Calcium-binding proteins in electroplax and skeletal muscle comparison of the parvalbumin and phosphodiesterase activator protein of Electrophorus electricus

Biochh~ica et Biop/o'sica Acre, 439 (1976) 316 325 ~'~ Elsevier Scientific Publishing Company. Amsterdam

I'rmted in The Netherlands

BBA 37418 CALCIUM-BINDING MUSCLE

PROTEINS

IN

ELECTROPLAX

AND

SKELETAL

C O M P A R I S O N OF T H E P A R V A L B U M I N A N D P H O S P H O D I E S T E R A S E A C T I V A T O R P R O T E I N OF E L E C T R O P H O R U S E L E C T R I C U S

STEVEN R. CHILDERS and FRANK L. SIEGEL Departments ~f Physiological Chemistry and Pediatrics, University of Wisconsin Center ]'or Health Sciences, Madison, Wisc. 53706 (U.S.A.)

(Received February 23rd, 1976)

SUMMARY A soluble calcium-binding protein has been isolated from the red skeletal muscle of the electric eel (Electrophorus electricus). The purification procedure involved ammonium sulfate precipitation, gel filtration on Sephadex G-75 in the presence of 45Ca2+, and chromatography on QAE-Sephadex. This procedure resulted in the isolation of a protein which was homogeneous upon polyacrylamide gel electrophoresis. The calcium-binding protein was found to be a typical parvalbumin by the following criteria: (1) molecular weight of l l 000; (2) pl of 4.7; (3) 1.9 tool Ca 2+ bound per tool protein; Kd of approx, l0 -v M; (4) no detectable phosphorus: (5) amino acid composition included nine residues of phenylalanine, single arginine, and no tyrosine or tryptophan; (6) emax at 259 nm; (7) 260 rim:280 nm absorbance ratio of 4.78. Only one parvalbumin could be detected in muscle. Immunoprecipitation assay revealed that the parvalbumin was a major soluble component of skeletal muscle (0.10 mg/mg soluble protein), but could not be detected in liver, kidney, brain, spleen, heart or electroplax. Comparison of the parvalbumin with a calcium-binding protein previously isolated from electroplax revealed that the two proteins were different as judged by a variety of chemical criteria. These results suggest that during embryological development of electroplax the parvalbumin is lost and that it is not required for the function of electric tissue.

INTRODUCTION Calcium has been shown to participate in several important synaptic events and therefore to play a key role in neurotransmission. The release of transmitters from vesicular stores [1, 2], the binding of transmitters to post-synaptic receptors [3], and the activities of such enzymes as tyrosine hydroxylase [4], adenylate cyclase [5, 6], and cyclic nucleotide phosphodiesterase [7, 8, 9] all appear to be calcium dependent. The investigation of the role played by calcium in the regulation of synaptic events in brain is complicated by the cellular heterogeneity of this tissue; nevertheless

317 it has been established that the calcium-dependent activities of adenylate cyclase and cyclic nucleotide phosphodiesterase in brain are stimulated by a calcium-binding protein (calcium-dependent regulator) in brain [5, 7], and calcium-sensitive contractile proteix~s have been implicated to play a key role in neurotransmitter release [10]. In an effort to clarify the roles of calcium-binding proteins in synaptic actions we have utilized electroplax of Electrophorus electricus as a model of the cholinergic synapse uncomplicated by the presence of mixtures of other types of neurons. We have recently reported the isolation from electroplax of a low molecular weight, acidic calcium-binding protein which is identical to the mammalian activator protein of phosphodiesterase [11]. Since electroplax is embryologically derived from skeletal muscle and contains the muscle protein tropomyosin [12], and since fish skeletal muscle contains low molecular weight, acidic calcium-binding proteins called parvalbumins [13], it became necessary to determine whether the electroplax calciumbinding protein was a parvalbumin. To resolve this question we have isolated the parvalbumin from skeletal muscle of Electrophorus electricus and compared the chemical and physical properties of the parvalbumin with those of the calciumbinding protein previously isolated from electroplax. MATERIALS AND METHODS

