Volume 9, number 2
MOLECULAR & CELLULAR BIOCHEMISTRY
November 30, 1975
M E M B R A N E B O U N D AND S O L U B L E A D E N O S I N E T R I P H O S P H A T A S E OF E S C H E R I C H I A COLI K 12. KINETIC P R O P E R T I E S OF T H E BASAL AND T R Y P S I N - S T I M U L A T E D ACTI.VITIES Jos6 CARREIRA and Emilio M U l q O Z
Instituto de Biologia Celular, C.S.I.C., Madrid-6, Spain (Received October 4, 1974)
Summary Basal and trypsin-stimulated adenosine triphosphatase activities of Escherichia coli K 12 have been characterized at pH 7.5 in the membranebound state and in a soluble form of the enzyme. The saturation curve for Mg 2 +/ATP = 1/2 was hyperbolic with the membrane-bound enzyme and sigmoidal with the soluble enzyme. Trypsin did not modify the shape of the curves. The kinetic parameters were for the membrane-bound ATPase: apparent K m = 2.5 mM, Vmax (minus trypsin) = 1.6/~mol-min- ~.mg protein- ~ Vmax (plus trypsin) = 2.44/~mol.min-1.mg protein- 1; for the soluble ATPase: [So.5] = 1.2 mM, Vmax ( - trypsin) = 4 #mol.min- 1.mg protein- ~; Vmax ( + trypsin) = 6.6 #mol.min- l-mg protein- a. Hill plot analysis showed a single slope for the membrane-bound ATPase (n = 0.92) but two slopes were obtained for the soluble enzyme (n = 0.98 and 1.87). It may suggest the existence of an initial positive cooperativity at low substrate concentrations followed by a lack of cooperativity at high ATP concentrations. Excess of free ATP and Mg 2+ inhibited the ATPase but excess of Mg/ATP (1/2) did not. Saturation for ATP at constant Mg 2+ concentration (4 mM) showed two sites (groups) with different Kms: at low ATP the values were 0.38 and 1.4 mM for the membrane-bound and soluble enzyme; at high ATP concentrations they were 17 and 20 mu, respectively. Mg 1+ saturation at constant ATP (8 mu) revealed michaelian kinetics for the membrane-bound ATPase and
sigmoid one for the protein in soluble state. When the ATPase was assayed in presence of trypsin we obtained higher K m values for Mg 2+. These results might suggest that trypsin stimulates E. coli ATPase by acting on some site(s) involved in Mg 1+ binding. Adenosine diphosphate and inorganic phosphate (Pi) act as competitive inhibitors of Escherichia coli ATPase. The Ki values for Pi were 1.6 _+ 0.1 mM for the membrane-bound ATPase and 1.3 _+ 0.1 mM for the enzyme in soluble form, the Ki values for ADP being 1.7 mM and 0.75 mM for the membrane-bound and soluble ATPase, respectively. Hill plots of the activity of the soluble enzyme in presence of ADP showed that ADP decreased the interaction coefficient at ATP concentrations below its K m value. Trypsin did not modify the mechanism of inhibition or the inhibition constants. Dicyclohexylcarbodiimide (0.4 rnM) inhibited the membrane-bound enzyme by 60-70 % but concentrations 100 times higher did not affect the residual activity nor the soluble ATPase. This inhibition was independent of trypsin. Sodium azide (20/~M) inhibited both states of E. coli ATPase by 50 %. Concentrations 25-fold higher were required for complete inhibition. Ouabain, atebrin and oligomycin did not affect the bacterial ATPase.
Introduction Adenosine triphosphatases (ATPases) (EC 3.6.1.3) are a family of membrane-bound enzymes
Dr. W. Junk b.v. Publishers - The Hague, The Netherlands
85
involved in a series of energy-transduction processes such as oxidative phosphorylation 1, photophosphorylation 2, transport 3 and contraction 4. Some of these enzymes have been obtained in soluble form by membrane washing with defined buffers (see for example ref. 5). The solubilization allowed the purification and structural characterization of ATPases from various sources. The great structural similarity between mitochondrial, chloroplast and bacterial ATPases 6 and the correlation among contractile and membrane ATPases have been substantiated recently 7. However, ATPases behaved differently with regard to ATP hydrolysis depending upon their source z'8 and/or state 9,1°. The reactivity of membrane-bound ATPases of mitochondria, chloroplasts and Mierococcus lysodeiktieus showed a certain degree of latency 2'11'12 Recently, MITCHELL and co-workers 13 have reported the virtual inhability of Escherichia coli M L 308-225 vesicles to hydrolyze ATP. These enzyme preparations were stimulated by various treatments: trypsin 2'11, heating2,12, dithiothreitol a4, changes in the environmental concentration of divalent metal ions 15,16, detergents 13, or solubilization 11. The latency of mitochondrial and chloroplasts ATPases appeared to be due to the presence of a minor subunit of the ATPases that acts as an ATPase inhibitor 1v,18. The mechanism of ATPase latency in Micrococcus lysodeikticus is still unknown. It may be due to a simple problem of enzyme crypticity or, alternatively, may reflect a mechanism of regulation for the possible polyfunctional role of ATPase in prokaryotic organisms as proposed by Mulqoz and coworkers 19,2°. The hydrolysis of ATP to ADP and Pi catalyzed by ATPases is usually coupled to the active transport of solutes 21'22 under anaerobic conditions or may reflect the uncoupling of the synthetic function of the protein in aerobiosis. The complexity of these processes imply the existence of regulatory mechanisms which may depend on intrinsic properties of the protein or on the physical state of the enzyme through its interaction with non-catalytic proteins. The end products of the reaction, ADP and Pi, appear to be good candidates for modulating the hydrolytic properties of ATPases. In this context, the ATPases from various sources showed a reduction in the rate of ATP hydrolysis by ADP 2'1°-14. It was admitted that the inhibition by ADP was 86
