Vol. 254. No. 10, Issue of May 25, pp. 4296-4303, 1979 Printed in U.S.A.

Chicken Parvalbumin COMPARISON WITH PARVALBUMIN-LIKE PROTEIN AND THREE OTHER COMPONENTS (A4r = 8,000 to 13,000) *

(Received for publication, January 9, 1978, and in revised form, November 15, 1978)

Claus W. Heizmann and Emanuel E. Strehler From the Institute for Cell Biology, Swiss Federal Institute of Technology, Hcnggerberg, 8093 Ziirich, Switzerland

Procedures for a rapid isolation and purification of parvalbumin (& = 12,600), parvalbumin-like protein (&fr = 12,800), and three other polypeptides with molec- ular weights of 12,400 (Component l), 11,700 (Compo- nent 2), and 8,000, respectively, from chicken leg mus- cle, are described.

A direct comparison of parvalbumin with these other proteins showed distinct differences in the amino acid compositions, charge, and immunological behavior. Parvalbumin has two high affinity sites for Caa+ with aK Mas 5 lo-’ M (Blum, H. E., Lehky, P., Kohler, L., Stein, E. A., and Fischer, E. H. (1977) J. Biol. Chem 262,2834- 2838), in contrast to parvalbumin-like protein, Compo- nents 1 and 2, and the 1M, = 8,000 protein, where only low affinity sites for Ca2+ could be detected (KDi.. > lo-’ m). From our results it is concluded that the co-ex- tracted proteins do not constitute isoproteins of par- valbumin. The very low affinity for Ca” suggests that these proteins are not involved in processes of Ca2+ transport or Ca2+ regulation as proposed for parval- bumin.

Parvalbumin could not be localized within isolated myofibrils and also did not accumulate in primary my- ogenic cell cultures together with proteins forming the myofibrillar structure. Parvalbumin was not even de- tected in myotubee in which myofibrils and sarcoplas- matic reticulum were already assembled and function- ing. Parvalbumin {or cross-reacting material) was de- tected in leg muscle and brain 1 day after hatching of the chick.

Possible roles for parvalbumin are discussed.

Parvalbumins are water-soluble, Ca”-binding proteins with a molecular weight of approximately 12,000. At one time parvalbumins were thought to exist only in lower vertebrates, but several recent reports have described their isolation and characterization from skeletal muscles of higher vertebrates (l-6). Despite intensive investigations of the physicochemical properties, including the elucidation of the three-dimensional structure of carp parvalbumin III (7, S), no physiological role has yet been established for parvalbumins.

The purification of parvalbumin (M, = 12,600) from chicken leg muscle proved to be rather difficult due to the additional presence of a parvalbumin-like protein (MI = 12,800) (9), two heat-acid-labile components (Component 1: M, = 12,400; Component 2, M, = 11,700) and of an M, = 8,000 protein.

* This work was supported by the Swiss National Science Foun- dation Grant 3.575.75. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.&C. Section 1734 solely to indicate this fact.

These findings raised the possibility that these proteins might be isoproteins of parvalbumin. Such proteins have been found earlier in muscles of fuh (lo-13), frog (14, 15), and turtle (1). Therefore, parvalbumin was directly compared to parvalbu- min-like protein, Components 1 and 2 as well as the M, = 8,000 protein with respect to molecular weight, charge, amino acid composition, Ca2+ affinity, and immunological behavior.

New information, potentially relevant to the biological role of parvalbumin in muscle, could also be obtained by studying its localization within isolated myofibrils, its accumulation in primary myogenic cell cultures, as well as its appearance in muscles during the development of the chicken.

MATERIALS AND METHODS

Chicken muscles were obtained from Kneuss, Maegenwil, Switzer- land. All solutions contained phenylmethane sulfonyl fluoride and Trasylol (Bayer) as protease inhibitors. The concentrations were 0.1 mM and 30 kallikrein-inactivator units/ml, respectively.

Isolation of Parvalbumin and the M, = 8,000 Protein-In the final steps of the purification of parvalbumin (6), the M, = 8,000 component was separated on a DEAE-cellulose column (20 X 1 cm) in 2 mM Tris- HCl, pH 7, using a linear gradient of 0 to 300 mM NaCl (Fig. 1). Five to ten milligrams of the M, = 8,000 protein (eluted at 10 to 20 mM NaCl, Peak I) and 10 to 20 mg of parvalbumin (eluted at 20 to 30 mM salt, Peak II) were obtained from 1 kg of leg muscle.

Isolation of Parvalbumin-like Protein-Parvalbumin-like protein could be isolated applying a procedure described earlier (9). Due to its heat and acid stability, however, it could more easily be obtained in the course of the parvalbumin purification (6), where it was eluted from the DEAE-cellulose column (2.8 x 35 cm) with 40 to 60 mM sodium acetate, pH 5.7. The protein was dialyzed against 30 volumes (twice) of distilled water and lyophilized. The powder was dissolved in 1 to 2 ml of 10 w imidazole/HCI, pH 6.8, and the parvalbumin- like protein purified on a Sephadex G-75 column (100 X 3 cm). Approximately 30 mg/kg of homogeneous protein was obtained. Forty milligrams per kg could be isolated by the procedure described earlier (9). Parvalbumin-like proteins isolated by either procedure were identical in electrophoretic mobilities, amino acid compositions, and immunological properties. The isolation of Components 1 and 2 followed the initial steps described earlier for parvalbumin-like pro- tein (9). The proteins, dissolved in 10 loll sodium acetate, pH 5.7, were seuarated on a DEAE-cellulose column (40 X 5 cm). Parvalbu- min was not retained on the column; it could be isolated from the wash by heat and acid treatment and finally purified as described (6).

