Proc. Nat. Acad. Sci. USAVol. 71, No. 6, pp. 2198-2202, June 1974

A Phosphate-Acceptor Protein Related to Parvalbuminsin Dogfish Skeletal Muscle

(Ca-binding protein/phosphorylation)

HUBERT E. BLUM, SITIVAD POCINWONG, AND EDMOND H. FISCHER*

Department of Biochemistry, University of Washington, Seattle, Wash. 98195

Contributed by Edmond H. Fischer, March 4, 1974

ABSTRACT A phosphate-acceptor protein was isolatedfrom the skeletal muscle of the Pacific dogfish (Squalusacanthias) displaying properties extremely similar to thoseof the parvalbumins, i.e., the low-molecular-weight,soluble, Ca-binding muscle proteins found in fish andamphibians. It has the same characteristic UV spectrum,strong affinity for calcium, and immunological cross-reactivity with antibodies against homogeneous dogfishparvalbumin. Although it was isolated in three states ofaggregation with molecular weights of about 350,000,75,000, and 25,000, all species dissociate in Na dodecyl sul-fate into subunits of 11,000 and 13,000 molecular weight.Furthermore, whereas no phosphorylation of parvalbu-mins could be demonstrated under any experimental con-ditions, the aggregated forms could be readily phos-phorylated by a cyclic AMP-independent dogfish proteinkinase, but not by phosphorylase kinase. One acid-stableand base-labile phosphate group was introduced per sub-unit which could be rapidly released by a dogfish proteinphosphatase, but only very slowly if at all by phosphorylasephosphatase. It is speculated that this "phosphate-accep-tor protein" might represent a physiologically active formof the parvalbumins.

As part of our continuing interest in the evolutionary aspectsof intracellular control mechanisms, work was initiated a fewyears ago on several enzymes and contractile proteins from thePacific dogfish (Squalus acanthiast, a very primitive verte-brate that separated from the main line of evolution about 450million years ago). Little was known at that time about themechanism by which this organism controls glycogen break-down in relation to either hormonal stimulation or musclecontraction; enzymes of the phosphorylase system and con-tractile proteins such as troponin, tropomyosin, actin, ormyosin had not been isolated or characterized.

In the course of this study, dogfish muscle phosphorylase,phosphorylase kinase and phosphatase, glycogen synthetase, aprotein kinase, and a protein phosphatase have been system-atically investigated, as well as the contractile proteins men-tioned above. Among various properties observed, one wasparticularly difficult to comprehend: contrary to the acceptedmechanism by which phosphorylase activation occurs inmammalian systems (1, 2), no activation of phosphorylasekinase by the protein kinase could be demonstrated and, there-fore, no physiological function could be ascribed to this latterenzyme. To resolve this dilemma, a search was initiated for amuscle protein that could serve as an endogenous substratefor the dogfish protein kinase.

This manuscript describes the isolation and characterizationof such a phosphate-acceptor protein. Unexpectedly, thematerial obtained had almost identical physical, chemical,and immunological properties to those of the parvalbumins(i.e., the low-molecular-weight, soluble, Ca-binding muscleproteins abundantly present in fish and amphibian musclethat display an unusual UV spectrum due to a high phenyl-alanine to tyrosine and tryptophan ratio). Parvalbumins havebeen extensively studied (3, 4); their amino-acid sequence(5-7) and x-ray crystallographic structure (8-11) have beendetermined. Paradoxically, however, no phosphorylation ofthe pure parvalbumins could be demonstrated, suggestingsome basic difference between these molecules.

MATERIALS AND METHODS

Dogfish (Squalus acanthias) were netted in the waters ofPuget Sound and kept for up to 2 months in a 3000-gallon (11.4X 107-cm3), donut-shaped tank with circulating sea water,kindly provided by Dr. A. C. DeLacy, College of Fisheries,University of Washington. The fish were killed by decapita-tion immediately before each experiment; only the backmuscle was used.Sephadex was obtained from Pharmacia, Piscataway, N.J.

and DE-52 cellulose from Whatman. 45CaC12 was purchasedfrom New England Nuclear Corp. (specific radioactivity about15 mCi/mg). [-y-32P]ATP (specific radioactivity about 300mCi/mmole) was prepared according to Glynn and Chappell(12) from carrier-free 32P, (International Nuclear Corp.);12P-labeled protamine was prepared enzymatically by incuba-tion with purified dogfish protein kinase, 10 mM Mg2+, and0.5 mM [32P]ATP at pH 6.5; excess ATP was removed bySephadex G-25 chromatography. Calcium binding was deter-mined according to Briggs and Fleishman (13).