competitive, although there was no total agreement at this respect 23-26. On the other hand, very few studies have been carried out on the inhibition by Pi. Nevertheless, it was concluded that this inhibition was of the same type as that of ADP 27. The ATPase system of E. coli has been the object of several recent studies 8,13'24,25,28-32. In previous work, we described a membrane system of E. coli K 12 able to hydrolyze externally added ATP 8. We also reported the complete solubilization of the ATPase by EDTA-alkaline buffers 8. Both, the membrane-bound and 'soluble' enzyme exhibited the same degree of stimulation by trypsin amounting to about 100 %8. These results suggested a mechanism of control for the unmasking of ATPase activity in E. coli. Furthermore, they seemed to rule out the hypothesis that trypsin stimulation resulted from a crypticity of ATPase in E. coli membranes. These interesting properties prompted us to compare the kinetic properties of the 'soluble' and membrane-bound ATPase when assayed with and without trypsin. Through such studies, it is hoped to gain information on the mechanism of ATPase latency in this organism and on the regulation of the expression of E. coli membrane ATPase activity. Methods
Reagents Adenosine triphosphate (Na2ATP) was purchased from PL Biochemicals. Tris (hydroxymethyl)aminomethane, MgC12 and activated charcoal of the highest quality available were from Merck. EDTA, disodium salt was from Fisher Scientific Co. Trypsin (EC 3.4.4.4), lyozyme (EC 3.6.1.3) and deoxyribonuclease (EC 3.1.4.5) were obtained from Calbiochem. Dicyclohexylcarbodiimide (DCCD) and N a H 2 P O 4 (Pi) were from Merck. Sodium azide (NAN3) was obtained from BDH Ltd. Chemicals. Oligomycin and atebrin were purchased from Sigma Chem. Co. Ouabain was from Calbiochem. Adenosine triphosphate 7_32p, sodium salt (specific activity 1-2 Ci/mmol) was purchased from Amersham, The Radiochemical Centre. Microorganism and membrane preparation Escherichia coli K 12, number 414 (Hfr, T h r - ) of the collection of Dr. J. PUIG (Facultad de
Ciencias, Universidad de los Andes, M6rida, Venezuela), was used in these studies and grown as described previously 33. The preparation of membrane 'ghosts' was carried out by a modification of the procedure of KABAC~ 34. The modification involved the sequential treatment of exponentially growing E. coli cells with E D T A and lysozyme instead of their simultaneous treatment with both agents. The characterization of the membrane 'ghost' fraction, i.e. membranes possessing a size similar to that of the bacterial cell, has been reported elsewhere 33. A TPase preparations
Membranes were kept as pellets at 0 ° for 1 week and resuspended in 30 mM Tris-HC1 (pH 7.5) 1 mM MgC12 at about 1 mg protein/ml for ATPase assays. ATPase was solubilized by resuspending the membrane preparation in 3 mM E D T A - 50 mM Tris (pH 9.0) at about 1 mg protein/ml. After standing 15 min at r o o m temperature, the membrane suspensions were centrifuged at 27000 × 9, in a Sorvall RC-2B for 20 min at r o o m temperature. The supernatants were considered as the soluble ATPase. Confirmation of the soluble nature of these fractions was obtained by centrifuging at 150,000 × 9 for 60 min. The ATPase activity of the supernatants at 150,000 × 9 and 27,000 × g showed identical kinetic behaviour. In subsequent studies, we have considered the supernatants of 27,000 × 9 as soluble ATPase. Because of the high enzyme lability in solution (8, and AZOCAR, O., unpublished observations), ATPase assays with the soluble fraction were performed within 2 hours after its extraction from the membrane.
reagents of Pi determination according to VA_UBUTASand RACKER2. Appropriate blanks were run each time. In m a n y experiments, the results were confirmed by using ATP-';-32p (specific activity in the assay 0.031 #Ci/#mol). In this case, the reaction was stopped by the addition of 0.5 ml of 10 ~ activated charcoal in 0.2 M KC1-HC1 (pH 1.6). The charcoal was removed by centrifugation at 4,000 x 9 for 10 min and aliquots (0.1 ml) of the supernatants were added to Bray's solution 35 for scintillation counting in a M a r k II (Nuclear Chicago). Blanks were prepared by adding 0.5 ml charcoal as above to samples without enzyme or to samples with boiled enzyme. One unit of activity is defined as the amount of enzyme able to liberate 1 #mol Pi per min at 37 °. Specific activity is expressed in units per mg of protein. Variations in the assay conditions during the kinetic studies are specified in the text and in the legends of figures. ADP, Pi and N a N 3 at the concentrations indicated in the legends of the respective figures, were added to the reaction mixture in 100 #1 30 mM Tris-HC1 (pH 7.5). D C C D was added in 10 #1 methanol and oligomycin in 10 #1 ethanol. These concentrations of alcohol did not show any effect on the rate of ATP hydrolysis. The effect of inhibitors was directly tested on the assay mixture without preincubation with the enzyme. When the inhibition of Pi was studied by the colorimetric procedure, appropriate blanks were run to correct for the excess of Pi in the samples. The protein content was measured by the method of LOWRY et al. 36 using bovine serum albumin as standard.
