[6] Purification of novel calcium-binding proteins from bovine brain

68

ISOLATION/CHARACTERIZATION

OF Ca-BINDING

PROTEINS

[6]

We have also developed a protocol for the rapid purification of calmodulin and calmodulin-binding proteins, which is well suited to overcome the inherent difficulties of working with lower eukaryotes imposed by the high level of proteases and extracellular matrix components elicited by these organisms. 31 W. Y. Cheung, J. Biol. Chem. 246, 2859 (1971). 32 M. Yazawa, K. Yagi, H. Toda, M. Kondo, K. Narita, R. Yamazaki, K. Sobue, S. Kakiuchi, S. Nagao, and Y. Nozawa, Biochem. Biophys. Res. Commun. 99, 1051 (1981). 33 D. R. Marshak, M. Clarke, D. M. Roberts, and D. M. Watterson, Biochemistry 23, 2891 (1984). 34 T. J. Lukas, D. B. Iverson, M. Schleicher, and D. M. Watterson, Plant Physiol. 75, 788 (1984).

[6] P u r i f i c a t i o n o f N o v e l C a l c i u m - B i n d i n g P r o t e i n s f r o m Bovine Brain B y MASAAKI TOKUDA, N A V I N C . K H A N N A , and D A V I D MORTON WAISMAN

Introduction Calcium has been reported as a second messenger in hormone action by a wide body of evidence. Fundamental to this second messenger system are the Ca2+-binding proteins, which function as intracellular receptors for second messenger Ca 2+. While many Ca2+-binding proteins have been isolated and characterized, it is surprising, considering their importance, that as yet a systematic search for Ca2+-binding proteins has not been undertaken. Recently we have reexamined and modified the Chelex-100 competitive Ca2+-binding assay ~to ensure maximum linearity and sensitivity, and using this assay as a probe for the detection of Ca2+-binding proteins we have begun a systematic analysis of tissue 100,000 g supernatants. Specifically, we have resolved the Ca2+-binding activity in the 100,000 g supernatants of bovine brain, heart, and liver 2,3 by a procedure involving l D. M. Waisman and H. Rasmussen, Cell Calcium 4, 89 (1983). 2 D. M. Waisman, J. Smallwood, D. Lafreniere, and H. Rasmussen, Biochem. Biophys. Res. Commun. 116, 435 (1983). 3 D. M. Waisman, J. Smallwood, D. Lafreniere, and H. Rasmussen, Biochem. Biophys. Res. Comman. 119, 440 (1984).

METHODS IN ENZYMOLOGY, VOL. 139

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

[6]

NOVEL Ca-BINDING PROTEINS FROM BOVINE BRAIN

69

DEAE-cellulose, hydroxylapatite, and gel permeation chromatography. These studies demonstrated that the majority of the Ca2+-binding activity, detected by the Chelex assay, was not associated with calmodulin activity and based on a comparison of the apparent Mr of the Ca2+-binding activity peaks with those of characterized CaZ+-binding proteins we suggested that the tissue extracts might contain uncharacterized (novel) Ca2÷-bind ing proteins. Recently, 3,4 we have purified and characterized a novel CaZ+-binding protein responsible for the major Ca2+-binding activity peak of bovine liver. We have also been successful in the purification and characterization of two novel Ca2+-binding proteins of bovine brain. 5.6 In the present report the purification of two novel bovine brain Ca z+binding proteins, CAB-27 and CAB-48, is detailed. In addition, the strengths and weaknesses of the Chelex assay as a probe for the measurement of Ca2+-binding activity in tissue homogenates are discussed. Purification of Novel Calcium-Binding Proteins from Bovine Brain--CAB-27 and CAB-48

Preparation of Bovine 100,000 g Supernatant Fresh bovine brain was obtained from a local slaughterhouse. Connective tissue was dissected away, and the tissue was rinsed thoroughly with ice-cold distilled water and frozen immediately at - 2 0 °. One kilogram of frozen tissue was chopped and then further minced in a meat grinder. The mince was mixed with 4 liters of ice-cold buffer containing 40 mM TrisHCI (pH 8.0), phenylmethylsulfonyl fluoride (0.5 mM), soybean trypsin inhibitor (5 mg/liter), benzamidine (10 mM), leupeptin (5 mg/liter), diisopropylfluorophosphate (1.0 mM), and DTT (I.0 mM) and then homogenized in a Waring blender. After addition of 50 ml of Chelex-100 slurry, the homogenate was centrifuged at 20,000 g for 30 min and the supernatant was centrifuged at 100,000 g for 60 min.

