Properties of the Membrane-Bound Enzyme

JOURNAL

OF

BACTERIOLOGY, Aug. 1974,

p.

Vol. 119, No. 2 Printed in U.S.A.

593-601

Copyright ( 1974 American Society for Microbiology

Membrane Adenosine Triphosphatase of Micrococcus lysodeikticus: Effect of Millimolar Mg2+ in Modulating the Properties of the Membrane-Bound Enzyme MERCEDES LASTRAS AND EMILIO MUNOZ Consejo Superior Investigaciones Cientificas, Institute of Cellular Biology, Velizquez, 144, Madrid-6, Spain Received for publication 22 February 1974

The latency of Micrococcus lysodeikticus membrane-bound Mg2+-adenosine triphosphatase (ATPase) is expressed by the ratio of its activity assayed in the presence of trypsin ("total") versus the activity assayed in absence of the protease ("basal"). By isolating membranes in the presence of variable concentrations of Mg2" (50 mM, 10 mM, or none) and by washing them with different Mg2+- and ethylenediaminetetraacetic acid-containing tris(hydroxymethyl)aminomethane-hydrochloride buffers (pH 7.5), we showed that the enzyme latency was dependent on the environmental concentration of this divalent metal ion. Mg2+ bound to at least two classes of sites. The binding of Mg2+ to low-affinity sites (saturation at approximately 40 mM external Mg2+) induced a high basal ATPase activity, whereas its binding to medium-affinity sites (saturation at about 2 mM Mg2+) correlated with low basal activity and a very high stimulation by trypsin. Membranes with tightly bound Mg2+ (high affinity?) revealed an intermediate behavior for the latency of M. lysodeikticus ATPase. The Mg2+/Ca2" antagonism as activators of the membrane ATPase was not directly related to Mg2` binding by the membranes. The efficiency of the ATPase release from M. lysodeikticus membrane by 3 mM tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 7.5) was inversely proportional to the concentration of external and/or bound Mg2+. Deoxycholate (DOC) (1%) solubilized the ATPase from all types of membrane. All the soluble ATPases behaved as Ca2+-ATPases, but the DOC-soluble fractions showed degrees of latency like those of the original membranes. The DOC-soluble ATPase preparation revealed a vesicular structure and complex protein patterns by sodium dodecyl sulfate gel electrophoresis. We propose that ATPase latency is modulated via a Mg2+-ATPase-membrane complex. The adenosine triphosphatase (ATPase) (EC 3.6.1.3) bound to standard membranes (19, 22) of Micrococcus lysodeikticus behaves as an "extrinsic" or peripheral protein, according to Singer and Nicolson (26), since it can be released into solution by ionic manipulation of the membranes (16, 18). The soluble protein was apparently lipid free (19), and behaved as a single molecular species (1, 19). However, the soluble ATPase differed from the membranebound enzyme with respect to latency and cation-activation responses (19). By measuring the latter parameters, we observed the existence of different states of the membrane-bound enzyme (11). These states were dependent on Mg2" and were partially reversible (11, 12). The estimation of a 20-mM intracellular Mg2" concentration indicated the possible physiological significance of these findings (11). Furthermore,

the latency of the enzyme, i.e., its stimulation by trypsin, could not be explained by the molecular properties of the soluble purified enzyme (1). These results suggest that the latency of the membrane-bound M. lysodeikticus ATPase and its response to cations as activators reflect a possible modulation of the enzyme through its association with the membrane ("allotopy"). If this were true, M. lysodeikticus ATPase would offer one of the clearest examples of the regulatory significance of such a mechanism. This possibility is particularly exciting if we take into account the "extrinsic" or "detachable" character (18) of M. lysodeikticus ATPase as membrane protein. To explore this property further, we studied the effect of millimolar Mg2" concentrations on the enzyme latency and cation-activation re-

