- Email: [email protected]
Lactate dehydrogenase from the caudal muscle of the shrimp Palaemon serratus: purification and characterization
Comp. Biochem. Physiol. Vol. 68B. pp. 65 to 70
0305-0491/81/0101-006550200/0
© Pergamon Press Ltd 1981. Printed in Great Britain
LACTATE DEHYDROGENASE FROM THE CAUDAL MUSCLE OF THE SHRIMP PALAEMON SERRATUS: PURIFICATION AND CHARACTERIZATION M. T. THEBAULT,A. BERN1CARD and J. F. LENNON Laboratory of Marine Biology, College de France, BP 38, 29182 Concarneau, France
(Received 2 May 1980) 1. Palaemon serratus lactate dehydrogenase (L-lactate: NAD ÷ oxidoreductase, EC 1.1.1.27) has been purified over 900 fold with an overall yield of 10-15~o. Gradient polyacrylamide gel electrophoresis yielded two bands, both exhibiting LDH activity. SDS gel electrophoresis yielded one single protein band corresponding to a monomer molecular weight of 36,000. The molecular weight of the tetramer was 140,000. 2. The purified enzyme exhibits non-Michaelian kinetic behaviour and displays negative cooperativity. The K,, values are 0.24 mM and 16 mM respectively for pyruvate and lactate and are independent of temperature in a 10 to 30°C range. The enzyme is inhibited by 3-acetyl-pyridine NAD in a non competitive way. 3. The kinetic properties of the enzyme are discussed with respect to the possibility of conformational changes. Abstract
Concarneau. The caudal muscle was dissected (about 200 g fresh weight)and frozen at -10°C. Sodium pyruvate, NAD, NADH, D-lactate, Phenazine methosulfate (PMS), MTT tetrazolium, 3-acetyl-pyridine NAD, were obtained from Sigma, and L-lactic acid from Merck.
INTRODUCTION
Lactate dehydrogenases from a wide variety of animals and tissues have been studied in great detail. Those of Crustaceans (Gleason etal., 1971) are generally tetrameric. A few enzymes catalyse the conservation of D-lactate (Balanus nubilis, Ellington & Long, 1979), although the majority are specific for L-lactate. Kinetic studies of these enzymes show the existence of two types of reactions: one is inhibited by an excess of pyruvate in vitro, essentially in aerobic tissues, and the second converts pyruvate to lactate in anaerobic tissues. The distinction between M a n d H types appears to be difficult as in the case of the L D H from vertebrates, particularly for organisms which possess a single enzyme having to a d a p t to aerobic a n d anaerobic metabolisms. Traush (1976) has shown that in the tail muscle of Homarus vulgaris, L D H could function like the M type or the H type, according to temperature. These kinetic properties, allow the maintenance of a good level of anaerobic metabolism for relatively high temperatures. Concerning the L D H of Palaemon serratus, we have previously studied the properties relative to temperature variations (Thebault & Le Gal, 1977, 1978; Thebault et al., 1980). The existence of compensation mechanisms for the enzyme substrate affinity was demonstrated. We have also described non-Michaelien kinetics. Substrate saturation curves allow the m e a s u r e m e n t of two values of app. Kin. These studies were performed with low substrate concentrations in order to analyze the regulatory properties of the enzyme. In this paper we study some of the properties of the purified enzyme under optimal conditions.
Methods LDH was assayed in a Gilford recording spectrophotometer. The LDH activity for pyruvate reduction was assayed in a system containing NADH (from 0.05 mM to 0.5 mM) and sodium pyruvate (from 0.05 mM to 0.5 raM) with 0.1 M sodium phosphate buffer (pH 7.0). LDH activity for lactate oxidation was assayed in a system containing NAD (from 0.5 M to 2 mM) and sodium lactate (from 5 mM to 100 mM) with 0.1 M glycine-NaOH buffer (pH 10). The total volume of each assay was 2 ml. Protein determination was carried out by the method of Lowry et al. (1951). Analytical gel electrophoresis Polyacrylamide gel electrophoresis was performed according to the procedure of Shuster (1971 ). Electrophoresis was carried out at 4°C in 6 ~ polyacrylamide gels (d = 6 mm l = 160 mm) with Tris glycine buffer (pH 8.0). A constant current of l mA per gel was applied for 15 hr. The proteins were stained with Amido Black or Coomassie Blue R-250. The LDH was located with a specific coloration using 1 mg/ml MTT, 0.1 mg/ml PMS, l mg/ml NAD and 0.5 M L-lactate, in 0.05 M sodium phosphate buffer (pH 7.6). After incubation the gels were fixed in acetic acid and scanned at 540 nm using an Isco gel scanner. Sodium dodecyl sulfate (SDS) polyacrylamide gel eiectrophoresis was performed according to the method of Lambin (1978). Bovine serum albumin (68,000), aspartate transcarbamylase (17,000, 33,000), Rabbit M4 LDH (35,000) as reference proteins, and the LDH of Palaemon serratus were incubated at 100°C in 0.1 M sodium phosphate buffer (pH 7.0) containing 0.2~ SDS and 3~o Mercaptoethanol for 20sec. Gradient polyacrylamide Gradipore gels 5-20~o were used for the electrophoresis carried out at room temperature for 3 hr at 150 V.
