E u r . J . Biochern. A?. 235-2.55 (1978)

The Binding of Calcium and Magnesium to Sarcoplasmic Reticulum Vesicles as Studied by Manganese Electron Paramagnetic Resonance Hans Robert KALHITZER, Dietniar STEHLIK, and Wilhelm HASSELBACH Max-Planck-lnstitut for Medical Research, Departments of Molecular Physics and Physiology. Heidelberg (Received May 6 . 1977)

The binding pattern of the biologically relevant ions calcium and magnesium has been investigated via the binding of the ion-analogue manganese. The binding parameters of manganese are obtained conveniently by standard electron paramagnetic resonance techniques, the binding of calcium and magnesium is inferred from competition experiments. The quantitative analysis was carried out with a computer program which was able to treat the competition of three kinds of ions for three classes of independent and one class of cooperative binding sites. I t was possible to correlate specific binding classes with biological functions of the sarcoplasmic reticulum membrane. The magnesium and manganese specific binding class of medium affinity is related to the catalytic function of these ions in the ATP-splitting. For the first time a manganese and calcium specific clash of cooperative binding sites has been observed and it can be related to the inhibition of the calcium transport at high concentrations of manganese or calcium. From the binding results a model can be developed which allows a full description of the role of the divalent ions and their specific binding sites during calcium transport.

The action of the contractible actin-myosin filaments in skeletal muscles is known to be controlled by the uptake and release of calcium ions into and out of the sarcoplasmic reticulum. Hence, the structure and function of the sarcoplasmic reticulum has been the subject of intensive research during the past decades, see recent reviews [ l - 31. Isolated fragments of the sarcoplasmic reticulum membrane reconstitute themselves in the form of closed vesicles [4] with a diameter of 60-300 nm centered around 80- 100 nm. They provide an excellent system to study in ~itr-othe structural and functional properties of the ATP-dependent calcium transport. The membrane structure is relatively protein-rich; 6 5 O . b proteins and 35% lipids with only about 304;, of the accessible surface occupied by the lipids. 70"d of the protein content is due t o a calcium and magnesium dependent ATPase with a molecular weight close to 100000; tryptic fragments between 50000 and 60000 have been observed [5,6]. Among the other proteins are the calsequestrin (5-200;), M , 45000-55000) and a high affinity calcium binding protein (about l O'?:,, M , about 50000) [ I -3.7.81. ~~

Ahhreritriion. EPR. electron paramagnetic resonance.

The ATPase enables the sarcoplasmic reticulum to pump two calcium ions in the presence of magnesium at the cost of the splitting of one molecule of ATP. The pump can also run backwards. The important role of the divalent ions in the function of the ATPase asks for detailed information concerning the ionic binding pattern. So far, however, consistent results are only available with respect to the strong binding sites. High affinity, calcium-specific binding sitcs are well established for the ATPase. The association constants range from 0.8 to 5 . 0 lo6 ~ M - ' [1.2,7- 121; the number of binding sites varies between 0.5 and 2.1 per ATPase unit. In addition, weaker calcium binding sites are reported in the literature [l, 2,7 - 1 ?]. The evaluated parameters differ so much that n o conclusions can be drawn yet. Agreement again can be stated with respect to a magnesium binding site with an association constant of less than 3 x 104 M I . There are some reports suggesting that in the presence of ATP the binding constant of magnesium approaches the order of magnitude of that of calcium [9,13]. The intention of the present work is to complete the information on the calcium and magnesium binding pattern with the hope of providing a conclusive

246

correlation with the functional requirements of the sarcoplasmic reticulum membrane. Our binding studies make use of the substrate analogue manganese which is able to replace calcium and magnesium in its biologically relevant sites. It is known that manganese can substitute for magnesium with respect to the ATPase activity [14,15]. Furthermore it has been demonstrated before that mitochondrial membranes can transport manganese as well as calcium [16]. Substrate analogues can be very helpful in resolving different classes of binding sites. Suppose the naturally used ions bind so strongly at high affinity sites that weaker binding sites are completely covered up in binding studies, then an ion analogue with a lower affinity to the strong sites might open up the chance to investigate the other binding classes with lower affinity. Finally, manganese has been chosen for its physical property to be paramagnetic. This permits the application of paramagnetic resonance methods. In this work the reaction equilibrium between sarcoplasmic reticulum vesicles and manganese ions in competion with calcium and magnesium ions has been investigated by determining the free manganese concentration with standard EPR techniques. The alternative method of the manganese-induced spin-relaxation enhancement of the water-protons has also been applied [I71 (Kiihn and Stehlik, unpublished results). I t provides useful additional information on particular binding sites. However, it turns out to be unable to resolve the full binding pattern. This is in part due to the limited differentiation at very low manganese concentrations but seems also related to the fact that the method relies on a fast exchange of the water molecules between the solvent and the hydration spheres of the bound manganese. This paper deals with the following questions. Firstly, how many binding classes d o cxist in sarcoplasmic vesicles for calcium and magnesium ions? What are the binding parameters'? Are the binding sites independent or interacting? Secondly, are the individual binding sites specific with respect to the investigated ions calcium, magnesium or manganese? Thirdly, is it possible to correlate the observed binding pattern with the information available about the reaction mechanism of the calcium transport and the structure of the membrane-bound proteins?