Rabbit-muscle phosphorylase b (EC 2.4.1.1) (14), phos-phorylase kinase (EC 2.7.1.38) (15), phosphorylase phos-phatase (EC 3.1.3.17) (16), and protein kinase (EC 2.7.1.37)[DEAE-cellulose fraction II (15)] were isolated as described.Dogfish phosphorylase b was purified according to Cohen et al.(17), and parvalbumin according to a modification of theprocedure of Pechrre et al. (18).

Dogfish protein kinase was purified by a combination of acid(pH 5.8) and protamine sulfate (0.05%) precipitations fol-lowed by chromatography on DE-52, DEAE-Sephadex A-50,and Sephadex G-100 (1300-fold purification, approximately20% yield). The final material had a specific activity of 12nmoles of phosphate incorporated per min per mg of proteinat pH 6.5 using protamine sulfate (Grade 1, Sigma) as sub-

2198

Abbreviation: TN-C, the calcium-binding subunit of troponin.* To whom reprint requests should be addressed.t Previously referred to as Squalus sucklii or suckleyi.

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P-Acceptor Protein Related to Parvalbumin 2199

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ELUTION VOLUME

FiG. 1. Sephadex G-150 chromatography elution profile ofP-acceptor protein. Elution was carried out with a 50 mM1 Tris-HCl-200 mMA KCl-2 mMI EDTA buffer, pH 6.8. Fractions (2 ml)were collected at a flow rate of 15 ml/hr. Values for cpm shouldbe multiplied by 10-3.

strate; no requirement for any cyclic nucleotide could bedemonstrated, nor was the enzyme inhibited by the regulatorysubunit of rabbit protein kinase (19) or by the heat-stableinhibitor previously described (20). It displayed only one

major band upon gel electrophoresis, and had a molecularweight of about 46,000, as estimated by gel electrophoresis inthe presence of Na dodecyl sulfate and Sephadex G-100chromatography. A protein phosphatase different from phos-phorylase phosphatase purified with the protein kinase up tothe Sephadex G-100 step, but emerged early during elution.The pooled phosphatase fractions had a specific activity of1 nmole of Pi released from 32P-labeled protamine per minper mg of protein at pH 6.5. Dogfish phosphorylase kinase was

purified by ammonium sulfate fractionation followed bychromatography on DE-52 cellulose and Sepharose 4B. Ayeast protein phosphokinase from Saccharomyces cerevisiae,purified 17,000-fold to homogeneity, was kindly provided byDr. Lerch. Antibodies against dogfish parvalbumin were ob-tained by immunizing white female rabbits (New Zealand)as described by Chase (21).

RESULTS

Purification of Dogfish Muscle P-Acceptor Protein. Twoprocedures are described, one mild and slightly more elaboratethan the other; the second is drastic but rapid and leads tomuch higher yields. No difference in physical or chemicalproperties could be found between the two final products.

Unless otherwise stated, all steps were carried out at 4°.Fresh or recently frozen dogfish skeletal muscle (2 kg) was

ground in a meat grinder; 2.5 volumes of 4mM EDTA, pH 7.0,was added, and the suspension was homogenized for 1 min in a

Waring Blendor at full speed. The homogenate was centrifugedfor 30 min at 14,000 X g and the supernatant solution iy-ascollected (crude extract).

Procedure 1: The crude extract was acidified to pH 5 byaddition of 1 N acetic acid and centrifuged at 14,000 X g for30 min. The supernatant was adjusted to pH 7.5, precipitatedwith ammonium sulfate to 0.6 saturation, and recentrifuged.The pellet obtained was suspended in about 100 ml of 10 mMTris - HCl-0.1 mM EDTA, pH 7.5, dialyzed overnight againstthe same solution, then (after clarification by centrifugation)

1 2 3 4 5

FIG. 2. Twelve percent polyacrylamide gel electrophoresisin the presence of 0.1% Na dodecyl sulfate (22) of fractions A, B,and C obtained from Sephadex G-150 chromatography (see Fig.1) and of dogfish parvalbumin. Approximately 30 ,ug of each pro-tein was applied to the gels. (1) Fraction A; (2) fraction B; (3)fraction C; (4) dogfish parvalbumin; (5) mixture of 30 ug each offraction C and dogfish parvalbumin.

