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A 96-well plate assay for the study of calmodulin-activated Ca2+-pumping ATPase from red-cell membranes
Biochemical Education ELSEVIER
Biochemical Education 26 (1998) 176-181
A 96-well plate assay for the study of calmodulin-activated Ca2’-pumping ATPase from red-cell membranes Arthur
D. Conigrave”,
Michael
B. Morrish
Departments of “Biochemistry and hPharmacy, University of Sydney, Sydney, NS W 2006, Australia
Abstract A simple multi-well assay is described which can be used to assess the activity of the plasma membrane Ca2+ pump in human erythrocytes. The effect on activity of Ca’+, calmodulin, and the two reagents in combination can be quantified through the detection of inorganic phosphate released from ATP. The malachite green assay and a microplate reader are used to measure the concentration of released inorganic phosphate. The experiment can be completed by tertiary level biochemistry students in under 90 min and can stimulate a range of tutorial discussion topics including Ca’+ homeostasis in cells, enzyme structure and kinetics, the role of allosteric effecters and their mechanism of action, protein purification, and data analysis. 0 1998 IUBMB. Published by Elsevier Science Ltd. All rights reserved
1. Introduction Calmodulin is present in the cytoplasm of all mammalian cells. It is a key biochemical component by virtue of its ability to bind Ca2+ in the submicromolar to micromolar concentration range, and provides a link in certain signal transduction pathways between an elevation in intracellular Ca2+ concentration produced by receptor activation and the activation of key intracellular enzymes. These enzymes include myosin light chain kinase, cyclic AMP phosphodiesterase, nitric oxide synthase, and the plasma-membrane Ca’+-transporting ATPase. Calmodulin (Mr 17 kDa) is a dumb-bell shaped molecule with a central flexible bar that connects two highly folded domains at the N- and C-termini. In total, the molecule has four Ca2+-binding loops (EF hands); two Ca2+ binding loops in the N-terminal domain and two in the C-terminal domain. Ca”-ATPase (M, 125 kDa) is an integral membrane protein that operates as a Ca2+ pump. It hydrolyses MgATP to MgADP and inorganic phosphate (P,) in the is tightly presence of Ca2+. In vivo, this hydrolysis coupled to Ca2+ transport from the inside to the outside of the cell. Pump activity, and hence ATP hydrolysis, is increased in the presence of an elevated intracellular Ca2+ concentration. Elevated intracellular Ca2 + concentration also results in an increased concentration of Ca’+-calmodulin complex in the cell. The complex binds to the Ca2+-ATPase, further stimulating pump activity. Therefore, Ca2+ has two roles in this process: (i) as the
species that is transported out of the cell, and (ii) as a direct and indirect modulator of pump activity. Calcium exists in both bound and free forms in the cytoplasm. The bulk of cytoplasmic Ca2+ (0.1 mM) is bound to macromolecules such as proteins and other organic anions such as citrate. Less than 0.1% ( -0.1 PM) is free in the resting cell. However, after receptor activation the total Ca2+ concentration rises, and the free Ca2+ concentration can reach 1.0 ,uM or more. This is sufficient to greatly increase the concentration of the Ca2’-calmodulin complex and activate the Ca’+-ATPase. The Ca2+-ATPase may be assayed using the ammonium molybdate-malachite green method [l]. Hydrolysis of ATP by the enzyme releases Pi which forms with ammonium molybdate, and the complexes malachite green acts as an indicator for this reaction. The absorbance maximum occurs at _ 650 nm.
2. Preparation 2.1. Preparation of erythrocyte membranes Cell membranes are prepared from human erythrocytes that have been screened and found to be negative for HIV, hepatitis B and hepatitis C. All procedures are performed by the Preparation Room staff. We routinely find that the yield of membranes obtained from 200 ml of packed red blood cells (equivalent to one unit of whole
0307-4412/98/$19.00 + 0.00 0 1998 IUBMB. Published by Elsevier Science Ltd. All rights reserved. PII: SO307-4412(98)00029-h
A. D. Conigrave, M. B. Morris/Biochemical
blood) is sufficient to run experiments in about two hundred 96-well plates. For good yields, the procedure outlined below should be followed closely. In particular, all rotors and buffers should be kept at 4°C and centrifuge tubes should be immersed in ice before and after centrifuge runs.