MATERIALS A N D METHODS Biologiccil Prepurritions

The vesicles were prepared from rabbit muscle according to the standard preparation procedure described by Makinose and Hasselbach [18,19]. The

Binding of M n 2 + , Ca2' and M$+ to Sarcoplasmic Reticulum

protein concentration of the vesicle preparation has been determined with the Biuret method. Typical protein concentrations between 36 and 48 mg/ml were available for further treatment. All preparations showed typical ATPase activity and calcium uptake as described in the next section. The concentration of the divalent ions Ca2+ and Mg2+ in the initial preparation could be reduced by dialysis against a buffer solution (0.02 M Tris-HCI pH 7.2) only when it contained an ion exchanger (Dowex 50 at 0.1 M KCI). The remaining concentration of divalent ions has been checked regularly using the atomic absorption method. By extensive dialysis it was possible to reduce the ion content to 0.4-0.9 MgZ+ and 1.3-2.6 Ca2+ per ATPase unit, depending on the individual vesicle preparation. 'Cholate-vesicles' have been prepared by solubilization of native vesicles with 0.3 mg deoxycholate per mg protein followed by 5 - 10-fold dilution by 0.1 M KCI and centrifugation at 50000 rev./min for one hour [20]. The protein composition of the sample has been checked with gel-clectrophoresis which showed a strong reduction of all protein bands except for the one assigned to the ATPase. The cholate-vesicles retain the ATPase activity. The titration of the divalent ions has been carried out by preparing a separate sample for each set of ion concentrations. The ion concentration was varied by adding small amounts of a 0.1 M or 0.01 M ion-buffer solution to the vesicle preparation. The precise ion concentration has been determined gravimetrically with an accuracy of better than ? 1.So/:,. Samples have been prepared for EPRmeasurements by filling calibrated 100-p1capillary tubes. In order to avoid degradation at the high protein concentration used, samples have been stored at 3 'C immediately after preparation and only shortly warmed up for measurements. With this precaution much better reproducibility and accuracy has been achieved during the rather lengthy measurements of a full concentration dependence. E . y p c r i m m t i l Procw1urc.s

The actual experiment determines the concentration of free manganese via the amplitude of the EPR signal which is calibrated against data from aequeous solutions of chosen manganese concentration with identical sample geometries. The method relies on the following assumptions. The EPR signal of the free manganese shows identical line shapes both in the biological sample and in the calibration standard. The signal of the bound manganese can be neglected in the region of the well known six-line spectrum of free manganese. The former condition can be easily fulfilled by choosing the suitable manganese solution in the

247

H. R. Kalbitzer, D. Stehlik, and W. Hasselbach -

calibrating sample. The second assumption needs specific confirmation. Generally it is known that the EPR spectrum of manganese bound in metal-enzyme complexes is not contributing in the region of the free MnZ ' spectrum due to broadening. However, spectra of bound manganese have been reported recently [21] which exhibit strong similarities with the free M n Z + spectrum. In order to exclude possible errors from a bound manganese contribution to the EPR signal the following test experiment has been performed. A vesicle preparation has been dialyzed against the same buffer solution containing a preset manganese concentration. Thus the concentration of free manganese in the solution is determined by that one in the external solvent. Any contribution of bound manganese to the EPR signal in the free M n 2 + region would show up as increased signal amplitude for the vesiclcs containing sample as compared to the reference sample taken from the external solvent. This test has been carried out for a concentration corresponding to 0.5 and 3 bound manganese per ATPase unit. The increase of the EPR signal in the vesicle sample. averaged over the six lines, was found to be S";, and 4",,, respectively. This deviation is considered to be insignificant since temperature differences between the EPR experiment and the dialysis could not be completely avoided. All EPR measurements were performed with a Varian E-line spectrometer. A time-averaging computer has been added to improve the signal to noise ratio and to subtract the broad background signal of the sample-free cavity. The sample temperature has been stabilized to 33.5 f 0 . 5 '-'Cwith a gas flow system. Furthermore. cart has been taken to assure a well centered and reproducible sample position. For weak signals all six lines of the free manganese spectrum have been used to evaluate an average signal amp1it ude.