applied to a 4 X 25-cm column of DE-52 cellulose equilibratedin the same buffer. Elution was carried out with a NaCl gra-dient from 0 to 500 mM. The fractions containing the P-acceptor protein (as determined by their uptake of 32p uponincubation with [32P]ATP, Mg2+, and protein kinase) andemerging at about 250 mM NaCl were collected, adjusted topH 5.7, and dialyzed overnight against 10 mM Na acetate-0.1 mM EDTA buffer, pH 5.7. This solution was then chroma-tographed through a second DE-52 column; on applying a 10-500 mM gradient of Na acetate, pH 5.7, the acceptor proteinemerged at about 50 mM salt; the pooled fractions were con-centrated by vacuum dialysis and applied to a 2 X 90-cmSephadex G-150 column. The elution profile is illustrated inFig. 1. Fractions under peaks A, B, and C were pooled sep-arately.

Procedure 11: The crude extract was adjusted to pHf 7.0 with5 N NH4 OH, heated to 800 under vigorous stirring over a 30-min period, then immediately cooled to 100 in an ice bath.The flocculent suspension was filtered through a fluted filterpaper, and the resulting solution was precipitated by additionof one-sixth volume of cold 100% trichloroacetic acid. The pre-cipitate was collected by centrifugation, and dialyzed ex-tensively against 10 mMI Na acetate, pH 5.7. This solution wasthen chromatographed through DE-52 cellulose and SephadexG-150 as described under Procedure I. Essentially, the sameelution pattern was obtained from Procedures I and II, whichyielded approximately 50 and 300 mg of pure material, respec-tively, per kilogram of muscle.

Physical Properties. Fractions A, B, and C were essentiallyhomogeneous as judged by 7.5% polyacrylamide gel electro-phoresis; sedimentation velocity and equilibrium experimentsin the ultracentrifuge gave molecular weights of about 350,000,75,000 and 25,000, respectively. On the other hand, whensubjected to gel electrophoresis in the presence of 0.1% Nadodecyl sulfate, all three fractions dissociated into subunitsof molecular weight about 11,000 and 13,000 (Fig. 2). It is notknown whether the faint slower band seen on some of the gelsis a contaminant or results from aggregation of the smallercomponents.

Proc. Nat. Acad. Sci: USA 71 (1974)

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FIG. 3. (A) Absorption spectrum of homogeneous dogfish parvalbumin (5 mg/ml in 20 mM Na glycerophosphate, 1 mMI ED)TA, pH6.8). (B) Absorption spectra of fractions A, B, and C under identical conditions.

Absorption Spectra of fractions A, B, and C are illustratedin Fig. 3; also shown is the very similar absorption spectrumof pure dogfish parvalbumin. As can be seen, all three frac-tions displayed the same vibrational structures in the 260-nmregion of the spectrum expected of proteins having a highphenylalanine to tyrosine and tryptophan ratio.

Phosphorylation and Dephosphorylation of the Phosphate-Acceptor Protein. When incubated with 10 mM Mg2+, 0.5 mMy-labeled [32P]ATP, and purified dogfish (or rabbit) proteinkinase, fractions A, B, and C showed an immediate incorpora-tion of 32p in a trichloroacetic acid-precipitable form (Fig. 4A).The reaction reached completion when about 1.0 mole ofphosphate was introduced per 12,000 g of protein, suggestingone phosphorylation site per subunit. By subjecting the 32p_labeled protein to Na dodecyl sulfate gel electrophoresisfollowed by radioautography, both the 11,000- and 13,000-molecular-weight subunits were radioactive.The bound phosphate is acid-stable and base-labile, with 3%

Pi being liberated by 0.25 N HCl in 24 hr at 200 as opposedto 80% by 0.25 N NaOH. This behavior suggests the presenceof a seryl-P or threonyl-P bond. Neither purified rabbit nordogfish muscle phosphorylase kinase could catalyze thisphosphorylation; on the other hand, a recently isolated yeastprotein phosphokinase was just as effective as the dogfish pro-tein kinase. For the dogfish enzyme, relative rates of phos-phorylation of protein substrates were: protamine, 100; P-acceptor protein, 75; phosvitin, 20; histone II A, 10; and casein5. Dephosphorylation of the P-acceptor protein was readilyachieved by a dogfish protein phosphatase (see Methods),but not by purified phosphorylase phosphatase (Fig. 4B).