(1) 200 ml ice-cold packed red blood cells (or one unit
(2)
(3)
(4)
(5)
(6)
(7)
(8)
of whole blood) should be distributed evenly between eight 50 ml SS34 screw-top rotor tubes. Fill tubes with ice-cold Wash Buffer A (150 mM NaCl, 10 mM Tris, pH 7.5 at 25°C). Wash twice by centrifugation (1 min at 10000 rpm with the brake set ‘on’) and carefully remove the floating huffy coat from the surface and side of each tube by aspiration each time. The buffy coat contains white cells that are a source of proteases. Shake the tubes vigorously before the second centrifugation to remove adherent red and white cells. Resuspend cells by addition of ice-cold Lysis Buffer (5 mM Tris, pH 8 at 25°C 1 mM EDTA) and shake vigorously. Transfer the contents to six GSA centrifuge tubes and fill these tubes with Lysis Buffer (total of l-l .5 1 required). Cap tubes and invert several times. Sediment membranes by centrifugation (15 min at 9000 rpm, brake ‘off). The pellet is large, loose and difficult to see, occupying -l/3 of the volume. Slowly pour off -l/3 of the supernatant in one smooth action, disturbing the pellet as little as possible. Refill with Lysis Buffer and shake to resuspend any unlysed red cells. Wash with Lysis Buffer a further 4-5 times. After the second or third wash the red-cell membrane pellet can be easily seen. A small, white, tightly compacted pellet that underlies the larger red-cell membrane pellet can also be seen now. It contains lipidaceous material including white cells and should be removed by aspiration. By the end of the procedure, the red-cell membranes should be creamy white. Pink membranes may contain large amounts of endogenous calmodulin. Finally, wash twice with ice-cold Wash Buffer B (150 mM KCl, 10 mM Tris, 0.1 mM EGTA, pH 7.5 at 25°C) with centrifugation after each wash as described in step 4. To 20 ~1 membranes add 100 ~1 10% SDS and 880 ~1 water. Read the A,,, against an appropriate blank (20 ,~l Wash Buffer B, 880 ~1 water, 100 ,~l 10% SDS). Determine the membrane protein concentration using an extinction coefficient of 1.44 1g-’ cm-’ [2] taking into account the 50-fold dilution factor. Resuspend membranes to a concentration of l-l.5 mg ml-’ by the addition of Wash Buffer B
Education
26 (1998) 176-181
177
that contains 0.1 mM ouabain to inhibit endogenous Na+/K’-ATPase activity. (The addition of ouabain is optional so long as K+-rich solutions are used.) (9) The membranes should then be stored in aliquots at -80°C and can be kept for several weeks. One 10 ml aliquot is sufficient for more than thirty 96-well plates. (10) On the day of the practical class, one aliquot of membranes should be thawed and then diluted lo-fold in 150 mM KC1 to achieve a final membrane-protein concentration of 0.1-0.15 mg ml-‘). 2.2. Processing solutions 1
These should also be prepared prior to the commencement of the practical class:
(1) Malachite green hydrochloride (M Stock; 0.045% in (4 (3)
(4)
ii;
(7)
(8)
water, 3 1; Sigma) Ammonium molybdate (A Stock; 4.2% in 4 M HCl, 1 1) Prepare AM Stock by adding 11 of A stock to 3 1of M stock. Filter through a Whatman No 1 filter paper. Solutions l-3 are stable at room temperature. AM stock should be yellow-brown but can appear green if the concentration of HCl is low. To check this, dilute an aliquot of stock HCl and measure the PH. Each day make up AMT Colour Reagent by adding 120 ,~l Tween-20 (Sigma) to 200 ml AM Stock. 20% sodium citrate.2H,O (500 ml) Sodium phosphate standards in water with Pi concentrations of 0, 0.01, 0.02, 0.03, 0.04, 0.06, 0.08 and 0.10 mM. Incubation Buffer (150 mM KCl, 9 mM MgCl,, 3 mM ATP, 1.5 mM EGTA, 60 mM HEPES, pH 7.2) is used as a concentrated stock to generate the Ca’+-containing buffers (see below). The Incubation buffer is diluted 3-fold in the assay by the addition of equal volumes of calmodulin-containing solutions and, finally, membranes. Calcium-containing buffers: See Table 1 for the method used to calculate the total Ca*+ concentrations required to achieve designated free Ca2+ concentrations. Solutions A-H are prepared by the addition of 1 M CaCl, to 50 ml aliquots of Incubation Buffer to yield various final (96-well plate) concentrations of Ca*+. The additions of CaCl, take into account the 3-fold dilution of Solutions A-H required for the final assay: A, no addition; B, 36 ~1 (final free Ca*+ 0.1 PM); C, 55 ~1 (final free Ca*+ 0.3 PM); D, 68 ~1 (final free Ca*’ 1.0 PM); E, 73 ~1 (final free Ca*+ 3.0 ,uM); F, 77 ~1 (final free Ca2+ 10 ,uM); G, 82 ~1 (final free Ca2+ 30 PM); H, 99 ~1
A. D. Conigrave, M. B. Morris/Biochemical Education 26 (1998) 176-181
178
Table I Calculated total Ca’+ concentrations required to achieve designated free Ca’+ concentrations for the assay described in the text. The total Ca*+ concentrations were calculated using a rearrangement of the equations described by Fabiato [12] and the FindRoot function in a program written for Mathematics (Version 3.0 for Macintosh). A pH of 7.2, and total concentrations of EGTA (0.5 mM), ATP (1.0 mM), and MgCl, (3.0 mM) were fixed in these calculations. The following molar association constants [13] were used in the calculations: Ca(EGTA)*-, 1 x 10”. Ca(HEGTA)), 2 x lo<; Mg( EGTA)’ , 2 x lo<; Mg(H;GTA) , 2 x 10’; H(EGTA)‘~, 3 x 10’; H(HEGTA)’ , 8 x 10’; Mg(ATP)‘-, 7 x 10’; Mg(HATP)), 1 x 10’; Ca(ATP)* -, 1 x 10J; Ca(HATP) ~, 1 x 10’; H(ATP)‘-, 3 x 10h; H(HATP)‘-, 1 x lo4
Calmodulin
3
increases
4
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7
8
9
10
11
12
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Free Ca*+ concentration
Required total Ca’+ concentration
(PM)
7
Nil 239 367 452 487 511 547 658
(final free Ca’+ 100 PM). These buffers should be stored at -20°C until required. (9) Calmodulin-containing solutions: Solutions l-8 are prepared by the addition of 100 PM calmodulin (bovine brain, Calbiochem) to 5 ml aliquots of water to yield final (96-well plate) concentrations. The additions take into account the 3-fold dilution of Solutions l-8 required for the final assay: 1, no addition; 2,0.75 ~1 (final calmodulin 5 nM); 3, 1.5 ~1 (final calmodulin 10 nM); 4, 3 ~1 (final calmodulin 20 nM); 5, 7.5 ~1 (final calmodulin 50 nM); 6, 15 ~1 (final calmodulin 100 nM); 7,30 ~1 (final calmodulin 200 nM); 8, 75 ~1 (final calmodulin 500 nM). These solutions should be stored at -20°C until required.
3. Experimental Equipment requirements include g-channel pipettes (20-200 pl), 96-well plates and a microplate reader with a filter in the range 600-700 nm. We routinely use a 650 nm filter. The following steps are all performed by the students on the day of the experiment. 3.1. Preparation
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(PM)
of 96-well plates
pipette, transfer 40 ~1 of (1) Using a multi-channel Calcium Buffer Solutions A-H into the first eight wells of Rows A-H of the plate, respectively (Fig. 1). (That is, Solution A to wells 1-8 of Row A, Solution B to wells l-8 of Row B, etc.)