RESULTS jC1mg~iiw.wBinding t o Nut ire

Surcqh.miic

Vrsicles

The EPR data render the concentration of free manganese as a function of the total manganese concentration in a solution with a fixed concentration of sarcoplasmic vesicles. Unfortunately, the results differ considerably from one vesicle preparation to the next. However, the dilyerences are only quantitative, not qualitative. Hence, a complete set of experiments has been performed on a single preparation, whenever possible. Following the standard preparation procedure. samples have been prepared for measurements in three ways: (a) no further treatment to remove remaining divalent ions, (b) extensive dialysis against

E 30000

7

A

1

[-

I L

5

7 Mn2'

10

15

bound per ATPase unit

Fig. 1. Scuic/icrrc/ plot of iitr nrcrrigutrese hmthn!: 10 surcoplr.si?ric wsicles. X;.Y, the concentration of hound manganese per ATPase unit (.ildivided h y the concentration ol' tree mangaiie\e ( v ) 15 plotted a s R function of .i.. (000) Standarci prepaixtion wit11 nnrnia~ rest concentration ofdivalent ions. type ii. (0.0) Different standard preparation. dialyzed 24 h agciinst ion-free butfsr (0.02 M Ti-isHCI. pH 7.1. 0 1 M KCI). type h

the buffer Tris-HCI, (c) extensive dialyzis against this buffer containing an ion complexer (Dowex 50). The data are presented using the Scatchard plot [22,23]. Note, however, that the plot will not be used for the quantitative analysis. I t is merely useful to discuss thc results qualitatively. The abscissa of this plot is given by the concentration of bound manganese relative to that of the ATPase unit to which 70",, of the protein weight and a molecular weight of 100000 are assigned. The quantity x/x is plotted as a function of X where .v represents the measured molar concentration of free manganese. Fig. 1 presents the Scatchard plot for preparations of type a and b. respectively. Fig.? gives the results for two different preparations of type c. Fig.? demonstrates that the essential features remain unchanged from one preparation to the other. However, there are considerable quantitativc difierences. the origin of which is not understood. Nevertheless we can state that the results are reproducible in the general sense. Comparing Fig. 1 and 2 a stronginfluenceofremaining divalent ions is noted. Fig. 1 can be described a5 reproducing the results of Fig. 2 except for the region of low X values where the strong manganese-binding sites show up. As we will see later small residual concentration? of Mg" and Ciiz+ are sufficient to block the strong binding sites for manganese. Note that the

x

248

Binding of Mn2+.Ca2+ and MgZc to Sarcoplasmic Reticulum

- 30000

-r

7

.

r.

5 a,

. 2

c

c

20000 &

h

*< L

a G)

n L

0 3

p

10000

OE

5.

r.

V, concentration of competing ion per ATPase unit

12

0

A

5

? ,

10

15

Mn2' bound per ATPase unit

Fig. 3. Replrcc~rne~n/ 14 mungunrse hound fo strrcopkisrtiic~reticu/rtm resides by cnlcirtm ( 0 ) nnd mnngnesium (0). rcy~ecrirely.The starting point ( * ) for the titration with the competing ions is specified by the following concentrations: [Mgf'] = 0.18 m M ; [Ca;'] = 0.79 m M ; [Mn: '1 = 0.24 m M : [protein] = 38.6 mg,ml tcmperiiture = 23.5 C

Fig. 2. Scufchurd plor of fhc mtinguncw binding 10 wrc~oplusmic rc~.riclc~.r (is Fig. I bur u i r h clif&wrr prt~pcrrtifionsof 1ypc c. hor/z diulyzeti / o r 12 h tr,?nirisr DOHKY 50 in the hiifier so/urion. The following rest-concentration have been determined at 23.5 ' C with the atomic absorption method: (000)[ M d + ] = 0 . 2 7 mM. [Caf']=0.59 mM, [protcin] = 46 m g , ml. (0.0) [Mgf ' ] = 0.17 niM. [Caf' 1 = 0.35 m M , [protein] = 48 mg ml

Replacement o f Mmngunese by Magnesium m d Calcium Now, the concentration of free and bound manganese is measured as a function of the M$ and Ca2 concentration at fixed concentrations of total manganese and sarcoplasmic reticulum protein. The most significant effect of the replacement experiments is shown in Fig.3. The concentration of bound manganese is plotted as a function of the total concentration Y of the competing ion, both per ATPase unit. Magnesium replaces manganese in its binding sites in the usual manner as indicated by the decrease of the concentration of bound manganese with increasing concentration of magnesium. In contrast, the concentration of bound manganese increases initially with the addition of calcium. Only at higher concentrations of calcium the manganese binding levels off in the normal behavior. This surprising result finds a plausible explanation when we assume that only calcium is able to replace manganese in the cooperative binding sites while magnesium has only a negligible affinity to these sites. In order to present the data in a concise way the original data as a function of the concentration of the competing ion (as in Fig. 3) for sets of fixed concentrations of manganese and protein are extrapolated to obtain Scatchard-plots with the concentrations of bound and free manganese being the running parameters while the total concentration Y of the competing ion per ATPase unit is extrapolated to a set of fixed parameters. When the experimental data are rearranged this way, neglecting the differences found for different +

