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  INTESTINAL CALCIUM-BINDING PROTEIN Chemical Composition, Affinity for Calcium, and Some Related Properties of the Vitamin D Dependent Calcium-Binding Protein? Paul J. Breddermant and Robert H. Wasserman* ABSTRACT: The concentration of a vitamin D dependent intestinal high-affinity Ca-binding protein (CaBP) is known to be highly correlated with the vitamin D dependent enhance- ment of intestinal Ca absorption. Purified CaBP from chick duodenal mucosa was analyzed for lipids, glycoprotein carbo- hydrate components, amino acid composition, and Ca-binding properties. It was free of lipid, carbohydrate, phosphorus, and other ash-producing substances. The molecular weight from amino acid composition and sodium dodecyl sulfate poly- acrylamide gel electrophoresis was near 28,000. CaBP con- tains only three half-cystine residues. Several spectrophoto- metric methods, including a new three-wavelength method, indicated the presence of two tryptophan residues. Polar residues make up 5 z of the 242 residues and 61 residues contain side-chain carboxyl groups. The calculated isoelectric point is 4.2 and the average charge per residue, 0.384. The computed partial specific volume and molecular volume were V tamin D, a sterol which undergoes metabolic conversion to more active forms in the liver and kidney, is required by several species for the optimal intestinal absorption of Ca and the vitamin influences Ca metabolism in kidney, bone, and the avian shell gland. Its metabolism and its metabolic effects have been recently summarized (Wasserman and Corradino, 1971 ; Wasserman and Taylor, 1972). The direct correspondence between the magnitude of absorption of Ca and the concentration of CaBPl in chick intestinal mucosa under a variety of physiological and nutritional situations (Wasserman et af. 1974) indicates CaBP to be intimately related to the vitamin D dependent translocation of calcium. Although intestinal Ca-binding proteins have been identified in a number of other species, including the human (Wasser- man et al., 1974), the function of these proteins is still un- determined. t From the Department of Physical Biology, New York State Veteri- nary College, Cornell University, Ithaca, New York 14850. Receiued August 20, 1973. Supported by U. S. Public Health Service Grant AM- 4652, U. S. Atomic Energy Commission Contract AT(ll-1)-3167, and the National Institutes of Health Training Grant 5-T01-DE-009. The sodium dodecyl sulfate electrophoresis studies were done at the Univer- sity of Rochester by Paul J. Bredderman supported by the National Institutes of Health Training Grant 5-T01-DE00175 and under contract with the U. S. Atomic Energy Commission at the University of Roches- ter Atomic Energy Project and has been assigned Report No. UR- 3490-360. $ Present address: Department of Radiation Biology and Biophysics, School of Medicine and Dentistry, University of Rochester, Rochester, N. Y. 14642. Abbreviations used are: CaBP, vitamin D dependent calcium- binding protein; NANA, N-acetylneuraminic acid; PAS, periodic acid- Schiff reagent; Pipes, piperazine-N,N-bis 2-ethanesulfonic acid); Tm temperature of maximum melting. 0.734 g and -34,000 A3, espectively. ey o (pH 6.5) at 280 nm was 9.03. A study of the thermal stability of CaBP indicated that its immunoreactivity, high-affinity binding of Ca and electrophoretic mobility were unchanged after a heat treatment of up to -80° but declined precipitously between 80 and 90'. Equilibrium dialysis studies revealed that Ca was bound exchangeably at four strong Ca-binding sites with apparent intrinsic association constant, ki, of 2 X lo6 M-' in 0.15 M KC1 (pH 6.8). Based on published competitive binding data (Ingersoll, R. J., and Wasserman, R. H. (1971), J. Biol. Chem. 246, 2808), the log ki for several divalent cations were calculated to be: Ca, 6.30; Cd, 5.10; Sr, 4.39- 4.58; Mn, 4.37; Zn, 3.71; Ba, 3.18-3.24; Co, 2.84; Mg, 2.44. Binding affinity appears to be related to the crystal ionic radius of these various cations. Additional Ca binding ap- peared