Materials. Electric eels (E. electricus) were obtained live from Paramount Research Supply Co. (Ardsley, N.Y.) and were usually about 4 ft in length. 45CaCl, (15.9 Ci/g) was purchased from New England Nuclear. Acrylamide (electrophoresis grade) was obtained from Eastman Chemicals, while (NH4)2SO4 and Tris were ultrapure reagents from Schwarz-Mann. Ampholine ampholytes (pH 3-6) were products of LKB. All other chemicals were reagent grade. Extraction of parvalbumin from eel skeletal muscle. Following decapitation, eels were placed on ice and muscle was dissected from electroplax. Approx. 1 kg of skeletal muscle could be obtained from each eel. A portion of the muscle, along with other organs, was immediately frozen at 70 °C. Subsequent experiments showed that calcium-binding activity in muscle was stable at --70 °C for at least 1 year. To isolate the calcium-binding protein, muscle was minced with scissors, homogenized for 2 min in a Waring blendor with four volumes of 50 mM Tris. HC1, pH 7.4, and soluble proteins, precipitating between 70 and 100[~/,, saturated (NH4)2SO 4, were extracted as previously described [11 ]. This mixture of muscle proteins was lyophilized and stored at 70 °C. Aliquots of the lyophilized powder were then taken for subsequent purification. PurOqcation of the parvalbumin. Approx. 200 mg of lyophilized soluble muscle proteins were dissolved in 5 ml of 50 mM Tris. HCI, pH 7.4, and centrifuged at 10 000 >~ g for I0 rain to remove residual particulate matter. The sample was incubated with 25 ~uCi of 4SCaC12 for 30 rain and applied to a column (2.5 ~,: 100 cm) of Sephadex G-75 which had been equilibrated with 50 mM Tris. HCI, pH 7.4. The column was eluted with upward flow of the same buffer, 2.5-ml fractions were collected, and protein was determined with 0. l-ml aliquots by the method of Lowry et al. [14]. Radioactivity was determined in a Packard TriCarb liquid scintillation spectrometer using 0.1-ml aliquots in 10 ml of Bray's scintillator [15]. The peak of calcium-binding activity from Sephadex G-75 was pooled, con-

318 centrated in a Diaflo apparatus utilizing Amicon PM-10 membranes, and applied to a column (2 ~< 25 era) ofQAE-Sephadex A-50 which had been swollen and equilibrated in 50 mM Tris- HC1, pH 7.4. The column was eluted with the same buffer, 4-ml fractions were collected, and the absorbance at 254 nm monitored. Following elution of the non-adsorbed material, the column was eluted with a linear gradient consisting of 250 ml of 50 mM Tris. HCI, pH 7.4, and 250 ml of 50 mM Tris. HC1, pH 7.4, containing 0.1 M NaC1. Each protein peak was assayed for calcium-binding activity by equilibrium dialysis in the presence of 45CaC~z. Electroplax calcium-binding protein. Calcium-binding protein was isolated and purified from eel electroplax as described previously [11], using ammonium sulfate precipitation, boiling treatment, Ecteola-cellulose chromatography, and gel filtration on Sephadex G-100. Polyacrylamide gel electrophoresis. Muscle and electroplax protein samples were analyzed on 15 ~ polyacrylamide gels according to the method of Davis [16]. Polyacrylamide gels (10~,~) containing 0.1 ..... ~,, sodium dodecyl sulfate were used for molecular weight determination, with pepsin, trypsin, ovalbumin, myoglobin, cytochrome c, and a-chymotrypsin utilized as standards according to the method of Dunker and Rueckert [17]. Isoelectric focusing. The isoelectric point of the purified parvalbumin was determined by isoelectric focusing in 15~/o~ polyacrylamide gels by the method of Wrigley [18], using pH 3-6 Ampholine ampholytes. Calcium measurements. In order to measure calcium bound to the parvalbumin, the purified protein was dialyzed against 50 mM Tris. HCI containing 10 mM CaCl2. Non-specifically bound calcium and buffer were removed by passage of the protein through a column (1.5 7~ 50 cm) of Sephadex G-25 equilibrated with distilled water. This procedure corrected for any high affinity bound calcium lost during the purification process. Calcium was measured by atomic absorption using a Perkin-Elmer model 305B spectrometer. Calcium-binding experiments were conducted using Chelex resin according to the method of Briggs and Fleishman [19]. Determination of the number of calcium-binding proteins in eel muscle. Skeletal muscle crude homogenate was incubated with 10/~Ci of 4SCaC12 for 30 min, then applied to duplicate 15 ~ polyacrylamide gels and subjected to electrophoresis at pH 8.3. One gel was stained with l o//oAmido-Schwarz and destained in 7 j,% acetic acid. The second gel was frozen, sliced, and radioactivity was determined as previously described [20]. lmmunologicalprocedures. Antiserum to the purified parvalbumin was prepared in rabbits by two injections at 10-day intervals in multiple subcutaneous sites of 2 mg protein in stable suspension with complete Freund's adjuvant (Difco). 1 week later each rabbit was given an intravenous injection of 100/zg protein; this was repeated the following week, and the rabbits were bled 10 days later. Quantitative immunoprecipitation assay of eel parvalbumin in eel tissues was performed by the method of Maurer [21]; precipitated protein was determined by the method of Lowry et al. [14] using rabbit )~-globulin as a standard. The maximum sensitivity of the assay was 500 ng of parvalbumin and the equivalence point was approx. 5/tg parvalbumin. Eel tissues were prepared for assay by homogenization and ammonium sulfate precipitalion as described earlier for eel skeletal muscle. Both crude homogenates and 70-100 ~'il (NH~)2SO~-precipitated fractions were assayed for the presence of parvalbumin.