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LASTRAS AND

sponse of M. Iysodeikticus ATPase. We attempted to find a quantitative explanation of the Mg2" effect and to study its role in influencing the interaction between ATPase and the rest of the membrane complex. We studied some properties of deoxycholate (DOC)-soluble ATPase fractions as model systems for understanding the mechanisms responsible for enzyme latency and its stimulation by trypsin. MATERIALS AND METHODS Chemicals. Adenosine triphosphate (ATP), disodium salt was a product of Pabst Laboratories Biochemicals; polyvinyl alcohol was from Doesder (Barcelona, Spain); Titan yellow, tris(hydroxymethyl)aminomethane (Tris), and amido black 10 were purchased from Merck; lysozyme (EC 3.2.1.17), deoxyribonuclease I (EC 3.1.4.5) and ,B-mercaptoethanol were from Calbiochem; sodium dodecyl sulfate (SDS; sodium laurylsulfate) and DOC were obtained from Fisher Scientific Co. Acrylamide, N,N'-methylenebisacrylamide and N, N, N', N'-tetramethylenediamine were purchased from Fluka and Buchs. All other reagents were of the highest purity grade available. Microorganism and preparation of membranes. M. Iysodeikticus (NCTC 2665) was used throughout these studies. Membranes were isolated by the conventional procedures described in detail elsewhere (16, 19, 22). They can be briefly summarized as follows: stationary-phase cells (25 to 30 mg [dry weight] per ml of 50 mM Tris-hydrochoride buffer, pH 7.5) were treated with lysozyme (0.2 mg/ml) and then lysed in the presence of various amounts of Mg2", depending on the type of membrane preparation (50 mM in types A and C, 10 mM for type B, and no Mg2" in type D) (11). Deoxyribonuclease (40 jig/ml) was added to reduce the viscosity of the lysates. Membranes were spun down in a Sorvall RC2-B refrigerated centrifuge at 27,000 x g for 40 min. The membranes so deposited were then washed five times with 50 mM Tris-hydrochloride buffer, pH 7.5, containing 50 mM MgCl2 (type A), 10 mM MgCl2 (type B), or no Mg2" (type C), or with 100 mM Tris-hydrochloride buffer, pH 7.5 (type D). ATPase activity. ATPase activity was measured by the liberation of Pi from ATP in a reaction mixture containing, in a final volume of 500 Mliters: 4 ,mol of ATP, 15 to 20 umol of Tris-hydrochloride buffer, pH 7.5, 100 Aliters of membrane suspension (about 100 to 300 jig of protein). In most instances, this mixture was supplemented with either 2 Amol of MgCl2 or 4 jimol of CaCl2 (19). Where stated, trypsin was added at 0.5 mg/ml. Incubations were carried out at 37 + 1 C for 30 (membranes) and 15 (soluble fractions) min and stopped by immersion in an ice-cooled water bath and rapid addition of the reagents for Pi determination (29). Under these conditions, the time course of trypsin activation was apparently the same for all types of membranes and identical to that previously reported (19). One unit of enzyme activity is defined as that amount able to liberate 1 nmol of Pi per min in these experimental conditions. Owing to the differ-

MU1&OZ

J. BACTERIOL.

ences in protein content between the various membrane and enzyme preparations, ATPase activity is given in units per unit of volume of each preparation, instead of in specifc activity. Analytical determinations. Protein was estimated by the method of Lowry et al. (14), with bovine serum albumin as standard. Magnesium was determined by the colorimetric method of Orange and Rhein (20) and directly applied to soluble samples or modified for its application to membrane fractions. This modification included a prior mineralization of the samples by heating first for 1 h at 100 C and then 2 h at 165 C with a mixture of H2SO4 and 70% HC104 (1:2.3, vol/vol). After the pyrolysis, the samples were diluted with an appropriate volume of water and assayed as for soluble samples. Good proportionality and reproducibility was found in the range from 2 to 10 Mg of Mg2". The Mg2" content of membrane fractions is expressed as microequivalents per milliliter of initial suspension to correct for variations in protein concentration from one preparation to another. Solubilization of ATPase. Attempts to release ATPase into solution were carried out by suspending the membranes in the original volume of the suspension with either 3 mM Tris-hydrochloride buffer, pH 7.5, or 50 mM Tris-hydrochloride buffer of the same pH containing 1% DOC. After 10 to 15 min at room temperature, the samples were centrifuged in a Sorvall RC2-B centrifuge at 27,000 x g for 40 min at 4 C. The resulting supernatants were considered as the soluble fractions. Polyacrylamide electrophoresis. Samples were electrophoresed by the system of Davis (5) without stacking gels. Gels (0.6 by 14 cm) were used and run (2.5 to 3 h) at a constant current 5 mA/gel. Electrophoresis under dissociating conditions was performed in the same system with 0.1% SDS - 0.05% fl-mercaptoethanol. Samples were pretreated with 1% SDS + 0.5% mercaptoethanol at 85 C for 5 min. Bromophenol blue was used as tracking dye. Proteins were stained with amido black (6).

