Eur. J . Biochem. 97,407-413 (1979)
Dissociation and Aggregation of Lactic Dehydrogenase by High Hydrostatic Pressure Gerhard SCHMID, Hans-Dietrich LUDEMANN, and Rainer JAENICKE lnstitut fur Biophysik und Physikalische Biochemic, Universitat Regensburg
(Received December 22, 1978)
As shown by earlier experiments high hydrostatic pressure affects the catalytic function of lactic dehydrogenase from rabbit muscle. In the presence of substrates denaturation occurs, whereas in the absence of substrates and - SH-protecting reagents oxidation of sulfhydryl groups takes place [Schmid, G., Liidemann, H.-D. & Jaenicke, R. (1975) Biophys. Chem. 3, 90-98; (1978) Euv. J . Biochem. 86, 21 9 - 2241. Avoiding oxidation effects by reducing conditions in the solvent medium and by chelation of heavy metal ions, the remaining high-pressure effects consist of dissociation of the native quaternary structure into subunits followed by aggregation. Both reactions are influenced by temperature and enzyme concentration. Short incubation ( 5 10 min) at pH 6.0- 8.5 and pressures of 0.3 - 1.O kbdr causes dissociation which is reversed at normal pressure. At 5 "C the activation volume is found to be A V * = - 62 2 3 cm3 . mol-'. Above 1.2 kbar irreversible aggregation takes place; the reaction is favoured by low temperature and decreased pH. The activation volume 3 cm3 . mol-'. for the aggregation process at 5 "C is A V* = - 97 The results may be described by a reaction sequence comprising pressure-induced dissociation of the native enzyme into its subunits followed by subunit aggregation to form inactive highmolecular-weight particles.
High hydrostatic pressure represents a common parameter in the biosphere [l]. It affects cellular processes on various levels. A qualitative explanation of the effects can be deduced from changes of ionization and/or solvation of mplecules involved in reactions of biological significance. For macromolecules in aqueous solution large volumes of reaction and activation have been reported so that shifts of equilibria and kinetic effects become equally interpretable [2]. As shown by previous experiments enzymes may exhibit high-pressure activation or deactivation [3- 71. The latter observation has been explained by a variety of mechanisms, including conformational changes ('pressure denaturation', cf. [5,6]), changes of kinetic constants [8,9], chemical modification (e.g. oxidation of -SH-groups, cf. [lo]), or changes of the native quaternary structure [6,7] etc. In the case of oligorneric enzymes dissociation into inactive subunits has been proposed as a general mechanism of pressure deactivation 1171. The hypothesis has been experimentally corroborated by pressure-induced hybridization of isoenzymes of lactic dehydrogenase [4,11]. In these experiments the coenzyme has been found to inhibit dissociation so that hybrid formation does no longer occur in the presence of NADH. ____~ Enzyme. Lactic dehydrogenase (EC 1.1.1.27).
The hybridization experiments may be used to indicate subunit dissociation qualitatively. They are insufficient for a quantitative treatment. In order to gain deeper insight into the kinetic and equilibirum properties of the dissociation - association reaction under high hydrostatic pressure the enzymatic activity of lactic dehydrogenase from rabbit muscle was investigated in the range between 1 and 2000 bar. Though the highest pressures found in the biosphere are around 1200 bar it appears sensible to extend the measurements well in excess of this pressure in order to enlarge the observable effects, and thus to gain a more detailed insight into the origin of the effects found at biologically relevant pressures.
MATERIALS AND METHODS Lactic dehydrogenase from rabbit muscle (band V) and NADH were purchased from Boehringer (Mannheim). Dithiothreitol was a product of Calbiochem (Luzern). All other reagents were of A-grade purity (Merck, Darmstadt) ; quartz-bidistilled water was used throughout. Stock solutions of the enzyme in potassium phosphate buffer, pH 7.0, Z = 0.1 M were prepared by desalting the ammonium sulphate suspension (10 mg/ml) on a short Sephadex G-25 column
408
(1 x 5 cm) at 25 'C. Enzymatic activity was determined at 366 nm in the same buffer, and in the presence of 0.71 mM pyruvate and 0.18 mM NADH using a recording Eppendorf spectrophotometer with thermostated cuvettes. Concentrations were calculated from A2s0 = 1.40 cm2 . mg-' [12]; the specific activity was 540 I.U./mg. In order to prevent oxidation of essential sulfhydryl groups the high-pressure experiments were performed in phosphate buffer I = 0.1 M containing 1 mM EDTA and 5 mM dithiothreitol [lo]. The pressure-generating equipment was the conventional 1/8 in (3.2 mm) valve and capillary system with a hydraulic pump and a thermostated autoclave (0.d. 80 mm; length 260 mm) from High Pressure Equipment (Erie, Pa., U.S.A.). Bourdon gauges of quality class 0.6 (A. Wiegand, Klingenberg, F.R.G.) were used. Ethylene glycol with dissolved 2.7 dibromofluorescein (60 mg/l) served as the pressure-transmitting medium. Contamination of the samples with this medium of the order of 5 parts/million could be detected fluorimetrically using a Hitachi-Perkin-Elmer MPF-2A spectrofluorimeter. The enzyme solutions were incubated at varying pressure in thin-walled teflon containers which were previously described [lo]. Pressures up to 1500 bar were generated within 5 s by connecting the filled autoclave to a second one that had been pressurized before to a defined value. Minor final adjustments of pressure were achieved with the hydraulic pump. At higher pressure the time necessary to reach the desired final value was 20 s. The temperature change caused by adiabatic compression was of the order of 2 "C/kbar. The temperature within the autoclave was monitored by a miniature chromelalumel thermocouple (Philips, Industrieelektronik, Hamburg) silver-soldered into a metal cone seal. Turbidity measurements were performed at 320 nm in a thermostated high-pressure cuvette with sapphire windows described elsewhere [6]. The cuvette was mounted in the optical path of a Gilford 2400 S singlebeam spectrophotometer. The kinetics of reactivation were analyzed by taking aliquots of the enzyme solution after release of pressure at defined times. RESULTS Kinetics of Deactivation and Reactivation In order to investigate the kinetics of deactivation, the enzyme (c = 2.5 pg/ml) was incubated under pressure at 25 "C in phosphate buffer, I = 0.1 M for different times. 5 min after release of pressure the enzymatic activity was determined. As shown for two selected pH values (pH 7.0 and pH 8.5) the enzyme is deactivated after 10 rnin to a constant final value which depends on the applied pressure (Fig. 1A). At 1000 bar the residual activity remained constant over a period of
High-pressure Dissociation and Aggregation of Lactic Dehydrogenase
25 "C
A
i
loo
t
+*-+-+&.
1.O kbar
.a....r-J....o......Q .....0..
