Comp. Biochem. Physiol., 1975, Vol. 52B, pp. 67 to 74. Pergamon Press. Printed in Great Britain

PRESSURE EFFECTS O N ENZYME STRUCTURE A N D F U N C T I O N IN VITRO A N D U N D E R SIMULATED IN VIVO CONDITIONS PHILIP S. Low AND GEORGE N. SOMERO

Scripps Institution of Oceanography, Box 1529, La Jolla, CA 92037, U.S.A. (Received 11 October 1974)

Abstract--1. The sources and magnitudes of pressure effects on enzymic structure and function are discussed. 2. Data suggest that, except for enzyme-ligand interactions, enzymes of abyssal organisms are highly similar to the homologous enzymes of shallow water and terrestrial species. 3. The magnitude of pressure effects on enzymic processes is critically dependent on the experimental conditions used, including pH, ionic strength, ionic composition, ligand concentrations and temperature. The more closely in vitro conditions approach the intracellular milieu of the enzyme, the smaller is the effect of pressure on many enzymic properties.

INTRODUCTION

ALTHOUGH the effects of hydrostatic pressure on many behavioral, morphological and physiological characteristics of marine organisms are well understood (see the volumes edited by Sleigh & MacDonald, 1972; Zimmerman, 1970), we are still without a firm understanding of how pressure affects enzyme structure and function in marine species under in vivo conditions. The primitive state of our knowledge about pressure--enzyme interactions derives from at least three factors. First, logistical difficulties have prevented extensive work with enzymes from deepsea species. Second, results from studies of pressure effects on highly purified enzymes of terrestrial species and on model compounds such as amino acid side-chain analogues have tended to give conflicting results in terms of predicting the magnitudes of pressure effects experienced by marine species in situ. Third, there exist serious deficiencies in our knowledge of how pressure affects the velocities of enzymic reactions since the many loci at which pressure can affect reaction rates under intracellular conditions have generally not been taken into account in most kinetic studies. The purpose of this essay is to integrate much of the best currently available information stemming from model compound studies and work with purified enzymes with modern pressure theories to provide a better basis for (i) predicting the nature of pressure stresses on, and pressure adaptations by, enzymes of marine organisms; and (ii) designing experiments in which the results of in vitro analysis can be meaningfully extrapolated to in vivo conditions. T H E P H Y S I C A L BASIS O F PRESSURE EFFECTS

All pressure effects, whether they be on chemical equilibria or on reaction rates, are due to volume changes which occur in the chemical process. If the volume of the system containing reaction products 67

differs from the volume of the system containing reactants, pressure will affect the equilibrium state of the reaction. If the volume of the system containing the ground state reactants differs from the volume of the system containing the activated (transition state) reactants, then pressure will affect the rate of the reaction. At constant temperature and pressure these relationships are given by equations 1_3 below: (1)

All = AE + PAV

AG° = A E + P A V -

TAS

AG~. = AE~. + PAVe. - TASk.

(2) (3)

Equation (1) states that the enthalpy change during a reaction is due to the change in internal energy (E) plus the pressure-volume work performed. Equation (2) expresses the effect of pressure-volume work on the standard free energy change (AG°) of a reaction. Equation (3) states the formally analogous effect of pressure-volume work on the free energy of activation (AG:~) of a reaction, i.e. the difference in flee energy between ground-state and activated reactants. A positive AV or AV:~ will lead to an increase in AG° or AG,, respectively, under increased pressure. Thus, if in the equilibrium A ~-- B the volume of the system containing B is less than the volume of the system containing A, elevated pressure will favor conversion of A to B. Similarly, if the volume of the system containing the activated enzyme-substrate complex (ES~:) is less than the volume of the system containing the ground state complex (ES), pressure will accelerate the reaction velocity. A positive AV:~ will lead to rate inhibition at high pressures. Only if AV or AV:~ are equal to zero will an equilibrium or rate, respectively, be pressure independent. The net effect of a change in pressure on the velocities of enzymic reactions will be determined by volume changes which occur during substrate binding events and during the activation event (Laidler, 1951).

68

PHILIP S. LOW AND GEORGE N. SOMERO

Under conditions of saturating substrate concentration the effect of pressure on an enzymic reaction rate is due solely to the sign and magnitude of AV:~ (assuming no pressure denaturation of the enzyme). However, under non-saturating, i.e. physiological, substrate concentrations, the net effect of pressure on reaction velocity is due to the sum of the volume changes which occur during the substrate binding steps and the activation event.

