Human salivary immunoglobulin A. Some immunological and physicochemical characteristics

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BBA 35394 HUMAN SALIVARY IMMUNOGLOBULIN A. SOME IMMUNOLOGICAL AND PHYSICOCHEMICAL CHARACTERISTICS

J. HURLIMANN*, M. WALDESBI~HL AND C. ZUBER Institut Universitaire de Biochimie, Rue du Bugnon 21, lOO 5 L a u s a n n e (Switzerland)

(Received January I3th, 1969)

SUMMARY H u m a n ascitic and salivary immunoglobulin A (IgA) were isolated and compared for their immunological and physicochemical properties. Salivary IgA, with a sedimentation coefficient of II.O S, had a molecular weight of 39 ° ooo; it consisted of two molecules of ascitic type IgA (mol. wt. 17o ooo) bound to one molecule of secretory piece (mol. wt. 5o ooo). Reduction and alkylation (pH 8.2) split the salivary IgA into fragments corresponding to ascitic IgA molecules and a secretory piece. Reduction and alkylation (pH 8.2) split a portion of ascitic IgA into fragments of a molecular weight of about 6o ooo of which some corresponded to heavy chains and others to dimers of L chains. Treatment with urea or guanidine dissociated the salivary IgA into two fragments of about the same molecular weight; the first corresponded to ascitic type IgA, the second to molecules formed b y two a-chains bound to the secretory piece. The secretory piece, isolated b y reduction and alkylation, had an electrophoretic mobility faster than that of salivary IgA. It possessed no antigenic determinants common to L chains but only determinants specific for secretions.

INTRODUCTION Of the immunoglobulins present in various secretions, immunoglobulin A (IgA) is the most concentrated1, 2. Secretory IgA has a higher sedimentation coefficient than serum IgA 3 and possesses antigenic determinants not present in serum IgA. These determinants are present in a supplementary fragment referred to as a transport piecO, secretory piece 5, as-chain s or T chain 7. It has been proposed that secretory IgA consists of several molecules of serum IgA bound to one molecule of secretory piece. Secretory IgA has been found in colostrum of rabbits v-9 and in saliva3, 4, colostrum 1°-12, bronchial secretionsl3,14, nasal secretions 15-17, tears15, TM, gastric juicO8,19 and urogenital secretions 20-22 of man. Antibody activity 23-25 and even autoantibody activity ~° has been shown in human secretory IgA. Abbreviation: IgA, immunoglobulin A. * Present address: Institut de Pathologic, H6pital Cantonal, iOli Lausanne, Switzerland. Biochim. Biophys, Acta,

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Despite the interest in secretory IgA, several points concerning its structure have not been solved. Some investigators have found that the secretory piece in human colostrum has antigenic determinants common to L chains 1°, while others claim that secretory piece does not possess such determinants12, 26. There are two structural models proposed for secretory IgA. One, for IgA from human colostrum, consists of three molecules of serum IgA (mol. wt. 17o ooo) bound to one molecule of transport piece (mol. wt. 5 ° 000) TM.The other, for IgA from rabbit colostrum, consists of two molecules of serum IgA (rnol. wt. 17o ooo) bound to one or two T chains (tool. wt. 24 000) 7. In addition to this uncertainty concerning the overall structure, it is not clear how in secretory IgA, the transport piece is attached to serum IgA. In the present studies, human salivary IgA and human ascitic IgA (which is similar to serum IgA) were isolated. These two IgA preparations, one of secretory type and the other of serum type, were characterized and compared by polyacrylamide-gel electrophoresis, double diffusion in agar, immunoelectrophoresis, ultracentrifugation and filtration on Sephadex. The effects of reduction, alkylation and denaturation also were examined. MATERIALS AND METHODS