Most surprisingly, despite the remarkable similarity of thestructural and immunological (see below) properties of theP-acceptor protein and parvalbumin, no phosphorylation ofthis latter protein could be demonstrated under any experi-mental condition, including whether or not it was in a nativeor denatured state, Ca±+-containing, or Ca2+-free. Pretreat-ment with the protein phosphatase to remove unlabeled phos-phate groups that might have occupied the sites susceptibleto phosphorylation did not help.

Calcium-Binding Properties. Since the main feature ofparvalbumins is that they bind strongly two equivalents ofCa2+ per mole [KdiS, about 2 X 10-7 Ml (23) ], the interactionof calcium with the P-acceptor protein was examined. Fig. 5shows that the high-molecular-weight acceptor protein be-haved almost identically to the parvalbumins with respect tothe amount of calcium bound aid dissociation constant (Kdi8sabout 5 X 10- M).Immunological Properties. Fractions A, B, and C displayed

strong immunological crossreactivities with antibodies pre-pared against homogeneous dogfish parvalbumin (Fig. 6A).Mixing any one of these fractions with parvalbumin resultedin a single precipitin line (Fig. 6B), indicating that the sameantigen was involved. This conclusion was further supportedby the complete fusion (no spurring) of the P-acceptor proteinand parvalbumin precipitin lines shown in Fig. 6C.

Identical immunological crossreactivities were displayedby the phosphorylated and nonphosphorylated acceptorprotein; when 32P-labeled material was used in the immuno-diffusion test, radioactivity was localized in the precipitinlines, as revealed by autoradiography. The immunological

Proc. Nat. Acad. Sci. USA 71 (1974)

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P-Acceptor Protein Related to Parvalbumin 2201

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Fie. 4. (A) Phosphorylation of acceptor protein (1 mg/ml) bypurified dogfish protein kinase (0, 2 gg/ml) and phosphorylasekinase (0, 10 ,g/ml). The reaction was carried out at 300 in a 50mM Tris * HCl-1 mM KH2PO4 buffer, pH 6.5, containing 20 mMNaF, 2 mM theophylline, 0.2 mM\1 EDTA, 10 mM M\1gC12, and0.5 mM [7-_32PJATP. Aliquots (0.1 ml) were removed, added to0.2 ml of bovine-serum albumin (10 mg/ml), and precipitatedwith 1.;5 ml of 15% trichloroacetic acid. After centrifugation,pellets were dissolved in 0.5 ml of 0.1 N NaOH and reprecipitatedwith 1.5 ml of trichloroacetic acid. After the protein was dissolvedand reprecipitated once more, the final pellet was dissolved in 1ml of 88% formic acid. This solution was counted in 10 ml ofscintillant (8 g of Omnifluor and 12.5 g of naphthalene per liter ofdioxane). (B) lDephosphorylation of 32P-labeled acceptor protein(1 mg/ml) by 20 pg/ml of dogfish protein phosphatase (-) or 5IAg/ml of purified phosphorylase phosphatase (0) in 50 mM\I Tris-HCl, pH 7.0.

specificity of the antiserum against dogfish parvalbumin (orthe P-acceptor protein) was demonstrated by the fact that no

crossreactivity was observed with either hake or frog parv-

albumin (Fig. 6D).

DISCUSSION

Both the P-acceptor protein and parvalbumins are found inskeletal muscle in sizable amounts. Remarkable similaritiesin their subunit structure, spectral behavior, Ca2+-bindingproperty, and immunological crossreactivity provide over-

whelming evidence for their close relationship. The only dif-ferences so far observed are that (a) the P-acceptor protein isisolated in various states of aggregation and (b) it can be co-

valently modified by phosphorylation or dephosphorylation.Aggregation could be readily explained on the basis of an

SH---S-S interchange since dogfish parvalbumin contains one

half-cystinyl residue. Changes in the degree of associationhave been noticed to vary with the presence or absence ofreducing agents; a possible aggregation of parvalbumins undercertain conditions has been suggested (25).The absence of phosphorylation of the low-molecular-weight

parvalbumins is more difficult to understand. This behaviorcould result from differences in either conformation or primarystructure between the two proteins. Must the acceptor pro-

tein be in an oligomeric state to accept phosphate? As of now,the smallest species found to be phosphorylated was the pro-

tein dimer with a molecular weight of about 25,000. Alter-natively, the phosphate-accepting ability could depend on

calcium content, this divalent cation acting either directly

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FIG. 3. Calcium-binding measurements of dogfish P-acceptorprotein (-) and parvalbumin (5) according to Briggs and Fleish-man (13). Included as controls were buffer alone (0), bovine-serum albumin (A), and 50 juS EGTA (0). All proteins were pres-ent at 0.1 mg/ml in 30 mM1 imidazole buffer, pH 6.7, containing 2mM iMgCl2 and 50 MM KCl.

