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Enzymes of coccidid: Purification and properties of l -lactate dehydrogenase from Eimeria stiedae
EXPERIMENTAL
PARASITOLOGY
Enzymes
%?, 390-402
of Coccidia: Purification Dehydrogenase from John
Regional
(1972)
Parasite
Agricultural
C. Frandsen
and
and Properties Eimeria stiedae’ Judith
of L-Lactate
A. Cooper
Research Laboratory, Veterinary Sciences Research Divbion, Research Service, u.& Department of Agriculture, Auburn, Alabama 86880
(Submitted for publication, 17 January 1972) FRANDSEN, JOHN C., AND COOPER, JUDITH A. 1972. Enzymes of coccidia: Purification and properties of ~-lactate dehydrogenase from Eimeria s&due. Experimental Parasitology 32,390402. The initial extensive purification and characterization of lactate dehydrogenase (EC 1.1.1.27) from sporozoa was accomplished. Fractional precipitation with ammonium sulfate, followed by chromatography on DEAE-Sephdex@ yielded a 500-1900-fold purification of the enzyme from unsporulated Rimeria stiedae oocysts. One isoenzynm was demonstrated by electrophoresis in polyacrylamide gel. The subunits of this isoenzyme were dissociated and recombined to form another active isoenzyme. The rate of conversion of pyruvate to lactate by this enzyme was strongly influenced by specific anions. In I?/2 0.1 glycine-sodium glycinate buffer at 40 C, the .R, for lactate was 3.03 X 10ea M and the Km for NAD* was 3.34 X lo-’ M. The enzyme accepted NAD and certain of its analogs as coenzymes. The 3-acetylpyridine analog of NADP served as coenzyme, whereas NADP did not. The enzyme was peculiar because it accepted a-NAD as coenzyme. A pyruvate-NAD complex apparently did not compete with NADH for the same binding site on the enzyme molecule. The rate of conversion of pyruvate to lactate was maximal at 40 C. The effect of hydrogen ion concentration on the rate of interconversion of pyruvate and lactate varied according to the buffer system in which the reaction occurred. INDEX DESCRIPTORS : Enzymes; Coccidia; Lactate dehydrogenase (EC 1.1.1.27) ; Eimeria stiedae; EC 1.1.1.27 (lactate dehydrogenase) ; Chromatography, ion exchange; Electrophoresis, disc.
Lactate dehydrogenase (EC 1.1.1.27) has been purified and characterized from numerous species of vertebrates and from certain species of protozoa and bacteria, but 1Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable. ‘ABBREVIATIONS: 3-AAD, 3-acetylpyridine-adenine dinucleotide, oxidized form; 3AADH, 3-acetylpyridine-adenine dinucleotide, reduced form; 3-AADP, 3-acetylpyridine-adenine dinucleotide phosphate, oxidized form; 3-AHD, 3-acetylpyridine-hypoxanthine dinucleotide, oxidized form ; EDTA, ethylenediaminetetraaeetic acid; EsLDH, lactate dehydrogenase from E. stiedae; LDH, lactate dehydrogenase; NAD, nicotin1973 by Academic Press, Inc. Copyright All rights oP reproduction in zmy form reserved.
to our knowledge the present report represents the first extensive purification and characterization of this enzyme from a sporozoan. This report is another contribution in our continuing series of investigations of the biochemistry of Eimeria stiedae, the amide-adenine dinucleotide, oxidized form; NADH, nicotinamide-adenine dinucleotide, reduced form ; NHD, nicotinamide-hypoxanthine dinucIeotide, oxidized form; NHDH, nicotinamidehypoxanthine dinucleotide, reduced form ; NADP, nicotinamide-adenine dinucleotide phosphate, oxidized form ; NADPH, nicotinamide-adenine dinucleotide phosphate, reduced form; 3-PAD, 3-pyridinealdehyde-adenine dinucleotide, oxidized form ; 2-SHEtOH, 2-mercaptoethanol; TAD, thionicotinamide-adenine dinucleotide, oxidized form. 390
Eimeriu
stiedae: L-LACTATE DEHYDROGENASE
coccidium of the liver of the domestic rabbit, The intracellular sites of E. stie&e lactate dehydrogenase (EsLDH) activity have been reported (Frandsen 1968, 1970), and some preliminary results of our biochemical investigations of this enzyme were presented in abstract form (Frandsen and Cooper 1970,197l). The isoenzymic composition of LDH was recently reviewed by Latner and Skillen (1968). The nomenclature we have used for the isoenzymes is that of the Committee on Enzymes of the International Union of Biochemistry (Webb 1964). Our investigations of the isoenzymic composition of EsLDH had two objectives: (1) the determination of the isoenzymic composition ;oer se of this enzyme in order to compare it with the isoenzymic composition reported for LDH’s from other species, and (2) the study of the molecular unit composition of EsLDH via dissociation and recombination. The intention in pursuing t,he second objective was to produce dissociation and recombination of the EsLDH alone, and then subsequently to produce hybrid molecules consisting of subunits from EsLDH combined with subunits of LDH from a vertebrate. Kaplan et al. (1968) investigated the effect of prior incubation with pyridine nucleotides on the rate of interconversion of pyruvate and lactate by chicken LDH. The effect of such prior incubation on reaction rates with EsLDH was studied to obt,ain comparative information. MATERIALS
AND METHOW
Purification
Unsporulated oocysts of E. stiedae were obtained from the livers of rabbits via the initial stages (i.e., to include flotation in sodium chloride solution and subsequent washing in water) of the procedure of Wagenbach et aZ. (1966). All subsequent procedures were carried out at O-4 C. The oocysts were stored as necessary in 2% potassium dichromate solution or in deionized
391
water containing a crystal of thymol. The oocysts were subsequently washed free of the potassium dichromate or thymol and then suspended in 0.1 M sodium phosphate buffer (pH 7.5) containing 0.01 M Z-mercaptoethanoi. This buffer mixture served as the vehicle in all subsequent manipulations unless otherwise noted. The suspended oocysts were passed through a French pressure cell at lO,OOO-17,000psig. The homogenate was then centrifuged at 10,800 g for 30 min, the sediment was resuspended in buffer mixture, and this resuspended sediment was again passed through the French cell in the same manner as before. The centrifugation procedure was repeated and the sediment was discarded. A 1% solution of protamine sulfate (from salmon) in the buffer mixture was added dropwise with constant stirring at the rat,e of Z Iiter of protamine suIfate solution per 60 g protein. The solution was allowed to stand overnight, and it was then centrifuged at 13,300 g for 30 min. The sediment was discarded, and more of the 1% protamine sulfate buffer solution was added at the rate of 1 liter per 30 g of protein. After standing overnight, the solution was centrifuged at 27,000 g for 2030 min, and the sediment was discarded. Sufficient crystalline ammonium sulfate was added to the supernatant Auid to bring it to 70% of saturation. iZfter 12-19 hr, the extract was centrifuged at 27,000 g for 15-30 min. The sediment was resuspended in the buffer mixture, and the supernatant fluid was discarded. The resuspended sediment was stirred for approx 3 hr, and it was then centrifuged at 27,000 g for 20 min. The supernatant fluid now contained the enzyme, and it was at this point that the E~so/E260 ratio exceeded unity for the first time. The sediment was discarded, and the supernatant. fluid was brought to 50% of saturation with crystalline ammonium sulfate. This solution was allowed to st.and overnight, was then centrifuged at 13,300 9 for 30 min, the sediment
FRANDSEN
392
AKD COOPER
cific activity remained quite constant for many weeks at 0 C. The specific activity of more highly purified preparations decayed much more rapidly, however, and neither the rate of decay nor the activity was affected by dialysis against fresh buffer mixture. Further purification, to specific activities greater than 500, could be achieved by’ passing the enzyme at a specific activity of approx 70 through a 2.5 x 40 cm column of DEAE A-50 Sephadex.@ The Sephadex@ was prepared according to the manufacturer’s instructions and equilibrated with r/2 0.02 phosphate buffer (pH 7.5) 0.01 M for 2-SHEtOH. The enzyme in ammonium sulfate solution was desalted and placed in the 1’/2 0.02 phosphate buffer (pH 7.5) via dialysis, inserted in the column with approx 300 ml of this buffer, and the column was then developed with a linear gradient formed from 2 liters of buffer and 2 liters of buffer containing 0.2 M sodium chloride. All the solution contained 0.01 M 2-SHEtOH. Although this development produced enzyme of high specific activity, it was highly diluted and required concentration. Experiments are in progress to determine a better elution method to produce a more concentrated solution. The dilute enzyme solution was concentrated using polyacrylamide gel beads. A representative purification series is presented as Table I.
was discarded, and sufficient crystalline ammonium sulfate was then added to bring the supernatant fluid to 60% of saturation. After 3 hr the centrifugation procedure was repeated, the sediment was discarded, and the supernatant fluid was then brought to 65% of saturation with crystalline ammonium sulfate. This solution was allowed to stand overnight and was then centrifuged at 27,000 g for 30 min. The sediment was discarded and crystalline ammonium sulfate was added very slowly to the supernatant fluid until floe appeared. The procedure of centrifugation and addition of ammonium sulfate to the supernatant fluid was repeated as necessary until that point was reached where the specific activity of the supernatant fluid was in the range of 65 to 70. If the percentage of saturation with ammonium sulfate exceeded a certain value between 65 and 70, the enzyme would precipitate. If this precipitation occurred, the enzyme could be recovered via suspension of the sediment in the buffer mixture and subsequent repetition of the above fractional precipitation series. The pH of the solution during the addition of ammonium sulfate was maintained in the range 7.0-7.5 via addition of ammonium hydroxide. Activity of the enzyme was maintained best in 50%saturated ammonium sulfate provided the precipitated nonenzyme protein had been removed via centrifugation. In this environment the speTABLE Purijication Procedure
Initial extract 1st precipitation Protamine sulfate 2nd precipitation Protamine sulfate (NH&S04 70% sat (NHJzSOI 55% sat (NH&SOa 69% sat
Vol (ml)
of EsLDH
Concentration (units/ml)
I from 4 X 109 Oocysts
Total units (X 103)
Protein b-s/ml)
Specific activity (units/mg)
Yield (%)
Purification
83
60.2
5
152
28.4
4
7.68
3.7
80
3.7
186 27 28 28
21.6 125 111 103
4 3 3 2.9
4.5 9.86 5.5 1.54
4.8 12.7 20.2 67
80 60 60 58
4.8 12.7 20.2 67
58
1
100
1
Eimeria Isoenzymic
StifXb:
L-LACTATE DEHYDROGENASE
Composition
The technique of isoenzyme separation used was disc electrophoresis in polyacrylamide gel (Omstein 1964; Davis 1964)) basically the method of Diete and Lubrano (1967). The following techniques were employed to obtain dissociation and recombination of LDH subunits. All enzyme solutions were in 0.1 M sodium phosphate buffer (pH 7.5) 1 mM for 2-SHEtOH ; and, except as noted, all operations were performed at 04 C. (1) A modification of the procedures of iUarkert (1963)) Chilson et al. (1964)) and Clausen and Hustrulid (1968) using 1 M sodium chloride and EsLDH alone and EsLDH plus LD1 (pure H4 enzyme from chick heart,, courtesy of Professor N. 0. Kaplan and his colleagues). (2) EsLDH solution was dialyzed overnight against a 6 M solution of sodium chloride (Chilson et al. 1964). Following this dialysis, the salt concentration was reduced by further dialysis against phosphate buffer. All solutions were 1 mM for 2SHEtOH. (3) EsLDH alone and EsLDH plus Hq LDH (ex Kaplan et al., vide supra) in buffer were made 0.12 M for 2-SHEtOH and 10.5 M for urea according to the procedures of Epstein et al. (1964). The diluted solutions were then incubated for 5 hr at 24 C, stored at 4 C for 12 hr, and then concentrated with Aquacide I (Calbiochem, Los Angeles, CA). Samples were withdrawn from the concentrated solutions over a 72hr period and assayed for activity. (4) A solution of EsLDH in buffer was made 0.9 M for sodium chloride via slow addition of the salt. The solution was quickly frozen with carbon dioxide snow and then immediately allowed to thaw at room temperature. It was then dialyzed for 12 hr at 4 C against buffer. All solutions were 1 mM for 2-SHEtOH. As an alternate method, NADH to 1.6 X low4 M was added to the initial enzyme solution.