+ ._
s
g
o
+:..+:::f-..& o
......+..,.+...........__
-DO
50
220
Time (min) 5
'C
L0.75kbar
0
I
50 100 220 Time of incubation at high pressure (rnin)
Fig. 1. Deaclivafion kinutIcs of lactic dehj~.irogenase front rcihbit muscle at varying temperature, p H , and pressure. Phosphate buffer, I = 0.1 M, plus 1 mM EDTA and 5 mM dithiothreitol; enzyme concentration 2.5 pg/ml. Optical test 5 min after release of pressure. (A) 25'C; measurements at varying pH and pressure: pH 6.0 (+), p H 7.0 (a),pH 8.5 ( 0 ) ;1.0 kbar (--), 1.7 kbar (......), 2.0 kbar (-.-.-). (B) 5 ' C ; measurements at indicated pressure; pH 7.0
4 h. At 1700 bar and 2000 bar a further decrease of activity of the enzyme occurred after 30 min. In a buffer of pH 6.0 a plateau of deactivation was observed at 1000 bar only. The decrease of the enzymatic activity to a fixed value points to an equilibrium of deactivation, which is reached after 10 min. In the case of a reversible pressure-dependent equilibrium reactivation after release of pressure is expected. As illustrated in Fig.2 the activity of the pressurized enzyme is partially restored at normal pressures. The final values of reactivation after 95 h are in the range of 80%. After 10 min incubation at 1200 bar identical kinetics of reactivation are observed in buffers with pH 8.5, pH 7.0 and pH 6.5. On the other hand, incubation at 1700 bar leads to a significant decrease of the rate and extent of reactivation with decreasing pH. Experiments concerning the effect of enzyme concentration on reactivation were unsuccessful, since lactic dehydrogenase from rabbit muscle at low enzyme concentration (c < 2 pg/ml) does not show a sufficiently high stability required for the determination of the order of the reactivation reaction (cf. 113-151).
G. Schmid, H.-D. Liidemann, and R. Jaenicke
409
Additional information regarding the mechanism of pressure deactivation should be provided by temperature-dependent measurements. As shown in Fig. 1 B the kinetics of deactivation are changed significantly at 5 "C compared to the previously mentioned experiments at 25 "C: both the extent and the rate of deactivation turn out to be enhanced. Moreover two different processes are clearly separable. Contrary to the situation at 25°C the second process is strongly favoured if high pressure is applied at lower temperature. Owing to the enhanced deactivation rate at 5 "C the first process is detectable already at lower pressures. Fig. 3 summarizes the deactivaton data after 5 min, from which an activation volume A V* = - 62 k 3 cm3 . mol-' is derived.
*-------
/1.2
kbar $.o-
/
U+
1.7kbar
20
10
30
Time (rnin)
Fig. 2. Reactivation kinetics of lactic dehydrogenase from rabbit muscle at I kbar, after pressure incubation at 1.2 kbar and 1.7 kbar respectively. Enzyme concentration 25 pg/ml; solvent conditions as in Fig. 1. Incubation at high pressure: 10 min pH 6.0 (+), pH 7.0 (01,PH 8.5 (0)
I "
0
Efect of Pressure, p H and Enzyme Concentrution on the Amount of Deactivation The results given before lead to the conclusion that the deactivation of lactic dehydrogenase by high hydrostatic pressure is partially reversible. Therefore the activity measured 5 min after pressure release is determined by the amount of enzyme which remained active under pressure and the fraction reactivated within the 5 min after pressure release. Studying the effects of pressure, pH and concentration on this value yields further information on the mechanism of deactivation. Influence of Pressure. The analysis of the deactivation kinetics shows that within 10 min a constant value is obtained. The data in Fig.4 demonstrate the influence of pressure on this step at three different pH values. In the range from 300 bar to 1200 bar the effects of pressure are identical and independent of the pH of the buffer; from 1000 bar to 1200 bar the pressure profile of enzymatic activity displays a plateau. At pressures above 1200 bar a second step can be observed which is favoured at acidic pH. Znfluence of p H . Experiments concerning the influence of the pH on the deactivation over the whole range of stability show that the limit of the stability of the enzyme in the acidic pH range is shifted towards higher pH values (Fig.4, insert a). In addition the cooperativity of the transition from the native to the deactivated state of the enzyme changes significantly with pressure. This effect cannot be attributed to pressure-induced pH changes which under the given experimental conditions d o not exceed about - 0.4 pH unit/kbar [16]. Influence of Enzyme Concentration. The influence of enzyme concentration is expected to give additional
Pressure (kbar)
0.5
1.0
15 .
Pressure (kbar)
Fig.3. High-pressure deactivation of lactic dehydrogenase from rabbit muscle. Phosphate buffer pH 7.0, I = 0.1 M plus 1 mM EDTA and 5 mM dithiothreitol; enzyme concentration 2.5 pg/ml, 5 "C. Incubation at given pressures: 5 min. Optical test 5 min after release of pressure. Linearization according to In v = d p ( A V Q / R T )+ In k + In t with v = In ( ~ J C ~ ) ~
High-pressure Dissociation and Aggregation of Lactic Dehydrogenase
410
0.5
1.0 Pressure (kbar)
1.5
2.0
Fig. 4. Effcyt of liigh pressure on the enzymatic actfvlry of lactic deiiydr-ogenasefrom rahbif muscle at varying p H and enzyme concentration. Phosphate buffer, I = 0.1 M, plus 1 m M EDTA and 5 mM dithiothreitol, 25"C, enzyme concentration, 2.5 pg/rnl. Incubation at high pressure: 10 min. Optical test 5 min after release of pressure. ( + ) pH 6.0; ( 0 )p H 7.0; (0)pH 8.5. Inserts. Effect of (a) pH, and (b) enzyme concentration at varying pressure (bar): (a) enzyme concentration 2.5 pg/ml, (b) pH 7.0
1200 bar in the activity versus pressure profile for the more diluted solutions is lowered (Fig. 5).
