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Bovine intestinal calcium-binding proteins
Biochimica et 13iophysica Acta, 3i 7 (i973) 172 ~80 c~ E l s e v i e r Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m
BBA
- P r i n t e d in The Neth(rla.nds
36462
BOVINE INTESTINAL CALCIUM-BINDING PROTEINS P U R I F I C A T I O N AND SOME PROPERTII~S
C. S. F U L L M E ] { AND R. 1t. W A S S E I < M A N
Departme~l of l~hysical Biology, New York State VeterDmrv College, a~d The (;radzmh, School oj Nutrition, Cornel[ Uni~'ersilV, Ithaca, N . Y . 1485o (U.S.A.) ( R e c e i v e d V e b r u a r v 2nd, 1073)
SUMMARY
~. Three separate calcium-binding proteins, atl similar to the vitamin D adependent calcium-binding protein of the chick, have been isolated in high purity from bovine intestinal nmcosa. All three proteins exhibit rapid electrophoretic migration rates on acrylamide gels and possess high affinities for calcium. One protein (major component) is always present in greater amounts than either of the others (termed "minor A component" and "minor B component"). 2. The molecular weight of the major bovine component was found to be about I . I . lO4 by calibrated gel filtration column chrolnatography. 3. The calcium-binding activity of partially purified preparations was not affected by trypsin digestion but was rapidly and conlpletely destroyed by incubation with pronase. Furthermore, moderate heat treatment of the supernatant solution from mucosal homogenates did not appreciably reduce calcium-binding activity . f this fraction. 4. An ilmnunnassay system for bovine calciunl-binding protein has been developed and shown to be specific for the three bovine components. Using this nmthod, calcium-binding protein has also been detected in the bovine kidney. 5. Preliminary results have indicated that, fi)llowing prolonged st,rage, one of the bovine components (inajor) gives rise to the remaining two (minor A and minor B). This degradation occurs in purified preparations as well as in the crude supernatant solution and m a y indicate that only one of the components is native to the intestine.
INTRODUCTION
A unique intestinal calcium-binding protein was first observed in the supernatant solution from nmcosal homogenates of the chick 1. This protein was found to be absent from the intestines of rachitic animals but appeared shortly following
BOVINE INTESTINAL CALCIUM-BINDING PROTEINS
173
administration of vitamin D 3 and increased in amount coincident with increased calcium absorption 2,3. Furthermore, pulse-labeling experiments revealed that stimulation of the rate of synthesis of intestinal calcium-binding protein by vitamin D 3 occurred before there was any substantial effect on the calcium transport process ~. The synthesis of the protein due to vitamin D 3 was inhibited by prior administration of actinomycin D 5, as is the enhanced absorption of calcium T M . These results lend support to the theory that calcium-binding protein is, in part, responsible for the intestinal translocation of calcium. Chick calcium-binding protein has an estimated molecular weight of 28 ooo by gel filtration (ref. II and Bredderman, P. J. and Wasserman, R. H., unpublished) and possesses four high affinity calcium-binding sites with an average affinity constant of approx, lO 6 M-1 (Bredderman, P. J. and Wasserman, R. H., unpublished). It has been shown to be present in all segments of the small intestine, the colon, and the kidney of the chick TM, and in the uterus (shell gland) of the laying hen 13. Calcium-binding protein, localized by fluorescent antibody techniques, appears in the brush border region of the intestinal absorptive cells as well as in the goblet cells of the chick duodenum la. Calcium-binding proteins have also been detected in the rat 15-17, dog TM, monkey 19, pig 2°, human2°, 21 and guinea pig (Fullmer, C. S. and Wasserman, R. H., unpublished). In the latter three species, as well as the chick (Fullmer, C.S. and Wasserman, R. H., unpublished), it appears that more than one calciumbinding component is present in the intestinal mucosa (refs 20 and 21 and unpublished results). The rat intestinal calcium-binding protein has also been shown to be vitamin D-dependent and has a molecular weight of about 8000 by sedimentation equilibrium and 13 ooo by gel filtration iv. Recent studies on other mammalian calcium-binding proteins, including the porcine ~°, human 2°, guinea pig (Fullmer, C. S. and Wasserman, R. H., unpublished), equine (Fullmer, C. S. and Wasserman, R. H., unpublished) and bovine, have revealed similar estimates for molecular weights in the range of II ooo-13 ooo. Preliminary calcium-binding studies on the porcine protein have shown an affinity constant similar to that of the chick calcium-binding protein ~°. The present paper reports on the isolation and purification of three separate calcium-binding proteins from bovine intestinal mucosa, although two of these may be derived from a single native protein. Earlier unpublished studies have shown that the calcium-binding activity of the bovine mucosa is vitamin D3-dependent, and it is presently believed that the isolated bovine proteins are analogous to the vitamin D-dependent proteins of the chick and rat. Also presented is a preliminary description of some of the properties of the bovine intestinal calcium-binding proteins as well as a description of the development of an immunoassay procedure. METHODS
Purification of bovine intestinal calcium-binding protein Bovine small intestine, including duodenum and some jejunum, was obtained from animals at local slaughterhouses immediately following sacrifice. The purification procedure was essentially that developed by Wasserman et al. 11, with slight modification.
I74
C.S.
FULLMFA{, R. H. W A S S E R M A N
The intestine was cut into 4-inch segments, slit lengthwise, rinsed with ice cold o.9~o saline, and carefully blotted. The mucosal tissue (about 2oo g) was scraped from the underlying muscle layers with a glass microscope slide on a cold glass stage and homogenized in a Waring blender in buffer nfixture (I :4, w/v) for three 3o-.~ periods, with a I-nfin cooling interval between each period. The composition of tile buffer mixture was o.o137 M Tris, o.I2 M Nat1, 4.74 mM KCl, and o.oI(~'o/#mercaptoethanol, pH 7.4. The homogenate was filtered once through four layers of cheesecloth and centrifugated at 33 3oo × g (12 ooo rev./min) for 45 lnin in a refrigerated centrifuge. The supernatant fluid was recovered and subjected to the following tmrifieation procedure. All subsequent steps were performed at 4 °C and the above-mentioned buffer was used unless otherwise specified. (NH4)2SO 4 precipitation. The supernatant solution was transferred to a large erlenmeyer flask and solid (NH4)2SO 4 was added periodically, with stirring, over a period of 2-3 h to a final concentration of o.4o g/ml. Stirring was continued for an additional 2 h, after which the suspension was centrifuged as described for the homogenate. The resulting (NH4)2SO4-soluble supernatant solution was concentrated to a volume of IOO ml by membrane ultrafiltration (Model 2ooo Diafiltration Apparatus, Amicon Corp., Lexington, Mass.) equipped with a UM-2 membrane (approximate mol. wt cut-off of IOOO). The concentrated solution was then diluted with 2 1 of the buffer and reconcentrated. This was repeated twice to reduce (N H4)2SO4 to a negligible concentration. After the last dilution and concentration step, the solution was concentrated further to a volume of 2o ml. Gelfiltration column chromatography. The concentrated (NH4)2SO4-soluble supernatant solution was subjected to gel filtration colunm chromatography on Sephadex G-Ioo (Pharmacia, Uppsala, Sweden), employing a 5.0 cm × 87 cm colunm (Pharmacia). The buffer was driven in the upward direction at a rate of o.5 ml/min by means of a peristaltic pump; 5.3-ml fractions were collected. Fractions were assayed for calcium-binding activity (Mini-Chelex procedure) and protein 22. ]'lie protein peak found to contain calcium-binding activity was further monitored by analytical acrylamide disc gel electrophoresis. The fractions were pooled in such a manner as to maximize the recovery of the calcium-binding protein band while minimizing the presence of contaminating bands of similar eleetrophoretic mobility. The pooled fractions were concentrated to a final volume of 4 ml by membrane ultrafiltration as before. Preparative disco~tinuous gel dectrophoresis. The concentrated protein solution from Step 2 was divided into two equal volumes, each of which was run separately on a preparative disc: acrylamide gel electrophoresis apparatus (Canal Industries, Rockville, Md.). The column employed was a PS2/32o with a cross-sectional area of 320 mm 2. A three gel system was employed which consisted of sample, stacking, and separating gels. The separating gel was prepared with 2o°,,/) linear acrvlamide,, o.o3 o.,/o bisacrylamide (i.e. N,N'-methylene bisacrylamide), and imidazole--HC1 buffer (pH 7.8-8.o) and stacked to a height of 5 cm. The stacking or spacer gel was prepared with 3.5'~, linear acrylamide in o.72'~i) Tris-HC1 buffer (pH 6.6-6.8), o.I(~, bisacrylamide, and was stacked to a height of
BOVINE INTESTINAL CALCIUM-BINDING PROTEINS
175
I cm. The sample gel was identical in composition to the spacer gel with the exception that the protein sample solution (in elution buffer) replaced an equal volume of the water in the spacer gel formulation. Both sample and stacking gels were polymerized photometrically by fluorescent light for 20-30 min. The anode, cathode, and elution buffers were identical in composition and consisted of 0.2% Tris and 0.72% glycine (pH 8.I-8.2), with o.o1%/~-mercaptoethanol. The operating current was maintained at 12 mA until the sample reached the separating gel, at which time it was increased to 18 mA. Elution buffer flow rate was 5.0 ml/min and 5-ml fractions were collected. All runs were performed at 4 °C by means of the inner and outer water-jacketed gel column. The pooled fractions were concentrated by membrane ultrafiltration, as before, and desired buffer changes were effected by the repeated addition of replacement buffer to the dialysis cell.