393
(5) EsLDH in buffer was dialyzed for 3 days against 0.1 M Tris-HCl buffer (pH 7.5) which was 1 mM for EDTA and 0.1 M for 2-SHEtOH (Chilson et al. 196513). The EsLDH remained active throughout this procedure. The solution was then made 9.6 M for urea by addition of a saturated solution of urea in buffer. After loss of enzymic activity, the enzyme solution was diluted 20-fold with buffer containing 1 mM 2SHEtOH and 1.3 mM NADH. This solution was allowed to stand at room temperature with hourly assays for LDH activity. (6’1 This method was based on the work of Epstein et ~2. (1964) and Appella and ?vIarkert (1961) using guanidine hydrochloride. (7) This was a modification of procedure 4. Sufficient sodium chloride was added to the enzyme solution to make it 1.8,3.6, and, finally, 5 M. An aliquot of each of the two lower concentrations was slowly frozen t,wice at -20 C and thawed at room temperature. The 5 M solution was frozen twice at -140 C and thawed each time at 4 C. (8) Lithium chloride as per the method of Chilson et al. (1966). (9) Concentrated hydrochloric acid was added as per the method of Clausen and Hustrulid (1968). (10) Sodium thiosulfate and sodium iodide as per the method of Chilson et at. (1965a, b) . Substrate Specificity The n-isomer of NAD was obtained through the courtesy of P-L Biochemicals, Inc., Milwaukee, WI. Its freedom from the p-isomer was proven by its inability to serve as coenzyme for the oxidat,ion of lactate by a commercial preparation of rabbit muscle LDH which had high activity with the p-isomer. The complete reaction mixture contained in addition to EsLDH r/2 0.1 glycine-sodium glycinate buffer (pH lO.O), 0.1 M L-lactate, and 8 x 10e4 M NAD. The reactions were conducted in triplicate at. 40 C with experimental controls to
394
FRANDSEN
AND
40 X lop5 M was used at constant lactate concentrations of 6,12, and 18 mlM.
establish the absence of reduction of NAD in the absence of enzyme and/or of lactate and the absence of reduction of a-NAD by commercial rabbit muscle LDH.
Coenzymes Investigations were conducted to determine the efficacy of various analogs of NAD as coenzymes for EsLDH. All reactions were run in triplicate and were monitored at 40 C, normal body temperature for’ the rabbit. The following analogs, all at 8 X 1O-4 M, were utilized in studies of the oxidation of lactate : nicotinamide-adenine dinucleotide phosphate (NADP) , 3-acetylpyridine-adenine dinucleotide phosphate (3-AADP) , 3-pyridinealdehyde-adenine dinucleotide (3-PAD), thionicotinamideadenine dinucleotide (TAD), 3-acetylpyridine-adenine dinucleotide (3-AAD), nicotinamide-hypoxanthine dinucleotide and 3-acetylpyridine-hypoxanCM=)), thine dinucleotide (3-AHD) . In addition, the reduced forms of NADP, NHD, and 3-AAD were tested for their efficacy as coenzymes in the reduction of pyruvate. The nomenclature used here for the pyridine nucleotides is that recommended by the Enzyme Commission of the International Union of Biochemistry (Dixon 1960). For the studies of the oxidation of lactate, the reactions were followed at two different acidities and at different substrate concentrations, as shown in Table II. For the reactions at pH 7.5, 0.05 M potassium
Specific Anion Effects
The effects of specific anions on the rate of reduction of pyruvate by EsLDH were ascertained by monitoring the reaction rate at 25 C in a series of buffers of constant ionic strength (0.2) and pH (6.5). In order to avoid extraneous ionic effects, neutral salts were not used to adjust ionic strengths. All assays were conducted in triplicate, using the same batch of enzyme on the same day. The ionic strength, pH, and buffers were selected to permit comparison with the data obtained on anionic effects on beef heart LDH by Winer and Schwert (1958). Kinetics
The Michaelis constants for NAD and lactate were determined by the method of Florini and Vestling (1957). All assays were performed in quintuplicate at 40 C in 1?/2 0.1 glycine-sodium glycinate buffer (pH 9.6). Neutralized hydroxylamine at 0.01 M was used as a pyruvate trap. Lithium L-lactate in concentrations of 3, 6, 12, 18, and 24 mM was used at constant NAD concentrations of 9, 18, and 36 X 10Y5 M. NAD in concentrations of 6, 9, 18, 36, and TABLE
~~
Eficacies
Pyridine nucleotide
~~ NAD NADP 3-AADP 3-PAD TAD 3-AAD NHD 3-AHD
of Pyridine
Nucleotides
m-lactate 0.013 M in phosphate buffer (pH 7.5)
COOPER
II
as Coenzymes for Oxidation m-lactate 0.1 M in phosphate buffer (pH 7.5)
of Lactate
with
EsLDH
Glycine buffer (pH 9.6)
Rate
Comparative rate
Rate
Comparative rate
Rate
Comparative rate
19.8 0
100 0
74.78 0 10.37 59.4 66.8 621 27.1 341
100 0 13.6 78.9 89.4 830 36.2 45.6
11.9 0
100 0
No data 17.63 80.25 421 14.19
89.1 404 2125 21.19 No data
No data 0.177 1.34 14.7 107.4 193.3 1467 1.44 10.9 No data
Eimeria
Stiedae: L-LACTATE DEHYDROGENASE
phosphate buffer r/2 0.087 was used in the medium, whereas for the reactions at pH 9.6 glycine-sodium glycinate buffer I’/2 0.1 was used. These buffers were chosen to facilitate comparison of the results with those published by Bonavita and Guarneri (1962), Kaplan et al. (1956), and Kaplan et al. (1960). Preincubation
with NAD and Pyruvate
Three sets of assays were performed, with three assays per set. The conditions of the respective assays were: temperature, 25 C; buffer, 0.05 M potassium phosphate (pH 7.5) ; pyruvate concentrations, 3 x low4 M; NAD concentration, 4.3 X 10V5 M; NADH concentration, 2.6 X 10e4 M. For the first set of assays, the reaction mixture consisted of buffer, pyruvate, NADH, and enzyme. For the second set of assays, the react,ion mixture consisted of NAD plus the same ingredients as for the first set of assays. Addition of the nucleotides was essentially simultaneous, and the reaction was init,iated by the addition of enzyme. For the third set of assays, enzyme was added to a mixture of buffer, pyruvate, and NAD. For this third set of assays, the reaction mixture (with enzyme) was incubated at room temperature (23 C) for 15 min. The reaction was then initiated by addition of NADH.
395
7.5-8.7, and glycine-sodium glycinate buffer, r/2 0.1 for the range pH 8.7-10.0. Hydroxylamine (neutralized) at a concentration of 0.01 M was used in all assay mixtures as a pyruvate trap, lactate was 0.1 M, NAD was 8 X 10L4 M. All assays were performed in triplicate at 25 C using the same batch of enzyme for each buffer system on the same day. The increase in absorbance at 340 nm was monitored for 90 set, and the pH was determined electrometrically at the conclusion of the assay. For studies of the reduction of pyruvate, two buffer systems were used: acetate buffer, r/2 0.1 for the pH range 3.96-6.0, and arsenate buffer, r/2 0.1 for the pH range 6-O-8.2. Pyruvate was 3 x 1O-4 M and NADH was 1.6 X 10B4 M. The assays were performed in triplicate at 25 C, and the pH was determined electrometrically at the conclusion of the 90-set monitoring of the reduction in absorbance at 340 nm. RESULTS
Isoenzymic Composition
Our electrophoretic investigations have established that the LDH from unsporulated oocysts of E. stiedae is isoenzymically homogeneous, migrating electrophoretically in the relative position of LD4. Each of the techniques described in Materials and Methods to obtain dissociation Reaction Rate as a Function of Temperaand recombination of LDH subunits were ture repeated several times, with different In the standard assay mixture [V&Z.,0.05 batches of EsLDH. In no case was combiM phosphate buffer (pH 7.5), 3.1 X 1O-4 nation of EsLDH subunits with H subunits 1M pyruvate, 1.6 x lOA M NADH], the from chick heart detected. The process of rate of conversion of pyruvate to lactate dissociation and recombination of EsLDH was monitored in triplicate at temperatures subunits was observed only twice, once with procedure 4 when t,he enzyme solution conof 25,30,35,40, and 45 C. tained NADH (Fig. I), and once with proReaction Rate as a Function of pH cedure 7 when the sodium chloride concenFor studies of the oxidation of lactate, tration had been brought to 5 M. In both three different buffer systems were used to cases, only one additional band was procover the pH range concerned: arsenate duced on electrophoresis; once this band buffer, r/2 0.1 for the range pH 6.15-7.79, was nearer the cathode than the normal sinTris-HCl buffer, r/2 0.2 for the range pH gle band, and the other time it was nearer
396
J?RANDSENAND COOPER
C--------L---
i 1 NATIVE
“Coenzyme Analogs,” EsLDH accepted certain pyridine nucleotides other than NAD as coenzymes.
/,
NAD Isomers
+-
+
I
iI
I
i-------------’
1 NATIVE
I I- --------w----
+
I
&-
FIG. 1. Zymogram, polyacrylamide gel disc electrophoresis showing new band formed by recombined subunits of EsLDH after freezing in sodium chloride solution (procedure 4 in text).