remaining divalent ions are bound rather tightly, they cannot be removed by dialysis against the ion-free buffer solution. The results of Fig.l and 2 show regions with a positive slope. This can only be accounted for by cooperative binding [24]. Therefore, a first inspection of the results of Fig.? yields at least three classes of binding sites. A class of strong binding sites as indicated by the steep negative slope at very low B values, a class of cooperative binding sites as manifested by the bell-shaped superposition on the whole curve in the central region of the Scatchard plot. and a class of weak binding sites responsible for the tail in the Scatchard plot at high 1 values. The contributions of the individual binding classes are rather well separated in the Scatchard-plot indicating good chances for a quantitative cvaluation of the relevant binding parameters. However, the comparison of Fig. I and 2 demonstrates the important role of remaining divalent ions, i . ~Mg2 . * and Ca2 . Hence, the quantitative evaluation of the manganese parameters is meaningful only when the influence of the unremoved Mg2+ and Ca2* can be taken into account. Therefore we describe first the series of experiments in which manganese is replaced in its binding sites by magnesium and calcium, respectively.

+

x

249

H R Kalbitzer. D Stehlik, and W Hasselbach

I

I

Al

il

E

--

30000

l

1

0

, J

& Q n

&

10000

E

LAPLA

OO

5

10

x , V n 2 ' bcmd per A-Pase

Fig. 4. S~crrchtrr.rl-plot.so/ thr

15

LAP,

OO

5

10

15

j , M n 2 ' boma per ATPase dn t

unl!

/or u .scl of .fi.rcc/ mrrgnc~siictir('iJticeiilrcrtrons per A I ' f ' u ~ t , uiiil. ( 1 , s obltriric,i/ h j , c~strupolutioti from rcplucrmenr studies ofmun,qcmese lit. mugnesiuiii (like Fig. 3 ) . For further description see the main text. (A) Experimental results. solid line represents the data of Fig.2 as a reference with no magnesium added to the initial preparation. (H)Theoretical curves using the binding model discussed in the text

I

tiiutigutiese hrtit/rng

A

i

0

0

n

&

c

IOOcin-

o i A . -I 0

S

p,Mn"

10

15

bound per ATPase unit

Fig. 5 . Set 01 Scnl(~/iurrlplorrtrmlogoic.c 10 Fig, 4 hut f o r the r.c~pluc.rtiic~nt of mtrnXuncse h j , ccrlc i u i ~ (. A ) bxperimental results including the same reference line as in Fig.4A. (B) Theoretical curves using the binding model discussed in the text

vesicle preparations one obtains a set of Scatchardplots as shown in Fig.4A for the replacement of manganese by magnesium and in Fig. 5A by calcium. The solid line in both figures represents the data of Fig. 2 as a reference with no competing ions added to the starting preparation. Instead of the data points a

numerical fit has been used which will be discussed later on. The different binding pattern of magnesium and calcium as mentioned in connection with Fig. 3 is even more apparent in Fig. 4 and 5. Starting again with a qualitative evaluation the comparison of Fig.4A and

250

SA results in the following conclusions. Firstly, for small X values. i.e. in the region of strong binding, replacement of manganese is indicated by both competing ions, magnesium and calcium, respectively. Secondly, calcium clearly competes about the cooperative binding sites. In contrast, competition of magnesium is nearly negligible as indicated by the unan‘ected bell-shaped contribution to the Scatchardplot as the magnesium concentration is increased. Therefore. the class of cooperative binding sites is concluded to be calcium specific. Thirdly, for large 2-values, i.r. in the region of weak binding, manganese is replaced by magnesium and calcium comparatively.

Qucintittitire Antilj~siso f ’ th” Bintliiig Resulrs

Due to the remaining concentrations of magnesium and calcium in the starting preparation we only obtained data concerning the binding of manganese to sarcoplasmic reticulum vesicles in the presence of competing ions. Therefore, it became necessary to analyse all data simultaneously. Obviously, the binding pattern is too complicated to be analyzed on the basis of the presented data alone. Fortunately, it is possible to start with a set of model assumptions for the binding pattern because independent data are available as well as functional requirements described in the introduction. Two classes of independent strong binding sites can be considered as well established. Firstly, a calcium-specific class with about two binding sites per ATPase unit. responsible for the calcium binding prior to the uptake. Secondly, a magnesium-specific class with about one binding site per ATPase unit. associated with the catalysis of the ATP splitting. I n addition, the qualitative evaluation ofthe present binding studies requires the consideration of a class of moderately strong, cooperative. calcium-specific binding sites. probably associated with the calcium release and a class of unspecilic weak binding sites. Given this model for the binding pattern a quantitative description is needed for the following problem: three species of ions compete for four classes of binding sites. one of them with cooperative binding sites. The mathematical formalism as well as the fitting procedures based on the work of Wyman [?8,29] are described elsewhere [30]. The important points concerning the numerical strategy can be summarized as follows. A total of about 350 data points has been fitted simultaneously. The binding parameters o f the strong binding sites are relatively well known for calcium and magnesium from independent measurements. This compensates to a great deal for the limited accuracy of the present data in the region of strong binding as well as for the inaccuracy in the determination of the residual concentration of magnesium and calcium in