DEAE-Cellulose Chromatography of Bovine Brain 100,000 g Supernatant The supernatant was diluted into 5 vol of 40 mM Tris-HCl (pH 8.0), 1.0 mM DTT, and 800 ml of packed DEAE-cellulose was then added. The mixture was stirred rapidly for 2 hr, and then filtered through a coarse sintered glass funnel. The resultant slurry was washed with 10 liters of 40 4 D. M. Waisman, B. Salimath, and J. Anderson, J. Biol. Chem. 260, 1652 (1985). 5 D. M. Waisman, J. Muranyi, and M. Ahmed, FEBS Lett. 164, 80 (1983). 6 D. M. Waisman, M. Tokuda, S. Morys, L. T. Buckland, and T. Clark, Biochem. Biophys. Res. Commun. 128, 1138 (1985).

70

ISOLATION/CHARACTERIZATION OF Ca-BINDING PROTEINS

[6]

I BRA! N 100,000xg SUPERNATAN T I

I

A

~ oJs

~o5

i

B [ T

o-. o

~

/:

~

X°

|g

~ o

~

~

g ~

o

~

~°

'°°i

'°°

o~

FaACT,~ N u b i a ,a.erlo.

~u.a~R

I HEAT TREATMENT(80"C, 2 rain) I

]

E

2,0

I

!

l,s

o:

z

?+ o

o

o

so

eo

;'o

eo

FRACT+ON mUl+eEq

s

i,;tl

o,~ E

o

i

i

i

+o

so

:m

40

,

so

so

To

FAACTIOII N u u s l a

io

io

io~

L

[6]

NOVEL Ca-BINDING PROTEINS FROM BOVINE BRAIN

71

mM Tris-HCl (pH 8.0), 1 mM DTT, and poured into a 5.0 × 60 cm column. Protein was eluted with 4.4 liters of 40 mM Tris-HCl (pH 8.0), 1.0 mM DTT containing linear concentration gradient of 0-0.5 M NaCI. The resulting fractions were analyzed for both calcium-binding activity (using the Chelex-100 competitive calcium-binding assay ~and calmodulin activity (assayed by phosphodiesterase activation7). Fractions from relevant peaks (peaks I-III) of calcium-binding activity were pooled (Fig. 1A) and subsequently calcium-binding proteins responsible for the calcium activity peaks were identified and purified, as depicted in Fig. 1.

Purification of CAB-27 from Peak I Step 1: Hydroxylapatite Chromatography of Peak I. The pooled peak I was dialyzed against buffer A (1.0 mM sodium phosphate buffer, pH 6.8, 1 mM DTT), and applied to a 5.0 × 30 cm column of hydroxylapatite previously equilibrated with buffer A. The column was washed with two column volumes of buffer A and eluted with 2 liters of buffer A containing linear phosphate gradient from 1.0 to 100 mM. Fractions (25 ml) were collected and analyzed for calcium-binding activity by Chelex assay (Fig. 1B). Only a single peak of calcium-binding activity was resolved between 50 and 80 mM phosphate concentration. The unbound fraction did not contain detectable calcium-binding activity. Step 2: Heat Treatment of the Calcium-Binding Activity Peak of Hydroxylapatite Chromatography. As shown in Fig. 1, the calcium-binding activity peak was then heated at 80° for 2 min and the denatured protein was removed by centrifugation at 30,000 g for 30 min. The supernatant was checked for calcium-binding activity by Chelex assay, and 80-85% activity was retained in the supernatant. Step 3: HPLC Gel Permeation Chromatography of the Heat-Treated Fraction. The supernatant was concentrated by ultrafiltration (PM 10; Amicon Corp.) and dialyzed against buffer B (40 mM MOPS, pH 7.1,150 mM NaCI, and 1 mM DTT), applied to a high-performance gel permeation liquid chromatography column (TSK-G3000 SW, LKB), and eluted with 7 T. S. Teo, T. H. Wang, and J. H. Wang, J. Biol. Chem. 248, 588 (1973).