Empty
Fig. 1. Layout of the 96-well plate. The plate is set up as shown with free Ca2+ concentration (O-100 PM) increasing from Row A to Row H, and calmodulin concentration (O-500 nM) increasing from Column 1 to Column 8. Columns 9 and 10 are empty. The phosphate standards (O-10 nmoliwell) are pipetted into columns 11 and 12 as shown.
(2) Using
a multi-channel pipette, transfer 40 ~1 of Calmodulin Solutions 1-8 into all wells of Columns l-8 of the plate, respectively (Fig. 1). (That is, Solution 1 to all wells of Column 1, Solution 2 to all wells of Column 2, etc.) pipette to transfer 100 ~1 of (3) Use a multi-channel phosphate standard solutions A-H to Rows A-H, respectively, of columns 11 and 12 only (Fig. 1). (That is, Std A to Row A, Std B to Row B, etc.) The final quantities of Pi in Rows A-H are 0, 1,2, 3,4, 6, 8, and 10 nmol, respectively. 3.2. Reactions the reaction, use a multi-channel (1) To commence pipette to transfer 40 ~1 of membrane suspension to all wells of Columns l-8. Begin with Column 1 and finish with Column 8. Mixing in each column is achieved by re-aspirating the contents of the wells two or three times with the pipette. The membrane suspension is not added to the phosphate standards. The 96-well plates are covered with Glad-Wrap (or Saran Wrap) and incubations are performed for 30-40 min in a 37°C oven. (2) After 30-40 min (the time should be noted), the plates are removed from the oven and the following steps are performed at room temperature. The reactions in each column of wells are stopped in (150 ~1 per sequence by the addition ofAMTsolution well). Then immediately add 20% sodium citrate solution (25 ~1 per well) to each column in sequence, making sure that the content of each well is thoroughly mixed. The plates are then allowed to stand for 15-60 min to permit further colour development prior to reading them in a microplate reader.
A. D. Conigrave, M. B. MorrislBiochemical Education 26 (1998) 176-181
3.3. Reading the plates
70
179
Calmodulin 0.5
1
The plates are placed in a microplate reader and the absorbances read at 650 nm. The standard curve for Pi is used to determine the specific ATPase activity (nmol Pi per mg protein per min) for each well. Ca’+-independent ATPase activity is determined by finding the mean activity in wells Al-S. This value should be subtracted from the activities obtained in other wells to find the Ca”-stimulated ATPase activity.
No Calmodulin
4. Results
Figure 2 shows a typical standard curve for Pi. This curve is linear over an absorbance range of O-3 using the Titertek microplate reader used here. The data are readily fitted by linear regression. Fig. 3 shows a typical plot of ATPase specific activity as a function of free Ca*+ concentration, after correction for Ca*‘-independent activity. In the absence of calmodulin, ATPase activity is activated by Ca2+ with an apparent Km of about 5 PM. As shown, calmodulin activates the ATPase in a concentration dependent fashion from -5 to 500 nM. Activation of the ATPase takes the form of an increase in V,,, and a small but important shift in Km from -5 to - 1 PM. Figure 4 shows the concentration dependence of calmodulin stimulation of Ca*+-ATPase activity, when the free Ca” concentration is fixed at 3 ,uM.
5. Discussion
Calmodulin was first identified as an activator of cyclic AMP phosphodiesterase [3,4]. It has since been shown to
Free Ca2+ Concentration (FM) Fig. 3. Ca*’ concentration dependence and the effect of calmodulin on Ca’+-ATPase activity. Red-cell membranes (4 pg) were incubated for 30 min at 37°C in an incubator in the presence of Ca*‘/EGTA buffers and various concentrations of calmodulin (in nM): zero (0);5 (0); 10 (D); 20 (A); 50 (D); 100 (v); 200 (0); 500 (B) as described in the Experimental section.
participate in a wide spectrum of signal transduction pathways; e.g., those for glycogen breakdown, smooth muscle contraction, and nitric oxide generation (for reviews see refs [5-71). It was first shown to activate the plasma membrane Ca*+-ATPase of erythrocytes [8] and then of other cells (e.g. ref [9]). Detailed knowledge of
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3
4
5
6
7
6
9
10
Inorganic phosphate (nmol)
Fig. 2. Standard curve for inorganic phosphate. Inorganic phosphate standards (O-10 nmol/well) were prepared in MilliQ water and then transferred as duplicates (o,=) into columns 11 and 12 of a 96-well plate. AMT solution was added, followed by sodium citrate solution (20%) as described in the Experimental section. The plate was then read in a Titertek microplate reader at 650 nm. The solid line represents the fit to the data by linear regression.
100 200 300 400 Calmodulin Concentration (nM)
500
Fig. 4. Concentration dependence of the stimulatory effect of calmodulin on Ca*‘-ATPase. Red-cell membranes (4 pg) were incubated for 30 min at 37°C in an incubator at a free Ca’+ concentration of 3 PM and various concentrations of calmodulin (in nM). The solid line represents the fit to the data by non-linear regression (Deltagraph for Macintosh) using the equation A = k,+k,cl(k,+c), where A is the specific activity of the enzyme, c is the calmodulin concentration, k, is the calmodulin-independent specific activity, k, is the maximal calmodulin-dependent specific activity, and kJ is the apparent Km for calmodulin. The returned values of the parameters were k, = 24 nmol rng-’ min. ‘, kz = 34 nmol rng-’ min.‘, and k, (apparent Km) = 26 nM.
180
A. D. Conigrave, M. B. MorrislBiochemical
the structure and reaction mechanism of the Ca*+-ATPase is now available [lO,ll]. The microplate assay described here was adapted from that described previously [2]. The method may be readily adapted to the analysis of other membrane ATPases (e.g. the plasma membrane Na’/K’-ATPase or Mg*‘-ATPase).
