Energetics of Streptococcal Growth Inhibition by Hydrostatic Pressure

Growth of Streptococcus faecalis in complex media with various fuel sources appeared to be limited by the rate of supply of adenosine-5′ -triphosphate (ATP) at 1 atm and also under 408 atm of hydrostatic pressure. Growth under pressure was energetically inefficient, as indicated by an average cell yield for exponentially growing cultures of only 10.7 g (dry weight) per mol of ATP produced compared with a 1-atm value of 15.6. Use of ATP for pressure-volume work or for turnover of protein, peptidoglycan, or stable ribonucleic acid (RNA) did not appear to be significant causes of growth inefficiency under pressure. In addition, there did not seem to be an increased ATP requirement for ion uptake because cells growing at 408 atm had significantly lower internal K⁺ levels than did those growing at 1 atm. Pressure did stimulate the membrane adenosine triphosphatase (ATPase) or S. faecalis at ATP concentrations greater than 0.5 mM. Intracellular ATP levels were found to vary during the culture cycle from about 2.5 μmol/ml of cytoplasmic water for lag-phase or stationary-phase cells to maxima for exponentially growing cells of about 7.5 μmol/ml at 1 atm and 5.5 μmol/ml at 408 atm. N,N′-dicyclohexylcarbodiimide at a 10 μM concentration improved growth efficiency under pressure, as did Mg²⁺ or Ca²⁺ ions at 50 mM concentration. These agents also enhanced ATP pooling, and it seemed that at least part of the growth inefficiency under pressure was due to increased ATPase activity. In all, it appeared that S. faecalis growing under pressure has somewhat reduced ATP supply but significantly increased demand and that the inhibitory effects of pressure can be interpreted largely in terms of ATP supply and demand.     

Pressure Sensitivity of Streptococcal Growth in Relation to Catabolism

The sensitivity of Streptococcus faecalis growth to hydrostatic pressures ranging up to 550 atm was found to depend on the source of adenosine triphosphate for growth. Barotolerance of cultures growing in a complex medium with ribose as major catabolite appeared to be determined primarily by the pressure sensitivity of ribose-degrading enzymes. Apparent activation volumes for growth were nearly identical to those for lactate production from ribose, and yield coefficients per mole of ribose degraded were relatively independent of pressure. In contrast, cultures with glucose as main catabolite were less sensitive to pressure; glycolysis was less severely restricted under high pressure than was growth, and yield coefficients declined with pressure, especially above 400 atm. Thus, two distinct types of barotolerance could be defined-one dominated by catabolic reactions and one dominated by noncatabolic reactions. The results of experiments with a series of other catabolites further supported the view that catabolic reactions can determine streptococcal barotolerance. We also found that growing, glucose-degrading cultures increased in volume under pressure in the same manner that they do at 1 atm. Thus, it appeared that the bacterium has no alternative means of carrying out glycolysis under pressure without dilatation. Also, the observation that cultures grown under pressure did not contain abnormally large or morphologically deformed cells suggested that pressure did not inhibit cell division more than cell growth.     

ATP inactivates hydrolysis of the K+-sensitive phosphoenzyme of kidney Na+,K+-transport ATPase and activates that of muscle sarcoplasmic reticulum Ca2+-transport ATPase

In order to characterize low affinity ATP-binding sites of renal (Na+,K+) ATPase and sarcoplasmic reticulum (Ca2+)ATPase, the effects of ATP on the splitting of the K+-sensitive phosphoenzymes were compared. ATP inactivated the dephosphorylation in the case of (Na+,K+)ATPase at relatively high concentrations, while activating it in the case of (Ca2+)ATPase. When various nucleotides were tested in place of ATP, inactivators of (Na+,K+)ATPase were found to be activators in (Ca2+)ATPase, with a few exceptions. In the absence of Mg2+, the half-maximum concentration of ATP for the inhibition or for the activation was about 0.35 mM or 0.25 mM, respectively. These values are comparable to the previously reported Km or the dissociation constant of the low affinity ATP site estimated from the steady-state kinetics of the stimulation of ATP hydrolysis or from binding measurements. By increasing the concentration of Mg2+, but not Na+, the effect of ATP on the phosphoenzyme of (Na+,K+)ATPase was reduced. On the other hand, Mg2+ did not modify the effect of ATP on the phosphoenzyme of (Ca2+)ATPase. During (Na+,K+)ATPase turnover, the low affinity ATP site appeared to be exposed in the phosphorylated form of the enzyme, but the magnesium-complexed ATP interacted poorly with the reactive K+-sensitive phosphoenzyme, which has a tightly bound magnesium, probably because of interaction between the divalent cations. In the presence of physiological levels of Mg2+ and K+, ATP appeared to bind to the (Na+,K+)ATPase only after the dephosphorylation, while it binds to the (Ca2+)-ATPase before the dephosphorylation to activate the turnover.     