Protein determination Total protein concentration was determined by the procedure of Lowry et al. 2~ with a Technicon auto analyzer (Technicon Co., Chauncey, N.Y.). Protein standards were prepared from lyophilized bovine serum albumin (Nutritional Biochemicals Co.) and a linear response from 0-750 #g/ml was obtained.
Assay for calcium-binding activity The calcium-binding assay was based on that of Briggs and Fleischman ~ as modified by Wasserman et al.lk Calcium-binding activity was expressed as either percent 45Ca in the supernatant (corresponds to protein-bound calcium) or as the ratio of "Ca bound to protein/~Ca bound to resin n.
Analytical acrylamide disc gel electrophoresis Analytical discontinuous electrophoresis on acrylamide was conducted, using a modified method of Ornstein and Davis 24. Cyanogum-4i gelling agent (E.C. Apparatus Corp.) was dissolved in the electrode buffer solution to a concentration of 1o% and O.olml T E M E D (N,N,N',N'-tetramethylethylenediamine, Eastman Chemicals) was added for each gram of Cyanogum. The solution was filtered and, immediately prior to use, o.oi g (NH4)2S~O s was dissolved per gram Cyanogum. The separating gels were poured to a height of 4.5 cm in 0.5 cm x 6.5 cm glass columns and allowed to polymerize. The spacer gel used was identical to that described for preparative electrophoresis and was stacked to a height of 0. 5 cm. Samples were applied to the gels as a dense sucrose solution along with 5 #1 of 0.05% Bromphenol blue tracking dye. Operating current was maintained at 2 mA per gel column and electrophoresis was terminated when the tracking dye reached the end of the column. The electrode buffer system consisted of Tris (I.8%), Na2EDTA (o.925%), and boric acid (o.o55%), p H 8.45. Gels were stained b y immersion in a 0.05% solution of Amido black loB dissolved in 7% aqueous acetic acid for a period of 30 min. Destaining was effected by suspending the stained gels in a solution of acetic acid-methanol-water (I :4 :IO, by vol.) with frequent changes of this mixture until the unbound dye was removed.
Immuno assay procedures Bovine intestinal calcium-binding protein (minor A component, cf. Results)
176
(;. S. F U L L M E R , R. H. W A S S E R M A N
was purified by methods already described. The purified protein solution was emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously at 8-1o sites on the back and once in each hind foot pad of four New Zealand White rabbits (I.I mg protein/rabbit). 23 days following the ilfitial injection, all animals were challenged by intravenous injection of 3oo #g protein (minor .K component). The challenge was repeated at 35 days post-injection and, 7 days later, all animals were bled by ear vein puncture. The serum was obtained and stored frozen, under nitrogen, in sealed ampoules. Immunoelectrophoresis and double diffusion analyses were conducted in 1.5% agar with barbital buffer (pH 8.6), according to the method of Scheidegger '-'a. In the case of immunoelectrophoresis, disc electrophoresis was perfornled in IO°,o acrylamide as previously described. The acrylamide gels were then sliced longitudinally and half was stained with Amido black loB. The other half was embedded in agar and the protein bands allowed to diffuse outward against antiserum placed in a slot in the agar.
Molecular weight determination The estimate ~f the molecular weight fl~r bovine calcium-binding protein was determined by gel filtration colunm chromatography, using a modification of the methods of Andrews ~ and Roubel and TappeW. Glass columns, 2. 5 cm :. IOO cm, were packed with Sephadex G-75 (Pharmacia) to a height of 85 cm and eluted by upward flow with Tris buffer, p H 7.4. Calibration was accomplished both before and after the actual determination by applying 3.0 mg each of cvtochrome c (Sigma Chemical Co., tool. wt 12 5oo), chymotrypsinogen A (Sigma, tool. wt 25 ooo), ovalbumin (Mann Res. Lab., moh wt 45 ooo) and insulin (Sigma, tool. wt 57oo). For each calibration run, and fl~r the actual determination, I.O ml of saturated Dextran Blue solution (Pharmacia) was included as an indication of the void volume of the colmnn. The major bovine calcium-binding protein component (I.O rag) was then applied to the calibrated colunm and the relative elution volumes expressed as tile ratio of elution wflume to column wild volmne (Ve/Vo) ratio.
Enzyme experiment.s In order to further establish tile protein nature of bovine intestinal calciumbinding protein, the effect of proteolytie enzymes on the calcium-binding activity of the preparation was assessed. Bovine calcium-binding protein, partially purified by or (NH4)2SO 4 fractionation and gel filtration on Sephadex G-ioo, was incubated 4 ° /o in the presence of either trypsin or pronase, incubation was conducted at 38 °C in stoppered reaction flasks in a shaking type water bath. The protein concentration of the calcium-binding protein solution was 374 #g/ml and to this, 5o #g/ml of enzyme solution was added. The total reaction volume was 4 nil. Reaction flasks containing trypsin only, pronase only, and protein solution only were incubated as controls. The binding of Ca by trypsin and pronase was found to be negligible. Aliquots of the reaction mixtures were removed at various time intervals, cooled immediately, and and assayed for calcium-binding activity by the Chelex-Ioo procedure.
177
BOVINE INTESTINAL CALCIUM-BINDING PROTEINS
RESULTS
I n order to establish i n i t i a l l y the presence of a c a l c i u m - b i n d i n g c o m p o n e n t in bovine i n t e s t i n a l mucosa, t h e original s u p e r n a t a n t , n o t s u b j e c t e d to (NH,)2SO * fractionation, was a p p l i e d to a 5.0 cm × 8 7 cm S e p h a d e x G - i o o column. The elution profile (Fig. i) i n d i c a t e d t h a t the c a l c i u m - b i n d i n g a c t i v i t y was associated p r i m a r i l y with a specific p r o t e i n p e a k a n d in the region of r e l a t i v e l y low molecular weight substances. A p e a k of lesser b i n d i n g a c t i v i t y was associated with the column v o i d v o l u m e a n d could, m o s t likely, be a t t r i b u t e d to non-specific p r o t e i n binding. A n imp o r t a n t o b s e r v a t i o n from t h e d a t a p r e s e n t e d in Fig. I was t h e initial i n d i c a t i o n of a b i m o d a l c a l c i u m - b i n d i n g a c t i v i t y peak. 1.6
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Fig. i. Separation of crude supernatant proteins by gel filtration on Sephadex G-ioo. The column was 5.o cm × ioo cm and the packed gel height 87 cm. Elution was performed with Tris-HC1 buffer (pH 7-4) at 4 °C and 5.3-ml fractions were collected. Protein was determined by the procedure of Lowry et a l . 22 and calcium-binding activity by the Chelex-ioo assay method n. Fig. 2. Separation of the minor A (Peak I) and major (Peak II) components of bovine calciumbinding protein by preparative acrylamide disc gel electrophoresis. 2-ml fractions were collected. Starting material was the pooled calcium-binding activity peak from Sephadex G-ioo gel filtration of the crude supernatant fraction. The material was not subjected to (NH4)zSO4 precipitation. F r a c t i o n s from t h e p r e p a r a t i v e disc gel electrophoresis were a s s a y e d for calc i u m - b i n d i n g a c t i v i t y a n d t h e results are shown in Fig. 2. These results confirmed the existence of two c a l c i u m - b i n d i n g c o m p o n e n t s which could be s e p a r a t e d comp l e t e l y from each other b y this procedure. The (NH4)ISO 4 f r a c t i o n a t i o n step was then included in t h e general purification procedure. The results (Fig. 3) i n d i c a t e d t h a t considerable purification was achieved and, furthermore, t h a t t h e b i m o d a l n a t u r e of t h e c a l c i u m - b i n d i n g a c t i v i t y p e a k was not altered. Fig. 4 shows the p r e p a r a t i v e gel electrophoresis elution profile following (NH4)2SO a f r a c t i o n a t i o n a n d gel filtration. A comparison of the results shown in F i g s 2 a n d 4 i n d i c a t e t h a t there was no significant difference in t h e elution profile as a result of (NH4)2SO a t r e a t m e n t , c o n t r a r y to the finding of Drescher a n d D e L u c a 17 t h a t such t r e a t m e n t alters t h e r a t c a l c i u m - b i n d i n g protein,
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trig. 3. Separation of the (Nt-14)2S()4 soluble proteins by gel filtration on Sephadex (; too. ('ohmm conditions were identical to those described in the legend for Fig. r. Fig. 4- Separation of the minor A (Peak 1) and the major (Peak 11) components of bovine calcium binding protein by preparative acrylamide disc gel electrophoresis. Starting material was the pooled calcium-binding activity peak from Sephadex G-ioo gel filtration of the (NH4)2S()4 soluble supernatant fraction. Column conditions were identical to those described in the legend for Fig. 2.