RESULTS Activity and latency of membrane-bound ATPase from M. lysodeikticus. The latency of the ATPase is indicated by the extent of its stimulation by trypsin as compared with the activity assayed in the absence of the protease (basal ATPase). Theoretically the activity assayed in the presence of trypsin, being the expression of the total or potential ATPase bound to membrane, should be the same for all assays and types of membranes. However, as we shall show in this report, exceptions can be found. Table 1 illustrates the levels of basal ATPase and of the activity assayed in the presence of trypsin, as well as the ratio between the two activities in different types of M. lysodeikticus membranes. The highest basal activity was exhibited by membranes isolated in the presence of Mg2" (types A and B), and these

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MG2+ EFFECT ON M. LYSODEIKTICUS ATPASE

preparations also showed the lowest degree of latency. Conversely, the high stimulation by trypsin is observed for membranes C and D, which otherwise exhibited low basal ATPase activity. Note the different figures of total ATPase for membranes A and D. These differences (see above) do not reflect a partial solubilization of the ATPase from membranes of type A but a lack of optimal conditions in the assay for these membranes due to the excess of Mg2". It is also worth noting that the trypsin stimulation of ATPase from membranes of type A is similar to that found for most of the crude-soluble Ca2+-ATPase preparations (1, 19). Hence, it seems reasonable to conclude that the expression of M. lysodeikticus ATPase is dependent

on the state of the association of the enzyme with the membrane which is influenced by the Mg2+ concentration during membrane isolation. To confirm this, we examined the effect on the ATPase of the progressive removal of Mg2+ from membranes by washing (Table 2). As one would expect, Mg2+ depletion induced by washing resulted in a decrease in the basal ATPase activity and a concomitant increase in the latency of the enzyme. Within the experimental error, it is worth noting that similar values were obtained for most of the assays of ATPase in the presence of trypsin (total ATPase). Several points deserve further comment. First, the washing of type A membranes with 50 mM Tris containing 5 or 1 mM Mg2+ yielded a maximal TABLE 1. Activitv and latency of the ATPase bound basal activity while exhibiting a low increase in to Micrococcus Ivsodeikticus membranes isolated in the stimulation by trypsin. Second, the washing the presence of variable amounts of Mg2+ with Mg2+-free buffers of membranes isolated in the presence of low Mg2+ (types C and D) did ATPase activitvb not essentially modify the characteristics of the bound ATPase. Third, the action of Trypsin Membrane - Trvpsin +Trypsin stimulation prepna (basal) ethylenediaminetetraacetic acid (EDTA) on (U/ml of O (U/mi of (trvpsin/ Mg2+-depleted membranes reversed the situa(u/menion suspension) suspension) basal) tion, increasing the basal ATPase activity and decreasing the latency of the enzyme. These A 147.0 225.0 1.5 results suggest that M. lysodeikticus memB 166.5 416.3 2.5 C 86.7 407.5 4.7 brane-bound ATPase is controlled by Mg2+ D 31.0 310.0 10.0 binding to the membrane. To investigate this possibility, we measured a4 mM MgCl2 was added to enzyme assays with the binding of Mg2+ to the different types of membranes B, C, and D. Membrane A received no membranes and to differentially washed memMgC12b Results are the means from three different experi- branes and correlated it with the properties of ments. their membrane-bound ATPase (Fig. 1). A

TABLE 2. Effect of membrane washing with different buffers on the activity and latency of Mg2+-ATPase bound to Micrococcus lysodeikticus mem branes Membrane prepn

Wasbing buffer" (pH 7.5

-T~psin

ATPase activity'

+Trypsin (total) - Trypsin (basal) (U/ml of suspension) (U/ml of suspension)

Trypsin stimulation (total/basal)