100
Aggregation under High Hydrostatic Pressure
I
0
05
Lo
I
1.5
1
2D
Pressure (kbar)
Fig. 5. effect of enzyme c~oncrnri~ifrotr on higlz-prr.s.sur-e deacfivuti~m of lactic dehydrogenase from rahhit muscle. Phosphate buffer, I = 0.1 M plus 1 mM EDTA and 5 mM dithiothreitol, 25°C. Incubation at high pressure. 10 min. Optical test 5 min after release of pressure. p H 7.0 (closed symbols), p H 8.5 (open symbols)
information regarding the deactivation mechanism. In the case of pressure-induced unfolding, concentration effects are not to be expected since transconformation in general obeys first-order kinetics. However, formation of 'wrong aggregates' [17] may affect the time course and the extent of renaturation [18,39]. As taken from Fig. 4 (insert b) activity measured 5 min after release of the pressure increases with increasing concentration. The plateau between 1000 bar and
From the results mentioned above the conclusion has been drawn that deactivation is a consecutive twostep reaction. With respect to the second process preliminary experiments suggested aggregation to be involved. Therefore, turbidity measurements at 320 nm ( 5 "C, pH 7.0) were performed. As shown in Fig. 6, A32" of the enzyme solution at c = 250 pg/ml slowly increases if a pressure of 1500 bar was applied (first phase). After 2 h a rapid increase of the turbidity is observed. This second phase reaches a final value after 5 h. At higher pressures the aggregation reaction starts earlier and the rate of aggregation is found to be enhanced. During the second phase the turbidity is increased linearly with time. The slope dA320/dtcan be used as a measure of the rate of aggregation [20]. From the experimental data this rate is found to depend exponentially on pressure. Linearization in a semilogarithmic plot gives an activation volume of AV* = - 97 5 cm3 . mol-' (Fig.6, insert). In order to investigate the effect of temperature on the aggregation at high hydrostatic pressure the time course of the turbidity (A320) at pH 7.0 and 1500 bar was monitored. As shown in Fig.7A the rate of aggregation decreases with increasing temperature. On the other hand the first phase of the aggregation
G. Schrnid, H.-D. Ludernann. and R. Jaenicke
41 1
Time (min)
Fig.6. Eflbct qf high pressure on the aggregation kinetics of lactic deli.rdrogma.sefrom rabbit muscle. Phosphate buffer pH 7.0, I = 0.1 M plus 1 mM EDTA and 5 mM dithiothreitol; enzyme concentration 250 pg/rnl, 5°C. Absorbance at 320 nm was measured in an optical cell of 12 mm path-length. (0)1.7 kbar; (A) 1.6 kbar; (0)1.5 kbar. Insert. Linearization of the initial velocity (v, = dA32,Jh) as a function of pressure leads to A = - 97 3 cm' . mol- I
v+
4t
250 LlQ/mi
/-
A
_k
500 v a h
process remains unperturbed by changing the temperature. Plotting the data in a van? Hoff diagram yields a straight line; from its slope an apparent enthalpy of activation AH* = - 22 2 3 kcal . mol-' (92 12 kJ . mol-') is derived. Variation of the enzyme concentration strongly affects the pattern of aggregation and the rate. At low concentrations the sigmoidal second phase of aggregation is not observable within the time of measurement (Fig. 7 B). On the other hand the rate of aggregation in the first phase is increased significantly. DISCUSSION
' -/
"
0
100
200 Time (min)
300
Fig. 7. Effect of enzyme concentration and temperature on the aggregation kinetics of lactic dehydrogenase from rabbit muscle. Phosphate buffer pH 7.0, I = 0.1 M plus 1 m M EDTA and 5 mM dithiothreitol, pressure 1.5 kbar. Concentration dependence (A) was measured at 5 C ; (a)25 pg/rnl; (0)50 pg/ml; (A) 100 pg/ml; ( 0 ) 250 pg/ml: ( 0 ) 500 pg/ml. Temperature dependence (B) was measured at 250 pgjml; ( 0 ) 5 ? C ; (0) 10°C; (0)15'C; (A)25°C. The specific turbidity ( T =~ Ajzo/mg ~ enzyme), and the absorbance at 320 nm (A3*")were monitored in an optical cell of 12 mm pdthlength
The physical properties of water change only slightly at hydrostatic pressures up to 2000 bar. At temperatures above 273 K, the correlation time, the self-diffusion coefficient, and the infrared spectra show only small deviations from the normal liquid behaviour. The magnitude of the pressure-induced changes in the physical quantities observed are comparable to temperature changes of a few degrees 1211. Comparably small effects are found in the pressure dependence of aqueous solutions of hydrophobic organic substances [221. Therefore, it appears permissible to treat the influence of high hydrostatic pressure in the given pressure range as a slight perturbation of the water structure comparable to a temperature change of approximately 10 K. The native conformation of proteins is the result of the interaction of the dissolved protein with the surrounding water. It is thus in principle impossible to separate conformational
High-pressure Dissociation and Aggregation of Lactic Dehydrogenase
412
changes of the protein in an unambiguous way from the influence of changes in the water structure. As shown by the preceding results lactic dehydrogenase from rabbit muscle is deactivated at high hydrostatic pressure. Since earlier experiments had proved that the oxidation of sulfhydryl groups may cause deactivation of the enzyme [I 01 the present investigation was performed under conditions excluding oxidation. The enzyme was incubated under high pressure at 25 'C for different periods of time and at different pH values. 5 min after releasing the pressure, the enzymatic activity was measured. At pH 7.0 and pH 8.5 a plateau value of deactivation is obtained in a time range from 10 min to 30 min. The amount of deactivation depends on pressure (Fig. 1). The occurrence of a plateau value strongly suggests a pressure-dependent equilibrium of deactivation. This can be characterized by the measured final value of the activity only if reactivation can be excluded. As shown in Fig. 2 the reactivation at 25 'C and normal pressure occurs comparatively fast so that the plateau values in Fig.1 reflect the contributions of both the equilibrium activity under pressure, and the amount of reactivation after pressure release. If this mechanism is valid one would expect that, after having reached equilibrium, further incubation at high pressure has no influence on the plateau value of enzymatic activity. At 1000 bar this holds over the whole range of pH and time. At higher pressures a second deactivation process is suggested by a further decrease of the enzymatic activity. This second step is favoured at decreased pH. The rate of reactivation is decreased drastically at low temperature [13,23], as shown by kinetic studies concerning the reconstitution of lactic dehydrogenase after dissociation and denaturation, e.g. at acid pH. Therefore, the investigation of the kinetics of pressure deactivation at low temperature ( 5 'C) will be disturbed to a lesser extent by reactivation after pressure release. Using this approach the data in Fig.1 clearly exhibit two distinct deactivation steps the second of which is favoured at low temperature. The kinetic results may be summarized by the following reaction scheme
NGDr
+
Dz.
(1)
At pressures up to 1000 bar the native enzyme N is denatured reversibly to an intermediate state DI ; at higher pressures a second, irreversible (or very slowly reversible) step follows which is favoured by low temperature and low pH. The effects of pressure, concentration, and pH on the deactivation are in accordance with the given model. Considering the effect of pressure in the range between 1000 bar and 1200 bar ig.4) a plateau of the residual activity is obtained w ich indicates that the enzyme is transformed to the inactive state D1 in a
T
