Transport In Sarcoplasmic Reticulum Research Articles

Action of Nitrogen Oxides and Hydrogen Peroxide on Ca2+ Transport in Sarcoplasmic Reticulum of Permeabilized Myocytes of Utera

Action of Nitrogen Oxides and Hydrogen Peroxide on Ca2+ Transport in Sarcoplasmic Reticulum of Permeabilized Myocytes of Utera

Mechanisms of reduced mitochondrial Ca2+ accumulation in failing hamster heart

Mitochondrial Ca(2+) plays important roles in the regulation of energy metabolism and cellular Ca(2+) homeostasis. In this study, we characterized mitochondrial Ca(2+) accumulation in Syrian hamster hearts with hereditary cardiomyopathy (strain BIO 14.6). Exposure of isolated mitochondria from 70 nM to 30 microM Ca(2+) ([Ca(2+)](o)) caused a concentration-dependent increase in intramitochondrial Ca(2+) concentrations ([Ca(2+)](m)). The [Ca(2+)](m) was significantly lower in cardiomyopathic (CMP) hamsters than in healthy hamsters when [Ca(2+)](o) was higher than 1 microM and a decrease of about 52% was detected at [Ca(2+)](o) of 30 microM (916 +/- 67 nM vs 1,932 +/- 132 nM in control). A possible mechanism responsible for the decreased mitochondrial Ca(2+) uptake in CMP hamsters is the depolarization of mitochondrial membrane potential (Delta psi (m)). Using a tetraphenylphosphonium (TPP(+)) electrode, the measured Delta psi (m) in failing heart mitochondria was -136 +/- 1.5 mV compared with -159 +/- 1.3 mV in controls. Analyses of mitochondrial respiratory chain demonstrated a significant impairment of complex I and complex IV activities in failing heart mitochondria. In summary, a less negative Delta psi (m) resulting from defects in the respiratory chain may lead to attenuated mitochondrial Ca(2+) accumulation, which in turn may contribute to the depressed energy production and myocardial contractility in this model of heart failure. In addition to other known impairments of ion transport in sarcoplasmic reticulum and plasma membrane, results from this paper on mitochondrial dysfunctions expand our understanding of the molecular mechanisms leading to heart failure.

Thyroid hormone regulation of calcium cycling proteins.

Alterations in thyroid hormone levels have a profound impact on myocardial contractility, speed of relaxation, cardiac output, and heart rate. The mechanisms for these changes include altered expression of several key proteins, involved in the regulation of intracellular calcium homeostasis. Most notably, increases in thyroid hormone and the coordinated increases in cardiac contractile parameters are marked by increases in the levels of the sarcoplasmic reticulum (SR) Ca2+-adenosine triphosphatase (ATPase) and decreases in its inhibitor, phospholamban. These changes at the protein level result in enhanced SR calcium transport and myocyte calcium cycling, leading to increases in the force and rates of contraction as well as relaxation rates at the organ level. However, decreases in thyroid hormone levels are associated with opposite alterations in these two proteins, leading to reduced myocyte calcium handling capacity and lower cardiac contractility. Furthermore, changes in the relative ratio of phospholamban/Ca2+-ATPase correlate with changes in the affinity of the SR Ca2+-transport system and relaxation rates in beating hearts. These findings suggest that thyroid hormone directly regulates SR protein levels and thus, cardiac function.

Preliminary investigation of sequence-independent DNA binding proteins in rat skeletal muscle sarcoplasmic reticulum and their function

To observe the binding of plasmid DNA to non-nuclear DNA binding proteins in sarcoplasmic reticulum (SR) and the effects of this binding on SR function, sarcoplasmic reticulum proteins in rat skeletal muscle were isolated by differential centrifuge and sucrose density-gradient centrifuge. The results showed that there are two sequence-independent DNA binding proteins in SR proteins, the molecular weights of which are 83 and 58 ku, respectively. Ca(2+) uptake and release of SR were remarkably promoted by the binding of plasmid DNA to DNA binding proteins in SR, the mechanism is probably through increasing of Ca(2+)-ATPase activity in SR and changing of character of Ca(2+) release channel ryanodine receptors induced by the binding. These results suggest that there exist DNA binding proteins in SR and its binding to DNA may affect Ca(2+) transport of SR.