Regulation of intracellular ATP concentration under conditions of reduced ATP consumption in pancreatic islets.

ATP is the most important factor in glucose-induced insulin secretion in pancreatic beta-cells, but examination of intracellular differences in ATP concentration is difficult because ATP production and consumption occur simultaneously. In the present study, we measured the ATP concentration under the condition of a reduced ATP requirement by omitting extracellular Ca(2+) and inhibiting Na-K ATPase. The ATP concentration in islets incubated with 16.7 mM glucose in the absence of Ca(2+) for 30 min was increased by about 1. 9-fold more than in the presence of Ca(2+). The increment was extracellular Ca(2+)-dependent, and was completely abolished by the metabolic inhibitors dinitrophenol and iodoacetic acid. The Ca channel blockers including nitrendipine and Ni(2+) did not affect the ATP concentration in islets incubated with 16.7 mM glucose in the presence of Ca(2+). However, when thapsigargin and suramin, inhibitors of Ca-ATPase at the endoplasmic reticulum, were added to Ca channel blockers in the presence of ambient Ca(2+), the intraislet ATP content was increased, similarly to that under Ca-free conditions. But thapsigargin did not further augment the ATP concentration in the islet with 16.7 mM glucose in the absence of Ca(2+). On the other hand, the suppression of Na-K ATPase by ouabain rather reduced the ATP concentration augmented by omission of extracellular Ca(2+). In addition, vanadate, a blocker of Ca-ATPase at the plasma membrane, failed to increase the ATP concentration in the islets. These data suggest that the increment of ATP concentration in the absence of Ca(2+) is attributable to the reduced ATP requirement due to stopping of the Ca-ATPase activity at the endoplasmic reticulum, and that the intracellular ATP concentration is differently regulated by Na-K ATPase at plasma membrane and by Ca-ATPase at endoplasmic reticulum.     

Plasma membrane Mg(2+)-ATPase of Pachysolen tannophilus: characterization and role in alcohol tolerance.

Following cell fractionation in sucrose density gradients, plasma membrane Mg(2+)-ATPase from Pachysolen tannophilus was studied. The ATPase displayed an apparent Km for ATP of 1.42 mM and was inhibited by high concentrations of Mg2+. The inhibitory effects of ethanol, 1-propanol, 1-butanol, and benzyl alcohol on Mg(2+)-ATPase were evaluated, and the concentration of each alcohol that inhibited ATPase activity by 50% (IC50) was determined. The IC50 decreased as the chain length of the alcohol increased. Moreover, the IC50 for ATPase activity was similar to the IC50 for growth rate, suggesting an association between impaired growth and ATPase inhibition. Almost complete inhibition of ATPase activity occurred at temperatures approaching 60 degrees C, and the optimal temperature was around 44 degrees C for ATPase from both control and ethanol-treated cells. Inclusion of 50 mM MgCl2 or CaCl2 in the medium did not rescue cells from the deleterious effects of ethanol.     

Plasma membrane Mg(2+)-ATPase of Pachysolen tannophilus: characterization and role in alcohol tolerance.

Following cell fractionation in sucrose density gradients, plasma membrane Mg(2+)-ATPase from Pachysolen tannophilus was studied. The ATPase displayed an apparent Km for ATP of 1.42 mM and was inhibited by high concentrations of Mg2+. The inhibitory effects of ethanol, 1-propanol, 1-butanol, and benzyl alcohol on Mg(2+)-ATPase were evaluated, and the concentration of each alcohol that inhibited ATPase activity by 50% (IC50) was determined. The IC50 decreased as the chain length of the alcohol increased. Moreover, the IC50 for ATPase activity was similar to the IC50 for growth rate, suggesting an association between impaired growth and ATPase inhibition. Almost complete inhibition of ATPase activity occurred at temperatures approaching 60 degrees C, and the optimal temperature was around 44 degrees C for ATPase from both control and ethanol-treated cells. Inclusion of 50 mM MgCl2 or CaCl2 in the medium did not rescue cells from the deleterious effects of ethanol.     