abruptly when the concentration of free Ca2+ eached 3 x 10-3~. This paper presents the results of detailed analyses of the lipid, carbohydrate, and amino acid composition of chick CaBP which expand upon, and provide refinements and modifications to, previously published preliminary results (Wasserman et al., 1968). Also presented are the results of equilibrium dialysis studies of the protein's Ca-binding prop- erties along with estimates of its binding affinity for several other divalent cations, based on their competitive inhibition of Ca binding as published by Ingersoll and Wasserman (1971). Materials and Methods Inorganic, organic, and biochemicals were, when available, of reagent grade or better. Guanidine hydrochloride was a spectroscopic grade from Mann Research. The regenerated cellulose dialysis membrane was pretreated as recommended by Craig (1967), including treatment with The purification of CaBP was outlined by Wasserman et af (1968), incorporating several minor modifications; among them was the use of sodium azide (0.0273 and mercapto- ethanol (1 mM) in all solutions. Protein was determined by the method of Lowry et al. (1951) with crystalline bovine serum albumin as standard and using the experimentally determined factor of 1.04 to obtain CaBP concentration. Nitrogen was determined by the sealed-tube method of Jacobs (1964). For the lipid analyses, CaBP was extracted with 20 volumes (v/v) of 3:l ethanol- ether (Boyd, 1936). Qualitative analysis of the extracts for neutral lipids and phospholipids was by thin-layer chromatog- raphy (tlc). Cholesterol and cholesterol esters were estimated by the method of Zak et al. (1954). Free fatty acids were BIOCHEMISTRY, VOL. 13, NO. 8, 1974 1687 M EDTA.  BREDDERMAN AND WASSERMAN determined by gas-liquid chromatography. Lipid phosphorus analysis was based on the methods of Bartlett (1959) and Morrison (1964). For all analyses, glassware was prerinsed with 2 : 1 chloroform-methanol and the results were expressed as the difference between the amount of lipid in the CaBP extract and in the blank extract. Total carbohydrate was measured by the method of Dubois et ul. (1956) on CaBP passed through a 10 p Millipore filter. The method of Gibbons (1955) was employed for methylpentose analysis. Total hexos- amine was estimated according to Cessi and Piliego (1960) on samples hydrolyzed for 4 hr at 100 in 4 N HCI under nitrogen at a CaBP concentration of 0.1%. The method of Warren (1959) was employed to estimate free sialic acid in samples hydrolyzed in 0.1 N H 04 at 80 for 1 hr using syn- thetic NANA as standard. Sialic acid was also estimated by the method of Svennerholm (1957), without prior hydrolysis. Each of these carbohydrate analyses was performed with the proper internal controls to correct for carbohydrate destruc- tion, protein interference? and nonspecific absorption, all of which proved to be minor. For the analysis of tryptophan and tyrosine, bovine serum albumin, a-chymotrypsinogin A, and lysozyme were used as controls and several spectrophotometric methods were em- ployed (Goodwin and Morton, 1946; Bencze and Schmid, 1957; Edelhoch, 1967), including a new three-wavelength method to be described in detail elsewhere (P, J. Bredderman, in preparation). Total half-cystine and methionine were determined accord- ing to Moore (1963) on performic acid oxidized CaBP hy- drolyzed for 20, 24, 48, and 72 hr. Amide nitrogen was esti- mated as outlined by Wilcox (1967) and also from linear extrapolation to zero time of hydrolysis, based on the am- monia content of CaBP hydrolysates prepared for amino acid analysis. This latter method was not very satisfactory because the variability in the ammonia values among hy- drolysates made extrapolation difficult. A third method for amide nitrogen came from the observation that the fluctuations in the concentrations of four amino acids (serine, threonine, methionine, and tyrosine) accounted quantitatively for the wide fluctuations in the ammonia content of the various CaBP hydrolysates, the sum of these five components being essen- tially constant