6. lbtorial
discussion
This experiment lends itself to wide-ranging that can include the following points:
discussion
What is the role of the Ca*‘-ATPase in normal intracellular Ca2+ homeostasis? This introduction can include values for normal extracellular and intracellular free Ca2+ concentrations ([Ca’+],), and a discussion of why cells cannot tolerate chronically elevated [Ca’+],. You can include discussion of why the pump needs to be constitutively expressed and must operate at all times. The pumping of free Ca2+ coupled to the hydrolysis of ATP is stimulated by free Ca*’ itself (Fig. 3). What advantages does this provide for the cell in terms of calcium homeostasis, the switching on and off of signalling pathways, and the use of energy within the cell? Also discuss what concentrations of calcium were required in the experiment to stimulate ATP hydrolysis, and how these concentrations compare to physiologically relevant [Ca’+], (0.1-10 PM) required for stimulating activity in viva. Ca*+-bound calmodulin further stimulates Ca*‘-ATPase activity (Figs 3 and 4). What advantages are there for the cell in having this extra layer of control which, in this case, is coupled to the rise in [Ca’+],? (Points to note here are the integral role that calmodulin plays in controlling many, but not all, signalling pathways within cells, and noting the principle that most enzymes are subjected to multiple levels of control.) The above discussion on the physiological role of the Ca*+-ATPase pump and the modulation of its activity by Ca*+ and calmodulin can lead naturally to a discussion of: The structure of the Ca*+-ATPase and its organization into eight transmembrane domains. The reaction mechanism of Ca*+ pumping and its coupling to ATP hydrolysis. The Ca’+-ATPase is an E,-E, type (or P-type) ATPase. The mechanism by which the Ca*‘-calmodulin complex and other activators (e.g. protein kinase C and phosphatidylserine) activate the enzyme by interacting with an autoinhibitory domain in the Ca*‘-ATPase.
Education
26 (1998) 176-181
0 The mechanism of activation of calmodulin itself through Ca*+ binding to the four high-affinity EF-hand (helix-loop-helix) binding sites. The scope of the discussion can now be broadened, desired, to the following related topics:
if
Applications of the interaction between calmodulin and the Ca2+-ATPase, such as purification of the Ca*+-ATPase by calmodulin-affinity chromatography, or the determination of cellular calmodulin levels. Other Ca*+ pumps, such as the sarcoplasmic reticulum Ca*‘-ATPase, and their roles in cells. Other calmodulin-sensitive enzymes. The roles of other ATPases of the plasma membrane (e.g. Na’/K’-ATPase and Mg*‘-ATPase). The experiment itself includes routine data manipulation, and regression analysis is an important component. Points for discussion include: What are the factors that indicate that a straight-line fit to the standard curve (Fig. 2) is appropriate (sums of squares of the residuals, correlation coefficient, random distribution of residuals, the value of the slope and intercept, and the errors on these returned parameter values). Many students mistakenly believe that a standard curve must always be linear and should be forced to pass through the origin (ie, with the intercept value fixed at zero). What equation should be used to fit the data in Fig. 4 and, again, what are the objective criteria by which a good fit can be assessed? You can also discuss the many reasons why it is unacceptable (given the ready availability of computerised regression programs) to ‘linearise’ the data in Fig. 4 in order to fit with a straight line.
Acknowledgements We would like to thank Professor Philip Kuchel for his help in developing the program in Muthematica for calculating the total calcium concentrations required to obtain designated free Ca2+ concentrations. Mrs Julie Ferenczi provided excellent technical assistance.
References [II P.A. Lanzetta, VI
L.J. Alvarez, P.S. Reinach, O.A. improved assay for nanomole amounts of inorganic Analytical Biochemistry 100 (1979) 95-97. M.B. Morris, G. Monteith, B.D. Roufogalis, The ATP-dependent shape change of human erythrocyte lates with an inhibition of Mg”-ATPase activity by
Candia, An phosphate, inhibition of ghosts correfluoride and
A. D. Con&rave, M. B. MorrisiBiochemical Education 26 (1998) 176-181
[3]
[4]
[5] [6] [7] [8]
aluminofluoride complexes, Journal of Cellular Biochemistry 48 (1992) 356-366. W.Y. Cheung, Cyclic 3’S’-nucleotide phosphodiesterase. Demonstration of an activator, Biochemistry and Biophysics Research Communications 38 (1970) 533-538. S. Kakiuchi, R. Yamazaki, Calcium dependent phosphodiesterase activity and its activating factor (PAF) from brain studies on cyclic 3’,5’-nucleotide phosphodiesterase, Biochemistry and Biophysics Research Communications 41 (1970) 1104-1110. W.Y. Cheung, Calmodulin plays a pivotal role in cellular regulation, Science 207 (1980) 19-27. B.E. Finn, S. Forsen, The evolving model of calmodulin structure, function and activation, Structure 3 (1995) 7-l 1. A.R. Rhoads, F. Friedberg, Sequence motifs for calmodulin recognition, FASEB Journal 11 (1997) 331-340. F.F. Vincenzi, T.R. Hinds, B.U. Raess, Calmodulin and the plasma membrane calcium pump, Annals of the N.Y. Academy of Sciences 356 (1980) 232-244.
181
[9] A.D. Conigrave, M. Treiman, T. Saermark, N.A. Thorn, Stimulation by calmodulin of Ca*+ uptake and (Ca*+-Mg*+) ATPase activity in membrane fractions from ox neurohypophyses, Cell Calcium 2 (1981) 125-136. [lo] E. Carafoli, Biogenesis: plasma membrane calcium ATPase: 15 years of work on the purified enzyme, FASEB Journal 8 (1994) 993-1002. [ 1l] G.R. Monteith, B.D. Roufogalis, The plasma membrane calcium pump a physiological perspective on its regulation, Cell Calcium 18 (1995) 459-470. [ 121 A. Fabiato, Computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands, Methods in Enzymology 157 (1988) 378-417. [13] D. Goldstein, Calculation of the concentration of the free cations and cation-ligand complexes in solutions containing multiple divalent cations and ligands, Biophysics Journal 26 (1979) 235-242.