319

Other methods. Nitrogen analysis of the purified protein was accomplished by the method o f J o h n s o n [22]; this analysis provided a factor of 5.2 for the conversion of absorbance at 260 n m to mg/ml o f purified protein. Phosphorus content of the protein was determined on ashed, neutralized samples by the method of Chen et al. [23]. The ultraviolet spectrum of the parvalbumin was determined on a Varian Model 635 spectrophotometer, while amino acid composition was analyzed as previously described [20]. RESULTS

Purification of the eel muscle calcium-binding protein. Since fish parvalbumins bind 2 mol of calcium with high affinity [24] it was decided to detect and to monitor the purification of such a protein from eel muscle by its ability to bind 45CaZ+. A crude mixture o f soluble muscle proteins (precipitated between 70 and 100 ~ saturated (NH4)2SO4) was incubated with 45CaClz and subjected to gel filtration on Sephadex G-75. The results (Fig. 1) showed a significant peak of 45CaZ+-binding activity e]uting with the low molecular weight muscle proteins. This calcium-binding peak was further purified by c h r o m a t o g r a p h y on QAE-Sephadex (Fig. 2). Equilibrium dialysis in the presence o f 4sCaClz showed that only the second protein peak possessed significant calcium-binding activity. Polyacrylamide gel electrophoresis (pH 8.3) of the second peak from QAE-Sephadex revealed a single protein band. Polyacrylamide gel electro-

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Fig. 2. Chromatography of calcium-binding peak from Sephadex G-75 on QAE-Sephadex. The calcium-binding peak from Sephadex G-75 was pooled, concentrated to 2 ml with an Amicon PM-10 Diaflo apparatus, applied to a column (2 × 25 cm) of QAE-Sephadex, and eluted with 50 mM Tris. HCI, pH 7.4. Following elution of the first peak, a linear gradient of 0-0.1 M NaCI in the same buffer was begun. Fraction volume was 4 ml. All significant calcium-binding activity eluted with Peak H. ------, absorbance at 254 nm : , [NaCI].

phoresis at pH 4.5 and in the presence of sodium dodecyl sulfate at pH 8.3 also revealed only one protein band in the sample. Properties of the eel muscle calcium-binding protein. Molecular weight of the muscle protein was estimated at 11 000 on sodium dodecyl sulfate-polyacrylamide gels: the minimum molecular weight, calculated from the amino acid composition, assuming one residue of arginine, was 11 648. Calcium measurements of the protein revealed 1.9 mol calcium bound per mol protein. Prolonged dialysis of the protein (24 h) against distilled water reduced the calcium content to 0.92 mol per mol protein. The dissociation constant for calcium binding could not be exactly determined because of difficulties in reducing the level of free [Ca 2+] in the region o f high affinity binding. The best estimates from Chelex resin experiments revealed high affinity binding with a dissociation constant of approx. 10 -7 M. The ultraviolet spectrum for the purified eel muscle protein revealed a maximum absorbance peak at 259 nm, with smaller peaks at 253, 265, and 269 nm. These results are identical to spectra obtained for parvalbumins from fish [13] and rabbit [25, 26]. Comparison qf eel muscle and electroplax calcium-binding proteins. The amino acid compositions of the two calcium-binding proteins are presented in Table I. A m o n g other differences, the electroplax protein did not possess the typical parvalbumin composition seen in the case of the muscle protein (i.e. 0 tyrosine, 0 tryptophan, 9 phenylalanine, 1 arginine). A comparison of the properties of the two eel calcium-binding proteins with those o f the fish parvalbumins is seen in Table II. The eel muscle protein shared several properties with the fish parvalbumin, including calcium-binding, molecular