Presence of a Na+-stimulated P-type ATPase in the plasma membrane of the alkaliphilic halotolerant cyanobacterium Aphanothece halophytica

Aphanothece cells could take up Na(+) and this uptake was strongly inhibited by the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP). Cells preloaded with Na(+) exhibited Na(+) extrusion ability upon energizing with glucose. Na(+) was also taken up by the plasma membranes supplied with ATP and the uptake was abolished by gramicidin D, monensin or Na(+)-ionophore. Orthovanadate and CCCP strongly inhibited Na(+) uptake, whereas N, N'-dicyclohexylcarbodiimide (DCCD) slightly inhibited the uptake. Plasma membranes could hydrolyse ATP in the presence of Na(+) but not with K(+), Ca(2+) and Li(+). The K(m) values for ATP and Na(+) were 1.66+/-0.12 and 25.0+/-1.8 mM, respectively, whereas the V(max) value was 0.66+/-0.05 mumol min(-1) mg(-1). Mg(2+) was required for ATPase activity whose optimal pH was 7.5. The ATPase was insensitive to N-ethylmaleimide, nitrate, thiocyanate, azide and ouabain, but was substantially inhibited by orthovanadate and DCCD. Amiloride, a Na(+)/H(+) antiporter inhibitor, and CCCP showed little or no effect. Gramicidin D and monensin stimulated ATPase activity. All these results suggest the existence of a P-type Na(+)-stimulated ATPase in Aphanothece halophytica. Plasma membranes from cells grown under salt stress condition showed higher ATPase activity than those from cells grown under nonstress condition.     

(Na+ + K+)- and Na+-stimulated Mg2+-dependent ATPase activities in kidney of sea bass (Dicentrarchus labrax L.)

1. Sea bass kidney microsomal preparations contain two Mg2+ dependent ATPase activities: the ouabain-sensitive (Na+ + K+)-ATPase and an ouabain-insensitive Na+-ATPase, requiring different assay conditions. The (Na+ + K+)-ATPase under the optimal conditions of pH 7.0, 100 mM Na+, 25 mM K+, 10 mM Mg2+, 5 mM ATP exhibits an average specific activity (S.A.) of 59 mumol Pi/mg protein per hr whereas the Na+-ATPase under the conditions of pH 6.0, 40 mM Na+, 1.5 mM MgATP, 1 mM ouabain has a maximal S.A. of 13.9 mumol Pi/mg protein per hr. 2. The (Na+ + K+)-ATPase is specifically inhibited by ouabain and vanadate; the Na+-ATPase specifically by ethacrynic acid and preferentially by frusemide; both activities are similarly inhibited by Ca2+. 3. The (Na+ + K+)-ATPase is specific for ATP and Na+, whereas the Na+-ATPase hydrolyzes other substrates in the efficiency order ATP greater than GTP greater than CTP greater than UTP and can be activated also by K+, NH4+ or Li+. 4. Minor differences between the two activities lie in the affinity for Na+, Mg2+, ATP and in the thermosensitivity. 5. The comparison between the two activities and with what has been reported in the literature only partly agree with our findings. It tentatively suggests that on the one hand two separate enzymes exist which are related to Na+ transport and, on the other, a distinct modulation in vivo in different tissues.     

Induction of Superoxide Dismutase by Molecular Oxygen

Oxygen induces superoxide dismutase in Streptococcus faecalis and in Escherichia coli B. S. faecalis grown under 20 atm of O(2) had 16 times more of this enzyme than did anaerobically grown cells. In the case of E. coli, changing the conditions of growth from anaerobic to 5 atm of O(2) caused a 25-fold increase in the level of superoxide dismutase. Induction of this enzyme was a response to O(2) rather than to pressure, since 20 atm of N(2) was without effect. Induction of superoxide dismutase was a rapid process, and half of the maximal level was reached within 90 min after N(2)-grown cells of S. faecalis were exposed to 20 atm of O(2) at 37 C. S. faecalis did not contain perceptible levels of catalase under any of the growth conditions investigated by Stanier, Doudoroff, and Adelberg (23), and the concentration of catalase in E. coli was not affected by the presence of O(2) during growth. S. faecalis, which had been grown under 100% O(2) and which therefore contained an elevated level of superoxide dismutase, was more resistant of 46 atm of O(2) than were cells which had been grown under N(2). E. coli grown under N(2) contained as much superoxide dismutase as did S. faecalis grown under 1 atm of O(2). The E. coli which had been grown under N(2) was as resistant to the deleterious effects of 50 atm of O(2) as was S. faecalis which had been grown under 1 atm of O(2). These results are consistent with the proposal that the peroxide radical is an important agent of the toxicity of oxygen and that superoxide dismutase may be a component of the systems which have been evolved to deal with this potential toxicity.     