Parvalbumin-like protein and Components 1 and 2 were retained and could be eluted together at 30 to 80 m salt, applying a linear gradient (10 to 500 mM sodium acetate, volume 2 liters). Fractions containing mainly the low molecular weight components were pooled, dialyzed for 20 h against 30 volumes of distilled water, and lyophilized. The powder was dissolved in 1 ml of 10 mu imidazole/HCl, pH 6.8, insoluble material removed by centrifugation (10 min at 15,000 x g), and separation carried out on a Sephadex G-75 column (100 x 3 cm). Fractions containing parvalbumin-like protein and Components 1 and 2 were pooled and dialyzed for 96 h against 30 volumes of 10 mM sodium acetate, pH 5.7. Separation was achieved on a second DEAE- cellulose column (10 x 1.3 cm), equilibrated in 10 mu sodium acetate, pH 5.7, by applying a shallower gradient (IO to 300 mM sodium

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*r.m CSRIDIENT FRACTION NUMBER

FIG. 1. Separation of parvalbumin and of the M, = 8,000 protein on a DEAE-cellulose column (20 x 1 cm) equilibrated in 2 mu Tris- HCl, pH 7.0. Twenty milligrams of protein were applied. Proteins were eluted with a linear salt gradient (0 to 300 mu NaCl, 300 ml). Peak Z, M, = 8,ooO protein; Peak ZZ, parvalbumin.

acetate, 300 ml) (Fig. 2). Homogeneous parvalbumin-like protein was obtained from Fractions 20 to 27, Component 1 from Fractions 29 to 32, and Component 2 from Fractions 34 to 38 (Fig. 2).

Stability of the Proteins-Parvalbumin, parvalbumin-like protein, and the M, = 8,000 protein were stable when stored in 2 mu Tris- HCl, pH 7, at -20°C. Homogeneous Components 1 and 2 showed considerable instability under the same conditions.

Electrophoresis-Polyacrylamide gel electrophoreses were carried out either as described by Ornstein and Davis (16) or in the presence of sodium dodecyl sulfate according to the method of Shapiro and Maize1 (17). Electrophoreses on slab gels were performed following the procedure of Laemmli (18).

Parvalburnin, parvalbumin-like protein, Components 1 and 2, and the M, = 8,ooO protein were identified during purification on poly- acrylamide gels in the presence of SDS’ by comparison with cyto- chrome c (M, = 12,000).

Protein Concentration-Protein was determined using the biuret method (19), or in case of the homogeneous proteins by their A%,,; the value for parvalbumin-like protein was 7.3 (9); for Component 1, 7.0; for Component 2, 7.4; and for the M, = 8,000 protein, 11.4; for parvalbumin, an A%,,,, of 1.85 w&s determined (6).

Determination of the Molecular Weights-The subunit molecular weights were estimated from SDS-polyacrylamide (15%) slab gel electrophoresis (18), using the following proteins as markers: a-amy- lase (hog), G-actin (rabbit), lactate dehydrogenase (pig), chymotryp- sinogen A (bovine), troponin-C (rabbit), and cytochrome c (horse), assuming subunit molecular weights of 50,000, 42,000, 36,000, 25,000, 18,000, and 12,400, respectively. The determination of the molecular weights of the native proteins was done by gel filtration on Sephadex G-100 according to the method of Andrews (20). The following marker proteins were used: bovine serum albumin (monomer, M, = 68,000), ovalbumin (45,000), chymotrypsinogen A (25,ooO) and cytochrome c Gwm.

Amino Acid Analyses-Amino acid analyses were performed on a Beckman model 121 analyzer. The samples were hydrolyzed in 6 N HCl at 1lO’C in sealed ampoules. Hydrolysis in 2 N KOH was also performed to test for the presence of y-carboxyglutamic acid (21).

Calcium Analysis-For all experiments, containers and pipettes were of polypropylene, previously soaked in 0.1 M HCl, and then rinsed with quartz-distilled water (containing less than 10m7 M Ca”).

Ca*‘-binding experiments were performed by equilibrium dialysis in a 50 mu imidazole/HCl buffer, pH 6.7, at 4’C, using Spectrapor dialysis membranes (Spectrum Medical Industries, Inc.); molecular weight cut-off 6,000 to 8,000). The dialysis membranes were washed extensively (48 h) with ion-free water (quartz-distilled). Prior to equilibrium dialysis, samples of proteins were dialyzed for 24 h against 1,000 volumes of 50 mM imidazole/HCl, pH 6.7, at 4°C (containing less than 1O-7 M Ca”). One hundred fiity microliters of the protein solution (concentrations were 5 X 10m5 M and 8 X 10m5 M) and 150 ~1 of Ca2+ buffer (concentrations of Ca*+ varied between 1 X 10m6 M and

1 The abbreviations used are: SDS, sodium dodecyl sulfate; tropo- nin-C (TN-C), calcium-binding subunit of troponin; EGTA, ethylene- glycol-bis(2-aminoethyl ether)-ZV,ZV’-tetraacetic acid.

r

FIG. 2. Separation of parvalbumin-like protein and of Components 1 and 2 by DEAE-cellulose chromatography in 10 mM sodium acetate, pH 5.7 (column size 10 x 1.3 cm). Twenty milligrams of protein were loaded. Proteins were eluted with a linear salt gradient (10 to 300 mM sodium acetate, 300 ml).

5 x 10e2 M) were placed on opposite sides of the dialyzing membrane, separating the chambers of a microdialysis cell. Dialysis cells were sealed and gently rotated for 1 h in a Dianorm shaker (Prochimie) at 4°C. This time period was shown in initial experiments to be sufficient for equilibrium to be reached between the concentrations of Ca*+ on both sides of the dialysis membrane.