321 TABLE 1 A M I N O ACID COMPOSITIONS OF EEL MUSCLE AND ELECTROPLAX CALCIUMBINDING PROTEINS Amino acid

Aspartic acid I-hreonine Serine Proline Glutamic acid Glycine Alanine Valine Cysteine Methionine lsoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Tryptophan Arginine

Mol amino acid per mol protein Muscle

Electroplax a

12.8 4.7 4.6 0.0 7.6 7.3 14.6 3.1 0.0 0.7 3.6 7.2 0.0 8.6 I 1.9 0.6 0.0 b 0.9

15.6 8.4 3.5 2.6 19.0 7.9 7.5 4.5 0.0 2.9 6.7 10.0 0.9 6.0 6.0 1.1 3.9

" From reference I1. b Determined spectrophotometrically by the method of Spande and Witkop[31]. TABLE 11 COMPARISON OF EEL MUSCLE CALCIUM-BINDING PROTEIN WITH FISH PARVALBUMINS AND ELECTROPLAX CALCIUM-BINDING PROTEIN Property

Fish parvalbumins a

Eel muscle protein

Electroplax protein

tool Ca 2+ bound Ka for Ca 2+ binding Molecular weight pl Phosphorus e259 .... ( m M - l ' c m -~) 260 nm :280 nm ratio

2 10 -v M 11 000-12 000 4-5 0 1.9-2.3 ~> 1

1.9 Approx. 10 -7 M 11 000 4.7 0 2.24 4.78

3 2.1- 10 -s M 13 000 4.3 1 per tool protein 8.97 0.88

~ Adapted from Demaille et al. [13]. w e i g h t , i s o e l e c t r i c p o i n t , p h o s p h o r u s c o n t e n t , m o l a r a b s o r p t i v i t y coefficient, a n d 260 n m : 2 8 0 n m a b s o r b a n c e r a t i o . T h e r e f o r e , t h e eel m u s c l e p r o t e i n c o u l d be c o n s i d e r e d as a t y p i c a l m u s c l e p a r v a l b u m i n . T h e eel e l e c t r o p l a x c a l c i u m - b i n d i n g p r o t e i n , a l t h o u g h similar with regards to m o l e c u l a r weight a n d acidic pl, was different f r o m t h e p a r v a l b u m i n s in c a l c i u m - b i n d i n g , p h o s p h o r u s c o n t e n t , m o l a r a b s o r p t i v i t y , a n d 260 n m : 2 8 0 n m a b s o r b a n c e r a t i o . T h e r e f o r e , t h e e l e c t r o p l a x p r o t e i n is n o t a p a r r albumin.

322

Number of parvalbumins hi eel skeletal muscle. Parvalbumins were assayed in skeletal muscle on the basis of their calcium binding in 15°~, polyacrylamide gels. Results of the experiment (Fig. 3) showed only one peak of ~SCaZ+-binding activity in the soluble muscle preparation. This peak corresponded to the band of the purified muscle protein. Therefore the protein was the only parvalbumin present in eel skeletal muscle. A similar experiment performed with electroplax homogenates suggested that the electroplax protein was the only soluble calcium-binding protein in that tissue.

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Fig. 3. Analysis of parvalbumins in eel muscle by polyacrylamide gel electrophoresis of proteinbound calcium. Duplicate samples (approx. 150/tg) of proteins from the supernatant of muscle homogenate were incubated with 0.5/~Ci of 4SCaClz, layered on gels with 5 ~ sucrose, and fractionated by electrophoresis at pH 8.3. One gel was stained with 1~ Amido-Schwarz and destained in 7 % acetic acid. A second gel was sliced into l-ram sections, and each section was dissolved in 0.2 ml of 30~ H202 at 50 °C, treated with 1 ml of Nuclear Chicago solubilizer, and counted in 10 ml of a (0.55 %, w/v) solution of permablend in toluene. Top gel: purified parvalbumin; bottom gel: duplicate stained gel of muscle supernatant.

Tissue distribution of the eel parvalbumin. Antiserum prepared against the purified parvalbumin reacted with skeletal muscle homogenates on Ouchterlony double diffusion plates, but did not react with homogenates of eel brain, spleen, liver, kidney, or electroplax. A similar experiment using 70-100 ~ saturated (NH4)zSO4 precipitates of eel tissue supernatants revealed precipitin lines with only skeletal muscle (Fig. 4).