FiG. 1. Purification procedure of CAB-27 and CAB-48. All steps were performed as described in the text. In the left column, the purification of CAB-27 is shown and in the right column, that of CAB-48 is shown. (A) DEAE-cellulose chromatography of bovine brain 100,000 g supernatant; (B) hydroxylapatite chromatography of peak 1; (C) HPLC gel permeation chromatography (TSK-G3000 SW) of the calcium-binding peak from hydroxylapatite chromatography; (D) hydroxylapatite chromatography of peak I1; (E) HPLC gel permeation chromatography (TSK-G3000 SW) of peak lla.

72

[6]

ISOLATION/CHARACTERIZATION OF Ca-BINDING PROTEINS

buffer B at a flow rate of 30 ml/hr. Fractions (3.0 ml) were collected and assayed for calcium-binding activity (Fig. 1C). A single peak of calciumbinding activity migrating with an apparent Mr 40,000 was resolved. This peak was identified as a novel calcium-binding protein, CAB-27. Summary of the Purification of CAB-27. SDS-PAGE analysis of the CAB-27-containing fractions at various stages of purification is shown in Fig. 2. Results from a representative purification scheme are summarized in Table I. From 1 kg of bovine brain 33 mg of CAB-27 was obtained. This represents a purification of 312.5-fold with a yield of 25.1%.

Purification of CAB-48from Peak H Step I: Hydroxylapatite Chromatography of Peak 11. Peak I1 from DEAE-cellulose chromatography was pooled and dialyzed against buffer

150.0K J J

116.OK 97.4K 66.2K 45.0K 29.0K

P

20.1K - a

b c

d e

f

g h

i

j

FIG. 2. SPS-polyacrylamide electrophoretic analysis at different stages of CAB-27 and CAB-48 purification. Aliquots of pooled fractions obtained during CAB-27 and CAB-48 purification were subjected to 5-20% SDS-polyacrylamide gel electrophoresis. (a,j) Molecular weight standards; (b) first peak of TSK-G3000 SW of peak Ila; (c) hydroxylapatite peak lla; (d) DEAE-cellulose peak II; (e) bovine brain 100,000 g supernatant; (f) DEAE-cellulose peak I; (g) hydroxylapatite of peak I; (h) after heat treatment; (i) TSK-G3000 SW of peak I.

[6]

73

NOVEL Ca-BINDING PROTEINS FROM BOVINE BRAIN TABLE I PURIFICATION OF CAB-27 AND CAB-48"

Purification step 1. 2. 3. 4. 5.

100,000 g supematant DEAE-cellulose Hydroxylapatite Heat treatment TSK-G3000 SW

Total protein (mg) b

Amount of CAB-27 (mg)"

Yield (%)

Purification (fold)

41,020 3,025 176 66 33

132 82 53 42 33

100 62.3 40.3 31.6 25.1

1.0 8.5 71.6 149.6 312.5

100 75.3 47.3 30.1

1.0 5.1 250.7 440.5

Amount of CAB-48 (mgy 1. 2. 3. 4.

100,000 g supematant DEAE-cellulose Hydroxylapatite TSK-G3000 SW

41,020 2,037 83 28

93 70 44 28

" Purification is from ! kg of bovine brain. b Protein is determined according to M. M. Bradford [Anal. Biochem. 72, 248 (1976)], using bovine serum albumin as a standard. As determined by densitometric analysis of SDS-PAGE.