The mean reaction rates expressed in micromoles of nucleotide reduced per minute were 13.7 for /3-NAD and 0.4 for a-NAD, indicating the EsLDH catalyzed the reduction of a-NAD at approx 3% of the rate at which it catalyzed the reduction of P-NAD under the experimental conditions. A review of the literature indicates that this ability of EsLDH to use a-NAD as coenzyme is unusual (vide Kaplan and Sarma 1970). Specific Anion Effects
The data are presented in Fig. 2, where the results obtained with EsLDH are dithe anode than the normal single band. Nu- rectly compared with those of Winer and merous efforts to repeat this dissociation Schwert (1958) for beef heart LDH. The and recombination have failed. Conse- suitability of benzoate buffer for the two quently, only sodium chloride followed by LDH’s was strikingly different. Whereas freezing has produced dissociation and re- the maximal rate for beef heart LDH was combination of EsLDH, and then only on two occasions out of many. When the enzyme was exposed to high concentrations of urea, a gradual decline in activity to zero usually, but not invariably, occurred. If this loss of activity is int,erpreted as evidence of molecular unfolding, I I I I i 70.5 then the EsLDH molecule did unfold. HISTIDINE /V&./W The enzyme survived the acid treatment PHOSPHATE w,$$&$!,j 1 1 1 0139 method (procedure 9) ; electrophoresis PHTHALATE may.4 again producing only the normal single PYROPHOSPHATE wm 1 1 I,! IO187 band. The sodium thiosulfate-sodium ioSVCCINATE -pi39 dide procedure (procedure 10) produced loss of activity. 0 4 8 12 14 v
Substrate Specificity
EsLDH was specific for L-( +) lactate. The presence of D-(-) lactate concurrently with L-(+) lactate did not alter the rate of oxidation of the L-( i-) lactate, nor did prior exposure to n-( -) lactate influence the rate at which the enzyme subsequently oxidized L- (+) lactate. As discussed under
un ESLDH hz9 BEEF HEART LDH -.----V WITH TRIS-HCL
Xl0
(WINER 8 BUFFER
SCHWERT)
FIG. 2. Comparative rates of reduction of pyruvate by EsLDH and by beef heart LDH (Winer and Schwert 1958) in different buffer systems at I?/2 0.2 and 25 C. Percentages represent rate with EsLDH compared with rate with beef heart LDH. V (velocity) figures are expressed in terms of V with TrisHCl buffer = 199.
Eimeria
stiedae: L-LACTATE
in histidine buffer, this being the only buffer producing a more rapid rate than that produced by Tris-HCl, the maximal rate for EsLDH was produced by citrate buffer. Arsenate, phosphate, and pyrophosphate buffers also produced more rapid rates with EsLDH than that rate produced with Tris-HCl.
DEHYDROGENASE
397
increased again as the pH was raised from 7.5 to 9.6. (3) The comparative rate with 3-AAD
decreased nearly 3-fold at pH 7.5 as the lactate concentration increased from 0.013 to 0.1 M. This comparative rate then nearly doubled as the pH increased to 9.6 while the lactate concentration remained constant. The different ionic environments at these Kinetics two acidities must be remembered lest one The work of Coulson and Rabin (1969) attribute the different comparative reaction demonstrated that only the keto form of rates to pH effects alone. Studies of the efficacies of the three repyruvate may serve as a substrate for LDH duced pyridine nucleotides as coenzymes for as obtained from pig heart. Because prepathe reduction of pyruvate were conducted in rations of pyruvate contain a variable and 0.05 M potassium phosphate buffer (pH changing amount of the enol form of the 7.5) at a pyruvate concentration of 3 X acid, it would appear impossible to deter1O-4 M. The results of these investigations mine accurately the kinetic constants of the are summarized in Table IV. pyruvate to lactate reaction unless the conKaplan et al. (1960) have investigated centration of keto pyruvate were known the relative rates of the interconversion of precisely during the course of the reactions lactate and pyruvate by lactate dehydrounder experimental conditions. As pergenases at different substrate concentraformed by Coulson and Rabin (1969), we tions and with different coenzymes from a unsuccessfully attempted preparation of variety of vertebrates and invertebrates. pure keto pyruvate via the procedure of von For comparison with the data published by Korff (1964). Until we are able to obtain these authors, the rate ratios for EsLDH known concentrations of keto pyruvate for the requisite experiments, we will not at- for the oxidation of lactate were determined tempt determination of the Michaelis con- in phosphate buffer at pH 7.5 at two lactate concentrations; the low concentration was stants for NADH and pyruvate of EsLDH. 0.013 1M and the high concentration was 0.1 Under the experimental conditions specified in “Materials and Met,hods,” the Michaelis M. These ratios are given in Table III, calculated from the data provided in Table II. constants for lactate and NAD were 3.03 x Rate ratios for the reduction of pyruvate by 10M2M and 3.34 X 10F4 M, respectively. EsLDH with the reduced forms of nicotinamide adenine dinucleotide and nicotinCoenzymes amide hypoxanthine dinucleotide at concenComparisons of the rates vi.+a-vis those trations of 3 x 10B5 M with two different with NAD under different conditions of concentrations of pyruvate (low = 3 X 1O-4 assay revealed the following items of inter- M, high = 3 x 1O-3 M) are provided in est (Table II) : Table V, from which they also may be com(1) The rate with 3-PAD diminished pared with the data provided by Kaplan et profoundly as the acidity decreased from al. (1960). As suggested by these authors pH 7.5 to pH 9.6. (p. 392), “the catalytic technique may be (2) The rate with TAD relative to that most useful in discriminating differences bewith NAD decreased nearly 5-fold at pH tween enzymes having the same function.” 7.5 as the lactate concentration increased There are available data on these properties from 0.013 to 0.1 M. This comparative rate in respect to so comparatively few species
398
FRANDSEN AND COOPEB
EsLDH the reaction rate for the reduction of pyruvate after preincubation with pyruTwo Different Concentrations (High, 0.1 M; Low, vate and NAD was not lower than that for 0.013M) with Diferent Pyridine Nucleotides at the other two conditions of assay (vide Ma8 X 10-’ M. All data from Table ZZ terials and Methods), the rates for all nine Rate ratios Pyridine nucleotides individual assays being essentially the same. These authors concluded that their 0.047 NAD (low)/3-AAD (low) data suggested that a pyruvate-NAD com1.26 NAD (high)/3-AAD (high) plex competed with NADH for the same 0.265 NAD (low)/NAD (high) binding site on the chicken LDH molecule. 0.678 3-AAD (low)/3-AAD (high) 0.297 3-PAD (low)/3-PAD (high) On the same bases, our data would suggest 1.2 TAD (low)/TAD (high) that such competition either did not occur 0.032 NAD (low)/3-AAD (high) under the conditions of assay with EsLDH, 1.122 NAD (low)/3-PAD (low) or that such competition was so ephemeral 1.26 NAD (high)/3-PAD (high) 0.334 as to be undetectable by the monitoring NAD (low)/3-PAD (high) 5.25 3-AAD (low)/TAD (low) system.
TABLE III Relative Reaction Rates for Oxidation of Lactate at
3.98 21.3
NAD (high)/NAD (low) 3-AAD (low)/NAD (low)
Reaction Rate as a Function of Temperature
TABLE IV Eficacies of P&.%ne Nucleotides as Coenzymes for Reduction of Pgruvate with EsLDH* Molarity of pyridine nucleotide
Pyridine nucleotide
4 x 10-s 4 x 10-s
NADH NHDH NADH
1.6 X Kr4 1.6 X W4
3-AADH (1Relative
Rates:
NADH/3-AADH
fimoles of Proporpyridine tionate nucleotide rate of oxidized oxidation of per pyridine minute nucleotide 17.85
100
2.42 10.81 9.24
NADH/NHDH
13.6
TABLE V Reduction of Pyruvate with EsLDH [NADH and NHDH at 3 X 10-s M as Coenqme] Reaction Rates as Function of Pyruvate Concentrations& Coenzyme
100
85.5 =
7.37,
= 1.17.
that it would be premature to speculate as to the significance of these findings. As this is the first LDH from sporozoa to be so characterized, the catalytic properties with regard to pyridine nucleotides were recorded so that they will be available for
comparison and analysis at that future time LDH’s from the animal kingdom as a whole have been characterized to permit rational analysis from an evolutionary standpoint. Unlike the results reported with chicken LDH by Kaplan et al. (1968), with when sufficient
As illustrated by the graph in Fig. 3, the rate of conversion of pyruvate to lactate was most rapid at 4.0C, which, perhaps incidentally, is also the normal body temperature of the rabbit host. It is of interest
NADH NHDH NADH NHDH NADH
NHDH
Pyruvate concentration @f)
pmoles of coenzyme oxidized per minute
Proportionate rates
1 x 10-a 1 x 10-2 3 x 10-a 3 x 10-a 3 x 10-4 3 x 10-d
20.75
100 45.1 100
9.36 17.7
8.23 12.2
2.74
46.5 100
22.4
a Comparative Rates of Oxidation of Coenzymes : NADH (0.003M Pyruvate) NADH (0.OC!Q3 M Pyruvate) = 1’45 NADH (0.0003M Pyruvate)
NHDH (0.0603M Pyruvste) = 4*46 NHDH
(0.003 M Pyruvate) = o 46 NADH (0.003M Pyruvate) . NHDH (0.003M Pyruvate) = 3 o NHDH (0.0003M Pyruvate) ’
Eimeria
stiedae: L-LACTATE
DEHYDROGENASE
399
that the rates at 5 C below and above this optimal temperature for the reaction were only 79 and 76%, respectively, of the maximal rate. Reaction Rate as a Function
of pH
As shown in Fig. 4, the rate of oxidation of lactate increased quite uniformly with decrease in hydrogen ion concentration to approx pH 8.7, where, in glycine-sodium glycinate buffer, at least, it became quite constant as the pH increased to 10.0. The differences in rates between the buffer systems reflected specific anion effects, which were pronounced with this enzyme (vide supra), and the differences in enzyme concentration and other properties of the different batches of enzyme used with the buffer systems. The important information to be obtained from this figure is the shape of the plot for each buffer system. For the arsenate and Tris buffer systems this plot described a steady, though not always uniform, increase of rate with increase of pH. For the glycine buffer system, the reaction rate changed little as the pH increased from 8.7 to 10.0.
110 90 2 i 3
70
8 3 ‘2. I? oz IOn z”
/
GLYCINE
8
0'
6
6.8
7.6
8.4
9.2
IO
PH
FIQ. 4. Oxidation of lactate by EsLDH tl~l a function of pH in different btier systems at 25 C. Studies for each buffer system were conducted on the same day with the same enzyme preparation, but properties of the enzyme preparations were not necessarily the 8ame for all the buffer systems, and conclusiona should not be drawn from these data regarding rates in different buiTer systems at common pH.
In the case of the reduction of pyruvate, again, differences in absolute rate between the buffers caused by specific anion effects and differences in the enzyme batches were apparent, but, in contrast to the situation with the oxidation of lactate, within each buffer system the rate of the reaction decreased as the pH increased, and the maximum rate of reduction of pyruvate by EsLDH in acetate buffer appeared to be below pH 4.0. DISCUSSION
Isoenzgmic Composition
30 1 20
30 40 50 DEGREES CENTIGRADE
, 60
FIG. 3. Rate of conversion of pyruvate to lectate by EsLDH a.8 a function of temperature in 0.05 M phosphate buffer (pH 7.5).