A TPase assay
The standard assay system contained 4 #tool ATP, 2 #mol Mg 2+ and 12 #mol Tris-HC1 (pH 7.5) in 400 #1. The reaction was started by the addition of 100 #1 containing either 80 #g of membrane protein or 25 #g of soluble protein. Where stated, trypsin was added to the assay before the ATPase preparations at a concentration of 0.2 mg/ml or with the modifications indicated in the legend of the figures. Incubations were carried out at 37 ° for 5 min. During this time, the reactions proceeded linearly. The reaction was stopped by the addition of the
Experimental Figure 1 illustrates the dependence of membranebound ATPase activity upon the protein concentration. The velocity is linear up to 125 130 #g protein/0.5 ml reaction mixture. Figure 2 shows parallel results for the soluble ATPase. In this case the linearity was maintained at protein concentrations below 40-45 #g in the reaction mixture. All these results were independent on the presence of trypsin. Subsequent work was carried 87
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Protein ()Jg) Fig. 1. Influence of protein concentration on the rate of ATP hydrolysis by the m e m b r a n e - b o u n d ATPase of Escherichia coll. The conditions for assay are given in the text (see Experimental). The protein refers to the a m o u n t of total m e m b r a n e protein put in the assay mixture: © © basal ATPase (minus trypsin); O - - O plus trypsin (0.2 mg/ml assay mixture); • - - • plus trypsin added to the assay mixture at the same concentrations as that of the membrane preparation.
out with 80-90 #g protein in the assay for the m e m b r a n e - b o u n d ATPase and 25 30 #g for the soluble enzyme. Studies on the effect of Mg 2 ÷ concentration on ATPase activity showed that the optimal activity was reached at a Mg 2 +/ATP ratio near to 1/2,
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Fig. 3. Double reciprocal plots of initial rate data for the m e m b r a n e - b o u n d ATPase of E. coli. The experimental conditions are given in the text. Substrate refers to Mg 2 +/ATP = 1/2, the actual concentrations used for the plot were those of ATP: © - - © basal ATPase; O - - O ATPase assayed with trypsin, The inset shows schematically the direct curve for substrate saturation.
as found by other workers 24'25'28 32. We then studied the saturation for this substrate for the m e m b r a n e - b o u n d and the solubilized enzyme. The range of A T P concentrations was 0.18 raM, Mg 2+ concentrations being 0.05 4 mM. The membrane-bound ATPase obeyed Michaelis-Menten kinetics when assayed either in presence or in absence of trypsin (see Fig. 3). However, the E D T A soluble ATPase gave a sigmoid curve for substrate saturation (see inset in Fig. 4), therefore, suggesting the existence of
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Protein ( ~ g ) Fig. 2. Influence of protein concentration on the rate of A T P hydrolysis by the soluble ATPase of E. coli. The conditions for ATPase assay and the nature of ATPase preparation are described in the text. The protein refers to the a m o u n t of soluble protein put in the assay medium: © - - © basal ATPase (minus trypsin); O - - O plus trypsin (0.2 mg/ml assay mixture); • • plus trypsin (increasing concentrations to equal the protein concentration of the soluble fraction).
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cooperativity between different active sites. These results were independent of trypsin. The same apparent K m value of 2.5 mM for the membranebound ATPase (Fig. 3) was obtained independently of the presence of trypsin in the assay. However, the Vmax values were respectively: 1.6 # m o l . m i n - < m g - 1 for the membrane ATPase assayed without trypsin and 2.44 #mol.min-1. m g - a for the ATPase assayed with trypsin (Fig. 3). The estimated kinetic parameters for the solubilized enzyme were: a [So.5] = 1.2 mM and Vmax values of 4 (without trypsin) and 6.6 (plus trypsin). Trypsin thus affects the hydrolysis rate of ATP without apparently altering the affinity of the enzyme for the substrate. The most likely mechanism of trypsin action appears to be consequence of the hydrolytic cleavage of some ATPase component(s), although we can not rule out a simple effect of protein-protein interaction, owing to the high concentrations of protease used (see Figs. 1 and 2). The small difference found in K m values for the m e m b r a n e - b o u n d and soluble ATPase was constant in 10 different experiments performed with various preparations. This finding, together with the different shape of the substrate saturation curve, may reflect pecularities of the two states of the ATPase (see Discussion). Figure 5 illustrates the Hill plots for E. coli Z5
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ATPase in both types of preparation. On the one hand, the 0.92 Hill coefficient calculated for the m e m b r a n e - b o u n d ATPase suggests that there is no interaction between the active sites in this state of the enzyme. On the other hand, the Hill plot of the soluble enzyme shows two slopes. The change in slope occurs at a substrate concentration about the [So.5] of the enzyme. The value of 1.87 tends to indicate a positive cooperativity between the active sites. The existence of two slopes in a Hill plot is very unusual 37. According t o COOK a n d KOSHLAND 37 w e may assume an initial positive cooperativity at low substrate concentrations, followed by a lack of cooperativity (negative cooperativity?) at substrate concentrations higher than those exhibiting halfsaturation for E. coli ATPase. As one could expect from previous results, trypsin did not influence these parameters. In attempts to individualize the role of each component of this complex substrate, we measured the influence of increased ATP concentrations (range 0.1-8 mM) at a constant Mg 2+ concentration (4 raM). The results are shown in Figures 6 and 7. Both the m e m b r a n e - b o u n d enzyme a n d the soluble ATPase followed Michaelis kinetics with two classes of saturation constants. The kinetic parameters are summarized in Table 1. The apparent K m value at low A T P concentrations for the particulate enzyme is very similar to that reported by Hanson and Kennedy for the m e m b r a n e - b o u n d ATPase from E. coli and a purified form of the enzyme, both assayed with excess of Mg 2 + ions. The apparent K m of 89
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soluble enzyme obtained in these conditions was higher than that of the membrane ATPase, which contrasts with results obtained with variable concentrations of Mg 2 +/ATP. The apparent K m values at high concentrations of ATP were very similar for both forms of the ATPase. These studies were not carried out in presence of trypsin due to the high sensitivity of the enzyme to trypsin digestion in presence of high Mg 2 + and incomplete enzyme protection by ATP (CARREIRA, J. and ROJAS, M., unpublished observations). The Michaelis kinetics of the soluble ATPase in these conditions may suggest that ATP did not contribute to the cooperativity found in previous experiments (see Fig. 4). Moreover, free ATP or Mg 2+ inhibited E. coli ATPase, whereas high Table 1 Kinetic parameters for A T P of the soluble and membraneb o u n d ATPase of Escherichia coli in function of the experimental A T P concentrations at a constant Mg 2 + concentration (4 mM). gmax
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Fig. 8. Double reciprocal plot of Mg 2 + saturation for the m e m b r a n e - b o u n d ATPase of E. coll. The plot is of the reciprocal initial rates versus 1/Mg 2 + at constant ATP concentration (8 mM). For experimental details see the text: • • basal activity; O - - O activity assayed with trypsin.