reversible fashion. At pressures beyond 1200 bar the second step of deactivation D1 D2 gains significance which causes the yield of reactivation to be drastically reduced. The occurrence of a second deactivation step can also be derived from the effect of pressure on the pH dependence of the enzymatic activity (Fig. 4). At 1000 bar the stability limit of the enzyme in the acidic pH range is found to be similar to the one observed at normal pressure. This is caused by the denaturation of the native quaternary structure at pH < 5.0 [24]. At 1700 bar and 2000 bar the denaturation increases at acidic pH which indicates two different mechanisms of denaturation. The effect of the enzyme concentration provides additional information regarding the deactivation mechanism. In the case of a pressure-induced dissociation equilibrium, concentration effects are expected to be of importance. On the other hand, no significant concentration dependence is expected if pressure induces only unfolding. As shown in Fig.4 and 5 lowering of the enzyme concentration leads to a significant decrease of the measured activity. This can be explained by both a concentration-dependent shift of the dissociation equilibrium and the concentrationdependent decrease of the rate of reassociation after the release of pressure. This implies that pressureinduced dissociation of the native quaternary structure of the enzyme participates in the first step of deactivation. At high pressure (p 2 1500 bar) formation of macroscopic aggregates follows the previously mentioned dissociation (Fig.6 and 7). This is in accordance with earlier observations which indicated high pressure denaturation to cause irreversible aggregation [25271. In the present experiments aggregation turns out to be favoured by low temperature; the kinetics were found to be biphasic. A similar time course observed for the heat aggregation of proteins 1281 has been shown to obey a sequential mechanism with the initial formation of low-molecular-weight particles followed by the formation of high aggregates. The second phase in the pressure-induced aggregation of lactic dehydrogenase vanishes at low enzyme concentrations ( e 5 100 pg/ml). The first phase is observed over the whole range of concentration. From this we conclude that aggregation is involved in the mechanism of pressure deactivation. Aggregation and deactivation refer to different steps in the overall scheme given in Eqn (I), so that different kinetic patterns are expected for the two processes. There are strong indications that aggregation parallels the second step of deactivation, since both reactions are enhanced at low temperatures, and both deactivation and aggregation are accelerated in the same range of pressure. The close correlation of irreversible deactivation and aggregation has been previously observed in connection with related reconstitution experiments [14,17,18]. --f
G. Schmid, H.-D. Ludemann, and R. Jaenicke
Linear reaction sequences are accelerated with increasing temperature. On the other hand, branched reactions (e.g. a pre-established equilibrium) may show the opposite effect of temperature. The results given in Fig.7B show a negative temperature coefficient. This observation corroborates the proposed mechanism consisting of deactivation as a first reversible step followed by irreversible aggregation. If the backward reaction of the equilibrium N + D 1 is enhanced by an increase of temperature the steadystate concentration of D1 will be lowered causing the aggregation to be slowed down. The first phase of aggregation (cf. Fig.7A) is characterized by an inverse proportionality with respect to enzyme concentration. This observation is in accordance with the assumption that dissociation precedes the irreversible aggregation reaction. Considering the enzyme subunits to represent the aggregating entities the rate of aggregation will depend on the degree of dissociaton of the native quaternary structure. Dissociation, however, is enhanced if the total concentration of the enzyme is lowered. Summarizing the foregoing results, two different conformational states, D1 and Dz, may be considered important in the overall process of pressure-dependent deactivation. The active tetrameric enzyme is reversibly dissociated into inactive subunits (D,), with increasing pressure and temperature as relevant parameters shifting the equilibrium towards dissociation. The subunits formed are metastable with respect to their state of association, and tend to form high aggregates (Dz). The given two-step mechanism appears to be a more general phenomenon which has been observed for a number of systems in the process of dissociation-association [14,15,17- 19,29-321. Previous experiments have shown that lactic dehydrogenase in the presence of coenzyme and substrate is stable towards pressures up to 1000 bar [6]. On the other hand, under conditions in vivo enzymes do not operate under saturating substrate concentrations [33]. Therefore, the present dissociation aggregation mechanism has to be considered a possible mechanism of deactivation under biological high-pressure conditions, e.g. in the deep sea. Obviously, adaptation to high-pressure biotopes must aim at the selection of a stable quaternary structure of oligomeric enzymes. This investigation was supported by grants of the Deutsche For.rchungsgrinrin.rc./taft(Ja 78/20), and the Verbund der Cliemisclzcn Industrie. G.S. was a recipient of a fellowship of the Friedrich Ehert Stifiung.
413
REFERENCES 1 . Bruun, A. F. (1956) A'uture (Lond.) 177. 1105-1108. 2. Johnson, F. H., Eyring, H. & Stover, B. J . (1974) Thc~the or^, uf Rule Procrsses in Biology and Medicine, pp. 273 - 369. Wiley, New York. 3. Sleigh, A. M. & MacDonald, A . G. (eds) (1972) The €//rct of Pressure on Organi.srns, Symp. Soc. E.xp. Biol. 26. Cambridge University Press, Cambridge. 4. Jaenicke, R. 5: Koberstein, R. (1971) FEBS L e t [ . 17, 351 -354. 5. Hawley, S . A. (1971) Bioc,liemistry, 10, 2436-2442. 6. Schmid, G., Ludemann, H.-D. & Jaenicke, R. (1975) Biop1ir.s. Chen7. 3, 90-98. 7. Penniston, I. T. (1971) Arch. Biochem. Biopli,rs. 142, 322-332. 8. Low, P. S . S: Somero, G . N . (1975) Proc. Nut/ Acurl. .%i. U.S.A. 72,3305-3309. 9. Morild, E. (1977) Bioiphis. Cliem. 6, 351 -362. 10. Schmid, G., Ludemann, H.-D. & Jaenicke, R. (1978) Eur. J . Biochem. 86, 219-224. I 1. Jaenicke, R. (1970) in Pj,ridine A'uc/eotic/t. Depmdent Dchjclro~ m u s r s(Sund, H., ed.) pp. 71 -90, Springer-Verlag. Berlin. Heidelberg, New York. 12. Jaenicke, R. & Knof, S. (1968) Eur. J . Biochcw?. 4, 157-163. 13. Rudolph, R. & Jaenicke, R . (1976) Eur. J . Biocl7em. 63. 40941 7. 14. Jaenicke, R. 9r Rudolph, R. (1977) in 2nd In/. Sjw7p. Pvidinr Nucleotide Dqwm'ent Del?ydrogenases (Sund, H., ed.) pp. 351 -367. W. de Gruyter, Berlin. 15. Jaenicke, R. (1978) A'uturii.issen.schu~ten,65, 569- 577. 16. Neumann, R. C., Jr, Kauzmann, W. & Zipp, A. (1973) J . Chem. Phys. 77, 2687- 2691. 17. Teipel, J . W . & Koshland, D. E., J r (1971) Biochcn7i.sfr.1~. 10. 792 - 805. 18. ZettlmeiRl. G., Rudolph, R. & Jaenicke, K. (1979) ELII..J . Bioc/7en7. in the press. 39. ZettlmeiBl, G. (1978) Thesis, Regensburg University. 20. Jaenicke, R. (1964) Ber. Bunsenges. Phis. Chem. 68, 857-862. 21. Todheide, K. (1972) in Wufer, A Comprehmsiw Trc2atisc2 (Franks, F., ed.) vol. 1, pp. 463ff., Plenum Press, New York. 22. Schneider, G. M. (1973) in Water, A Comprc4cwsivr Treatise (Franks, F.?ed.) vol. 2, pp. 381 ff., Plenum Press. New York. 23. Rudolph, R., Heider, I., Westhof, E. & Jaenicke. R. (1977) Biochrmistr>., 16, 3384- 3390. 24. Lowell, S.J . S: Winzor, D. J. (1974) Biochen~isrry,13, 35273537. 25. Suzuki, K., Miyosawa, Y . S: Suzuki, C. (1963) Arch. Biochrn~ Bi(1phy.s. 101. 225-228, 102. 367-372. 26. Joly, M . (1965) A Physico-Chemical Approach 10 fhe D~nurur~ilion uf Proleins, pp. 12ff., Academic Press. London, New York. 27. Zimmermann, A. M. (ed.) (1970) High Pre.rsure E(fec1.s 01) Cellulur Processes, Academic Press, New York. 28. Stauff, J., Barthel, H., Jaenicke, R., Krekel, R. & Ulilein, E. (1961) K ~ l l ~ i d - 178, Z . 128- 142. 29. Elodi, P. & Jecsai, G. (1960) Actu Pliysiol. Hung. 17, 165- 182. 30. Jaenicke, R. & Pfleiderer, G. (1 962) Biochim. Bioplij..~.Actu. 60, 615-629. 31. Payens, T. A . J. & Heremans, K. (1969) Bioplyni 345. 32. Rudolph, R. (1977) Thesis, Regensburg University. 33. Atkinson, D . E. (1966) Annu. Rev. Biocf7em. 35, 85-124.