Seasonal effects on Ca 2+ transport of sarcoplasmic reticulum and on meat quality of pigs with different malignant hyperthermia status

The intracellular Ca 2+ concentration is mainly regulated by the sarcoplasmic reticulum (SR). A disturbed regulation leads to an increased metabolism and ultimately to inferior meat quality. In this study, pigs with normal and mutated calcium release channels (referred to as MHR and MHS, respectively) were used. One batch of pigs was slaughtered in winter months and another in summer months. The intention was to elucidate the impact of season on the Ca 2+ transport of SR and on meat quality in dependence of the MH-status. The results show an impact of MH-status and of season on the meat quality with inferior values in summer months, and in MHS pigs. Immediately after slaughter the rate of Ca 2+ sequestration of the SR with biochemically closed and with opened calcium release channels (CRC) was not different between experimental groups. However, with basic CRC (not biochemically manipulated) the Ca 2+ uptake rate of MHR muscle of summer pigs was significantly reduced and at the level of that of MHS pigs. The results indicate an impact of summer season on the ability of SR to regulate Ca 2+ concentration properly even in pigs with a normal CRC. This disturbed Ca 2+ regulation is correlated with diminished meat quality.

Influence of dietary n-3 fatty acids on the membrane properties of skeletal muscle in pigs

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Ca2+ diffusion and sarcoplasmic reticulum transport both contribute to [Ca2+]i decline during Ca2+ sparks in rat ventricular myocytes.

1. We sought to evaluate the contribution of the sarcoplasmic reticulum (SR) Ca2+ pump (vs. diffusion) to the kinetics of [Ca24]i decline during Ca2+ sparks, which are due to spontaneous local SR Ca2+ release, in isolated rat ventricular myocytes measured using fluo-3 and laser scanning confocal microscopy. 2. Resting Ca2+ sparks were compared before (control) and after the SR Ca2(+)-ATPase was either completely blocked by 5 microM thapsigargin (TG) or stimulated by isoprenaline. Na(+)-Ca2+ exchange was blocked using Na(+)-free, Ca(2+)-free solution (0 Na+, O Ca2+) and conditions were arranged so that the SR Ca2+ content was the same under all conditions when Ca2+ sparks were measured. 3. The control Ca2+ spark amplitude (281 +/- 13 nM) was not changed by TG (270 +/- 21 nM) or isoprenaline (302 +/- 10 nM). However, the time constant of [Ca2+]i decline was significantly slower in the presence of TG (29.3 +/- 4.3 ms) compared with control (21.6 +/- 1.5 ms) and faster with isoprenaline (14.5 +/- 0.9 ms), but in all cases was much faster than the global [Ca2+]i decline during a control twitch (177 +/- 10 ms). 4. The spatial spread of Ca2+ during the Ca2+ spark was also influenced by the SR Ca2+ pump. The apparent 'space constant' of the Ca2+ sparks was longest when the SR Ca2+ pump was blocked, intermediate in control and shortest with isoprenaline. 5. We conclude that while Ca2+ diffusion from the source of Ca2+ release is the dominant process in local [Ca2+]i decline during the Ca2+ spark, Ca2+ transport by the SR contributes significantly to both the kinetics and spatial distribution of [Ca2+]i during the Ca2+ spark.

Reciprocal changes in the postnatal expression of the sarcolemmal Na +-Ca 2+-exchanger and SERCA2 in rat heart

The aim of this study was to examine the relationship between sarcolemmal Na(+)-Ca2+ exchangers and sarcoplasmic reticulum (SR) Ca(2+) -ATPase (SERCA2) expression and the developmental differences in cardiac Ca2+ handling. Postnatal steady-state mRNA and protein levels were analysed in rat ventricular myocardium by Northern and immunoblot analysis, respectively. This was compared to Na+ gradient-induced and SR oxalate-supported Ca2 transport in isolated membranes. Na(+)-Ca2+ exchanger mRNA declined by 75% between day 1 and 30, whereas SR Ca2+ ATPase mRNA levels increased by 97% during this period. The Na(+)-Ca2+ exchanger mRNA/Ca(2+)-ATPase mRNA ratio was found to be inversely related to post-natal age. The changes in mRNA levels were associated with corresponding developmental differences in the Ca2+ transport activities of the respective membrane proteins. In crude membranes, the Na(+)-dependent Ca2+ transport activity (at 75 microM Ca2+) declined gradually (P < 0.01; mean +/- S.E.) from 17.7 +/- 2.4 nmoles Ca2+/g wet tissue/2s at day 1-3 (n = 5) to a value of 4.2 +/- 1.1 at day 40 (n =4). Conversely, SR Ca2+ uptake increased (P < 0.01) 2.6-fold during this period. The inversely related changes in the post-natal expression and function of the Na(+)-Ca2+ exchanger and SR Ca(2+)-ATPase suggest a coordinated control at the pretranslational level of the cellular Ca2+ transport processes mediated by the two membrane proteins.

