Biochem. J. (1987) 242, 245-251 (Printed in Great Britain)

Comparison of calregulins from vertebrate liversNavin C. KHANNA, Masaaki TOKUDA and David Morton WAISMANDepartment of Medical Biochemistry, University of Calgary, Calgary, Alberta T2N 4N1, Canada

Calregulins were purified from bovine, rabbit and chicken liver, and their structural properties werecompared. Significant differences between the three calregulins include a lower Mr for chicken calregulin(57000) than for rabbit and bovine calregulin (63000), and the glycosylation of only bovine calregulin.Amino acid composition and peptide maps of the three calregulins were very similar. No major differenceswere detected in the Ca2+-binding properties of the three proteins. Zn2+-induced changes in calregulinconformation and hydrophobicity monitored by intrinsic protein fluorescence and the hydrophobicfluorescent probe 8-anilino-1-naphthalenesulphonate were very similar, suggesting that the Zn2+-dependentincrease in the hydrophobicity of bovine, rabbit and chicken calregulin was conserved. These studies morefully define what is a calregulin, demonstrate that calregulin is a relatively invariant constituent of vertebrateliver, and indicate that calregulin structure has been highly conserved in bovine, chicken and rabbit liver.

INTRODUCTIONPrevious studies in our laboratory (Waisman et al.,

1985) have documented the presence of a Mr-63000Ca2+-binding protein, called calregulin (Khanna &Waisman, 1986; Khanna et al., 1987), in the 100000 gsupernatant of a variety of bovine tissues. That thisprotein might be an important intracellular receptor forsecond-messenger Ca2+ has been suggested by the highconcentration of calregulin in bovine tissues (Khanna &Waisman, 1986) and by the high affinity of binding ofCa2+ under physiological conditions (Khanna et al.,1986). In our previous report (Khanna et al., 1986) thepresence of distinct and specific ligand-binding sites onbovine calregulin for Ca2+ and Zn2+ were documented.Measurement of Ca2+- and Zn2+-induced changes intryptophan fluorescence suggested that both Ca2+ andZn2+ induced distinct conformational changes in bovinecalregulin. Studies with the fluorescent hydrophobicprobe ANS suggested that Zn2+, but not Ca2 , inducedan increase in the hydrophobicity of bovine calregulin(Khanna et al., 1986). This result presented thepossibility that mechanistically calregulin might act in amanner similar to calmodulin, whose Ca2+-dependentincrease in hydrophobicity has been shown to be anecessary step in the activation of many enzymes by thisprotein (LaPorte et al., 1980).As part of a systematic study of the structure and

evolution of calregulin, we have isolated calregulin frombovine, chicken and rabbit livers, using the sameisolation protocol for each species. We report here acomparison of the physico-chemical and structuralproperties of the liver calregulins from these species.These studies more fully define what constitutes acalregulin and demonstrate that the physicochemicaland structural properties of calregulin from a singletissue have been highly conserved during vertebrateevolution.

EXPERIMENTAL

MaterialsAll chemicals were reagent grade, unless specified

otherwise. Deionized water was used throughout. Chelex

100 was obtained from Bio-Rad. 45CaC12 (20 mCi/mgof Ca2+) was purchased from Amersham Corp.Staphylococcus aureus V8 proteinase was obtained fromPierce Chemical Co. Hepes, Mops and ANS were ob-tained from Sigma.

Purification of calregulinCalregulins from bovine, chicken and rabbit livers

were purified by the procedure of Waisman et al. (1985).Ca2+ binding of purified calregulins

This was determined by equilibrium dialysis. Cal-regulin was first dialysed overnight against 1000 vol. ofa solution containing 150 mM-KCl, 10 mM-Mops(pH 7.1),3 mM-MgCl2, 1.0 mM-dithiothreitol and 0.1 mm-EGTA, to remove bound Ca2+ from the proteins. Thedialysed proteins were then used for equilibrium dialysisas follows: a 0.5 ml portion of protein was dialysed withshaking for 48 h at 4 °C against 100 ml of the samesolution, containing various amounts of CaCl2 and45Ca2+ (5 #uCi) to achieve the desired free Ca2+concentration. The solutions outside and inside thedialysis tubing were removed, the A278 was determined,and the protein concentration calculated from theabsorption coefficient. Samples of these solutions weresubjected to liquid-scintillation spectrometry, and thetotal Ca2+ concentration was calculated from thecontaminating Ca2+ (1.0 /zM) determined by atomicabsorption, plus the amount of Ca2+ added. Theassociation constants for metal and H+ binding ofEGTAwere based on values measured by Fabiato (1981).