concentrations of Mg 2 +/ATP (1/2) were not inhibitory. These results indicate the existence of sites with different affinities for ATP, probably depending on Mg 2 +/ATP ratios. The complexity of these results tends to suggest that Mg/ATP might not be the true substrate of E. coli ATPase. In a subsequent step, we studied the effect of Mg 2 + concentrations of the velocity of ATP hydrolysis at constant ATP (8 ms). In these experimental conditions (see Figs. 8 and 9), the membrane bound enzyme and the soluble enzyme behaved differently. The former showed Mg 2+ saturation curve yielding K m = 1.2 mM, whereas the latter revealed a sigmoid curve (approximate [So.5] = 1.1 mM). Trypsin modified these parameters thereby increasing the Km value to 7 mM for the membrane-bound ATPase and to 2 mM for the EDTA-soluble enzyme. Vmax values were affected in a similar way. These results, taken together indicate again that Mg 2 +/ATP is not the true substrate for E. coli ATPase. The decrease in affinity for Mg 2+ of E. coli ATPase induced by trypsin suggests the existence of some site(s) directly involved in Mg 2 + binding. This(these) site(s) appear(s) to be the point of specific attack by trypsin under the protective conditions where stimulation of the ATPase occurs. Dixon plots for the inhibition by ADP of the membrane-bound and soluble ATPase are
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shown in Figures 10 and 11, respectively. The lines intersect in the second quadrant over the abscissa axis confirming that ADP acts as a pure competitive inhibitor of E. coli ATPase. Ki values of 1.7 mM and 0.75 mM were calculated
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by this procedure for the membrane-bound and soluble enzyme, respectively. As in previous cases, these constants were not modified by trypsin (results not shown). Similar results were obtained from double reciprocal plots. Figure 12 compares the different Hill plots of the EDTA-soluble ATPase assayed under the conditions described in Figure 11. As in Figure 5, two slopes are seen in these plots. ADP decreases the interaction coefficient in the first part of the curve before the change in slope. After this point,
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Fig. 1 i. Dixon plots of ADP inhibition of the soluble ATPase of E . coli assayed in absence of trypsin. The data are plotted as reciprocal initial velocities v e r s u s ADP concentrations at fixed initial concentration of Mg2+/ATP = 1/2: © - - O 8 mM ATP; ~ - - / X 4.8 mM ATP; F 1 - - D 1.6 mM ATP. For experimental details see the text. The same results were obtained for the soluble ATPase assayed with trypsin.
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Fig. 15. Concentration dependence of the inhibition of Escherichia coli ATPase by dicyclohexylcarbodiimide
Fig. 13. Double reciprocal plots of Pi inhibition data for the basal membrane-bound ATPase of Escherichia coli. The plots are the reciprocal initial rates versus I/ATP (Mg 2 +/ATP = 1/2) at constant Pi concentrations: O---O no Pi; A A 2 mM Pi; F3---[~ 4 mM Pi. The experimental details are described in the text. Similar results were obtained for the activity assayed with trypsin.
(DCCD). The inhibitor was added to the assay mixture as indicated in Experimental. The data correspond to the activity assayed in presence of trypsin and are referred to 1 ml of enzyme preparation for facilitating comparative purposes: @ @ membrane-bound ATPase; • - • soluble ATPase. Similar results were obtained for the basal activities.
the interaction coefficient appears to be the same, independently of the concentration of the inhibitor. This could suggest that A D P affects in different way the sites involved in the recognition of substrate at low and high concentrations. The effect of Pi on the membrane-bound and
soluble ATPase is illustrated in Figures 13 and 14, respectively. These graphs indicate that Pi acts as a competitive inhibitor in both cases. The same effect of Pi was observed on the ATPase assayed in presence of trypsin. Ki values of 1.6 _+ 0.1 mM and 1.3 + 0.1 mM were approximated from these results for the m e m b r a n e - b o u n d and soluble ATPase, respectively. The concentration dependence effect of D C C D on the membrane-bound and soluble ATPase from E. coli is shown in Figure 15. D C C D inhibited the m e m b r a n e - b o u n d activity but not the soluble enzyme. It is worth noting that low concentrations of D C C D (0.4 mM) reduced about 60 70 ~o of the m e m b r a n e - b o u n d activity, whereas a concentration 100 times higher did not significantly affect the residual activity. The effect of D C C D appeared to be independent of the presence of trypsin. On the other hand, sodium azide inhibited both states of E. coli ATPase. Figure 16 shows the concentration dependence of this effect. Concentrations as low as 20 #M inhibited 50 ~o of the activity in any of its states, and concentrations 25 times higher inhibited 100 ~o of the activity. The mechanism of inhibition by azide of the m e m b r a n e - b o u n d ATPase was apparently
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Is] Fig. 14. Double reciprocal plots of Pi inhibition data for the basal soluble ATPase of E. coli. The plots are the reciprocal initial rates versus 1/ATP (MgZ+/ATP = 1/2) at constant Pi concentrations: © © no Pi; A - - A 2 mM Pi; U]---[2] 4 mM Pi. Experimental details in the text, Similar results were obtained for the trypsin stimulated soluble ATPase.
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S o d i u m azide,pM Fig. 16. Concentration dependence of the inhibition of E. coli ATPase by sodium azide. As in Figure 15, the data correspond to trypsin stimulated activities. For other details see the text and legend of Figure 8: • • membrane-bound ATPase; A - - & soluble ATPase. The same results were obtained for the activity assayed without trypsin.
competitive for the membrane-bound enzyme but showed a mixed type of inhibition for the EDTA-soluble enzyme (see Discussion). A Ki value of the order of 20-30 #M could be approximated from these experiments for the soluble enzyme. The Ki values for the membrane-bound enzyme were erratic and varied with the concentration of the inhibitor. Specific inhibitors of other ATPase systems such as ouabain 3s, oligomycin 9'39 and atebrin26,40 did not influence E. coli ATPase in any of its states working at the concentration range used for all the other inhibitors.