A

50 mM Tris-5 mM MgCl2 50 mM Tris-1 mM MgCl2 50 mM Tris 50 mM Tris-5 mM EDTA

227 220 178 124

454 440 391.6 310

B

50 mM Tris 50 mM Tris-5 mM EDTA

140 45

532 450

3.8 10.0

C


58 33

493 495

8.5 15.0

D


47 125

446.5 437.5

2.0 2.0 2.2 2.5

9.5 3.5

Membranes were washed by centrifugation after being suspended in the appropriate buffer. bATPase was determined in the presence of 4 mM MgCl2. The EDTA present in some assay mixtures was previously titrated with MgCl2. Results are the means of at least duplicate experiments. a

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ered it of interest to examine the possible

'a ioo-

I

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IL

7

regulation of this property of the ATPase by the

Mg2".

Activienvironmental concentration of ties were measured by the standard assay, as t I described under Materials and Methods, sup16 with either 4 mM MgCl2 or 8 mM plemented I CaCl2, and the ratios of Ca2+ versus Mg2+ I activities were calculated (Table 3). Their analIs /0 ysis is difficult, but several points emerge. The I ATPase activity assayed in the presence of trypsin (total) and the basal activity differ with 2wa respect to their Ca2+/Mg2+ antagonism. This 24 52 40 16 81 confirms that both parameters of ATPase activExternal [Mg"a] mM ity do reflect distinct functional states of the FIG 1. Binding of Mg2+ by Micrococcus lysodeik- protein. With few exceptions, Mg2` was a better ticus membranes in function of the environmental activator than Ca2+ for the membrane-bound conce?ntration of this divalent metal ion. The histo- enzyme (Table 3). It is worth noting that all gram in the upper part shows the correlation between exceptions correspond to the basal ATPase. the sstates of membrane-bound ATPase from M. There was no correlation between the ATPase . 2 .~~~~~~~o of M2. Mug2. ~,22 lysodo'eikticus and the concentration stimulation response to Ca`+ or Mg2+ as activators and the of trypsin; in absence assayed 3, A TPcise concentration of bound Mg2+ (Table 3). This is trypsin. o of thE eATPassayedin ATPase byabsenceyof in contrast with the results described above on trypsin. the latency of the enzyme. was obtained. This, for binding asic curve biph, Differential solubilization of M. lysodeikthere fore, indicates the existence of more than one c lass of sites for Mg2+ binding. One seems to ticus ATPase bound to different types of membrane. The effect of Mg2+ concentration in eorre Q.nond to VX low affinitv ,> LV a site(s) .XssLAAJ-" (saturation O>WI'Oj of %,lIIV0JjVII% membrane ml of of at about 16 ,ueq Mg2" per TABLE 3. Ca2+/Mg2+ antagonism as activators of suspension); another can be related to a site(s) Micrococcus lysodeikticus ATPase bound to different of medium affinity (saturation at 6 Aeq per ml of types of membranea suspension). The existence of a third type of Mg2+ Ca2+/Mg2+ ATPase site(s), which binds Mg2+ very tightly, may also activity bound be inferred from the amount of Mg2` still MemWashing buffer (iieq/ml brane of D and and of C types in membranes present -Tryp- +Tryp(pH 7.5) ofss pen of susprepn sin sin thoroughly washed membranes of type A. The pension) (basal) (total) bound Mg2" fraction of tightly existence of a has been recently demonstrated in Mycoplasma 0.32 0.70 16 A membranes (10) by a different experimental 0.43 0.51 13.4 50 mM Tris-5 mM approach. More interesting is the fact that a Mg2+ certain correlation seems to exist between the 0.59 0.50 8.8 50 mM Tris-1 mM Mg2+ concentration of Mg2` bound to membranes 0.56 0.75 6 50 mM Tris-5 mM and the properties of their ATPase (Fig. 1). The EDTA high basal membrane-bound ATPase and the concomitant low level of its trypsin stimulation 0.95 0.67 6 B coincided with the binding of metal ion to the 0.56 0.74 5.6 mM Tris 50 the On the other of hand, low affinity. site(s) 0.67 1.78 4.5 Tris-5 mM mM 50 low basal ATPase and the high stimulation by EDTA trypsin are seemingly coincident with the binding of Mg2" to the site(s) of medium affinity, 0.64 C 5.4 0.65 with a likely maximum at its saturation. These 0.80 0.63 5 50 mM Tris 4.2 1.47 0.57 50 mM Tris-5 mM results confirm the idea of a regulation of EDTA membrane-bound ATPase from M. lysodeikticus by the environmental Mg2` concentration. 0.36 0.37 3.2 Ca2+/Mg2+ antagonism as activators of D 50 mM Tris 0.63 1.30 3 of M. lysodeikticus ATPase. The requirement 0.63 ND" 0.64 50 mM Tris-5 mM M. lysodeikticus ATPase for Ca2+ and Mg2+ EDTA as activators apparently varies with the physical a state of the protein (19). Ca2+ and Mg2+ beResults are the means of duplicate experiments. haved as antagonists in this respect. We considb ND, Not done. i