Binding of Mn”. Ca” and M e z t to Sarcoplasmic Reticulum

the starting preparation. The number of ion binding sites per ATPase unit has been varied only in integer values. The latter assumption was considered to be the least critical possibility to further reduce the number of open parameters. Concerning the strong independent binding sites the data d o not permit a better resolution anyway and reliable independent values are available. For the large number of weak binding sites the assumption is also irrelevant. Hence, it concerns mostly the results with respect to the cooperative sites. When the number of cooperative binding sites was left as an open parameter as well, the values for the binding constants and the Ilill coefficient d o not change by more than 15”,, with no improvement of the least square error. With respect to the data accuracy the open parameters have to be arranged in the following sequence of importance. Firstly, the binding parameters of the cooperative sites, i.e. the Hill coefficient, the three association constants for each of the three ions and the number of binding sites. Secondly, the association constants of manganese for the strong binding sites while those for magnesium and calcium agree well with reported data. Thirdly, the binding parameters for the weak binding sites which have a considerable influence on the numerical results although they are much less important. Finally, the number of binding sites in each class which initially is taken to be the same for all three ion species. Although the numerical analysis has been carried out by minimization of the root mean square error to all data points it is desirable to have graphical comparison of experimental and numerical results. This has been attempted in Fig.4 and 5 with the Scatchard plots described before. Most satisfying seems the fact that the theoretical curves in Fig.4B reproduce well the replacement of manganese by magnesium at the strong binding sites while the bell-shaped contribution to the Scatchard plot remains unafTected by the addition of magnesium. The calcium-specific replacement of manganese in the cooperative sites shows up in Fig.5B by the characteristic shift of the maximum into the initial slope with increasing calcium concentration. The binding parameters obtained from the fitting procedure are summarized in Table 1. They are classified according to the four classes of binding sites, three of them independent, termed I , , I,, and I,, and one cooperative, denoted C, . The specific characteristics of each binding class for a particular ion can be seen from the relative association constants. Clearly, class I, has to be called caicium-specific and defines the high affinity calcium sites ofthe sarcoplasmicvesicles. Class I, ismagnesiumspecific in relation to calcium. Note. however. that manganese binds about four times stronger than

251

H. R. Kalbitzer, D. Stehlik. and W. Hasselbach Table I . The binding of diiialent ions to native sarcoplasmic reticulum iwsicles Binding class

