Molecular and Biochemical Parasitology, 4 (1981) 29- 38 Elsevier/North-Holland Biomedical Press

29

PROPERTIES OF ~-HYDROXYACID DEHYDROGENASE ISOZYMES FROM TR YPANOSOMA CR UZI

CARLOS CORONEL, LEONOR E. ROVAI, NELIA M. GEREZ DE BURGOS, CARLOS BURGOS and ANTONIO BLANCO

Cdtedra de Qu[mica Biol6gica, Facultad de Ciencias M~dicas, Universidad Nacional de C6rdoba, 5000 C6rdoba, Argentina

(Received 5 February 1981; accepted 1 April 1981)

Whole cell extracts of culture epimastigotes of Trypanosoma cruzi (Tulahu~n strain) have a-hydro- xyaeid dehydrogenase activity wkieh catalyzes the NAD-linkod reaction a-ketoacid ~ a-hydroxyaeid, with a variety of substrates. Two molecular forms of the enzyme have been separated by means of gel electrophoresi~ These isozymes were partially purified by DEAE-CelIuloso 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 substrate~ The lowest K m 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 K m value were recorded with a- keto-isoeaproate as substrate.

Key words: Trypanosoma cruzi, a-Hydroxyacid dehydrogenase, lsozymes.

INTRODUCTION

Previous work in this laboratory had demonstrated ot-hydroxyacid dehydrogenase

activity in whole cell extracts of epimastigotes o f Trypanosoma cruzi [1] . The enzyme

catalyzes the NADqinked reaction a-ketoacid ~ a-hydroxyacid with a variety o f linear

or branched carbon chain substrates. For some of these, like pyruvate and ot-ketoiso-

caproate, curves obtained by plott ing enzymic activity o f crude extracts against concen-

trat ion o f substrate were hyperbolic. For a-ketobutyrate , 0t-ketovalerate, a-ketoisovale-

rate and a-ketocaproate, the curves showed a bimodal character and two different values

of K m and V could be calculated [1]. This atypical behaviour suggested the possible

existence, in crude extracts of the parasite, o f more than one enzyme with similar or over-

lapping substrate specificity.

This possibility was confirmed by means of gel electrophoresis, which demonstrated

two molecular forms showing different substrate affinity and heat stabili ty [2]. The two

0166-6851/81/0000-0000/$02.75 © 1981 Elsevier/North-Holland Biomedical Press

30

forms could also be separated by column chromatography on DEAE cellulose [3], a method which allowed purified enzymes to be obtained.

This paper presents studies on physico-chemical properties of the two isozymes of a-hydroxyacid dehydrogenase from T. cruzi.

MATERIALS AND METHODS

Organisms. Epimastigotes of T. cruzi (Tulahu6n strain) were cultivated in a biphasic medium composed of: a) Solid phase: 5 g bovine liver paste; 5 g beef heart proteo- lysate; 3 g beef meat extract (MC 6); 10 g casein (acid hydrolysate MC 7); (all these products were obtained from INORPSA, Buenos Aires, Argentina); 5 g NaC1; 0.4 g KC1; 20 g agar (Soriano, Buenos Aires, Argentina); 10 g disodium phosphate; 0.02 g hemin; distilled water to 1000 ml. (b) Liquid phase. 30 g tryptose-phosphate broth (Difco, U.S.A.); 10 g glucose; 5 g bovine liver paste; 5 g beef heart proteolysate; 0.02 g hemin; and distilled water to 1000 ml. Parasites were collected from the liquid phase after 5 days of culture. At this stage, the growth is exponential. The liquid phase was centri- fuged at 3000 X g for 15 min and the pellet washed twice with about 10 vols. of 0.25 M sucrose solution containing 5 mM KC1.

Homogenates. The pellet of washed parasites was suspended in 4 vols. of 0.25 M sucrose/ 5 mM KC1 solution and homogenized with the microattachment of an Omni-mixer Sorvall at 30 000 rpm for 3 min at 4°C. The disruption of parasites was monitored by micro- scopic examination of the resulting suspension. The preparation was frozen a t - 2 0 ° C until experiments were performed (usually 16-24 h later). There was no loss of activity in the homogenates after storage at -20°C for up to 2 months. Immediately before study, the suspension was thawed and centrifuged at 20 000 X g for 20 min at 4°C. The supernatant was used for the analyses.