Samples were withdrawn after incubation, appropriately diluted, and the concentrations of Ca2+ determined on a Perkin-Elmer atomic absorption spectrophotometer. Suprapure CaCl* (Merck) was used to construct standard curves. Binding constants were calculated using the computer program described by PliBka and Sachs (22).

Antibody Preparation-Antibodies against chicken parvalbumin were obtained by injecting rabbits intradermally with an emulsion of 0.3 mg of parvalbumin, dissolved in 1 ml of 0.9% NaCl, and mixed with 1 ml of Freund’s complete adjuvant.

A second immunization (0.3 mg of parvalbumin, but mixed with Freund’s incomplete adjuvant) was carried out 1 week later. Four weeks later a final intramuscular injection (1 mg of parvalbumin mixed with 1 ml of Freund’s incomplete adjuvant) was given. The IgG fraction of the serum was obtained by employing the procedure described by Givol et al. (23).

The preparation of the anti-parvalbumin-like protein serum was described previously (9).

Ouchterlony Double Zmmunodiffusion-Ouchterlony double im- munodiffusion was performed as described (24). Antiserum (25 pl/ well) was added 6 to 8 h before antigen samples (25 $1, were applied. Precipitin lines were stained as described (9). The titer of anti- parvalbumin serum was 1:2 when determined on Ouchterlony plates.

Localization Study in Isolated Myofibrils-Myoiibrils were pre- pared according to the method of Kundrat and Pepe (25) with the precautions given by Heizmann et al. (26). The procedure for the localization studies was described earlier (9, 26-28). The anti-parval- bumin serum, the anti-parvalbumin IgG fraction, and the fluorescein- conjugated goat-anti-rabbit IgG (Grand Island Biological Co.) were diluted 1:50,1:25, and 1:200, respectively, in relaxing buffer (containing 0.1 M KCl, 1 mM EGTA, 5 mM EDTA, 1 mM dithiothreitol, 0.1% bovine serum albumin, pH 7).

Cell Cultures-Primary cell cultures of myogenic cells from leg and breast muscles of 11-day chicken embryos were prepared as described (29). The cultures were plated at a nominal density of 4 x lo5 cells/ plate. Cultures were washed and fixed as described elsewhere (30). Immunofluorescence studies in cultures were done as described (31). The cultures were incubated with anti-panralbumin serum, and, as an internal control, with anti-MM-creatine kinase serum (both diluted 1: 40), followed by incubation with fluorescein-labeled goat antibodies against rabbit IgG (diluted 1:70).

RESULTS

Purity and Electrophoretic Properties--The purified pro- tein components were homogeneous as judged from polyacryl-

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abc d e f g h-i k indicates dye front.

I m

amide gel electrophoresis in the presence and absence of SDS (Fig. 3).

The migration of parvalbumin, parvalbumin-like protein, and of Components 1 and 2 on 10% gels under denaturing conditions was nearly identical (Fig. 3, a to d).

When separation, however, was performed on 15% poly- acrylamide slab gels according to the method of Laemmli (18), small but distinct variations in their migration could be ob- tained resulting from differences in the molecular weights of the polypeptide chains (see below, Fig. 4 and Table I). When purified proteins were separated on polyacrylamide (7.5%) gels in the absence of SDS different migration rates for parvalbumin and parvalbumin-like protein were found. Par- valbumin-like protein migrated faster than the low molecular weight Components 1 and 2 (Fig. 3, f to K).

Determination of Molecular Weights-The molecular weights of the polypeptide chains were estimated from slab gel electrophoresis (15%) in the presence of 0.1% SDS. A linear plot of mobility uersus log molecular weight was obtained from the set of marker proteins described under “Materials and Methods.” The molecular weights of the polypeptide chains were estimated from the graph (Fig. 4) and the results are summarized in Table I, Column 1. The molecular weights of the native proteins were estimated by gel filtration on Sephadex G-100 as described under “Materials and Methods.” A linear plot of elution volume against log molecular weight was obtained from the set of marker proteins. The molecular weights of pa&albumin-like protein and of the M, = 8,000 component are given in Table I, Column 2, and are compared to that of parvalbumin. Identical molecular weights under denaturing and nondenaturing conditions were obtained for parvalbumin, parvalbumin-like protein, and the M, = 8,000 component thus demonstrating that these proteins are mon- omeric.

FIG. 4. Determination of the molecular weight of the polypeptide chains of parvalbumin, parvalbumin-like protein (PUP), Compo- nents 1 and 2 and of the M, = 8,000 protein from 15% polyacrylamide slab gel electrophoresis. The molecular weights of the marker proteins are given under “Materials and Methods.” LDH, lactate dehydrogen- aae.

Calcium Analysis-The Ca’+-binding properties of parval- bumin and the other low molecular weight proteins are sum- marized in Table II. Only parvalbumin had two high affinity sites for Ca2+/molecule whereas all other proteins showed only low affinity for this metal ion. Dialysis of the proteins against 2 mM EGTA (dissolved in 2 mM Tris-HCl, 0.1 M KCl, pH 7) for 2 h resulted in the complete removal of the metal ion from all proteins except from parvalbumin. Only one Ca2+ is removed from parvalbumin under these conditions. The displacement of the 2nd ion required prolonged dialysis against 2 mM EGTA (72 h) or against 2 mu EGTA, 20 mM

MgC12 (2 h). Ekctrophoretic Mobilities in the Presence and Absence of

Calcium-Electrophoresis was performed on polyacrylamide

FIG. 3. Ten per cent polyacrylamide gels in the presence of SDS: (a) parval- bumin (10 ag), (b) parvaIbumin-like pro- tein (30 pgg), (c) Component 1 (10 pg), (4 Component 2 (10 j.ig), (e) cytocbrome c (2Opg, M, = 12,000). 7.5% polyacrylamide gels in the absence of SDS: (f) par&- bumin (30 pg), (g) parvalbumin-like pro- tein (10 pg), (A) Component 1 (10 j.ig), (i) Component 2 (10 pgg). (A) mixture of parvalbumin-like protein, Components 1 and 2 (3 pg each). 15% polyacrylamide slab gel in the presence of SDS: (I) M, = 8,009 protein (3 pgj. 7.5% polyacryl- amide slab gel in the absence of deter- gent: (m) M, = 8,000 protein (3 pg). Mi- gration is from top to bottom. Arrow