The a n a l y t i c a l a c r y l a m i d e electrophoretic p a t t e r n s at various stages of the purification p r o c e d u r e are shown in Fig. 5- The s t a c k i n g a n d s e p a r a t i n g gel f o r m u lations, in this case, were identical to those described for the p r e p a r a t i v e electrophoresis a n d samples were a p p l i e d in sucrose solution. These results i n d i c a t e d t h a t the two b i n d i n g p e a k s from the p r e p a r a t i v e electrophoresis step could be a t t r i b u t e d to two d i s t i n c t p r o t e i n components. W h i l e b o t h of these c o m p o n e n t s exhibit r a p i d electrophoretic m i g r a t i o n rates, the difference is s u b s t a n t i a l . The choice of t h e descriptive terms, m i n o r A a n d m a j o r c o m p o n e n t s , corresponding to the first a n d second peaks, respectively, resulted from the observ a t i o n t h a t the a m o u n t of p r o t e i n associated w i t h the first p e a k was always less t h a n t h a t associated with t h e second peak. The specific a c t i v i t y a n d r e l a t i v e specific a c t i v i t y of fractions from the various purification steps are shown in Table I.
Molecular weight determination The results of molecular weight d e t e r m i n a t i o n b y c a l i b r a t e d gel filtration are shown in Fig. 6, a n d indicate the a p p r o x i m a t e m o l e c u l a r weight of the bovine m a j o r c o m p o n e n t to be I I ooo.
Enzyme experiments The results are shown in Irig. 7 a n d i n d i c a t e t h a t pronase a c t e d r a p i d l y to almost c o m p l e t e l y d e s t r o y b i n d i n g a c t i v i t y , whereas t r y p s i n h a d little or no effect.
Alterations upon storage Purified m a j o r bovine c a l c i u m - b i n d i n g protein, stored at - 20 °C in a nitrogen a t m o s p h e r e in sealed ampoules, e x h i b i t e d a single discreet b a n d on a c r y l a m i d e gel electrophoresis for a period of at least one y e a r following purification. Storage of an
179
BOVINE INTESTINAL CALCIUM-BINDING PROTEINS
Fig. 5. 2o% acrylamide analytical disc gels at various stages of the purification procedure. Solid arrow indicates position of the major component and open arrow, position of minor A component. (I) crude supernatant fraction from mucosal homogenate (I2OO/~g protein), (2) (NH4)2SO 4 soluble s u p e r n a t a n t fraction (75opg protein), (3) pooled calcium-binding activity from gel filtration on Sephadex G-ioo (2oo/~g protein), (4) purified minor A component eluted from preparative disc electrophoresis (combined fractions from Peak I in Fig. 4, (15/*g protein), (5) purified major component eluted from preparative disc electrophoresis (combined fractions from Peak II in Fig. 4 (15/~g protein). In each case, o.i ml of protein solution was subjected to electrophoresis. Migration was toward the anode (bottom). TABLEI PURIFICATION OF BOVINE CALCIUM-BINDING PROTEIN
Fraction
(NH,)zSO, Specific activity*
Crude (NH4)8SO4 Gel filtration Preparative disc electrophoresis Minor A comp. Major comp.
No (NH4)2SO 4 Relative specific activity**
Specific activity*
Relative specific activity**
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I.OO 9.14 22.60
0.44 -3.13
i.oo -7.11
46.31 62.89
lOO.24 136.13
21.o9 22.84
47.93 51.91
* Specific activity values expressed as the *Ca bound to protein/*Ca bound to resin per mg protein. ** Relative specific activity values based on the original s u p e r n a t a n t solution. i d e n t i c a l p r o t e i n s o l u t i o n a t 4 °C for a 3 - m o n t h p e r i o d r e s u l t e d i n t h e a p p e a r a n c e o f three easily distinguishable protein bands when subjected to the same electrophoretic conditions. One band corresponded to the original major component, another to the minor A component, and the third, to a more rapidly migrating species (designated
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Fig. 6. Molecular weight estimation of the major component of bovine cal(ium-binding protein as determined by calibrated gel filtration on Sephadex (;-75. (See text for details). Fig. 7. The effect of digestion by trypsin and pronase on the calcium-binding activity of a partially purified bovine calcium-binding protein preparation. Incubation was performed at 38 'C for the indicated time periods and enzvme concentration was 5° Hg/ml of protein solution. Calciumbinding activity expressed as *~'a bound to protein/"Ca bound to rcsm (*('al'rl'('al~). CaBt'. calcium-binding protein.
as " m i n o r B component"), not previously identified. This third c()mp~ment retains calcium-binding activity and is immunologically reactive with antiserum prepared against the minor A component. Following a 6-month period of storage at 4 °C, tile b a n d corresponding to the major component had disappeared completely while the two more rapidly migrating species remained. In addition, the purified minor A component stored at 4 °C for the same 3- and 6-month periods showed no evidence of alteration. The acrylamide gel eleetrophoretograms of both lnajor and minor A components stored for 3- and 6-month periods at -2o "C and 4 ( i are presented in Fig. 8. The new protein (minor B component) was observed t{~ be present at various stages of the isolation procedure and was subsequently purified to apparent homogeneity. An electrophoretic comparison of this protein along with the purified maior and minor components is presented in Vig. 9-
Puri~v and specificity of the antiserum The specificity and purity of the bovine calcium-binding protein antisermn was tested by two methods. Initially, in order to determine specificity and reactivity, the three purified bovine components were tested by immunodiffusion, using micro Ouehterlony double diffusion plates, against the antiserum. Diffusion was allowed to continue with subsequent formation of preeipitin lines (Fig. IO), thereby establishing the reactivity of all three components with the antiserum. Yurthermore, fusion
BOVINE INTESTINAL CALCIUM-BINDING PROTEINS
181
Fig. 8. A n a l y t i c a l a c r y l a m i d e gel electrophoresis of purified m a j o r a n d m i n o r A bovine c o m p o n e n t s following s t o r a g e a t - - 2 0 °C a n d 4 °C. (I) M a j o r c o m p o n e n t stored for 6 m o n t h s at - - 2 o °C, (2) Minor A c o m p o n e n t stored for 6 m o n t h s a t - - 2 0 °C, (3) Major a n d m i n o r A c o m p o n e n t s s t o r e d for 6 m o n t h s a t - - 2 0 °C a n d applied to t h e s a m e gel to indicate relative migration, (4) M a j o r c o m p o n e n t stored for 3 m o n t h s at 4 °C, (5) Minor A c o m p o n e n t stored for 3 m o n t h s a t 4 °C, (6) M a j o r c o m p o n e n t stored for 6 m o n t h s a t 4 °C, (7) Minor c o m p o n e n t stored for 6 m o n t h s at 4 °C. M i g r a t i o n w a s t o w a r d t h e a n o d e (bottom).
C Fig. 9. A n a l y t i c a l a c r y l a m i d e gel electrophoresis of t h e t h r e e p u r i f e d bovine c o m p o n e n t s . (A) M a j o r c o m p o n e n t , (B) Minor A c o m p o n e n t , (C) Minor B c o m p o n e n t . Gels were p u r p o s e l y overloaded to d e m o n s t r a t e t h e e x t r e m e h o m o g e n e i t y of each c o m p o n e n t .
I82
C . S . FULLMER, R. H. WASSERMAN
Fig. to. I)ouble immunodiffusion analysis of the thrc'e purified bovine c o m p o n e n t s in 1.5".i~ agar. Bovine calcium-binding protein a n t i s e r u m p r e p a r e d in response to the m i n o r A c o m p o n e n t was placed in the center well and the three bovine c o m p o n e n t s placed in the o u t e r wells as indicated.
of the adjacent precipitin lines, with no evidence of spur formation, indicated that the components were serologically identical. The immunoelectrophoretic procedure was performed on supernatant s()lution from the mucosal homogenate to indicate antiserum purity (Fig. IIa), and on a mixture of the three purified components to show specificity (Fig. I zb). It should be noted that the major bovine component appears to be the only component present in fresh supernatant fluid (Fig. IIa). This was the usual finding with supernatant solution that was subjected to immunoelectrophoresis immediately following preparation. This suggests that the formation of the two other calcium-binding protein components is a time dependent process and reflects degradarive reactions. Refrigeration of the fresh supernatant solution for several days led to the appearance of the secondary protein bands which resulted in precipitin a r c s
AI BI C l
11a
11b
Fig. l I. l m m u n o e l e c t r o p h o r e s i s performed on fresh bovine mucosal s u p e r n a t a n t solution. A r r o w indicates position of the m a j o r calcium-binding protein band in the stained gel (left) and the corres p o n d i n g precipitin arc in the embedded gel (right). The presence of a single precipitin arc indicates a high degree of a n t i s e r u m purity. (b) l m m u n o e l e c t r o p h o r e s i s performed on a m i x t u r e of the three purified bovine components. Stained protein b a n d s (left) corresponding to the m a j o r (A), m i n o r A (B), and m i n o r B (C) c o m p o n e n t s as well as three separate, distinct precipitin arcs (right) are shown.
