the pathways of carbohydrate metabolism. Several possibilities could account for the wide variations in specific activities of certain enzymes examined in the course of this study. There is considerable difficulty in standardizing the age and physiologic state of cells of different species in batch cultures which were used for the preparation of cell-free extracts. As reported earlier (41 ), variations in specific activity depending upon the age of the cells even within the same species have been shown at least with regard to phosphofructokinase and pyruvate kinase. This may reflect differential rates of protein turnover in vivo. Also, one cannot overlook the possibility of differential rates of proteolytic activity in crude cell-free extracts from different species which can account for differential rates of decay of specific enzymes. An equally important consideration is the fact that promastigotes of Leishmania are eukaryotic with internal compartmentalization due to the presence of organelles. The procedure of rupturing cells by sonication used in the present study did not ensure dis- ruption of all organelles; thus considerable inconsistency in the degree of structural damage could be expected. This would in- variably result in significant variation in the extraction of com- partmentalized enzymes, as are many enzymes of the TCA cycle. The emphasis in this study was primarily on qualitative rather than quantitative aspects. No effort was made to localize the intracellular distribution of various enzymes, whether soluble or bound. Negative results, however, were checked using both cytosol and particulate fractions.

The presence of the 3 irreversible enzymes in the Embden- Meyerhof pathway, hexokinase, phosphofructokinase, and pyruvate kinase, indicates that as in most other organisms where this pathway is operative, the leishmanias also have the steps at which this pathway is regulated. These are well established points of allosteric regulation of this pathway (55, 64) and play a crucial role in controlling carbohydrate metabolism. In a previous communication, we have shown that as in most other organisms, the pyruvate kinase of L. tropica is regulated by AMP and fructose-1, 6-diphosphate (FDP) both of which activate the enzyme (41 ). Lactate dehydrogenase could not be demonstrated in any of the species investigated. The results were always negative although the enzyme was assayed in both directions using NAD, AP-NAD, and flavin mononucleotide as cofactors. A

number of lactate dehydrogenases are now known to be allo- sterically activated by FDP (69). Even when assayed in the presence of various concentrations of FDP, the results were con- sistently negative. Bacchi et al. (2 ) reported the extreme lability of lactate dehydrogenase from Trypanosoma conorhini and Crithidia fasciculata which was prevented by the presence of L-cysteine. Inclusion of 10 mM cysteine in the saline during the preparation of cell-free extracts and in the assay mixtures, also failed to show any lactate dehydrogenase activity. Krassner (34) had previously reported the absence of this enzyme in L . donovani and L . tarentolae.

In organisms with a complete glycolytic pathway, the lactate dehydrogenase mediated reduction of pyruvate to lactate is an important mechanism for the regeneration of NAD. The ap- parent lack of lactate dehydrogenase prompted the search for an alternate route for the reoxidation of NADH generated during the oxidation of 3-phosphoglyceraldehyde. This could be accomplished through the glycerophosphate dehydrogenase reaction in which dihydroxyacetone phosphate, a product of the aldolase reaction, could assume the role of the hydrogen ac- ceptor and become reduced to glycerol phosphate, which could subsequently be hydrolyzed to yield glycerol and inorganic phosphate. Whether this pathway plays any role in the physiology of Leishmania promastigotes or not, has not been determined although it is reported to be functional in Trypanosoma hippicum (28) and Trypanosoma rhodesiense (25, 26). It is conceivable that in cells grown aerobically (as were those in the present work), this pathway is of negligible importance, since reoxidation of NADH could be achieved through the terminal electron transport chain. Glycerophosphate dehydrogenase, however, is involved in the aerobic utilization of glycerol by whole cells

The ability of various species of Leishmania to utilize substrates such as citrate, a-ketoglutarate, and succinate as well as the identification of certain enzymes of the TCA cycle led to the general acceptance of a functional TCA cycle in these organisms (7, 21 ). I t should be noted, however, that the ability to utilize intermediates of the TCA cycle does not necessarily indicate the occurrence of a complete cycle, since they could be used as precursors of compounds, such as amino acids, not involved in the TCA cycle. Positive proof for a complete TCA cycle in Leishmania was lacking, since a-ketoglutarate dehydrogenase, a crucial enzyme in the cycle, was not demonstrated in any species. The accumulation of succinate reported in many species (67) could easily be accounted for by the carboxylation of pyruvate to oxaloacetate and a reversal of malate dehydrogenase, fumarase, and succinate dehydrogenase activities. Such a pathway exists in a number of invertebrates (29). Pyruvate entering the TCA cycle as acetyl CoA could reach a-ketoglutarate which could then be converted to glutamic acid through the glutamate dehydrogenase present in all 4 species examined during this study. Thus, even if a-ketoglutarate dehydrogenase were lacking, one could explain the utilization of a number of intermediary metabolites. The presence of a-ketoglutarate dehydrogenase, however, has been clearly established (present study) providing direct evidence for a complete TCA cycle. The difficulty of demonstrating this enzyme in cell-free extracts in the absence of TPP suggests the possibility that in organisms where this enzyme is thought to be absent, inclusion of TPP in the reaction mixture might reveal its presence. Glutamate dehydrogenase and a-ketoglutarate dehydrogenase would appar- ently compete for the common substrate a-ketoglutarate. Until more is known about the controls of these 2 enzymes in Leish- mania, it is not possible to predict whether a-ketoglutarate is

(71).


further oxidized through the TCA cycle or if it is reduced to glutamic acid. I t must be recognized, however, that glutamate dehydrogenase could also oxidize glutamic acid to a-ketoglutarate thus providing TCA cycle intermediates. This is quite relevant for Leishmania grown in complex media rich in amino acids and other reduced substrates. Thus, the a-ketoglutarate dehydrogenase and glutamate dehydrogenase may in fact com- plement each other rather than compete for a common substrate. Although an active isocitrate dehydrogenase is present, it is NADP-specific (30; present work). No NAD-linked activity was detected, as is also the case in Trypanosoma cruzi ( l ) , Fasciola heflatica (47) , as well as in the nematodes Ascaris lum bricoides (45) and Trichinella spiralis (24) .

The principal enzyme for heterotrophic CO, fixation in all 4 species of Leishmania is pyruvate carboxylase. CO, fixation was demonstrated in cell-free extracts when PEP was used instead of pyruvate. It should, however, be noted that crude extracts contain an active pyruvate kinase which rapidly converts PEP to pyruvate. It was, therefore, not possible to establish whether there is also a PEP carboxylase. We could not find any evidence of CO, fixation through the malic enzyme which mediates the carboxylation of pyruvate to malate. This enzyme is reversible in most mammalian systems but in a number of bacteria it functions only in the direction of malate to pyruvate (70) . In the species of Leishmania examined in the present study, the enzyme mediating the reaction from malate to pyruvate was found but not one catalyzing the reaction from pyruvate to malate. Thus, this NADP-linked enzyme can only be regarded as a dehydrogenase carrying out an oxidative decarboxylation of malate to pyruvate. C02 fixation through the malic enzyme could not be confirmed even in L. donovani in which it was reported by Chatterjee & Datta (15) . I t should, however, be pointed out that Chatterjee & Datta assayed the reaction from malate to pyruvate but failed to demonstrate the reaction in the opposite direction. Therefore, no evidence of carboxylation of pyruvate to malate by the malic enzyme is indicated by their results but rather only the decarboxylation of malate to pyruvate as we have observed in the present study.

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Enzymes of Carbohydrate Metabolism in Four Human Species of Leishmania: A Comparative Survey

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