for all hydrolysates. This suggested that amide nitrogen could be estimated more precisely as the total ammonia in each hydrolysate minus that ammonia arising from non-protein sources and from the destruction of these four amino acids. Amino acids other than tryptophan, tyrosine, methionine, and half-cystine were determined by ion-exchange chromatog- raphy (Moore and Stein, 1963) on a Beckman Model 120C analyzer. Two batches of CaBP were analyzed; for each batch, two samples were hydrolyzed for each of four hydrolysis times (12, 24,48, and 72 hr) in sealed degassed tubes at 110 1 in 6 N HCI at a final protein concentration of 0.1 %. Hydrolysis blanks were used to correct for non-protein ammonia. Hydrochloric acid was removed from the hydrolysis tubes by rotary evaporation at 40' and 5 mm. From plots of amino acid concentration cs. the hours of hydrolysis, serine and threonine, which showed gradual destruction during hydrolysis, were determined as the linearly extrapolated zero- time values, whereas all other acids were determined from the plateau values. The molecular weight of CaBP was estimated from the amino acid composition by the method of Nyman and Lindskog (1964), and the results were utilized to calculate the amino acid residue nearest integer values. The molecular 1688 BIOCHEMISTRY, OL. 13, NO. 8 1974 weight was also estimated by sodium dodecyl sulfate poly- acrylamide gel electrophoresis, using 16 standard proteins ranging in molecular weight from 11,700 to 67,000 and em- ploying the sodium dodecyl sulfate polyacrylamide gel electro- phoresis system of Fairbanks et ctl. (1971) T = 11.1 x = 0.9%).? To assess the thermal stability of CaBP, homogenates of duodenal mucosa of vitamin D repleted chicks were prepared in 1 :4 (w/v) dilution with Tris-buffered saline (pH 7.4). Aliquots of supernatant from a 30 min, 78,000g centrifugation were treated for 10 min at temperatures ranging from 25 to 100 and recentrifuged. As indications of the amount and quality of the CdBP in the resulting supernatants, the CaBP band on analytical polyacrylamide gel electrophoresis was scanned densitometrically, the high-affinity Ca-binding activity was measured using equilibrium dialysis and tracer ITa, and the amount of immunoreactive CaBP was assessed by radial immunoassay, kindly performed by Dr. A. N. Taylor, using specific rabbit antibody. As part of the study of the Ca- binding properties of CaBP, liquid scintillation methodology was used to measure 15Ca in Bray's solution (Bray, 1960). Quench corrections were not required. Total Ca was measured by atomic absorption spectrometry with standards prepared from a certified Ca standard (Fisher Scientific). To measure the exchangeability of the bound Ca, purified CaBP (0.700 mg/ml) was first preequilibrated at 4 with 4 X M Pipes (pH 6.8). Then. starting with equal concentrations of I5Ca in retentate and dialysate, the specific radioactivity of both solutions and the protein concentration of the retentate were monitored at 12-hr intervals during a 180-hr dialysis at 4'. Exchangeability was assessed from the ratio of the specific activities of calcium in each compartment. In a second study the affinity and binding capacity of CaBP for Ca were determined by equilibrium dialysis, employing plastic dialysis cells with 1-ml chambers and a range of equilibrium ionic Ca concentrations, (Ca)f, from 2 X lo--? to 1 x M in 0.15 M KCI-10-3 M Pipes (pH 6.8) with a CaBP concentration of 0.700 mgjml. Dialysis was for 48 hr at 4 , about twice the time required for equilibration. The Ca contamination in the KCI-Pipes buffer was estimated by the method of additions (Dickson and Johnson, 1966) to be about 2.5 X 10-7 hi, in close agreement with the results of others (Nanninga and Kempen, 1971), and con- stituted a negligible fraction of the total Ca in almost all cells. The data were plotted as described by Scatchard (1949). The Xi and n obtained above for the high-affinity Ca-binding sites of CaBP were used in conjunction with previously pub- lished data (Ingersoll and Wasserman, 1971) to estimate thc affinity of other