.1

1

20

X

H

El,stimul

4A

lvv

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VOL. 119, 1974

membranes on the solubilization of M. lysodeikticus ATPase was tested by the efficiency of washing the ditferent types of membrane with 3 mM Tris-hydrochloride buffer, pH 7.5 ("shock wash") (Table 4). Results for membranes of type D are not shown, since they were similar to those for membranes C and have been previously reported (16, 17). The shock wash treatment of membranes A did not release into solution any ATPase activity, measured in the presence or absence of trypsin with either Ca2+ or Mg2+ as activators. A small percentage of ATPase units was solubilized by identical treatment of membranes isolated in the presence of 10 mM MgCl2 (type B). The shock wash treatment of membranes with low Mg2+ content gave a marked release of ATPase activity into solution. This solubilization was accompanied by an increase in the total number of enzyme units (i.e., the percentages of recovery in the soluble fractions are higher than 100% of the initial

membrane-bound activity). In all cases, the shock wash-soluble ATPase behaved as a Ca+ATPase, slightly stimulated by trypsin. These results confirm and extend previous findings (1, 17, 19). The ease of ATPase solubilization appears to be inversely related to the amount of Mg2+ present in membranes. The increase in efficiency of shock wash solubilization of ATPase by prewashing the membranes with Mg2+-free buffers is illustrated in Table 5. The washing of Mg2+ from membranes of type B improved the yield of ATPase solubilized by the low-ionic-strength buffer. Again all soluble ATPases, independently of the nature of starting material, behaved

as

Ca21_

ATPase with low stimulation by trypsin. These properties are thus inherent in this soluble state of the protein and therefore suggest that they reflect true "allotopic" properties of the enzyme.

Solubilization of membrane-bound ATPase

TABLE 4. Release of membrane-bound ATPase from different types of Micrococcus lysodeikticus membranes by washing with 3 mM Tris-hydrochloride buffer, pH 7.5 ATPase activity Protein (%)

Fraction

Membranes A ......... Shock wash soluble

.........

Membranes B ......... Shock wash soluble

.........

Membranes C ..... Shock wash soluble

.......

.......

.........

Metal ion added

+ Trypsin Trypsin (total) (U/ml stimulation of fraction) (total/basal)

Percent

100 4-8 4-8

Mg2+ Ca2+ Mg2+

220.0 0 0

1.5

100 0 0

100 22 22

Mg2+ Ca2+

Mg2+

420.4 78.9 37.6

2.5 1.4 1.4

100 18.8 8.9

100 28-35 28-35

Mg2+ Ca2+ Mg2+

410.8 1,300.0 619.0

4.7 1.4 1.4

100 316.4 150.7

TABLE 5. Increase in the solubilization of Micrococcus lysodeikticus ATPase by 3 mM Tris-hydrochloride buffer, pH 7.5, after Mg2+ washing of membranes isolated in presence of 10

mM MgC12 ATPase activity Fraction

Membrane B washed in 50 mM Tris-hydrochloride ........... Shock wash soluble .............

Membrane B washed in 50 mM Tris-5mM EDTA ............ Shock wash soluble .............