BOVINE INTESTINAL CALCIUM-BINDING PROTEINS
183
corresponding to the two more rapidly migrating binding components and seems to parallel the degradation which occurred both during and following isolation of the major component. Furthermore, in the initial stages of purification, the concentration of the maior bovine component is sufficiently great that its precipitin arc overwhelms the lower area of the gel such that resolution of the two more rapidly migrating components becomes obscured. DISCUSSION
Three separate calcium-binding proteins have been isolated in high purity from bovine intestinal mucosa. The procedure employed for purification was similar to that devised for the chick calcium-binding protein, and included (NH4)2SO 4 fractionation, gel filtration column chromatography on Sephadex G-Ioo, and preparative discontinuous gel eleetrophoresis in 20% acrylamide. Prior to employing the entire separation procedure, supernatant solution from the mucosal homogenate was subjected to gel filtration on Sephadex G-Ioo. The results indicated a substantial calcium-binding activity peak which appeared to be bimodal in nature. Preparative electrophoresis of the proteins in this area confirmed the existence of two components, each possessing a high degree of affinity for radiocalcium. (NH4)2SO 4 fractionation was found to provide an excellent means of reducing the extraneous protein content (that not associated with calcium-binding activity) of the supernatant solution and made possible the isolation of increased amounts of calcium-binding protein. Also, (NH4)2SO * removed several closely associated contaminating proteins, while exerting no observable adverse effects on the quality of the recovered calcium-binding protein. Drescher and DeLuca 17 noted an accelerated loss of binding activity subsequent to heat and (NH4)2SO 4 fractionation procedures during isolation of the analogous rat protein and suggested that the use of such procedures may be responsible for altering the native state of calcium-binding protein. These investigators further suggested that the appearance of a second binding factor noted in previous studies on the rat protein may have resulted from such procedures. The results of the present study clearly indicate that the presence of more than one bovine calcium-binding factor cannot be attributed to (NH,)2SO 4 fractionation and that the appearance of the two more rapidly migrating components seems to be more dependent upon degradative reactions that occur during the course of the purification procedure. Prolonged storage of the purified major component at 4 "C resulted in a progressive loss of this component with the concomitant appearance of the minor A component and eventually to an even more rapidly migrating protein band on acrylamide electrophoresis. This new component (minor B) has since been isolated and purified by the standard procedure and found to possess an affinity for calcium similar to that of the major and minor A components. The exact relationship of these three components and the nature of their formation is not presently known, although similar conversions have been noted with the calcium-binding proteins from the chick and the guinea pig (Fullmer, C. S. and Wasserman, R. H., unpublished). The evidence at hand, however, would suggest that the minor A and minor B components are actually fragments or altered forms of the native major component. This hypothesis
I8 4
c.s.
FULLMER, R. H. WASSERMAN
is strengthened by the fact that, when very fresh supernatant solution is subjected to immunoelectrophoresis, only tile major bovine component is present. Other investigators have noted the appearance of more than one cakqumbinding component from intestinal supernatant solutions. Alpers c t a l . "~ have suggested the presence of two separate calcium-binding proteins isolated from human duodenal mucosa. While only one of these proteins has been isolated in pure form, it appears that these may correspond to the major and minor A bovine components. Hitchman and Harrison ~° have also isolated two calcium-binding components from porcine intestine. These proteins have been thoroughly separated and both bind calcium with high affinity. As mentioned earlier, studies in this laboratory have shown the presence of at least two calcium-binding components from the intestines of chick, guinea pig, and pig, in addition to the bovine. An immunological assay system has recently been developed as an aid in identifying and quantitating bovine calcium-binding protein. The choice of the minor A component to elicit antibody response resulted from the obserwttion that this protein was not subject to breakdown upon storage and, at this point, it was important to assure the presence of only a single protein species for injection. However, subsequent investigation revealed that all three bovine colnponents were immunologically reactive with this antiserum and that they were, in fact, serologically identical. Similarly, antiserum prepared in response to a recently discovered minor chick calcium-binding protein component, reacted equally well when tested against both the major and minor chick components (unpublished). To date, no chick component corresponding to the bovine minor B component has been identified. hnnmnologic cross-reactivity has not been observed between the chick and bovine calcium-binding proteins, t;urthermore, no reactivity was seen when either chick or bovine antiserum was tested against mucosal supernatant solutions from the dog, pig, horse, guinea pig, or human. A weak preeipitin reaction was observed when the bovine antiserum was tested against bovine kidney homogenate. This latter observation is especially important since chick calcium-binding protein has beeu detected, both immunologically and electrophoretically, in chick kidney pretlarations p-'. Another interesting finding is that the chick antiserum reacts with intestinal supernatant solutions from frogs, toads, and turtles (unpublished); however, no specific calcium-binding activity could be detected in these fractions as vet. The boviue antiserum elicited no response with these amphibian species. The preliminary molecular weight estimate of the major bovine component by calibrated gel filtration yielded a value of about ~I ooo. This value is in close agreement with those observed for other mammalian species, but is considerably smaller than the 28 ooo molecular weight value for the chick calcium-binding protein. Schaehter lG reported a value of 13 ooo for the rat calcium-binding protein, while Drescher and DeImca ~v arrived at a value between 8ooo and 13 ooo, depending on procedure, for the same species. Hitchman and Harrison "° found intestinal calciumbinding activity of the rat, pig, and human to be in the range of Iz ooo 13 ooo molecular weight and, at the same time, estimated the size of the chick protein to be about 25 ooo. Studies from this laboratory (unpublished) have shown that calcium-binding proteins isolated from the guinea pig, horse, and pig are quite similar in size to the bovine calcium-binding protein, with a molecular weight in the range of z I ooo 13 ooo. Although most of these determinations have relied on calibrated gel filtration me-
BOVINE INTESTINAL CALCIUM-BINDING PROTEINS
185
thods and have not been substantiated by ultracentrifugal studies or amino acid analyses, it seems certain that the mammalian calcium-binding proteins are significantly smaller in size than the analogous chick protein. Incubation of partially purified preparations of bovine calcium-binding protein with trypsin or pronase indicated that, while trypsin exerted a minimal effect, pronase rapidly and completely destroyed the calcium-binding activity of these preparations. Wasserman and Taylor 1 noted, in contrast, that the calcium-binding activity of supernatant solutions from chick mucosal homogenates was sensitive to relatively short incubation periods with trypsin. These workers later noted, however, that the calcium-binding activity of canine supernatant solutions was sensitive to pronase but not to trypsin is. The reasons for these species differences remain obscure, but might bear speculation. Trypsin exhibits rather specific cleavage of proteins. Pronase, on the other hand, exhibits little specificity and results in considerably more extensive hydrolysis than trypsin. It is also conceivable that trypsin does effect cleavage of calcium-binding protein but that these points of cleavage are far enough removed from the active binding site(s) of the molecule so that calcium-binding activity is not impaired. ACKNOWLEDGEMENTS
This investigation was supported by U.S.P.H.S. Grant AM-o4652 and U.S.A. E.C. Contract AT(II-I)-3167. C. S. Fullmer was supported by U.S.P.H.S. Training Grant 5-ToI-GM-oo8866 and I-ToI-AMo5684-o2.
REFERENCES I 2 3 4 5 6 7 8 9 io II 12 13 14 15 16 17 18 19 20 21 22
Wasserman, R. H. and Taylor, A. N. (1966) Science 152, 791-793 Taylor, A. N. and Wasserman, R. H. (1965) Nature 198, 248-25o Ebel, J. G., Taylor, A. N. and Wasserman, R. H. (1969) Am. J. Clin. Nutr. 22, 431-436 MacGregor, R. R., Hamilton, J. w . and Cohn, D. V. (197 o) Biochim. Biophys. Acta 222, 482 49 ° Corradino, R. A. and Wasserman, R. H. (1968) Arch. Biochem. Biophys. 126, 957-96o Zull, J. E., Czarnowska-Misztal, E. and DeLuca, H. F. (1966) Proc. Natl. Acad. Sci. U.S. 55, 177 184 Harrison, H. E. and Harrison, H. C. (1966) Proc. Soc. Exp. Biol. Med. 121, 312-317 Norman, A. W. (1966) Am. J. Physiol. 211, 829-834 Stohs, S. J., Zull, J. E. and DeLuca, H. F. (1967) Biochemistry 6, 13o4-131o Haussler, M. R. and Norman, A. W. (1967) Arch. Biochem. Biophys. 118, 145-153 Wasserman, R. H., Corradino, R. A. and Taylor, A. N. (1968) J. Biol. Chem. 243, 3978-398~ Taylor, A. N. and Wasserman, R. H. (1967) Arch. Biochem. Biophys. 119, 536-54 o Corradino, R. A., Wasserman, R. H., Pubols, M. H. and Chang, S. I. (1968) Arch. Biochem. Biophys. 125, 378-380 Taylor, A. N. and Wasserman, R. H. (197o) J. Histochem. Cylochem. 18, lO7-115 Kallfelz, F. A., Taylor, A. N. and Wasserman, R. H. (1967) Proc. Soc. Exp. Biol. Med. 125, 54-58 Schachter, D. (1969) in Biological Membranes (Dowben, R. M., ed.), p. lO3, Little and Brown, Boston Drescher, D. and DeLuca, H. F. (1971) Biochemistry IO, 23o2-23o6 Taylor, A. N., Wasserman, R. H. and Jowsey, J. (1968) Fed. Proc. 27, 675 Wasserman, R. H. and Taylor, A. N. (1971) Proc. Soc. Exp. Biol. Med. 136, 25-28 Hitchman, A. J. W. and Harrison, J. E. (1972) Can. J. Biochem. 5o, 758-765 Alpers, D. H., Lee, S. W. and Avioli, L. V. (1972) Gastroenterology 62, 559-564 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275
186 23 24 25 26 27
c . S . FULLMER, R. H. WASSERMAN
Briggs, F. N. a n d F l e i s c h m a n , M. (1965) J. Gem Physiol. 49, 131--149 Davis, B. J. (1964) A n n . N . Y . dcad. Sci. 121, 404-427 Scheidegger, J. J. (1955) lnl. drch. ,4llerg. Appl. I m m u n . 7, I o y - t o 7 Andrews, P. (1964) Biochem. J . 91, 222-233 Roubel, \V. R. a n d Tappcl, A. L. (i964) A~al. Biochem. 9, 2 t t 2 1 0
BBA
- P r i n t e d in The Neth(rla.nds
36462
BOVINE INTESTINAL CALCIUM-BINDING PROTEINS P U R I F I C A T I O N AND SOME PROPERTII~S
C. S. F U L L M E ] { AND R. 1t. W A S S E I < M A N
Departme~l of l~hysical Biology, New York State VeterDmrv College, a~d The (;radzmh, School oj Nutrition, Cornel[ Uni~'ersilV, Ithaca, N . Y . 1485o (U.S.A.) ( R e c e i v e d V e b r u a r v 2nd, 1073)
SUMMARY
~. Three separate calcium-binding proteins, atl similar to the vitamin D adependent calcium-binding protein of the chick, have been isolated in high purity from bovine intestinal nmcosa. All three proteins exhibit rapid electrophoretic migration rates on acrylamide gels and possess high affinities for calcium. One protein (major component) is always present in greater amounts than either of the others (termed "minor A component" and "minor B component"). 2. The molecular weight of the major bovine component was found to be about I . I . lO4 by calibrated gel filtration column chrolnatography. 3. The calcium-binding activity of partially purified preparations was not affected by trypsin digestion but was rapidly and conlpletely destroyed by incubation with pronase. Furthermore, moderate heat treatment of the supernatant solution from mucosal homogenates did not appreciably reduce calcium-binding activity . f this fraction. 4. An ilmnunnassay system for bovine calciunl-binding protein has been developed and shown to be specific for the three bovine components. Using this nmthod, calcium-binding protein has also been detected in the bovine kidney. 5. Preliminary results have indicated that, fi)llowing prolonged st,rage, one of the bovine components (inajor) gives rise to the remaining two (minor A and minor B). This degradation occurs in purified preparations as well as in the crude supernatant solution and m a y indicate that only one of the components is native to the intestine.