Biochemical Education 26 (1998) 176-181
A 96-well plate assay for the study of calmodulin-activated Ca2’-pumping ATPase from red-cell membranes Arthur
D. Conigrave”,
Michael
B. Morrish
Departments of “Biochemistry and hPharmacy, University of Sydney, Sydney, NS W 2006, Australia
Abstract A simple multi-well assay is described which can be used to assess the activity of the plasma membrane Ca2+ pump in human erythrocytes. The effect on activity of Ca’+, calmodulin, and the two reagents in combination can be quantified through the detection of inorganic phosphate released from ATP. The malachite green assay and a microplate reader are used to measure the concentration of released inorganic phosphate. The experiment can be completed by tertiary level biochemistry students in under 90 min and can stimulate a range of tutorial discussion topics including Ca’+ homeostasis in cells, enzyme structure and kinetics, the role of allosteric effecters and their mechanism of action, protein purification, and data analysis. 0 1998 IUBMB. Published by Elsevier Science Ltd. All rights reserved
1. Introduction Calmodulin is present in the cytoplasm of all mammalian cells. It is a key biochemical component by virtue of its ability to bind Ca2+ in the submicromolar to micromolar concentration range, and provides a link in certain signal transduction pathways between an elevation in intracellular Ca2+ concentration produced by receptor activation and the activation of key intracellular enzymes. These enzymes include myosin light chain kinase, cyclic AMP phosphodiesterase, nitric oxide synthase, and the plasma-membrane Ca’+-transporting ATPase. Calmodulin (Mr 17 kDa) is a dumb-bell shaped molecule with a central flexible bar that connects two highly folded domains at the N- and C-termini. In total, the molecule has four Ca2+-binding loops (EF hands); two Ca2+ binding loops in the N-terminal domain and two in the C-terminal domain. Ca”-ATPase (M, 125 kDa) is an integral membrane protein that operates as a Ca2+ pump. It hydrolyses MgATP to MgADP and inorganic phosphate (P,) in the is tightly presence of Ca2+. In vivo, this hydrolysis coupled to Ca2+ transport from the inside to the outside of the cell. Pump activity, and hence ATP hydrolysis, is increased in the presence of an elevated intracellular Ca2+ concentration. Elevated intracellular Ca2 + concentration also results in an increased concentration of Ca’+-calmodulin complex in the cell. The complex binds to the Ca2+-ATPase, further stimulating pump activity. Therefore, Ca2+ has two roles in this process: (i) as the
species that is transported out of the cell, and (ii) as a direct and indirect modulator of pump activity. Calcium exists in both bound and free forms in the cytoplasm. The bulk of cytoplasmic Ca2+ (0.1 mM) is bound to macromolecules such as proteins and other organic anions such as citrate. Less than 0.1% ( -0.1 PM) is free in the resting cell. However, after receptor activation the total Ca2+ concentration rises, and the free Ca2+ concentration can reach 1.0 ,uM or more. This is sufficient to greatly increase the concentration of the Ca2’-calmodulin complex and activate the Ca’+-ATPase. The Ca2+-ATPase may be assayed using the ammonium molybdate-malachite green method [l]. Hydrolysis of ATP by the enzyme releases Pi which forms with ammonium molybdate, and the complexes malachite green acts as an indicator for this reaction. The absorbance maximum occurs at _ 650 nm.
2. Preparation 2.1. Preparation of erythrocyte membranes Cell membranes are prepared from human erythrocytes that have been screened and found to be negative for HIV, hepatitis B and hepatitis C. All procedures are performed by the Preparation Room staff. We routinely find that the yield of membranes obtained from 200 ml of packed red blood cells (equivalent to one unit of whole
0307-4412/98/$19.00 + 0.00 0 1998 IUBMB. Published by Elsevier Science Ltd. All rights reserved. PII: SO307-4412(98)00029-h
A. D. Conigrave, M. B. Morris/Biochemical
blood) is sufficient to run experiments in about two hundred 96-well plates. For good yields, the procedure outlined below should be followed closely. In particular, all rotors and buffers should be kept at 4°C and centrifuge tubes should be immersed in ice before and after centrifuge runs.