Amino acid analysisCalregulins were dialysed overnight against two

changes of 1000 vol. of 0.04 M-NaHCO3, and portionsof the protein solution were transferred to hydrolysistubes containing 0.1% phenol and 0.02% 2-mercapto-ethanol. Norleucine (10 nmol) was used as an internalstandard to correct for losses of protein. Hydrolysis wascarried out at 110 °C for 24, 48 and 72 h. The calculatedvalues were based on mean values from eight deter-minations. Performic acid oxidation was performed bythe method of Hirs (1967). Tryptophan determinations

Abbreviation used: ANS, 8-anilinonaphthalene-1-sulphonic acid.

Vol. 242

245

N. C. Khanna, M. Tokuda and D. M. Waisman

were performed by the method of Simpson et al. (1976).Recoveries of tryptophan were typically 90-95%.Protein sequencing

Calregulin from chicken and rabbit livers wassequenced on an Applied Biosystems 470A gas-phaseprotein sequencer by using methanolic-HCI conversionand reagents from Applied Biosystems. Biobrene (30 ul;Applied Biosystems' Polybrene) was precycled for threecycles (2 x 0.2 nfil degradation cycles plus 1 x 0.2 nvacdegradation cycle) before each run (0.2 nfil and 0.2 nvacare standard programme cycles supplied by AppliedBiosystems for Polybrene pre-conditioning and proteindegradation respectively). The amino acid phenylthio-hydantoins from the sequencer were identified by h.p.l.c.by using a Varian 401 data system, a Waters WISP 710Bautosampler and a Waters 440 two-channel u.v. detector(254 and 313 nm). A Beckman Ultrasphere ODS column(5 ,im; 0.46 cm x 25 cm) with a Whatman Col: Pell ODSguard column (0.21 cm x 5.0 cm) was eluted by the25 min sodium acetate (pH 5.2, 15-50 cm)/acetonitrileprogramme at 40 °C, at a flow rate of 1.0 ml/min. Allamino acid phenylthiohydantoins except the lysine/phenylalanine pair were well resolved, and that ofpyridylethyl-cysteine was eluted in front of that ofmethionine. Norleucine phenylthiohydantoin was usedas an internal standard in each sequencer vial.

Peptide mappingPeptide mapping by limited proteolysis was performed

by the method of Cleveland et al. (1977). Purified proteinwas dissolved at approx. 0.5 mg/ml in sample buffer,containing 0.1 M-Tris/HCl, pH 6.8, 0.5% SDS, 100%(v/v) glycerol and 0.001% Bromophenol Blue. Thesamples were then heated to 100 °C for 2 min. Proteolyticdigestions were carried out at 37 °C for 30 min byaddition of Staph. aureus V8 protease (10 ,g/ml). Afteraddition of 2-mercaptoethanol and SDS to finalconcentrations of 10% and 2% (v/v) respectively,proteolysis was stopped by boiling the samples for 2 min.About 20-30 ,1 (10-20 ,Cg) of each sample were loadedinto a sample well of the 15% -acrylamide gel, which wasrun in the normal manner, and stained with CoomassieBlue.

Fluorescence measurementsThese were performed in a Perkin-Elmer MPF 4

spectrofluorimeter in water-jacketted cuvette holders at25 °C operated in the normal mode. All fluorescencemeasurements were done with protein solutions in 10 mm-Mops/3 mM-MgCl2/150 mM-KCl, pH 7.1. Zn2+ titra-tions were performed by sequentially adding smallportions of ZnCl2 from a stock solution to proteinsolutions and to a blank solution containing no proteinand allowing the sample to equilibrate after eachaddition, for 10 min. Titration measurements were madeat an excitation wavelength of 286 nm and an emissionwavelength of 345 nm. The binding of the hydrophobicfluorescent probe ANS was monitored by its fluor-escence enhancement. Again, the ANS was added fromconcentrated stock solution (10 nm in water) to cuvettescontaining calregulin or buffer only, and the fluorescenceof the solution was measured at the emission maximum,480 nm, after excitation at 360 nm. The effects of Ca2+and Zn2+ on the binding of ANS to calregulin wereevaluated by performing titrations and analysing spectra