Electrophoresis. Starch gel electrophoresis was performed with a vertical device [4], using the Tris-borate-EDTA buffer system described by Markert and Faulhaber [5]. Disc gel electrophoresis on polyacrylamide gel was performed as described by Davis [6]. Acryl- amide concentration for the ffme pore gel was 6 g% instead of 7.5 g%. A 0.3-ml sample containing about 2 mg of protein and a drop of saturated sucrose solution was inserted directly on the spacer gel. After the run, gels were submerged in a staining solution similar to that used by Blanco et ai. [7] for lactate dehydrogenase, substituting different a-hydroxyacids for lactate in the mixture.

Enzyme assay. The reagent mixture contained, in a final volume of 3 ml: 0.115 mM NADH; 100 mM sodium phosphate buffer pH 7.4; a-ketoacid as neutral sodium salt (concentrations indicated in Results); enzyme preparation, diluted with sodium phos- phate buffer 0.1 M (pH 7.4) in order to obtain an absorbance change at 340 nm of 0.050-0.080 per min with a 5 mM concentration of substrate. Assays were incubated

31

at 37°C in a thermostat-controlled cuvette holder. Change in absorption at 340 nm was recorded during a 4-min period. One unit of enzyme is defined as the amount con- verting 1/a_rnol NAD per rain in the assay conditions.

Purification of molecular form~ Partial purification of the molecuhr forms of ot-hydroxy- acid dehydrogenase was performed by DEAE cellulose column chromatography. About 18 ml of parasite extract containing 3.5 mg of total protein per ml were added to a column (19 cm X 1.7 cm diameter) previously equilibrated with 0.01 M sodium phos- phate buffer (pH 7.4). Elution was performed with a stepwise pH and NaC1 gradient by adding successively 45 ml of each of the following solutions: (I) 0.01 M sodium phos- phate buffer (pH 7.4); (II) 0.01 M sodium phosphate buffer (pH 7.2) with 1 mM NaC1; (III) 0.01 M sodium phosphate buffer (pH 7.0) with 2 mM NaC1; (IV) 0.01 M sodium phosphate buffer (pH 6.8) with 3 mM NaCI; (V) 0.01 M sodium phosphate buffer (pH 6.6) with 4 mM NaC1; (VI) 0.01 M sodium phosphate buffer (pH 6.4) with 5 mM NaC1; and (VII) 0.01 M sodium phosphate buffer (pH 6.2) with 10 mM NaCI. Effluents were collected in 4-ml fractions. All tubes of eluates corresponding to one peak of enzyme activity were pooled. The enzyme was then precipitated by addition of ammonium sulfate to 70% saturation, sedimented by centrifugation for 20 min at 10 000 X g, and finally redissolved in a small volume of 0.1 M sodium phosphate buffer, pH 7.4. All operations were conducted at 4°C.

Determination of molecular size. Molecular weights of enzymes in crude extracts and purified preparations was estimated following the method of Hedrick and Smith [8]. The molecular weight-slope relation was established by utilizing the following protein standards: ovalbumin, lactate dehydrogenase (from beef heart) and human serum albumin (Sigma, St. Louis, MO, U.S.A.).

Chemicals. NAD, NADH, pyruvate (2-oxopropanoate), a-ketobutyrate (2-oxobutanoate), t~-ketovalerate (2-oxopentanoate), a-ketoisovalerate (2-oxo-3-methylbutanoate), t~-keto- /3-methylvalerate (2-oxo-3-methylpentanoate), a-ketoisocaproate (2-oxo-4-methylpen- tanoate), DL-a-hydroxybutyrate (DL-2-hydroxybutanoate), DL-a-hydroxyvalerate (DL- 2-hydroxypentanoate), D L-ot-hydroxy-/~-methylvaler ate (DL-2-hydroxy-3-methylpen- tanoate), all as sodium salts, L-lactate (2-hydroxypropanoate) as lithium salt, ot-keto- caproic (2-oxo-hexanoic), DL-a-hydroxyisovaleric (DL-2-hydroxy-3-methylbutanoic), DL-ot-hydroxycaproic (DL-2-hydroxyhexanoic), DL-a-hydroxyisocaproic (DL-2-hydroxy- 4-methylpentanoic), as free acids, were purchased from Sigma (St. Louis, MO, U.S.A.). The free acids were neutralized with NaOH solution immediately before they were added to the reagent mixture.

