A C T A U N I V E R S I T A T I S L O D Z I E N S I S
FOLIA BIOCHIMICA ET BIOPHYSICA 11, 1996
Janusz Błasiak
KINETIC PROPERTIES OF THE (Ca2+ + Mg2*) -ATPASE BOUND TO AND EXTRACTED FROM
THE PIG ERYTHROCYTE MEMBRANE
The activity of the pig erythrocyte membrane (Ca2+ + Mg2+)-ATPase was investigated. Two kinds of enzyme preparations were used: enzyme bound to the fragments of membrane and extracted from them. Both enzyme preparations exhibited biphasic aubstrate curves displaying the existence of two functional active sites with high and low affinity to ATP. Also the relationship between the activity of bound enzyme and Ca2+ concentration was biphasic. The activity reached maximum at about 20 fiM then dropped progressively as the Ca2+ concentration was raised. Obtained data indicate that the dependence of the pig erythrocyte membrane (Ca2+ + Mg2+)-ATPase on both ATP and free Ca2+ concentration as well as kinetic parameters of the enzyme are similar to those obtained for human erythrocytes.
1. INTRODUCTION
(C a2+ + M g2+)-A T P ase from erythrocyte was the first discovered enzyme of the plasm a m em brane acting as a calcium pum p [1]. It is now established that low level o f calcium ions inside hum an erythrocyte [2] is m aintained by an active extrusion of them [3]. The energy necessary for this process is supplied by the hydrolysis o f A TP located on the internal surface o f the m em brane [3].
(C a2+ + M g2+)-A T P ase o f the hum an erythrocyte has been well characterized [4], but there are little inform ation on the properties o f the enzyme o f pig red cell m em brane. In the present w ork the basal (i.e. independent o f calm odulin) activity o f pig erythrocyte m em brane (Ca2 + + M g2+)-A TPase was investigated. Two kinds of m em brane preparations were used: the enzyme bound to fragments o f m em brane and extracted from them by means o f detergent.
2. MATERIALS AND METHODS
The m ethod of A u [5] was employed to obtain pig erythrocyte membranes containing (Ca2+ + M g2+)-A TPase. Briefly, washed erythrocytes were lysed with 8 vol. of 10 m M Tris, 1 m M ED TA (pH 7.4), then washed five times with the same buffer before three m ore washes with 10 mM T ris-H C l (pH 7.4). M em brane suspensions were adjusted to about 2 mg protein/m l and were kept at -75°C before use. W hen an extracted enzyme was used, the m em brane fragments were treated for 10 min at 4°C with 50 m M imidazole, 100 m M N aCl, 5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 0.5% T riton X-100, 1% Tween 20, 0.1% phosphatidylcholine (pH 7.4). A fter the treatm ent, the m ixture was centrifuged at 100,000 g for 60 min to sediment residual m em branes and the supernatant containing the ATPase was concentrated by ultrafiltration.
Protein was estimated by the protein-dye binding m ethod o f B r a d f o r d [6] with bovine serum album in as the standard.
T he activity o f (Ca2+ + M g2+)-A T P ase was determ ined spectrop- hotom etrically on the basis o f am ount of inorganic phosphate (Pt) released during an enzymatic hydrolysis o f ATP [7]. Briefly, m em brane preparations were preincubated with 0.1 m M EG TA for 60 m in at 37°C and washed three times with 20 m M Hepes buffer (pH 7.4). The washed m em brane were incubated for 30 m in at 37°C in a buffer containing 2 m M A T P, 55 m M T ris-H C l (pH 7.2), 66 m M NaCl, 1 mM M gCl2, 0.1 mM oubain, 0.1 m M EG TA plus sufficient am ount of CaCl2 to give a final free C a2+ concentration of 19.1 ¡ M . The concentrations of both A T P and free calcium were changed when required. A t the end o f the incubation the reaction was stopped by the addition of the colour developing reagent. The activity of the enzyme was expressed in /¿mol P( released per ho ur per m g of protein. From the total am ount of P; released during the incubation period the am ount o f P, resulting from C a2+-independent hydrolysis o f A T P was subtracted.
Inorganic phosphorus was determined by the sensitive m alachite green m ethod [8] as described by L a n z e t t a et al. [9].
Free calcium levels in the presence of EG TA and A T P, and M gATP concentrations were calculated using the binding constants given by R o o n e y and L e e [10].
One-way analysis o f variance was used in the statistical analysis. The differences between m eans were com pared by the Scheffe’s m ultiple com parison test [11].