INTRODUCTION
A unique intestinal calcium-binding protein was first observed in the supernatant solution from nmcosal homogenates of the chick 1. This protein was found to be absent from the intestines of rachitic animals but appeared shortly following
BOVINE INTESTINAL CALCIUM-BINDING PROTEINS
173
administration of vitamin D 3 and increased in amount coincident with increased calcium absorption 2,3. Furthermore, pulse-labeling experiments revealed that stimulation of the rate of synthesis of intestinal calcium-binding protein by vitamin D 3 occurred before there was any substantial effect on the calcium transport process ~. The synthesis of the protein due to vitamin D 3 was inhibited by prior administration of actinomycin D 5, as is the enhanced absorption of calcium T M . These results lend support to the theory that calcium-binding protein is, in part, responsible for the intestinal translocation of calcium. Chick calcium-binding protein has an estimated molecular weight of 28 ooo by gel filtration (ref. II and Bredderman, P. J. and Wasserman, R. H., unpublished) and possesses four high affinity calcium-binding sites with an average affinity constant of approx, lO 6 M-1 (Bredderman, P. J. and Wasserman, R. H., unpublished). It has been shown to be present in all segments of the small intestine, the colon, and the kidney of the chick TM, and in the uterus (shell gland) of the laying hen 13. Calcium-binding protein, localized by fluorescent antibody techniques, appears in the brush border region of the intestinal absorptive cells as well as in the goblet cells of the chick duodenum la. Calcium-binding proteins have also been detected in the rat 15-17, dog TM, monkey 19, pig 2°, human2°, 21 and guinea pig (Fullmer, C. S. and Wasserman, R. H., unpublished). In the latter three species, as well as the chick (Fullmer, C.S. and Wasserman, R. H., unpublished), it appears that more than one calciumbinding component is present in the intestinal mucosa (refs 20 and 21 and unpublished results). The rat intestinal calcium-binding protein has also been shown to be vitamin D-dependent and has a molecular weight of about 8000 by sedimentation equilibrium and 13 ooo by gel filtration iv. Recent studies on other mammalian calcium-binding proteins, including the porcine ~°, human 2°, guinea pig (Fullmer, C. S. and Wasserman, R. H., unpublished), equine (Fullmer, C. S. and Wasserman, R. H., unpublished) and bovine, have revealed similar estimates for molecular weights in the range of II ooo-13 ooo. Preliminary calcium-binding studies on the porcine protein have shown an affinity constant similar to that of the chick calcium-binding protein ~°. The present paper reports on the isolation and purification of three separate calcium-binding proteins from bovine intestinal mucosa, although two of these may be derived from a single native protein. Earlier unpublished studies have shown that the calcium-binding activity of the bovine mucosa is vitamin D3-dependent, and it is presently believed that the isolated bovine proteins are analogous to the vitamin D-dependent proteins of the chick and rat. Also presented is a preliminary description of some of the properties of the bovine intestinal calcium-binding proteins as well as a description of the development of an immunoassay procedure. METHODS
Purification of bovine intestinal calcium-binding protein Bovine small intestine, including duodenum and some jejunum, was obtained from animals at local slaughterhouses immediately following sacrifice. The purification procedure was essentially that developed by Wasserman et al. 11, with slight modification.
I74
C.S.
FULLMFA{, R. H. W A S S E R M A N
The intestine was cut into 4-inch segments, slit lengthwise, rinsed with ice cold o.9~o saline, and carefully blotted. The mucosal tissue (about 2oo g) was scraped from the underlying muscle layers with a glass microscope slide on a cold glass stage and homogenized in a Waring blender in buffer nfixture (I :4, w/v) for three 3o-.~ periods, with a I-nfin cooling interval between each period. The composition of tile buffer mixture was o.o137 M Tris, o.I2 M Nat1, 4.74 mM KCl, and o.oI(~'o/#mercaptoethanol, pH 7.4. The homogenate was filtered once through four layers of cheesecloth and centrifugated at 33 3oo × g (12 ooo rev./min) for 45 lnin in a refrigerated centrifuge. The supernatant fluid was recovered and subjected to the following tmrifieation procedure. All subsequent steps were performed at 4 °C and the above-mentioned buffer was used unless otherwise specified. (NH4)2SO 4 precipitation. The supernatant solution was transferred to a large erlenmeyer flask and solid (NH4)2SO 4 was added periodically, with stirring, over a period of 2-3 h to a final concentration of o.4o g/ml. Stirring was continued for an additional 2 h, after which the suspension was centrifuged as described for the homogenate. The resulting (NH4)2SO4-soluble supernatant solution was concentrated to a volume of IOO ml by membrane ultrafiltration (Model 2ooo Diafiltration Apparatus, Amicon Corp., Lexington, Mass.) equipped with a UM-2 membrane (approximate mol. wt cut-off of IOOO). The concentrated solution was then diluted with 2 1 of the buffer and reconcentrated. This was repeated twice to reduce (N H4)2SO4 to a negligible concentration. After the last dilution and concentration step, the solution was concentrated further to a volume of 2o ml. Gelfiltration column chromatography. The concentrated (NH4)2SO4-soluble supernatant solution was subjected to gel filtration colunm chromatography on Sephadex G-Ioo (Pharmacia, Uppsala, Sweden), employing a 5.0 cm × 87 cm colunm (Pharmacia). The buffer was driven in the upward direction at a rate of o.5 ml/min by means of a peristaltic pump; 5.3-ml fractions were collected. Fractions were assayed for calcium-binding activity (Mini-Chelex procedure) and protein 22. ]'lie protein peak found to contain calcium-binding activity was further monitored by analytical acrylamide disc gel electrophoresis. The fractions were pooled in such a manner as to maximize the recovery of the calcium-binding protein band while minimizing the presence of contaminating bands of similar eleetrophoretic mobility. The pooled fractions were concentrated to a final volume of 4 ml by membrane ultrafiltration as before. Preparative disco~tinuous gel dectrophoresis. The concentrated protein solution from Step 2 was divided into two equal volumes, each of which was run separately on a preparative disc: acrylamide gel electrophoresis apparatus (Canal Industries, Rockville, Md.). The column employed was a PS2/32o with a cross-sectional area of 320 mm 2. A three gel system was employed which consisted of sample, stacking, and separating gels. The separating gel was prepared with 2o°,,/) linear acrvlamide,, o.o3 o.,/o bisacrylamide (i.e. N,N'-methylene bisacrylamide), and imidazole--HC1 buffer (pH 7.8-8.o) and stacked to a height of 5 cm. The stacking or spacer gel was prepared with 3.5'~, linear acrylamide in o.72'~i) Tris-HC1 buffer (pH 6.6-6.8), o.I(~, bisacrylamide, and was stacked to a height of
BOVINE INTESTINAL CALCIUM-BINDING PROTEINS
175
I cm. The sample gel was identical in composition to the spacer gel with the exception that the protein sample solution (in elution buffer) replaced an equal volume of the water in the spacer gel formulation. Both sample and stacking gels were polymerized photometrically by fluorescent light for 20-30 min. The anode, cathode, and elution buffers were identical in composition and consisted of 0.2% Tris and 0.72% glycine (pH 8.I-8.2), with o.o1%/~-mercaptoethanol. The operating current was maintained at 12 mA until the sample reached the separating gel, at which time it was increased to 18 mA. Elution buffer flow rate was 5.0 ml/min and 5-ml fractions were collected. All runs were performed at 4 °C by means of the inner and outer water-jacketed gel column. The pooled fractions were concentrated by membrane ultrafiltration, as before, and desired buffer changes were effected by the repeated addition of replacement buffer to the dialysis cell.