divalent cations for these sites. The experi- mental data of Ingersoll and Wasserman (1971) indicated the degree to which various cations (Sr2+, Cd'.. . Mn'., 2n'4, Ba'+, CoZT, MgZ7) t M inhibited Ca binding when Cab was 5 X M using an equilibrium dialysis procedure. The k, for each of these cations was calculated from thc following expression M CaCI? in 0.15 M KCI and ____ ____ ~- . ... ~~ 2 The gels are described by the notation of Hjertcn (19621, whcrc T = percentage (w v) of acrylalnide moiiomers nii C = pcrccntciyc (w w) of N,~'-methS.lcnebisacr~l~ll~id~ xpressctl as pcr ccnt of total u eight of moiioiiicrs.  INTESTINAL CALCIUM-BINDING PROTEIN OIIIIIIIII where kiCa and kix are the apparent intrinsic association constants for Ca and the competing divalent cation, X, respectively; CY is the fractional inhibition of Ca binding at (Ca)f and (X)f; and 7 is the average number of Ca ions bound to the n high-affinity sites of CaBP at (Ca)f in the absence of the competing divalent cation. The derivation of the above equation is straightforward and omitted here because of limitation of space. However, it should be pointed out that the assumptions underlying the derivation are, as follows : (1) the competing ion has no allosteric effects on the protein; (2) the competition is of equal intensity at all n sites; (3) the values of (Ca)f remain the same whether or not the competing ion is present; and (4) all of the unbound competing ion is present in solution as the free divalent cation. The validity of assumption (3) was assured by the experimental design which used a large volume ratio of dialysate to retentate. About 25 % of the CdZ+ robably was present as the CdOH+ and CdC1+ complexes (SillCn and Martell, 1971), but this has only a minor effect on the degree of competitive inhibition by CdZ+ nd on its computed association constant. Results Lipid Analysis. Both 90-mg batches of CaBP yielded in- distinguishable neutral lipid patterns on tlc. However, quan- titative analysis indicated that the CaBP contained less than 0.1 x ipid, much less than a 1 1 lipid: protein molecular ratio. The absence of phospholipid was also indicated by direct elemental analysis of CaBP which showed that less than 0.02 x was present. Carl~ohydrate nalysis. The amounts of CaBP tested in each of the several analyses would have allowed a 1 : 1 carbo- hydrate : CaBP molecular ratio to be easily detected. In each case, however, the absorption observed was close to the limits of sensitivity of the assay and only about twice that produced by the ultrafiltrate of the CaBP solution, thereby indicating the absence of carbohydrate as an integral part of the molecule. We also observed that the CaBP band on polyacrylamide gel electrophoresis of freshly prepared 70’ heat-treated mucosal homogenates reacted negatively to PAS staining (Fairbanks et a[., 1971), even when very large amounts of CaBP were present. Tryptophan and Tyrosine Analysis. The method of Goodwin and Morton (1946) and that of Bencze and Schmid (1957) gave unreliable estimates of tryptophan and tyrosine in the three control proteins analyzed. The simultaneous determina- tion of these two amino acids in 6 M guanidine hydrochloride (Edelhoch, 1967) overestimated tryptophan and slightly underestimated tyrosine in bovine serum albumin, possibly as a result of the need to correct for “irrelevant absorption” in the albumin. When tyrosine was estimated independently prior to the determination of tryptophan (Edelhoch, 1967), correct estimates were obtained for all three control proteins. However, the three-wavelength method for tryptophan gave the most accurate values. It was concluded that CaBP con- tains two residues of tryptophan and eight residues of tyrosine per molecule. HaCf-cystine and Methionine Analysis. After correcting the cysteic acid content of performic acid oxidized CaBP for the 94.7 conversion of cystine to cysteic acid determined experi- mentally, the average plateau values of the ratios between the amounts of cysteic acid and each of six stable amino acids resulted in an estimate of 2.58 half-cystine