MATERIAL A N D M E T H O D S
Material Shrimps Palaemon serratus, were collected in the bay of 65
66
M.T. THEBAULT, A. BERNICARD and J. F. LENNON 280
nm abs
340nmabs
~
3o
~o
IO
"N,. i
20
30
50
Vml
Fig. 1. AffÉnity chromatography on 5' AMP Sepharose. t 280 nm absorption; (3 LDH activity at 340 nm.
The mixture was then stirred for 1 hr and centrifuged for 15 min. This procedure eliminated muscle pigments. Sephadex G200 chromatography. The extract was filtered on an Amicon XM 50 membrane in order to concentrate the enzyme fraction and the extract was applied on a Sephadex G200 column previously equilibrated with the extraction buffer. Affinity chromatography. The extract was further purified by affinity chromatography on 5' AMP Sepharose. The column was equilibrate first with 0.01 M sodium phosphate buffer (pH 7.0) with 0.25 mM PMSF and 0.5 M mercaptoethanol. The column was eluted with the same buffer containing 0.25 mM NADH. 0.4ml fractions were collected. The purified LDH was then concentrated on an Amicon XM 50 membrane. The purification factor reached at the final stage of the purification is routinely over 900 for an average yield of
]0-15Vo. The final step involved affinity chromatography on 5' Amp Sepharose (Fig. 1) and resulted in a major increase in specific actiyity (Table 1). The purified enzyme was electrophoresed on a poly acrylamide gel gradient 5-20% for the control of the purification (Fig. 3) with Amido Black. During and after purification, the LDH from the caudal muscle showed a two banded profile with polyacrylamide gel electrophoresis (Fig. 2).
U V spectrum The ultraviolet absorption spectrum was performed with a Cary 14 spectrophotometer. Fluorescence spectrum The fluorescence spectrum was performed with an Aminco-Bowman ratio spectrofluorimeter with an exitation light at 280 nm. Enzyme purification All preparation procedures were carried out at 0-4°C. Crude extract. 200 g of caudal muscle were homogenized in 5 vol of 0.05 M sodium phosphate buffer (pH 7.5) containing 0.5 mM PMSF and 0.25 mM mercaptoethanol in an Ultraturrax for 60 sec. The homogenate was centrifuged for 20 rain at 37,000g and the supernatant was collected. The pellet was then homogenized and centrifuged in the same conditions. The two supernatants were pooled and saved. Ammonium sulfate fractionation. The supernatant was brought to 60~ saturation with powdered (NH4)2SO4. The solution was stirred for 45 rain. and centrifuged at 12,000 0 for 30min. The pellet was suspended in a minimum volume of the extraction buffer and dialyzed against the same buffer overnight, Hydroxyapatite batch. Hydroxyapatite, prepared in the laboratory according to the method of Tiseliues-Bernardi et al. (1956) and extracts were mixed volume to volume.
Fig. 2. 6~o polyacrylamide gel electrophoresis of purified LDH of Palaemon serratus caudal muscle stained with Amido Black.
Table I. Control of the purification
Extract Crude extract Hydroxyapatite (NH4)2SO4 Biogel P 200 5' AMP Sepharose
A/ml AOD 340 rm/mn/ml 3.8 2.15 33.2 40 145
Protein (mg/ml)
Specific Activity A/ml/mg prot sol.
Volume (ml)
10.7 4.8 12.1 7.4 0.45
0.36 0.45 2.74 5.4 322.2
1010 1650 90 40 4
Total Activity A/ml × Vml 3850 3547.5 2990 1600 580
Rendt
(%)
100 92.14 77.66 41.56 13.3
LDH from shrimp muscle
67
A~
alO
300
290
280 270
260
250
240 rim
Fig. 5. Ultraviolet spectrum of LDH in 0.01 M sodium phosphate buffer pH 7.5 at 18°C.
Fig. 3. Polyacrylamide gel gradient (5-20%) stained with Amido Black.
The purified enzyme was kept in 0.01 M sodium phosphate buffer (pH 7.0) with 20% glycerol. The activity remained almost constant for about 2 months; it was however not possible to keep the enzyme by freezing, for it was immediately inactivated.
firmed by gel gradient electrophoresis (5-20%). Here again it is possible to separate two distinct bands (Fig. 3). In contrast, the SDS gradient electrophoresis yield one single band corresponding to a molecular weight of 35,000 (Fig. 4).
u.v. Spectrum The u.v. spectrum of an 0.05 mg solution of enzyme shows a shoulder at 295-300nm like the Human heart LDH (Nisselbaum & Bodansky, 1961) (Fig. 5), which corresponds to the absorption of tryptophane.
RESULTS
Physicochemical properties
Fluorescence spectrum
The molecular weight of the purified enzyme has been measured by chromatography on Sephadex G200. The value obtained for the native enzyme ranges around 140,000. This value is further con-
The wavelength of the emission maximum of the native enzyme was at 335 nm, approx similar to other dehydrogenases, which correspond to tryptophane fluorescence (Fig. 6). IOC
8C
6C
8 c
~_ 4c
Wavelength
Fig. 4. Polyacrylamide gel gradient (5-20%) electrophoresis in dissociating conditions (SDS) stained with Coomassie Blue.
Fig. 6. Emission spectrum of LDH in 0.01 M sodium phosphate buffer pH 7:5 at the excitation wavelength of 280 nm, at 18°C.