SOURCES A N D M A G N I T U D E S OF VOLUME CHANGES IN BIOCHEMICAL PROCESSES

Since all pressure effects derive from volume changes, it is important to determine the probable sources and magnitudes of volume changes which may accompany biological processes. A very important point to stress at this juncture is that the entire system proteins, low molecular weight solutes (substrates, inorganic ions, etc.) and water--must be considered in interpreting and predicting volume changes. The reason for this is that the density (volume) of water in the neighborhood of hydrophobic and hydrophilic molecules is often severely altered. Thus in many enzymic reactions a major source of volume changes lies in the alterations in water density which occur when amino acid sidechains and ligands (substrates, cofactors and modulators) are added to or removed from the surrounding cellular water. In addition to hydrophilic and hydrophobic group mediated effects on solvent density, the density of an enzyme molecule itself may change during one or more events of the catalytic process. Let us now examine the magnitudes of these different classes of volume change.

1. Hydrophilic contributions to A V and AV~.

Charged or highly polar molecules orient water in a denser arrangement than that present in so-called "bulk" water. Volume decreases in excess of - 1 0 cm3/mole due to electrostriction of water are common in ionization reactions, and volume changes resulting from exposure of a polar group such as O H can exceed 5 cma/mole (Table 1). Because many enzymic processes such as ligand binding, activation and denaturation occur with an accompanying change in polarity or ionization, the structures and functions of enzymes can be expected to display significant pressure sensitivities. The net volume change in each case will, of course, depend on the algebraic sum of the volume changes resulting from the various ionization and polarization reactions. 2. Hydrophobic contributions to A V and A V {

The effect of hydrophobic molecules on the structure and volume of vicinal water is complex, and depends very'highly on the proximity of neighboring non-polar groups (Table 1 ; Brandts et al., 1970; Zipp & Kauzmann, 1973). Model compound studies of the behavior of hydrophobic groups in water seem to suggest conflicting and confusing explanations of how the presence of non-polar molecules or amino acid side-chains will affect water volume. In the simplest type of model compound studies, a non-polar molecule such as ethane has been transferred from an organic solvent such as hexane to pure water (Table 1). This transfer reaction has been taken as an analogue of the process which occurs when a hydrophobic amino acid side-chain is taken from the protein's interior and exposed to water. This model assumes that the exposed hydrophobic side-chain is completely solvated by water, and that neither the

Table 1. Important sources of volume changes in biological processes Process Hydrogen bond formation 1 H ydrophobic interactions CH a in hexane---~ CH4 in water e C6H6 (pure liquid)--* C6H6 in water-' H20 (pure)---~ H:O (dilute) in l,l,l-trichloroethane 3 H20 (pure)---, H20 (dilute) in CC13 Ionic interactions Lysine (neutral) ---, Lysine ( + )1 Acetic acid --, Acetate- + H ÷' Glutamic acid ~ Glutamate- + H ÷' Mg-ATP complex--, ATP + Mg + +' Change in exposure of polar yroup Methanol in CC14---~Methanol in H20s Ethanol in CC14--, Ethanol in H205 n-propanol in CCI 4 ~ n-propanol in HEO5 Methanol (pure)---, Methanol in H20 ~' Ethanol (pure)--~Ethanol in H206 n-propanol (pure)--, n-propanol in H2 O6 Helix-coil transition 7

Volume change (cm3/mole) - ( 3 5) -22-7 -6.2 +4.3 + 13-6 - 26.4 -11-8 -12.7 -22 -7-1 -4-9 -6.4 - 2.44 -3.4 -4.52 +1

1 Suzuki & Taniguchi (1972). 2 Kauzmann (1959). 3 Masterton & Seiler (1968). * Rainford et al. (1965). 5 Values calculated from data given by Duboc (1969) and Friedman & Scheraga (1965). 6 Friedman & Scheraga (1965). 7 Noguchi (1966).

