erythrocyte origin (Coelho-Sampaio et al., 1994; see Fig. 8). ... Pi, (de Meis and Inesi, 1988; Jorge-Harcia et al., 1988), it may be that the binding of Pi to the ...
Bioscience Reports, Vol. 16, No. 2, 1996
REVIEW
Catalytic and Structural Modifications of Sarcoplasmic Reticulum and Plasma Membrane (Ca a§ + Mga§ Induced by Organic Solutes that Accumulate in Living Systems Adalberto Vieyra
This review is dedicated to Prof. Carlos Chagas Filho, founder of the Institute of Biophysics, on the occasion of its 50th anniversary. Received October 27, 1995 Organic solutes such as urea, methylamines, polyols and amino acid can accumulate in the cytoplasm of cells to compensate for hyperosmotic conditions in the external medium. Whereas urea is considered to be typical of solutes that destabilize structure and function of proteins, methylamines, polyols and some amino acids appear to have the opposite effect, and can also compensate for the perturbing effects of urea. These effects have been extensively analyzed for a variety of proteins in terms of global changes in enzyme structure and acceleration or inhibition of overall reaction rates. Here the influence of these solutes on sarcoplasmic reticulum and plasma membrane (Ca2++ Mg2+)ATPases is reviewed. The focus is on the changes induced by "perturbing" and "stabilizing" solutes at specific steps of the catalytic cycles of these enzymes, which can run forward (leading to ATP hydrolysis) and backward (leading to ATP synthesis). Structural changes promoted by osmolytes 'are correlated with functional changes, especially those that are related to energy coupling. KEY WORDS: ATPase; calcium; magnesium; plasma membrane; polyols; organic solutes; sarcoplas-
mic raticulum; urea.
INTRODUCTION Unicellular organisms and specialized tissues in multicellular organisms that are adapted tO the presence of high concentrations of salts and/or urea ("perturbing" solutes) have developed the ability to accumulate certain low molecular-weight, osmotically active organic solutes (the so-called "stabilizing" osmolytes) (Yancey et al., 1982; Somero, 1986; Garcia-Perez and Burg, 1991; Burg, 1995). High levels Departamento de Bioquimica Medica, Instituto de Ci~ncias Biomedicas, Universidade Federal de Rio de Janeiro 21941-590, Rio de Janeiro, Brazil. 115
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of salts and urea within the cells can inhibit metabolic 6nzymes as a result of deleterious effects on their structure and dynamic properties (Mashino and Fridovich, 1987). In contrast, polyols, methylamines and some amino acids and amino acid derivatives ("non-perturbing" or "stabilizing" solutes) counteract the destabilizing actions of those solutes by accumulating in parallel with them, or in some cases accumulate instead of the more deleterious solutes (Yancey et al., 1982; Somero, 1986; Bagnasco et al., 1986; Yancey and Burg, 1989, 1990; Nakanishi et aL, 1992; Lin and Timasheff, 1994; Burg, 1995). Special cases of osmotic adaptation have been described in systems that are chaiienged with drastic modifications in the availability of water during development or due to seasonal changes in aqueous environments (Yancey et al., 1982; Somero 1986; Kinne, 1993). Terrestrial mammals, having conquered the transition from water to dry land during evolution, eliminate a concentrated urine as the result of osmotic equilibration between the fluid of collecting tubules and the medullary interstitium, a region where NaC1 and urea are highly concentrated under physiological conditions (Bankir and de Rouffignac, 1985; Bagnasco et al., 1986; Balaban and Burg, 1987; Burg, 1995). All of these organisms accumulate different "stabilizing" solutes in various combinations (Yancey et al., 1982; Garcia-Perez and Burg, 1991; Burg, 1995). It has been proposed that "stabilizing" solutes protect enzyme activity because they are compatible with macromolecular structure and function and do not interact with substrates and cofactors. In contrast, "perturbing" solutes are considered to interact with ligands and also with active and/or regulatory sites of the enzymes. Somero and coworkers (Yancey et al., 1982; Somero, 1986) were the first to point out that several animal species accumulate "perturbing" (urea) and "stabilizing" solutes (mainly methylamines) in an approximately 2 to 1 molar ratio, and that this ratio appears to have been conserved during evolution. Later, Mashino and Fridovich (1987) found that methylamine derivatives, principally trimethylamine-N-oxide (TMAO), favor a more compact protein structure, but do not always cancel the effect of urea on enzyme activity, indicating that the most compact structure does not necessarily correspond to a more active conformation. As mentioned above, high concentrations of urea and salts are commonly found in mammalian kidneys and in the tissues of several fishes in which the high osmolarity of