Ouabain-insensitive Na+ stimulation of a microsomal Mg2+ -ATPase in gills of sea bass (Dicentrarchus labrax L.)

Bass gill microsomal preparations contain a Mg2+-dependent Na+-stimulated ATPase activity in the absence of K+, whose characteristics are compared with those of the (Na+ + K+)-ATPase of the same preparations. The activity at 30 degrees C is 11.3 mumol Pi X mg-1 protein X hr-1 under optimal conditions (5 mM MgATP, 75 mM Na+, 75 mM HEPES, pH 6.0) and exhibits a lower pH optimum than the (Na+ + K+)-ATPase. The Na+ stimulation of ATPase is only 17% inhibited by 10-3M ouabain and completely abolished by 2.5 mM ethacrinic acid which on the contrary cause, respectively, 100% and 34% inhibition of the (Na+ + K+)-ATPase. Both Na+-and (Na+ + K+)-stimulated activities can hydrolyze nucleotides other than ATP in the efficiency order ATP greater than CTP greater than UTP greater than GTP and ATP greater than CTP greater than GPT greater than UTP, respectively. In the presence of 10(-3)M ouabain millimolar concentrations of K+ ion lower the Na+ activation (90% inhibition at 40 mM K+). The Na+-ATPase is less sensitive than (Na+ + K+)-ATPase to the Ca2+ induced inhibition as the former is only 57.5% inhibited by a concentration of 1 X 10(-2)M which completely suppresses the latter. The thermosensitivity follows the order Mg2+--greater than (Na+ + K+)--greater than Na+-ATPase. A similar break of the Arrhenius plot of the three enzymes is found. Only some of these characteristics do coincide with those of a Na+-ATPase described elsewhere. A presumptive physiological role of Na+-ATPase activity in seawater adapted teleost gills is suggested.     

A high affinity calcium-stimulated magnesium-dependent ATPase in rat liver plasma membranes. Dependence of an endogenous protein activator distinct from calmodulin

A Mg-dependent adenosine triphosphatase (ATPase) activated by submicromolar free Ca2+ was identified in detergent-dispersed rat liver plasma membranes after fractionation by concanavalin A-Ultrogel chromatography. Further resolution by DE-52 chromatography resulted in the separation of an activator from the enzyme. The activator, although sensitive to trypsin hydrolysis, was distinct from calmodulin for it was degraded by boiling for 2 min, and its action was not sensitive to trifluoperazine; in addition, calmodulin at concentrations ranging from 0.25 ng-25 micrograms/assay had no effect on enzyme activity. Ca2+ activation followed a cooperative mechanism (nH = 1.4), half-maximal activation occurring at 13 +/- 5 nM free Ca2+. ATP, ITP, GTP, CTP, UPT, and ADP displayed similar affinities for the enzyme; K0.5 for ATP was 21+/- 9 microM. However, the highest hydrolysis rate (20 mumol of Pi/mg of protein/10 min) was observed at 0.25 mM ATP. For all the substrates tested kinetic studies indicated that two interacting catalytic sites were involved. Half-maximal activity of the enzyme required less than 12 microM total Mg2+. This low requirement for Mg2+ of the high affinity (Ca2+-Mg2+)ATPase was probably the major kinetic difference between this activity and the nonspecific (Ca2+ or Mg2+)ATPase. In fact, definition of new assay conditions, i.e. a low ATP concentration (0.25 mM) and the absence of added Mg2+, allowed us to reveal the (Ca2+-Mg2+)ATPase activity in native rat liver plasma membranes. This enzyme belongs to the class of plasma membrane (Ca2+-Mg2+)ATPases dependent on submicromolar free Ca2+ probably responsible for extrusion of intracellular Ca2+.     

Enzymatic adaptation by bacteria under pressure.