A and applied on a 5.0 x 30 cm column of hydroxylapatite previously equilibrated with buffer A. The column was washed with two column volumes of buffer A and eluted with 4 liters of buffer A containing two different linear phosphate gradients (0-2.5 liters: 10-200 mM; 2.5-4.0 liters: 200-400 mM). Fractions (20 ml) were collected and analyzed for calcium-binding activity by Chelex assay (Fig. 1D). Two major calciumbinding peaks were resolved. The first peak (peak IIa) was eluted between 60 and I00 mM phosphate concentration, and the second peak (peak lib) was eluted between 120 and 160 mM. Both peaks were individually pooled, concentrated by ultrafiltration (PM 10), and dialyzed against buffer B. Step 2: HPLC Gel Performance Chromatography of Peak fla. Peak IIa of hydroxylapatite chromatography was applied to the high-performance gel permeation liquid chromatography column (TSK-G3000 SW). The column was eluted with buffer B at a flow rate of 30 ml/hr and fractions (3.0 ml) were collected and assayed for calcium-binding activity and calcineurin activity 8 (Fig. 1E). Analysis of peak IIa by gel permeation chromatography resolved two calcium-binding activity peaks migrating C. J. Pallen and J. H. Wang, J. Biol. Chem. 258, 8550 (1983).

74

ISOLATION/CHARACTERIZATION OF Ca-BINDING PROTEINS

[6]

T A B L E II CHARACTERIZATION OF CAB-27 AND CAB-48

Properties

CAB-27

CAB-48

Molecular weight by SDS-PAGE Amount of protein/kg brain (mg) Extinction coefficient (e2761%) Percentage of acidic residuesa Percentage of basic residuesa Isoelectric point (IpH) Kd for Ca2+ (IJ,M ) b Maximum Ca2÷ binding (mol Ca2+/mol protein) Kd for Zn2+ (I~M)b Maximum Zn2+ binding (mol Zn2+/mol protein)

27,000 132 5.722 26 13 4.8 0.22 2.0 26.5 2.0

48,000 93 9.661 24 12 4.7 15.4 1.0 38.8 1.0

a Calculated from amino acid composition analysis. b Measured by equilibrium dialysis in the presence of 150 mM KCI, 3 mM MgCI2.

with apparent Mr 75,000 and 72,000 (Fig. 5). The higher molecular weight peak was identified as a novel calcium-binding protein, CAB-48. Summary o f the Purification of CAB-48. S D S - P A G E analysis of the CAB-48 containing fractions at various stages o f purification is shown in Fig. 2. Results from a representative purification scheme are summarized in Table I. F r o m 1 kg o f bovine brain 28 mg of CAB-48 was obtained. This represents a purification of 440.5-fold with a yield of 30.1%.

Characterization of CAB-27 and CAB-48 The summary of the characterization study of CAB-27 and CAB-48 is shown in Table II. Both proteins are different from each other and they appear to bind Ca z+ under physiological conditions. Characterization of the Chelex Competitive Calcium-Binding Assay as a Tool to Detect Calcium-Binding Proteins The Chelex-100 is a resin of styrene divinylbenzene with acetate as its functional group. The resin binds a variety of divalent cations including calcium and has been used to determine the stability constants of several cation-binding substances. 9-1~ Waisman and Rasmussen I have carried out extensive analysis of this assay system and reported its use for the detec9 j. Shubert, J. Physiol. Colloid Chem. 52, 340 (1948). lop. j. Bredeerman and R. H. Wasserman, Biochemistry 13, 1687 (1974). IID. J. Hartshorne and H. U. Pyan, Biochim. Biophys. Aeta 229, 698 (1971).

[6]