Mr(X 10-3)

(a) (b)

67.0 _

45.0o

30.0

20.1 _w

14.4 _

A B CFig. 1. Polyacrylamide-gel

regulins

D B' Cl Dtelectrophoresis of purified cal-

Panel (a) shows SDS/polyacrylamide-gel electrophoresisof the purified bovine (B), rabbit (C) and chickencalregulin (D); panel (b) shows the non-denaturingpolyacrylamide-gel electrophoresis of bovine (B'), chicken(C') and rabbit calregulin (D'). Positions of Mr markersare indicated on the left.

in the presence and absence of metal ion. All titrationdata were corrected for the fluorescence of the blank andfor dilution.

Gel electrophoresisElectrophoresis in gels containing SDS was performed

in a slab-gel apparatus, by using the system described byLaemmli (1970). For analysis of peptides generated byproteolysis, 15% (w/v) acrylamide gels [75:2 (w/w)acrylamide/bisacrylamide] were routinely used. Non-denaturing gel electrophoresis was performed by theLaemmli (1970) method, except that SDS was omittedfrom the system and unboiled samples were used foranalysis. Gels were stained in a solution containing (finalconcentrations) 0.1% Coomassie Blue, 50% (v/v)methanol and 10% (v/v) acetic acid, and destained bydiffusion in 5% methanol/10% acetic acid.

Determination of total carbohydrateProtein solutions were filtered through a 0.45 ,sm-

pore-size Millipore filter. Total carbohydrate content ofbovine, chicken and rabbit calregulins was measured bythe method ofDubois et al. (1956). In brief, 0.5 ml ofeachprotein sample (1 mg/ml) was mixed with 0.5 ml of 0.5%phenol. After thorough mixing, the colour was developedby the rapid addition of 2.5 ml of conc. H2S04. Thesamples were incubated at 50 °C for 15 min and, aftercooling, their A495 was measured. Glucose (100 ,g/ml)was used as a standard.

Fluorescein isothiocyanate-linked lectins (concana-valin A, wheat-germ, soya-bean, lentil, asparagus, pea,ricin I and gorse seed) were used as probes to evaluatethe glycosylated nature of the purified calregulins. Thepurified calregulins dotted on a nitrocellulose sheet wereincubated at room temperature for 1 h with 50 mM-Hepes(pH 7.6) containing 3% (w/v) bovine serum albumin,0. 15 M-NaCl and 0.1 % Triton X-100. The sheets were

1987

246

Vertebrate calregulins

V

Fig. 2. Ca2+ binding by calregulins

The experiment was carried out by equilibrium dialysisas described in the Experimental section. The mediumused contained 150 mM-KCI, 10 mM-Mops (pH 7.1),3 mM-MgC12, 1 mM-dithiothreitol and 0.1 mM-EGTA.Scatchard plots of data: V, mol of Ca2+ bound/mol ofcalregulin; C, free [Ca2+] (,UM). (M) Bovine, (A) chickenand (0) rabbit calregulin.

washed with the above filter without albumin, and wereincubated separately with various lectins for 1 h at roomtemperature. After extensive washing with the samebuffer (without albumin), the sheets were evaluatedunder u.v. light.

Protein determinationProtein concentrations were determined by the method

of Bradford (1976). Calregulin concentration wasestimated by its absorption coefficient.

RESULTSPurification of calregulins

Calregulins from bovine, chicken and rabbit liverswere purified to electrophoretic homogeneity by thepreviously published procedure (Waisman et al., 1985).Fig. l(a) shows SDS/polyacrylamide-gel electrophoresisof the purified calregulins. Bovine and rabbit calregulins(lanes B and C) have Mr 63000, whereas chickencalregulin has Mr 57000 (lane D). Since the extensive useof proteolytic inhibitors has failed to allow theobservation of a higher-Mr form of chicken calregulin, itis unlikely that the Mr-57000 protein is a proteolyticfragment of a higher-Mr form of the protein. Fig. l(b)shows non-denaturing polyacrylamide-gel electro-phoresis of bovine, chicken and rabbit calregulins: thethree calregulins migrate similarly under these conditions,indicating a similar mass-to-charge ratio of theseproteins.