A study of enzymic adaptation under hydrostatic pressure by moderately barotolerant bacteria that can grow at pressure up to about 500 atm revealed that some adaptive processes are relatively insensitive to pressure, whereas others are sufficiently barosensitive to compromise survival capacity in situations requiring adaptation to new substrates under pressure. Examples of the former include adaptation of Escherichia coli to arabinose catabolism for growth and adaptation of Streptococcus faecalis to catabolism of lactose, ribose, or maltose. Examples of the latter include derepression of the lac operon in Escherichia coli and induction of penicillinase synthesis by Bacillus licheniformis. For both these barosensitive systems, pressure had little effect on enzyme levels in constitutive strains or in bacteria that had previously been induced at 1 atm. Moreover, it had no detectable effect on penicillinase secretion. However, pressures of 300 to 400 atm were found to reduce markedly rates and extents of enzyme synthesis by bacteria undergoing derepression or adaptation. This inhibitory effect of pressure was reflected in greater barosensitivity with extended lag and slower growth of initially unadapted Escherichia coli cells inoculated into minimal medium with lactose as sole source of carbon and fuel, and by major reductions in the minimal inhibitory concentrations of penicillin G for unadapted B. licheniformis cells inoculated into complex, antibiotic-containing media. Cyclic adenosine 5'-monophosphate did not reverse pressure inhibition of derepression of the lac operon, and catabolite repression was complete under pressure. However, derepression of the lac operon was more sensitive to pressure at low concentrations of inducer than at high concentrations. Apparent volume changes for derepression were 94 and 60 ml/mol at inducer concentrations of about 0.5 and 5 mM, respectively. Pressure was found not to be inhibitory for uptake of beta-galactosides; in fact, it was somewhat stimulatory. Therefore, results were interpreted in terms of inducer binding and subsequent conversion of an operator-inducer-repressor complex to inactive repressor and operator. Both reactions appeared to result in an increase in volume, the former more so than the latter. We found also that 200 atm was actually stimulatory for growth of Escherichia coli in minimal media, and the bacterium was in a sense barophilic.     

Kinetics of a nucleotide pyrophosphatase in the plasma membrane of the fat cell

Fat cells from rat and rabbit hydrolyzed externally applied adenosine triphosphate at a rate of about 1.8 nmol times mg(-1) cells times min(-1) corresponding to about 0.3 mumol times mg(-1) protein tinus min(-1). Similar activities were found in cell homogenates. In purified adipocyte plasma membranes the rate of hydrolysis was about 1.8 mumol times mg(-1) protein times min(-1). The hydrolytic activity was dependent on divalent metal ions. Mg(2+), Mn(2+) and Ca(2+) gave highest activities. The activity was maximal at about equimolar concentrations of M(2+) and ATP. Km for MgATP was about 0.23 mM and for CaATP about 0.36 mM. Combinations of Mg(2+) and Ca(2+), or of Mg(2+), Na(+) and K(+) gave similar activities as did Mg(2+) only. At concentrations of 1 mM the following nucleotides were hydrolyzed with a decreasing rate: ATP > ITP > GTP > UTP = CTP. In isolated fat cells the beta-adrenergic drug isoproterenol and insulin slightly increased the rate of hydrolysis of external ATP, while the alpha-effector clonidine was inhibitory. The results suggest that a major portion of the ATP hydrolytic activity of the fat cell plasma membrane represents a nucleotide pyrophosphatase activity with access to externally applied ATP.     

Isolation and characteristics of scallop sarcoplasmic reticulum with calcium transport activity.

Sarcoplasmic reticulum with calcium transport activity has been isolated from the cross-striated adductor muscle of the scallop, which lives in cold (< or = 20 degrees C) sea water, by using pH 7.0 buffer solution both to homogenize the tissue and to sediment the membrane fraction. The yield of the preparation was 60-100 mg protein from 100 g of the scallop muscle. Ca(2+)-activated ATPase protein of about 100 kDa accounted for 40-50% of the protein preparation. The maximum activities of ATP-dependent, oxalate-facilitated calcium accumulation and Ca(2+)-ATPase were observed at a pH of about 7.0 and temperature of 20-30 degrees C, and their values were about 2 mumol Ca2+/mg of protein/min and about 3 mumol ATP hydrolysis/mg of protein/min, respectively. At 0 degree C, 10-20% of these activities was maintained, while at 37 degrees C, the activities were irreversibly lost. The Ca(2+)-ATPase activity was half-maximally activated at about 0.3 microM [Ca2+]. The ATPase activity exhibited non-Michaelian behavior with respect to ATP, with two different Km values of approximately 10 microM and 0.1-0.3 mM. GTP, CTP, and ITP were also hydrolyzed by the preparation at a rate of 10-30% of that of ATP. The preparation was stored at -80 degrees C with retention of function for about a year.     