Pressure effects on enzyme structure and function peptide backbone nor the neighboring amino acid side-chains are capable of interfering with the formation of water "icebergs" around the exposed hydrophobic group. These highly simplified model compound studies predict very large volume decreases during hydrophobic side-chain exposure (Table 1) due to the high density of ordered water around the nonpolar group (Kauzmann, 1959). There is, however, an alternate model for describing the process of hydrophobic side-chain exposure to water. If the exposed hydrophobic group is pictured as existing in a "pocket" on the protein surface where neighboring amino acid side-chains and the polypeptide backbone can sterically hinder the formation of dense "icebergs" of water around the hydrophobic group, then a different interpretation of water-protein interactions is necessary. We must now consider the volume changes which can accompany the addition of relatively small amounts of water to a concentrated organic phase. Masterton & Seiler (1968) showed that the apparent partial molar volume of water in a nonpolar solvent is greater than in bulk water (Table 1). The expansion of water in an area of concentrated non-polar groups has also been demonstrated in model compound studies. Using a synthetic polypeptide having a high density of hydrophobic residues, Boje & Hvidt (1972) observed small positive volume changes during unfolding of the molecule in water. These findings suggest that the density of neighboring hydrophobic moieties hindered solvation of each nonpolar side-chain, and that only a small number of water molecules were capable of filling the large void spaces between the side-chains. In conclusion, the exposure of hydrophobic groups to water can lead to volume increases or decreases, depending on the types and concentrations of adjacent hydrophobic groups. Model compound studies may therefore give us a poor basis for predicting, quantitatively, the volume changes which occur during protein denaturation. Where model study data may have their greatest validity and predictability is in the case of enzyme-ligand interactions, where the common model system conditions of low molecular weight and high dilution are more nearly matched. 3. Protein structural contributions to AV and AV~ Volume changes may occur during enzymic processes in the absence of any exposures/withdrawls of charged, polar or hydrophobic groups to/from water. Conformational changes during substrate binding (Koshtand & Neet, 1968), modulator interactions (Kayne & Suelter, 1968), the activation event (Low & Somero, 1974), and denaturation (Zipp & Kauzmann, 1973) necessarily involve rearrangements of the amino acid residues of the enzyme. Such structural rearrangements may often involve changes in the "packing density" of amino acid side-chains within the interior of the protein, i.e. the volume occupied by the protein may change during each of these processes. We have in fact observed structural contributions to the activation volumes of enzymic reactions ranging up to 32"5 cma/mole (Low & Somero, 1975). E N Z Y M I C PROCESSES INHERENTLY SENSITIVE TO PRESSURE

Volume changes involving enzymes can occur during one or more of the following processes: (i) forma-

69

tion of weak bonds between enzymes and ligands, (ii) conformational changes during ligand binding and during the activation event, (iii) subunit association/ dissociation reactions, and (iv) loss of tertiary structure during denaturation. It is important to consider the possible importance of pressure-volume effects at each of these loci when interpreting the effect of pressure on an overall enzymic process. In the following paragraphs we will examine existing data in each of these areas and discuss likely pressure effects in vivo and the requirements for, and the nature of, pressure adaptation mechanisms. 1. Enzyme-ligand interactions There are two possible sources of volume changes during the formation of enzyme-ligand complexes. First, due to the highly charged and polar nature of virtually all metabolic intermediates, the release of electrostricted water during ligand binding should be a nearly universal phenomenon. Binding reactions which involve hydrophobic interactions should also be characterized by significant volume changes, albeit the signs of the volume changes will depend on the numbers and types of adjacent groups on the protein surface, as discussed above. A second, and kinetically indistinguishable, source of volume changes during ligand binding stems from protein conformational changes. These volume changes may be due to alterations in the internal "packing" of the protein or to changes in the numbers and types of amino acid side-chains exposed on the protein surface, either at or remote from the ligand binding sites. These alterations in side-chain exposure will promote volume changes by affecting the densit~¢ of neighboring water molecules. This source of volume changes may play an important role in establishing the pressure sensitivities of binding events since most ligand binding reactions appear to be accompanied by structural changes in the enzyme. Because of the critical importance of ligand binding events in controlling rates of enzymic activity in vivo, one might predict that pressure adaptation by deepsea or vertically migrating marine organisms would involve reduction in the pressure-sensitivities of substrate and modulator binding reactions. Let us then examine the available information dealing with pressure effects on these equilibria to determine (i) if enzyme-ligand interactions are pressure sensitive, and, if so, (ii) if the enzymes of abyssal species differ in their ligand binding sensitivities from the enzymes of shallow water and terrestrial species. For pyruvate kinases of different marine teleosts we observed generally similar effects of pressure on the apparent Km values of phosphoenolpyruvate (PEP) for most PKs examined. Between pressures of 1 and 204 atm the apparent K m of PEP varied by no more than approximately 35~o for the PKs of the two deepwater fishes and the surface species, Trematomus borchgrevinki (Table 2). Only Scorpaena P K displayed a relatively large change in apparent K m over this range of pressures. We do not feel that any pattern of pressure "adaptation" is apparent in these data. A comparison of lactate dehydrogenases (LDH's) of different species revealed that the effect of pressure on the apparent Km of pyruvate was virtually identical for all homologues of the enzyme up to pressures