Table 1. Amino acid composition (mol %) of liver calregulins

Amino acid Bovine Rabbit Chicken

Aspartic acidThreonineSerineGlutamic acidProlineGlycineAlanineHalf-cystineValineMethionineIsoleucineLeucineTyrosinePhenylalanineHistidineLysineArginineTryptophanNo. of residues

16.53.73.7

17.76.46.84.60.54.20.94.44.43.54.62.09.52.44.4547

15.73.73.7

16.86.06.44.80.74.90.74.24.83.34.41.6

10.13.15.1547

15.73.93.9

17.76.76.94.11.04.51.03.94.13.04.91.8

10.03.93.3492

Ca2+-binding properties of calregulinFig. 2 presents Scatchard (1949) plots for Ca2+

binding to bovine, chicken and rabbit calregulins.Analysis of the data reveals that, in the presence of3.0 mM-MgCl2 and 150 mM-KCl, all the calregulins bind1.0 mol of Ca2+/mol of protein, with apparent Kd valuesof 0.05 /M (bovine) and 0.03 /M (chicken and rabbit).

Ca2+-binding studies were also performed in theabsence of MgCl2 (results not shown), but no significantdifferences were observed between the values for bindingof Ca2+ to any calregulin in the presence or absence ofMgCl2. These results indicate that the Ca2+-bindingproperties of calregulin are phylogenetically conserved inthese three species.

Amino acid composition and sequence of calregulinsThe amino acid compositions of bovine, chicken and

rabbit calregulins are shown in Table 1; they are verysimilar. All the proteins contain very similar amounts ofacidic, basic and aromatic residues. However, the mol %of tryptophan appears to differ significantly in bovine(4.4%), chicken (3.3%) and rabbit (5.1%) calregulins.The high contents of tryptophan account for theunusually high A280. The absorption coefficients (61c) ofbovine, chicken and rabbit calregulins are 17.8, 17.8 and18.8 respectively. The pl values of bovine (4.15), rabbit(4.15) and chicken (4.20) calregulins indicate their acidicnature.The N-terminal amino acid sequence of the first 15

amino acids of chicken and rabbit calregulins is shownin Fig. 3. The bovine calregulin could not be sequenced,owing to its blocked N-terminus. The amino acids atpositions 2, 6-8 and 11-15 are identical in chicken andrabbit calregulins. Moreover, positions 5 and 9 show aconserved substitution of amino acids. Such a closehomology in the amino acid sequence of chicken andrabbit calregulins is consistent with its highly conservedphylogenetic nature.

Vol. 242

247

N. C. Khanna, M. Tokuda and D. M. Waisman

Chicken: Gly -Pro -Ala~Gln~-Phe -Phe-Lys-Glu -Glu-Lys -Leu-Asp-Gly-Asp-Gly

Rabbit: Glu Pro Vali-ValitTyr Phe-Lys-Glu Gln-Phe Leu-Asp-Gly-Asp-Gly

Fig. 3. N-Terminal amino acid sequences of chicken and rabbit calregulinsBoxes with continuous lines indicate homology, and those with broken lines indicate conservative substitutions.

Carbohydrate analysis of calregulinsAnalysis of the total carbohydrate content of bovine,

rabbit and chicken calregulins was estimated by thephenol/H2SO4 method (Dubois et al., 1956). Bovinecalregulin contained 16 ,g of carbohydrate/mg ofprotein. In contrast, rabbit and chicken calregulin did notcontain any detectable sugar residues. Lectins were alsoused as a probe to identify the sugar residues of bovinecalregulin (see the Experimental section). Of seven lectinstested, only concanavalin A bound to bovine calregulin.Rabbit and chicken calregulins failed to interact with anyof the lectins tested. We have shown that about 90O% ofbovine calregulin is glycosylated and binds to aconcanavalin A-agarose affinity column (Khanna et al.,1986). However, a small population of bovinecalregulin (0.5-1.0%) is unglycosylated, and can beresolved from the glycosylated calregulin by concanavalinA-agarose affinity chromatography. Concanavalin-A-

M1(,. 10 i)

67.0

45.0

30.0

20.1

14.4

A B C

Fig. 4. Peptide maps of calregulinsD E

Bovine, chicken and rabbit calregulins, each at aconcentration of 0.5 mg/ml in sample buffer (see theExperimental section), were incubated at 100 °C for 2 min.After cooling, proteolytic digestions were carried out at37 °C for 30 min by addition of V8 protease (10 jug/ml).About 20-30 ,1 of each sample was subjected toSDS/15%-polyacrylamide-gel electrophoresis. Lanes: A,Mr ( X 10-3) markers; B, glycosylated bovine calregulin; C,non-glycosylated bovine calregulin; D, chicken calregulin;E, rabbit calregulin.

binding proteins are reported to be asparagine-linked pro-teins rich in a-D-mannopyranosyl, a-D-glycopyranosyland sterically related residues.