32

RESULTS

Electrophoretic patterns. Starch gels stained by using a-hydroxyvalerate as substrate revealed two zones of enzymic activity which move toward the anode (Fig. 1C). The fastest migrating component appears as a single band, while the slow region comprises multiple sub-fractions. When lactate and a-hydroxybutyrate were used as substrates in the staining mixture, only the fast band appeared (Fig. 1A and B). The slow region showed up as a light dye precipitate when the staining with a-hydroxybutyrate was prolonged for 3 h or more. Staining with a-hydroxyisocaproate reveals exclusively the slow region, composed of one band intensely stained and two minor subfractions migrat- ing immediately in front of it (Fig. 1E). These bands are also, but less intensely, stained with a-hydroxycaproate (Fig. 1D), a-hydroxyisovalerate and a-hydroxy-/~-methylvalerate as substrates. The three subfractions of the slow region showed identical substrate specifi- city, as judged by the electrophoretic patterns developed with different a-hydroxyacids.

The results obtained using polyacrylamide gels were similar to those described for

starch gel electrophoresis. The fast band will be designated isozyme I, and the slow complex will be considered

as one component and designated isozyme II. Successive studies with the same preparation showed a progressive increase in the

Fig. 1. Starch gel electrophoretic patterns of a-hydroxyacid dehydrogenase from total extracts of T. cruzi. Stained with (A) lactate as substrate; (B) a-hydroxybutyrate; (C) a-hydroxyvalerate; (D) t~-hydroxyeaproate; (E) a-hydroxyisocaproate. Roman numerals on the fight indicate the position of the corresponding isozyme.

33

relative activity of the minor bands as time of storage was prolonged, suggesting that these minor bands may originate from the principal one.

Chromatography. Figure 2 presents the elution profiles for total protein and for ot-hydro- xyacid dehydrogenase activity assayed with 5 mM ,~-ketoisocaproate and 5 mM ot-keto- butyrate as substrates. Two peaks of activity are clearly separated. The first was collected in tubes 18-32, and the second came out between fractions 35 and 44. In the particular experiment represented in Fig. 2, tubes 21-27 and tubes 36-43 were pooled separately in order to proceed with the ammonium sulfate precipitation. Specific activity of the enzyme recovered from the first pool was 3.0 U per mg of protein, assayed with 5 mM t~-ketoisocaproate as substrate and that for the second pool was 1.1 U per mg of protein, assayed with 5 mM o~-ketobutyrate.

When analyzed by electrophoresis and stained for enzymic activity, the protein col- lected in the first peak exhibited the complex described as isozyme II, e.g., a main band and two additional fractions migrating immediately in front. The enzyme obtained from the second peak showed the fast band or isozyme I. In both cases, there was no contami- nation with the other molecular form.

Catalytic properties. By plotting initial activity of purified enzyme against concentration

3 6 0 -

.50¢

E =o O4 .24o-

.o6o

I II III IV V Vl

/9 11

I •

5 I0 15 20 25 30 55 40 45 50

TUBE NUMBER

E B v

.120 )--

_>

.o9o '~

>.- .060 ~1

ul

030

Fig. 2. DEAE-cellulose column chromatography of total extracts of T. cruzL Profiles of 0--% pro- tein; e - - e . c~-hydroxyacid dehydrogenase activity assayed with 5 mM o~ketoisocaproate; - - - - , a-hydroxyacid dehydrogenase activity assayed with 5 ram c~-ketobutyrate. Arrows on top indicate the changes of eluting solution. Roman numerals correspond with those of the solutions described in Materials and Methoda