68
M.T. THEBAULT,A. BERNICARDand J. F. LENNON Table 3. Values of the Hill number for Palaemon serratus LDH
Vu/mn
Substrate 5OO
30C
I0¢
,
,
6
i
8
no
pH
Fig. 7. Effect of pH on the activity of the LDH: O with pyruvate as substrate; • with lactate as substrate. Initial velocity is plotted against increasing pH values.
Catalytic properties Effect of pH. Fig. 7 shows the pH profile between pH 6.0 to pH 11 for the forward and the reverse reactions. An optimum at pH 9.5 was found for the lactate oxidation and at pH 7.8 for the pyruvate reduction. Specificity. The LDH of the caudal muscle of Palaemon serratus appears to be specific of L-lactate acid. There is no activity with D-lactate as substrate. Kinetic properties. The values of the apparent Michaelis constants (KIn) were determined from the Lineweaver-Burk plots (Table 2); at saturation of coenzyme (2raM NAD and 0.2mM NADH) we measured two values of K,, for lactate and pyruvate as we had previously observed with low concentrations of coenzymes (Thebault & Le Gal (1978). There is only one value of K,, for N A D and N A D H (with 100 mM lactate and 1 m M pyruvate). Owing to the fact that two distinct parts are observed in the substrate saturation curves, we can measure two values for lactate and pyruvate. The Hill numbers have been determined graphically for the forward and reverse reaction, using the relation:
v. yo-yo.
-
-
=
n log
S.
For low concentrations of substrate the Hill number is less than 1. For high concentrations of substrate, the Hill number is greater than 1. There is only one value for N A D and N A D H which is approx 1.20 and 1.80 (Table 3). The two Hill numbers observed with lactate and pyruvate can be set in relation with the two K,, values Table 2. Values of apparent K,. for Palaemon serratus LDH Substrate Pyruvate Lactate NAD NADH
K,. (mM) at 20°C 0.24 16 0.24 0.17
0.086 3.70 ---
Pyruvate Lactate NAD NADH
Hill number 0.85 0.77 1.17 1.80
2.16 1.32 --
previously determined. Similar results were obtained with the Human heart LDH (Nisselbaum & Bodansky, 1961) and with several enzymes (Alaskan King Crab pyruvate kinase, Somero, 1969); (cytidine triphosphate synthetase, Teipel & Koshland, 1969). According to Conway & Koshland (1968), these curves are the result of a negative cooperativity, where successive fixations of substrate could produce binding sites with a different conformation, having a lower affinity, but a high turnover number. This could explain the negative cooperativity observed with low substrate concentrations and the positive cooperativity observed when the substrate concentrations increased. However, the enzyme was a better catalyst with pyruvate as substrate, and we observed a substrate inhibition above 5 mM pyruvate, similar to the H LDH of vertebrates (Fig. 8). These results are in agreement with those of Traush (1976) and Gade (1979). The variation of the K,, values, for substrate concentrations greater than the value of the K,,, as a function of the incubation temperature, showed a temperature independence from 10 to 30°C (Fig. 9). When the concentration of substrate were lower (about the K,, value), the temperature modulation was more effective (Thebault & Le Gal, 1979; Thebault et al., 1979).
Effect of inhibitors The effect of 3-acetyl pyridine NAD, and NAD analogue, was studiedby increasing inhibitor concentration, with lactate as substrate. The apparent Ki and the K,, had the same value, and the inhibition was non-competitive (Fig. 10). We can notice that the presence of this inhibitor does not suppress the break in the Lineweaver-Burk plot for a value of lactate equal to the K,. for this substrate. The negative cooperativity is then maintained. The effect of urea investigated, the inhibitory effect of urea, expressed with respect to the activity in the Spe Act
/ "%'°~° / PYR mM
Fig. 8. Saturation curves for the pyruvate at 15°C. Initial velocity is plotted against increasing pyruvate concentrations.
69
LDH from shrimp muscle
KmmM
main conclusions: the first one is that the enzyme is likely to be a tetramer as the subunit molecular weight observed in the presence of SDS of 35,000 leads a final molecular weight of 140,000. The second one is that this apparent lack of homogeneity of the purified enzyme as judged by polyacrylamide gel electrophoresis does not appear to be consequence of a fundamental difference in the primary sequence of the two species. Our results show a substrate stereospecificity for L-lactate and catalytic properties similar to that observed in the muscle type LDH with respect to the K,, values for lactate and pyruvate and the pH optimum, and in the heart type LDH for the vertebrates with respect to the inhibition by pyruvate. This is the case of many LDH so far studied in marine invertebrates (Molluscs and Crustaceans). This kinetic behaviour is consistent with the lack of isoenzymes in different tissues. In Palaemon serratus the same enzyme would have to function like the vertebrate M and H type of LDH. We have previously reported (Thebault & Le Gal, 1978; Thebault et al., 1979) a temperature compensation process of the apparent Km with respect to the variations of external temperature. The amount of the apparent Km values with non saturation values of coenzymes did not vary significantly throughout the temperature range of the natural habitat of the shrimp, and increased abruptly near the lethal limit for this species. This is figured by a U-shaped curve of the apparent Kr, variation versus the assay temperature. When the coenzyme is utilized at saturation, the Km is temperature independent from 8 to 30°C. Hochachka & Somero (1976) showed the importance of non-saturating values of substrate for the modulation of enzyme activity. We have previously observed a break in the Lineweaver-Burk plots occurring at low substrate concentration. The Hill plots with pyruvate or lactate as substrate exhibits a break corresponding to the Km value for this substrate. At coenzyme saturation, these breaks are less obvious. We have described this phenomenon as the result of conformational changes of the enzyme (Thebault & Le Gal, 1977). An increase in buffer molarity, assay temperature and substrate concentration leads to an
50
J 25
•
,b
--o /
20
~)
I."[*
410
(a)
KmmM
__O IO
20
30
I.T*
40
(b) Fig. 9. Variations of the Km values as a function of the incubation temperature: (a) lactate as substrate; (b) pyruvate as substrate.
presence of the inhibitor, increased slowly until a concentration of 2 H urea. Then, the inhibition became very important (Fig. 11).