Discussion The membrane ATPase of E. coli is being actively investigated in several laboratories s,2
MOLECULAR & CELLULAR BIOCHEMISTRY
November 30, 1975
M E M B R A N E B O U N D AND S O L U B L E A D E N O S I N E T R I P H O S P H A T A S E OF E S C H E R I C H I A COLI K 12. KINETIC P R O P E R T I E S OF T H E BASAL AND T R Y P S I N - S T I M U L A T E D ACTI.VITIES Jos6 CARREIRA and Emilio M U l q O Z
Instituto de Biologia Celular, C.S.I.C., Madrid-6, Spain (Received October 4, 1974)
Summary Basal and trypsin-stimulated adenosine triphosphatase activities of Escherichia coli K 12 have been characterized at pH 7.5 in the membranebound state and in a soluble form of the enzyme. The saturation curve for Mg 2 +/ATP = 1/2 was hyperbolic with the membrane-bound enzyme and sigmoidal with the soluble enzyme. Trypsin did not modify the shape of the curves. The kinetic parameters were for the membrane-bound ATPase: apparent K m = 2.5 mM, Vmax (minus trypsin) = 1.6/~mol-min- ~.mg protein- ~ Vmax (plus trypsin) = 2.44/~mol.min-1.mg protein- 1; for the soluble ATPase: [So.5] = 1.2 mM, Vmax ( - trypsin) = 4 #mol.min- 1.mg protein- ~; Vmax ( + trypsin) = 6.6 #mol.min- l-mg protein- a. Hill plot analysis showed a single slope for the membrane-bound ATPase (n = 0.92) but two slopes were obtained for the soluble enzyme (n = 0.98 and 1.87). It may suggest the existence of an initial positive cooperativity at low substrate concentrations followed by a lack of cooperativity at high ATP concentrations. Excess of free ATP and Mg 2+ inhibited the ATPase but excess of Mg/ATP (1/2) did not. Saturation for ATP at constant Mg 2+ concentration (4 mM) showed two sites (groups) with different Kms: at low ATP the values were 0.38 and 1.4 mM for the membrane-bound and soluble enzyme; at high ATP concentrations they were 17 and 20 mu, respectively. Mg 1+ saturation at constant ATP (8 mu) revealed michaelian kinetics for the membrane-bound ATPase and
sigmoid one for the protein in soluble state. When the ATPase was assayed in presence of trypsin we obtained higher K m values for Mg 2+. These results might suggest that trypsin stimulates E. coli ATPase by acting on some site(s) involved in Mg 1+ binding. Adenosine diphosphate and inorganic phosphate (Pi) act as competitive inhibitors of Escherichia coli ATPase. The Ki values for Pi were 1.6 _+ 0.1 mM for the membrane-bound ATPase and 1.3 _+ 0.1 mM for the enzyme in soluble form, the Ki values for ADP being 1.7 mM and 0.75 mM for the membrane-bound and soluble ATPase, respectively. Hill plots of the activity of the soluble enzyme in presence of ADP showed that ADP decreased the interaction coefficient at ATP concentrations below its K m value. Trypsin did not modify the mechanism of inhibition or the inhibition constants. Dicyclohexylcarbodiimide (0.4 rnM) inhibited the membrane-bound enzyme by 60-70 % but concentrations 100 times higher did not affect the residual activity nor the soluble ATPase. This inhibition was independent of trypsin. Sodium azide (20/~M) inhibited both states of E. coli ATPase by 50 %. Concentrations 25-fold higher were required for complete inhibition. Ouabain, atebrin and oligomycin did not affect the bacterial ATPase.
Introduction Adenosine triphosphatases (ATPases) (EC 3.6.1.3) are a family of membrane-bound enzymes
Dr. W. Junk b.v. Publishers - The Hague, The Netherlands
85
involved in a series of energy-transduction processes such as oxidative phosphorylation 1, photophosphorylation 2, transport 3 and contraction 4. Some of these enzymes have been obtained in soluble form by membrane washing with defined buffers (see for example ref. 5). The solubilization allowed the purification and structural characterization of ATPases from various sources. The great structural similarity between mitochondrial, chloroplast and bacterial ATPases 6 and the correlation among contractile and membrane ATPases have been substantiated recently 7. However, ATPases behaved differently with regard to ATP hydrolysis depending upon their source z'8 and/or state 9,1°. The reactivity of membrane-bound ATPases of mitochondria, chloroplasts and Mierococcus lysodeiktieus showed a certain degree of latency 2'11'12 Recently, MITCHELL and co-workers 13 have reported the virtual inhability of Escherichia coli M L 308-225 vesicles to hydrolyze ATP. These enzyme preparations were stimulated by various treatments: trypsin 2'11, heating2,12, dithiothreitol a4, changes in the environmental concentration of divalent metal ions 15,16, detergents 13, or solubilization 11. The latency of mitochondrial and chloroplasts ATPases appeared to be due to the presence of a minor subunit of the ATPases that acts as an ATPase inhibitor 1v,18. The mechanism of ATPase latency in Micrococcus lysodeikticus is still unknown. It may be due to a simple problem of enzyme crypticity or, alternatively, may reflect a mechanism of regulation for the possible polyfunctional role of ATPase in prokaryotic organisms as proposed by Mulqoz and coworkers 19,2°. The hydrolysis of ATP to ADP and Pi catalyzed by ATPases is usually coupled to the active transport of solutes 21'22 under anaerobic conditions or may reflect the uncoupling of the synthetic function of the protein in aerobiosis. The complexity of these processes imply the existence of regulatory mechanisms which may depend on intrinsic properties of the protein or on the physical state of the enzyme through its interaction with non-catalytic proteins. The end products of the reaction, ADP and Pi, appear to be good candidates for modulating the hydrolytic properties of ATPases. In this context, the ATPases from various sources showed a reduction in the rate of ATP hydrolysis by ADP 2'1°-14. It was admitted that the inhibition by ADP was 86