Effect of Taurine and Methionine on Sarcoplasmic Reticular Ca2+ Transport and Phospholipid Methyltransferase Activity

Summary: Perfusion of rat hearts with buffer containing 300 μM L-methionine led to a decrease in Ca2+-induced Ca2+ release in sarcoplasmic reticulum (SR) but an increase in calcium-independent Ca2+ release from junctional SR. These effects of L-methionine were not altered by exposure of the hearts to high levels of extracellular taurine, but changes in the size of the intracellular taurine pool appear to modulate calcium transport in SR through two mechanisms. First, millimolar concentrations of taurine can directly promote release of calcium from 45Ca2+-loaded junctional SR vesicles. Second, taurine serves as an inhibitor of SR phospholipid methyltransferase, an enzyme that appears to be responsible for methionine-mediated loss in Ca2+-induced Ca2+ release activity and promotion of Ca2+-independent Ca2+ release. The data imply that modulation of the intracellular taurine pool may affect cellular calcium homeostasis and myocardial contractile function. This may be important in development of the cardiomyopathy linked to taurine deficiency.

Effect of Taurine and Methionine on Sarcoplasmic Reticular Ca2+ Transport and Phospholipid Methyltransferase Activity

Perfusion of rat hearts with buffer containing 300 microM L-methionine led to a decrease in Ca(2+)-induced Ca2+ release in sarcoplasmic reticulum (SR) but an increase in calcium-independent Ca2+ release from junctional SR. These effects of L-methionine were not altered by exposure of the hearts to high levels of extracellular taurine, but changes in the size of the intracellular taurine pool appear to modulate calcium transport in SR through two mechanisms. First, millimolar concentrations of taurine can directly promote release of calcium from 45Ca(2+)-loaded junctional SR vesicles. Second, taurine serves as an inhibitor of SR phospholipid methyltransferase, an enzyme that appears to be responsible for methionine-mediated loss in Ca(2+)-induced Ca2+ release activity and promotion of Ca(2+)-independent Ca2+ release. The data imply that modulation of the intracellular taurine pool may affect cellular calcium homeostasis and myocardial contractile function. This may be important in development of the cardiomyopathy linked to taurine deficiency.

Protein kinase inhibitors reduce SR Ca transport in permeabilized cardiac myocytes

Phosphorylation of the sarcoplasmic reticulum (SR) protein phospholamban by adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) and Ca-calmodulin-dependent protein kinase (CaM-KII) stimulates Ca-adenosinetriphosphatase (ATPase) activity and SR Ca transport, but the role of CaM-KII-dependent phosphorylation is not well defined. We studied the PKA- and CaM-KII-dependent regulation of SR Ca transport in digitonin-permeabilized rabbit ventricular myocytes. SR Ca uptake and free Ca concentration were measured on line with indo 1 and Ca electrodes in the presence of 20 microM ruthenium red and 10 mM oxalate. neither N5,2'-w-dibutyryl-cAMP (up to 500 microM) nor the nonhydrolyzable cAMP agonist adenosine 3'5'-cyclic monophosphorothioate sodium salt (Sp-cAMP[S]; up to 275 microM) affected the maximum uptake rate (Vmax) or the dissociation constant (Kd) for Ca uptake. However, the PKA inhibitor H-89 significantly increased Kd (e.g., from 307 +/- 67 to 826 +/- 62 nM Ca at 40-65 microM H-89) without significantly affecting Vmax. Both CaM-KII inhibitors, KN-62 (60 microM) and a CaM-KII inhibitory peptide (10 microM), significantly decreased Vmax from 11.95 +/- 0.5 to 9.48 +/- 0.6 nmol.mg-1.min-1 and from 10.95 +/- 1.72 to 7.37 +/- 0.94 nmol.mg-1.min-1, respectively, without consistently changing Kd. The effects of H-89 on Kd and of KN-62 on Vmax were prevented by a monoclonal antibody to phospholamban 2D12 (consistent with the antibody removing the inhibitory effect of phospholamban on the SR Ca-ATPase).(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiac Contractile and Calcium Transport Function after Burn Injury in Adult and Aged Guinea Pigs