70

PHILIP S. Low AND GEORGEN. SOMERO Table 2. The influence of pressure on the apparent Michaelis constant (Kin) of phosphoenolpyruvate (PEP) for pyruvate kinases of four marine teleosts K m

1 atm.

Trematomus borchgrevinki

of PEP (x 10-s M) 204 atm. 408 atm.

3.47

4.71

5-83

4.8

1.04

4.5

2.15

2.95

3.75

2-61

2.67

4.51

(shallow)

Scorpaena gutatta (shallow)

Coryphaenoides acrolepis (deep water)

Sebastolobus altivelis (deep water)

Pyruvate kinase activity was measured spectrophotometrically in a Pye-Unicam SP1800 ultraviolet spectrophotometer, equipped with an Aminco high-pressure cell (Mustafa et al., 1971), by monitoring the decrease in extinction at 340 nm due to NADH oxidation as a function of time. The assay solution contained, in a total volume of 5.0 ml, TrisHCI buffer (25 mM, pH 7.5 at the assay temperature); 1 mM ADP, 80 KC1, 10 mM Mg(C1)2, 0.05 mM Fructosediphosphate, varying concentrations of PEP, 0.12 mM NADH, excess lactate dehydrogenase activity, and pyruvate kinase (added last to start the reaction). The enzyme from Scorpaena was purified to homogeneity. The activities of the other enzymes were measured using supernatant fractions of tissue homogenates (1:7 in 0.01 M Tris/HC1 buffer, pH 7'5) of white skeletal muscle spun at 15,000 x g for 20min. Apparent K m values were determined by computer, using a regression of [substrate]/velocity vs [substrate] (Dowd & Riggs, 1965). Temperature of all assays was 5 + 0"05°C.

of approximately 400 atm (Fig. 1). This range of pressures, which corresponds to a depth range of 4000 meters, encompasses the major portion of the range of pressures experienced by marine teleosts. Thus it is largely outside of the biological pressure range of fishes that apparent differences in substrate binding sensitivities to pressure are observed (Fig. 1). At higher pressure, only the L D H of the abyssal species, Coryphaenoides acrolepsis, continues to display a linear relationship between pressure and apparent Km of pyruvate. IO--

• Halibut

omus

I

I

200

i

I

400 Pressure,

~

I

600

atm

Fig. 1. The effect of pressure on the apparent Michaelis constant (Kin) of pyruvate of lactate dehydrogenases from three marine teleosts adapted to similar temperatures but widely different pressures. Trematomus borch(Irevinki is an Antarctic species which lives at -1.86°C; Coryphaenoides acrolepis (rattail) is a deep benthic species which lives at 2-4°C (Phleger, 1971). The east coast halibut (Hippoglossus hippoglossus) is a benthic species not subjected to extreme pressures. Lactate dehydrogenase activities were measured as described in Table 3. Apparent Km values were determined by computer using plots of [substrate]/velocity vs [substrate] (Dowd & Riggs, 1965).