competitive, although there was no total agreement at this respect 23-26. On the other hand, very few studies have been carried out on the inhibition by Pi. Nevertheless, it was concluded that this inhibition was of the same type as that of ADP 27. The ATPase system of E. coli has been the object of several recent studies 8,13'24,25,28-32. In previous work, we described a membrane system of E. coli K 12 able to hydrolyze externally added ATP 8. We also reported the complete solubilization of the ATPase by EDTA-alkaline buffers 8. Both, the membrane-bound and 'soluble' enzyme exhibited the same degree of stimulation by trypsin amounting to about 100 %8. These results suggested a mechanism of control for the unmasking of ATPase activity in E. coli. Furthermore, they seemed to rule out the hypothesis that trypsin stimulation resulted from a crypticity of ATPase in E. coli membranes. These interesting properties prompted us to compare the kinetic properties of the 'soluble' and membrane-bound ATPase when assayed with and without trypsin. Through such studies, it is hoped to gain information on the mechanism of ATPase latency in this organism and on the regulation of the expression of E. coli membrane ATPase activity. Methods
Reagents Adenosine triphosphate (Na2ATP) was purchased from PL Biochemicals. Tris (hydroxymethyl)aminomethane, MgC12 and activated charcoal of the highest quality available were from Merck. EDTA, disodium salt was from Fisher Scientific Co. Trypsin (EC 3.4.4.4), lyozyme (EC 3.6.1.3) and deoxyribonuclease (EC 3.1.4.5) were obtained from Calbiochem. Dicyclohexylcarbodiimide (DCCD) and N a H 2 P O 4 (Pi) were from Merck. Sodium azide (NAN3) was obtained from BDH Ltd. Chemicals. Oligomycin and atebrin were purchased from Sigma Chem. Co. Ouabain was from Calbiochem. Adenosine triphosphate 7_32p, sodium salt (specific activity 1-2 Ci/mmol) was purchased from Amersham, The Radiochemical Centre. Microorganism and membrane preparation Escherichia coli K 12, number 414 (Hfr, T h r - ) of the collection of Dr. J. PUIG (Facultad de
Ciencias, Universidad de los Andes, M6rida, Venezuela), was used in these studies and grown as described previously 33. The preparation of membrane 'ghosts' was carried out by a modification of the procedure of KABAC~ 34. The modification involved the sequential treatment of exponentially growing E. coli cells with E D T A and lysozyme instead of their simultaneous treatment with both agents. The characterization of the membrane 'ghost' fraction, i.e. membranes possessing a size similar to that of the bacterial cell, has been reported elsewhere 33. A TPase preparations
Membranes were kept as pellets at 0 ° for 1 week and resuspended in 30 mM Tris-HC1 (pH 7.5) 1 mM MgC12 at about 1 mg protein/ml for ATPase assays. ATPase was solubilized by resuspending the membrane preparation in 3 mM E D T A - 50 mM Tris (pH 9.0) at about 1 mg protein/ml. After standing 15 min at r o o m temperature, the membrane suspensions were centrifuged at 27000 × 9, in a Sorvall RC-2B for 20 min at r o o m temperature. The supernatants were considered as the soluble ATPase. Confirmation of the soluble nature of these fractions was obtained by centrifuging at 150,000 × 9 for 60 min. The ATPase activity of the supernatants at 150,000 × 9 and 27,000 × g showed identical kinetic behaviour. In subsequent studies, we have considered the supernatants of 27,000 × 9 as soluble ATPase. Because of the high enzyme lability in solution (8, and AZOCAR, O., unpublished observations), ATPase assays with the soluble fraction were performed within 2 hours after its extraction from the membrane.
reagents of Pi determination according to VA_UBUTASand RACKER2. Appropriate blanks were run each time. In m a n y experiments, the results were confirmed by using ATP-';-32p (specific activity in the assay 0.031 #Ci/#mol). In this case, the reaction was stopped by the addition of 0.5 ml of 10 ~ activated charcoal in 0.2 M KC1-HC1 (pH 1.6). The charcoal was removed by centrifugation at 4,000 x 9 for 10 min and aliquots (0.1 ml) of the supernatants were added to Bray's solution 35 for scintillation counting in a M a r k II (Nuclear Chicago). Blanks were prepared by adding 0.5 ml charcoal as above to samples without enzyme or to samples with boiled enzyme. One unit of activity is defined as the amount of enzyme able to liberate 1 #mol Pi per min at 37 °. Specific activity is expressed in units per mg of protein. Variations in the assay conditions during the kinetic studies are specified in the text and in the legends of figures. ADP, Pi and N a N 3 at the concentrations indicated in the legends of the respective figures, were added to the reaction mixture in 100 #1 30 mM Tris-HC1 (pH 7.5). D C C D was added in 10 #1 methanol and oligomycin in 10 #1 ethanol. These concentrations of alcohol did not show any effect on the rate of ATP hydrolysis. The effect of inhibitors was directly tested on the assay mixture without preincubation with the enzyme. When the inhibition of Pi was studied by the colorimetric procedure, appropriate blanks were run to correct for the excess of Pi in the samples. The protein content was measured by the method of LOWRY et al. 36 using bovine serum albumin as standard.