TABLE I Molecular weights of the polypeptide chains and of the native

protein comvonents M, of thEhs$peptide M, of the native

orotein Parvalbumin ParvaIbumin-like protein Component 1 Component 2 M, = 8,000 protein

12,609 * 309 12,000” 12,806 f 200 12,609 i 9006 12,400 rt 200 Not determined 11,700 f 500 Not determined 8,000 f 500 9,000 * 1,000

o Obtained from sedimentation equilibrium studies (1). *A value of 12,000 was obtained in sedimentation equilibrium

experiments (9).

TABLE II Calcium- binding aarameters

mol ca=+/mo1 mol/l mol Ca”/mol protein protein mid/l

ParvaIbumir? 2.1 SW6 Several approx. IO-’ Parvalbumin- None 8-12 >10-3

like protein’ Component 1’ None 8-12 >10-3 Component 2’ None 8-12 >lo-j M, = 8,000 None 6-10 >W3

protein’ a n, number of binding sites; Ko, dissociation constant. * Determined by partition chromatography and equilibrium dialysis

(1). ’ Determined by equilibrium dialysis under identical conditions as

in Ref. 1.

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slab gels (7.5%) at pH 8.6 in the absence of SDS. Parvalbumin showed a relative mobility of 0.39 f 0.01 in

the presence of 0.1 mM CaCb. In the Ca2+-free state (2 mM EGTA) a shift in mobility to 0.65 + 0.01 was observed. In contrast, the migration of parvalbumin-like protein, Compo- nents 1 and 2 and of the il4, = 8,000 protein remained un- changed under both conditions. The change in mobility of parvalbumin was that which could be expected for a lower net negative charge resulting from the binding of Ca’+. A similar effect has been described for a troponin-C-like protein, iso- lated from bovine brain (32). The opposite effect (slower migration in the Ca2+-free state) was reported for troponin-C from rabbit white skeletal muscle by Head and Perry (33), possibly reflecting a change in conformation of the protein molecule. A change in conformation was, however, also re- ported for parvalbumin from circular dichroism measure- ments (34,35).

Amino Acid Analysis-The amino acid analysis data for parvalbumin and parvalbumin-like protein were reported ear- lier (6,9), and considerable differences in the contents of, e.g. cysteine, phenylalanine, and tryptophan were obtained. The compositions of the M, = 8,000 protein and of Components 1 and 2 are shown in Table III. Components 1 and 2 differ from parvalbumin (6) as well as from parvalbumin-like protein (9) mainly in the low contents of lysine and phenylalanine and the high level of proline. The M, = 8,000 protein is clearly different from parvalbumin, parvalbumin-like protein, or low molecular weight Components 1 and 2. The possibility of the M, = 8,000 protein being a breakdown product of the larger parvalbumin molecule can be eliminated due to the additional presence of a tryptophan and the higher content of tyrosine. This is also supported by the lack of an immunological cross- reactivity of these proteins (see below). A direct comparison of the amino acid analysis of parvalbumin-like protein, Com- ponents 1 and 2, and of the M, = 8,000 protein with the sequence analysis of troponin-C (37) from chicken shows that none of these proteins are fragments of the larger TN-C molecule, due to, for example, the additional presence of

TABLE III Amino acid compositions

Amino acid residue Component 1” Component 2” M, = 8,000 pro- tein

Lysine 6.2 8.5 9.1 Hi&dine 4.4 1.2 1.3 Argiuine 5.5 2.7 1.1 Aspartic acid 9.4 8.3 7.3 Threonined 3.5 7.0 3.0 Serined 6.6 10.7 5.5 Glutamic acid 13.6 14.1 10.5 Proline 7.4 4.3 2.3 Glycine 12.6 12.6 6.6 Abmine 9.5 7.9 8.3 Valine 4.4 7.2 5.0 Methionine 1.7 2.4 2.3 Isoleucine 7.5 4.5 1.3 Leucine 11.1 6.9 4.2 Tyrosine 2.1 1.9 2.8 Phenylabmine 3.2 3.5 1.7 Tryptophan’ 1.2 1.4 1.2

Total 109.9 105.1 73.5

“Average of 24-, 4%, and 72-h hydrolyses assuming a molecular weight of 12,400.

b 24-h hydrolysis based on a molecular weight of 11,700. ’ 24-h hydrolysis assuming a molecular weight of 8,000. d Extrapolated to zero time of hydrolysis or by adding 5% to the

swine and 10% to the threonine value when only 24-h hydrolysis was performed.

’ Determined spectrophotometrically (36).

FIG. 5. Double immunodiffusion test of anti-parvalbumin-IgG (center well) against (a) parvalbumin, (6) 4 mru EDTA extract (pH 7) from adult leg muscle (5 mg/ml), (c) parvalbumin-like protein, (4 Component 1, (e) Component 2, ( f) M, = 8,009 protein. The concen- trations of the purified proteins were 0.3 mg/ml.

tyrosine and tryptophan not found in chicken TN-C. The content of y-carboxyglutamic acid was also determined

for parvalbumin since this amino acid had been found previ- ously in vitamin K-dependent, calcium-binding plasma pro- teins participating in blood coagulation (38-40). Less than 0.04 residue of y-carboxyglutamic acid were found, a result also reported for parvalbumin-like protein (9).