Assuming EsLDH to be composed of 3 M subunits and 1 H subunit, as LDI from vertebrates is, then it should be possible to cause dissociation and recombination of the molecular subunits, with such recombination resulting in the appearance of additional bands on electrophoresis. These additional bands would correspond to isoen-
400
FRANDSEN
AND
COOPER
with several LDH’s from various sources 3-AAD was the most efficient coenzyme for the oxidation of lactate. As demonstrated here by the greater efficacy of 3-AHD as coenzyme compared with NHD, as well as by the paramount efficacy of 3-AAD, it was the replacement of the amino group on the third carbon of the pyridine ring by the -COCHs of acetylpyridine which was somehow responsible for marked increase of efficiency as a coenzyme. Kaplan and Sarma (1970) have discussed the physicochemical bases of the efficacies of various pyridine nucleotides as coenzymes for LDH. It is intriguing in the present case that whereas NADP and NADPH will not serve as coenzyme for EsLDH, 3AADP will so serve. This is another illustration of the fitness-producing power of the acetylpyridine ring. Because contamination of our 3-AADP by 3-AAD could explain the apparent suitability of 3-AADP as a coenzyme, the purity of our 3-AADP was checked by ascending paper chromatography on Whatman j$l paper, as kindly suggested by Dr. Coenz ymes J. M. Siegel of P-L Biochemicals, Inc. The From the data on EsLDH, several con- solvent systems were (1) isobutyric acid/ clusions may be drawn. This enzyme did ammonium hydroxide/water, 66/l/33 (pH not require a nicotinamide ring of its coen- 3.7) ; and (2) ethanol/l.0 M ammonium aczymes, accepting substitution on the third etate, 7/3 (pH 7.5). These chromatographic carbon atom of the pyridine ring of -CHO, procedures confirmed the purity of our 3-COCHa, and -CSNH2 for the -CONH2 AADP. of nicotinamide. That the --NH2 of adenine was not essential is demonstrated by the Reaction Rate as a Function of pH The general picture for the rates of interacceptability of NHD, with its hydroxyl group at the C6 position of the purine ring, conversion of lactate and pyruvate as funcas a coenzyme. The rate of oxidation of tions of the pH by EsLDH is generally as lactate with NHD was only about one- would be expected from studies on these quarter to one-third that with NAD at pH properties of LDH from other organisms. 7.5, and one-tenth that at pH 9.6, however, The principal point of difference between’ indicating that for some reason NHD was our results and those of Winer and Schwert (1958) working with beef heart LDH is not as eflicient a coenzyme as was NAD. It is noteworthy that when the -CONH, of that they observed a marked reduction in the nicotinamide ring of NHD was replaced the rate of oxidation of lactate as the pH with -COCHQ to form 3-AHD, the rate of increased from 9.0 to 10.0, whereas we observed a slight increase in the rate from pH’ oxidation of lactate increased la-fold. A survey of the literature revealed that 9.0 to pH 9.3 and then a slight decrease in zymes LDI, LDZ, LD3 and LDs. The LDI isoenzyme would be expected to be present in greatest amount due to incomplete dissociation, and the other isoenzymes in decreasing amounts would be LDr,, LDS, LDZ, and LD1. If the subunits of EsLDH are similar to the subunits of vertebrate LDH isoenzymes, then it should be possible to form hybrid active tetramers. Inability to produce hybrids between the subunits of different LDH’s has been reported previously (Salthe et al. 1965)) and it may indeed be that subunits of EsLDH cannot combine with H subunits from chick heart LDH to produce active units of enzyme. The apparent unusual tenacity of attachment of subunits of EsLDH, their resistance to most of the treatments which other workers have found successful in dissociating subunits of other LDH’s, their lack of hybridization with H subunits from chick heart LDH, and the anomalous electrophoretic results may indicate that EsLDH is of different subunit construction than the LDH’s so characterized from vertebrates.
Eimeriu
stiedue: L-LACTATE DEHYDROGENASE
401
the rate as the pH increased further to 10.0. This difference might be accounted for by the differences in the buffer systems used. We used glycine-sodium glycinate buffer in this pH range whereas Winer and Schwert used Tris-HCI buffer.
CHILSON, 0. P., COSTELLO,L. A., AND KAPLAN, N.
Physiological
1006-1014. CHILSON, 0. P., KITTO, G. B., PUDLES, J., AND inactivation KAPLAN, N. 0. 1966. Reversible
Function
Since the work of Warburg (1924), biochemists have been especially interested in those cells which denend on LDH for replenishment of the NAD pool in glycolysiS, for in such cells LDH would be a vital enzyme. We have established (unpublished data) cytochemically that unsporulated oocysts of E. stiedae contain a-glycerophosphate dehydrogenase, however; and replenishment of the intracellular pool of NAD may consequently be accomplished through the reduction of dihydroxyacetone phosphate by cytoplasmic a-glycerophosphate dehydrogenase as in certain mammalian cells. ACKNOWLEDGMENT The authors acknowledge the generous gift of H, LDH from chick heart by Professor N. 0. Kaplan and Dr. F. Stolzenbach of the University of California at San Diego, the generous gift of c+NAD by Dr. J. Siegel of P-L Biochemicals, Inc., Milwaukee, WI, and the expert technical assistance of Mr. Mark R. Kearse, Mr. Thomas H. Ennis, Mr. William A. Wilder, Miss Margaret 0. Haven, and Miss Judy Jones.
REFERENCES APELLA, E., AND MARKERT, C. L. 1961. Dissociation of lactate guanidine Biophysicial 171-176.
dehydrogenase into subunits hydrochloride. Biochemical Research Communications
BONAVITA,V., AND GUARNERI,R. 1962. Lactic
with and 6,
dehydrogenase isozymes in the nervous tissue. I. The reaction of isozymes with diphosphopyridine nucleotide analogues and their inhibition by sodium metabisulfite. Biochimica et Biophysics Acta 59,634-642. CHILSON, 0. P., COSTELLO,L. A., AND KAPLAN, N. 0.1964. Hybridization of lactic dehydrogenases in vitro by concentrated sodium chloride and by reversible inactivation in urea. Journal of Molecular Biology 10,349-352.
0. 1965a. Studies on the mechanism of hybridization of lactic dehydrogenases in vitro. Biochemistry 4 271-281. CHILSON, 0. P., KITTO, G. B., AND KAPLAN, N. 0. 1965b. Factors affecting the reversible dissociation of dehydrogenases. Proceedings of the National Academy of Sciences U5. 53,
of dehydrogenases. Journal of Biological Chemistry 241,2431-2445. CLAUSEN, J., AND HUSTRULID, R. 1968. Factors affecting recombination of lactate dehydrogenase isoenzyme subunits. Biochimica et Biophysics Acta 167,221-226. COULSON, C. J., AND RABIN, B. R. 1969. Inhibition of lactate dehydrogenase by high concentrations of pyruvate: the nature and removal of the inhibitor. FEBS Letters 3,333-337. DAVIS, B. J. 1964. Disc electrophoresis-II. Method and application to human serum proteins. Annals of the New York Academy of Sciences
121,404-427. DIETZ, A. A., AND LUBRANO,T. 1967. Separation and quantitation of lactic dehydrogenase isoenzymes by disc electrophoresis. Analytical Biochemistry 20,246-257. DIXON, M. 1960. Nomenclature of the nicotinamide nucleotide coenzymes. Nature 188,
464-466. EPSTEIN, C. J., CARTER,M. M., AND GOLDBERGEB, R. F. 1964. Reversible denaturation of rabbitmuscle lactate dehydrogenase. Biochimica et Biophysics Acta 92,391-394. FLORINI, J. R., ANDVESTLINC,C. S. 1957. Graphical determination of the dissociation constants for two-substrate enzyme systems. Biochimica et Biophysics Acta 25,575-578. FRANDSEN,J. C. 1968. Eimeria stiedae: Cytochemical identification of acid and alkaline phosphatases, carboxylic ester hydrolases, and succinate, lactate, and glucose&phosphate dehydrogenases in endogenous stages from rabbit tissues. Experimental Parasitology 23, 393411. FRANDSEN,J. C. 1970. Eimeria .&due: Cytochemical identification of enzymes and lipids in sporozoites and endogenous stages. Experimental Parasitology 27, 106115. FRANDSEN,J. C., AND COOPER,J. A. 1970. Some properties of L-lactate dehydrogenase from Eimeria stiedae (Protozoa: Coccidia). ASB Bulletin 17,43. FRANDSEN,J. C., AND COOPER,J. A. 1971. Some properties of n-lactate dehydrogenase from
402
FRANDSEN
Eimeria stiedae (Protozoa : Coccidia) . II. Further studies of the semi-purified enzyme. ASB Bulletin 18,35. KAPLAN, N. O., CIOTTZ, M. M., HAMOLSKY, M., AND B~BER, R. E. 1960. Molecular heterogeneity and evolution of enzymes. Science 131, 392
AND COOPER
isozymes: dissociation and recombination of subunits. Science 140,1329-1330. ORNSTEIN, L. 1964. Disc electrophoresis-I. Background and theory. Annals of the New York Academy of Sciences 121,321-%t9. SALTHE, S. N., CHILSON, 0. P., AND KAPLAN, N. 0. 397. 1965. Hybridization of lactic dehydrogenase KAPLAN, N. O., CI~II, M. M., AND STOLZENBACH, in vivo and in vitro. Nature 207,723726. F. E. 1956. Reaction of pyridine nucleotide VON KORFF, R. W. 1964. Pyruvate-C”, purity and stability. Analytical Biochemistry 8, 17~ analogues with dehydrogenaees. Journal of Biological Chemistry 221,833-8~. 178. KAPLAN, N. O., EVERSE, J., AND ADM~AAL, J. 1968. WAQENBACH, G. E., CHALLEY, J. R., AND BURNS, W. Significance of substrate inhibition of deC. 1966. A method for purifying coccidian hydrogenases. Annals of the New York Acadoocysts employing Clorox and sulfuric acidemy ofSciences 151,400412. dichromate solution. Journal of Parasitology 52,1222. KAPU, N. O., AND SARMA, R. H. 1970. The structure of pyridine coenzymes as related to bindWARBURQ,0. 1924. “Uber den Stoffwechsel der ing. In “Pyridine Nucleotide-dependent DeTumoren.” Springer-Verlag, Berlin. hydrogenases” (H. Sund, ed.), pp. 39-56. WEBB, E. C. 1964. Nomenclature of multiple Springer-Verlag, Berlin. enzyme forms. Nature 203,821. LATNER, A. L., AND SKILLEN, A. W. 1968. “IsoenWINER, A. D., AND SCHWERT, G. W. 1958. Lactic zymes in Biology and Medicine.” Academic dehydrogenases. IV. The influence of pH on the kinetics of the reaction. Journal of BioPress, New York. MARKEBT, C. L. 1963. Lactate dehydrogenase logical Chemistry 231,1065-1083.