A TPase assay
The standard assay system contained 4 #tool ATP, 2 #mol Mg 2+ and 12 #mol Tris-HC1 (pH 7.5) in 400 #1. The reaction was started by the addition of 100 #1 containing either 80 #g of membrane protein or 25 #g of soluble protein. Where stated, trypsin was added to the assay before the ATPase preparations at a concentration of 0.2 mg/ml or with the modifications indicated in the legend of the figures. Incubations were carried out at 37 ° for 5 min. During this time, the reactions proceeded linearly. The reaction was stopped by the addition of the
Experimental Figure 1 illustrates the dependence of membranebound ATPase activity upon the protein concentration. The velocity is linear up to 125 130 #g protein/0.5 ml reaction mixture. Figure 2 shows parallel results for the soluble ATPase. In this case the linearity was maintained at protein concentrations below 40-45 #g in the reaction mixture. All these results were independent on the presence of trypsin. Subsequent work was carried 87
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Protein ()Jg) Fig. 1. Influence of protein concentration on the rate of ATP hydrolysis by the m e m b r a n e - b o u n d ATPase of Escherichia coll. The conditions for assay are given in the text (see Experimental). The protein refers to the a m o u n t of total m e m b r a n e protein put in the assay mixture: © © basal ATPase (minus trypsin); O - - O plus trypsin (0.2 mg/ml assay mixture); • - - • plus trypsin added to the assay mixture at the same concentrations as that of the membrane preparation.
out with 80-90 #g protein in the assay for the m e m b r a n e - b o u n d ATPase and 25 30 #g for the soluble enzyme. Studies on the effect of Mg 2 ÷ concentration on ATPase activity showed that the optimal activity was reached at a Mg 2 +/ATP ratio near to 1/2,
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Fig. 3. Double reciprocal plots of initial rate data for the m e m b r a n e - b o u n d ATPase of E. coli. The experimental conditions are given in the text. Substrate refers to Mg 2 +/ATP = 1/2, the actual concentrations used for the plot were those of ATP: © - - © basal ATPase; O - - O ATPase assayed with trypsin, The inset shows schematically the direct curve for substrate saturation.
as found by other workers 24'25'28 32. We then studied the saturation for this substrate for the m e m b r a n e - b o u n d and the solubilized enzyme. The range of A T P concentrations was 0.18 raM, Mg 2+ concentrations being 0.05 4 mM. The membrane-bound ATPase obeyed Michaelis-Menten kinetics when assayed either in presence or in absence of trypsin (see Fig. 3). However, the E D T A soluble ATPase gave a sigmoid curve for substrate saturation (see inset in Fig. 4), therefore, suggesting the existence of
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Protein ( ~ g ) Fig. 2. Influence of protein concentration on the rate of A T P hydrolysis by the soluble ATPase of E. coli. The conditions for ATPase assay and the nature of ATPase preparation are described in the text. The protein refers to the a m o u n t of soluble protein put in the assay medium: © - - © basal ATPase (minus trypsin); O - - O plus trypsin (0.2 mg/ml assay mixture); • • plus trypsin (increasing concentrations to equal the protein concentration of the soluble fraction).
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cooperativity between different active sites. These results were independent of trypsin. The same apparent K m value of 2.5 mM for the membranebound ATPase (Fig. 3) was obtained independently of the presence of trypsin in the assay. However, the Vmax values were respectively: 1.6 # m o l . m i n - < m g - 1 for the membrane ATPase assayed without trypsin and 2.44 #mol.min-1. m g - a for the ATPase assayed with trypsin (Fig. 3). The estimated kinetic parameters for the solubilized enzyme were: a [So.5] = 1.2 mM and Vmax values of 4 (without trypsin) and 6.6 (plus trypsin). Trypsin thus affects the hydrolysis rate of ATP without apparently altering the affinity of the enzyme for the substrate. The most likely mechanism of trypsin action appears to be consequence of the hydrolytic cleavage of some ATPase component(s), although we can not rule out a simple effect of protein-protein interaction, owing to the high concentrations of protease used (see Figs. 1 and 2). The small difference found in K m values for the m e m b r a n e - b o u n d and soluble ATPase was constant in 10 different experiments performed with various preparations. This finding, together with the different shape of the substrate saturation curve, may reflect pecularities of the two states of the ATPase (see Discussion). Figure 5 illustrates the Hill plots for E. coli Z5
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ATPase in both types of preparation. On the one hand, the 0.92 Hill coefficient calculated for the m e m b r a n e - b o u n d ATPase suggests that there is no interaction between the active sites in this state of the enzyme. On the other hand, the Hill plot of the soluble enzyme shows two slopes. The change in slope occurs at a substrate concentration about the [So.5] of the enzyme. The value of 1.87 tends to indicate a positive cooperativity between the active sites. The existence of two slopes in a Hill plot is very unusual 37. According t o COOK a n d KOSHLAND 37 w e may assume an initial positive cooperativity at low substrate concentrations, followed by a lack of cooperativity (negative cooperativity?) at substrate concentrations higher than those exhibiting halfsaturation for E. coli ATPase. As one could expect from previous results, trypsin did not influence these parameters. In attempts to individualize the role of each component of this complex substrate, we measured the influence of increased ATP concentrations (range 0.1-8 mM) at a constant Mg 2+ concentration (4 raM). The results are shown in Figures 6 and 7. Both the m e m b r a n e - b o u n d enzyme a n d the soluble ATPase followed Michaelis kinetics with two classes of saturation constants. The kinetic parameters are summarized in Table 1. The apparent K m value at low A T P concentrations for the particulate enzyme is very similar to that reported by Hanson and Kennedy for the m e m b r a n e - b o u n d ATPase from E. coli and a purified form of the enzyme, both assayed with excess of Mg 2 + ions. The apparent K m of 89
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soluble enzyme obtained in these conditions was higher than that of the membrane ATPase, which contrasts with results obtained with variable concentrations of Mg 2 +/ATP. The apparent K m values at high concentrations of ATP were very similar for both forms of the ATPase. These studies were not carried out in presence of trypsin due to the high sensitivity of the enzyme to trypsin digestion in presence of high Mg 2 + and incomplete enzyme protection by ATP (CARREIRA, J. and ROJAS, M., unpublished observations). The Michaelis kinetics of the soluble ATPase in these conditions may suggest that ATP did not contribute to the cooperativity found in previous experiments (see Fig. 4). Moreover, free ATP or Mg 2+ inhibited E. coli ATPase, whereas high Table 1 Kinetic parameters for A T P of the soluble and membraneb o u n d ATPase of Escherichia coli in function of the experimental A T P concentrations at a constant Mg 2 + concentration (4 mM). gmax
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Fig. 8. Double reciprocal plot of Mg 2 + saturation for the m e m b r a n e - b o u n d ATPase of E. coll. The plot is of the reciprocal initial rates versus 1/Mg 2 + at constant ATP concentration (8 mM). For experimental details see the text: • • basal activity; O - - O activity assayed with trypsin.