Protein (%)

Metal ion added

+ Trypsin

Trypsin

(U/ml of fraction)

stimulation (total/basal)

Percent

100 25-27 25-27

Mg2+ Ca2+ Mg2+

550 640 300.5

3.6 1.3 1.3

100 116.4 54.6

100 25 25

Mg2+

480 793 377.6

10.0 1.1 1.1

100 165.2 78.7

Ca2+ Mg2+

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from different types of M. lysodeikticus membrane by DOC. Shock wash treatment of type A membranes did not release into solution any ATPase activity, even after prewashing the membranes with Mg2"-free or EDTA-containing buffers. We therefore attempted to solubilize the ATPase bound to membranes of type A by using detergents. Deoxycholate was selected on the basis of a previous report by Salton and Nachbar (23). The solubilization of ATPase by DOC from various types of membrane is shown in Table 6. The characteristics of these DOCsoluble fractions with respect to cation-activation response and to enzyme latency are also given. DOC solubilized the ATPase from all classes of membrane. All the DOC-soluble enzyme used Ca2" as a better activator than Mg2+ and yielded similar figures for total Ca2+-ATPase. However, unlike the shock wash ATPase (Tables 4 and 5), the DOC-soluble fractions showed great variations in the latency of their activity; i.e., trypsin stimulation of the Ca2+-APase from the different DOC fractions ranged from 77.6 to 1.1. Interestingly enough, the latency seemed to be directly proportional to the concentration of Mg2+ originally present in each membrane preparation. Another interesting property of the DOC-soluble ATPases is that, again unlike the shock wash enzymes, their Ca2+/Mg2+ antagonism varied, depending upon the type of membrane from which they were derived. In these DOC-soluble fractions, the trypsin stimulation of their Mg2+ ATPase

did not parallel that of their Ca2" activity; trypsin stimulation of Mg2+-ATPase was practically constant for all DOC fractions. Hence, the DOC-soluble ATPase fractions differ from the shock wash preparations. The difference might be either an artifact produced by the presence of detergent or actually reflect new states of the soluble enzyme. If the first alternative is true, this would invalidate any comparison between the two types of preparations. Anyhow, the comparison between different DOC-soluble fractions would be still valuable. In this regard, the DOC-soluble fractions from membranes A and C appear to offer a good system for studying the latency of the enzyme (compare the figures for trypsin stimulation of both fractions in Table 6). The electrophoretic profiles of these two fractions are illustrated in Fig. 2. Both preparations had similar complex patterns of about 18 polypeptides, whose molecular weights ranged from 150,000 to 15,000 as revealed by SDS-mercaptoethanol gel electrophoresis (compare gels 1 and 2 in Fig. 2). A similar complex pattern but with different relative polypeptide intensities is found in gel 3 in the same figure (type C or "standard" membranes). In spite of differences in loading and migration, note the exact correspondence (as indicated by the arrows) in the relative mobilities of most polypeptide groups from the three gels. When observed under the electron microscope by negative staining with ammonium molybdate (16), both preparations

TABLE 6. Properties of the A TPase activity of DOC-soluble fractions from different types of Micrococcus lysodeikticus membranes ATPase activitya

Fraction

Membrane protein (%)

......... 56 DOC-soluble, membrane A ........ ......... 56 DOC-soluble, membrane A ........ DOC-soluble, membrane A washed in ........... 55 Tris-hydrochloride ......... DOC-soluble, membrane A washed in 55 Tris-hydrochloride ......... ........... DOC-soluble, membrane A washed in 69 Tris-EDTA ........................... DOC-soluble, membrane A washed in Tris-EDTA ........................... 69 DOC-soluble, membrane B .62 62 ......... DOC-soluble, membrane B ........ 75 ......... DOC-soluble, membrane C ........ 75 ......... DOC-soluble, membrane C ........

+ Trypsin

(U/mi of

Trypin Trypsin

suspense

stimulation

Mg2+ Ca2+

126.5 520

2.1 77.6

Mg2+

200

Ca2+

596.3

29.7

Mg2+

313.3

2.8

Ca2+

673.5 267 600 403.4 520

5.1 4.1 27.3 1.8 1.1

Metal ion added

Mg2+ Ca2+

Mg2+ Ca2+

2.7

aATPase was assayed in the presence of 0.2% DOC since the detergent was not dialyzed. The dialysis did not essentially modify the results.