PARASITOLOGY
Enzymes
%?, 390-402
of Coccidia: Purification Dehydrogenase from John
Regional
(1972)
Parasite
Agricultural
C. Frandsen
and
and Properties Eimeria stiedae’ Judith
of L-Lactate
A. Cooper
Research Laboratory, Veterinary Sciences Research Divbion, Research Service, u.& Department of Agriculture, Auburn, Alabama 86880
(Submitted for publication, 17 January 1972) FRANDSEN, JOHN C., AND COOPER, JUDITH A. 1972. Enzymes of coccidia: Purification and properties of ~-lactate dehydrogenase from Eimeria s&due. Experimental Parasitology 32,390402. The initial extensive purification and characterization of lactate dehydrogenase (EC 1.1.1.27) from sporozoa was accomplished. Fractional precipitation with ammonium sulfate, followed by chromatography on DEAE-Sephdex@ yielded a 500-1900-fold purification of the enzyme from unsporulated Rimeria stiedae oocysts. One isoenzynm was demonstrated by electrophoresis in polyacrylamide gel. The subunits of this isoenzyme were dissociated and recombined to form another active isoenzyme. The rate of conversion of pyruvate to lactate by this enzyme was strongly influenced by specific anions. In I?/2 0.1 glycine-sodium glycinate buffer at 40 C, the .R, for lactate was 3.03 X 10ea M and the Km for NAD* was 3.34 X lo-’ M. The enzyme accepted NAD and certain of its analogs as coenzymes. The 3-acetylpyridine analog of NADP served as coenzyme, whereas NADP did not. The enzyme was peculiar because it accepted a-NAD as coenzyme. A pyruvate-NAD complex apparently did not compete with NADH for the same binding site on the enzyme molecule. The rate of conversion of pyruvate to lactate was maximal at 40 C. The effect of hydrogen ion concentration on the rate of interconversion of pyruvate and lactate varied according to the buffer system in which the reaction occurred. INDEX DESCRIPTORS : Enzymes; Coccidia; Lactate dehydrogenase (EC 1.1.1.27) ; Eimeria stiedae; EC 1.1.1.27 (lactate dehydrogenase) ; Chromatography, ion exchange; Electrophoresis, disc.
Lactate dehydrogenase (EC 1.1.1.27) has been purified and characterized from numerous species of vertebrates and from certain species of protozoa and bacteria, but 1Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable. ‘ABBREVIATIONS: 3-AAD, 3-acetylpyridine-adenine dinucleotide, oxidized form; 3AADH, 3-acetylpyridine-adenine dinucleotide, reduced form; 3-AADP, 3-acetylpyridine-adenine dinucleotide phosphate, oxidized form; 3-AHD, 3-acetylpyridine-hypoxanthine dinucleotide, oxidized form ; EDTA, ethylenediaminetetraaeetic acid; EsLDH, lactate dehydrogenase from E. stiedae; LDH, lactate dehydrogenase; NAD, nicotin1973 by Academic Press, Inc. Copyright All rights oP reproduction in zmy form reserved.
to our knowledge the present report represents the first extensive purification and characterization of this enzyme from a sporozoan. This report is another contribution in our continuing series of investigations of the biochemistry of Eimeria stiedae, the amide-adenine dinucleotide, oxidized form; NADH, nicotinamide-adenine dinucleotide, reduced form ; NHD, nicotinamide-hypoxanthine dinucIeotide, oxidized form; NHDH, nicotinamidehypoxanthine dinucleotide, reduced form ; NADP, nicotinamide-adenine dinucleotide phosphate, oxidized form ; NADPH, nicotinamide-adenine dinucleotide phosphate, reduced form; 3-PAD, 3-pyridinealdehyde-adenine dinucleotide, oxidized form ; 2-SHEtOH, 2-mercaptoethanol; TAD, thionicotinamide-adenine dinucleotide, oxidized form. 390
Eimeriu
stiedae: L-LACTATE DEHYDROGENASE
coccidium of the liver of the domestic rabbit, The intracellular sites of E. stie&e lactate dehydrogenase (EsLDH) activity have been reported (Frandsen 1968, 1970), and some preliminary results of our biochemical investigations of this enzyme were presented in abstract form (Frandsen and Cooper 1970,197l). The isoenzymic composition of LDH was recently reviewed by Latner and Skillen (1968). The nomenclature we have used for the isoenzymes is that of the Committee on Enzymes of the International Union of Biochemistry (Webb 1964). Our investigations of the isoenzymic composition of EsLDH had two objectives: (1) the determination of the isoenzymic composition ;oer se of this enzyme in order to compare it with the isoenzymic composition reported for LDH’s from other species, and (2) the study of the molecular unit composition of EsLDH via dissociation and recombination. The intention in pursuing t,he second objective was to produce dissociation and recombination of the EsLDH alone, and then subsequently to produce hybrid molecules consisting of subunits from EsLDH combined with subunits of LDH from a vertebrate. Kaplan et al. (1968) investigated the effect of prior incubation with pyridine nucleotides on the rate of interconversion of pyruvate and lactate by chicken LDH. The effect of such prior incubation on reaction rates with EsLDH was studied to obt,ain comparative information. MATERIALS
AND METHOW
Purification
Unsporulated oocysts of E. stiedae were obtained from the livers of rabbits via the initial stages (i.e., to include flotation in sodium chloride solution and subsequent washing in water) of the procedure of Wagenbach et aZ. (1966). All subsequent procedures were carried out at O-4 C. The oocysts were stored as necessary in 2% potassium dichromate solution or in deionized
391
water containing a crystal of thymol. The oocysts were subsequently washed free of the potassium dichromate or thymol and then suspended in 0.1 M sodium phosphate buffer (pH 7.5) containing 0.01 M Z-mercaptoethanoi. This buffer mixture served as the vehicle in all subsequent manipulations unless otherwise noted. The suspended oocysts were passed through a French pressure cell at lO,OOO-17,000psig. The homogenate was then centrifuged at 10,800 g for 30 min, the sediment was resuspended in buffer mixture, and this resuspended sediment was again passed through the French cell in the same manner as before. The centrifugation procedure was repeated and the sediment was discarded. A 1% solution of protamine sulfate (from salmon) in the buffer mixture was added dropwise with constant stirring at the rat,e of Z Iiter of protamine suIfate solution per 60 g protein. The solution was allowed to stand overnight, and it was then centrifuged at 13,300 g for 30 min. The sediment was discarded, and more of the 1% protamine sulfate buffer solution was added at the rate of 1 liter per 30 g of protein. After standing overnight, the solution was centrifuged at 27,000 g for 2030 min, and the sediment was discarded. Sufficient crystalline ammonium sulfate was added to the supernatant Auid to bring it to 70% of saturation. iZfter 12-19 hr, the extract was centrifuged at 27,000 g for 15-30 min. The sediment was resuspended in the buffer mixture, and the supernatant fluid was discarded. The resuspended sediment was stirred for approx 3 hr, and it was then centrifuged at 27,000 g for 20 min. The supernatant fluid now contained the enzyme, and it was at this point that the E~so/E260 ratio exceeded unity for the first time. The sediment was discarded, and the supernatant. fluid was brought to 50% of saturation with crystalline ammonium sulfate. This solution was allowed to st.and overnight, was then centrifuged at 13,300 9 for 30 min, the sediment
FRANDSEN
392
AKD COOPER
cific activity remained quite constant for many weeks at 0 C. The specific activity of more highly purified preparations decayed much more rapidly, however, and neither the rate of decay nor the activity was affected by dialysis against fresh buffer mixture. Further purification, to specific activities greater than 500, could be achieved by’ passing the enzyme at a specific activity of approx 70 through a 2.5 x 40 cm column of DEAE A-50 Sephadex.@ The Sephadex@ was prepared according to the manufacturer’s instructions and equilibrated with r/2 0.02 phosphate buffer (pH 7.5) 0.01 M for 2-SHEtOH. The enzyme in ammonium sulfate solution was desalted and placed in the 1’/2 0.02 phosphate buffer (pH 7.5) via dialysis, inserted in the column with approx 300 ml of this buffer, and the column was then developed with a linear gradient formed from 2 liters of buffer and 2 liters of buffer containing 0.2 M sodium chloride. All the solution contained 0.01 M 2-SHEtOH. Although this development produced enzyme of high specific activity, it was highly diluted and required concentration. Experiments are in progress to determine a better elution method to produce a more concentrated solution. The dilute enzyme solution was concentrated using polyacrylamide gel beads. A representative purification series is presented as Table I.
was discarded, and sufficient crystalline ammonium sulfate was then added to bring the supernatant fluid to 60% of saturation. After 3 hr the centrifugation procedure was repeated, the sediment was discarded, and the supernatant fluid was then brought to 65% of saturation with crystalline ammonium sulfate. This solution was allowed to stand overnight and was then centrifuged at 27,000 g for 30 min. The sediment was discarded and crystalline ammonium sulfate was added very slowly to the supernatant fluid until floe appeared. The procedure of centrifugation and addition of ammonium sulfate to the supernatant fluid was repeated as necessary until that point was reached where the specific activity of the supernatant fluid was in the range of 65 to 70. If the percentage of saturation with ammonium sulfate exceeded a certain value between 65 and 70, the enzyme would precipitate. If this precipitation occurred, the enzyme could be recovered via suspension of the sediment in the buffer mixture and subsequent repetition of the above fractional precipitation series. The pH of the solution during the addition of ammonium sulfate was maintained in the range 7.0-7.5 via addition of ammonium hydroxide. Activity of the enzyme was maintained best in 50%saturated ammonium sulfate provided the precipitated nonenzyme protein had been removed via centrifugation. In this environment the speTABLE Purijication Procedure
Initial extract 1st precipitation Protamine sulfate 2nd precipitation Protamine sulfate (NH&S04 70% sat (NHJzSOI 55% sat (NH&SOa 69% sat
Vol (ml)
of EsLDH
Concentration (units/ml)
I from 4 X 109 Oocysts
Total units (X 103)
Protein b-s/ml)
Specific activity (units/mg)
Yield (%)
Purification
83
60.2
5
152
28.4
4
7.68
3.7
80
3.7
186 27 28 28
21.6 125 111 103
4 3 3 2.9
4.5 9.86 5.5 1.54
4.8 12.7 20.2 67
80 60 60 58
4.8 12.7 20.2 67
58
1
100
1
Eimeria Isoenzymic
StifXb:
L-LACTATE DEHYDROGENASE
Composition
The technique of isoenzyme separation used was disc electrophoresis in polyacrylamide gel (Omstein 1964; Davis 1964)) basically the method of Diete and Lubrano (1967). The following techniques were employed to obtain dissociation and recombination of LDH subunits. All enzyme solutions were in 0.1 M sodium phosphate buffer (pH 7.5) 1 mM for 2-SHEtOH ; and, except as noted, all operations were performed at 04 C. (1) A modification of the procedures of iUarkert (1963)) Chilson et al. (1964)) and Clausen and Hustrulid (1968) using 1 M sodium chloride and EsLDH alone and EsLDH plus LD1 (pure H4 enzyme from chick heart,, courtesy of Professor N. 0. Kaplan and his colleagues). (2) EsLDH solution was dialyzed overnight against a 6 M solution of sodium chloride (Chilson et al. 1964). Following this dialysis, the salt concentration was reduced by further dialysis against phosphate buffer. All solutions were 1 mM for 2SHEtOH. (3) EsLDH alone and EsLDH plus Hq LDH (ex Kaplan et al., vide supra) in buffer were made 0.12 M for 2-SHEtOH and 10.5 M for urea according to the procedures of Epstein et al. (1964). The diluted solutions were then incubated for 5 hr at 24 C, stored at 4 C for 12 hr, and then concentrated with Aquacide I (Calbiochem, Los Angeles, CA). Samples were withdrawn from the concentrated solutions over a 72hr period and assayed for activity. (4) A solution of EsLDH in buffer was made 0.9 M for sodium chloride via slow addition of the salt. The solution was quickly frozen with carbon dioxide snow and then immediately allowed to thaw at room temperature. It was then dialyzed for 12 hr at 4 C against buffer. All solutions were 1 mM for 2-SHEtOH. As an alternate method, NADH to 1.6 X low4 M was added to the initial enzyme solution.