Peptide mappingSimilar patterns were obtained when proteolytic

digests of bovine, chicken and rabbit calregulins wereanalysed by SDS/ 150%-polyacrylamide-gel electro-phoresis. Fig. 4 shows the V8-proteinase-cleaved patternof bovine (lanes B and C), chicken (lane D) and rabbit(lane E) calregulins. Lanes B and C compare with thepeptide pattern of the glycosylated population of bovinecalregulin (lane B) and that of the uiiglycosylated popula-tion (lane C). The peptide pattern of unglycosylatedbovine calregulin (lane C) is very similar to that ofchicken (lane D) and rabbit calregulin (lane E). In allthree species there is one major band ofMr about 18 000,which seems to be more resistant to V8 protease. Thisband also seems to be preserved in the glycosylatedpopulation of bovine calregulin, but it has a lowermobility, possibly owing to the presence of sugarresidues.

Increased intrinsic fluorescence of calregulins on bindingZn2 +

Fig. 5 showsthe intrinsicand Zn2+-inducedfluorescence-emission spectra of bovine, chicken and rabbit cal-regulins. When excited at 286 nm (where predominantlytryptophan absorbs), the tyrosine contribution to theemission spectra (emission maximum 303 nm) wasnegligible, and the emission maximum at 334 nm wastypical of tryptophan. Addition of Zn2+ (0.5 mM) causesa dose-dependent increase of about 2-fold in intrinsicfluorescence intensity of all three calregulins, with ared-shift of about 11 nm in Amax of bovine and chickencalregulin and 10 nm for rabbit calregulin. With similarprotein concentrations, chicken and rabbit calregulins(0.1 mg/ml) exhibit 3-fold higher intrinsic tryptophanemission fluorescence as compared to bovine calregulin(Figs. 5a, 5b and 5c). Addition of 1 mM-EDTA to theprotein solution containing 0.5 mM-Zn2+ reversed theZn2+-induced increase in fluorescence intensity and thewavelength shift of the emission spectrum of all threecalregulins. Titration curves of the Zn2+-induced shift inintrinsic Amax of bovine, chicken and rabbit calregulinsare shown in Fig. 5(d). Approx. 8-20 ,tM-Zn2+ wasrequired to produce 50o% of the maximal effect of theZn2+-induced shift in Amax of bovine, chicken and rabbitcalregulins. The shift in Amax. of intrinsic proteinfluorescence of all three calregulins was complete atabout 100/LM-Zn2+

Zn2+-induced enhancement of ANS fluorescenceANS is an anionic amphiphile used as a probe for

hydrophobic regions of proteins (Fairclough & Canter,1978). Fig. 6 shows the Zn2+-induced enhancement ofANS fluorescence by chicken (a), rabbit (b) and bovine

1987

248

IWMNW ..'XX-..

': rsZ.:

41M

KuiwwAw:

Vertebrate calregulins

80

.tCc

.4

a)c04o

U-

20

0

25

20

C._c

CDa,c

10blo0

LL

U)

0)

LL.

450

Wavelength (nm)

EEx

mE0

4-

Ul

450

Wavelength (nm)

[Zn2"] (#M)

300 350 400Wavelength (nm)

Fig. 5. Increased intrinsic fluorescence of calregulins on binding Zn2+

Emission spectra are shown of calregulins (a, chicken; b, rabbit; c, bovine calregulin) in 10 mM-Mops (pH 7.1)/3 mM-MgCl2/150 mM-KCl at excitation wavelength of 286 nm. The protein solutions contained no Zn2+ (a), 0.5 mM-Zn2+ (b) or0.5 mM-Zn2++1 mM-EDTA (c). Panel (d) shows the titration curve of the Zn2+-induced shift in intrinsic Amax of bovine (x),chicken (A) and rabbit (v) calregulins.