(1) 200 ml ice-cold packed red blood cells (or one unit
(2)
(3)
(4)
(5)
(6)
(7)
(8)
of whole blood) should be distributed evenly between eight 50 ml SS34 screw-top rotor tubes. Fill tubes with ice-cold Wash Buffer A (150 mM NaCl, 10 mM Tris, pH 7.5 at 25°C). Wash twice by centrifugation (1 min at 10000 rpm with the brake set ‘on’) and carefully remove the floating huffy coat from the surface and side of each tube by aspiration each time. The buffy coat contains white cells that are a source of proteases. Shake the tubes vigorously before the second centrifugation to remove adherent red and white cells. Resuspend cells by addition of ice-cold Lysis Buffer (5 mM Tris, pH 8 at 25°C 1 mM EDTA) and shake vigorously. Transfer the contents to six GSA centrifuge tubes and fill these tubes with Lysis Buffer (total of l-l .5 1 required). Cap tubes and invert several times. Sediment membranes by centrifugation (15 min at 9000 rpm, brake ‘off). The pellet is large, loose and difficult to see, occupying -l/3 of the volume. Slowly pour off -l/3 of the supernatant in one smooth action, disturbing the pellet as little as possible. Refill with Lysis Buffer and shake to resuspend any unlysed red cells. Wash with Lysis Buffer a further 4-5 times. After the second or third wash the red-cell membrane pellet can be easily seen. A small, white, tightly compacted pellet that underlies the larger red-cell membrane pellet can also be seen now. It contains lipidaceous material including white cells and should be removed by aspiration. By the end of the procedure, the red-cell membranes should be creamy white. Pink membranes may contain large amounts of endogenous calmodulin. Finally, wash twice with ice-cold Wash Buffer B (150 mM KCl, 10 mM Tris, 0.1 mM EGTA, pH 7.5 at 25°C) with centrifugation after each wash as described in step 4. To 20 ~1 membranes add 100 ~1 10% SDS and 880 ~1 water. Read the A,,, against an appropriate blank (20 ,~l Wash Buffer B, 880 ~1 water, 100 ,~l 10% SDS). Determine the membrane protein concentration using an extinction coefficient of 1.44 1g-’ cm-’ [2] taking into account the 50-fold dilution factor. Resuspend membranes to a concentration of l-l.5 mg ml-’ by the addition of Wash Buffer B
Education
26 (1998) 176-181
177
that contains 0.1 mM ouabain to inhibit endogenous Na+/K’-ATPase activity. (The addition of ouabain is optional so long as K+-rich solutions are used.) (9) The membranes should then be stored in aliquots at -80°C and can be kept for several weeks. One 10 ml aliquot is sufficient for more than thirty 96-well plates. (10) On the day of the practical class, one aliquot of membranes should be thawed and then diluted lo-fold in 150 mM KC1 to achieve a final membrane-protein concentration of 0.1-0.15 mg ml-‘). 2.2. Processing solutions 1
These should also be prepared prior to the commencement of the practical class:
(1) Malachite green hydrochloride (M Stock; 0.045% in (4 (3)
(4)
ii;
(7)
(8)
water, 3 1; Sigma) Ammonium molybdate (A Stock; 4.2% in 4 M HCl, 1 1) Prepare AM Stock by adding 11 of A stock to 3 1of M stock. Filter through a Whatman No 1 filter paper. Solutions l-3 are stable at room temperature. AM stock should be yellow-brown but can appear green if the concentration of HCl is low. To check this, dilute an aliquot of stock HCl and measure the PH. Each day make up AMT Colour Reagent by adding 120 ,~l Tween-20 (Sigma) to 200 ml AM Stock. 20% sodium citrate.2H,O (500 ml) Sodium phosphate standards in water with Pi concentrations of 0, 0.01, 0.02, 0.03, 0.04, 0.06, 0.08 and 0.10 mM. Incubation Buffer (150 mM KCl, 9 mM MgCl,, 3 mM ATP, 1.5 mM EGTA, 60 mM HEPES, pH 7.2) is used as a concentrated stock to generate the Ca’+-containing buffers (see below). The Incubation buffer is diluted 3-fold in the assay by the addition of equal volumes of calmodulin-containing solutions and, finally, membranes. Calcium-containing buffers: See Table 1 for the method used to calculate the total Ca*+ concentrations required to achieve designated free Ca2+ concentrations. Solutions A-H are prepared by the addition of 1 M CaCl, to 50 ml aliquots of Incubation Buffer to yield various final (96-well plate) concentrations of Ca*+. The additions of CaCl, take into account the 3-fold dilution of Solutions A-H required for the final assay: A, no addition; B, 36 ~1 (final free Ca*+ 0.1 PM); C, 55 ~1 (final free Ca*+ 0.3 PM); D, 68 ~1 (final free Ca*’ 1.0 PM); E, 73 ~1 (final free Ca*+ 3.0 ,uM); F, 77 ~1 (final free Ca2+ 10 ,uM); G, 82 ~1 (final free Ca2+ 30 PM); H, 99 ~1
A. D. Conigrave, M. B. Morris/Biochemical Education 26 (1998) 176-181
178
Table I Calculated total Ca’+ concentrations required to achieve designated free Ca’+ concentrations for the assay described in the text. The total Ca*+ concentrations were calculated using a rearrangement of the equations described by Fabiato [12] and the FindRoot function in a program written for Mathematics (Version 3.0 for Macintosh). A pH of 7.2, and total concentrations of EGTA (0.5 mM), ATP (1.0 mM), and MgCl, (3.0 mM) were fixed in these calculations. The following molar association constants [13] were used in the calculations: Ca(EGTA)*-, 1 x 10”. Ca(HEGTA)), 2 x lo<; Mg( EGTA)’ , 2 x lo<; Mg(H;GTA) , 2 x 10’; H(EGTA)‘~, 3 x 10’; H(HEGTA)’ , 8 x 10’; Mg(ATP)‘-, 7 x 10’; Mg(HATP)), 1 x 10’; Ca(ATP)* -, 1 x 10J; Ca(HATP) ~, 1 x 10’; H(ATP)‘-, 3 x 10h; H(HATP)‘-, 1 x lo4
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(final free Ca’+ 100 PM). These buffers should be stored at -20°C until required. (9) Calmodulin-containing solutions: Solutions l-8 are prepared by the addition of 100 PM calmodulin (bovine brain, Calbiochem) to 5 ml aliquots of water to yield final (96-well plate) concentrations. The additions take into account the 3-fold dilution of Solutions l-8 required for the final assay: 1, no addition; 2,0.75 ~1 (final calmodulin 5 nM); 3, 1.5 ~1 (final calmodulin 10 nM); 4, 3 ~1 (final calmodulin 20 nM); 5, 7.5 ~1 (final calmodulin 50 nM); 6, 15 ~1 (final calmodulin 100 nM); 7,30 ~1 (final calmodulin 200 nM); 8, 75 ~1 (final calmodulin 500 nM). These solutions should be stored at -20°C until required.
3. Experimental Equipment requirements include g-channel pipettes (20-200 pl), 96-well plates and a microplate reader with a filter in the range 600-700 nm. We routinely use a 650 nm filter. The following steps are all performed by the students on the day of the experiment. 3.1. Preparation
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pipette, transfer 40 ~1 of (1) Using a multi-channel Calcium Buffer Solutions A-H into the first eight wells of Rows A-H of the plate, respectively (Fig. 1). (That is, Solution A to wells 1-8 of Row A, Solution B to wells l-8 of Row B, etc.)
