A rapid and precise assay for calmodulin and cyclic AMP phosphodiesterase on a centrifugal analyser

Clinica Chimica Acta, 164 (1987) 305-313 Elsevier

305

CCA 03777

A rapid and precise assay for calmodulin and cyclic AMP phosphodiesterase on a centrifugal analyser S.W. Walker, S.W. Milner and A.F. Howie lJniversi@ Department (Received

4 August

of Clinical Chemistry, The Royal Infirmary, Edinburgh, Scotland (UK)

1986; revision

received 18 December

Key worak Calmodulin;

1986; accepted

Cyclic AMP; Phosphodiesterase;

after revision

5 January

1987)

Calcium

An assay for cyclic AMP phosphodiesterase in biological samples is described. The method is a continuous, spectrophotometric assay adapted for use on a centrifugal analyser. Using the calmodulin-activated cyclic AMP phosphodiesterase prepared from pig brain by DEAE-cellulose chromatography, and a standard calmodulin preparation from the same source, a rapid assay for calmodulin is described. The calmodulin assay has a working range of 2 to 6 mg/l calmodulin in the sample. The within-batch CV is < 5% and the between-batch CV is 7.0% or less.

Introduction

Many of the actions of intracellular calcium ions may depend upon their binding to calmodulin [l]. Binding occurs at a calcium concentration characteristic of the activated cell and the complex of calcium and calmodulin is able to activate a wide range of receptor proteins; these receptor proteins include several key, regulatory enzymes and structural cell proteins, believed to be responsible for the changes in cell function brought about by the initial stimulus-evoked rise in intracellular [Ca2’] [l]. There is increasing interest in possible abnormalities in intracellular [Ca2’] and/or calmodulin in various disease states which include, for example, essential hypertension [2], psoriasis [3] and cystic fibrosis [4]. There are several assays available for calmodulin, some of which ultimately depend upon the ability of calcium-calmodulin to activate the enzyme cyclic AMP phosphodiesterase (PDE) [5]. A continuous, spectrophotometric assay for cyclic AMP PDE and calmodulin has been optimised by Chock and Huang [5]. In this assay, PDE activity is measured by its coupling to the myokinase, pyruvate kinase 0009-8981/87/$03.50

0 1987 Elsevier Science Publishers

B.V. (Biomedical

Division)

306

and lactate dehydrogenase reactions. A standard curve for activation of cyclic AMP PDE by calmodulin is then constructed. Cyclic AMP + AMP AMP + ATP + 2 ADP

(PDE) (myokinase)

ADP + PEP + ATP + pyruvate

(pyruvate kinase)

Pyruvate + NADH + NAD + lactate

(lactate dehydrogenase)

We describe here how the optimised assay of Chock and Huang [5] can be readily adapted for use on a cent~fug~ analyser, using partially purified PDE from pig brain and purified calmodulin from the same source. The ease of preparation of the pig brain PDE and calmodulin, together with the speed of centrifugal analysers, should widen the scope for assay of both cyclic AMP PDE and calmodulin. Materials The following chemicals were obtained from the Sigma Chemical Co., Poole, Dorset, UK: Tris (hydroxymethyl)aminomethane (Tris), 4-(2-hydroxyethyl)-lpiper~ne-ethanesulphonic acid (HEPES), pheny~ethanesulphonyl fluoride (PMSF), pepstatin A, e~ylene~y~l-his-( ~-~n~thyl ether)&, N, N ‘, N’ tetraacetic acid (EGTA), adenosine 5’-t~phosphate (ATP), adenosine 3’ : 5’-cyclic monophosphate (cyclic AMP), phosph~enol)py~vate (PEP), /3-nicotinamide adenine dinucleotide (reduced form) (NADH), myokinase (grade III from rabbit muscle), pyruvate kinase (type III from rabbit muscle), L-lactate dehydrogenase (type III from bovine heart) and Coomassie Brilliant Blue (G250). Phenyl-Sepharose was from Pharmacia, Milton Keynes, Bucks., UK and DEAE-cellulose (Whatman DE-52) from Whatman, Maidstone, Kent, UK. Acrylamide, bisacrylamide and N, N, N ‘, N’-tetramethylethylenediamine were from Bio-Rad Laboratories, Watford, Herts, UK. All other chemicals were of analytical grade and obtained from BDH Chemicals, Poole, Dorset, UK. Methods Preparation of cyclic AMP phosphodiesterase (FDE) The cyclic AMP PDE from pig brain which is activated by Ca-calmodulin was prepared from 175 g of pig brain. The tissue was freshly obtained from the abattoir and homogenised in 3 ~0150 mmol/l Tris-HCl (pH 7.5 at 4” C), 1 mmol/l EGTA and containing 0.2 mmol/l PMSF and 50 pg/l pepstatin A (buffer A). The homogenate was centrifuged at 12000 X g for 1 h at 4 o C, and the supernatant from this step subsequently centrifuged at 100000 x g for 1 h at 4” C. The supematant from the high-speed ~~fugation was dialysed overnight against buffer A and then c~omato~aphed on DUE-cellulose (bed vol 300 ml), pre-~ui~brat~ with buffer A. The column was eluted with a linear NaCl gradient, collecting 5 ml fractions and