249

(c) calregulins. Chicken calregulin blue-shifted theAmax of ANS from 520 nm to 485 nm (Fig. 6a).Addition of Zn2+ sequentially blue-shifted the Amax. ofthe ANS-protein complex from 485 to 465 nm andproduced about a 3-4-fold enhancement of ANSfluorescence intensity. Rabbit calregulin produced a shiftin ANS emission maximum from 520 to 490 nm (Fig. 6b).Again, the sequential addition of Zn2+ blue-shifted theAmax ofANS-protein complex from 490 to 470 nm, with3-4-fold enhancement of ANS fluorescence intensity(Fig. 6b). Bovine calregulin blue-shifted the ANSemission spectra from 520 to 500 nm (Fig. 6c).Sequential addition of 0.5 nM-Zn2+ produced a shift inAmax. of ANSprotein complex from 500 to about480 nm, with a 2-3-fold enhancement of fluorescenceintensity. The Zn2+-induced effect on the ANS emissionspectrum was reversible by EDTA. Fig. 6(d) shows thetitration curve of Zn2.+-induced shift in Amax. of

ANS-protein complex. About 16-32 /sM-Zn2+ was re-quired to produce 50% of the maximal effect of theZn2+-induced shift in Amax of ANS-protein complex.Rabbit calregulin required about 110IO/M-Zn2+ to producea similar effect in the Zn2+-induced shift in Amax ofANS-protein complex.

DISCUSSIONPrevious studies from our laboratory have documented

the presence of a Mr-63 000 Ca2+-binding protein inbovine liver (Waisman et al., 1985). This protein, calledcalregulin (Khanna & Waisman, 1986; Khanna et al.,1986, 1987), was quantified by radioimmunoassay in avariety of bovine tissues (Khanna & Waisman, 1986),and it was shown that calregulin exists in particularlyhigh amounts in pancreas (540 ,g/g of tissue), liver(375 ,ug/g of tissue) and testis (256 #ug/g of tissue). The

Vol. 242

N. C. Khanna, M. Tokuda and D. M. Waisman

Wavelength (nm)

ca)

cJXC00

0

U-

420 460 500 540 580 620Wavelength (nm)

20

15

cnc

40

c

LL

10

5

E

._x

EcoE0

wU

[Zn2 ] (#M)O . . . .

420 460 500 540 580 620Wavelength (nm)

Fig. 6. Effect of cakregulin and Zn2+ on the fluorescence emission spectra of ANS

Emission spectra are shown of ANS (25 /uM) in 10 mM-Mops (pH 7.1)/150 mM-KCI/3 mM-MgCl2 at excitation wavelength of360 nm, with calregulin: (a) chicken; (b) rabbit; (c) bovine. Panel (d) shows the titration curve of the Zn2+-induced shift in ANSAmax by bovine (x), chicken (A) and rabbit (A) calregulin. The protein solutions contained no Zn2+ (a), 0.5 mM-Zn2+ (b),or 0.5 mM-Zn2++ 1 mM-EDTA (c). Trace d shows the emission spectrum ofANS; 10 ,uM-ANS was used with bovine calregulin.

only bovine tissue shown not to contain calregulin wasthe erythrocyte. Considering the presence of largeamounts of calregulin in bovine tissues and the affinityand specificity of Ca2+ binding (Kd = 0.05 /tM), wesuggested that the calregulin might be an importantintracellular receptor for Ca2+ (Waisman et al., 1985;Khanna et al., 1986).

In a previous report (Khanna et al., 1986), we haveexamined the effects of metal binding on the confor-mation of bovine calregulin. It was shown that bovinecalregulin binds both Ca2+ and Zn2+ at distinct andspecific sites. Ca2+ binding to calregulin resulted in themovement of tryptophan away from the solvent,whereas Zn2+ binding caused a movement of tryptophaninto the solvent and exposure of a hydrophobic domain.Although the cytosolic free concentration of Zn2+ is

unknown, the total cytosolic concentration of Zn2+ hasbeen estimated at about 200 /SM (Thiers & Vallee, 1957),and the cytosolic free Ca2+ concentration is known tobe about 50 nm (Snowdowne & Borle, 1984). It istherefore reasonable to propose that under physiologicalconditions calregulin binds both Ca2+ and Zn2+.