NOVEL Ca-BINDING PROTEINS FROM BOVINE BRAIN

75

tion of calcium-binding proteins. The Chelex assay system has many advantages over the traditional methods for detection of calcium-binding proteins. It offers higher sensitivity and is able to detect even the heatlabile calcium-binding activity. The Millipore filtration method ~2 is less sensitive as protein may be lost through the Millipore filters. The equilibrium dialysis method 13is very time consuming and has a high background value. The 45Ca2+ autoradiographic analysis 6 can only detect the heatstable components of calcium-binding activity of crude extracts. Results presented in this study indicate that the Chelex assay can be effectively used for the qualitative detection of calcium-binding activity in tissue extracts or biological fluids. Necessary precautions and the limitations of the assay system are also discussed. Procedure of the Chelex Assay. 45Ca2+ (5 mCi/ml) was obtained from New England Nuclear. Double-distilled deionized water was used to prepare all buffers. Two-milliliter plastic tubes were obtained from Nunc (55 x 11, PS SH 3-61239), and Chelex-100 resin (100-200 mesh) from Bio-Rad Laboratories. The resin was washed thoroughly before use with 1 M HCI, adjusted to pH 7.4 with NaOH, and washed extensively with 40 mM TrisHCI (pH 7.4) and resuspended in buffer of the same composition at a resin-to-liquid ratio of 1/10. The slurry was stirred rapidly before addition. To a final reaction mixture volume of 1.0 ml were added variable concentrations of test substance, 45CAC12(0.5-1.0 × 106 cpm final in assay), and 25 ~1 of Chelex-100 resin. To assure that the binding equilibrium is attained, the mixture was incubated at room temperature with continued rotatory agitation. After 30 min, the tubes were centrifuged at 3000 g for 5 min and 0. l-ml aliquots of the supernatants were added to 5 ml of Scinti Verse I (Fischer Scientific, Canada) and were subjected to liquid scintillation spectrometry. A control sample of Chelex-100 and reaction media without the protein fraction were included in each assay. The results of the Chelex assay are expressed a s 45Ca2+ bound (cpm in test supernatant-cpm in control). Typically the control supernatant contains 2-4% of added radioactivity. Dose-Response Curve of Ca2+-Binding Proteins. Purified calmodulin, CAB-27, and CAB-48 were used to evaluate the linearity and sensitivity of the Chelex assay. As shown in Fig. 3, the assay is linear from 0.05 to 1.0/xM protein concentration. Since these proteins differ in their affinities and in binding capacities for calcium, the assay exhibits different sensitivity to each protein. The protein with a high Kd value and/or large calciumbinding capacity is more sensitive in the Chelex assay than the protein with a low Kd value and/or small calcium-binding capacity. In an earlier iz A. Martonosi and R. Feretos, J. Biol. Chem. 239, 648 (1964). 13 y . p. M y e r and J. A. Scheilman, Biochim. Biophys. Acta 55, 361 (1962).

76

ISOLATION/CHARACTERIZATION OF Ca-BINDING PROTEINS

[6]

120 ~-'-I00 b 13-

c~60 Z

~ 4o ~ 2o 0o

0.5

1.0

1.5 2D 2.5 PROTEIN(pM)

3.0

3.5

4.0

FIG. 3. Dose-response curve of calcium-binding substances. The Chelex competitive calcium-binding assay was performed to check the sensitivity of this assay by using calmodulin ( A - - & ) , CAB-27 ( I - - t ) , and CAB-48 (rq--E]). Assay condition was described in the text. All proteins were dialyzed against 5 mM Tris-HCl (pH 7.4) before the assay. From the supernatant, 0.1-ml aliquots were taken and counted in the liquid scintillation counter.

report, 1 we have demonstrated a qualitative correlation between the stability constants of Ca 2÷ binding (K0 and the concentration of binder required to displace 50% total counts (As0~). While the Chelex assay appears to be of use for the detection of Ca2+-binding activity of pure proteins, it is also linear with crude tissue homogenates) Effect of Salts on the Chelex Assay. The ionic strength of the test sample has a profound effect on the sensitivity of the assay (Fig. 4). 10

8 Z O

\

%. \

"lk

J

"'-9..

,,

~r v-

t~ 4

8, LL

2

°0

i

~

;

;

lb 17 iX 1; 1; 20

CONCENTRATION(mM) FIo. 4. Effects of several salts on the Chelex assay. The effects of NaCI ( © - - © ) , KCI (Q---O), and phosphate ( & - - - A ) on the Chelex-100 assay were determined. Fold stimulation was calculated as follows:

Fold stimulation =

cpm in the presence of 10/~g of calmodulin

cpm in the absence of calmodulin

[6]