307

measuring the Na content of every 5th fraction by flame photometry. Cyclic AMP PDE activity was measured in every 2nd fraction, with and without the addition of 0.2 pmol/l calmodulin. On DEAE-cellulose chromatography the calmodulin activated PDE is well separated on the NaCl gradient from endogenous calmodulin, which is an acidic protein (pl approximately 4.l)and elutes at a NaCl concentration > 200 mmol/l Wallace et al [6]. Measurement of cyclic AMP PDE activity The Cobas Fara (Roche) centrifugal analyser was programmed to measure cyclic AMP PDE by a method based upon the optimised assay described by Chock and Huang [5]. A sample volume of 15 ~1 is used (with 15 ~1 of diluent) and 110 1.11of working reagent. After an incubation of 120 s, the reaction is initiated by the addition of 10 ~1 of the substrate cyclic AMP. The reaction is measured at 37 o C and, following the addition of the substrate, the absorbance at 340 nm monitored every 5 s, between 50 and 100 s, measuring the rate by linear regression analysis. A summary of the centrifugal analyser parameters is shown in Table I. The working reagent is made up freshly by adding together (1) 5 ml 0.13 mol/l HEPES buffer (pH 8.0 at 37’ C) containing 13 mmol/l MgCl, and 0.52 mmol/l EGTA (final concentrations in the assay, 0.05 mol/l HEPES, 5 mmol/l MgCl, and 0.2 mmol/l EGTA); (2) 4 ml 6.5 mmol/l CaCl, (final assay concentration, 2 mmol/l CaCl,); (3) 300 ~10.1 mol/l PEP; (4) 200 ~1 NADH (10 mg/ml); (5) 50 ~1 0.1 mmol/l ATP. To 4 ml of working reagent are added 50 ~1 of the linking enzyme solution, a mixture of equal volumes of pyruvate kinase (5 mg/ml), LDH (12 mg/ml) and myokinase (5 mg/ml). The 4 ml of working reagent which results is sufficient for at least 30 estimations. Cyclic AMP substrate is made up freshly by adding 200 ~10.1 mol/l cyclic AMP to 2.46 ml H,O. This gives a final assay concentration of 500 pmol/l cyclic AMP.