residues/ molecule of CaBP. In a similar manner, it was determined FIGURE 1 : Estimation of molecular weight of calcium-binding protein from amino acid composition by the method of Nyman and Lindskog (1964). The upper curve includes weighted devia- tions of half-cystine residues and the lower curve does not. Both yield, as best estimate, a mol wt of slightly less than 28.000. that CaBP contains eight methionine residues per mol wt 28,000. Amide Nitrogen Analysis. The method described by Wilcox (1967) indicated 18.36 amide groups/mol wt 28,000. By extrap- olation, the ammonia in the two sets of CaBP hydrolysates yielded values of 22.7 and 21.4 residues. These were rather uncertain, however, and the results obtained in this manner are generally considered maximum amide values. By the third method of amide analysis (see Materials and Methods), values of 17.1 and 17.2 residues were obtained for the two sets of hydrolysates. These latter values, because they too would appear to be maximum values, were accepted as the best estimates. Ocerall Amino Acid Analysis. Table I is a summary of the amino acid analysis. Of the 108 + 2.2 pg (SE) of protein nitro- gen present in the samples, 107 pg is accounted for by the composition shown. The composition shown also accounts for 101.3 of the protein determined by the method of Lowry et al. (1951). Molecular Weight Determinations. Both the estimate of the molecular weight from the amino acid composition (Figure 1 by the procedure of Nyman and Lindskog (1964) and the estimate by sodium dodecyl sulfate polyacrylamide gel electrophoresis (Figure 2) indicate a value of close to 28,000 about 1000. This value is in accord with the value of 28,000 obtained previously by molecular exclusion chromatography (Wasserman et al., 1968). However, when the computed partial specific volume (Table I) was used to recalculate the three sedimentation equilibrium molecular weight estimates (Wasserman et al., 1968), only one of these values (24,972 1,518 (=kSE); 25,247 =k 1,671; 27,462 =t 1,339) is in reason- able agreement with the other independent estimates. Isoelectric Point. The point of zero net charge of CaBP was computed from the amino acid composition using average BIOCHEMISTRY, VOL. 13, NO. 8, 1974 1689  BREDDERMAN AND WASSERMAN f for CoBPq FIGURE 2: Molecular weight determination of calcium-binding protein by sodium dodecyl sulfate polyacrylamide gel electro- phoresis. The reference proteins and their molecular weights are, as follows: 1, cytochrome c (equine, 11,700); 2, ribo- nticlease A (bovine, 13,700); 3, lysozyme (egg white, 14,300): 4, myoglobin (whale, 17,200); 5, soybean trypsin inhibitor (Kunitz. 21,500); 6, papain, 23,400; 7, a-chymotrypsinogen A (bovine, 25,700); 8, elastase (porcine, 25,900); 9. carbonic anhydrase (bovine erythrocyte, 30,000); 10, carboxypeptidase A (bovine, 34,400): 1 glyceraldehyde-3-phosphate dehydrogenase (rabbit muscle, 35,700); 12, aldolase (rabbit muscle. 40.000); 13, ovalbumin (45,000); 14, L-glutamic acid dehydrogenase (bovine, 56,100): 15, catalase (bovine erythrocyte, 57,500); 16, bovine serum albumin (67,000). RF values are mobilities relative to the tracking dye. Pyronin-Y. pK, values for aspartic (3.85) and glutamic (4.4) side chains and the end-group carboxyl (3.1) (Cohn and Edsall, 1943) after arbitrarily assigning the amide groups to an equal per- centage of the two types of side-chain carboxyls. The estimate, 4.2, was the same as the experimentally determined isoelectric point reported by Ingersoll and Wasserman (1971). Extinction CoefJicient. The percentage nitrogen (1 5.4), calculated from the nearest integer residue values, along with total nitrogen and 280-nm absorption values of CaBP in water at pH 6.5, were used to calculate an el ,, 1 cm value of 9.03. Thermal Stability of' CaBP. Figure 3 shows that a large fraction of the total protein in mucosal homogenates started precipitating at about 40 , with only about 33% remaining soluble at 60 and only about 15% soluble at 100 . However, CaBP appeared to be unaffected up to almost SO , based on electrophoretic