concentrations of Mg 2 +/ATP (1/2) were not inhibitory. These results indicate the existence of sites with different affinities for ATP, probably depending on Mg 2 +/ATP ratios. The complexity of these results tends to suggest that Mg/ATP might not be the true substrate of E. coli ATPase. In a subsequent step, we studied the effect of Mg 2 + concentrations of the velocity of ATP hydrolysis at constant ATP (8 ms). In these experimental conditions (see Figs. 8 and 9), the membrane bound enzyme and the soluble enzyme behaved differently. The former showed Mg 2+ saturation curve yielding K m = 1.2 mM, whereas the latter revealed a sigmoid curve (approximate [So.5] = 1.1 mM). Trypsin modified these parameters thereby increasing the Km value to 7 mM for the membrane-bound ATPase and to 2 mM for the EDTA-soluble enzyme. Vmax values were affected in a similar way. These results, taken together indicate again that Mg 2 +/ATP is not the true substrate for E. coli ATPase. The decrease in affinity for Mg 2+ of E. coli ATPase induced by trypsin suggests the existence of some site(s) directly involved in Mg 2 + binding. This(these) site(s) appear(s) to be the point of specific attack by trypsin under the protective conditions where stimulation of the ATPase occurs. Dixon plots for the inhibition by ADP of the membrane-bound and soluble ATPase are
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shown in Figures 10 and 11, respectively. The lines intersect in the second quadrant over the abscissa axis confirming that ADP acts as a pure competitive inhibitor of E. coli ATPase. Ki values of 1.7 mM and 0.75 mM were calculated
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by this procedure for the membrane-bound and soluble enzyme, respectively. As in previous cases, these constants were not modified by trypsin (results not shown). Similar results were obtained from double reciprocal plots. Figure 12 compares the different Hill plots of the EDTA-soluble ATPase assayed under the conditions described in Figure 11. As in Figure 5, two slopes are seen in these plots. ADP decreases the interaction coefficient in the first part of the curve before the change in slope. After this point,
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Fig. 13. Double reciprocal plots of Pi inhibition data for the basal membrane-bound ATPase of Escherichia coli. The plots are the reciprocal initial rates versus I/ATP (Mg 2 +/ATP = 1/2) at constant Pi concentrations: O---O no Pi; A A 2 mM Pi; F3---[~ 4 mM Pi. The experimental details are described in the text. Similar results were obtained for the activity assayed with trypsin.
(DCCD). The inhibitor was added to the assay mixture as indicated in Experimental. The data correspond to the activity assayed in presence of trypsin and are referred to 1 ml of enzyme preparation for facilitating comparative purposes: @ @ membrane-bound ATPase; • - • soluble ATPase. Similar results were obtained for the basal activities.
the interaction coefficient appears to be the same, independently of the concentration of the inhibitor. This could suggest that A D P affects in different way the sites involved in the recognition of substrate at low and high concentrations. The effect of Pi on the membrane-bound and
soluble ATPase is illustrated in Figures 13 and 14, respectively. These graphs indicate that Pi acts as a competitive inhibitor in both cases. The same effect of Pi was observed on the ATPase assayed in presence of trypsin. Ki values of 1.6 _+ 0.1 mM and 1.3 + 0.1 mM were approximated from these results for the m e m b r a n e - b o u n d and soluble ATPase, respectively. The concentration dependence effect of D C C D on the membrane-bound and soluble ATPase from E. coli is shown in Figure 15. D C C D inhibited the m e m b r a n e - b o u n d activity but not the soluble enzyme. It is worth noting that low concentrations of D C C D (0.4 mM) reduced about 60 70 ~o of the m e m b r a n e - b o u n d activity, whereas a concentration 100 times higher did not significantly affect the residual activity. The effect of D C C D appeared to be independent of the presence of trypsin. On the other hand, sodium azide inhibited both states of E. coli ATPase. Figure 16 shows the concentration dependence of this effect. Concentrations as low as 20 #M inhibited 50 ~o of the activity in any of its states, and concentrations 25 times higher inhibited 100 ~o of the activity. The mechanism of inhibition by azide of the m e m b r a n e - b o u n d ATPase was apparently
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Is] Fig. 14. Double reciprocal plots of Pi inhibition data for the basal soluble ATPase of E. coli. The plots are the reciprocal initial rates versus 1/ATP (MgZ+/ATP = 1/2) at constant Pi concentrations: © © no Pi; A - - A 2 mM Pi; U]---[2] 4 mM Pi. Experimental details in the text, Similar results were obtained for the trypsin stimulated soluble ATPase.
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S o d i u m azide,pM Fig. 16. Concentration dependence of the inhibition of E. coli ATPase by sodium azide. As in Figure 15, the data correspond to trypsin stimulated activities. For other details see the text and legend of Figure 8: • • membrane-bound ATPase; A - - & soluble ATPase. The same results were obtained for the activity assayed without trypsin.
competitive for the membrane-bound enzyme but showed a mixed type of inhibition for the EDTA-soluble enzyme (see Discussion). A Ki value of the order of 20-30 #M could be approximated from these experiments for the soluble enzyme. The Ki values for the membrane-bound enzyme were erratic and varied with the concentration of the inhibitor. Specific inhibitors of other ATPase systems such as ouabain 3s, oligomycin 9'39 and atebrin26,40 did not influence E. coli ATPase in any of its states working at the concentration range used for all the other inhibitors.
Discussion The membrane ATPase of E. coli is being actively investigated in several laboratories s,2