A third enzyme for which pressure effects on enzyme-substrate interactions have been studied is acetylcholinesterase (ACHE) (Hochachka, 1974). For this enzyme marked differences were observed between the homologues from shallow water and deepsea fishes. Pressure disruption of acetylcholine (ACh) binding by AChE was found for the enzyme of a surface fish, but not for the AChE of a deepsea fish, Antimora. Experiments using substrate analogues and competitive inhibitors showed that the hydrophobic contribution to stabilizing the AChE-ACh complex was reduced in the deepsea form, and that the electrostatic interaction between enzyme and substrate was of relatively greater importance in the deepsea species. The latter finding is, of course, paradoxical, since electrostatic interactions, like hydrophobic interactions, are disrupted by high pressures (Table 1). What these observations indicate is that the pressure effects on enzyme-ligand interactions are determined by more than the single chemical reaction between ligand and binding site. Coupled, simultaneous volume changes elsewhere on or within the enzyme could compensate for volume changes at ligand binding sites. Thus if ligand binding involves hydrophobic or electrostatic interactions at the ligand binding site, the volume increase associated with this event could be counteracted if, for example, a charged amino acid side-chain were exposed to water elsewhere on the enzyme molecule concurrent with ligand binding. Since the net volume changes will be the algebraic sum of all volume changes occurring, coupled volume changes of this type could enable an enzyme to gain complete control of its ligand binding volumes. Additional examples of pressure adaptation during ligand binding events are described by Hochachka & Somero (1973).

Pressure effects on enzyme structure and function

2. Activation processes Conformational changes during the generation of the activated enzyme-substrate complex (ES:~) from the ground state ES complex can lead to substantial volume changes. Consider, for example, an enzymic reaction in which a dissociated glutamic acid sidechain is exposed to water in the ground state but is withdrawn away from water during formation of the activated complex. A volume increase of around 12 cma/mole enzyme would be expected as the electrostricted solvent shell surrounding the carboxylate moiety expanded into the bulk water (Table 1). Negative activation volumes could result from exposure of a charged or highly polar side-chain during formation of the ES~ complex. The movement of hydrophobic residues could also lead to volume changes during the activation event (Table 1). In terms of pressure-adaptive changes in enzymes with regard to activation volumes, one might predict that enzymes of abyssal and vertically-migrating species would differ from the homologous enzymes of non-pressure-stressed organisms in two ways. First, reductions in the absolute values of activation volumes would render enzymic reaction rates less pressure sensitive at all substrate concentrations. We might therefore expect to find lower absolute values for the AV:~ of a particular reaction in deepsea or vertically migrating forms. Second, since abyssal temperatures are very low (near 2-4°C), a deepsea organism might simultaneously adapt to temperature and pressure by developing enzymes which have large negative activation volumes. The pressure enhancement of reaction rates would tend to compensate for the decelerating effects of low temperatures.

Table 3. Activation volumes (AVe) for various pyruvate kinase and lactate dehydrogenase reactions Activation volume (cma/mole)

Pyruvate kinase* Trematomus borchgrevinki (shallow, Antarctic) Scorpaena #utatta (shallow, temperate) Coryphaenoides acrolepis (deep water) Sebastolobus altivelis (deep water) Lactate dehydrogenase~ Trematomus borchgrevinki Hippoglossus hippoglossus Coryphaenoides acrolepis Rabbit

71

An examination of available data leads us to conclude that neither of these expectations is realized (Table 3). AV:~ values for LDH reactions of different fishes are nearly identical. For PK, the magnitude of AV~ shows no reduction in deepsea forms; on the contrary, the PK of the shallow water fish, Trematomus, seems "better" for abyssal function than the PK of the deepsea fish, Coryphaenoides. It appears, therefore, that the size of the activation volume of a reaction does not serve as "raw material" for adapting an enzyme to high and constant pressures.

3. Subunit association-dissociation equilibria The stabilizing energy which holds subunits together in multimeric enzymes is due to weak chemical bonds such as hydrophobic interactions and electrostatic interactions. Since bonds of these types form/ break with changes in volume (Table I), one would predict that the equilibrium between aggregated and dissociated subunits of an enzyme would display a pressure sensitivity. Indeed, Penniston (1971) has suggested that the disrupting effects of high pressures on multimeric enzymes may be so severe as to preclude the existence of multimeric enzymes in deepsea species. The data shown in Fig. 2 suggest that multimeric enzymes are fully capable of withstanding the pressures characteristic of the abyssal regions. LDH and PK are known to be tetrameric proteins in all multicellular organisms examined to date. It seems extremely unlikely that a deepsea fish would, or indeed could, revert to a monomeric form of either enzyme. In fact, the electrophoretic patterns observed for several enzymes of Coryphaenoides are completely consistent with the existence of multimeric structures identical to those of enzymes of non-abyssal organisms (Somero & Soul6, 1974).