It is well known that the divalent cation Ca2+ is involved in the regulation of muscle contraction. Among all the proteins examined in this work, only parvalbumin showed a compara- bly high affinity for this metal ion as troponin C (41). There- fore, immunological and developmental studies were per- formed in more detail only with parvalbumin and the com- parison restricted to parvalbumin-like protein, the protein of greatest similarity in several physicochemical properties.

Immunological Properties-The specialty of the anti-par- valbumin IgG fraction was tested in the immunodiffusion test using parvalbumin and crude leg muscle extract as antigens (Fig. 5, a and b). In both cases a single precipitin line was obtained. Parvalbumin and parvalbumin-like protein are im- munologically distinct (6). There was no cross-reactivity of anti-parvalbumin IgG with Components 1 and 2 when freshly purified proteins were used (Fig. 5). A faint precipitin line, however, was observed against highly degraded Component 2 (Fig. 5e). Fig. 5, c and fshows that anti-parvalbumin IgG (or serum) cross-reacted neither with the purified parvalbumin- like protein nor with the M, = 8,000 protein, and not even after denaturation of these proteins in an 0.1% SDS/phos- phate buffer, pH 7.2.

Distribution in Different Tissues-It seemed worthwhile to examine whether chicken parvalbumin could also be detected (by immunological methods) in tissues other than muscle. Earlier investigations (1, 42,43) already showed that parval- bumins isolated from several species (including rabbit) were not found in all types of muscles. Spleen, liver, heart, breast and leg muscles, gizxard, brain, pancreas, and kidney were dissected from adult chickens, minced, suspended in 2.5 vol- umes of 4 mM EDTA, pH 7, and homogenized in a Potter- Elvehjem (glass-Teflon) homogenizer (employing three 1-min periods of homogenization). The extracts were finally centri- fuged for 30 min at 16,000 x g. The supernatants (1 to 3 mg/ ml) were tested qualitatively for parvalbumin in Ouchterlony double diffusion tests against anti-parvalbumin IgG fraction. Precipitin lines were formed only against extracts from leg, white and red back muscles, and brain, but not against those from breast, cardiac, and smooth muscles. In contrast parval- bumin-like protein was detected in most tissues tested (9). The precipitin lines fused completely without showing any spurs indicating cross-reactivity and immunological identity (not shown). Neither anti-parvalbumin nor anti-parvalbumin- like protein serum cross-reacted with extracts from human or rabbit skeletal muscle. This is not surprising because it is

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4300 Low Molecular Weight Muscle Proteins, Cat+-binding Parvalbumin

known that parvalbumins from different species, despite their similarities in amino acid compositions and their high degree of homology, are usually immunologically distinct (1).

Localization Study in Relaxed and Contracted Myofi- brils-The indirect immunofluorescence method was used for a study of the localization of parvalbumin in relaxed and strongly contracted myofibrils, as had been done earlier for the parvalbumin-like protein (9, 26). No fluorescence over background level was observed under these conditions when the isolated myofibrils were incubated with anti-parvalbumin IgG (Fig 6, A and B). The localization of parvalbumin in relaxed myofibrils was directly compared to that of p-actinin and actin under the same conditions. A strong fluorescence in the I-band region was observed (Fig. 6, C and D), as expected for actin and fi-actinin. Controls with pre-immune serum were treated under identical conditions, as with immune sera (Fig. 6E). The results are in agreement with localization studies done with carp parvalbumin, where the protein appeared evenly distributed in carp white muscle, and not bound to distinct structural regions (44, 45). Myofibrils stained for parvalbumin-like protein (9, 26) showed, however, a regular pattern of fluorescent cross-striation within the I-band of relaxed myofibrils, a result also found for a Ca2’-binding protein from crayfiih (44, 45). There was also no binding of parvalbumin to strongly contracted myotibrils (Fig. 6B).

FIG. 6. Localization of parvalbumin in myofibrils isolated from leg muscle. Staining was performed by the indirect immunofluorescence technique. (A) relaxed and (B) contracted myotibrils. Fluorescence is compared to that of relaxed myofibrils incubated with (C) anti-/?- actinin and (D) anti-actin serum. As a control, myofibrils were incu- bated with preimmune serum (E). The exposures on the left show the phase contrast and on the right the fluorescence of the same tibril.

Accumulation Studies in Primary Cultures of Differentiat- ing Chicken Leg and Breast Muscle Cells-Multinucleated muscle cells are formed after fusion of mononucleated myo- blasts and fusion is accompanied by an accelerated synthesis of myofibrillar proteins (46-50). The biogenesis of sarco- plasmic reticulum in cultured chicken muscle cells has been studied by Ezerman and Ishikawa (51). Formation of sarco- plasmic reticulum begins around fusion. Accumulation of pro- teins such as myosin, myoglobin, creatine kinase, phosphoryl- ase, and adenylate kinase, as well as the development of sarcoplasmic reticulum, appear simultaneously (49,50,52-58). The same has been found also for the accumulation of par- valbumin-like protein (59). Innervation is not required for these events to occur (51, 60).

In order to get some information on the possible role of parvalbumin in muscle cells, the accumulation of this muscle- specific, Ca2’-binding protein, was studied in primary my- ogenic cell cultures.

Fig. 7, A to C, shows micrographs of 48-h cultures of chicken

FIG. 7. Immunofluorescent staining of a 48-h standard medium culture. Three different regions of the same culture plate were reacted with anti-parvalbumin (A), anti-MM-creatine kinase (for comparison) (B), and control serum (C). Immunofluorescent staining of a 7-day culture with (D) anti-parvalbumin, (E) anti-MM-creatine kmase, and (Fl control serum. Arrows indicate cross-striation. Exposures on the left show the phase contrast and on the right fluorescence.