the internal milieu balances that of the sea water (Yancey et at., 1982; Somero, 1986; Bagnasco et al., 1986; Yancey and Burg, 1989, 1990; Garcia-Perez and Burg, 1991). In addition, it has been demonstrated that kidney tissues accumulate methylamines and polyols in physiological and pathological conditions (Balaba_n a_nd Knepper, 1983; Somero, 1986; Bagnasco et at., 1986; Burg and Kador, 1988; Yancey and Burg, 1989, 1990; Garcia-Perez and Burg, 1991; Burg, 1995). These solutes predominate in the internal medulla where high concentrations of urea and salts participate in the formation of hypertonic urine. More recently, glycerophosphorylcholine was also found to be metabolized in the renal cortex (Gulland et al., 1988; Garcia-Perez and Burg, 1991). The natural occurrence of these high concentrations of "perturbing" and "non-perturbing" solutes in intracellular and extracellular compartments of mammal tissues, implies exposure of membrane-bound enzymes--including
Organic Solutes and (Ca2+ + Mg2§ ATPases
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ion-transporting ATPases--to environments assymetrically enriched with those molecules. In addition, erythrocytes and other cells that circulate through renal tissue are challenged by the presence of high solute concentrations in the intravascular compartment. Since fluctuations of cytosolic Ca 2+ in all kinds of mammalian cells are fine-tuned regulated by the (Ca 2+ + Mg2+)ATPase (Carafoli, 1991, 1992; Penniston and Enyedi, 1994), and these fluctuations are associated with changes in intracellular organic osmolytes during cell volume regulation (Pierce and Politis, 1990), the renal plasma membrane Ca 2+ pump becomes an interesting subject for studying osmolyte effects on ion-transporting enzymes. The erythrocyte plasma membrane (Ca2+ + Mg2+)ATPase has the additional advantage that it can be purified to homogeneity (Niggli et al., 1979), thus allowing investigation of osmolyte effects on enzyme structure. The extensively studied sarcoplasmic reticulum (Ca2+ + Mg2+)ATPase is a good model for the study of the effects of solutes on intracellular Ca 2+ pumps. It is now apparent that the (Ca z+ +Mg2+)ATPases from sarcoplasmic reticulum and plasma membranes have their catalytic and regulatory properties drastically changed by "perturbing" and "stabilizing" solutes. Some solutes-especially those of the "stabilizing" family--can affect specific domains of ATPases rather than promote global and unspecific changes in the enzyme molecule. This review will focus on the effects of organic solutes on the catalysis by the (Ca 2§ + Mg2+)ATPases of the sarcoplasmic reticulum of skeletal muscle and the plasma membranes of red cells and kidney proximal tubules, with emphasis on the partial reactions involved in energy transduction. Implications for possible regulatory mechanisms will also be discussed. THE CATALYTIC CYCLE OF THE SARCOPLASMIC RETICULUM AND OF THE PLASMA MEMBRANE (CaZ++ MgZ+)ATPases Since other aspects of catalysis by (Ca 2+ + Mg2+)ATPases are covered in this collection and excellent reviews have been published elsewhere (de Meis, 1989; Carafoli, 1991, 1992; Missiaen et al., 1991; Carafoli and Chiesi, 1992; Penniston and Enyedi, 1994; Inesi et al., 1994), the reaction sequence of these pumps will be described only in outline. Although the enzyme from sarcoplasmic reticulum differs in some respects from those of plasmalemma (for example in molecular weight and regulatory properties), they share common features in their catalytic properties. Both of these (Ca2+ + MgZ+)ATPases are P-type ATPases, i.e. they form an acytphosphoprotein intermediate and alternate between two main conformations (El and E2) during the catalytic cycle (Fig. 1). It is accepted that Ca 2+ binds with high affinity to the E1 conformation at a site (or sites) accessible from the cytosol. They, an aspartyl residue is phosphorylated by ATP in a domain of the active site that is highly conserved among the different enzymes of the family (Green, 1989; Carafoli, 1991, 1992; Stokes et al., 1994). After ADP is released from the ATPase, Ca 2+ ions are occluded, and become inaccessible to the cytosol. Occlusion is associated with a large conformational transition in which the high-energy r a - P intermediate is converted to the low-energy r - P intermediate. This transition is accompanied by a large decrease in Ca 2+
118
Vieyra Ca 2+
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ADP
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Pi H20 Ca z§ Fig. 1. Reaction sequence of sarcoplasmic reticulum and plasma membrane (Ca2++ Mg2+)ATPases, -P and -P indicate high- and low-energy states of the acyl phosphoprotem linkage. The box around Ca in the first phosphorylated intermediateindicates occlusion of the cation. For description see text. Adapted from Carvalho et al. (1976), Pedersen and Carafoli (1987) and de Meis (1989).