DISCUSSION The LDH from the tail muscle of Palaemon serratus obtained with a 900-fold purification yields after polyacrylamide gel electrophresis, a two banded pattern. The two presumptive molecular species are, however, resolved in one unique band when electrophoresed under dissociation conditions. This result leads to two I
=~~~~~=i0.25
mM
0,8
°~ ° ~
I = 0.10 m M
o
°"
I=OmM
,
OI
,
0.2
I 0.3
I lact mM
Fig. 10. Lineweaver-Burke plot for lactate with increasing concentration of 3-acetyl-pyridine NAD.
70
M.T. THEBAULT, A. BERNICARDand J. F. LENNON REFERENCES
30
20
I
I
I
2
3
--I
4
Ureo Mol
Fig. 11. Inhibition by urea: initial velocity without urea versus velocity in the presence of urea is plotted against increasing urea concentration.
attenuation of the m a r k e d frontier between the two parts of the Hill plots, or an attenuation of the intermediary plateau of the saturation curves, affecting the conformational flexibility of the protein. The conformational flexibility of the caudal muscle L D H could be related to the presence of two b a n d s in the electrophoretic pattern of the purified enzyme. These two bands could be two conformational variants of the same protein. But they could be products of two different genes or epigenetic transformation too. The conformational flexibility of the muscle L D H of Palaemon serratus, depending to external factors (temperature or cellular factors, substrate or coenzyme concentration) could be a possible means of regulation of the enzyme activity in relation with the variations of the modifications of the environment. The study of the transconformations of the Palaemon serratus L D H is under investigation. Acknowled(tements--Helpful discussion with Dr Y. Le Gal is fully acknowledged. We are indebted to Professor B. S. C. Leadbeater for correcting the manuscript. This work has been supported by the Coll6ge de France and by the Centre National de la Recherche Scientifique.
CONWAV A. & KOSLAND D. E. (1968) Negative cooperativity in enzyme action. The binding of diphosphorydine nucleotide to glyceraldehyde-3-P dehydrogenase. Biochemistry, 7, 4011~-022. ELLINGTON W. R. & LONG G. L. (1978) Purification and characterization of a highly unusual tetrameric D-LDH from the muscle of the giant barnacle. Archs Bioehem. Biophys. 186, 265 274. GAOL G. (1979) L-lactate specific lactate dehydrogenase from the mantle muscle of the Squid Loligo vulgaris: purification and catalytic properties. Comp. Biochem. Physiol. 63B, 387-395. GLEASON F. n., PRICE J. S., MANN R. A. & STUART T. D. (1971) Lactate dehydrogenases from Crustaceans and Arachnids. Comp. Biochem. Physiol. 40B, 387 394. HOCHACHKA P. W. & SOMERO G. N. /1976) Adaptation to Environment, pp, 125 190 Butterworths, London. LAMBIN P. (1978) Reliability of molecular weight determination of proteins by polyacrylamide gradient gel electrophoresis in the presence of sodium dodecyl sulfate. Analyt. Biochem. 85, 114 125. LOWRY O. H., ROSENBROt!GH N. H. & RANDALL (1951) Protein measurement with the Folin-phenol reagent. J. biol. Chem. 193, 265-275. NISSELBAUM J. S. & BO|3ANSKYO. (1961) Purification and properties of Human heart LDH. J. biol. Chem. 236, 323-327. SntJSTER L. (1971) Preparation acrylamide gel electrophoresis. Meth. Enzym. XXll, 412 437. SOMERO G. N. (1969) Pyruvate kinase variants of the Alaskan King Crab. Biochem. J. 114, 237 241. TEIPEL J. & KOSLAND D. E. (1969) The significance of intermediary plateau regions in enzyme saturation. Biochemistry, 8, 4656~4663. THE~AULJ" M. T. & LEGAL Y. (1976) Studies on lactate dehydrogenase activity on the Shrimp Palaemon serratus. Part I, Characterisation of the enzymes from the caudal muscle. Biochem. Syst. Ecol. 5, 155-159. THEaAULT M. T. & LE GAL Y. (1978) Studies on lactatedehydrogenase activity in Palaemon serratus. Effect of the incubation temperature on the kinetic properties of thc enzyme system from the caudal muscle. Bioehem. Syst. Ecol. 6, 141-144. THEBAULT M. T., BERNICARD A. & LEGAL Y. (1980) Effect of acclimation on lactate dehydrogenase activity in Palaemon serratus. Comp. Biochem. Physiol. 65B, 357-361. TRAUSH G. (1976) Effect of temperature upon catalytic properties of lactate dehydrogenase in the lobster. Biochem. Syst. Ecol. 6, 65 68.