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FIG. 8. Accumulation of parvalbumin in leg (A/B) and brain (C/ 0) tissues of chicken embryos (A/C) and of chicken after hatching (B/D). Center wells contained anti-parvalbumin IgG, peripheral wells the 4 mru EDTA (pH 7) extracts (5 mg/ml). A, leg muscle extracts of ll- (a), 16- (b), and 20- to 21-day-old chicken embryos (hatching point) (c); B, leg muscle extracts of l- (a), 3- (b), and S-day-old chicken (c); C, brain extracts of ll- (a), 16- (b), and 20- to al-day-old embryos (c); D, brain extracts of l- (a), 3- (b), and S-day-old chicken (CL

leg muscle cells which were allowed to react with anti-parval- bumin serum (Fig. 7A), anti-MM-creatine kinase serum (Fig. 7B) and with pre-immune serum (Fig. 7C). Dilutions and incubations were done in the same way in all three cases. Fig. 7, D to F, demonstrates myotubes from 7-day-old cultures, containing assembled myofibrils (see arrows, Fig. 7E) within the myotubes, already capable of spontaneous contraction. Only background fluorescence could be observed when stained for parvalbumin (Fig. 70). This is in contrast to MM-creatine kinase, the presence of which could be clearly demonstrated by its strong fluorescence after only 2 days in cultures (Fig. 7B). Identical results were found using cell cultures obtained from breast muscles. The lack of accumulation of parvalbumin in cell cultures was further supported by experiments, done similarly to studies previously described for parvalbumin-like protein (9). Cells were extracted at different stages of devel- opment (with 4 mu EDTA, pH 7) and the supematanta (3 to 5 mg/ml) allowed to react on Ouchterlony plates against anti- parvalbumin serum. No parvalbumin (or cross-reacting ma- terial) could be detected in these cell extracts.

Accumulation in Embryonic and Postembryonic Chicken- Furthermore, we investigated the appearance of parvalbumin in leg and breast muscles during the ontogeny of the chicken. Leg and breast muscles as well as brain were dissected from ll- 16-, and 20-day-old embryos and of l- 3-, 5-, and 9- day-old chicken, and the tissues extracted with 4 mr+f EDTA, pH 7. The supematants, averaging 5 mg of protein/ml, were tested qualitatively for the presence of parvalbumin (or cross- reacting material) in Ouchterlony double diffusion tests against anti-parvalbumin serum (Fig. 8).

Parvalbumin was tit detected in leg muscles and in brain of l-day-old chicken, but not in embryonic tissues. No parval- bumin could, however, be detected in breast muscles of either embryonic or adult chicken.

DISCUSSION

The purpose of this study was a basic comparison of par- valbumin with the newly detected, low molecular weight

parvalbumin-like protein, Components 1 and 2, and the M, = 8,000 protein, all extracted under identical conditions from chicken leg muscle. Furthermore, an approach was under- taken to obtain more information on the physiological role of parvalbumin in muscle. The direct comparison of all these proteins showed differences in their molecular weights, charge, and immunological behavior. The amino acid compositions revealed several similarities between parvalbumin and parvalbumin-like protein (6) but those of Components 1 and 2 as well as the M, = 8,000 protein were clearly distinct in most amino acid residues.

Considerable differences have also been found in the affin- ities for Ca2+. Parvalbumin was found to be the only protein species with two high affinity sites for Ca2’ (Kw 5 10e6 M)

(1). Parvalbumin-like protein, Components 1 and 2, as well as the M, = 8,000 protein, showed only a low affinity for this metal ion, with a KD~, > 10e3 M.

From the results obtained, the parvalbumin-like protein and the other three components cannot be considered being isoproteins of parvalbumin, although such isoproteins have been detected in several other species (1, 10-15).

Parvalbumins are considered to be muscle proteins. Among all the tissues investigated, only brain, besides muscle, has been found to contain significant amounts of this Ca’+-binding protein. In contrast, parvalbumin-like protein could be found in muscle and non-muscle tissues (9). The function of the parvalbumins is not yet clear but the fact that they bind Ca2+ with an affinity comparable to that of TN-C (41) suggested that they may function as a Ca2+-trapping or -transport pro- tein. The findings of only low affinity sites for Ca2’ in the other proteins implies that they may have other functions than parvalbumin in uiuo and may not be involved in proc- esses of Ca2+ regulation, or transport, or both. Parvalbumins are, however, not found in all types of muscles; for example, they were not detected in breast muscle of the chicken. This is based on the assumption that parvalbumins in different tissues are immunologically identical. Their association mainly with fast, nerve impulse-activated skeletal muscle suggested a connection with the regulation of this type of muscle (61).

New information on the physiological role of parvalbumin could be obtained by studying 1) its localization within the isolated myofibrils, 2) its accumulation in primary cell cultures during myogenesis, and 3) its bulk accumulation in muscle during the development of the chick. Parvalbumin was neither bound to relaxed nor to strongly contracted myofibrils, sug- gesting that this protein is not a structural part of the con- tractile machinery itself. Parvalbumin was neither detected in myoblasts of primary myogenic cell cultures nor in 7-day-old myotubes, which already contain assembled myofibrils capa- ble of spontaneous contraction. Parvalbumin-like protein, however, could clearly be detected in myoblasta prior to fusion (59), using experimental conditions identical with those de- scribed here. The accumulation of myosin and actin, for example, and the appearance of the myofilamenta and of the sarcoplasmic reticulum approximately parallels fusion of the myoblasta in myogenic cell cultures (49,50,52-58), but is not dependent on fusion per se (62-64). These findings indicate that parvalbumin may not be essential for the assembly of the myotibrillar structure. Histochemically, however, cultured muscle fibers are different from those found in adult and embryonic muscles (65).