Cardiac dysfunction occurs after a major burn injury regardless of age; whether burn trauma causes greater myocardial contractile depression in the older subject due to reduced cardiac reserves that normally occur with adult aging is not known. The cellular basis for burn-induced cardiac dysfunction is not known, but several studies have suggested that alterations occur in the rate of Ca2+ delivery to the contractile proteins as well as in the rate of Ca2+ removal from the sarcoplasm by the sarcoplasmic reticulum (SR). To determine if agerelated differences in the cardiac contractile response to burn injury are associated with differences in SR Ca2+ transport, an isolated heart preparation was used to examine mechanical function, and myocardial homogenate preparations were used to assess SR Ca2+ transport. Guinea pigs from both age groups (adult, 6-8 months, and senescent, 34-36 months of age) were divided into two subgroups—control and 45% cutaneous scald burn. Cardiac dysfunction associated with adult aging alone was indicated by lower systolic pressure and lower rates of left ventricular (LV) pressure rise and fall, as well as decreased responses to isoproterenol, exogenous Ca2+, increased coronary flow rate, and electrical pacing. Myocardial depression in senescent control hearts was accompanied by a decreased maximal Ca2+ uptake in myocardial homogenates, suggesting that altered SR calcium transport may contribute to the diminished contractile function associated with aging. Burn injury impaired cardiac function in all animals regardless of age as evidenced by a leftward shift of LV function curves and altered responses to receptor- and nonreceptor-mediated inotropic interventions. However, the percentage change in cardiac function after burn injury was similar in both age groups compared to those of their respective controls. Significant alterations in SR Ca2+ transport were observed in myocardial homogenates isolated from both adult and senescent hearts after burn injury. Our data confirm that burn injury induced cardiac contractile dysfunction as well as alterations in SR Ca2+ transport function regardless of age, and we conclude that altered SR Ca2+ transport function contributes, in part, to the diminished cardiac function after burn injury.

The effect of myoinositol on the myocardial mechanics and calcium transport of sarcoplasmic reticulum in diabetes mellitus

The effect of myoinositol on the myocardial mechanics and calcium transport of sarcoplasmic reticulum in diabetes mellitus

Differential changes in left and right ventricular SR calcium transport in congestive heart failure.

To examine the status of sarcoplasmic reticulum (SR) with respect to Ca2+ transport in congestive heart failure due to myocardial infarction, the left coronary artery in rats was ligated for 4, 8, and 16 wk. The left heart function was assessed with an intraventricular pressure transducer, and SR membrane fractions from the right ventricle and the viable left ventricle were isolated for measuring the ATP-dependent Ca2+ uptake activities. In comparison to sham-operated controls, SR Ca2+ uptake activity was decreased in viable left ventricle of the experimental animals at 4, 8, and 16 wk. On the other hand, SR Ca2+ uptake activity in the right ventricle was increased at 4 and 8 wk, but no change was apparent at 16 wk of coronary occlusion. The decrease in SR Ca2+ uptake in left ventricle and increase in right ventricle were associated with corresponding changes in maximal velocity values without any alterations in the affinity for Ca2+. These opposite changes in the right and left ventricles were dependent on the scar size as well as time after inducing the myocardial infarction. The SR Ca(2+)-stimulated adenosinetriphosphatase activity was decreased in left ventricle and increased in the right ventricle from 4 wk experimental animals. The results suggest differential remodeling of the SR membranes with respect to Ca(2+)-pump mechanisms in left and right ventricles during the development of congestive heart failure.