393
(5) EsLDH in buffer was dialyzed for 3 days against 0.1 M Tris-HCl buffer (pH 7.5) which was 1 mM for EDTA and 0.1 M for 2-SHEtOH (Chilson et al. 196513). The EsLDH remained active throughout this procedure. The solution was then made 9.6 M for urea by addition of a saturated solution of urea in buffer. After loss of enzymic activity, the enzyme solution was diluted 20-fold with buffer containing 1 mM 2SHEtOH and 1.3 mM NADH. This solution was allowed to stand at room temperature with hourly assays for LDH activity. (6’1 This method was based on the work of Epstein et ~2. (1964) and Appella and ?vIarkert (1961) using guanidine hydrochloride. (7) This was a modification of procedure 4. Sufficient sodium chloride was added to the enzyme solution to make it 1.8,3.6, and, finally, 5 M. An aliquot of each of the two lower concentrations was slowly frozen t,wice at -20 C and thawed at room temperature. The 5 M solution was frozen twice at -140 C and thawed each time at 4 C. (8) Lithium chloride as per the method of Chilson et al. (1966). (9) Concentrated hydrochloric acid was added as per the method of Clausen and Hustrulid (1968). (10) Sodium thiosulfate and sodium iodide as per the method of Chilson et at. (1965a, b) . Substrate Specificity The n-isomer of NAD was obtained through the courtesy of P-L Biochemicals, Inc., Milwaukee, WI. Its freedom from the p-isomer was proven by its inability to serve as coenzyme for the oxidat,ion of lactate by a commercial preparation of rabbit muscle LDH which had high activity with the p-isomer. The complete reaction mixture contained in addition to EsLDH r/2 0.1 glycine-sodium glycinate buffer (pH lO.O), 0.1 M L-lactate, and 8 x 10e4 M NAD. The reactions were conducted in triplicate at. 40 C with experimental controls to
394
FRANDSEN
AND
40 X lop5 M was used at constant lactate concentrations of 6,12, and 18 mlM.
establish the absence of reduction of NAD in the absence of enzyme and/or of lactate and the absence of reduction of a-NAD by commercial rabbit muscle LDH.
Coenzymes Investigations were conducted to determine the efficacy of various analogs of NAD as coenzymes for EsLDH. All reactions were run in triplicate and were monitored at 40 C, normal body temperature for’ the rabbit. The following analogs, all at 8 X 1O-4 M, were utilized in studies of the oxidation of lactate : nicotinamide-adenine dinucleotide phosphate (NADP) , 3-acetylpyridine-adenine dinucleotide phosphate (3-AADP) , 3-pyridinealdehyde-adenine dinucleotide (3-PAD), thionicotinamideadenine dinucleotide (TAD), 3-acetylpyridine-adenine dinucleotide (3-AAD), nicotinamide-hypoxanthine dinucleotide and 3-acetylpyridine-hypoxanCM=)), thine dinucleotide (3-AHD) . In addition, the reduced forms of NADP, NHD, and 3-AAD were tested for their efficacy as coenzymes in the reduction of pyruvate. The nomenclature used here for the pyridine nucleotides is that recommended by the Enzyme Commission of the International Union of Biochemistry (Dixon 1960). For the studies of the oxidation of lactate, the reactions were followed at two different acidities and at different substrate concentrations, as shown in Table II. For the reactions at pH 7.5, 0.05 M potassium
Specific Anion Effects
The effects of specific anions on the rate of reduction of pyruvate by EsLDH were ascertained by monitoring the reaction rate at 25 C in a series of buffers of constant ionic strength (0.2) and pH (6.5). In order to avoid extraneous ionic effects, neutral salts were not used to adjust ionic strengths. All assays were conducted in triplicate, using the same batch of enzyme on the same day. The ionic strength, pH, and buffers were selected to permit comparison with the data obtained on anionic effects on beef heart LDH by Winer and Schwert (1958). Kinetics
The Michaelis constants for NAD and lactate were determined by the method of Florini and Vestling (1957). All assays were performed in quintuplicate at 40 C in 1?/2 0.1 glycine-sodium glycinate buffer (pH 9.6). Neutralized hydroxylamine at 0.01 M was used as a pyruvate trap. Lithium L-lactate in concentrations of 3, 6, 12, 18, and 24 mM was used at constant NAD concentrations of 9, 18, and 36 X 10Y5 M. NAD in concentrations of 6, 9, 18, 36, and TABLE
~~
Eficacies
Pyridine nucleotide
~~ NAD NADP 3-AADP 3-PAD TAD 3-AAD NHD 3-AHD
of Pyridine
Nucleotides
m-lactate 0.013 M in phosphate buffer (pH 7.5)
COOPER
II
as Coenzymes for Oxidation m-lactate 0.1 M in phosphate buffer (pH 7.5)
of Lactate
with
EsLDH
Glycine buffer (pH 9.6)
Rate
Comparative rate
Rate
Comparative rate
Rate
Comparative rate
19.8 0
100 0
74.78 0 10.37 59.4 66.8 621 27.1 341
100 0 13.6 78.9 89.4 830 36.2 45.6
11.9 0
100 0
No data 17.63 80.25 421 14.19
89.1 404 2125 21.19 No data
No data 0.177 1.34 14.7 107.4 193.3 1467 1.44 10.9 No data
Eimeria
Stiedae: L-LACTATE DEHYDROGENASE
phosphate buffer r/2 0.087 was used in the medium, whereas for the reactions at pH 9.6 glycine-sodium glycinate buffer I’/2 0.1 was used. These buffers were chosen to facilitate comparison of the results with those published by Bonavita and Guarneri (1962), Kaplan et al. (1956), and Kaplan et al. (1960). Preincubation
with NAD and Pyruvate
Three sets of assays were performed, with three assays per set. The conditions of the respective assays were: temperature, 25 C; buffer, 0.05 M potassium phosphate (pH 7.5) ; pyruvate concentrations, 3 x low4 M; NAD concentration, 4.3 X 10V5 M; NADH concentration, 2.6 X 10e4 M. For the first set of assays, the reaction mixture consisted of buffer, pyruvate, NADH, and enzyme. For the second set of assays, the react,ion mixture consisted of NAD plus the same ingredients as for the first set of assays. Addition of the nucleotides was essentially simultaneous, and the reaction was init,iated by the addition of enzyme. For the third set of assays, enzyme was added to a mixture of buffer, pyruvate, and NAD. For this third set of assays, the reaction mixture (with enzyme) was incubated at room temperature (23 C) for 15 min. The reaction was then initiated by addition of NADH.
395
7.5-8.7, and glycine-sodium glycinate buffer, r/2 0.1 for the range pH 8.7-10.0. Hydroxylamine (neutralized) at a concentration of 0.01 M was used in all assay mixtures as a pyruvate trap, lactate was 0.1 M, NAD was 8 X 10L4 M. All assays were performed in triplicate at 25 C using the same batch of enzyme for each buffer system on the same day. The increase in absorbance at 340 nm was monitored for 90 set, and the pH was determined electrometrically at the conclusion of the assay. For studies of the reduction of pyruvate, two buffer systems were used: acetate buffer, r/2 0.1 for the pH range 3.96-6.0, and arsenate buffer, r/2 0.1 for the pH range 6-O-8.2. Pyruvate was 3 x 1O-4 M and NADH was 1.6 X 10B4 M. The assays were performed in triplicate at 25 C, and the pH was determined electrometrically at the conclusion of the 90-set monitoring of the reduction in absorbance at 340 nm. RESULTS
Isoenzymic Composition
Our electrophoretic investigations have established that the LDH from unsporulated oocysts of E. stiedae is isoenzymically homogeneous, migrating electrophoretically in the relative position of LD4. Each of the techniques described in Materials and Methods to obtain dissociation Reaction Rate as a Function of Temperaand recombination of LDH subunits were ture repeated several times, with different In the standard assay mixture [V&Z.,0.05 batches of EsLDH. In no case was combiM phosphate buffer (pH 7.5), 3.1 X 1O-4 nation of EsLDH subunits with H subunits 1M pyruvate, 1.6 x lOA M NADH], the from chick heart detected. The process of rate of conversion of pyruvate to lactate dissociation and recombination of EsLDH was monitored in triplicate at temperatures subunits was observed only twice, once with procedure 4 when t,he enzyme solution conof 25,30,35,40, and 45 C. tained NADH (Fig. I), and once with proReaction Rate as a Function of pH cedure 7 when the sodium chloride concenFor studies of the oxidation of lactate, tration had been brought to 5 M. In both three different buffer systems were used to cases, only one additional band was procover the pH range concerned: arsenate duced on electrophoresis; once this band buffer, r/2 0.1 for the range pH 6.15-7.79, was nearer the cathode than the normal sinTris-HCl buffer, r/2 0.2 for the range pH gle band, and the other time it was nearer
396
J?RANDSENAND COOPER
C--------L---
i 1 NATIVE
“Coenzyme Analogs,” EsLDH accepted certain pyridine nucleotides other than NAD as coenzymes.
/,
NAD Isomers
+-
+
I
iI
I
i-------------’
1 NATIVE
I I- --------w----
+
I
&-
FIG. 1. Zymogram, polyacrylamide gel disc electrophoresis showing new band formed by recombined subunits of EsLDH after freezing in sodium chloride solution (procedure 4 in text).