G. Schmid. H.-D. Ludemann, and R. Jaenicke*. Institut fur Biophysik und Physikalische Biochemie, Fachbereich Biolopie und Vorklinische Medizin der Universitiit Regensburg, UniversititsstraBe 31, D-8400 Regensburg, Federal Republic of Germany
* To whom correspondence should be addressed
Dissociation and Aggregation of Lactic Dehydrogenase by High Hydrostatic Pressure Gerhard SCHMID, Hans-Dietrich LUDEMANN, and Rainer JAENICKE lnstitut fur Biophysik und Physikalische Biochemic, Universitat Regensburg
(Received December 22, 1978)
As shown by earlier experiments high hydrostatic pressure affects the catalytic function of lactic dehydrogenase from rabbit muscle. In the presence of substrates denaturation occurs, whereas in the absence of substrates and - SH-protecting reagents oxidation of sulfhydryl groups takes place [Schmid, G., Liidemann, H.-D. & Jaenicke, R. (1975) Biophys. Chem. 3, 90-98; (1978) Euv. J . Biochem. 86, 21 9 - 2241. Avoiding oxidation effects by reducing conditions in the solvent medium and by chelation of heavy metal ions, the remaining high-pressure effects consist of dissociation of the native quaternary structure into subunits followed by aggregation. Both reactions are influenced by temperature and enzyme concentration. Short incubation ( 5 10 min) at pH 6.0- 8.5 and pressures of 0.3 - 1.O kbdr causes dissociation which is reversed at normal pressure. At 5 "C the activation volume is found to be A V * = - 62 2 3 cm3 . mol-'. Above 1.2 kbar irreversible aggregation takes place; the reaction is favoured by low temperature and decreased pH. The activation volume 3 cm3 . mol-'. for the aggregation process at 5 "C is A V* = - 97 The results may be described by a reaction sequence comprising pressure-induced dissociation of the native enzyme into its subunits followed by subunit aggregation to form inactive highmolecular-weight particles.
High hydrostatic pressure represents a common parameter in the biosphere [l]. It affects cellular processes on various levels. A qualitative explanation of the effects can be deduced from changes of ionization and/or solvation of mplecules involved in reactions of biological significance. For macromolecules in aqueous solution large volumes of reaction and activation have been reported so that shifts of equilibria and kinetic effects become equally interpretable [2]. As shown by previous experiments enzymes may exhibit high-pressure activation or deactivation [3- 71. The latter observation has been explained by a variety of mechanisms, including conformational changes ('pressure denaturation', cf. [5,6]), changes of kinetic constants [8,9], chemical modification (e.g. oxidation of -SH-groups, cf. [lo]), or changes of the native quaternary structure [6,7] etc. In the case of oligorneric enzymes dissociation into inactive subunits has been proposed as a general mechanism of pressure deactivation 1171. The hypothesis has been experimentally corroborated by pressure-induced hybridization of isoenzymes of lactic dehydrogenase [4,11]. In these experiments the coenzyme has been found to inhibit dissociation so that hybrid formation does no longer occur in the presence of NADH. ____~ Enzyme. Lactic dehydrogenase (EC 1.1.1.27).
The hybridization experiments may be used to indicate subunit dissociation qualitatively. They are insufficient for a quantitative treatment. In order to gain deeper insight into the kinetic and equilibirum properties of the dissociation - association reaction under high hydrostatic pressure the enzymatic activity of lactic dehydrogenase from rabbit muscle was investigated in the range between 1 and 2000 bar. Though the highest pressures found in the biosphere are around 1200 bar it appears sensible to extend the measurements well in excess of this pressure in order to enlarge the observable effects, and thus to gain a more detailed insight into the origin of the effects found at biologically relevant pressures.
MATERIALS AND METHODS Lactic dehydrogenase from rabbit muscle (band V) and NADH were purchased from Boehringer (Mannheim). Dithiothreitol was a product of Calbiochem (Luzern). All other reagents were of A-grade purity (Merck, Darmstadt) ; quartz-bidistilled water was used throughout. Stock solutions of the enzyme in potassium phosphate buffer, pH 7.0, Z = 0.1 M were prepared by desalting the ammonium sulphate suspension (10 mg/ml) on a short Sephadex G-25 column
408
(1 x 5 cm) at 25 'C. Enzymatic activity was determined at 366 nm in the same buffer, and in the presence of 0.71 mM pyruvate and 0.18 mM NADH using a recording Eppendorf spectrophotometer with thermostated cuvettes. Concentrations were calculated from A2s0 = 1.40 cm2 . mg-' [12]; the specific activity was 540 I.U./mg. In order to prevent oxidation of essential sulfhydryl groups the high-pressure experiments were performed in phosphate buffer I = 0.1 M containing 1 mM EDTA and 5 mM dithiothreitol [lo]. The pressure-generating equipment was the conventional 1/8 in (3.2 mm) valve and capillary system with a hydraulic pump and a thermostated autoclave (0.d. 80 mm; length 260 mm) from High Pressure Equipment (Erie, Pa., U.S.A.). Bourdon gauges of quality class 0.6 (A. Wiegand, Klingenberg, F.R.G.) were used. Ethylene glycol with dissolved 2.7 dibromofluorescein (60 mg/l) served as the pressure-transmitting medium. Contamination of the samples with this medium of the order of 5 parts/million could be detected fluorimetrically using a Hitachi-Perkin-Elmer MPF-2A spectrofluorimeter. The enzyme solutions were incubated at varying pressure in thin-walled teflon containers which were previously described [lo]. Pressures up to 1500 bar were generated within 5 s by connecting the filled autoclave to a second one that had been pressurized before to a defined value. Minor final adjustments of pressure were achieved with the hydraulic pump. At higher pressure the time necessary to reach the desired final value was 20 s. The temperature change caused by adiabatic compression was of the order of 2 "C/kbar. The temperature within the autoclave was monitored by a miniature chromelalumel thermocouple (Philips, Industrieelektronik, Hamburg) silver-soldered into a metal cone seal. Turbidity measurements were performed at 320 nm in a thermostated high-pressure cuvette with sapphire windows described elsewhere [6]. The cuvette was mounted in the optical path of a Gilford 2400 S singlebeam spectrophotometer. The kinetics of reactivation were analyzed by taking aliquots of the enzyme solution after release of pressure at defined times. RESULTS Kinetics of Deactivation and Reactivation In order to investigate the kinetics of deactivation, the enzyme (c = 2.5 pg/ml) was incubated under pressure at 25 "C in phosphate buffer, I = 0.1 M for different times. 5 min after release of pressure the enzymatic activity was determined. As shown for two selected pH values (pH 7.0 and pH 8.5) the enzyme is deactivated after 10 rnin to a constant final value which depends on the applied pressure (Fig. 1A). At 1000 bar the residual activity remained constant over a period of
High-pressure Dissociation and Aggregation of Lactic Dehydrogenase
25 "C
A
i
loo
t
+*-+-+&.
1.O kbar
.a....r-J....o......Q .....0..