Number of binding sites per ATPase unit __

Association constant

Hill coefficient

~~~~~~~~~

Mg”

caZ

+

Mn2

+

~

M ’ ~

~~

Independent binding sites Cooperative binding sites

1,

2

13

1 23

c,

2

12

1 8x106 10x102 13x10’

23x10’ 60x10’ 85x10’

23Xl(y‘

54x10’

1 1 1

5 7 ~ 1 0 ~

58x102

10x104

4

magnesium. Class I, is associated with the unspecific weak binding of ions in agreement with comparable association constants for all three ions. Class C, is calcium-specific with respect to magnesium. However, manganese binds with about twice the association constants of calcium. In connection with the moderate association constants of manganese with respect to the strong bindingsites, the highest bindingconstant ofmanganese at the cooperative sites is very crucial for their detection which could not be performed by a measurement of the calcium binding alone. The strong calcium binding for the class I, completely covers up the cooperative binding in class C, which in turn dominates the Scatchard plot for manganese due to the favorable set of association constants.

Mungrirzese Binding to ‘Cholute’ Vesicles

The characteristic feature of this modification (see Materials and Methods) is a protein composition which consists almost exclusively of the ATPase unit with molecular weight of 100000while thecomposition of lipids is retained. The ATPase activity remains nearly unchanged but the ability to store calcium is lost. The manganese binding as shown in Fig.6 in the form of the Scatchard plot is strikingly different from the results for native vesicles (see Fig.2). Most important, no indication of cooperative binding is observed anymore. In addition, as expected. the number of weak binding sites is reduced. Assuming the same binding classes and parameters for calcium and magnesium as in the case of native vesicles (see Table 1 ) the fitting procedure renders the full curve in Fig.6 with the parameters collected in Table 2. Most significantly. the Hill coefficient is reduced to one. No cooperativity is left. Furthermore, the number of weak binding sites is reduced by about one third.

13x102

5

I0

15

y,Mn2+ bound per ATPase unit

Fig. 6. Scci/ckarcl-plo/ o/ /he ni~rii,~iinesc~ hinclitig l o ‘i~holir/r‘i w i i k . in Fix. I . The rest concentrations of divalent ions werc: [Me:+ = 0.22 m M ; [Ca:’]=0.29 m M ; [protein]=47.6 mgiml; temperature 23.5 C. Solid line represents a computer fit using the parameters collected in Table 2. Binding parameters of Mg’ ’ and Ca” are assumed to be the same as for native vesicles (Table I )

rrs

Binding class

Independent binding sites.

Number of Association Hill binding sites constant for coefficient per unit MnZ+

I, Iz

I,

Cooperative binding sites

c,

M-’ 1.3 . 10’

15

2 . 9 . 10, 5.1 . lo2

I I I

2

1.0. 10,

I

2 1

It should be noted that the data points of Fig.6 are satisfactorily fitted already with the assumption of two classes of indzpendent binding sites. Biological Tests The large number of open parameters used in the fitting procedure naturally raises serious doubts about

Binding of M n 2 + ,CaZt and M$+ to Sarcoplasmic Reticulum

0.5 rnM

I.OrnM

2.0

I

c

H

ea

1.5

F .

3 a,

m

1.0

1 I

a 3 N

s

0.1

0.5

1

[ Mn2+] (mM) Fig. 7. A TPuse artwiry (is 11 /iinrtion oj tlic total mtirigune.se concentration f o r sonirorrd w s i r l e s ciiti1v:ed against buffer .solution with D o n s s 5 0 f o r I2 h. [Protein]=O.l mgjml. [Ca2+]=0.1 mM. [ATP] = 5 mM. buffer: 0.02 M Tris-HCI. pH 7.1, 0.1 M KCI. Room temperature

the relevance of the parameter set which is obtained. It is therefore important to emphasize that in the present case the binding data can be checked by biological tests. The results of Table 1 predict quantitatively the effect of manganese on certain biological functions related to the calcium and magnesium concentrations. Most important, the corresponding experiments establish the correlation of the individual binding classes and the biological functions. A TPaw Activitj,

ATPase activity has been measured as described in [19]. The ATP-splitting is measured as a function of the magnesium concentration in the presence of a fixed calcium concentration and an excess of ATP. For a protein concentration of 0.05 mg/ml the maximum ATP-splitting is reached at magnesium concentrations of 2-4 mM with 0.1 mM calcium and 5 mM ATP [I]. If we relate the Mg'+-dependent ATPase activity to the I, binding class, Table 1 predicts a four-times stronger association for manganese. Hence, an activation of the ATPase by Mn2+can be expected for lower concentrations as compared to Mg2+. Indeed, the result in Fig.7 shows a maximum ATP-splitting at Mn2+concentrations of about 0.3 mM. A quantitative comparison is not possible because the relevant association constants could be those of the ion-ATP complexes. Anyway it can be concluded that manganese can substitute magnesium as a cofactor with respect to the ATPase activity, moreover, manganese stimulates ATPase activity more effectively than magnesium.

0

0

2

4

6

8

10

Time (min)

Fig. 8. C'ulriimi i(p/uhe us u juwtion of time after rotitart hetu,een surcopltrsmic vesicles tmd A TP for rarifnts manganese concentrations. Test conditions are: [protein]=0.05 mg/ml; [CaZ']=O.l mM. [ATP]=5 mM. [ M g Z C ] = 0 . 5mM. [oxalate]=S mM, buffer: Histidine 0.02 M. pH 7.0. KCI 0.04 M. room temperature. [Mn"] was (0) none; (A) 0.S m M ; ( A ) 1.0 m M ; (0) 2.0 m M ; (W) 3.0 m M ; ( 0 )5.0 mM