Protein determination Total protein concentration was determined by the procedure of Lowry et al. 2~ with a Technicon auto analyzer (Technicon Co., Chauncey, N.Y.). Protein standards were prepared from lyophilized bovine serum albumin (Nutritional Biochemicals Co.) and a linear response from 0-750 #g/ml was obtained.
Assay for calcium-binding activity The calcium-binding assay was based on that of Briggs and Fleischman ~ as modified by Wasserman et al.lk Calcium-binding activity was expressed as either percent 45Ca in the supernatant (corresponds to protein-bound calcium) or as the ratio of "Ca bound to protein/~Ca bound to resin n.
Analytical acrylamide disc gel electrophoresis Analytical discontinuous electrophoresis on acrylamide was conducted, using a modified method of Ornstein and Davis 24. Cyanogum-4i gelling agent (E.C. Apparatus Corp.) was dissolved in the electrode buffer solution to a concentration of 1o% and O.olml T E M E D (N,N,N',N'-tetramethylethylenediamine, Eastman Chemicals) was added for each gram of Cyanogum. The solution was filtered and, immediately prior to use, o.oi g (NH4)2S~O s was dissolved per gram Cyanogum. The separating gels were poured to a height of 4.5 cm in 0.5 cm x 6.5 cm glass columns and allowed to polymerize. The spacer gel used was identical to that described for preparative electrophoresis and was stacked to a height of 0. 5 cm. Samples were applied to the gels as a dense sucrose solution along with 5 #1 of 0.05% Bromphenol blue tracking dye. Operating current was maintained at 2 mA per gel column and electrophoresis was terminated when the tracking dye reached the end of the column. The electrode buffer system consisted of Tris (I.8%), Na2EDTA (o.925%), and boric acid (o.o55%), p H 8.45. Gels were stained b y immersion in a 0.05% solution of Amido black loB dissolved in 7% aqueous acetic acid for a period of 30 min. Destaining was effected by suspending the stained gels in a solution of acetic acid-methanol-water (I :4 :IO, by vol.) with frequent changes of this mixture until the unbound dye was removed.
Immuno assay procedures Bovine intestinal calcium-binding protein (minor A component, cf. Results)
176
(;. S. F U L L M E R , R. H. W A S S E R M A N
was purified by methods already described. The purified protein solution was emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously at 8-1o sites on the back and once in each hind foot pad of four New Zealand White rabbits (I.I mg protein/rabbit). 23 days following the ilfitial injection, all animals were challenged by intravenous injection of 3oo #g protein (minor .K component). The challenge was repeated at 35 days post-injection and, 7 days later, all animals were bled by ear vein puncture. The serum was obtained and stored frozen, under nitrogen, in sealed ampoules. Immunoelectrophoresis and double diffusion analyses were conducted in 1.5% agar with barbital buffer (pH 8.6), according to the method of Scheidegger '-'a. In the case of immunoelectrophoresis, disc electrophoresis was perfornled in IO°,o acrylamide as previously described. The acrylamide gels were then sliced longitudinally and half was stained with Amido black loB. The other half was embedded in agar and the protein bands allowed to diffuse outward against antiserum placed in a slot in the agar.
Molecular weight determination The estimate ~f the molecular weight fl~r bovine calcium-binding protein was determined by gel filtration colunm chromatography, using a modification of the methods of Andrews ~ and Roubel and TappeW. Glass columns, 2. 5 cm :. IOO cm, were packed with Sephadex G-75 (Pharmacia) to a height of 85 cm and eluted by upward flow with Tris buffer, p H 7.4. Calibration was accomplished both before and after the actual determination by applying 3.0 mg each of cvtochrome c (Sigma Chemical Co., tool. wt 12 5oo), chymotrypsinogen A (Sigma, tool. wt 25 ooo), ovalbumin (Mann Res. Lab., moh wt 45 ooo) and insulin (Sigma, tool. wt 57oo). For each calibration run, and fl~r the actual determination, I.O ml of saturated Dextran Blue solution (Pharmacia) was included as an indication of the void volume of the colmnn. The major bovine calcium-binding protein component (I.O rag) was then applied to the calibrated colunm and the relative elution volumes expressed as tile ratio of elution wflume to column wild volmne (Ve/Vo) ratio.
Enzyme experiment.s In order to further establish tile protein nature of bovine intestinal calciumbinding protein, the effect of proteolytie enzymes on the calcium-binding activity of the preparation was assessed. Bovine calcium-binding protein, partially purified by or (NH4)2SO 4 fractionation and gel filtration on Sephadex G-ioo, was incubated 4 ° /o in the presence of either trypsin or pronase, incubation was conducted at 38 °C in stoppered reaction flasks in a shaking type water bath. The protein concentration of the calcium-binding protein solution was 374 #g/ml and to this, 5o #g/ml of enzyme solution was added. The total reaction volume was 4 nil. Reaction flasks containing trypsin only, pronase only, and protein solution only were incubated as controls. The binding of Ca by trypsin and pronase was found to be negligible. Aliquots of the reaction mixtures were removed at various time intervals, cooled immediately, and and assayed for calcium-binding activity by the Chelex-Ioo procedure.
177
BOVINE INTESTINAL CALCIUM-BINDING PROTEINS
RESULTS
I n order to establish i n i t i a l l y the presence of a c a l c i u m - b i n d i n g c o m p o n e n t in bovine i n t e s t i n a l mucosa, t h e original s u p e r n a t a n t , n o t s u b j e c t e d to (NH,)2SO * fractionation, was a p p l i e d to a 5.0 cm × 8 7 cm S e p h a d e x G - i o o column. The elution profile (Fig. i) i n d i c a t e d t h a t the c a l c i u m - b i n d i n g a c t i v i t y was associated p r i m a r i l y with a specific p r o t e i n p e a k a n d in the region of r e l a t i v e l y low molecular weight substances. A p e a k of lesser b i n d i n g a c t i v i t y was associated with the column v o i d v o l u m e a n d could, m o s t likely, be a t t r i b u t e d to non-specific p r o t e i n binding. A n imp o r t a n t o b s e r v a t i o n from t h e d a t a p r e s e n t e d in Fig. I was t h e initial i n d i c a t i o n of a b i m o d a l c a l c i u m - b i n d i n g a c t i v i t y peak. 1.6
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Fig. i. Separation of crude supernatant proteins by gel filtration on Sephadex G-ioo. The column was 5.o cm × ioo cm and the packed gel height 87 cm. Elution was performed with Tris-HC1 buffer (pH 7-4) at 4 °C and 5.3-ml fractions were collected. Protein was determined by the procedure of Lowry et a l . 22 and calcium-binding activity by the Chelex-ioo assay method n. Fig. 2. Separation of the minor A (Peak I) and major (Peak II) components of bovine calciumbinding protein by preparative acrylamide disc gel electrophoresis. 2-ml fractions were collected. Starting material was the pooled calcium-binding activity peak from Sephadex G-ioo gel filtration of the crude supernatant fraction. The material was not subjected to (NH4)zSO4 precipitation. F r a c t i o n s from t h e p r e p a r a t i v e disc gel electrophoresis were a s s a y e d for calc i u m - b i n d i n g a c t i v i t y a n d t h e results are shown in Fig. 2. These results confirmed the existence of two c a l c i u m - b i n d i n g c o m p o n e n t s which could be s e p a r a t e d comp l e t e l y from each other b y this procedure. The (NH4)ISO 4 f r a c t i o n a t i o n step was then included in t h e general purification procedure. The results (Fig. 3) i n d i c a t e d t h a t considerable purification was achieved and, furthermore, t h a t t h e b i m o d a l n a t u r e of t h e c a l c i u m - b i n d i n g a c t i v i t y p e a k was not altered. Fig. 4 shows the p r e p a r a t i v e gel electrophoresis elution profile following (NH4)2SO a f r a c t i o n a t i o n a n d gel filtration. A comparison of the results shown in F i g s 2 a n d 4 i n d i c a t e t h a t there was no significant difference in t h e elution profile as a result of (NH4)2SO a t r e a t m e n t , c o n t r a r y to the finding of Drescher a n d D e L u c a 17 t h a t such t r e a t m e n t alters t h e r a t c a l c i u m - b i n d i n g protein,
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trig. 3. Separation of the (Nt-14)2S()4 soluble proteins by gel filtration on Sephadex (; too. ('ohmm conditions were identical to those described in the legend for Fig. r. Fig. 4- Separation of the minor A (Peak 1) and the major (Peak 11) components of bovine calcium binding protein by preparative acrylamide disc gel electrophoresis. Starting material was the pooled calcium-binding activity peak from Sephadex G-ioo gel filtration of the (NH4)2S()4 soluble supernatant fraction. Column conditions were identical to those described in the legend for Fig. 2.