NOVEL Ca-BINDING PROTEINS FROM BOVINE BRAIN

77

Increasing the ionic strength produces large decreases in sensitivity. Furthermore, the largest decreases in sensitivity due to ionic strength occur at the lower Chelex concentrations. Among various salts tested, phosphate had a strong inhibitory effect on the assay sensitivity, while sodium, potassium, and chloride ions had lesser effect. This result suggests the necessity of dialyzing the salt-containing fractions against the no-saltcontaining buffer before the assay. Effect of EDTA and EGTA on the Chelex Assay. Both EDTA and EGTA are able to bind calcium with high affinity, therefore the trace amounts of these compounds (<0.05 mM) are able to interfere with the assay (data not shown). Extensive dialysis of EDTA- or EGTA-containing fractions is recommended to achieve greater sensitivity and reliability of the assay. Effect of Chelex Concentration on Assay Sensitivity. Figure 5 shows that increasing the amount of Chelex in the assay results in a reduction in the sensitivity of the assay to a fixed concentration of calmodulin (1/zM). In order to optimize the sensitivity of the assay, it is necessary to determine the minimum quantity of Chelex required to give good separation between percentage total counts -+Ca2+-binding protein, i.e., calmodulin. If Chelex concentration in the assay is too low or too high (particularly with crude samples), the two curves approach each other and the resolution is lost. We have also found that increasing the concentration ofaSCa2+ (>5 × 106 cpm) in the assay does not increase the assay sensitivity. Summary of Chelex Competitive Calcium-Binding Assay. Based on these studies, we conclude that the Chelex assay is useful for the detection of calcium-binding proteins in the tissue homogenates. It is important to point out that the assay must be carried out in plastic tubes with rotatory agitation. The test sample should be extensively dialyzed to achieve lower ionic strength. The amount of Chelex to be used in the assay system should be carefully selected to maximize the sensitivity of

100' 'o

,~80 E

#

60

~= 20 0 0

20 40 60 BO 100 [HELEX-IOO(~t)

FIG. 5. Effect of Chelex concentration on a s s a y sensitivity. Calcium binding was a s s a y e d at a fixed 45Ca concentration (0.5 x 106 c p m in the assay) and various Chelex concentrations in the presence ( 0 ) or a b s e n c e (O) of 1 /zM calmodulin.

78

ISOLATION/CHARACTERIZATION OF Ca-BINDING PROTEINS

[6]

lOO 80 .~ 6C ~- 4C x .<

x 2C 0

20

/*0 60 CAB-27(~Jg)

80

FIG. 6. Inhibition of phospholipase A2 by purified CAB-27. Aliquots of purified CAB-27 were incubated with 0. I/zg bovine pancreas phospholipase A_~and the samples were assayed for phospholipase A2 activity using e-a-l-palmitoyl-2-[t4C] oleoylphosphatidylcholine as substrate. Antiphospholipase A2 activity of calmodulin (©), parvalbumin (A), CAB-48 Ix ), S-100 (V), and calregulin ([3) were assayed.

this assay in the detection of unknown calcium-binding substances in tissue extracts. Physiological Function of CAB-27 and CAB-48 Phospholipase Az inhibitory proteins have been identified in several cells including rat macrophages, 14 rat renal medullary cells, ~5 rabbit neutrophils,16 and bovine thymus.17 The predominant form of this inhibitory protein is a Mr 40,000 protein called lipocortin. While lipocortin has been localized to many tissues, it has been shown to be absent from brain tissue. 18 The presence of antiphospholipase Az brain proteins has never been directly addressed. As shown in Fig. 6, purified CAB-27 inhibits phospholipase A2 activity in a dose-dependent manner. In contrast, other brain CaZ+-binding proteins, such as CAB-48, calregulin, calmodulin, S100, and parvalbumin, had little effect on phospholipase A: activity. Our data establish CAB-27 as an in vitro regulator of phospholipase A2. In contrast, the physiological function of CAB-48 is unknown. t4 G. J. Blackwell, R. Carnuccio, M. DiRosa, R. J. Flower, C. J. S. Langham, L. Parente, P. Persico, N. C. Russell-Smith, and D. Stone, Br. J. Pharmacol. 76, 185 (1982). 15 F. Hirata, J. Biol. Chem. 256, 7730 (1981). 16 B. Rothhut, F. Russo-Marie, J. Wood, M. DiRosa, and R. J. Flower, Biochem. Biophys. Res. Commun. 117, 878 (1983). 17 C. Gupta, M. Katsumata, A. S. Goldman, R. Herold, and R. Piddington, Proc. Natl. Acad. Sci. 81, 1140 (1984). ts R. B. Pepinsky, L. K. Sinclair, J. L. Browning, R. J. Mattaliano, J. E. Smart, E. P. Chow, T. Falbel, A. Ribolini, J. L. Garwin, and B. P. Wallner, J. Biol. Chem. 261, 4239 (1986).