0305-0491/81/0101-006550200/0
© Pergamon Press Ltd 1981. Printed in Great Britain
LACTATE DEHYDROGENASE FROM THE CAUDAL MUSCLE OF THE SHRIMP PALAEMON SERRATUS: PURIFICATION AND CHARACTERIZATION M. T. THEBAULT,A. BERN1CARD and J. F. LENNON Laboratory of Marine Biology, College de France, BP 38, 29182 Concarneau, France
(Received 2 May 1980) 1. Palaemon serratus lactate dehydrogenase (L-lactate: NAD ÷ oxidoreductase, EC 1.1.1.27) has been purified over 900 fold with an overall yield of 10-15~o. Gradient polyacrylamide gel electrophoresis yielded two bands, both exhibiting LDH activity. SDS gel electrophoresis yielded one single protein band corresponding to a monomer molecular weight of 36,000. The molecular weight of the tetramer was 140,000. 2. The purified enzyme exhibits non-Michaelian kinetic behaviour and displays negative cooperativity. The K,, values are 0.24 mM and 16 mM respectively for pyruvate and lactate and are independent of temperature in a 10 to 30°C range. The enzyme is inhibited by 3-acetyl-pyridine NAD in a non competitive way. 3. The kinetic properties of the enzyme are discussed with respect to the possibility of conformational changes. Abstract
Concarneau. The caudal muscle was dissected (about 200 g fresh weight)and frozen at -10°C. Sodium pyruvate, NAD, NADH, D-lactate, Phenazine methosulfate (PMS), MTT tetrazolium, 3-acetyl-pyridine NAD, were obtained from Sigma, and L-lactic acid from Merck.
INTRODUCTION
Lactate dehydrogenases from a wide variety of animals and tissues have been studied in great detail. Those of Crustaceans (Gleason etal., 1971) are generally tetrameric. A few enzymes catalyse the conservation of D-lactate (Balanus nubilis, Ellington & Long, 1979), although the majority are specific for L-lactate. Kinetic studies of these enzymes show the existence of two types of reactions: one is inhibited by an excess of pyruvate in vitro, essentially in aerobic tissues, and the second converts pyruvate to lactate in anaerobic tissues. The distinction between M a n d H types appears to be difficult as in the case of the L D H from vertebrates, particularly for organisms which possess a single enzyme having to a d a p t to aerobic a n d anaerobic metabolisms. Traush (1976) has shown that in the tail muscle of Homarus vulgaris, L D H could function like the M type or the H type, according to temperature. These kinetic properties, allow the maintenance of a good level of anaerobic metabolism for relatively high temperatures. Concerning the L D H of Palaemon serratus, we have previously studied the properties relative to temperature variations (Thebault & Le Gal, 1977, 1978; Thebault et al., 1980). The existence of compensation mechanisms for the enzyme substrate affinity was demonstrated. We have also described non-Michaelien kinetics. Substrate saturation curves allow the m e a s u r e m e n t of two values of app. Kin. These studies were performed with low substrate concentrations in order to analyze the regulatory properties of the enzyme. In this paper we study some of the properties of the purified enzyme under optimal conditions.
Methods LDH was assayed in a Gilford recording spectrophotometer. The LDH activity for pyruvate reduction was assayed in a system containing NADH (from 0.05 mM to 0.5 mM) and sodium pyruvate (from 0.05 mM to 0.5 raM) with 0.1 M sodium phosphate buffer (pH 7.0). LDH activity for lactate oxidation was assayed in a system containing NAD (from 0.5 M to 2 mM) and sodium lactate (from 5 mM to 100 mM) with 0.1 M glycine-NaOH buffer (pH 10). The total volume of each assay was 2 ml. Protein determination was carried out by the method of Lowry et al. (1951). Analytical gel electrophoresis Polyacrylamide gel electrophoresis was performed according to the procedure of Shuster (1971 ). Electrophoresis was carried out at 4°C in 6 ~ polyacrylamide gels (d = 6 mm l = 160 mm) with Tris glycine buffer (pH 8.0). A constant current of l mA per gel was applied for 15 hr. The proteins were stained with Amido Black or Coomassie Blue R-250. The LDH was located with a specific coloration using 1 mg/ml MTT, 0.1 mg/ml PMS, l mg/ml NAD and 0.5 M L-lactate, in 0.05 M sodium phosphate buffer (pH 7.6). After incubation the gels were fixed in acetic acid and scanned at 540 nm using an Isco gel scanner. Sodium dodecyl sulfate (SDS) polyacrylamide gel eiectrophoresis was performed according to the method of Lambin (1978). Bovine serum albumin (68,000), aspartate transcarbamylase (17,000, 33,000), Rabbit M4 LDH (35,000) as reference proteins, and the LDH of Palaemon serratus were incubated at 100°C in 0.1 M sodium phosphate buffer (pH 7.0) containing 0.2~ SDS and 3~o Mercaptoethanol for 20sec. Gradient polyacrylamide Gradipore gels 5-20~o were used for the electrophoresis carried out at room temperature for 3 hr at 150 V.