Empty
Fig. 1. Layout of the 96-well plate. The plate is set up as shown with free Ca2+ concentration (O-100 PM) increasing from Row A to Row H, and calmodulin concentration (O-500 nM) increasing from Column 1 to Column 8. Columns 9 and 10 are empty. The phosphate standards (O-10 nmoliwell) are pipetted into columns 11 and 12 as shown.
(2) Using
a multi-channel pipette, transfer 40 ~1 of Calmodulin Solutions 1-8 into all wells of Columns l-8 of the plate, respectively (Fig. 1). (That is, Solution 1 to all wells of Column 1, Solution 2 to all wells of Column 2, etc.) pipette to transfer 100 ~1 of (3) Use a multi-channel phosphate standard solutions A-H to Rows A-H, respectively, of columns 11 and 12 only (Fig. 1). (That is, Std A to Row A, Std B to Row B, etc.) The final quantities of Pi in Rows A-H are 0, 1,2, 3,4, 6, 8, and 10 nmol, respectively. 3.2. Reactions the reaction, use a multi-channel (1) To commence pipette to transfer 40 ~1 of membrane suspension to all wells of Columns l-8. Begin with Column 1 and finish with Column 8. Mixing in each column is achieved by re-aspirating the contents of the wells two or three times with the pipette. The membrane suspension is not added to the phosphate standards. The 96-well plates are covered with Glad-Wrap (or Saran Wrap) and incubations are performed for 30-40 min in a 37°C oven. (2) After 30-40 min (the time should be noted), the plates are removed from the oven and the following steps are performed at room temperature. The reactions in each column of wells are stopped in (150 ~1 per sequence by the addition ofAMTsolution well). Then immediately add 20% sodium citrate solution (25 ~1 per well) to each column in sequence, making sure that the content of each well is thoroughly mixed. The plates are then allowed to stand for 15-60 min to permit further colour development prior to reading them in a microplate reader.
A. D. Conigrave, M. B. MorrislBiochemical Education 26 (1998) 176-181
3.3. Reading the plates
70
179
Calmodulin 0.5
1
The plates are placed in a microplate reader and the absorbances read at 650 nm. The standard curve for Pi is used to determine the specific ATPase activity (nmol Pi per mg protein per min) for each well. Ca’+-independent ATPase activity is determined by finding the mean activity in wells Al-S. This value should be subtracted from the activities obtained in other wells to find the Ca”-stimulated ATPase activity.
No Calmodulin
4. Results
Figure 2 shows a typical standard curve for Pi. This curve is linear over an absorbance range of O-3 using the Titertek microplate reader used here. The data are readily fitted by linear regression. Fig. 3 shows a typical plot of ATPase specific activity as a function of free Ca*+ concentration, after correction for Ca*‘-independent activity. In the absence of calmodulin, ATPase activity is activated by Ca2+ with an apparent Km of about 5 PM. As shown, calmodulin activates the ATPase in a concentration dependent fashion from -5 to 500 nM. Activation of the ATPase takes the form of an increase in V,,, and a small but important shift in Km from -5 to - 1 PM. Figure 4 shows the concentration dependence of calmodulin stimulation of Ca*+-ATPase activity, when the free Ca” concentration is fixed at 3 ,uM.
5. Discussion
Calmodulin was first identified as an activator of cyclic AMP phosphodiesterase [3,4]. It has since been shown to
Free Ca2+ Concentration (FM) Fig. 3. Ca*’ concentration dependence and the effect of calmodulin on Ca’+-ATPase activity. Red-cell membranes (4 pg) were incubated for 30 min at 37°C in an incubator in the presence of Ca*‘/EGTA buffers and various concentrations of calmodulin (in nM): zero (0);5 (0); 10 (D); 20 (A); 50 (D); 100 (v); 200 (0); 500 (B) as described in the Experimental section.
participate in a wide spectrum of signal transduction pathways; e.g., those for glycogen breakdown, smooth muscle contraction, and nitric oxide generation (for reviews see refs [5-71). It was first shown to activate the plasma membrane Ca*+-ATPase of erythrocytes [8] and then of other cells (e.g. ref [9]). Detailed knowledge of
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Fig. 2. Standard curve for inorganic phosphate. Inorganic phosphate standards (O-10 nmol/well) were prepared in MilliQ water and then transferred as duplicates (o,=) into columns 11 and 12 of a 96-well plate. AMT solution was added, followed by sodium citrate solution (20%) as described in the Experimental section. The plate was then read in a Titertek microplate reader at 650 nm. The solid line represents the fit to the data by linear regression.
100 200 300 400 Calmodulin Concentration (nM)
500
Fig. 4. Concentration dependence of the stimulatory effect of calmodulin on Ca*‘-ATPase. Red-cell membranes (4 pg) were incubated for 30 min at 37°C in an incubator at a free Ca’+ concentration of 3 PM and various concentrations of calmodulin (in nM). The solid line represents the fit to the data by non-linear regression (Deltagraph for Macintosh) using the equation A = k,+k,cl(k,+c), where A is the specific activity of the enzyme, c is the calmodulin concentration, k, is the calmodulin-independent specific activity, k, is the maximal calmodulin-dependent specific activity, and kJ is the apparent Km for calmodulin. The returned values of the parameters were k, = 24 nmol rng-’ min. ‘, kz = 34 nmol rng-’ min.‘, and k, (apparent Km) = 26 nM.
180
A. D. Conigrave, M. B. MorrislBiochemical
the structure and reaction mechanism of the Ca*+-ATPase is now available [lO,ll]. The microplate assay described here was adapted from that described previously [2]. The method may be readily adapted to the analysis of other membrane ATPases (e.g. the plasma membrane Na’/K’-ATPase or Mg*‘-ATPase).