mobility, immunoreactivity, and high-affinity Ca binding. (The low value for the electrophoresis of the control resulted from the protein overload on the gel.) Above 80° however, the values of all three of these parameters declined precipitously and then plateaued at 5-20 % of the 1690 BIOCHEMISTRY, VOL. 13, NO. 8, 1974 initial values. The temperature of maximum melting, T,,, would appear to be about 85'. Exchangeability of CaBP-Bound Calcium. After equilibrium was attained at 40 hr, the mean specific activity ratio (re- tentate : ialysate) for the subsequent 140-hr dialysis was 1.039 =t .019 (SE), indicative of the complete exchangeability of bound Ca. It was also calculated from atomic absorption measurements of the Ca in the dialysate and retentate and from protein determinations on the retentate (0.700 mgiml) that CaBP binds four Ca2T per molecule at 4.12 X M (Calf, in complete accord with the findings described below. It is noteworthy that direct elemental analysis of CaBP, indicating less than 0.01 % ash, is consistent with the presence of nonexchangeable metal ions in no more than a 1 :10 metal : CaBP molecular ratio. Calcium-Binding Capacity and Afinity of CaBP. Under the experimental conditions Donnan effects were insignificant over the entire (Ca)r range (2 x 10-7 to 1 x M). The ionic strength was essentially constant at 0.16 for (Ca)f 5 1 x M. Above this range, the increase in ionic strength due to an increase in CaCI? up to 1 X lo-' hi would have depressed the activity coefficient of Ca2'- by no more than about 2.2%. Ingersoll and Wasserman (1971) found that the degree of depression in Ca binding caused by increasing the ionic strength over the range of 0.02-0.15 was the same regardless of whether KCI or NaCl was used, suggesting that there is no specific binding of the Ki of the buffer to the Ca-binding sites. Figure 4 is a Scatchard plot of the results (Scatchard, 1949). The plotted values were obtained from 24 dialysis cells of two 12-cell experiments and cover 14 different Ca concentra- tions. The ordinate in Figure 4b, where (Ca)f for the plotted values ranges from 2.1 X 10-7 to 1.4 x hi, covers 250 times the range covered in Figure 4a, where (Ca)f for the plotted values ranges from 1.6 x IO-' to 1.0 x lo--'? i. From the slope in Figure 4b, the apparent intrinsic association constant, ki or the high-aflinity sites was estimated as 2 X IO6 k1-I (range 1.6 x lo6 o 2.5 X lo6 wl). The curve in the sub- figure intercepts the abscissa to yield a value of 4 for the number of high-affinity sites PI) per mol wt 2S,000. The slight downward curvature of the experimental points may indicate slight cooperativity among the four sites, but this is uncertain because of the size of the standard error estimates. Figure 4a shows that no more calcium becomes associated with the protein until a (Ca)f of 3 x lo+ xi is reached; above this concentration there is a sharp upturn in bound calcium. By subtracting the contribution of the four high-affinity sites, one should be left with a linear plot reflecting the relationship F,l(Ca)f = k,(n - 5) (2) for binding to a second set of preexisting identical, independent sites. Instead, however, a curvilinear plot (not shown) was obtained that passed through the origin at 3 X M (Cali as would occur if n for these sites went to zero at 3 X 10 - (Ca)f as ii went to zero. From extrapolation of the linear portion of this latter curve, the rough estimates of Xi 30 M--I and n l 32 were obtained for these low-affinity sites. Apparent Intrinsic Association Consta nt.r /or Otlrer Diva cml Cutions. The competitive inhibition data used to calculate these association constants (Ingersoll and Wasserman, 1971) are plotted in Figure 5 as a function of crystal ionic radius (Pauling, 1960). The log of the association constants obtained as described in Materials and Methods are: Ca, 6.30; Cd. 5.10; Sr, 4.39-4.58; Mn 4.37; Zn, 3.71; Ba, 3.18-3.24; CO, 2.84; Mg, 2.44. The range in the values for Ba and Sr rep- resent the results obtained at two concentrations of these ions.
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