Properties of the cyanobacterial coupling factor ATPase from Spirulina platensis. II. Activity of the purified and membrane-bound enzymes

Cyanobacterial (Spirulina platensis) photosynthetic membranes and isolated F1 ATPase were characterized with respect to ATP activity. The following results indicate that the regulation of expression of ATPase activity in Spirulina platensis is similar to that found in chloroplasts: the ATPase activity of Spirulina membranes and isolated F1 ATPase is mostly latent, a characteristic of chloroplast ATPase activity; treatments that elicit ATPase activity in higher plant chloroplast thylakoids and isolated chloroplast coupling factor (CF1) greatly stimulate the activity of Spirulina membranes and F1, and the cation specificity of chloroplast ATPase activity, e. g., light-induced membrane activity that is magnesium dependent and trypsin-activated CF1 activity that is calcium dependent, is also observed in Spirulina. Thus, an 8- to 15-fold increase in specific activity (to 13-15 mumol Pi min-1 mg chl-1) is obtained when Spirulina membranes are treated with trypsin (CaATPase) or with methanol (MgATPase): a light-induced, dithiothreitol-dependent MgATPase activity is also found in the membranes. Purified Spirulina F1 is a CaATPase when activated with trypsin (endogenous activity increases from 4 to 27-37 mumol Pi min-1 mg protein-1) or with dithiothreitol (5.6 mumol Pi min-1 mg-1), but a MgATPase when assayed with methanol (18-20 mumol Pi min-1 mg-1). The effects of varying calcium and ATP concentrations on the kinetics of trypsin-induced CaATPase activity of Spirulina F1 were examined. When the calcium concentration is varied at constant ATP concentration, the velocity plot shows a marked sigmoidicity. By varying Ca-ATP metal-nucleotide complex concentration at constant concentrations of free calcium or ATP, it is shown that the sigmoidicity is due to the effect of free ATP, which changes the Hill constant to 1.6 from 1.0 observed when the free calcium concentration is kept constant at 5 mM. Therefore not only is ATP an inhibitor but it is also an allosteric effector of Spirulina F1 ATPase activity. At 5 mM free calcium, the Km for teh Ca-ATP metal-nucleotide complex is 0.42 mM.     

Simulation of cardiac work transitions, in vitro: effects of simultaneous Ca2+ and ATPase additions on isolated porcine heart mitochondria

During increases in cardiac work there are net increases in cytosolic [Ca(2+)] and ATP hydrolysis by myofiliments and ion transport ATPases. However, it is still unclear what role Ca(2+)or the ATP hydrolysis products, ADP and Pi, have on the regulation of mitochondrial ATP production. In this study, work jumps were simulated by simultaneous additions of Ca(2+) and ATPase to porcine heart mitochondria. The net effects on the mitochondrial ATP production were monitored by simultaneously monitoring respiration (mVo2), [NADH], [ADP] and membrane potential (deltapsi) at 37 degrees C. Addition of exogenous ATPase (300 mlU.ml(-1))]ATP (3.4 mM) was used to generate a 'resting' background production of ADP. This resting metabolic rate was 200% higher than the quiescent rate while [NADH] and deltapsi were reduced. Subsequent ATPase additions (1.3IU.ml(-)) were made with varying amounts of Ca(2+)(0 to 535 nM) to simulate step increases in cardiac work. Ca(2+) additions increased mVo2 and depolarized deltapsi, and were consistent with an activation of Fo/F1)ATPase. In contrast, Ca(2+) reduced the [NADH] response to the ATPase addition, consistent with Ca(2+)-sensitive dehydrogenase activity (CaDH). The calculated free ADP response to ATPase decreased \2-fold in the presence of Ca(2+). The addition of 172nM free Ca(2+)] ATPase increased mVo2 by 300% (P<0.05, n=8) while deltapsi decreased by 14.9+/-0.1 mV without changes in [NADH] (P > or =0.05, n=8), consistent with working heart preparations. The addition of Ca(2+) and ATPase combined increased the mitochondrial ATP production rate with changes in deltapsi, NADH and [ADP], consistent with an activation of CaDH and F o /F(1)ATPase activity. These balancing effects of ATPase activity and [Ca(2+)] may explain several aspects of metabolic regulation in the heart during work transitions in vivo.     