0.~

O'E

10.5 15.0

0-4

23"5

I

l

l

I

23.5 LDH

0.8 K-

g

u

-0"43 -0"23 - 3.93 -4.20 o.4D

* Pyruvate kinase activity was measured as described in Table 2, except that the total concentration of potassium and ammonium ion was approximately 120 mM. Assay temperature was 5°C. t Lactate dehydrogenase activity was measured spectrophotometrically by following the decrease in optical density at 340 nm as a function of time. The assay mixture contained, in a total volume of 5"0 ml, 100 mM Tris/HC1 buffer, pH 7.5; 0.12 mM NADH, varying concentrations of pyruvate, and enzyme, added last. The assay temperature for the fish enzymes was 5°C; for the rabbit enzyme (the M 4 isozyme from skeletal muscle) the assay temperature was 15°C.

I

I

200

f Pressure,

I

400 ofm.

I

I

600

Fig. 2. The effect of pressure on the maximal velocities (Vm,x'S)of pyruvate kinase (PK) and lactate dehydrogenase (LDH) reactions of different marine teleosts. Assay conditions were as described in Tables 2 and 3. Potassium concentration in the PK assay solution was 80 mM. l : Scorpaena gutatta; A: Sebastolobus altivelis (deep benthic); O Trematomus borclglrevinki; [] Coryphaenoides acrolepis; • halibut (Hippoglossus hippoglossus).

72

PHILIP S. Low AND GEORGEN. SOMERO

Further evidence for the stability of quaternary structure at high pressures comes from the observation that upon depressurization of a reaction the maximal velocity returns immediately to its 1 atm value. This indicates that subunit depolymerization did not occur at high pressures since individual LDH and PK subunits are not active catalytically. It is also interesting that the different homologues of LDH are much more similar in terms of the pressure effects on maximal velocities than they are with regard to pressure-Kin relationships (Fig. 1). Perhaps subtle conformational changes are sufficient to alter enzyme-substrate interactions while having no effect on the catalytic events which follow formation of the ES complex.

4. Pressure denaturation of tertiary structure The results of early model compound studies which focused on simplified transfer reactions such as those discussed above led biochemists to predict that denaturation volume changes of the order of several hundreds of cm3/mole would accompany the unfolding of a protein's tertiary structure. Volume changes of this magnitude would render proteins very susceptible to denaturation at abyssal pressures. Evidence from several types of experiments suggests that such large volume changes do not accompany loss of tertiary structure. More "biological" model compound studies such as those of Boje & Hvidt (1972) have called into question the conclusions about protein denaturation volumes based on simplified amino acid side-chain transfer studies. Measurement of the actual volume changes which accompany the unfolding of monomeric proteins has revealed that volume changes upon denaturation are of the order of tens of cm3/mole, not hundreds of cm3/mole. In addition, the precise magnitude of the denaturation volume change may be highly dependent on the conditions of pH and temperature used in the experiment (Brandts et al., 1970; Zipp & Kauzmann, 1973). Lastly, kinetic data such as those shown in Figs. 1 and 2 argue strongly that abyssal pressures are not sufficient to cause the gross destruction of protein conformation--even in the case of enzymes from shallow water species. PRESSURE E F F E C T S A N D T H E

CATALYTIC MICROENVIRONMENT In virtually all enzyme studies one faces "uncertainty principles" arising from a lack of knowledge about how the enzyme's function under simplified in vitro conditions compares with its function within the cellular microenvironment it actually experiences. It is therefore not unwarranted to inquire about the roles which variation in ionic composition, ionic strength, pH, modulator concentrations, substrate concentrations, and enzyme concentrations might play in determining the pressure responses of an enzyme. As we shall find, most of these factors can exert profound influences on the pressure sensitivities of enzymes.

1. Ionic strenoth effects The most dramatic illustration we are aware of concerning the effects of the catalytic microenvironment on the pressure response of an enzyme is shown

~c E

+-

3(

Pressure effects on enzyme structure and function in vitro and under simulated in vivo conditions.

Comp. Biochem. Physiol., 1975, Vol. 52B, pp. 67 to 74. Pergamon Press. Printed in Great Britain PRESSURE EFFECTS O N ENZYME STRUCTURE A N D F U N C T...
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