Fig. 1. Pig erythrocyte membrane (Ca2+ + Mg2+)-ATPase activity as a function of ATP concentration. Open symbols - bound enzyme, solid symbols - extracted enzyme. Error bars denote SE, each experimental point is the mean of five replications. Insets - Lineweaver-Burke plots for ATP concentrations in the range 5-20 ¡iM (A) and 0.125-2 mM. Regression lines were calculated by means of the least square method. The R values were equal to 0.986 and
0.991 for bound and extracted enzyme respectively
3. RESULTS AND DISCUSSION
The activity of (C a2+ + M g2+)-A T Pase was measured as the func tion o f A T P concentration in the range 5 f i M - 2 m M . Results (Fig. 1) show th at the response o f the enzyme to A TP is biphasic. Reciprocal p lo ts o f (C a 2+ + M g2+)-A T P ase activity against A T P co n cen tratio n (Fig. 1, insets) in the range 5-20 fiM and 0.125-2 m M yield straight lines for both kinds o f enzyme preparations. The substrate curves in Fig. 1 can therefo re be represented by th e sum o f tw o M ichaelis - like equations V V V = V, + v2 = ---SL--- + — m2 1 I ^M2 1 + IIÜ L 1 + [S] [S]
where [S] is the concentration o f A T P, v, represents the rate o f the com ponent observed at low concentration of ATP which shows high-apparent affinity for A T P (KM1) and low maximum velocity (Vml), and v2 the rate of the com ponent observed at high concentration o f A T P with lower apparent affinity for A TP (KM2) and higher m aximum velocity (Vra2). The kinetic param eters for both kinds o f enzyme preparations are summarized in Tab. 1.
T a b l e 1 Kinetic parameters of (Ca2+ + Mg2+)-ATPase activity measured at two different concentration
ranges of ATP (mean ± SE; n = 5 in each experiment)
Enzyme ATP concentration range (/iM) V» (/anol 1/mg per h) k m (mM) Bound 5-20 0.099 ± 0.019 7.60 ± 1.37 125-2000 0.331 ± 0.043 217.4 ± 27.2 Extracted 5-20 0.079 ± 0.015 8.13 ± 1.46 125-2000 0.286 ± 0.026 194.6 ± 21.4
Biphasic A TP activation curves for the (Ca2+ + M g2+)-A T Pase has also been found in hum an erythrocytes [12-14], m em brane preparations from rat pancreatic islet cells [15], hum an lymphocytes [16] and rabbit sarcoplasm ic reticulum [17].
R i c h a r d s et a l. tested hum an erythrocyte m em branes for C a2+ + M g2+)-A TPase activity within a range o f A TP concentration from 0.5 to 4000 /¿M [18]. They obtained values of Vm equaled 0.0343 and 0.375 /im ol/m g/h for high and low affinity active site, respectively. K M was equal to 2.46 and 143 /iM. These values are of the same order as obtained in this work.
W hen the dependence o f the activity of (Ca2+ + M g2+)-A T Pase on th e co n cen tratio n of free C a2+ ions was tested the c o n c en tra tio n o f A T P was equal to 2 m M , whereas free C a2+ concentration varied from 0.5 /iM to 5 m M .
T he results are displayed in Fig. 2. T he curve relatin g (C a2+ M g2+)-A T Pase activity to free C a2+ is also biphasic, the activity reaches the m aximum centered at around 20 /iM free C a2+, and then drops to alm ost zero as C a2+ concentration rises. Similar dependence was obtained in the research on the hum an red cell m em brane [19-21], In order to explain the results o f the inhibition of the (Ca2+ + M g2+)-A T Pase from plasm a m em brane, it was considered th at the inhibitory species could be either C a2+ or CaA TP complex.
log free Ca2+ concentration [nM]
Fig. 2. The activity of the (Ca2+ + Mg2+)-ATPase bound to (open symbols) and extracted from (solid symbols) pig erythrocyte membrane as a function of free Ca2+ concentration. The ATP concentration was 2 mM in either case. Each point represents the mean of five
replications. Errors bars denote SE
D ata obtained in this work indicate th at the dependence of the activity o f the pig erythrocyte m em brane (C a2+ + M g2+)-A TPase on both A TP and free C a2+ is similar to th at obtained for hum an erythrocytes. Also kinetic param eters of the enzyme from pig red cells are close to the values typical for hum an being.
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Came in editorial office Department of Molecular
„Folia biochimica et biophysica” Genetics, University of Łódź 22.11.1993
Janusz Błasiak
WŁASNOŚCI KINETYCZNE (Ca2+ + MgJ+)-ATPAZY ZWIĄZANEJ Z BŁONAMI ERYTROCYTÓW ŚWINI I EKSTRAHOWANEJ Z NICH
Badano aktywność (Ca2+ ■+■ Mg2+)-ATPazy z błon erytrocytów świni. Używano dwóch rodzajów preparatów: enzymu związanego z fragmentami błon oraz enzymu ekstrahowanego z błon za pomocą detergentu. Obydwa preparaty enzymatyczne charakteryzowały się dwufazową zależnością aktywnośd od stężenia substratu, świadczącą o istnieniu dwóch funkcjonalnych centrów aktywnych: o niskim i wysokim powinowactwie do ATP. Również zależność aktywnośd enzymu od stężenia wolnych jonów Ca2+ miała charakter dwufazowy. Aktywność osiągała maksimum dla stężenia około 20 fiM, a następnie stopniowo malała, gdy stężenie Ca2+ rosło. Otrzymane rezultaty świadczą, że zależność aktywnośd (Ca2+ + Mg2+)-ATPazy błon erytrocytów świni od stężenia ATP i Ca2+, jak również charakteryzujące ją parametry kinetyczne są podobne do otrzymanych dla enzymu z błon erytrocytów ludzkich.