6. lbtorial
discussion
This experiment lends itself to wide-ranging that can include the following points:
discussion
What is the role of the Ca*‘-ATPase in normal intracellular Ca2+ homeostasis? This introduction can include values for normal extracellular and intracellular free Ca2+ concentrations ([Ca’+],), and a discussion of why cells cannot tolerate chronically elevated [Ca’+],. You can include discussion of why the pump needs to be constitutively expressed and must operate at all times. The pumping of free Ca2+ coupled to the hydrolysis of ATP is stimulated by free Ca*’ itself (Fig. 3). What advantages does this provide for the cell in terms of calcium homeostasis, the switching on and off of signalling pathways, and the use of energy within the cell? Also discuss what concentrations of calcium were required in the experiment to stimulate ATP hydrolysis, and how these concentrations compare to physiologically relevant [Ca’+], (0.1-10 PM) required for stimulating activity in viva. Ca*+-bound calmodulin further stimulates Ca*‘-ATPase activity (Figs 3 and 4). What advantages are there for the cell in having this extra layer of control which, in this case, is coupled to the rise in [Ca’+],? (Points to note here are the integral role that calmodulin plays in controlling many, but not all, signalling pathways within cells, and noting the principle that most enzymes are subjected to multiple levels of control.) The above discussion on the physiological role of the Ca*+-ATPase pump and the modulation of its activity by Ca*+ and calmodulin can lead naturally to a discussion of: The structure of the Ca*+-ATPase and its organization into eight transmembrane domains. The reaction mechanism of Ca*+ pumping and its coupling to ATP hydrolysis. The Ca’+-ATPase is an E,-E, type (or P-type) ATPase. The mechanism by which the Ca*‘-calmodulin complex and other activators (e.g. protein kinase C and phosphatidylserine) activate the enzyme by interacting with an autoinhibitory domain in the Ca*‘-ATPase.
Education
26 (1998) 176-181
0 The mechanism of activation of calmodulin itself through Ca*+ binding to the four high-affinity EF-hand (helix-loop-helix) binding sites. The scope of the discussion can now be broadened, desired, to the following related topics:
if
Applications of the interaction between calmodulin and the Ca2+-ATPase, such as purification of the Ca*+-ATPase by calmodulin-affinity chromatography, or the determination of cellular calmodulin levels. Other Ca*+ pumps, such as the sarcoplasmic reticulum Ca*‘-ATPase, and their roles in cells. Other calmodulin-sensitive enzymes. The roles of other ATPases of the plasma membrane (e.g. Na’/K’-ATPase and Mg*‘-ATPase). The experiment itself includes routine data manipulation, and regression analysis is an important component. Points for discussion include: What are the factors that indicate that a straight-line fit to the standard curve (Fig. 2) is appropriate (sums of squares of the residuals, correlation coefficient, random distribution of residuals, the value of the slope and intercept, and the errors on these returned parameter values). Many students mistakenly believe that a standard curve must always be linear and should be forced to pass through the origin (ie, with the intercept value fixed at zero). What equation should be used to fit the data in Fig. 4 and, again, what are the objective criteria by which a good fit can be assessed? You can also discuss the many reasons why it is unacceptable (given the ready availability of computerised regression programs) to ‘linearise’ the data in Fig. 4 in order to fit with a straight line.
Acknowledgements We would like to thank Professor Philip Kuchel for his help in developing the program in Muthematica for calculating the total calcium concentrations required to obtain designated free Ca2+ concentrations. Mrs Julie Ferenczi provided excellent technical assistance.
References [II P.A. Lanzetta, VI
L.J. Alvarez, P.S. Reinach, O.A. improved assay for nanomole amounts of inorganic Analytical Biochemistry 100 (1979) 95-97. M.B. Morris, G. Monteith, B.D. Roufogalis, The ATP-dependent shape change of human erythrocyte lates with an inhibition of Mg”-ATPase activity by
Candia, An phosphate, inhibition of ghosts correfluoride and
A. D. Con&rave, M. B. MorrisiBiochemical Education 26 (1998) 176-181
[3]
[4]
[5] [6] [7] [8]
aluminofluoride complexes, Journal of Cellular Biochemistry 48 (1992) 356-366. W.Y. Cheung, Cyclic 3’S’-nucleotide phosphodiesterase. Demonstration of an activator, Biochemistry and Biophysics Research Communications 38 (1970) 533-538. S. Kakiuchi, R. Yamazaki, Calcium dependent phosphodiesterase activity and its activating factor (PAF) from brain studies on cyclic 3’,5’-nucleotide phosphodiesterase, Biochemistry and Biophysics Research Communications 41 (1970) 1104-1110. W.Y. Cheung, Calmodulin plays a pivotal role in cellular regulation, Science 207 (1980) 19-27. B.E. Finn, S. Forsen, The evolving model of calmodulin structure, function and activation, Structure 3 (1995) 7-l 1. A.R. Rhoads, F. Friedberg, Sequence motifs for calmodulin recognition, FASEB Journal 11 (1997) 331-340. F.F. Vincenzi, T.R. Hinds, B.U. Raess, Calmodulin and the plasma membrane calcium pump, Annals of the N.Y. Academy of Sciences 356 (1980) 232-244.
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[9] A.D. Conigrave, M. Treiman, T. Saermark, N.A. Thorn, Stimulation by calmodulin of Ca*+ uptake and (Ca*+-Mg*+) ATPase activity in membrane fractions from ox neurohypophyses, Cell Calcium 2 (1981) 125-136. [lo] E. Carafoli, Biogenesis: plasma membrane calcium ATPase: 15 years of work on the purified enzyme, FASEB Journal 8 (1994) 993-1002. [ 1l] G.R. Monteith, B.D. Roufogalis, The plasma membrane calcium pump a physiological perspective on its regulation, Cell Calcium 18 (1995) 459-470. [ 121 A. Fabiato, Computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands, Methods in Enzymology 157 (1988) 378-417. [13] D. Goldstein, Calculation of the concentration of the free cations and cation-ligand complexes in solutions containing multiple divalent cations and ligands, Biophysics Journal 26 (1979) 235-242.