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599

Re I Mob. qalp-

a a

ATPase

S

0.41

0-49

J/

FAd. /I,V r

0.66 0.69 0.73 0.76 0.81 0.86 0.87 0.91 0.95

1

2

_

3

4

FIG. 2. Characterization by gel electrophoresis of DOC-soluble ATPase fractions from Micrococcus lysodeikticus. Gels 1 and 2 illustrate the SDS-polyacrylamide electrophoretic patterns of DOC-soluble fractions from membranes A and C as compared with that of the complete membranes of type C (gel 3); gel 4 shows the electrophoretic profile of DOC-soluble ATPase from type C under non-dissociating conditions. Migration was towards the anode (bottom of the gels).

revealed a membranous nature being formed by vesicular elements of heterogeneous and relatively small size (0.1 to 0.2 Am in diameter). The major difference found up to now for the two fractions is that ATPase protein can be resolved and identified by polyacrylamide electrophoresis of DOC-soluble fraction C under non-dissociating conditions (see-gel 4 in Fig. 2), whereas the protein could not be resolved by electrophoresis of the DOC-soluble fraction from membranes A (data not shown). DISCUSSION The presence of several dissociable groups with pK values of 4.96, 4.18, 3.60, and 3.09 in M.

lysodeikticus membranes (4), as for other membrane complexes, suggests that the binding of a metal ion must be a complex and unspecific process. Although our results are not totally comparable with those of Cutinelli et al. (4), we have shown that Mg2+ binding to M. lysodeikticus involves at least two sites with different affinities. Our results are strikingly similar in this respect to those reported for mitochondria (21) and sarcoplasmic reticulum, although differences in concentrations are also evident (27). Interestingly enough, our results demonstrated a direct dependence between metal ion binding and the states of ATPase activity. A high concentration of external Mg2+

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(binding to sites with low affinity) induces a lysodeikticus ATPase (e.g., its possible associahigh basal membrane-bound ATPase activity, tion with regulatory proteins) requires a tight whereas lower external concentrations of the enzyme-membrane complex with specific concation correlate with low levels of basal activity formation and/or interaction for the compoand very high stimulation by trypsin. It must be* nents. Since the presence of the only Mg2" noted that the number of ATPase units is fairly difference so far detected between theis different constant for most of the membrane preparathis divalent metal ion seems to tions assayed in presence of trypsin. This sug- preparations, such characteristics. Mg2+ likely modiimpart gests that trypsin probably affects only those fies the degree of the hydrophobic interaction ATPase molecules which have not exhibited and/or changes the type of interaction between their hydrolytic capacity. We may therefore the ATPase and the other membrane constituinfer that Mg2" (and/or other divalent cations) lipids and proteins. This might be a acts as an effector of the membrane-ATPase ents, long-range effect, because it is extended to complex regulating the dual role of this mem- soluble fractions. The possibility of a lipid brane protein in the hydrolysis (ATPase) or involvement in this mechanism is being synthesis of ATP (9, 17). The ATPase activity considered. Horvath and Sovak (8)now have reis probably involved in the movement of divalent cently proposed a role for regulatory bound cations. More work on the physiology of this enzymes via membrane coarctation calcium by bacterial ATPase system must be done before or other multivalent metal ions. The effect of the value of this suggestion can be ascertained. cations inducing organization in lipid Nevertheless, the system appears to be related bilayer model membranes haschanges also been reto those of mnitochondria (15), chloroplast (13), ported (2). and sarcoplasmic reticulum (7). On the other hand, the cation-activation On the other hand, we have not deeply response of M. lysodeikticus does not explored the effect of tightly bound Mg2+ (high appear to be strictly modulatedATPase and its by Mg2+ affinity?) on the properties of M. Iysodeikticus association with the membrane. This regulatory membrane ATPase, but the preliminary results property of the ATPase may be dependent on (Table 2) suggest a similar modulation of the short-range effects (modifications in the enATPase by Mg2" at much lower concentrations. zyme molecule or in the binding of some regulaAlthough our results do not explain the la- tors to the enzyme, or both). The of M. tency of M. Iysodeikticus ATPase and its subse- Iysodeikticus ATPase to divalentresponse cations does quent stimulation by trypsin, they suggest an seem in any event to agree with its proposed role allotopic mechanism. The term "aflotopy" was in cation movements. proposed by Racker to account for the altered We have shown that Mg2+ modulates some properties of mitochondrial and chloroplast properties of M. lysodeikticus ATPase in their soluble and membrane-bound bound ATPase. Our results can be membraneinterpreted form (E. Racker, Fed. Proc. 26:1335-1340, as a consequence of a variable distribution of 1967). However, as pointed out by Tzagoloff ATPase on either side of the membrane vesicles (28), the altered properties of these two systems from the different preparations. This asymmetseem to be a function of the self-association of ric distribution of ATPase molecules could alter the enzyme with specific regulatory proteins its accessibility to substrate and/or to divalent (inhibitors?) rather than a dependence on their cations. However, this possibility seems very presence in a membrane complex. A similar unlikely on the basis of the results recently situation has been observed in our laboratory reported Salton and co-workers (25) indicatfor Escherichia coli ATPase (3). Conversely, our ing that by ATPase is accessible to 125I labeling previous (1 ) and current studies on M. when "standard" membranes (i.e., types C and Iysodeikticus ATPase suggest that the associa- D of the present work) are reacted with laction of the enzyme with a certain number of toperoxidase-sodium 125I. In addition, our remembrane proteins or the presence of a mem- sults point out the reversibility (12) and/or the branous form of the ATPase may be necessary response of ATPase properties to a small but not sufficient requisites to account for a rapid of the environmental conditions. perturbation high stimulation of the enzyme by trypsin. Furthermore, membranes from types B, C, and Although we cannot yet rule out the possible D showed by negative staining the same appearimplication of some protein(s) in this property ance data) as that previously reof the ATPase, it is well to remember the slight ported(unpublished for "standard" (18). We stimulation observed with trypsin for most of its therefore propose that themembranes via soluble preparations (1, 24); the current evi- a Mg2+-ATPase-membraneregulation occurs the dicomplex, dence favors the idea that the latency of M. valent cation inducing conformational changes