affinity (three orders of magnitude or more). Subsequently, Ca 2+. is released outside the cell (in the case of plasma membrane) or to the luminal space (in the case of sarcoplasmic reticulum). After release of Ca 2+, the phosphorylated intermediate is cleaved by water and the resulting dephosphoenzyme Ez undergoes a conformational change that is accelerated by free Mgz+ and MgATP 2- (Vieyra and Caruso-Neves, 1993). This transition completes the coupled cycle of Ca a+ transport and ATP hydrolysis. These (Ca2+ + Mg2+)ATPases can also function in the backward direction. Partial reactions in the reverse mode (phosphorylation by Pi in the absence of Ca 2+ forming E2 - P; single cycles of ATP synthesis from E2 - P and ADP), as well as complete and continuous reversal cycles (ATPe-~3aPi exchange), have been described for both the sarcoplasmic reticulum and the plasma membrane (Ca 2+ + Mg2+)ATPase (Masuda and de Meis, 1973; Carvalho et al., 1976; de Meis et al., 1980; Chiesi et al., 1984; Vieyra et al., 1989, 1991). As described below, osmolytes can selectively modify different steps of the reaction cycle. These modifications, in some cases, may be associated with structural changes in specific domains of the ATPase molecule.
MODIFICATIONS BY ORGANIC SOLUTES OF PARTIAL REACTIONS CATALYZED BY THE SARCOPLASMIC RETICULUM (CaZ+ + MgZ+)ATPase Effects of Urea and Methylamines in Phosphorylation Events From the foregoing description of the reaction cycle (Fig. 1), it can be seen that phosphorylation by ATP in the presence of Ca :+ and phosphorylation by Pi in the absence of Ca z§ are critical steps in the forward and reverse cycles, respectively. A number of recent studies show contrasting effects of osmolytes on
Organic Solutes and (Ca 2§ + Mg 2+) ATPases
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Fig. 2. A: Effect of urea on calcium binding to sarcoplasmic reticulum (Ca2+ + Mg 2+) ATPase in the absence of ATP (O), and on enzyme phosphorylation with ATP in the presence of Ca z+ (9 B: Effect of urea on steady-state ATPase activity. Sarcoplasmic reticulum vesicles were incubated for 2 (9 or 30 (O) min with the solute. Taken from Jorge-Garcia et al. (1988), with permission.
these steps. Jorge-Garcia et al., (1988) have shown (Fig. 2A) that Ca 2+ binding (Fig. 1, step 1) and enzyme phosphorylation with ATP at saturating Ca z+ concentrations are not modified by urea up to 2 M. In contrast, the steady-state (Ca2++ MgZ+)ATPase activity was markedly inhibited with lower urea concentrations (Fig. 2B), suggesting an effect of the solute on the enzyme turnover and in the conversion of the unphosphorylated forms (Fig. 1, step 6). This observation is in line with the effect of urea in another different P-ATPase, the renal plasma membrane ( C a 2+ + MgZ+)ATPase (Vieyra and Caruso-Neves, 1993; see Fig. 5), but somewhat different with that found using the enzyme of erythrocyte origin (Coelho-Sampaio et al., 1994; see Fig. 8). Unlike phosphorylation of the enzyme by ATP, phosphorylation by Pi (Fig. 1, step 5, reversal) is progressively inhibited even by low concentrations of urea (Jorge-Garcia et aL, 1988). Since these concentrations also decreased affinity for Pi, (de Meis and Inesi, 1988; Jorge-Harcia et al., 1988), it may be that the binding of Pi to the aspartyl residue is more sensitive to the urea induced unfolding. The binding of ATP, which involves both its adenosine moiety and an upstream pocket (the FITC-binding domain) (Green, 1989; Stokes et al., 1994), may confer a more stable structure that resists the perturbations promoted by urea. Urea inhibition of the phosphorylation by Pi of the sarcoplasmic reticulum ATPase was blocked by the simultaneous addition of TMAO or betaine (de Meis and Inesi, ]988). In fact, the affinity for Pi was increased by TMAO alone (de Meis and Inesi, 1988), in line with the proposal that a more compact state of the enzyme domain that includes the phosphorylation site, may favor binding of Pi and its interaction with its aspartyl residue acceptor.