+ ._
s
g
o
+:..+:::f-..& o
......+..,.+...........__
-DO
50
220
Time (min) 5
'C
L0.75kbar
0
I
50 100 220 Time of incubation at high pressure (rnin)
Fig. 1. Deaclivafion kinutIcs of lactic dehj~.irogenase front rcihbit muscle at varying temperature, p H , and pressure. Phosphate buffer, I = 0.1 M, plus 1 mM EDTA and 5 mM dithiothreitol; enzyme concentration 2.5 pg/ml. Optical test 5 min after release of pressure. (A) 25'C; measurements at varying pH and pressure: pH 6.0 (+), p H 7.0 (a),pH 8.5 ( 0 ) ;1.0 kbar (--), 1.7 kbar (......), 2.0 kbar (-.-.-). (B) 5 ' C ; measurements at indicated pressure; pH 7.0
4 h. At 1700 bar and 2000 bar a further decrease of activity of the enzyme occurred after 30 min. In a buffer of pH 6.0 a plateau of deactivation was observed at 1000 bar only. The decrease of the enzymatic activity to a fixed value points to an equilibrium of deactivation, which is reached after 10 min. In the case of a reversible pressure-dependent equilibrium reactivation after release of pressure is expected. As illustrated in Fig.2 the activity of the pressurized enzyme is partially restored at normal pressures. The final values of reactivation after 95 h are in the range of 80%. After 10 min incubation at 1200 bar identical kinetics of reactivation are observed in buffers with pH 8.5, pH 7.0 and pH 6.5. On the other hand, incubation at 1700 bar leads to a significant decrease of the rate and extent of reactivation with decreasing pH. Experiments concerning the effect of enzyme concentration on reactivation were unsuccessful, since lactic dehydrogenase from rabbit muscle at low enzyme concentration (c < 2 pg/ml) does not show a sufficiently high stability required for the determination of the order of the reactivation reaction (cf. 113-151).
G. Schmid, H.-D. Liidemann, and R. Jaenicke
409
Additional information regarding the mechanism of pressure deactivation should be provided by temperature-dependent measurements. As shown in Fig. 1 B the kinetics of deactivation are changed significantly at 5 "C compared to the previously mentioned experiments at 25 "C: both the extent and the rate of deactivation turn out to be enhanced. Moreover two different processes are clearly separable. Contrary to the situation at 25°C the second process is strongly favoured if high pressure is applied at lower temperature. Owing to the enhanced deactivation rate at 5 "C the first process is detectable already at lower pressures. Fig. 3 summarizes the deactivaton data after 5 min, from which an activation volume A V* = - 62 k 3 cm3 . mol-' is derived.
*-------
/1.2
kbar $.o-
/
U+
1.7kbar
20
10
30
Time (rnin)
Fig. 2. Reactivation kinetics of lactic dehydrogenase from rabbit muscle at I kbar, after pressure incubation at 1.2 kbar and 1.7 kbar respectively. Enzyme concentration 25 pg/ml; solvent conditions as in Fig. 1. Incubation at high pressure: 10 min pH 6.0 (+), pH 7.0 (01,PH 8.5 (0)
I "
0
Efect of Pressure, p H and Enzyme Concentrution on the Amount of Deactivation The results given before lead to the conclusion that the deactivation of lactic dehydrogenase by high hydrostatic pressure is partially reversible. Therefore the activity measured 5 min after pressure release is determined by the amount of enzyme which remained active under pressure and the fraction reactivated within the 5 min after pressure release. Studying the effects of pressure, pH and concentration on this value yields further information on the mechanism of deactivation. Influence of Pressure. The analysis of the deactivation kinetics shows that within 10 min a constant value is obtained. The data in Fig.4 demonstrate the influence of pressure on this step at three different pH values. In the range from 300 bar to 1200 bar the effects of pressure are identical and independent of the pH of the buffer; from 1000 bar to 1200 bar the pressure profile of enzymatic activity displays a plateau. At pressures above 1200 bar a second step can be observed which is favoured at acidic pH. Znfluence of p H . Experiments concerning the influence of the pH on the deactivation over the whole range of stability show that the limit of the stability of the enzyme in the acidic pH range is shifted towards higher pH values (Fig.4, insert a). In addition the cooperativity of the transition from the native to the deactivated state of the enzyme changes significantly with pressure. This effect cannot be attributed to pressure-induced pH changes which under the given experimental conditions d o not exceed about - 0.4 pH unit/kbar [16]. Influence of Enzyme Concentration. The influence of enzyme concentration is expected to give additional
Pressure (kbar)
0.5
1.0
15 .
Pressure (kbar)
Fig.3. High-pressure deactivation of lactic dehydrogenase from rabbit muscle. Phosphate buffer pH 7.0, I = 0.1 M plus 1 mM EDTA and 5 mM dithiothreitol; enzyme concentration 2.5 pg/ml, 5 "C. Incubation at given pressures: 5 min. Optical test 5 min after release of pressure. Linearization according to In v = d p ( A V Q / R T )+ In k + In t with v = In ( ~ J C ~ ) ~
High-pressure Dissociation and Aggregation of Lactic Dehydrogenase
410
0.5
1.0 Pressure (kbar)
1.5
2.0
Fig. 4. Effcyt of liigh pressure on the enzymatic actfvlry of lactic deiiydr-ogenasefrom rahbif muscle at varying p H and enzyme concentration. Phosphate buffer, I = 0.1 M, plus 1 m M EDTA and 5 mM dithiothreitol, 25"C, enzyme concentration, 2.5 pg/rnl. Incubation at high pressure: 10 min. Optical test 5 min after release of pressure. ( + ) pH 6.0; ( 0 )p H 7.0; (0)pH 8.5. Inserts. Effect of (a) pH, and (b) enzyme concentration at varying pressure (bar): (a) enzyme concentration 2.5 pg/ml, (b) pH 7.0
1200 bar in the activity versus pressure profile for the more diluted solutions is lowered (Fig. 5).