H+ transport by reconstituted gastric (H+ + K+)-ATPase

Gastric (H+ + K+)-ATPase was reconstituted into artificial phosphatidylcholine/cholesterol liposomes by means of a freeze-thaw-sonication technique. Upon addition of MgATP, active H+ transport was observed, with a maximal rate of 2.1 mumol X mg-1 X min-1, requiring the presence of 100 mM K+ at the intravesicular site. However, in the absence of ATP an H+-K+ exchange with a maximal rate of 0.12 mumol X mg-1 X min-1 was measured, which could be inhibited by the well-known ATPase inhibitors vanadate and omeprazole, giving the first evidence of a passive K+-H+ exchange function of gastric (H+ + K+)-ATPase. An Na+-H+ exchange activity was also measured, which was fully inhibited by 1 mM amiloride. Simultaneous reconstitution of Na+/H+ antiport and (H+ + K+)-ATPase could explain why reconstituted ATPase appeared less cation-specific than the native enzyme (Rabon, E.C., Gunther, R.B., Soumarmon, A., Bassilian, B., Lewin, M.J.M. and Sachs, G. (1985) J. Biol. Chem. 260, 10200-10212).     

Partial purification and properties of (Na+ + K+)-ATPase from Potamon potamios.

1. The tissue distribution of the (Na+ + K+)-ATPase in the freshwater/land crab Potamon Potamios was studied. 2. Gills were found to display the highest total activity in the whole animal (47%) but the highest specific activity was detected in the heart (15.15 mumol Pi/mg protein/min). 3. All other organs tested were found to have low enzyme activity. 4. The freshwater/land crab ATPase enzyme was inhibited by ouabain with a Ki of 0.5 mM.Km values for ATP, Mg2+ and K+ were 1.4, 4.0 and 1.2 mM respectively. The enzyme also showed a break in the Arrhenius plot at 23 degrees C. 5. A purification method of microsomal ATPase is described involving ultracentrifugation and electrofocusing.     

Activation of the dynein adenosinetriphosphatase by cross-linking to microtubules

The microtubule-dynein complex consisting of 22S dynein from Tetrahymena cilia and MAP-free microtubules was subjected to treatment with various concentrations of 1-ethyl-3-[3-(dimethylamino)-propyl]carbodiimide (EDC), a zero-length cross-linker, at 28 degrees C for 1 h. Following cross-linking of the microtubule-dynein complex, nearly all of the ATPase activity cosedimented with the microtubules in the presence of ATP. Electron microscopic observation by negative staining revealed that, following treatment with 1 mM EDC, the complex did not dissociate in the presence of ATP, although the dynein decoration pattern was disordered. The complex treated with 3 mM EDC exhibited normal microtubule-dynein patterns even after the addition of ATP. The ATPase activity of the microtubule-dynein complex was enhanced about 30-fold by the treatment with 1-3 mM EDC. These results indicate that the ATPase activation was caused by the close proximity of the dynein ATPase sites to the microtubules and provide further support for the functional interaction of all three dynein heads with the microtubule. The maximal specific activity was 12 mumol min-1 (mg of dynein)-1, corresponding to a turnover rate of 150 s-1, which may be the rate-limiting step at infinite microtubule concentration and may represent the maximum rate of force production in the axoneme.     

The vanadate-sensitive ATPase of Streptococcus faecalis pumps potassium in a reconstituted system

The vanadate-sensitive ATPase of Streptococcus faecalis, purified to homogeneity, was reconstituted into soybean phospholipid vesicles in a functional state. Freeze-fracture electron micrographs revealed a relatively uniform population of unilamellar liposomes of 50-100 nm in diameter, with particles protruding from both fracture faces. Transport studies with 42K+ and with a K+-selective electrode showed that the ATP-ase catalyzes electrogenic potassium extrusion in proteoliposomes. The following parameters for potassium transport in the reconstituted system were determined: K+/ATP stoichiometry = 1, Km for potassium = 1.4 mM, Vmax = 0.1 mumol/min/mg. The ATPase could be activated by an electrical membrane potential, vesicle interior positive. This ATPase thus appears to function as a potential regulated, ATP-driven pump that serves in electrogenic potassium accumulation by the bacterial cell.