Effects of Polyols in the Phosphorylation by Pi As mentioned above, polyols can also accumulate in some prokaryotic and eukaryotic cells, increasing salt tolerance during water stress and conferring
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Fig. 3. Effects of trehalose (0) and sucrose (A) on the Pi dependence of the phosphorylation reaction of sarcoplasmic reticulum ATPase. Empty circles show the control without sugars (apparent Km for Pi > 10 mM). Panel B shows a double-reciprocal plot of the data shown in A (apparent Km values: 1.6 and 2.0mM with trehalose and sucrose, respectively). Taken from Chini et al. (1991), with permission.
protection against low temperatures (Yancey et aL, 1982; Somero, 1986; GarciaPerez and Burg, 1991). Glycerol, for example, is found in euryaline unicellular algae when they are grown in saturated NaC1 solutions. Glycerol is not needed for metabolism, but it promotes water retention and preserves the structure and function of macromolecules (Brown and Simpson, 1972). It has been proposed that sugars--and polyols in general--may have acted as compatible solutes in early stages of evolution (Yancey et aL, 1982; Somero, 1986; Kinne, 1993), protecting primitive living systems against the deleterious effects of the unfolding promoted by urea and/or salts. With the sarcoplasmic reticulum (Ca 2+ +Mg2+)ATPase, Chini and coworkers (Chini et al., 1991) observed that some monosaccharides and disaccharities mimic the effect of methylamines on phosphoenzyme formation and Pi affinity (Fig. 3). These effects, as well as those reported by de Meis and Inesi (1988), are similar to those obtained with dimethyl sulfoxide, a molecule with high dipolar moment that promotes desolvation of anions (Parker, 1962). It may be that in the presence of sugars the hydration shell of Pi diminishes, thus favoring its binding in the more hydrophobic catalytic site of the enzyme in the E2 conformation (de Meis, 1989). This pattern of activation by desolvation of reactants which is shared by polyols and methalamines appears to be characteristic in phosphoryl transfer reactions catalyzed by (Ca 2+ + Mg2+)ATPases. ORGANIC
S O L U T E S IN T H E C A T A L Y S I S B Y T H E P L A S M A M E M B R A N E (Ca 2+ + Mg2+)ATPase
Glycerol-induced Modifications in Kinetics and Structure
Using a purified preparation of red cell ( C a 2+ + Mg2+)ATPase, Benaim and de Meis (1989) showed that addition of glycerol mimics the effects of the natural
Organic Solutes and (Ca 2+ + Mg 2+) ATPases
121
activator calmodulin (Gopinath and Vincenzi, 1977; Jarrett and Penniston, 1977). Glycerol increased the affinity for Ca 2+ and the enzyme turnover, an effect that, as in the case of the ATPase from sarcoplasmic reticulum (de Meis and Inesi, 1988), was similar to that obtained with dimethyl sulfoxide. Since-it is believed that the enzyme domains involved in Ca 2+ binding and translocation are--at least in part--located in transmembrane segments (Carafoli, 1991, 1992; Stokes et al., 1994; Inesi et al., 1994), the authors proposed that the polyol modifies hydrophobic interactions of the polypeptide chain with the surrounding phospholipids. The same modifications may occur in the vicinity of the segments of the ATPase molecule that interact with the autoinhibitory calmodulin binding domain (Falchetto et al., 1992), thus favoring activation of the enzyme. It may be that the thermodynamically unfavorable exclusion of glycerol from enzyme domains is compensated for by a decrease in the area of water-protein contact (Gekko and Timasheff, 1981). Polyols interact with phospholipids and proteins (Arakawa and Timasheff, 1982), increasing non-polar interactions between folded protein domains and decreasing exposure of hydrophobic domains to the aqueous environment (Back et al., 1979). Thus, addition of glycerol could also enhance the self-association of (Ca z+ + Mg2+)ATPase molecules, a process that activates the enzyme (Kosk-Kosicka et al., 1989; Coelho-Sampaio et aL, 1991). Selfassociation may be responsible for the blue-shift in the intrinsic fluorescence of red cell ATPase that is induced by glycerol in the absence of C a 2+ (unpublished observations from this laboratory). In this condition, without glycerol, the monomeric state predominates (Coelho-Sampaio et al., 1991). The formation of dimers would be expected to decrease exposure of tryptophan residues located in the interface between ATPase subunits to the aqueous medium.