In order to investigate whether or not calregulin wasrestricted to bovine tissues, we attempted the purificationof calregulin from rabbit and chicken liver. Initially wewere unsuccessful in this by our rapid purificationprocedure (Khanna et al., 1987). However, using our

older purification procedure (Waisman et al., 1985) wepurified calregulin from rabbit and chicken liver. Ourrapid purification procedure for calregulin (Khanna et al.,1987) failed because rabbit and chicken calregulins,unlike bovine calregulin, are unglycosylated proteins and

1987

250

100

80

601Ct

0

U)

0

LL

Vertebrate calregulins 251

therefore do not bind to the concanavalin A-agarosecolumn.

In the present paper we have compared the amino acidcomposition and peptide maps of bovine, rabbit andchicken calregulin and shown that these proteins are verysimilar. Chicken calregulin (Mr 57000) is significantlysmaller than bovine and rabbit calregulin (Mr 63000).The major difference between the three calregulins is thepresence of sugar residues on bovine calregulin and theabsence of these sugar residues from rabbit and chickencalregulin. It was therefore important to establishwhether or not the glycosylation of bovine calregulinaffected its metal-binding properties.A comparison ofthe Ca2+- and Zn2+-binding properties

of the calregulins suggests that the selective glycosylationof bovine calregulin does not affect the response of theprotein to these metal ions. All three calregulins bindCa2+ with about the same affinity, and all threecalregulins show a Zn2+-dependent conformationalchange. The latter involves an increased hydrophobicityof the protein, as illustrated by the Zn2+-dependentincrease in ANS fluorescence and red-shift in the ANSfluorescence-emission maximum. Zn2+ binding alsoresults in a large increase in intrinsic protein fluorescenceand a 11 nm red-shift in emission maximum. Althoughthe intrinsic protein fluorescence of bovine calregulinappears to be lower than that of chicken and rabbitcalregulin, the magnitude of the Zn2+-induced change inAmax appears identical.

These studies indicate that the physical, chemicaland metal-ion-binding properties of bovine, rabbit andchicken calregulin are conserved, and that the selectiveglycosylation of bovine calregulin does not result indramatic changes in the metal-binding properties of theprotein. Although speculative, it is reasonable topropose that the Zn2+-dependent increase in the

hydrophobicity of the three calregulins represents ageneral mechanism by which vertebrate calregulin mayinteract with other proteins in a similar manner to theCa2+-dependent increase in the hydrophobicity ofcalmodulin.

This work was supported by a grant from the MedicalResearch Council of Canada. N.C.K. and M.T. are Fellowsof the Alberta Heritage Foundation for Medical Research.

REFERENCESBradford, M. M. (1976) Anal. Biochem. 72, 248-254Cleveland, D. W., Fisher, S. G., Kirschner, M. W. & Laemmli,

U. K. (1977) J. Biol. Chem. 252, 1102-1104Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. &

Smith, F. (1956) Anal. Chem. 28, 350-356Fabiato, A. (1981) J. Gen. Physiol. 78, 457-497Fairclough, R. H. & Canter, C. R. (1978) Methods Enzymol.

48, 347-379Hirs, C. H. W. (1967) Methods Enzymol. 11, 59-62Khanna, N. C. & Waisman, D. M. (1986) Biochemistry 25,

1078-1082Khanna, N. C., Tokuda, M. & Waisman, D. M. (1986) J. Biol.Chem. 261, 8883-8887

Khanna, N. C., Tokuda, M. & Waisman, D. M. (1987)Methods Enzymol. 139, 36-50

Laemmli, U. K. (1970) Nature (London) 227, 680-685LaPorte, D. C., Wierman, B. M. & Storm, D. R. (1980)

Biochemistry 19, 3814-3819Scatchard, G. (1949) Ann. N.Y. Acad Sci. 51, 660-672Simpson, R. J., Newberger, M. R. & Liu, T.-Y. (1976) J. Biol.Chem. 251, 1936-1940

Snowdowne, K. W. & Borle, A. B. (1984) Am. J. Physiol. 247,C396-C408

Thiers, R. E. & Vallee, B. L. (1957) J. Biol. Chem. 226,911-920

Waisman, D. M., Salimath, B. P. & Anderson, J. (1985) J. Biol.Chem. 260, 1652-1660

Received 23 July 1986/22 September 1986; accepted 27 October 1986

Vol. 242