on the site of phosphorylation, or indirectly, by bringingabout changes in tertiary or quaternary structure of the pro-tein. The structure of rabbit TN-C (the calcium-binding sub-unit of troponin) was shown to collapse upon removal ofcalcium (26); the same is true for the parvalbumins (27).The second possibility is that the P-acceptor protein con-

tains an additional group or fragment that accepts phosphatebut is missing from the parvalbumins. This group would prob-ably be covalently bound since it is released neither by Nadodecyl sulfate nor by trichloroacetic acid precipitation. IfS-£i->^St~~ ..........

FIG. 6. Immunodiffusion experiments in 1% agar prepared in

.50 mM'\ Tris.HCI, 150 mM\ NaCl, and 0.02%, Na azide, pH 7.5.

Protein bands were stained with Amnido black (24). All center

wells contained antiserum against homogeneous dogfish parval-

bumnin. Peripheral wells contained approximately 5 j~g each of:

Plate A: fractions A (1), B (2), and C (3), obtained from Sephadex

G-150 chromatography (see Fig. 1). Plate B: mixture of dogfish

parvalbumnin with fractions A (1), B (2), and C (3). Plate C: dog-

fish parvalbumnin (1) and fraction C (2). Plate D: parvalbuinins

from dogfish (1), frog (2), and hake (3).

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2202 Biochemistry: Blum et al.

this were so, parvalbumins could be regarded as modified-orperhaps even degraded-forms of the more native P-acceptorprotein.

In support of this hypothesis is the fact that no definitephysiological function has yet been ascribed to the parv-albumins. A relationship with the troponin system, par-

ticularly with TN-C, would first come to mind. A considerabledegree of homology was uncovered between the sequence ofrabbit TN-C atid sequences from fish and frog parvalbumins,providing strong evidence that these proteins are evolution-arily related (28, 29). While it has been proposed that the threehomologous regions found in parvalbumins might have arisenfrom a gene triplication (9, 10), the larger TN-C moleculecould have evolved by further duplication of the parvalbumingene (28, 29). Furthermore, when purified dogfish troponin or

TN-C was allowed to undergo proteolytic digestion in thenbsence of Ca2+ ions, fragments of about 12,000 molecularweight were obtained that displayed strong immunologicalorossreactivities with dogfish antiparvalbumin even thoughthe intact proteins did not crossreact (30). However, no

mechanistic relationship between these two systems has yetbeen established. Since no function has yet been found for theparvalbumins, it would be tempting to propose that the P-acceptor protein is the real physiologically active species. Avariety of functions could be imagined, such as its participa-tion in calcium storage or transport, or its involvement in a

regulatory process either alone or in conjunction with otherrpuscle proteins; even an enzymatic role has not been ex-

eluded. A dissociation constant for calcium of the order of0.1 ,gM is very similar to that obtained for ATPase involvedin calcium transport to and from the sarcoplasmic reticulum[< 0.1 uM (31)]. Considerable support could be given to anyone of these hypotheses if it were found that Ca2+-binding can

be modulated by phosphorylation-dephosphorylationI of theprotein, or vice versa.The finding of a new protein related to parvalbumin raised

an obvious question: even though no parvalbumins had beenfound hitherto in higher vertebrates, might the P-acceptorprotein be present? The answer is yes, which immediately sug-

gested that parvalbumins might also be present. Recentevidence from this laboratory indicates that this is indeed theease: parvalbumins have now been isolated and characterizedfrom the skeletal muscle of turtle, chicken, rabbit, and man

(32).

This work was supported by the National Institutes of Ar-thritis and Metabolic Diseases (AM 07902) NIH, USPHS; theNational Science Foundation (GD 20482); the Muscular Dys-trophy Association of America; and fellowships from the Studi-enstiftung des Deutschen Volkes and the Deutsche Forschungs-gemeinschaft to H.E.B. It was carried out in cooperation withthe laboratory of Dr. J. -F. Pechbre, Department of Macromo-lecular Biochemistry, CNRS, Montpellier, France, whose gift ofmaterial is gratefully acknowledged.

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