The mean reaction rates expressed in micromoles of nucleotide reduced per minute were 13.7 for /3-NAD and 0.4 for a-NAD, indicating the EsLDH catalyzed the reduction of a-NAD at approx 3% of the rate at which it catalyzed the reduction of P-NAD under the experimental conditions. A review of the literature indicates that this ability of EsLDH to use a-NAD as coenzyme is unusual (vide Kaplan and Sarma 1970). Specific Anion Effects
The data are presented in Fig. 2, where the results obtained with EsLDH are dithe anode than the normal single band. Nu- rectly compared with those of Winer and merous efforts to repeat this dissociation Schwert (1958) for beef heart LDH. The and recombination have failed. Conse- suitability of benzoate buffer for the two quently, only sodium chloride followed by LDH’s was strikingly different. Whereas freezing has produced dissociation and re- the maximal rate for beef heart LDH was combination of EsLDH, and then only on two occasions out of many. When the enzyme was exposed to high concentrations of urea, a gradual decline in activity to zero usually, but not invariably, occurred. If this loss of activity is int,erpreted as evidence of molecular unfolding, I I I I i 70.5 then the EsLDH molecule did unfold. HISTIDINE /V&./W The enzyme survived the acid treatment PHOSPHATE w,$$&$!,j 1 1 1 0139 method (procedure 9) ; electrophoresis PHTHALATE may.4 again producing only the normal single PYROPHOSPHATE wm 1 1 I,! IO187 band. The sodium thiosulfate-sodium ioSVCCINATE -pi39 dide procedure (procedure 10) produced loss of activity. 0 4 8 12 14 v
Substrate Specificity
EsLDH was specific for L-( +) lactate. The presence of D-(-) lactate concurrently with L-(+) lactate did not alter the rate of oxidation of the L-( i-) lactate, nor did prior exposure to n-( -) lactate influence the rate at which the enzyme subsequently oxidized L- (+) lactate. As discussed under
un ESLDH hz9 BEEF HEART LDH -.----V WITH TRIS-HCL
Xl0
(WINER 8 BUFFER
SCHWERT)
FIG. 2. Comparative rates of reduction of pyruvate by EsLDH and by beef heart LDH (Winer and Schwert 1958) in different buffer systems at I?/2 0.2 and 25 C. Percentages represent rate with EsLDH compared with rate with beef heart LDH. V (velocity) figures are expressed in terms of V with TrisHCl buffer = 199.
Eimeria
stiedae: L-LACTATE
in histidine buffer, this being the only buffer producing a more rapid rate than that produced by Tris-HCl, the maximal rate for EsLDH was produced by citrate buffer. Arsenate, phosphate, and pyrophosphate buffers also produced more rapid rates with EsLDH than that rate produced with Tris-HCl.
DEHYDROGENASE
397
increased again as the pH was raised from 7.5 to 9.6. (3) The comparative rate with 3-AAD
decreased nearly 3-fold at pH 7.5 as the lactate concentration increased from 0.013 to 0.1 M. This comparative rate then nearly doubled as the pH increased to 9.6 while the lactate concentration remained constant. The different ionic environments at these Kinetics two acidities must be remembered lest one The work of Coulson and Rabin (1969) attribute the different comparative reaction demonstrated that only the keto form of rates to pH effects alone. Studies of the efficacies of the three repyruvate may serve as a substrate for LDH duced pyridine nucleotides as coenzymes for as obtained from pig heart. Because prepathe reduction of pyruvate were conducted in rations of pyruvate contain a variable and 0.05 M potassium phosphate buffer (pH changing amount of the enol form of the 7.5) at a pyruvate concentration of 3 X acid, it would appear impossible to deter1O-4 M. The results of these investigations mine accurately the kinetic constants of the are summarized in Table IV. pyruvate to lactate reaction unless the conKaplan et al. (1960) have investigated centration of keto pyruvate were known the relative rates of the interconversion of precisely during the course of the reactions lactate and pyruvate by lactate dehydrounder experimental conditions. As pergenases at different substrate concentraformed by Coulson and Rabin (1969), we tions and with different coenzymes from a unsuccessfully attempted preparation of variety of vertebrates and invertebrates. pure keto pyruvate via the procedure of von For comparison with the data published by Korff (1964). Until we are able to obtain these authors, the rate ratios for EsLDH known concentrations of keto pyruvate for the requisite experiments, we will not at- for the oxidation of lactate were determined tempt determination of the Michaelis con- in phosphate buffer at pH 7.5 at two lactate concentrations; the low concentration was stants for NADH and pyruvate of EsLDH. 0.013 1M and the high concentration was 0.1 Under the experimental conditions specified in “Materials and Met,hods,” the Michaelis M. These ratios are given in Table III, calculated from the data provided in Table II. constants for lactate and NAD were 3.03 x Rate ratios for the reduction of pyruvate by 10M2M and 3.34 X 10F4 M, respectively. EsLDH with the reduced forms of nicotinamide adenine dinucleotide and nicotinCoenzymes amide hypoxanthine dinucleotide at concenComparisons of the rates vi.+a-vis those trations of 3 x 10B5 M with two different with NAD under different conditions of concentrations of pyruvate (low = 3 X 1O-4 assay revealed the following items of inter- M, high = 3 x 1O-3 M) are provided in est (Table II) : Table V, from which they also may be com(1) The rate with 3-PAD diminished pared with the data provided by Kaplan et profoundly as the acidity decreased from al. (1960). As suggested by these authors pH 7.5 to pH 9.6. (p. 392), “the catalytic technique may be (2) The rate with TAD relative to that most useful in discriminating differences bewith NAD decreased nearly 5-fold at pH tween enzymes having the same function.” 7.5 as the lactate concentration increased There are available data on these properties from 0.013 to 0.1 M. This comparative rate in respect to so comparatively few species
398
FRANDSEN AND COOPEB
EsLDH the reaction rate for the reduction of pyruvate after preincubation with pyruTwo Different Concentrations (High, 0.1 M; Low, vate and NAD was not lower than that for 0.013M) with Diferent Pyridine Nucleotides at the other two conditions of assay (vide Ma8 X 10-’ M. All data from Table ZZ terials and Methods), the rates for all nine Rate ratios Pyridine nucleotides individual assays being essentially the same. These authors concluded that their 0.047 NAD (low)/3-AAD (low) data suggested that a pyruvate-NAD com1.26 NAD (high)/3-AAD (high) plex competed with NADH for the same 0.265 NAD (low)/NAD (high) binding site on the chicken LDH molecule. 0.678 3-AAD (low)/3-AAD (high) 0.297 3-PAD (low)/3-PAD (high) On the same bases, our data would suggest 1.2 TAD (low)/TAD (high) that such competition either did not occur 0.032 NAD (low)/3-AAD (high) under the conditions of assay with EsLDH, 1.122 NAD (low)/3-PAD (low) or that such competition was so ephemeral 1.26 NAD (high)/3-PAD (high) 0.334 as to be undetectable by the monitoring NAD (low)/3-PAD (high) 5.25 3-AAD (low)/TAD (low) system.
TABLE III Relative Reaction Rates for Oxidation of Lactate at
3.98 21.3
NAD (high)/NAD (low) 3-AAD (low)/NAD (low)
Reaction Rate as a Function of Temperature
TABLE IV Eficacies of P&.%ne Nucleotides as Coenzymes for Reduction of Pgruvate with EsLDH* Molarity of pyridine nucleotide
Pyridine nucleotide
4 x 10-s 4 x 10-s
NADH NHDH NADH
1.6 X Kr4 1.6 X W4
3-AADH (1Relative
Rates:
NADH/3-AADH
fimoles of Proporpyridine tionate nucleotide rate of oxidized oxidation of per pyridine minute nucleotide 17.85
100
2.42 10.81 9.24
NADH/NHDH
13.6
TABLE V Reduction of Pyruvate with EsLDH [NADH and NHDH at 3 X 10-s M as Coenqme] Reaction Rates as Function of Pyruvate Concentrations& Coenzyme
100
85.5 =
7.37,
= 1.17.
that it would be premature to speculate as to the significance of these findings. As this is the first LDH from sporozoa to be so characterized, the catalytic properties with regard to pyridine nucleotides were recorded so that they will be available for
comparison and analysis at that future time LDH’s from the animal kingdom as a whole have been characterized to permit rational analysis from an evolutionary standpoint. Unlike the results reported with chicken LDH by Kaplan et al. (1968), with when sufficient
As illustrated by the graph in Fig. 3, the rate of conversion of pyruvate to lactate was most rapid at 4.0C, which, perhaps incidentally, is also the normal body temperature of the rabbit host. It is of interest
NADH NHDH NADH NHDH NADH
NHDH
Pyruvate concentration @f)
pmoles of coenzyme oxidized per minute
Proportionate rates
1 x 10-a 1 x 10-2 3 x 10-a 3 x 10-a 3 x 10-4 3 x 10-d
20.75
100 45.1 100
9.36 17.7
8.23 12.2
2.74
46.5 100
22.4
a Comparative Rates of Oxidation of Coenzymes : NADH (0.003M Pyruvate) NADH (0.OC!Q3 M Pyruvate) = 1’45 NADH (0.0003M Pyruvate)
NHDH (0.0603M Pyruvste) = 4*46 NHDH
(0.003 M Pyruvate) = o 46 NADH (0.003M Pyruvate) . NHDH (0.003M Pyruvate) = 3 o NHDH (0.0003M Pyruvate) ’
Eimeria
stiedae: L-LACTATE
DEHYDROGENASE
399
that the rates at 5 C below and above this optimal temperature for the reaction were only 79 and 76%, respectively, of the maximal rate. Reaction Rate as a Function
of pH
As shown in Fig. 4, the rate of oxidation of lactate increased quite uniformly with decrease in hydrogen ion concentration to approx pH 8.7, where, in glycine-sodium glycinate buffer, at least, it became quite constant as the pH increased to 10.0. The differences in rates between the buffer systems reflected specific anion effects, which were pronounced with this enzyme (vide supra), and the differences in enzyme concentration and other properties of the different batches of enzyme used with the buffer systems. The important information to be obtained from this figure is the shape of the plot for each buffer system. For the arsenate and Tris buffer systems this plot described a steady, though not always uniform, increase of rate with increase of pH. For the glycine buffer system, the reaction rate changed little as the pH increased from 8.7 to 10.0.
110 90 2 i 3
70
8 3 ‘2. I? oz IOn z”
/
GLYCINE
8
0'
6
6.8
7.6
8.4
9.2
IO
PH
FIQ. 4. Oxidation of lactate by EsLDH tl~l a function of pH in different btier systems at 25 C. Studies for each buffer system were conducted on the same day with the same enzyme preparation, but properties of the enzyme preparations were not necessarily the 8ame for all the buffer systems, and conclusiona should not be drawn from these data regarding rates in different buiTer systems at common pH.
In the case of the reduction of pyruvate, again, differences in absolute rate between the buffers caused by specific anion effects and differences in the enzyme batches were apparent, but, in contrast to the situation with the oxidation of lactate, within each buffer system the rate of the reaction decreased as the pH increased, and the maximum rate of reduction of pyruvate by EsLDH in acetate buffer appeared to be below pH 4.0. DISCUSSION
Isoenzgmic Composition
30 1 20
30 40 50 DEGREES CENTIGRADE
, 60
FIG. 3. Rate of conversion of pyruvate to lectate by EsLDH a.8 a function of temperature in 0.05 M phosphate buffer (pH 7.5).