[7]

CALMODULIN-DEPENDENT

PROTEIN

PHOSPHATASE

79

Acknowledgments Supported by a grant from the Medical Research Council of Canada. M.T. and N.C.K. are recipients of an Alberta Heritage Foundation for Medical Research Postdoctoral Fellowship.

[7] C a l m o d u l i n - D e p e n d e n t P r o t e i n P h o s p h a t a s e : Isolation o f S u b u n i t s and R e c o n s t i t u t i o n to H o l o e n z y m e

By DENNIS L. MERAT and WAI Ylu CHEUNG Calmodulin-dependent protein phosphatase, also referred to as modulator-binding protein, L2 CaM-BP80, 3 and calcineurin, 4 is a heterodimer consisting of subunit A (Mr 60,000) and subunit B (Mr 19,000). Subunit A holds the catalytic and calmodulin-binding sites 2.4.5 whereas subunit B, which has no phosphatase activity 5 and 35% sequence homology with calmodulin, 6 binds four Ca 2÷ ions with high affinity (Kd is about 1 ~M or less). 4

The enzyme has broad substrate specificity, removing phosphate groups from both protein 7-13 (phosphorylated on serine, threonine, or tyrosine residues) and nonprotein 14(such as p-nitrophenylphosphate) subi j. H. Wang and R. Desai, J. Biol. Chem. 252, 4175 (1977). 2 R. K. Sharma, R. Desai, D. M. Waismah, and J. H. Wang, J. Biol. Chem. 254, 4276 (1979). 3 R. W. Wallace, E. A. Tallant, and W. Y. Cheung, Biochemistry 19, 1831 (1980). 4 C. B. Klee, T. H. Crouch, and M. H. Krinks, Proc. Natl. Acad. Sci. U.S.A. 76, 6270 (1979). 5 M. A. Winkler, D. L. Merat, E. A. TaUant, S. Hawkins, and W. Y. Cheung, Proc. Natl. Acad. Sci. U.S.A. 81, 3054 (1984). 6 A. Aitken, C. B. Klee, and P. Cohen, Eur. J. Biochem. 139, 663 (1984). 7 A. A. Stewart, T. S. Ingebritsen, A. Manalan, C. B. Klee, and P. Cohen, FEBS Lett. 137, 80 (1982). 8 S.-D. Yang, E. A. Tallant, and W. Y. Cheung, Biochem. Biophys. Res. Commun. 106, 1419 (1982). 9 C. B. Klee, M. H. Krinks, A. S. Manalan, P. Cohen, and A. A. Stewart, this series, Vol. 102, p. 227. l0 j. Chernoff, M. A. Sells, and H.-C. Li, Biochem. Biophys. Res. Commun. 121, 141 (1984). i1 M. M. King, C. Y. Huang, P. B. Chock, A. C. Nairn, H. C. Hemmings, Jr., K.-F. J. Chan, and P. Greengard, J. Biol. Chem. 259, 8080 (1984). 12 C. J. Pallen, K. A. Valentine, J. H. Wang, and M. D. Hollenberg, Biochemistry 24, 4727 (1985). ~3 S. Goto, H. Yamamoto, K. Fukunaga, T. lwasa, Y. Matsukado, and E. Miyamoto, J. Neurochem. 45, 276 (1985). 14 C. J. Pallen and J. H. Wang, J. Biol. Chem. 258, 8550 (1983).

METHODS IN ENZYMOLOGY, VOL. 139

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.