100
Aggregation under High Hydrostatic Pressure
I
0
05
Lo
I
1.5
1
2D
Pressure (kbar)
Fig. 5. effect of enzyme c~oncrnri~ifrotr on higlz-prr.s.sur-e deacfivuti~m of lactic dehydrogenase from rahhit muscle. Phosphate buffer, I = 0.1 M plus 1 mM EDTA and 5 mM dithiothreitol, 25°C. Incubation at high pressure. 10 min. Optical test 5 min after release of pressure. p H 7.0 (closed symbols), p H 8.5 (open symbols)
information regarding the deactivation mechanism. In the case of pressure-induced unfolding, concentration effects are not to be expected since transconformation in general obeys first-order kinetics. However, formation of 'wrong aggregates' [17] may affect the time course and the extent of renaturation [18,39]. As taken from Fig. 4 (insert b) activity measured 5 min after release of the pressure increases with increasing concentration. The plateau between 1000 bar and
From the results mentioned above the conclusion has been drawn that deactivation is a consecutive twostep reaction. With respect to the second process preliminary experiments suggested aggregation to be involved. Therefore, turbidity measurements at 320 nm ( 5 "C, pH 7.0) were performed. As shown in Fig. 6, A32" of the enzyme solution at c = 250 pg/ml slowly increases if a pressure of 1500 bar was applied (first phase). After 2 h a rapid increase of the turbidity is observed. This second phase reaches a final value after 5 h. At higher pressures the aggregation reaction starts earlier and the rate of aggregation is found to be enhanced. During the second phase the turbidity is increased linearly with time. The slope dA320/dtcan be used as a measure of the rate of aggregation [20]. From the experimental data this rate is found to depend exponentially on pressure. Linearization in a semilogarithmic plot gives an activation volume of AV* = - 97 5 cm3 . mol-' (Fig.6, insert). In order to investigate the effect of temperature on the aggregation at high hydrostatic pressure the time course of the turbidity (A320) at pH 7.0 and 1500 bar was monitored. As shown in Fig.7A the rate of aggregation decreases with increasing temperature. On the other hand the first phase of the aggregation
G. Schrnid, H.-D. Ludernann. and R. Jaenicke
41 1
Time (min)
Fig.6. Eflbct qf high pressure on the aggregation kinetics of lactic deli.rdrogma.sefrom rabbit muscle. Phosphate buffer pH 7.0, I = 0.1 M plus 1 mM EDTA and 5 mM dithiothreitol; enzyme concentration 250 pg/rnl, 5°C. Absorbance at 320 nm was measured in an optical cell of 12 mm path-length. (0)1.7 kbar; (A) 1.6 kbar; (0)1.5 kbar. Insert. Linearization of the initial velocity (v, = dA32,Jh) as a function of pressure leads to A = - 97 3 cm' . mol- I
v+
4t
250 LlQ/mi
/-
A
_k
500 v a h
process remains unperturbed by changing the temperature. Plotting the data in a van? Hoff diagram yields a straight line; from its slope an apparent enthalpy of activation AH* = - 22 2 3 kcal . mol-' (92 12 kJ . mol-') is derived. Variation of the enzyme concentration strongly affects the pattern of aggregation and the rate. At low concentrations the sigmoidal second phase of aggregation is not observable within the time of measurement (Fig. 7 B). On the other hand the rate of aggregation in the first phase is increased significantly. DISCUSSION
' -/
"
0
100
200 Time (min)
300
Fig. 7. Effect of enzyme concentration and temperature on the aggregation kinetics of lactic dehydrogenase from rabbit muscle. Phosphate buffer pH 7.0, I = 0.1 M plus 1 m M EDTA and 5 mM dithiothreitol, pressure 1.5 kbar. Concentration dependence (A) was measured at 5 C ; (a)25 pg/rnl; (0)50 pg/ml; (A) 100 pg/ml; ( 0 ) 250 pg/ml: ( 0 ) 500 pg/ml. Temperature dependence (B) was measured at 250 pgjml; ( 0 ) 5 ? C ; (0) 10°C; (0)15'C; (A)25°C. The specific turbidity ( T =~ Ajzo/mg ~ enzyme), and the absorbance at 320 nm (A3*")were monitored in an optical cell of 12 mm pdthlength
The physical properties of water change only slightly at hydrostatic pressures up to 2000 bar. At temperatures above 273 K, the correlation time, the self-diffusion coefficient, and the infrared spectra show only small deviations from the normal liquid behaviour. The magnitude of the pressure-induced changes in the physical quantities observed are comparable to temperature changes of a few degrees 1211. Comparably small effects are found in the pressure dependence of aqueous solutions of hydrophobic organic substances [221. Therefore, it appears permissible to treat the influence of high hydrostatic pressure in the given pressure range as a slight perturbation of the water structure comparable to a temperature change of approximately 10 K. The native conformation of proteins is the result of the interaction of the dissolved protein with the surrounding water. It is thus in principle impossible to separate conformational
High-pressure Dissociation and Aggregation of Lactic Dehydrogenase
412
changes of the protein in an unambiguous way from the influence of changes in the water structure. As shown by the preceding results lactic dehydrogenase from rabbit muscle is deactivated at high hydrostatic pressure. Since earlier experiments had proved that the oxidation of sulfhydryl groups may cause deactivation of the enzyme [I 01 the present investigation was performed under conditions excluding oxidation. The enzyme was incubated under high pressure at 25 'C for different periods of time and at different pH values. 5 min after releasing the pressure, the enzymatic activity was measured. At pH 7.0 and pH 8.5 a plateau value of deactivation is obtained in a time range from 10 min to 30 min. The amount of deactivation depends on pressure (Fig. 1). The occurrence of a plateau value strongly suggests a pressure-dependent equilibrium of deactivation. This can be characterized by the measured final value of the activity only if reactivation can be excluded. As shown in Fig. 2 the reactivation at 25 'C and normal pressure occurs comparatively fast so that the plateau values in Fig.1 reflect the contributions of both the equilibrium activity under pressure, and the amount of reactivation after pressure release. If this mechanism is valid one would expect that, after having reached equilibrium, further incubation at high pressure has no influence on the plateau value of enzymatic activity. At 1000 bar this holds over the whole range of pH and time. At higher pressures a second deactivation process is suggested by a further decrease of the enzymatic activity. This second step is favoured at decreased pH. The rate of reactivation is decreased drastically at low temperature [13,23], as shown by kinetic studies concerning the reconstitution of lactic dehydrogenase after dissociation and denaturation, e.g. at acid pH. Therefore, the investigation of the kinetics of pressure deactivation at low temperature ( 5 'C) will be disturbed to a lesser extent by reactivation after pressure release. Using this approach the data in Fig.1 clearly exhibit two distinct deactivation steps the second of which is favoured at low temperature. The kinetic results may be summarized by the following reaction scheme
NGDr
+
Dz.
(1)
At pressures up to 1000 bar the native enzyme N is denatured reversibly to an intermediate state DI ; at higher pressures a second, irreversible (or very slowly reversible) step follows which is favoured by low temperature and low pH. The effects of pressure, concentration, and pH on the deactivation are in accordance with the given model. Considering the effect of pressure in the range between 1000 bar and 1200 bar ig.4) a plateau of the residual activity is obtained w ich indicates that the enzyme is transformed to the inactive state D1 in a
T