323

Fig. 4. Ouchterlony double diffusion assay of parvalbumin in eel tissues. Electric eel tissues were homogenized and the soluble portion was precipitated between 70 and 100~ (NH4)2SO4. The pellets were dissolved in and dialyzed against 50 mM Tris. HCI, pH 7.4 (protein concentration approx. 8 mg/ml). Aliquots from each tissue were placed in the side wells. Center well: antiserum to purified parvalbumin. Side wells: P, purified parvalbumin; S, spleen; L, liver; H, heart; M, skeletal muscle; K, kidney; B, brain; E, electroplax. More sensitive measurements of tissue levels of parvalbumin were made using quantitative immunoprecipitation. Results showed the parvalbumin in skeletal muscle at the level of 0.10 mg/mg protein. Levels of parvalbumin in all other eel tissues (brain, spleen, liver, kidney, heart and electroplax) were consistently beneath the level of sensitivity for the assay (i.e. <0.0004 mg/mg soluble protein). Therefore the eel parvalbumin appears to be localized in skeletal muscle only. Preliminary .studies have been conducted to determine cross-reactivity of the eel parvalbumin antiserum with muscle from other species. These results have demonstrated no cross-reactions with muscle from mollusk (barnacle), amphibian (frog), or mammalian (bovine) sources, but significant cross-reaction with white muscle of goldfish. DISCUSSION This report demonstrates that a parvalbumin can be purified from the soluble fraction of electric eel skeletal muscle by monitoring its calcium-binding activity during gel filtration on Sephadex G-75. Because of its high calcium-binding affinity (Kd approx. 10 -v M), the protein retains bound calcium during gel filtration. The 4sca 2+ gel filtration experiment thus permits the detection ofparvalbumins in relatively crude mixtures of proteins, unlike earlier parvalbumin assays which characterized these proteins by their unusually high 260 nm:280 nm absorbance ratio and which therefore can be used only in comparatively pure preparations [25, 27]. Calcium measurements revealed that although gel filtration on Sephadex G-25 did not remove any high affinity bound calcium (1.9 mol/mol protein), prolonged dialysis against water removed one of the two bound calciums. This result is equivalent to the effect seen by Donato and Martin [28] with carp parvalbumin following short dialysis periods against ethyleneglycol-bis-(fl-aminoethylether)-N,N'-tetraacetic acid.

324 Analysis of the purified calcium-binding protein revealed properties typical of the fish parvalbumins. Moreover, immunoprecipitation results showed that the protein was a major soluble component of eel skeletal muscle (0.10 rag/rag soluble prorein). Unlike the white muscle of other fish, which have been reported to contain several parvalbumins [29], electric eel red muscle contained only one parvalbumin, as measured by binding of 45Ca z+ to proteins in crude homogenates fractionated by polyacrylamide gel electrophoresis. The tissue distribution study of the eel parvalbumin revealed that the protein was present only in skeletal muscle. These results conflict with a previous immunological survey by Gosselin-Rey [30] who reported cross-reacted material in liver, kidney, and intestine, with smaller amounts in heart. Whether these differences represent species variation, differences in purity of antigen or different specificities of antiserum is not presently known. The principle reason for investigating parvalbumins in the electric eel was the possible use of the electric organ as a model of excitation-contraction coupling in a modified muscle tissue. Results of this study showed: (1) eel skeletal muscle parvalbumin was not present in electroplax; (2) although electroplax contained a soluble low molecular weight calcium-binding protein, it was not similar to the muscle protein and was not a typical parvalbumin; (3) electroplax homogenate did ~aot contain any other significant calcium-binding activity. These results suggest that in the process of developmental modification of muscle to electroplax, the parvalbumin has been lost. The possibility exists that the electroplax calcium-binding protein is a modified parvalbumin and performs the same function in electroplax that the parvalbumin does in skeletal muscle. This possibility, however, does not appear likely because: (1) the calcium-binding affinity of the electroplax protein is approximately two orders of magnitude lower than that of parvalbumins: (2) the electroplax protein is identical to the activator of mammalian cyclic nucleotide phosphodiesterase [I 1] ; although the electroplax protein can activate mammalian phosphodiesterase, the eel parvalbumin has no such activity (Childers, S. R., unpublished observations). It appears more likely that parvalbumin functions only in skeletal muscle, and not in either cardiac muscle or electroplax. This suggests that the parvalbumin is probably not directly involved in regulation of excitation-contraction coupling but instead may be a component of a skeletal muscle-specific calcium transport or calcium trapping system. ACKNOWLEDGEMENTS This study was supported in part by U.S. Public Health Service Grant NS 11652. S.R.C. acknowledges the support o f a predoctoral traineeship from the National Institutes of Health. We wish to thank Dr. Theo Gerritsen and Mr. Alan Hamstra for the amino acid analysis, Dr. S. Kornguth and Ms. Elaine Sunderland for the ultraviolet spectrum and Mr. James Campbell for excellent technical assistance. We also thank Dr. Jack C. Brooks for many helpful suggestions throughout this study. REFERENCES 1 Katz, B. and Miledi. R. (1965) Proc. R. Soc. Lond., Set. B, 161, 496-503 2 Douglas, W. W. (1968) Br. J. Pharmacol. 34, 451-474 3 Cohen, J. B., Weber, M. and Changeux, J.-P. (1974) Mol. Pharmacol. 10, 904-932