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13. Lin, D. C.. and P. S. Nobel. 1971. Control of photosynthesis by Mg2+. Arch. Biochem. Biophys. 145:622-632. 14. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 15. Loyter, A.. R. 0. Christiansen, H. Steensland. J. Saltzgaber, and E. Racker. 1969. Energy-linked ion translocation in submitochondrial particles. I. Ca"+ accumulation in submitochondrial particles. J. Biol. Chem. 244:4422-4427. 16. Munoz, E., J. H. Freer, D. J. Ellar, and M. R. J. Salton. 1968. Membrane-associated ATPase activity from Micrococcus lysodeikticus. Biochim. Biophys. Acta 150:531-533. 17. Mufioz, E., M. S. Nachbar, M. T. Schor, and M. R. J. ACKNOWLEDGMENTS Salton. 1968. Adenosine triphosphatase of Micrococcus Our thanks to M. R. J. Salton for critical reading of the Ivsodeikticus: selective release and relationship to manuscript. membrane structure. Biochem. Biophys. Res. ComThis work was supported in part by U.S. Public Health mun. 32:539-546. Service grant AI-08598 from the National Institute of Allergy E., M. R. J. Salton, and D. J. Ellar. 1970. The and Infectious Diseases. M. L. is indebted to the Ministerio 18. Mufioz, resolution and properties of some major components of de Educaci6n y Ciencia for a fellowship. Micrococcus Iysodeikticus cell membranes. p. 51-58. In F. Ponz and J. R. Villanueva (ed.), FEBS symposium, LITERATURE CITED vol. 20. Academic Press Inc., New York. 1. Andreu, J. M., J. A. Albendea, and E. Mufioz. 1973. 19. Mufioz, E., M. R. J. Salton. M. H. Ng, and M. T. Schor. 1969. Membrane adenosine triphosphatase of MicroMembrane adenosine triphosphatase of Micrococcus coccus Ivsodeikticus: purification, properties of the Ivsodeikticus: molecular properties of the purified en"soluble" enzyme and properties of the membranezyme unstimulated by trypsin. Eur. J. Biochem. bound enzyme. Eur. J. Biochem. 7:490-501. 37:505-515. 20. 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either directly on the ATPase molecule or indirectly through its interaction with other membrane components, which then influence the enzyme properties. Further studies on the elucidation of the activator or effector role of divalent cations as well as on the definition of the function of each protein component will be needed for understanding the mechanism of regulation of the M. lysodeikticus ATPase system (1, 24).