TABLE

I

Centrifugal

analyser

Measurement mode Blank Wavelength Temperature unit Sample volume Reagent volume Initial incubation Start reagent volume First reading time Number of readings Calculation

parameters Absorbance Reagent/diluent 34Omn 37OC AA/minx1000 15pldiluent15pl 110 pl 120 s 10 81 0.5 s 20 interval 5 s Linear regression readings lo-20

on

308

When measuring the PDE activity in fractions eluted from the DEAE-cell~ose column, the assay was carried out with and without 0.2 pmol/l calmodulin added. Those fractions containing calmodulin-activated PDE were pooled, subdivided into 5 ml portions and then in turn further divided into 500~~1portions which were then stored at - 20 ‘C for use in the subsequent calmodulin assay. Preparation of calmodulin

Calmodulin was prepared by the method of Gopalakrishna and Anderson [7] using hydrophobic interaction chromatography on phenyl-Sepharose. Pig brain is a suitable source of calmodulin. The purity of the calmodulin preparation was assessed by its migration as a single band on SDS-PAGE. The protein concentration of the purified calmodulin preparation was determined by the method of Bradford fS], and 2 ml aliquots of a standard solution of 10 mg/l prepared by diluting the stock solution with water. These aliquots were then stored at -2OOC. Assay for calmodulin

From the working standard (10 mg/l) solutions of 8, 7, 6, 5, 4, 3, 2 and 1 mg/l were made by dilution with water. The zero standard consisted of water alone. To obtain a standard curve, 100 ~1 of each calmodulin standard are mixed with 50 ~1 of the brain PDE preparation (protein content of 1.8 g/l) and 50 ~1 H,O added. Using the program listing shown in Table I the PDE activity (expressed as AA,,/min x 1000) is measured and the standard curve constructed. The preparation of tissue homogenates or fractions for use in the assay was carried out using buffer A, as used in the pu~fication scheme for brain PDE. To determine calmodulin in an unknown sample, especially where endogenous PDE activity is known to be present, a blank is essential; in this the brain PDE (50 ~1) is replaced by distilled water. To reduce the blank value to low levels, use is made of the heat-stability of calmodulin; PDE is thermolabile. Thus, 400 ~1 of sample are heated at 90°C for 30 s, and then clarified by centrifugation on a microfuge at 12000 x g for 2 min. Under these conditions there is insignificant loss of calmodulin activity (see ‘Results’). For a previously unknown sample, it is advisable to assay at a range of different sample dilutions in order to obtain at least one point on the working part of the standard curve. The sample is treated identically to the standards (100 ~1 sample + 50 ~1 brain PDE + 50 ~1 H,O) and the calmodulin activity expressed as ~g/mg protein of the original (unheated~ sample. Ca~modulin recovery The extent of calmodulin

loss during heating of samples was determined by a recovery experiment. To 200 ~1 of a high-speed supernatant prepared from bovine adrenal cortex were added 200 ~1 H,O or 200 ~1 of an aqueous solution of calmodulin (100 mg/l). Each sample was heated for 30 s at 90°C and then assayed as described above at dilutions of 1: 10 and 1: 20. The procedure was repeated but this time using a heating period of 1 min. After making the appropriate blank corrections for any endogenous PDE in the adrenal supematant, and after subtracting the calmodulin present in the adrenal sample to which no calmodulin had been

309

added, recoveries were calculated for the different heating periods at both dilutions used. Experiments were also undertaken to measure the rate of loss of endogenous PDE activity in a tissue sample when heated at 90°C.

Results

Preparation of calmodulin sensitive PDE The DEAE-cellulose profile for the high-speed supematant prepared from pig brain (Fig. 1) shows a prominent peak of calmodulin-activated PDE eluting at a NaCl concentration of approximately 150 mmol/l. This contrasts with the elution position of calmodulin itself, which emerges at a NaCl concentration of about 250 mmol/l (not shown). Standard curve for calmodulin activity Calmoduhn, purified from pig brain, was used in conjunction with the calmodulin-sensitive PDE to construct a standard curve (Fig. 2). This shows a narrow working range, between 2 and 6 mg/l calmodulin, deviating from linearity above and below these values, and reaching maximal activation by 8 mg/l cahnodulin. The curve shows good reproducibility between different runs, and appears to be

Fraction No.