The a n a l y t i c a l a c r y l a m i d e electrophoretic p a t t e r n s at various stages of the purification p r o c e d u r e are shown in Fig. 5- The s t a c k i n g a n d s e p a r a t i n g gel f o r m u lations, in this case, were identical to those described for the p r e p a r a t i v e electrophoresis a n d samples were a p p l i e d in sucrose solution. These results i n d i c a t e d t h a t the two b i n d i n g p e a k s from the p r e p a r a t i v e electrophoresis step could be a t t r i b u t e d to two d i s t i n c t p r o t e i n components. W h i l e b o t h of these c o m p o n e n t s exhibit r a p i d electrophoretic m i g r a t i o n rates, the difference is s u b s t a n t i a l . The choice of t h e descriptive terms, m i n o r A a n d m a j o r c o m p o n e n t s , corresponding to the first a n d second peaks, respectively, resulted from the observ a t i o n t h a t the a m o u n t of p r o t e i n associated w i t h the first p e a k was always less t h a n t h a t associated with t h e second peak. The specific a c t i v i t y a n d r e l a t i v e specific a c t i v i t y of fractions from the various purification steps are shown in Table I.
Molecular weight determination The results of molecular weight d e t e r m i n a t i o n b y c a l i b r a t e d gel filtration are shown in Fig. 6, a n d indicate the a p p r o x i m a t e m o l e c u l a r weight of the bovine m a j o r c o m p o n e n t to be I I ooo.
Enzyme experiments The results are shown in Irig. 7 a n d i n d i c a t e t h a t pronase a c t e d r a p i d l y to almost c o m p l e t e l y d e s t r o y b i n d i n g a c t i v i t y , whereas t r y p s i n h a d little or no effect.
Alterations upon storage Purified m a j o r bovine c a l c i u m - b i n d i n g protein, stored at - 20 °C in a nitrogen a t m o s p h e r e in sealed ampoules, e x h i b i t e d a single discreet b a n d on a c r y l a m i d e gel electrophoresis for a period of at least one y e a r following purification. Storage of an
179
BOVINE INTESTINAL CALCIUM-BINDING PROTEINS
Fig. 5. 2o% acrylamide analytical disc gels at various stages of the purification procedure. Solid arrow indicates position of the major component and open arrow, position of minor A component. (I) crude supernatant fraction from mucosal homogenate (I2OO/~g protein), (2) (NH4)2SO 4 soluble s u p e r n a t a n t fraction (75opg protein), (3) pooled calcium-binding activity from gel filtration on Sephadex G-ioo (2oo/~g protein), (4) purified minor A component eluted from preparative disc electrophoresis (combined fractions from Peak I in Fig. 4, (15/*g protein), (5) purified major component eluted from preparative disc electrophoresis (combined fractions from Peak II in Fig. 4 (15/~g protein). In each case, o.i ml of protein solution was subjected to electrophoresis. Migration was toward the anode (bottom). TABLEI PURIFICATION OF BOVINE CALCIUM-BINDING PROTEIN
Fraction
(NH,)zSO, Specific activity*
Crude (NH4)8SO4 Gel filtration Preparative disc electrophoresis Minor A comp. Major comp.
No (NH4)2SO 4 Relative specific activity**
Specific activity*
Relative specific activity**
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0.44 -3.13
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46.31 62.89
lOO.24 136.13
21.o9 22.84
47.93 51.91
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as " m i n o r B component"), not previously identified. This third c()mp~ment retains calcium-binding activity and is immunologically reactive with antiserum prepared against the minor A component. Following a 6-month period of storage at 4 °C, tile b a n d corresponding to the major component had disappeared completely while the two more rapidly migrating species remained. In addition, the purified minor A component stored at 4 °C for the same 3- and 6-month periods showed no evidence of alteration. The acrylamide gel eleetrophoretograms of both lnajor and minor A components stored for 3- and 6-month periods at -2o "C and 4 ( i are presented in Fig. 8. The new protein (minor B component) was observed t{~ be present at various stages of the isolation procedure and was subsequently purified to apparent homogeneity. An electrophoretic comparison of this protein along with the purified maior and minor components is presented in Vig. 9-
Puri~v and specificity of the antiserum The specificity and purity of the bovine calcium-binding protein antisermn was tested by two methods. Initially, in order to determine specificity and reactivity, the three purified bovine components were tested by immunodiffusion, using micro Ouehterlony double diffusion plates, against the antiserum. Diffusion was allowed to continue with subsequent formation of preeipitin lines (Fig. IO), thereby establishing the reactivity of all three components with the antiserum. Yurthermore, fusion
BOVINE INTESTINAL CALCIUM-BINDING PROTEINS
181
Fig. 8. A n a l y t i c a l a c r y l a m i d e gel electrophoresis of purified m a j o r a n d m i n o r A bovine c o m p o n e n t s following s t o r a g e a t - - 2 0 °C a n d 4 °C. (I) M a j o r c o m p o n e n t stored for 6 m o n t h s at - - 2 o °C, (2) Minor A c o m p o n e n t stored for 6 m o n t h s a t - - 2 0 °C, (3) Major a n d m i n o r A c o m p o n e n t s s t o r e d for 6 m o n t h s a t - - 2 0 °C a n d applied to t h e s a m e gel to indicate relative migration, (4) M a j o r c o m p o n e n t stored for 3 m o n t h s at 4 °C, (5) Minor A c o m p o n e n t stored for 3 m o n t h s a t 4 °C, (6) M a j o r c o m p o n e n t stored for 6 m o n t h s a t 4 °C, (7) Minor c o m p o n e n t stored for 6 m o n t h s at 4 °C. M i g r a t i o n w a s t o w a r d t h e a n o d e (bottom).
C Fig. 9. A n a l y t i c a l a c r y l a m i d e gel electrophoresis of t h e t h r e e p u r i f e d bovine c o m p o n e n t s . (A) M a j o r c o m p o n e n t , (B) Minor A c o m p o n e n t , (C) Minor B c o m p o n e n t . Gels were p u r p o s e l y overloaded to d e m o n s t r a t e t h e e x t r e m e h o m o g e n e i t y of each c o m p o n e n t .
I82
C . S . FULLMER, R. H. WASSERMAN
Fig. to. I)ouble immunodiffusion analysis of the thrc'e purified bovine c o m p o n e n t s in 1.5".i~ agar. Bovine calcium-binding protein a n t i s e r u m p r e p a r e d in response to the m i n o r A c o m p o n e n t was placed in the center well and the three bovine c o m p o n e n t s placed in the o u t e r wells as indicated.
of the adjacent precipitin lines, with no evidence of spur formation, indicated that the components were serologically identical. The immunoelectrophoretic procedure was performed on supernatant s()lution from the mucosal homogenate to indicate antiserum purity (Fig. IIa), and on a mixture of the three purified components to show specificity (Fig. I zb). It should be noted that the major bovine component appears to be the only component present in fresh supernatant fluid (Fig. IIa). This was the usual finding with supernatant solution that was subjected to immunoelectrophoresis immediately following preparation. This suggests that the formation of the two other calcium-binding protein components is a time dependent process and reflects degradarive reactions. Refrigeration of the fresh supernatant solution for several days led to the appearance of the secondary protein bands which resulted in precipitin a r c s
AI BI C l
11a
11b
Fig. l I. l m m u n o e l e c t r o p h o r e s i s performed on fresh bovine mucosal s u p e r n a t a n t solution. A r r o w indicates position of the m a j o r calcium-binding protein band in the stained gel (left) and the corres p o n d i n g precipitin arc in the embedded gel (right). The presence of a single precipitin arc indicates a high degree of a n t i s e r u m purity. (b) l m m u n o e l e c t r o p h o r e s i s performed on a m i x t u r e of the three purified bovine components. Stained protein b a n d s (left) corresponding to the m a j o r (A), m i n o r A (B), and m i n o r B (C) c o m p o n e n t s as well as three separate, distinct precipitin arcs (right) are shown.