Assuming EsLDH to be composed of 3 M subunits and 1 H subunit, as LDI from vertebrates is, then it should be possible to cause dissociation and recombination of the molecular subunits, with such recombination resulting in the appearance of additional bands on electrophoresis. These additional bands would correspond to isoen-
400
FRANDSEN
AND
COOPER
with several LDH’s from various sources 3-AAD was the most efficient coenzyme for the oxidation of lactate. As demonstrated here by the greater efficacy of 3-AHD as coenzyme compared with NHD, as well as by the paramount efficacy of 3-AAD, it was the replacement of the amino group on the third carbon of the pyridine ring by the -COCHs of acetylpyridine which was somehow responsible for marked increase of efficiency as a coenzyme. Kaplan and Sarma (1970) have discussed the physicochemical bases of the efficacies of various pyridine nucleotides as coenzymes for LDH. It is intriguing in the present case that whereas NADP and NADPH will not serve as coenzyme for EsLDH, 3AADP will so serve. This is another illustration of the fitness-producing power of the acetylpyridine ring. Because contamination of our 3-AADP by 3-AAD could explain the apparent suitability of 3-AADP as a coenzyme, the purity of our 3-AADP was checked by ascending paper chromatography on Whatman j$l paper, as kindly suggested by Dr. Coenz ymes J. M. Siegel of P-L Biochemicals, Inc. The From the data on EsLDH, several con- solvent systems were (1) isobutyric acid/ clusions may be drawn. This enzyme did ammonium hydroxide/water, 66/l/33 (pH not require a nicotinamide ring of its coen- 3.7) ; and (2) ethanol/l.0 M ammonium aczymes, accepting substitution on the third etate, 7/3 (pH 7.5). These chromatographic carbon atom of the pyridine ring of -CHO, procedures confirmed the purity of our 3-COCHa, and -CSNH2 for the -CONH2 AADP. of nicotinamide. That the --NH2 of adenine was not essential is demonstrated by the Reaction Rate as a Function of pH The general picture for the rates of interacceptability of NHD, with its hydroxyl group at the C6 position of the purine ring, conversion of lactate and pyruvate as funcas a coenzyme. The rate of oxidation of tions of the pH by EsLDH is generally as lactate with NHD was only about one- would be expected from studies on these quarter to one-third that with NAD at pH properties of LDH from other organisms. 7.5, and one-tenth that at pH 9.6, however, The principal point of difference between’ indicating that for some reason NHD was our results and those of Winer and Schwert (1958) working with beef heart LDH is not as eflicient a coenzyme as was NAD. It is noteworthy that when the -CONH, of that they observed a marked reduction in the nicotinamide ring of NHD was replaced the rate of oxidation of lactate as the pH with -COCHQ to form 3-AHD, the rate of increased from 9.0 to 10.0, whereas we observed a slight increase in the rate from pH’ oxidation of lactate increased la-fold. A survey of the literature revealed that 9.0 to pH 9.3 and then a slight decrease in zymes LDI, LDZ, LD3 and LDs. The LDI isoenzyme would be expected to be present in greatest amount due to incomplete dissociation, and the other isoenzymes in decreasing amounts would be LDr,, LDS, LDZ, and LD1. If the subunits of EsLDH are similar to the subunits of vertebrate LDH isoenzymes, then it should be possible to form hybrid active tetramers. Inability to produce hybrids between the subunits of different LDH’s has been reported previously (Salthe et al. 1965)) and it may indeed be that subunits of EsLDH cannot combine with H subunits from chick heart LDH to produce active units of enzyme. The apparent unusual tenacity of attachment of subunits of EsLDH, their resistance to most of the treatments which other workers have found successful in dissociating subunits of other LDH’s, their lack of hybridization with H subunits from chick heart LDH, and the anomalous electrophoretic results may indicate that EsLDH is of different subunit construction than the LDH’s so characterized from vertebrates.
Eimeriu
stiedue: L-LACTATE DEHYDROGENASE
401
the rate as the pH increased further to 10.0. This difference might be accounted for by the differences in the buffer systems used. We used glycine-sodium glycinate buffer in this pH range whereas Winer and Schwert used Tris-HCI buffer.
CHILSON, 0. P., COSTELLO,L. A., AND KAPLAN, N.
Physiological
1006-1014. CHILSON, 0. P., KITTO, G. B., PUDLES, J., AND inactivation KAPLAN, N. 0. 1966. Reversible
Function
Since the work of Warburg (1924), biochemists have been especially interested in those cells which denend on LDH for replenishment of the NAD pool in glycolysiS, for in such cells LDH would be a vital enzyme. We have established (unpublished data) cytochemically that unsporulated oocysts of E. stiedae contain a-glycerophosphate dehydrogenase, however; and replenishment of the intracellular pool of NAD may consequently be accomplished through the reduction of dihydroxyacetone phosphate by cytoplasmic a-glycerophosphate dehydrogenase as in certain mammalian cells. ACKNOWLEDGMENT The authors acknowledge the generous gift of H, LDH from chick heart by Professor N. 0. Kaplan and Dr. F. Stolzenbach of the University of California at San Diego, the generous gift of c+NAD by Dr. J. Siegel of P-L Biochemicals, Inc., Milwaukee, WI, and the expert technical assistance of Mr. Mark R. Kearse, Mr. Thomas H. Ennis, Mr. William A. Wilder, Miss Margaret 0. Haven, and Miss Judy Jones.
REFERENCES APELLA, E., AND MARKERT, C. L. 1961. Dissociation of lactate guanidine Biophysicial 171-176.
dehydrogenase into subunits hydrochloride. Biochemical Research Communications
BONAVITA,V., AND GUARNERI,R. 1962. Lactic
with and 6,
dehydrogenase isozymes in the nervous tissue. I. The reaction of isozymes with diphosphopyridine nucleotide analogues and their inhibition by sodium metabisulfite. Biochimica et Biophysics Acta 59,634-642. CHILSON, 0. P., COSTELLO,L. A., AND KAPLAN, N. 0.1964. Hybridization of lactic dehydrogenases in vitro by concentrated sodium chloride and by reversible inactivation in urea. Journal of Molecular Biology 10,349-352.
0. 1965a. Studies on the mechanism of hybridization of lactic dehydrogenases in vitro. Biochemistry 4 271-281. CHILSON, 0. P., KITTO, G. B., AND KAPLAN, N. 0. 1965b. Factors affecting the reversible dissociation of dehydrogenases. Proceedings of the National Academy of Sciences U5. 53,
of dehydrogenases. Journal of Biological Chemistry 241,2431-2445. CLAUSEN, J., AND HUSTRULID, R. 1968. Factors affecting recombination of lactate dehydrogenase isoenzyme subunits. Biochimica et Biophysics Acta 167,221-226. COULSON, C. J., AND RABIN, B. R. 1969. Inhibition of lactate dehydrogenase by high concentrations of pyruvate: the nature and removal of the inhibitor. FEBS Letters 3,333-337. DAVIS, B. J. 1964. Disc electrophoresis-II. Method and application to human serum proteins. Annals of the New York Academy of Sciences
121,404-427. DIETZ, A. A., AND LUBRANO,T. 1967. Separation and quantitation of lactic dehydrogenase isoenzymes by disc electrophoresis. Analytical Biochemistry 20,246-257. DIXON, M. 1960. Nomenclature of the nicotinamide nucleotide coenzymes. Nature 188,
464-466. EPSTEIN, C. J., CARTER,M. M., AND GOLDBERGEB, R. F. 1964. Reversible denaturation of rabbitmuscle lactate dehydrogenase. Biochimica et Biophysics Acta 92,391-394. FLORINI, J. R., ANDVESTLINC,C. S. 1957. Graphical determination of the dissociation constants for two-substrate enzyme systems. Biochimica et Biophysics Acta 25,575-578. FRANDSEN,J. C. 1968. Eimeria stiedae: Cytochemical identification of acid and alkaline phosphatases, carboxylic ester hydrolases, and succinate, lactate, and glucose&phosphate dehydrogenases in endogenous stages from rabbit tissues. Experimental Parasitology 23, 393411. FRANDSEN,J. C. 1970. Eimeria .&due: Cytochemical identification of enzymes and lipids in sporozoites and endogenous stages. Experimental Parasitology 27, 106115. FRANDSEN,J. C., AND COOPER,J. A. 1970. Some properties of L-lactate dehydrogenase from Eimeria stiedae (Protozoa: Coccidia). ASB Bulletin 17,43. FRANDSEN,J. C., AND COOPER,J. A. 1971. Some properties of n-lactate dehydrogenase from
402
FRANDSEN
Eimeria stiedae (Protozoa : Coccidia) . II. Further studies of the semi-purified enzyme. ASB Bulletin 18,35. KAPLAN, N. O., CIOTTZ, M. M., HAMOLSKY, M., AND B~BER, R. E. 1960. Molecular heterogeneity and evolution of enzymes. Science 131, 392
AND COOPER
isozymes: dissociation and recombination of subunits. Science 140,1329-1330. ORNSTEIN, L. 1964. Disc electrophoresis-I. Background and theory. Annals of the New York Academy of Sciences 121,321-%t9. SALTHE, S. N., CHILSON, 0. P., AND KAPLAN, N. 0. 397. 1965. Hybridization of lactic dehydrogenase KAPLAN, N. O., CI~II, M. M., AND STOLZENBACH, in vivo and in vitro. Nature 207,723726. F. E. 1956. Reaction of pyridine nucleotide VON KORFF, R. W. 1964. Pyruvate-C”, purity and stability. Analytical Biochemistry 8, 17~ analogues with dehydrogenaees. Journal of Biological Chemistry 221,833-8~. 178. KAPLAN, N. O., EVERSE, J., AND ADM~AAL, J. 1968. WAQENBACH, G. E., CHALLEY, J. R., AND BURNS, W. Significance of substrate inhibition of deC. 1966. A method for purifying coccidian hydrogenases. Annals of the New York Acadoocysts employing Clorox and sulfuric acidemy ofSciences 151,400412. dichromate solution. Journal of Parasitology 52,1222. KAPU, N. O., AND SARMA, R. H. 1970. The structure of pyridine coenzymes as related to bindWARBURQ,0. 1924. “Uber den Stoffwechsel der ing. In “Pyridine Nucleotide-dependent DeTumoren.” Springer-Verlag, Berlin. hydrogenases” (H. Sund, ed.), pp. 39-56. WEBB, E. C. 1964. Nomenclature of multiple Springer-Verlag, Berlin. enzyme forms. Nature 203,821. LATNER, A. L., AND SKILLEN, A. W. 1968. “IsoenWINER, A. D., AND SCHWERT, G. W. 1958. Lactic zymes in Biology and Medicine.” Academic dehydrogenases. IV. The influence of pH on the kinetics of the reaction. Journal of BioPress, New York. MARKEBT, C. L. 1963. Lactate dehydrogenase logical Chemistry 231,1065-1083.