MATERIAL A N D M E T H O D S
Material Shrimps Palaemon serratus, were collected in the bay of 65
66
M.T. THEBAULT, A. BERNICARD and J. F. LENNON 280
nm abs
340nmabs
~
3o
~o
IO
"N,. i
20
30
50
Vml
Fig. 1. AffÉnity chromatography on 5' AMP Sepharose. t 280 nm absorption; (3 LDH activity at 340 nm.
The mixture was then stirred for 1 hr and centrifuged for 15 min. This procedure eliminated muscle pigments. Sephadex G200 chromatography. The extract was filtered on an Amicon XM 50 membrane in order to concentrate the enzyme fraction and the extract was applied on a Sephadex G200 column previously equilibrated with the extraction buffer. Affinity chromatography. The extract was further purified by affinity chromatography on 5' AMP Sepharose. The column was equilibrate first with 0.01 M sodium phosphate buffer (pH 7.0) with 0.25 mM PMSF and 0.5 M mercaptoethanol. The column was eluted with the same buffer containing 0.25 mM NADH. 0.4ml fractions were collected. The purified LDH was then concentrated on an Amicon XM 50 membrane. The purification factor reached at the final stage of the purification is routinely over 900 for an average yield of
]0-15Vo. The final step involved affinity chromatography on 5' Amp Sepharose (Fig. 1) and resulted in a major increase in specific actiyity (Table 1). The purified enzyme was electrophoresed on a poly acrylamide gel gradient 5-20% for the control of the purification (Fig. 3) with Amido Black. During and after purification, the LDH from the caudal muscle showed a two banded profile with polyacrylamide gel electrophoresis (Fig. 2).
U V spectrum The ultraviolet absorption spectrum was performed with a Cary 14 spectrophotometer. Fluorescence spectrum The fluorescence spectrum was performed with an Aminco-Bowman ratio spectrofluorimeter with an exitation light at 280 nm. Enzyme purification All preparation procedures were carried out at 0-4°C. Crude extract. 200 g of caudal muscle were homogenized in 5 vol of 0.05 M sodium phosphate buffer (pH 7.5) containing 0.5 mM PMSF and 0.25 mM mercaptoethanol in an Ultraturrax for 60 sec. The homogenate was centrifuged for 20 rain at 37,000g and the supernatant was collected. The pellet was then homogenized and centrifuged in the same conditions. The two supernatants were pooled and saved. Ammonium sulfate fractionation. The supernatant was brought to 60~ saturation with powdered (NH4)2SO4. The solution was stirred for 45 rain. and centrifuged at 12,000 0 for 30min. The pellet was suspended in a minimum volume of the extraction buffer and dialyzed against the same buffer overnight, Hydroxyapatite batch. Hydroxyapatite, prepared in the laboratory according to the method of Tiseliues-Bernardi et al. (1956) and extracts were mixed volume to volume.
Fig. 2. 6~o polyacrylamide gel electrophoresis of purified LDH of Palaemon serratus caudal muscle stained with Amido Black.
Table I. Control of the purification
Extract Crude extract Hydroxyapatite (NH4)2SO4 Biogel P 200 5' AMP Sepharose
A/ml AOD 340 rm/mn/ml 3.8 2.15 33.2 40 145
Protein (mg/ml)
Specific Activity A/ml/mg prot sol.
Volume (ml)
10.7 4.8 12.1 7.4 0.45
0.36 0.45 2.74 5.4 322.2
1010 1650 90 40 4
Total Activity A/ml × Vml 3850 3547.5 2990 1600 580
Rendt
(%)
100 92.14 77.66 41.56 13.3
LDH from shrimp muscle
67
A~
alO
300
290
280 270
260
250
240 rim
Fig. 5. Ultraviolet spectrum of LDH in 0.01 M sodium phosphate buffer pH 7.5 at 18°C.
Fig. 3. Polyacrylamide gel gradient (5-20%) stained with Amido Black.
The purified enzyme was kept in 0.01 M sodium phosphate buffer (pH 7.0) with 20% glycerol. The activity remained almost constant for about 2 months; it was however not possible to keep the enzyme by freezing, for it was immediately inactivated.
firmed by gel gradient electrophoresis (5-20%). Here again it is possible to separate two distinct bands (Fig. 3). In contrast, the SDS gradient electrophoresis yield one single band corresponding to a molecular weight of 35,000 (Fig. 4).
u.v. Spectrum The u.v. spectrum of an 0.05 mg solution of enzyme shows a shoulder at 295-300nm like the Human heart LDH (Nisselbaum & Bodansky, 1961) (Fig. 5), which corresponds to the absorption of tryptophane.
RESULTS
Physicochemical properties
Fluorescence spectrum
The molecular weight of the purified enzyme has been measured by chromatography on Sephadex G200. The value obtained for the native enzyme ranges around 140,000. This value is further con-
The wavelength of the emission maximum of the native enzyme was at 335 nm, approx similar to other dehydrogenases, which correspond to tryptophane fluorescence (Fig. 6). IOC
8C
6C
8 c
~_ 4c
Wavelength
Fig. 4. Polyacrylamide gel gradient (5-20%) electrophoresis in dissociating conditions (SDS) stained with Coomassie Blue.
Fig. 6. Emission spectrum of LDH in 0.01 M sodium phosphate buffer pH 7:5 at the excitation wavelength of 280 nm, at 18°C.