BOVINE INTESTINAL CALCIUM-BINDING PROTEINS
183
corresponding to the two more rapidly migrating binding components and seems to parallel the degradation which occurred both during and following isolation of the major component. Furthermore, in the initial stages of purification, the concentration of the maior bovine component is sufficiently great that its precipitin arc overwhelms the lower area of the gel such that resolution of the two more rapidly migrating components becomes obscured. DISCUSSION
Three separate calcium-binding proteins have been isolated in high purity from bovine intestinal mucosa. The procedure employed for purification was similar to that devised for the chick calcium-binding protein, and included (NH4)2SO 4 fractionation, gel filtration column chromatography on Sephadex G-Ioo, and preparative discontinuous gel eleetrophoresis in 20% acrylamide. Prior to employing the entire separation procedure, supernatant solution from the mucosal homogenate was subjected to gel filtration on Sephadex G-Ioo. The results indicated a substantial calcium-binding activity peak which appeared to be bimodal in nature. Preparative electrophoresis of the proteins in this area confirmed the existence of two components, each possessing a high degree of affinity for radiocalcium. (NH4)2SO 4 fractionation was found to provide an excellent means of reducing the extraneous protein content (that not associated with calcium-binding activity) of the supernatant solution and made possible the isolation of increased amounts of calcium-binding protein. Also, (NH4)2SO * removed several closely associated contaminating proteins, while exerting no observable adverse effects on the quality of the recovered calcium-binding protein. Drescher and DeLuca 17 noted an accelerated loss of binding activity subsequent to heat and (NH4)2SO 4 fractionation procedures during isolation of the analogous rat protein and suggested that the use of such procedures may be responsible for altering the native state of calcium-binding protein. These investigators further suggested that the appearance of a second binding factor noted in previous studies on the rat protein may have resulted from such procedures. The results of the present study clearly indicate that the presence of more than one bovine calcium-binding factor cannot be attributed to (NH,)2SO 4 fractionation and that the appearance of the two more rapidly migrating components seems to be more dependent upon degradative reactions that occur during the course of the purification procedure. Prolonged storage of the purified major component at 4 "C resulted in a progressive loss of this component with the concomitant appearance of the minor A component and eventually to an even more rapidly migrating protein band on acrylamide electrophoresis. This new component (minor B) has since been isolated and purified by the standard procedure and found to possess an affinity for calcium similar to that of the major and minor A components. The exact relationship of these three components and the nature of their formation is not presently known, although similar conversions have been noted with the calcium-binding proteins from the chick and the guinea pig (Fullmer, C. S. and Wasserman, R. H., unpublished). The evidence at hand, however, would suggest that the minor A and minor B components are actually fragments or altered forms of the native major component. This hypothesis
I8 4
c.s.
FULLMER, R. H. WASSERMAN
is strengthened by the fact that, when very fresh supernatant solution is subjected to immunoelectrophoresis, only tile major bovine component is present. Other investigators have noted the appearance of more than one cakqumbinding component from intestinal supernatant solutions. Alpers c t a l . "~ have suggested the presence of two separate calcium-binding proteins isolated from human duodenal mucosa. While only one of these proteins has been isolated in pure form, it appears that these may correspond to the major and minor A bovine components. Hitchman and Harrison ~° have also isolated two calcium-binding components from porcine intestine. These proteins have been thoroughly separated and both bind calcium with high affinity. As mentioned earlier, studies in this laboratory have shown the presence of at least two calcium-binding components from the intestines of chick, guinea pig, and pig, in addition to the bovine. An immunological assay system has recently been developed as an aid in identifying and quantitating bovine calcium-binding protein. The choice of the minor A component to elicit antibody response resulted from the obserwttion that this protein was not subject to breakdown upon storage and, at this point, it was important to assure the presence of only a single protein species for injection. However, subsequent investigation revealed that all three bovine colnponents were immunologically reactive with this antiserum and that they were, in fact, serologically identical. Similarly, antiserum prepared in response to a recently discovered minor chick calcium-binding protein component, reacted equally well when tested against both the major and minor chick components (unpublished). To date, no chick component corresponding to the bovine minor B component has been identified. hnnmnologic cross-reactivity has not been observed between the chick and bovine calcium-binding proteins, t;urthermore, no reactivity was seen when either chick or bovine antiserum was tested against mucosal supernatant solutions from the dog, pig, horse, guinea pig, or human. A weak preeipitin reaction was observed when the bovine antiserum was tested against bovine kidney homogenate. This latter observation is especially important since chick calcium-binding protein has beeu detected, both immunologically and electrophoretically, in chick kidney pretlarations p-'. Another interesting finding is that the chick antiserum reacts with intestinal supernatant solutions from frogs, toads, and turtles (unpublished); however, no specific calcium-binding activity could be detected in these fractions as vet. The boviue antiserum elicited no response with these amphibian species. The preliminary molecular weight estimate of the major bovine component by calibrated gel filtration yielded a value of about ~I ooo. This value is in close agreement with those observed for other mammalian species, but is considerably smaller than the 28 ooo molecular weight value for the chick calcium-binding protein. Schaehter lG reported a value of 13 ooo for the rat calcium-binding protein, while Drescher and DeImca ~v arrived at a value between 8ooo and 13 ooo, depending on procedure, for the same species. Hitchman and Harrison "° found intestinal calciumbinding activity of the rat, pig, and human to be in the range of Iz ooo 13 ooo molecular weight and, at the same time, estimated the size of the chick protein to be about 25 ooo. Studies from this laboratory (unpublished) have shown that calcium-binding proteins isolated from the guinea pig, horse, and pig are quite similar in size to the bovine calcium-binding protein, with a molecular weight in the range of z I ooo 13 ooo. Although most of these determinations have relied on calibrated gel filtration me-
BOVINE INTESTINAL CALCIUM-BINDING PROTEINS
185
thods and have not been substantiated by ultracentrifugal studies or amino acid analyses, it seems certain that the mammalian calcium-binding proteins are significantly smaller in size than the analogous chick protein. Incubation of partially purified preparations of bovine calcium-binding protein with trypsin or pronase indicated that, while trypsin exerted a minimal effect, pronase rapidly and completely destroyed the calcium-binding activity of these preparations. Wasserman and Taylor 1 noted, in contrast, that the calcium-binding activity of supernatant solutions from chick mucosal homogenates was sensitive to relatively short incubation periods with trypsin. These workers later noted, however, that the calcium-binding activity of canine supernatant solutions was sensitive to pronase but not to trypsin is. The reasons for these species differences remain obscure, but might bear speculation. Trypsin exhibits rather specific cleavage of proteins. Pronase, on the other hand, exhibits little specificity and results in considerably more extensive hydrolysis than trypsin. It is also conceivable that trypsin does effect cleavage of calcium-binding protein but that these points of cleavage are far enough removed from the active binding site(s) of the molecule so that calcium-binding activity is not impaired. ACKNOWLEDGEMENTS
This investigation was supported by U.S.P.H.S. Grant AM-o4652 and U.S.A. E.C. Contract AT(II-I)-3167. C. S. Fullmer was supported by U.S.P.H.S. Training Grant 5-ToI-GM-oo8866 and I-ToI-AMo5684-o2.
REFERENCES I 2 3 4 5 6 7 8 9 io II 12 13 14 15 16 17 18 19 20 21 22
Wasserman, R. H. and Taylor, A. N. (1966) Science 152, 791-793 Taylor, A. N. and Wasserman, R. H. (1965) Nature 198, 248-25o Ebel, J. G., Taylor, A. N. and Wasserman, R. H. (1969) Am. J. Clin. Nutr. 22, 431-436 MacGregor, R. R., Hamilton, J. w . and Cohn, D. V. (197 o) Biochim. Biophys. Acta 222, 482 49 ° Corradino, R. A. and Wasserman, R. H. (1968) Arch. Biochem. Biophys. 126, 957-96o Zull, J. E., Czarnowska-Misztal, E. and DeLuca, H. F. (1966) Proc. Natl. Acad. Sci. U.S. 55, 177 184 Harrison, H. E. and Harrison, H. C. (1966) Proc. Soc. Exp. Biol. Med. 121, 312-317 Norman, A. W. (1966) Am. J. Physiol. 211, 829-834 Stohs, S. J., Zull, J. E. and DeLuca, H. F. (1967) Biochemistry 6, 13o4-131o Haussler, M. R. and Norman, A. W. (1967) Arch. Biochem. Biophys. 118, 145-153 Wasserman, R. H., Corradino, R. A. and Taylor, A. N. (1968) J. Biol. Chem. 243, 3978-398~ Taylor, A. N. and Wasserman, R. H. (1967) Arch. Biochem. Biophys. 119, 536-54 o Corradino, R. A., Wasserman, R. H., Pubols, M. H. and Chang, S. I. (1968) Arch. Biochem. Biophys. 125, 378-380 Taylor, A. N. and Wasserman, R. H. (197o) J. Histochem. Cylochem. 18, lO7-115 Kallfelz, F. A., Taylor, A. N. and Wasserman, R. H. (1967) Proc. Soc. Exp. Biol. Med. 125, 54-58 Schachter, D. (1969) in Biological Membranes (Dowben, R. M., ed.), p. lO3, Little and Brown, Boston Drescher, D. and DeLuca, H. F. (1971) Biochemistry IO, 23o2-23o6 Taylor, A. N., Wasserman, R. H. and Jowsey, J. (1968) Fed. Proc. 27, 675 Wasserman, R. H. and Taylor, A. N. (1971) Proc. Soc. Exp. Biol. Med. 136, 25-28 Hitchman, A. J. W. and Harrison, J. E. (1972) Can. J. Biochem. 5o, 758-765 Alpers, D. H., Lee, S. W. and Avioli, L. V. (1972) Gastroenterology 62, 559-564 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275
186 23 24 25 26 27
c . S . FULLMER, R. H. WASSERMAN
Briggs, F. N. a n d F l e i s c h m a n , M. (1965) J. Gem Physiol. 49, 131--149 Davis, B. J. (1964) A n n . N . Y . dcad. Sci. 121, 404-427 Scheidegger, J. J. (1955) lnl. drch. ,4llerg. Appl. I m m u n . 7, I o y - t o 7 Andrews, P. (1964) Biochem. J . 91, 222-233 Roubel, \V. R. a n d Tappcl, A. L. (i964) A~al. Biochem. 9, 2 t t 2 1 0