During the development of the chicken parvalbumin was detected earliest in leg muscle and brain 1 day after hatching. It is known that during early embryonic development chicken skeletal muscles are largely inactive and the microsomes are devoid of Ca2+ transport activity (66,67). The appearance of

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4302 Low Molecular Weight Muscle Proteins, Ca’+-binding Parvalbumin

Ca” transport and of the Cf+-sensitive ATPase around the time of hatching coincides with the increasing muscular activ- ity. Our results show that parvalbumin appeared at a rather late stage in muscle development.

All these results, including the findings that parvalbumin was not detected in breast and heart muscles of the chicken by the methods applied, are in agreement with the idea that parvalbumin may constitute a regulatory system which oper- ates mainly in fast muscles, so as to facilitate their cyclic relaxation (61,68). Although the present results are consistent with the idea that parvalbumin may be involved in processes associated with muscle contraction, this does not rule out the possibility that it could have other function(s) in muscle as well. Recent reports have shown that parvalbumin, purified from carp muscle, can regulate rat brain adenosine 3’:5’-mono- phosphate phosphodiesterase in a Ca*+-dependent manner; however, the amount of parvalbumin necessary for maximal activation was considerably higher than the amount of native Ca*‘-dependent regulator protein (69). An involvement of parvalbumin in the degradation of glycogen, regulated by the Ca*‘-binding enzyme, phosphorylase kinase, could also be considered. In this respect, parvalbumin could be thought to connect motility with other cellular events which are regulated by Ca*‘.

Acknowledgments-We wish to thank Dr. H. M. Eppenberger for supporting this work, Mrs. Z. Zanivan for carrying out the amino acid analyses, Dr. P. Hauschka for measuring the content of y-carboxyglu- tamic acid, Mrs. I. Hotz for excellent technical assistance, and Mrs. M. Leuzinger for typing this manuscript.

REFERENCES

1. Blum, H. E., Lehky, P., Kohler, L., Stein, E. A., and Fischer, E. H. (1977) J. Biol. Chem. 252,2834-2838

2. Lehky, P., Blum, H. E., Stein, E. A., and Fischer, E. H. (1974) J. Biol. Chem. 249,4332-4334

3. Pechere. J.-F. (1974) C. R. Hebd. Seances Acad. Sci. 278. 2577-

4

5.

6,

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18. 19. 20. 21.

2579 Enfield, D. L., Ericsson, L. H., Blum, H. E., Fischer, E. H., and

Neurath. H. (1975) Proc. Natl. Acad. Sci. U. S. A. 72. 1309- 1313

Capony, J.-P., Piia, C., and Pechere, J.-F. (1976) Eur. J. Biochem. 70, 123-135

Strehler, E. E., Eppenberger, H. M., and Heizmann, C. W. (1977) FEBS Lett. 78, 127-133

Kretsiuger, R. H., and Nockolds, C. E. (1973) J. Biol. Chem. 248, 3313-3326

Hendrickson, W. A., and Karle, J. (1973) J. Biol. Chem. 248, 3327-3334

Heizrnann, C. W., Hliuptle, M.-T., and Eppenberger, H. M. (1977) Eur. J. Biochem. 80,433-441

Pechi+re, J.-F., Capony, J.-P., and Demaille, J. (1973) SystemZooZ. 22,533~548

Closset, J. I., and Gerday, C. (1976) Comp. Biochem. Physiol. 55B. 537-542

Sullivan, B.. Bonaventura, J.,Bonaventura, C., Pennell, L.,Elliott, J., Bo&, R., and Lambie, W. (1975) J. Mol. Euol. 5, 103-116

GosselinRev. C., Piront, A., and Gerday, C. (1978) Biochim. Biophys. Acta 532,294-304

Pechkre, J.-F., and Capony, J.-P. (1969) Comp. B&hem. Physiol. 28, 1089-1102

56. Holland, P. C., and MacLennan, D. H. (1976) J. Biol. Chem. 251, 2030-2036

57. Zubrzycka, E., and MacLennan, D. H. (1976) J. Biol. Chem. 251, 7733-7738

Pechire, J.-F., Demaille, J., and Capony, J.-P. (1971) Biochim. 58. Jorgensen, A. O., Kalnins, V. I., Zubrzycka, E., and MacLennan, Biophys. Acta 236,391-408 D. H. (1977) J. Cell Biol. 74,287-298

Omstein, L., and Davis, R. J. (1963) Disc Gel Electrophoresis, 59. Baltisberger, E. (1977) DipZomarbeit, Swiss Federal Institute of DistiUation Products Industries, Rochester, N. Y. Technology, Zurich, Switzerland

Shapiro, A. L., and Maixel, J. V. (1969) Anal. Biochem. 29,505 60. Shimada, Y., Fischman, D. A., and Moscowa, A. A. (1967) J. Cell 514 Biol. 35.445-453

Liiemmli, U. K. (1970) Nature 227.680685 Layne, E. (1957) Methods Enzymol. 3,447-454 Andrews, P. (1964) B&hem. J. 91,222-232 Hauschka, P. V., Lian, J. B., and Gallop, P. M. (1975) Proc. Natl.

61. Pechbre, J.-F., Derancourt, J., and Haiech, J. (1977) FEBS Lett. 75,111-114

62. Emerson, C. P., Jr., and Beckner, S. K. (1975) J. Mol. Biol. 93, 431-447

Acad. Sci. U. S. A. 72,3925-3929 63. Moss, P. S., and Strohman, R. C. (1976) Dev. Biol. 48,431-437

22. 23.

24.

25. 26.

27.

28.

29.

30.

31.

32.

33. 34.

35.

36. 37. 38.

39.

40.

41.

42.