68
M.T. THEBAULT,A. BERNICARDand J. F. LENNON Table 3. Values of the Hill number for Palaemon serratus LDH
Vu/mn
Substrate 5OO
30C
I0¢
,
,
6
i
8
no
pH
Fig. 7. Effect of pH on the activity of the LDH: O with pyruvate as substrate; • with lactate as substrate. Initial velocity is plotted against increasing pH values.
Catalytic properties Effect of pH. Fig. 7 shows the pH profile between pH 6.0 to pH 11 for the forward and the reverse reactions. An optimum at pH 9.5 was found for the lactate oxidation and at pH 7.8 for the pyruvate reduction. Specificity. The LDH of the caudal muscle of Palaemon serratus appears to be specific of L-lactate acid. There is no activity with D-lactate as substrate. Kinetic properties. The values of the apparent Michaelis constants (KIn) were determined from the Lineweaver-Burk plots (Table 2); at saturation of coenzyme (2raM NAD and 0.2mM NADH) we measured two values of K,, for lactate and pyruvate as we had previously observed with low concentrations of coenzymes (Thebault & Le Gal (1978). There is only one value of K,, for N A D and N A D H (with 100 mM lactate and 1 m M pyruvate). Owing to the fact that two distinct parts are observed in the substrate saturation curves, we can measure two values for lactate and pyruvate. The Hill numbers have been determined graphically for the forward and reverse reaction, using the relation:
v. yo-yo.
-
-
=
n log
S.
For low concentrations of substrate the Hill number is less than 1. For high concentrations of substrate, the Hill number is greater than 1. There is only one value for N A D and N A D H which is approx 1.20 and 1.80 (Table 3). The two Hill numbers observed with lactate and pyruvate can be set in relation with the two K,, values Table 2. Values of apparent K,. for Palaemon serratus LDH Substrate Pyruvate Lactate NAD NADH
K,. (mM) at 20°C 0.24 16 0.24 0.17
0.086 3.70 ---
Pyruvate Lactate NAD NADH
Hill number 0.85 0.77 1.17 1.80
2.16 1.32 --
previously determined. Similar results were obtained with the Human heart LDH (Nisselbaum & Bodansky, 1961) and with several enzymes (Alaskan King Crab pyruvate kinase, Somero, 1969); (cytidine triphosphate synthetase, Teipel & Koshland, 1969). According to Conway & Koshland (1968), these curves are the result of a negative cooperativity, where successive fixations of substrate could produce binding sites with a different conformation, having a lower affinity, but a high turnover number. This could explain the negative cooperativity observed with low substrate concentrations and the positive cooperativity observed when the substrate concentrations increased. However, the enzyme was a better catalyst with pyruvate as substrate, and we observed a substrate inhibition above 5 mM pyruvate, similar to the H LDH of vertebrates (Fig. 8). These results are in agreement with those of Traush (1976) and Gade (1979). The variation of the K,, values, for substrate concentrations greater than the value of the K,,, as a function of the incubation temperature, showed a temperature independence from 10 to 30°C (Fig. 9). When the concentration of substrate were lower (about the K,, value), the temperature modulation was more effective (Thebault & Le Gal, 1979; Thebault et al., 1979).
Effect of inhibitors The effect of 3-acetyl pyridine NAD, and NAD analogue, was studiedby increasing inhibitor concentration, with lactate as substrate. The apparent Ki and the K,, had the same value, and the inhibition was non-competitive (Fig. 10). We can notice that the presence of this inhibitor does not suppress the break in the Lineweaver-Burk plot for a value of lactate equal to the K,. for this substrate. The negative cooperativity is then maintained. The effect of urea investigated, the inhibitory effect of urea, expressed with respect to the activity in the Spe Act
/ "%'°~° / PYR mM
Fig. 8. Saturation curves for the pyruvate at 15°C. Initial velocity is plotted against increasing pyruvate concentrations.
69
LDH from shrimp muscle
KmmM
main conclusions: the first one is that the enzyme is likely to be a tetramer as the subunit molecular weight observed in the presence of SDS of 35,000 leads a final molecular weight of 140,000. The second one is that this apparent lack of homogeneity of the purified enzyme as judged by polyacrylamide gel electrophoresis does not appear to be consequence of a fundamental difference in the primary sequence of the two species. Our results show a substrate stereospecificity for L-lactate and catalytic properties similar to that observed in the muscle type LDH with respect to the K,, values for lactate and pyruvate and the pH optimum, and in the heart type LDH for the vertebrates with respect to the inhibition by pyruvate. This is the case of many LDH so far studied in marine invertebrates (Molluscs and Crustaceans). This kinetic behaviour is consistent with the lack of isoenzymes in different tissues. In Palaemon serratus the same enzyme would have to function like the vertebrate M and H type of LDH. We have previously reported (Thebault & Le Gal, 1978; Thebault et al., 1979) a temperature compensation process of the apparent Km with respect to the variations of external temperature. The amount of the apparent Km values with non saturation values of coenzymes did not vary significantly throughout the temperature range of the natural habitat of the shrimp, and increased abruptly near the lethal limit for this species. This is figured by a U-shaped curve of the apparent Kr, variation versus the assay temperature. When the coenzyme is utilized at saturation, the Km is temperature independent from 8 to 30°C. Hochachka & Somero (1976) showed the importance of non-saturating values of substrate for the modulation of enzyme activity. We have previously observed a break in the Lineweaver-Burk plots occurring at low substrate concentration. The Hill plots with pyruvate or lactate as substrate exhibits a break corresponding to the Km value for this substrate. At coenzyme saturation, these breaks are less obvious. We have described this phenomenon as the result of conformational changes of the enzyme (Thebault & Le Gal, 1977). An increase in buffer molarity, assay temperature and substrate concentration leads to an
50
J 25
•
,b
--o /
20
~)
I."[*
410
(a)
KmmM
__O IO
20
30
I.T*
40
(b) Fig. 9. Variations of the Km values as a function of the incubation temperature: (a) lactate as substrate; (b) pyruvate as substrate.
presence of the inhibitor, increased slowly until a concentration of 2 H urea. Then, the inhibition became very important (Fig. 11).