Inhibition of rat cardiac calcium pump activity by cationic amphiphilic drugs

Cationic amphiphilic drugs such as imipramine, chlorimipramine, chlorpromazine, chlorphentermine, and chloroquine have been shown to cause myocardial toxicity including arrhythmia, cardiomyopathy, and myocarditis. Since calcium transport by sarcoplasmic reticulum (SR) plays a critical role in contraction-relaxation coupling of myocardium, the toxicity exhibited by these drugs could be due to their effects on this phenomenon. Hence, we have studied the effects in vitro and in vivo of imipramine, chlorimipramine, chlorpromazine, chloroquine, and chlorphentermine on cardiac SR 45Ca-uptake and Ca2+-ATPase in male Fisher-344 rats. For in vitro studies, cardiac SR was prepared from normal rat hearts. For in vivo studies, rats were treated with drugs by oral gavage (p.o) at a dose of 20 pair-fed. All these drugs in vitro inhibited the cardiac SR 45Ca-uptake to a varying extent. The order of potency is chlorimipramine > chlorpromazine > imipramine > chlorphentermine > chloroquine. The inhibitory pattern of these drugs on cardiac SR Ca2+-ATPase also followed the same trend. The chlorinated analogs of these drugs are more potent in inhibiting 45Ca-uptake as well as Ca2+-ATPase as compared to their nonchlorinated con-geners. In vivo, all the drugs except chloroquine inhibited the 45Ca-uptake and Ca2+-ATPase activity to a different extent. The inhibition of cardiac SR calcium pump activity was seen as early as 7 days of treatment with chlorimipramine whereas all the other drugs (imipramine, chlorpromazine, and chlorphentermine) inhibited this calcium pump only at 21 days of treatment. Although in vivo chloroquine-induced inhibition of calcium pump activity did not reach significant level, it seems that there may be an effect at higher concentrations. In summary, the present results indicate that these cationic amphiphilic drugs might be exerting myocardial toxicity due to changes in cellular Ca2+ homeostasis because of their effects on cardiac SR calcium transport phenomenon. © 1992 Wiley-Liss, Inc.

Impaired calcium uptake by cardiac sarcoplasmic reticulum and its underlying mechanism in endotoxin shock

Effects of endotoxin administration on the ATP-dependent Ca2+ uptake by canine cardiac sarcoplasmic reticulum (SR) were investigated. Results obtained 4 h after endotoxin administration show that ATP-dependent Ca2+ uptake by cardiac SR was decreased by 27-43% (p less than 0.05). Kinetic analysis indicates that the Vmax values for Ca2+ and for ATP were significantly decreased while the S0.5 and the Hill coefficient values were not affected during endotoxin shock. Magnesium (1-5 mM) stimulated while vanadate (25-250 microM) inhibited the ATP-dependent Ca2+ uptake, but the Mg(2+)-stimulated and the vanadate-inhibited activities remained significantly lower in the endotoxin-treated animals. Phosphorylation of SR by the exogenously added catalytic subunit of the cAMP-dependent protein kinase or by the addition of calmodulin stimulated the ATP-dependent Ca2+ uptake activities both in the control and endotoxin-injected dogs. However, the phosphorylation-stimulated activities remained significantly lower in the endotoxin-injected dogs. Dephosphorylation of SR decreased the ATP-dependent Ca2+ uptake, but the half-time required for the maximal dephosphorylation was reduced by 31% (p less than 0.05) 4 h post-endotoxin. These data indicate that endotoxin administration impairs the ATP-dependent Ca2+ uptake in canine cardiac SR and the endotoxin-induced impairment in the SR calcium transport is associated with a mechanism involving a defective phosphorylation and an accelerated dephosphorylation of SR membrane protein. Since ATP-dependent Ca2+ uptake by cardiac SR plays an important role in the regulation of the homeostatic levels of the contractile calcium, our findings may provide a biochemical explanation for myocardial dysfunction that occurs during endotoxin shock.

Ca2+-ATPase and function of sarcoplasmic reticulum during cardiac hypertrophy

The properties of the calcium pump system of sarcoplasmic reticulum (SR) were studied in a series of 34 rats subjected to cardiac overload and 19 sham-operated animals. Total homogenates of left ventricle were analyzed by measuring the oxalate-supported Ca2+ uptake rate, the steady-state level of the phosphorylated intermediate of Ca(2+)-adenosine triphosphatase (Ca(2+)-ATPase) (EP), and the amount of Ca(2+)-ATPase mRNA. All three parameters decreased gradually as a function of the relative left ventricular weight increase. The calcium-sensitivity curves showed that the velocity of Ca2+ transport in SR from the hypertrophied heart is diminished at low as well as optimal Ca2+ concentrations, with the dissociation constant (Kd) value for Ca2+ unchanged from that of the control preparation. Taken together with the results presented in our recent publication (De la Bastie, Levitsky, Mercadier, Marotte, Wisnewsky, Brovkovivh, Schwartz, and Lompré, Circ. Res. 66: 554-564, 1990), these data strongly indicate that differences in the Ca2+ pump activities of SR from normal and hypertrophied rat hearts are due to quantitative rather than qualitative changes of the Ca(2+)-ATPase protein.