325 4 Morgenroth, I11, V. H., Boadle-Biber, M. and Roth, R. H. (1974) Proc. Natl. Acad. Sci. U.S. 71, 4283-4287 5 Brostrom, C. O., Huang, Y.-C., Breckenridge, B. M. and Wolff, D. J. (1975) Proc. Natl. Acad. Sci. U.S. 72, 64-68 6 von Hungen, K. and Roberts, S. (1973) Nat. New Biol. 242, 58-60 7 Wolff, D. J. and Brostrom, C. O. (1974) Arch. Biochem. Biophys. 163, 349-358 8 Cheung, W. Y. (1971) J. Biol. Chem. 246, 2859-2869 9 Teo, T. S. and Wang, J. H. (1973) J. Biol. Chem. 248, 5950-5955 10 Berl, S., Puszkin, S. and Nicklas, W. J. (1972) Science 179, 441-446 11 Childers, S. R. and Siegel, F. L. (1975) Biochim. Biophys. Acta 405, 99-108 12 Kaminer, B. and Szonyi, E. (1972) J. Cell Biol. 55, 129a 13 Demaille, J., Dutruge, E., Capony, J-P. and Pech~re, J.-F. (1974) in Calcium Binding Proteins (Drabikowski, W., Strzelecka-Golaskewska, H. and Carafoli, E., eds.), pp. 643-677, Elsevier, Amsterdam 14 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193,265275 15 Bray, G. A. (1960) Anal. Biochem. 1, 279-285 16 Davis, B. J. (1964) Ann. N.Y. Acad. Sci. 121,404-427 17 Dunker, A. K. and Rueckert, R. R. (1969) J. Biol. Chem. 244, 5074-5080 18 Wrigley, C. W. (1971) in Methods in Enzymology (Jakoby, W. B., ed.), Vol. 22, pp. 559 564, Academic Press, New York 19 Briggs, F. N. and Fleishman, M. (1965) J. Gen. Physiol. 49, 131 149 20 Wolff, D. J. and Siegel, F. L. (1972) J. Biol. Chem. 247, 4180~4185 21 Maurer, P. H. (1971) in Methods in Immunology and Immunochemistry (Williams, C. A. and Chase, M. W., eds.), Vol. 3, pp. 1-58, Academic Press, New York 22 Johnson, M. J. (1941) J. Biol. Chem. 137, 575-586 23 Chen, Jr., P. S., Toribara, T. Y. and Warner, H. W. (1956) Anal. Chem. 28, 1756-1758 24 Benzonana, G., Capony, J.-P. and Pech~re, J.-F. (1972) Biochim. Biophys. Acta 278, 110-116 25 Lehky, P., Blum, H. E., Stein, E. A. and Fischer, E. H. (1974) J. Biol. Chem. 249, 4332-4334 26 Pech~re, J.-F. (1974) C.R. Acad. Sci. (Paris) 278, 2577-2579 27 Pech~re, J.-F., Capony, J.-P. and Ryden, L. (1971) Eur. J. Biochem. 23, 421 428 28 Donato, H. and Martin, R. B. (1974) Biochemistry 13, 4575 4579 29 Pech~re, J.-F., Capony, J.-P. and Demaille, J. (1972) System. Zool. 22, 533-548 30 Gosselin-Rey, C. (1972) in Calcium Binding Proteins (Drabikowski, W., Strzlecka-Golaszewska, H. and Carafoli, E., eds.), pp. 679-701, Elsevier, Amsterdam 31 Spande, T. F. and Witkop, B. (1967) in Methods in Enzymology (Hirs, C. H. W., ed.), Vol. I 1, pp. 498-506, Academic Press, New York