DISCUSSION The LDH from the tail muscle of Palaemon serratus obtained with a 900-fold purification yields after polyacrylamide gel electrophresis, a two banded pattern. The two presumptive molecular species are, however, resolved in one unique band when electrophoresed under dissociation conditions. This result leads to two I
=~~~~~=i0.25
mM
0,8
°~ ° ~
I = 0.10 m M
o
°"
I=OmM
,
OI
,
0.2
I 0.3
I lact mM
Fig. 10. Lineweaver-Burke plot for lactate with increasing concentration of 3-acetyl-pyridine NAD.
70
M.T. THEBAULT, A. BERNICARDand J. F. LENNON REFERENCES
30
20
I
I
I
2
3
--I
4
Ureo Mol
Fig. 11. Inhibition by urea: initial velocity without urea versus velocity in the presence of urea is plotted against increasing urea concentration.
attenuation of the m a r k e d frontier between the two parts of the Hill plots, or an attenuation of the intermediary plateau of the saturation curves, affecting the conformational flexibility of the protein. The conformational flexibility of the caudal muscle L D H could be related to the presence of two b a n d s in the electrophoretic pattern of the purified enzyme. These two bands could be two conformational variants of the same protein. But they could be products of two different genes or epigenetic transformation too. The conformational flexibility of the muscle L D H of Palaemon serratus, depending to external factors (temperature or cellular factors, substrate or coenzyme concentration) could be a possible means of regulation of the enzyme activity in relation with the variations of the modifications of the environment. The study of the transconformations of the Palaemon serratus L D H is under investigation. Acknowled(tements--Helpful discussion with Dr Y. Le Gal is fully acknowledged. We are indebted to Professor B. S. C. Leadbeater for correcting the manuscript. This work has been supported by the Coll6ge de France and by the Centre National de la Recherche Scientifique.
CONWAV A. & KOSLAND D. E. (1968) Negative cooperativity in enzyme action. The binding of diphosphorydine nucleotide to glyceraldehyde-3-P dehydrogenase. Biochemistry, 7, 4011~-022. ELLINGTON W. R. & LONG G. L. (1978) Purification and characterization of a highly unusual tetrameric D-LDH from the muscle of the giant barnacle. Archs Bioehem. Biophys. 186, 265 274. GAOL G. (1979) L-lactate specific lactate dehydrogenase from the mantle muscle of the Squid Loligo vulgaris: purification and catalytic properties. Comp. Biochem. Physiol. 63B, 387-395. GLEASON F. n., PRICE J. S., MANN R. A. & STUART T. D. (1971) Lactate dehydrogenases from Crustaceans and Arachnids. Comp. Biochem. Physiol. 40B, 387 394. HOCHACHKA P. W. & SOMERO G. N. /1976) Adaptation to Environment, pp, 125 190 Butterworths, London. LAMBIN P. (1978) Reliability of molecular weight determination of proteins by polyacrylamide gradient gel electrophoresis in the presence of sodium dodecyl sulfate. Analyt. Biochem. 85, 114 125. LOWRY O. H., ROSENBROt!GH N. H. & RANDALL (1951) Protein measurement with the Folin-phenol reagent. J. biol. Chem. 193, 265-275. NISSELBAUM J. S. & BO|3ANSKYO. (1961) Purification and properties of Human heart LDH. J. biol. Chem. 236, 323-327. SntJSTER L. (1971) Preparation acrylamide gel electrophoresis. Meth. Enzym. XXll, 412 437. SOMERO G. N. (1969) Pyruvate kinase variants of the Alaskan King Crab. Biochem. J. 114, 237 241. TEIPEL J. & KOSLAND D. E. (1969) The significance of intermediary plateau regions in enzyme saturation. Biochemistry, 8, 4656~4663. THE~AULJ" M. T. & LEGAL Y. (1976) Studies on lactate dehydrogenase activity on the Shrimp Palaemon serratus. Part I, Characterisation of the enzymes from the caudal muscle. Biochem. Syst. Ecol. 5, 155-159. THEaAULT M. T. & LE GAL Y. (1978) Studies on lactatedehydrogenase activity in Palaemon serratus. Effect of the incubation temperature on the kinetic properties of thc enzyme system from the caudal muscle. Bioehem. Syst. Ecol. 6, 141-144. THEBAULT M. T., BERNICARD A. & LEGAL Y. (1980) Effect of acclimation on lactate dehydrogenase activity in Palaemon serratus. Comp. Biochem. Physiol. 65B, 357-361. TRAUSH G. (1976) Effect of temperature upon catalytic properties of lactate dehydrogenase in the lobster. Biochem. Syst. Ecol. 6, 65 68.