Fig. 1. The DEAE-cellulose profile for the high-speed supematant from pig brain. The PDE activity is and without (Oshown with (0 -0) 0) added calmodulin, together with the NaCl gradient x). Fraction volume, 5 ml. (X-

Caknodulin Img/L)

Fig. 2. Standard curve for the measurement of cahnodulin based on the activation, in the presence of (Ca*+ 1, of pig brain PDE prepared as in Fig. 1. The three symbols drawn correspond to the standard curve constrncted on three separate occasions. The calmodulin ~n~ntration refers to the standard and not the final assay con~ntration.

reproducible between different preparations of PDE and calmodulin. Blank values for the standard calmodulin preparations alone (no PDE) were small. Preparation of the sample and calmodulin recovery It is advisable to reduce the high sample blank

activity which results from endogenous PDE activity, by first heating the sample, as explained in ‘Methods’. The time-course of endogenous PDE activity and calmodulin activity during heating at 90 o C, in a sample consisting of the high-speed supematant from bovine adrenal cortex, is shown in Fig. 3. The rapid decline in endogenous PDE activity by 30 s, and the much less steep decline in activity of the relatively heat-stable calmodulin, are shown. Based on such measurements, a heating time of 30 s was used in subsequent sample preparations. Recovery experiments confirmed the suitability of this approach to sample preparation. Calmodulin recoveries added to an adrenal sample at 50 mg/l were 97% and 102% (assayed at 1: 10 and 1: 20 dilutions, respectively) after sample heating for 30 s. Recoveries fell to 85% and 84% (assayed at 1: 10 and 1: 20 dilutions, respectively) if the heating time was increased to 60 s.

Heating time at 90°C (set) Fig. 3. A comparison of the heat-sensitivity of endogenous PDE activity (0) and calmodulin sample consisting of the high-speed supematant from bovine adrenal cortex.

(A)

in a

When a tissue sample is assayed for calmodulin activity, it is advisable to include several sample dilutions (after heating) in the assay. This entails little extra effort in the semi-automated assay described here, and helps to ensure that one or more assay points fall on the linear portion of the standard curve. We recommend that at least two points be established on the linear portion of the standard curve, as a check for parallelism, especially where the sample is from a previously untested tissue. In the example of the high-speed supernatant from adrenal cortex, values for calmodulin of 24 mg/l, 20 mg/l and 22 mg/l in the undiluted sample (after heating), were obtained from the standard curve when the sample was assayed at dilutions of 1: 5, 1: 8 and 1: 10, respectively. Calmodulin

assay precision

This was measured at levels of about 2 mg/l and 6 mg/l. The material used to assess precision was the cytosol fraction of bovine adrenal cortex, diluted to give assay values at the approximate level and subsequently divided into small (250 ~1) samples and stored at - 20 ’ C. The between-batch CV for the assay was 4.6% (n = 8) at a mean control value of 2.32 mg/l calmodulin, and 7.0% (n = 8) at a mean control value of 5.75 mg/l. The within-assay CV was below 5% at both levels. The worse precision at the higher value probably reflects its position on the standard curve, at which point the curve