34

of substrate, the curves shown in Figs. 3 and 4 were obtained. All curves were hyperbolic and there was no inhibition by substrate up to 5 mM concentration. Values of apparent K m and V calculated from these data, as well as values of V/K m ratios, which give an indication of relative catalytic efficiency with different substrates, are presented in Table I. Isozyme I showed very low activity with branched chain a-ketoacids of 5 and 6 carbon atoms, or with a-ketocaproate. Although the maximum velocity of isozyme I with pyruvate is relatively poor, the Km for this substrate is very low, and the value of V/KM

ratio is higher than those for a-ketobutyrate and a-ketovalerate. Isozyme II is strikingly active against ct-ketoisocaproate and showed practically no activity with pyruvate. Both isozymes use specifically NAD as coenzyme; activity with NADP was less than 10% of that obtained with NAD. The reverse reaction (a-hydroxyacid ~ ot-ketoacid)is catalyzed by both isozymes. Although D-a-hydroxyacids were not assayed, it is likely that L-iso- mers are preferred, since activity with L-substrates was always higher than that with

racemic (D L) compounds.

Molecular size. Plots of log (R m X 100) against acrylamide concentration gave different slops for isozymes I and II indicating that they differ in net charge and in molecular size (Fig. 5). The three subfractions comprising isozyme II were identical from the point of view of molecular size. By using ovalbumin (MW 45 000), lactate dehydrogenase (from

0 .3 -

i=

_ . 0 . 2 - > I -

_o Z

0,1

I I I I I .I .2 .5 1.0 2 . 0 5 .0

S U B S T R A T E C O N C E N T R A T I O N (raM)

Fig. 3. Initial activity of purified c~-hydsoxyacid dehydrogenase isozyme I from T. cruzi plotted against substrate concentration, e - - e , ~-ketobutyrate; A_..~, ~-ketovalerate; o - - % pyruvato: Concentrations of substrate used were 0.1, 0.2, 0.5, 1.0, 2.0 and 5.0 raM. All points represent activity of the same amount of enzyme~

35

4O

>,

,< ----- 20

5o!

/ t

/ .

/ s n

• o . /

. , .2 .5 ,~ 2.o ~!o SUBSTRATF CONCENTRATION (mM)

Fig. 4. Initial activity of purified ~,-hydroxyacid dehydrogenase isozyme II from Z cruzi plotted against substrate concen t r a t ion . . - - e , ceketoisocaproate; ~--A, ¢x-ketocaproate; m---.-m, a-ketovalerate; 0--% a-ketobutyrate; v---o, a-ketoisovalerate; ~-...-a, ~-keto-~-methylvalerate. ConcentratiOns of substrate used were 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 and 5.0 nuM. All points represent activity of the same amount of enzyme.

TABLE I

Apparent K m and V values for a-hydroxyacid dehydrogenase isozymes from Trypanosoma cruzi.

Substrate K m //" V/K m (mM) (U/ml)

Isozyme I

Pymvate 0.05 0.05 1 a-Ketobutyrate 0.47 0.36 0.76 ~,-Ketovalerate 0.5 0.066 0.13

Isozyme II

a-Ketobutyrate 5.0 0.142 0.028 a-Ketovalorate 2.0 0.48 0.24 a-Ketoisovalerate 1.1 0.035 0.032 a-Keto-~-me thylvaler ate 1.0 0.032 0.032 ~-Ketocaproate 0. 8 0.96 1.2 ¢~-Ketoisocaproate 0.4 0.99 2.48

36

19c

18C

17C 0

g ~60

cS _o 150

140

,91 I I I I / 3 4 5 6 7 B

GEL CONCENTRATION %

Fig. 5. Effect of different gel concentra t ions on the electrophoret ic mobil i ty of ~ h y d r o x y a c i d dehy- drogenase, isozyme I (o) and isozyme II (*). Negative slopes were 0.85 for isozyme I and 0.7 for

isozyme IL

beef heart) (MW 135 000), human albumin monomer (MW 66 500) and trirner (MW 199 500) as standard proteins, the molecular weight estimated from the slope-molecular weight relation was 85 500 for isozyme I and 60 500 for isozyme II.