Pliska, V., and Sachs, H. (1974) Eur. J. Biochem. 41.229-239 Givol, D., Weinstein, Y., Gore&i, M., and Wiecheck, M. (1970)

Biochem. Biophys. Res. Commun. 38,825-830 Ouchterlony, 0. (1967) in Handbook of Experimental Immunol-

ogy (Weir, D. M., ed) Blackwell Scientific Publications, Oxford Ku&rat, F., and Pepe, F. A. (1971) J. Cell Biol. 48.340-347 Heizmann, C. W., Blauenstein, I. E., and Eppenberger, H. M.

(1978) Experientia 34,38-40 Heizmann, C. W., and Hauptle, M.-T. (1977) Eur. J. Biochem.

80,443-451 Heizmann, C. W., and Eppenberger, H. M. (1978) J. Biol. Chem.

253,270-277 Turner, D. C., Maier, V., and Eppenberger, H. M. (1974) Deu.

Biol. 37,63-89 Gohhnan, R. D., Lazarides, E., PolIack, R., and Weber, K. (1975)

Exp. Cell Res. 90, 333-344 Turner, D. C., Gmiir, R., Lebherz, H. G., Siegrist, M., and Eppen-

berger, H. M. (1976) Dev. Biol. 48.284-307 Amphlett, G. W., Vanaman, T. C., and Perry, S. V. (1976) FEBS

Lett. 72,163-168 Head, J. F., and Perry, S. V. (1974) Biochem. J. 137, 145-154 Donato, H., Jr., and Martin, R. B. (1974) Biochemistry 13,4575-

4579 Closset, J. I., and Gerday, C. (1975) Biochim. Biophys. Acta 405,

228-235 Bencze, W. L., and S&mid, K. (1957) Anal. Chem. 29, 1193-1196 Wilkinson. J. M. (1976) FEBS Lett. 70. 254-256 Stenflo, J:, Fernlund, P., Egan, W., and Roepstorff, P. (1974)

Proc. Natl. Acad. Sci. U. S. A. 71,2730-2733 Nelsestuen, G. L., Zytkovicz, T. H., and Howard, J. B. (1974) J.

Biol. Chem. 249,6347-6350 Magnusson, S., Sottrup-Jensen, L., Petersen, T. E., Morris, H. R.,

and Dell. A. (1974) FEBS Lett. 44.189-193 Potter, J. D., and Gergely, J. (1975) J. Biol. Chem. 250, 4628-

4633 Gosselin-Rey, C. (1974) in Calcium Binding Proteins (Drabi-

kowski, W., Strzelecka-Golaszewska, H., and Carafoli, E., eds) pp. 643-677, PWN-Polish Scientific Publishers, Warsaw, and Elsevier, Amsterdam

43.

44.

45.

46.

Baron, G., Demaille, J., and Dutruge, E. (1975) FEBS Lett. 56, 156-160

Benzonana, G., Wnuk, W., Cox, J. A., and Gabbiani, G. (1975) Experientia 31,716

Benzonana, G., Wnuk, W., Cox, J. A., and Gabbiani, G. (1977) Histochemistry 51.335-341

47.

48.

49.

Konigsberg, I. g. (1965) in Molecular and Cellular Aspects of Development (Bell, E., ed) pp. 116-134, Harper and Row Pub- lishers, New York

Hauschka, S. D. (1972) in Growth, Nutrition and Metabolism of Cells in Culture (Rothblat. G. H.. and Cristofalo. V. J., eds) Vol. 2, pp. 67-131,‘Academic Press; New York

Holtzer, H., Rubinstein, N., Fellini, S., Yeoh, G., Chi, J., and Okayama, M. (1975) Q. Rev. Biophys. 8,4-40

Buckingham, M. E. (1977) in International Review of Biochem- istry, Biochemistry of Cell Differentiation II (Paul, J., ed) Vol. 15, pp. 269-332, University Park Press, Baltimore

Devlin, R. B., and Emerson, C. P., Jr. (1978) Cell 13,599-611 Ezerman, E. B., and Ishikawa, H. (1967) J. Cell Biol. 35,405-420

52. Shainberg, A., Yagil, G., and Yaffe, D. (1971) Dev. Biol. 25. l-29 53. Paterson, B.. and Strohman, R. C. (1972) Dev. Biol. 29, 113-138 54. Kagen, L. J., and Freedman, A. (1974) Exp. Cell Res. 88,135-142 55. Tarikas, H., and Schubert, D. (1974) Proc. Natl. Acad. Sci. U. S.

A. 71.2377-2381

50. 51.

by guest on July 9, 2018http://w

ww

.jbc.org/D

ownloaded from

Low Molecular Weight Muscle Proteins, Ca’+- binding Parvalbumin 4303

64. Vertel, B. M., and F&&man, D. A. (1973) Dev. Biol. 48,438-446 66. Gillis, J. M., and Gerday, C. (1977) in Calcium Binding Proteins 65. Askanss, V., Shafig, S. A., and Milhorat, A. T. (1972) Exp. Neurol. and Calcium Function (Wasserman, R. H., Corradino, R. A.,

37,21a-230 Carafoli, E., Kretainger, R. H., MacLennan, D. H., and Siegel, 66. Boland, R., Martonosi, A., and Tillack, T. W. (1974) J. Biol. F. L., eds) pp. 193-196, Elsevier North-Holland, Inc., Amster-

Chem. 249,612-623 dam 67. Tillack, T. W., Boland, R., and Martonosi, A. (1974) J. Biol. 69. Potter, J. D., Dedman, J. R., and Means, A. R. (1977) J. Biol.

Chem. 249,624-633 Chem. 252,560%5611

by guest on July 9, 2018http://w

ww

.jbc.org/D

ownloaded from

C W Heizmann and E E Strehlerother components (Mr = 8,000 to 13,000).

Chicken parvalbumin. Comparison with parvalbumin-like protein and three

1979, 254:4296-4303.J. Biol. Chem. 

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