The Effects of Glycerol are Modified by C a 2+ Binding: Uncoupling and Inhibition
It is interesting to note that glycerol effects on the plasma membrane (Ca 2+ + Mg2+)ATPase appear to be modulated by the occupancy of C a 2+ binding sites. At subsaturating Ca 2+ concentrations, glycerol stimulates ATP hydrolysis and a very high-affinity component of the Ca 2+ curve is revealed. Stimulation of ATP hydrolysis, however, is accompanied by inhibition of Ca 2+ uptake, indicating uncoupling of the pump (Sola-Penna et al., 1995b). This is evidence that the enzyme domains that link C a 2+ binding sites with those involved in ATP binding and breakdown, are modified by variations in their interactions with water. Sola-Penna et al., (1995b) demonstrated that at Ca 2+ concentraions high enough to saturate the high-affinity sites, glycerol concentrations above 10% (v/v) inhibit Ca 2+ flux and CaZ+-dependent ATP hydrolysis in parallel. Thus despite its similarity with calmodulin in activating the hydrolytic activity, glycerol differs in another respect: unlike calmodulin, it does not stimulate Ca 2+ transport (Gopinath and Vincenzi, 1977; Jarrett and Penniston, 1977). Uncoupling of C a 2+ from ATP hydrolysis appears to be a general feature of
122
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[ Urea] (M) Fig. 4. Sorbitoland mannitolprotection against urea inhibition of renal plasma membrane (Caz+ +Mg2+)ATPase activity. Assays were in the absence (D) or in the presence (11)of 0.4 M sorbitol (A) or 0.4 M mannitol (B). Taken from Sola-Penna et al. (1995a), with permission. polyols, since trehatose, sorbitol and mannitol inhibit the ATP-dependent Ca 2+ flux across the plasma membrane without modifying (Ca 2+ +MgZ+)ATPase activity (Sola-Penna et al., 1994, 1995a). Interestingly, the presence of polyols cancels the inhibition promoted by urea (Fig. 4) (Sola-Penna et aL, 1995a), raising the possibility that polyols that accumulate in renal tissue could protect renal enzymes when urea and NaC1 become highly concentrated. It remains to be seen whether the apparent lack of effect of polyols on ATP binding and breakdown is a general property that was evolved to preserve phosphoryl transfer reactions of soluble and membrane-bound enzymes in renal tissue during osmotic stress.
Solute Effects are Modified by Energy-Donor Ligands, Medium pH and Cofactors Solute effects on plasma membrane (Ca2+ + Mg2+)ATPase are also dependent on other physiological ligands such as free Mg2+ and the MgATP 2- complex. Free Mg> and the MgATP 2- complex activate the enzyme by binding to nonidentical and independent regulatory sites (Vieyra and Caruso-Neves, 1993). Stimulation of (Ca2+ + MgZ+)ATPase by both ligands (Fig. 5A) is abolished in the presence of non-denaturating urea concentrations (Fig. 5B), indicating that in a partially unfolded state, the ATPase molecule loses its ability to recognize these ligands at their regulatory binding domains. This effect of urea is thus similar to that shown for enzyme turnover of the sarcoplasmic reticulum ATPase (JorgeGarcia et al., 1988), which is regulated by high concentrations of ATP at a low-affinity site. TMAO inhibits the activity at all concentrations of free Mg 2+ and MgAtP 2- complex (Fig. 5C), confirming the proposal by Mashino and Fridovich (1987) that the more compact state of an enzyme is not necessarily the most active. Since inhibition by TMAO was more pronounced with the lower concentration of MgATP 2-, it may be that the enzyme is more sensitive to the methylamine when the nucleotide regulatory binding site is empty. Another TMAO effect appears to be blocking of free Mg2§ activation (Fig. 5C), indicating a specific interaction with protein domains involved in Mg 2§ binding. When
Organic Solutes and (Ca2+ + Mg2+) ATPases
123
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