312

is already beginning to flatten off. In the absence of a full precision profile, it is recommended that samples be diluted to give at least one concentration value falling between 2 and 6 mg/l. Discussion Several methods for the measurement of calmodulin in biological samples have been reported. These include a ra~o~~oassay for calmodulin [9], but ~fficulti~ have been experienced in raising ~~-~fi~ty antibodies against the native protein; this is a problem which may be related to the high degree of conservation of amino acid sequence between diverse species for this protein [lo]. Most of the remaining methods depend upon the fact that certain forms of cyclic AMP PDE are activated by cahnodulin in the presence of calmodulin. The shortcomings of the methods based on this principle are discussed by Chock and Huang [5]. The advantages of the calmodulin assay described here include both cost and speed. Considerable reduction in the overall cost is achieved by preparing both calmodulin and cyclic AMP PDE from pig brain. A major advantage is the use of a centrifugal analyser, which significantly improves the convenience and speed of the original optimised method of Chock and Huang [5]. In addition, the method shows good repr~ucib~ty of the standard curve on a day-to-day basis and acceptable precision. Disadvantages of this assay include the narrow working range of the standard curve and the high blank value of tissue samples. However, the speed of the assay allows a range of sample dilutions to be readily assayed to ensure one or more points are located within the working range. A high blank value for the sample is a feature of all calmodulin assays which use activation of cyclic AMP PDE, since this enzyme is of widespread distribution. Our results confirm that a short period of sample heating will destroy most endogenous cyclic AMP PDE activity, without significantly reducing calmodulin levels. The centrifugal anaIyser can also be used to assay cyclic AMP PDE itself, irrespective of the presence of cahnodulin. Indeed, the #n~bution of ~almodu~ to total cyclic AMP PDE activity in a tissue sample can be measured by assaying with Ca2+ present and then in the presence of an excess of EGTA. The difference between the two values is then a measure of cyclic AMP PDE activity dependent upon Ca*+ and calmodulin. ‘However, the method lacks the sensitivity of the radioisotope assay [ll], and we have found that detailed kinetic studies are hampered by the complex linking enzyme system. The discovery of calmodulin by Cheung [12] and Kakiuchi et al (131 can now be seen to be a landmark in our understanding of intracellular calcium signalling. The increasing awareness of possible abnormalities in intracellular calcium in disease states f14], and the central importance of calmodulin demand, suitable assays for this key, regulatory protein. We believe that the calmodulin assay described here offers significant advantages in speed, cost and ease of assay, as compared with other techniques previously described.

313

Acknowledgement We would like to thank Professor L.G. Whitby for his encouragement work and for critically reading the manuscript.

of this

References 1 Klee CB, Crouch TH, Richman RH. Calmodulin. Ann Rev Biochem 1981;49:489-515. 2 Swales JD. Ion transport in hypertension. Biosci Rep 1982;2:967-990. 3 Van der Kerkhof PCM, Van Erp PET. Cahnodulin levels are greatly elevated in the psoriatic lesion. Br J Dermatol 1983;108:217-218. 4 Gnegy ME, Erickson RP, Markovac J. increased calmodulin in cultured skin fibroblasts from patients with cystic fibrosis. B&hem Med 1981;26:294-298. 5 Chock SP, Huang CY. An optimised continuous assay for cyclic AMP phosphodiesterase and caimodulin. Anal Biochem 1984;138:34-43. 6 Wallace RW, Tallant EA, Cheung WY. Assay, preparation and properties of c~rn~u~n. In: Cheung WY, ed. Calcium of cell function, Vol. 1. New York: Academic Press, 1980;13-19. apptication for 7 ~op~a~s~a R, Anderson WB. Ca ‘+-induced hy~ophobi~ site on ~rn~~rn: pu~fication of calmodulin by phenyl-~pharose affinity c~omat~aphy. Biochem Biophys Res Commun 1982;104:830-836. 8 Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein dye binding. Anal B&hem 1976;72:248-254. 9 Chafouleas JM, Dedman JR, Munjall RP, Means AR. Calmodulin. Development and application of a sensitive radioimmunoassay. J Biol Chem 1979;254:10262-10267. 10 Biber A, Hempel K, Radioimmunoassay of oxidised calmoduhn in chloramine-T treated tissue samples. J Clin Chem Clin Biochem 1984;22:185-188. 11 Thompson WJ, Epstein PM, Strada SJ. Purification and characterisation of high-affinity cyclic adenosine monophosphate phosphodiesterase from dog kidney. Bicohemistry 1979;18:5228-5237. 12 Cheung WY. Cyclic 3’,5’-nucleotide phosphodiesterase. Demonstration of an activator. Biochem Biophys Res Commun 1970;38:533-538. 13 Kakiuchi S, Yamazaki R, Nakajima H. Properties of a heat-stable phosphodiesterase activating factor isolated from brain extract. Prc Jpn Acad 19;46:587-592. 14 Tomlinson S, MacNeil S, Walker SW, Ollis CA, Merritt JE, Brown BL. Calmodulin and cell function. Clin Sci 1984;66:497-508.