Calcium Uptake The most significant test concerns the manganese influence on the calcium uptake by sarcoplasmic vesicles. In the typical experiment (see Fig. 8) the calcium uptake is measured by filtration through Sartorius filters (0.45 pm) as a function of time after ATP and vesicles have been added together [15]. The reference in Fig. 8 is chosen to be the uptake behavior in the presence of a moderate magnesium concentration with no Mn2+.When manganese is added at low concentration, the calcium uptake is stimulated first, i.e. manganese is speeding up the calcium transport just as it is found with an increased calcium concentration. Further increase of the maganese concentration slows down the uptake until the calcium transport is stopped when the manganese concentration exceeds 5 mM. This value for the total inhibition of the calciumuptake together with the binding data of Table l 1 corresponds to a free manganese concentration of 0.1 2 mM and an occupation of 70% of the cooperative sites. Without manganese the experiment renders a

'

In addition the following association constants for the ion binding to the substrates ATP and oxalate have been considered: KM,-,,,,,=8.8 x 10" M - ' (311 K,, A l p = 3 . 2 x I d M - ' (311 K,,,-,,,=7.1 x l @ M - ' (321 K,,,, = 4 . 0 ~10'M-I [32] Kc-., = 1 . 0 lo3 ~ M-' [32] K," ox =7.9 x 103 M - ' [ X I

H. R. Kalbitzer. D. Stehlik. and W. Hasselbach

D

1 mM Ma’’

no M r 2 *

/

I

I

_ _ - - - O

1 mM Mg2+ 3 m M Mn2*

/---

-.-A

1 0 m M Mg2* 3 mM Mn2*

Fig. 9. Scrnre 17s Fig. 8 birr f o r rlirec JL’I.T of tricignrsiuin rind iricrngutiese [Protein) - 0.2 mg!ml, [Ca” ] = O . I mM. [ATPI = 1 IrtM. [osalatr] = I m M . buRrr: histidine 0.02 M. pH 7 0. KCI 0.04 M, room temperature coticenrrmions.

total inhibition of the calcium uptake when the concentration of free calcium is about 1 mM [l]. The two numbers agree satisfactorily with the relation of the association constants of manganese and calcium with respect to binding class C, . Fig.9 demonstrates that the inhibition of the calcium uptake by manganese cannot be lifted by an excess magnesium concentration. Hence, the alternative explanation of a reduced uptake by a slowed ATPase activity and/or lack of substrate is ruled out. From the results the most likely conclusion is that the calcium transport ceases when the cooperative binding sites in class C, are occupied by either calcium or manganese. With that result it is tempting to check whether manganese can be transported through the sarcoplasmic reticulum membrane. A control experiment with the radioactive isotope 54Mn2+ failed to show a measurable manganese uptake. However, this is not surprising. Table 1 shows that manganese binds weaker to the strong calcium binding sites as compared to the association at the cooperative sites: thus, the latter will be occupied first before a transport can occur.

DISCUSSION The results demonstrate the existence of at least four classes of binding sites for divalent ions in the sarcoplasmic reticulum. They differ distinctly with respect to their affinity as well as specificity for each of the ions manganese, magnesium and calcium (see

253

Table 1). Most important, clear evidence is found for a correlation of the various binding classes with specific biological activities of the system. The question arises now whether these results allow further conclusions concerning the identification of functional entities with structural and molecular units known in the sarcoplasmic reticulum membrane. First, however, we would like t o comment on the accuracy of the present binding studies affecting the relevance of the subsequent conclusions. The essential new feature in the whole binding pattern concerns the class of cooperative binding sites which have not been detected before. It should be stressed that due to the favorable binding parameters the method used reaches the highest accuracy in the region where manganese starts to bind to the cooperative sites for the given protein concentration. In addition, the binding parameters of both the strong and weak binding sites are different enough such that the effects of the cooperative binding can be seen well resolved. Of course, the evaluated parameters are only accurate within the chosen Hill approximation [28,29,30]. Relative to the most important cooperative binding the characteristics of the strong binding sites cannot be evaluated with great accuracy from the present data. This is also true for the weak and unspecific binding sites where it is not so important anyway. However, a wealth of independent data is available concerning the strong binding sites as mentioned in the introduction [1,2,7 - 121. The parameters of Table 1 are seen to fall right into the range of numbers available for the high affinity association constant and the number of binding sites for calcium and magnesium. The comparison of the binding parameters is limited anyway since the experimental conditions vary with respect to temperature, pH, ionic environment and the protein composition of the sample. Hence, the agreement between the ‘apparent’ binding constants is more than satisfying. The number of weak binding sites is relatively low ascomparedtosimilarstudieson a Na’, K-dependent ATPase [33]. This is attributed to the high concentration of monovalent ions in the present experiments which assures that nearly all charged groups are neutralized by the monovalent ions and hence they are not available for unspecific binding of the divalent ions. Now we turn to the assignment of the various binding sites to molecular units of the sarcoplasmic reticulum membrane. Based on solid evidence from other experiments [ I , 10,13,25-271 it is safe to assign the two calcium-specific high affinity sites to the main protein, the M$ ‘ , Ca2+-dependent ATPase. Furthermore it is obvious that they constitute the calcium binding sites necessary for the start of the uptake reaction steps.

‘54