Ca2+-ATPase and function of sarcoplasmic reticulum during cardiac hypertrophy

The properties of the calcium pump system of sarcoplasmic reticulum (SR) were studied in a series of 34 rats subjected to cardiac overload and 19 sham-operated animals. Total homogenates of left ventricle were analyzed by measuring the oxalate-supported Ca2+ uptake rate, the steady-state level of the phosphorylated intermediate of Ca2+-adenosine triphosphatase (Ca2+-ATPase) (E-P), and the amount of Ca2+-ATPase mRNA. All three parameters decreased gradually as a function of the relative left ventricular weight increase. The calcium-sensitivity curves showed that the velocity of Ca2+ transport in SR from the hypertrophied heart is diminished at low as well as optimal Ca2+ concentrations, with the dissociation constant (Kd) value for Ca2+ unchanged from that of the control preparation. Taken together with the results presented in our recent publication (De la Bastie, Levitsky, Mercadier, Marotte, Wisnewsky, Brovkovivh, Schwartz, and Lompré, Circ. Res. 66: 554–564, 1990), these data strongly indicate that differences in the Ca2+ pump activities of SR from normal and hypertrophied rat hearts are due to quantitative rather than qualitative changes of the Ca2+-ATPase protein. calcium ion uptake; calcium ion adenosine triphosphatase messenger ribonucleic acid; cardiac hypertrophy; monoclonal antibody

Calcium transport activity of rat cardiac sarcoplasmic reticulum isolated in the presence of dithiothreitol

We examined the effect of dithiothreitol (DTT) on the isolation and calcium transport properties of rat cardiac sarcoplasmic reticulum (SR). DTT at a concentration of 4 mM was included in the homogenization/initial isolation and the high-salt (KCI) buffers used in the preparation of the SR fraction. The inclusion of DTT in the SR isolation media did not affect the total amount of SR protein isolated from ventricular tissue. However, SR isolated in the presence of DTT exhibited a significantly increased ( p < 0.05) rate of calcium transport at free calcium concentrations ranging from 0.1 μm up to 2.0 μm, and a 26% increase in the apparent affinity of the SR calcium transport system for Ca 2+. DTT included in the calcium-transport reaction medium did not significantly alter the rate of SR calcium transport in SR preparations isolated in the absence or presence of DTT. These results demonstrate that functionally important sulfhydryl groups of the SR calcium transport pump may be oxidized during the isolation of the membrane fraction and that such oxidation can be reduced by including DTT in the isolation buffers.

Activation and binding volumes of the sarcoplasmic reticulum transport enzyme activated by calcium or strontium

The effect of pressure on the hydrolysis of dinitrophenyl phosphate (DnpP) and p-nitrophenyl phosphate (NpP) by the sarcoplasmatic reticulum transport enzyme in permeabilized and native closed vesicles activated by calcium or strontium, respectively, in aqueous and Me2SO-containing media has been studied. At atmospheric pressure, the enzyme in permeabilized vesicles, saturated with respect to substrates and activating ions, hydrolyzes DnpP ten times faster than NpP; for both substrates, calcium activation exceeds that by strontium only a little (20%). In aqueous media the enzyme displays, under all activating conditions, an almost identical curvilinear relationship between the logarithm of enzyme activity and pressure. The data were analysed on the basis of a simplified reaction scheme, in which two unidirectionally proceeding substrate-driven pressure-dependent reactions (k2, k4) cyclically transfer high-affinity into low-affinity binding sites which are assumed to be in equilibrium with either calcium or strontium. The fitting procedure yielded two sets of positive activation volumes delta V2* = 90-110 ml/mol and delta V4* = 15-25 ml/mol. Substrate specificity, as well as the effect of temperature, are exclusively localized in the pressure-independent rate constants k'2 and k'4. Considerable different pressure/activity relations characterized by a single activation volume of 20 ml/mol were obtained for the strongly suppressed substrate hydrolysis of native closed vesicles. At atmospheric pressure DnpP hydrolysis of open vesicles is inhibited by Me2SO, while NpP hydrolysis is considerably activated, irrespective of its activation by calcium or strontium. In the presence of 22.5% Me2SO, the activation volumes are reduced by 50-70 ml/mol. The rate constants of DnpP and NpP hydrolysis are either augmented or reduced by rising Me2SO concentrations, depending on the corresponding supporting substrate. Me2SO has only a slight effect on the pressure dependence of substrate hydrolysis by native vesicles. The small activation volume observed for the activity of native vesicles could be assigned on account of the simplified reaction scheme of the slow reaction step k4, by which the enzyme is transferred from its low-affinity into its high-affinity binding state. Volume changes connected with the binding of calcium or strontium to the luminal binding site of the enzyme were deduced from the observed activation volume and the computed volume change of the slow reaction step (delta V4*).