DISCUSSION

Data reported here confirm our previous assumption [1, 2] that a-hydroxyacid dehydrogenase from extracts of epimastigotes of T. cruzi (Tulahu6n strain) is composed of multiple molecular forms with overlapping substrate specificity. This finding explains the irregular catalytic behaviour of the enzyme in crude preparations [ 1 ]. Two isozymes have been separated and partially purified. Both forms catalyze the NAD-linked conver- sion, a-ketoacid ~ a-hydroxyacid, but they differ in molecular weight and affinity for

substrates. Isozyme I appears as a single component, as judged by electrophoretic and chromato-

graphic criteria. It is active against 3 -5 carbon atom linear chain substrates. Although its highest activity was recorded with a-ketobutyrate, the lowest Km was obtained with pyruvate as substrate. In fact, the K m for pyruvate is of the same order of magnitude as that of lactate dehydrogenase isozyme 1 (B4 or H4) and isozyme X (C4) from many mammalian species [9]. Isozyme I must be responsible for the weak lactate dehydro- genase activity found in T. cruzi extracts [10]. However, since lactate does not appear to be a quantitatively important metabolic product in the parasite, it is possible that the

37

enzyme mostly operates in the direction of lactate oxidation, enabling the organism to utilize exogenous lactate as an energy source.

The slow migrating fraction (isozyme II) showed maximum activity and lowest Km with ot-ketoisocaproate as substrate. It did not show activity against pymvate. Isozyme

II appeared heterogeneous after electrophoresis. The subfractions exhibited identical substrate specificity and molecular size. It is possible that the minor bands migrating

immediately in front of the main fraction are 'satellite' bands originated from the princi- pal one. Elucidation of this point requires additional studies. The a-ketoisocaproate, which apparently is the best substrate for isozyme II, is a physiological metabolite derived from leucine by transamination or oxidative deamination. Previous studies in this labora- tory demonstrated the existence of leucine aminotransferase in T. cruzi extracts [11]. This enzyme, at variance with that found in higher organisms [12, 13], has no activity with the other branched chain amino acids (isoleucine and valine). This correlates with the relative low activity of isozyme II against wketoacids derived from these amino acids (a-keto-/3-methylvalerate and a-ketoisovalerate).

It is interesting to note that the lactate dehydrogenase isozyme X (LDH X or C4) found in spermatozoa of many species of mammals and birds [9] shows a substrate spectrum similar to that of the a-hydroxyacid dehydrogenase from T. cruzi [14]. Find- ings by different groups of investigators indicate that LDH X may accomplish very specific functions in spermatozoa [15-17] and it has been proposed that it must be integrated in metabolic pathways providing energy for the motility of the flagellum. Whether the a-hydroxyacid dehydrogenase performs in T. cruzi a role similar to that of LDH X in spermatozoa remains to be elucidated.

ACKNOWLEDGEMENT

This work was supported in part by research grants from the Programa Nacional de Enfermedades End6micas, Secretaria de Estado de Ciencia y Tecnologla (SECYT) of Argentina. N.M.G. de B, C.B. and A.B. are Career Investigators of the Consejo Nacional de Investigaciones Cientfficas y T6cnicas (CONICET) of Argentina. C.C. is a Fellow from the CONICET.

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14 Blanco, A., Burgos, C., Gerez de Burgos, N.M. and Montamat, E.F. (1976) Properties of the testicular lactate dehydrogenase isoenzyme. Biochem. J. 153, 165-172.

15 Storey, B.T. and Kayne, F.J. (1977) Energy metabolism of spermatazoa. VI. Direct intramito- chondrial lactate oxidation by rabbit sperm mitochondria. Biol. Repro& 16, 549-566.

16 Van Dop, C., Hutson, S.M. and Lardy, H.A. (1977) Pyruvate metabolism in bovine epididymal spermatozoa. J. Biol. Chem. 252, 1303-1308.

17 Blanco, A. (1980) On the functional significance of LDH X. Johns Hopkins Med. J. 146, 231-

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