Similarly, the magnesium binding site of class I, should be assigned to the ATPase since its characteristics are retained in the ‘cholate’ vesicles. It is suggested to associate this binding site with the magnesium-dependent ATP-splitting of the system. The difficult task is the assignment of the cooperative calcium-specific sites. Three facts should be recalled in this connection. Firstly, the cooperativity observed in native sarcoplasmic vesicles is lost in ‘cholate’ vesicles where the low molecular weight proteins have been removed. Hence it is sensitive to either the protein composition or conformation. Secondly, the Hill coefficient of 4.0 when normalized to the ATPase concentration is about twice the number of cooperative binding sites per ATPase unit of molecular weight (1 00000). For independent macromolecules it has been shown [29] that the Hill coefficient should be equal or smaller than the number of cooperative binding sites of the macromolecular unit. Hence a single ATPase unit cannot provide the binding sites involved in the cooperativity. Thirdly, the affinity of the cooperative sites decreases in the sequence manganese, calcium and magnesium. Several alternative models can account for these observations. The cooperative sites can be assigned to the calsequestrin and the ‘high affinity calcium-binding protein’ which constitute possibly the same entity [7]. Since the low molecular weight proteins contribute less than 301;; to the total membrane protein, the number of cooperative sites per protein unit would be indeed larger than the observed Hill coefficient. The sequence of affinity constants would also agree with that obtained for isolated calsequestrin [7]. The cooperative binding sites could be also assigned to diflerent molecular units interacting with each other. The loss of cooperativity would correspond to the loss of this interaction in the modified membrane structure. Finally, a model involving just the ATPase molecules seems to be most attractive. In order to allow for the Hill coefficient of 4, a polymer of several ATPase 100000 M , units has to be postulated with the minimal size of a dimer. The loss of cooperativity in the ‘cholate’ vesicles corresponds to a break-up of the polymers. In this model the number of high affinity calcium sites would be the same as the number of cooperative sites which offers their interpretation as external calcium binding and internal release sites, respectively. In this context it is interesting to note that ATPase was found to split in subunits with molecular weights from 50000 to 60000 after mild treatment with trypsin [5,6]. Thus the possibility exists that the cooperativity is associated with the interaction of four, not necessarily equal, subunits, two of them constituting the well known ATPase unit, 100000 in molecular weight.

Binding of Mn”, Ca2+ and M g Z +to Sarcoplasmic Reticulum

In analogy to the hemoglobin molecule one would expect an interaction of the a2P2type [34]. Table 2 states that the binding sites of medium affinity are still found in ‘cholate’ vesicles. However, this is not conclusive to distinguish between the various models. The measurements are not sensitive with respect to this point. I t is tempting to identify the cooperative binding sites as the calcium release sites during transport. The strongest support comes from the observation that the calcium transport is inhibited by high calcium and manganese concentration and just when the cooperative sites are being occupied. Furthermore, measurements of the water proton relaxation enhancement [I 71 indicate a considerably reduced water exchange rate out of the hydration sphere of the cooperative sites. This is consistent with the concept of the cooperative sites being buried in the membrane as one might assume for the calcium release sites. In this context it might be relevant to consider two other observations. A Hill coefficient larger than two has been reported for the calcium transport [20,35]. Martonosi concludes the lateral association of ATPase molecules to form a channel for the calcium transport

WI.

Ikemoto [36] investigated the decay of the ATPase phosphate complex with purified ATPase at 72 C. The release of free phosphate was found to be inhibited when two calcium sites with an association constant of about 1 x lo3 M - ’ were occupied.

Conclusions The binding pattern observed in this paper is consistent with the following functional model of the sarcoplasmic reticulum membrane. Two high affinity calcium sites (class I, of Table 1) per ATPase unit are located at the outside of the membrane. They are responsible for the initial calcium binding prior to the uptake. The magnesium site (class I, of Table 1) corresponds to the catalytic function of this ion in the ATPase-splitting. The cooperative calcium sites (class C, of Table 1) are located in the interior of the membrane and constitue the calcium release sites during transport. The cooperativity explains the allosteric inhibition of the calcium transport at high calcium concentrations. Although many supporting facts can be given for this model it should be pointed out that the hard facts of this paper concern only the binding pattern of divalent ions and its relation to the important biological functions. The binding pattern could be completed by a class of cooperative calcium-specific binding sites which allows us to formulate a comprehensive association of the relevant biological functions involving divalent ions and specific binding sites.

H R Kalbitzer, D. Stehlik. and W Hasselbach

255

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H R K,ilbitzer Abteilung Molehul,iie Physih M,~\-Plnn~h-liiatitiit l u r Mediiinis~he1 orb~hiing, JdhnstraBe 29, D-6900 Heidelberg, Federal Republic of Ciernidny

D Stehlik, lnstitut fur Atom- und Festkorperphysik, Fd~hbereichPhysik der Freien Unicersitdt Berlin Konigin-Lur~s-Strale34A. D-1000 Berlin (West) 33-Dahlem W Hasselh IL ti Abteilung Physiologie. Max-Planck-Institut tur Medizinische Forschung. JahnatrdBc I Y . D-6900 Heidelberg. Federal Republic of Germany

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The binding of calcium and magnesium to sarcoplasmic reticulum vesicles as studied by manganese electron paramagnetic resonance.

E u r . J . Biochern. A?. 235-2.55 (1978) The Binding of Calcium and Magnesium to Sarcoplasmic Reticulum Vesicles as Studied by Manganese Electron Pa...
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