reversible fashion. At pressures beyond 1200 bar the second step of deactivation D1 D2 gains significance which causes the yield of reactivation to be drastically reduced. The occurrence of a second deactivation step can also be derived from the effect of pressure on the pH dependence of the enzymatic activity (Fig. 4). At 1000 bar the stability limit of the enzyme in the acidic pH range is found to be similar to the one observed at normal pressure. This is caused by the denaturation of the native quaternary structure at pH < 5.0 [24]. At 1700 bar and 2000 bar the denaturation increases at acidic pH which indicates two different mechanisms of denaturation. The effect of the enzyme concentration provides additional information regarding the deactivation mechanism. In the case of a pressure-induced dissociation equilibrium, concentration effects are expected to be of importance. On the other hand, no significant concentration dependence is expected if pressure induces only unfolding. As shown in Fig.4 and 5 lowering of the enzyme concentration leads to a significant decrease of the measured activity. This can be explained by both a concentration-dependent shift of the dissociation equilibrium and the concentrationdependent decrease of the rate of reassociation after the release of pressure. This implies that pressureinduced dissociation of the native quaternary structure of the enzyme participates in the first step of deactivation. At high pressure (p 2 1500 bar) formation of macroscopic aggregates follows the previously mentioned dissociation (Fig.6 and 7). This is in accordance with earlier observations which indicated high pressure denaturation to cause irreversible aggregation [25271. In the present experiments aggregation turns out to be favoured by low temperature; the kinetics were found to be biphasic. A similar time course observed for the heat aggregation of proteins 1281 has been shown to obey a sequential mechanism with the initial formation of low-molecular-weight particles followed by the formation of high aggregates. The second phase in the pressure-induced aggregation of lactic dehydrogenase vanishes at low enzyme concentrations ( e 5 100 pg/ml). The first phase is observed over the whole range of concentration. From this we conclude that aggregation is involved in the mechanism of pressure deactivation. Aggregation and deactivation refer to different steps in the overall scheme given in Eqn (I), so that different kinetic patterns are expected for the two processes. There are strong indications that aggregation parallels the second step of deactivation, since both reactions are enhanced at low temperatures, and both deactivation and aggregation are accelerated in the same range of pressure. The close correlation of irreversible deactivation and aggregation has been previously observed in connection with related reconstitution experiments [14,17,18]. --f
G. Schmid, H.-D. Ludemann, and R. Jaenicke
Linear reaction sequences are accelerated with increasing temperature. On the other hand, branched reactions (e.g. a pre-established equilibrium) may show the opposite effect of temperature. The results given in Fig.7B show a negative temperature coefficient. This observation corroborates the proposed mechanism consisting of deactivation as a first reversible step followed by irreversible aggregation. If the backward reaction of the equilibrium N + D 1 is enhanced by an increase of temperature the steadystate concentration of D1 will be lowered causing the aggregation to be slowed down. The first phase of aggregation (cf. Fig.7A) is characterized by an inverse proportionality with respect to enzyme concentration. This observation is in accordance with the assumption that dissociation precedes the irreversible aggregation reaction. Considering the enzyme subunits to represent the aggregating entities the rate of aggregation will depend on the degree of dissociaton of the native quaternary structure. Dissociation, however, is enhanced if the total concentration of the enzyme is lowered. Summarizing the foregoing results, two different conformational states, D1 and Dz, may be considered important in the overall process of pressure-dependent deactivation. The active tetrameric enzyme is reversibly dissociated into inactive subunits (D,), with increasing pressure and temperature as relevant parameters shifting the equilibrium towards dissociation. The subunits formed are metastable with respect to their state of association, and tend to form high aggregates (Dz). The given two-step mechanism appears to be a more general phenomenon which has been observed for a number of systems in the process of dissociation-association [14,15,17- 19,29-321. Previous experiments have shown that lactic dehydrogenase in the presence of coenzyme and substrate is stable towards pressures up to 1000 bar [6]. On the other hand, under conditions in vivo enzymes do not operate under saturating substrate concentrations [33]. Therefore, the present dissociation aggregation mechanism has to be considered a possible mechanism of deactivation under biological high-pressure conditions, e.g. in the deep sea. Obviously, adaptation to high-pressure biotopes must aim at the selection of a stable quaternary structure of oligomeric enzymes. This investigation was supported by grants of the Deutsche For.rchungsgrinrin.rc./taft(Ja 78/20), and the Verbund der Cliemisclzcn Industrie. G.S. was a recipient of a fellowship of the Friedrich Ehert Stifiung.
413
REFERENCES 1 . Bruun, A. F. (1956) A'uture (Lond.) 177. 1105-1108. 2. Johnson, F. H., Eyring, H. & Stover, B. J . (1974) Thc~the or^, uf Rule Procrsses in Biology and Medicine, pp. 273 - 369. Wiley, New York. 3. Sleigh, A. M. & MacDonald, A . G. (eds) (1972) The €//rct of Pressure on Organi.srns, Symp. Soc. E.xp. Biol. 26. Cambridge University Press, Cambridge. 4. Jaenicke, R. 5: Koberstein, R. (1971) FEBS L e t [ . 17, 351 -354. 5. Hawley, S . A. (1971) Bioc,liemistry, 10, 2436-2442. 6. Schmid, G., Ludemann, H.-D. & Jaenicke, R. (1975) Biop1ir.s. Chen7. 3, 90-98. 7. Penniston, I. T. (1971) Arch. Biochem. Biopli,rs. 142, 322-332. 8. Low, P. S . S: Somero, G . N . (1975) Proc. Nut/ Acurl. .%i. U.S.A. 72,3305-3309. 9. Morild, E. (1977) Bioiphis. Cliem. 6, 351 -362. 10. Schmid, G., Ludemann, H.-D. & Jaenicke, R. (1978) Eur. J . Biochem. 86, 219-224. I 1. Jaenicke, R. (1970) in Pj,ridine A'uc/eotic/t. Depmdent Dchjclro~ m u s r s(Sund, H., ed.) pp. 71 -90, Springer-Verlag. Berlin. Heidelberg, New York. 12. Jaenicke, R. & Knof, S. (1968) Eur. J . Biochcw?. 4, 157-163. 13. Rudolph, R. & Jaenicke, R . (1976) Eur. J . Biocl7em. 63. 40941 7. 14. Jaenicke, R. 9r Rudolph, R. (1977) in 2nd In/. Sjw7p. Pvidinr Nucleotide Dqwm'ent Del?ydrogenases (Sund, H., ed.) pp. 351 -367. W. de Gruyter, Berlin. 15. Jaenicke, R. (1978) A'uturii.issen.schu~ten,65, 569- 577. 16. Neumann, R. C., Jr, Kauzmann, W. & Zipp, A. (1973) J . Chem. Phys. 77, 2687- 2691. 17. Teipel, J . W . & Koshland, D. E., J r (1971) Biochcn7i.sfr.1~. 10. 792 - 805. 18. ZettlmeiRl. G., Rudolph, R. & Jaenicke, K. (1979) ELII..J . Bioc/7en7. in the press. 39. ZettlmeiBl, G. (1978) Thesis, Regensburg University. 20. Jaenicke, R. (1964) Ber. Bunsenges. Phis. Chem. 68, 857-862. 21. Todheide, K. (1972) in Wufer, A Comprehmsiw Trc2atisc2 (Franks, F., ed.) vol. 1, pp. 463ff., Plenum Press, New York. 22. Schneider, G. M. (1973) in Water, A Comprc4cwsivr Treatise (Franks, F.?ed.) vol. 2, pp. 381 ff., Plenum Press. New York. 23. Rudolph, R., Heider, I., Westhof, E. & Jaenicke. R. (1977) Biochrmistr>., 16, 3384- 3390. 24. Lowell, S.J . S: Winzor, D. J. (1974) Biochen~isrry,13, 35273537. 25. Suzuki, K., Miyosawa, Y . S: Suzuki, C. (1963) Arch. Biochrn~ Bi(1phy.s. 101. 225-228, 102. 367-372. 26. Joly, M . (1965) A Physico-Chemical Approach 10 fhe D~nurur~ilion uf Proleins, pp. 12ff., Academic Press. London, New York. 27. Zimmermann, A. M. (ed.) (1970) High Pre.rsure E(fec1.s 01) Cellulur Processes, Academic Press, New York. 28. Stauff, J., Barthel, H., Jaenicke, R., Krekel, R. & Ulilein, E. (1961) K ~ l l ~ i d - 178, Z . 128- 142. 29. Elodi, P. & Jecsai, G. (1960) Actu Pliysiol. Hung. 17, 165- 182. 30. Jaenicke, R. & Pfleiderer, G. (1 962) Biochim. Bioplij..~.Actu. 60, 615-629. 31. Payens, T. A . J. & Heremans, K. (1969) Bioplyni 345. 32. Rudolph, R. (1977) Thesis, Regensburg University. 33. Atkinson, D . E. (1966) Annu. Rev. Biocf7em. 35, 85-124.
G. Schmid. H.-D. Ludemann, and R. Jaenicke*. Institut fur Biophysik und Physikalische Biochemie, Fachbereich Biolopie und Vorklinische Medizin der Universitiit Regensburg, UniversititsstraBe 31, D-8400 Regensburg, Federal Republic of Germany
* To whom correspondence should be addressed