Saliva. After stimulation with chewing gum, saliva from normal adults was collected, pooled, dialyzed against distilled water and lyophilized. Saliva from a patient without IgA. This saliva was obtained from an adult whose serum contained no detectable IgA but a component which had antigenic determinants of the secretory piece; this component is hereafter termed "free piece". Ascitic fluid. Aseitic fluid was collected from patients with hepatic cirrhosis or cancer with peritoneal metastases. Antisera. Anti-saliva, anti-human serum, anti-ascitic IgA, anti-salivary IgA and anti-secretory piece sera were obtained using the immunization methods described in a previous paper ~7. Absorption. Antisera prepared against ascitic IgA and salivary IgA were absorbed with pure human immunoglobulin (IgG) to remove antibodies to L chains. A sample of anti-salivary IgA serum was absorbed with normal human sermn and analyzed by immunodiffusion. It revealed only the antigenic determinants of secretory piece and was called anti-piece serum. For absorption, an excess of antigen was added to the antiserum which was incubated at 37 ° for 0. 5 h, maintained at 4 ° overnight and then centrifuged at 3000 × g at 4 ° for 20 rain. Preparation of salivary IgA. Purification of salivary IgA was done as previously described 27. Briefly, the saliva was chromatographed on DEAE-cellulose according to the method of TOMASI et al. a. The IgA-rich fraction was passed through a CMcellulose column according to the method of MASSONet al. 2s and was filtered on Sephadex G-200. The first peak, containing the salivary IgA, was passed a second time through Sephadex G-200, dialyzed and lyophilized. Preparation of ascitic IgA. Ascitic IgA was purified according to the method of TOMASI et al. 3. Electrophoresis. Electrophoresis was carried out on microscope slides in 2 % agar with 0.025 M barbital buffer (pH 8.2). Horizontal starch-gel electrophoresis (pH 8.4) Biochim. Biophys. Acta, 18i (1969) 393-4o3

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was according to the method of SMITHIES29, and electrophoresis in polyacrylamide gel was by the technique of ORNSTEIN3°. On the latter, solutions for the lower and upper gels were prepared as described by WILLIAMS AND REISFELD 31, and the electrophoresis was carried out at p H 2.7 and 9.3 with and without urea. Double d~usion in agar. The microtechnique was used in 2% agar in 0.025 M barbital buffer (pH 8.2). Immunoelectrophoresis. The micromethod of SCHEIDEGGER32 was employed, using the L K B apparatus 6800 (LKB, Stockholm, Sweden) and a 0.025 M barbital buffer (pH 8.2). Protein determination. Protein concentrations were determined by use of FolinCiocalteu's phenol reagent 33 or b y absorbance measurements at 280 m#. Ultracentrifugation. Analytical ultracentrifugation was carried out in a Spinco model E ultracentrifuge (Beckman Instruments, Palo Alto, Calif.) in a titanium An-H rotor, with an aluminum single-sector cell, at 56 lOO-67 77 ° rev./min at 20 °. The observed values were corrected to standard conditions of water and 20 ° using a factor of i.o 3. Sedimentation was effected using four concentrations of protein in phosphatebuffered saline and was extrapolated to protein concentration zero. For diffusion the same rotor was used with a valve-type synthetic boundary cell at 15 220 rev./min at 20 °. Filtration on Sephadex. The Sephadex G-2oo column (2.4 cm × 12o cm, flow rate 12 ml/h, 3 ml per tube) and Sephadex G-Ioo column (2.4 cm × 12o cm, flow rate 12 ml/h, 3 ml per tube) were calibrated with proteins of known molecular weight according to ANDREWS 84. Reduction and alkylation, treatment with urea and guanidine. Reduction of salivary IgA under N 2 was made as described b y SOUTH et al. 4. Salivary IgA was treated with guanidine at room temperature b y dialysis for 24 h against 5 M guanidine purified b y charcoal absorption in o.i M Tris buffer (pH 8.0). The sample was applied to a column of Sephadex G-ioo equilibrated with 5 M guanidine. HC1 in o.I M Tris buffer (pH 8.0). The material was collected according to absorbance peaks at 28o m/,. Salivary IgA was treated with urea by dialysis at room temperature for 24 h against 8 M urea in 0.05 M glycine buffer (pH 8.4). The sample was analyzed b y polyacrylamide-gel electrophoresis in 8 M urea.

RESULTS

Purity of ascitic and salivary IgA 2o mg of salivary IgA was the average amount obtained per 2000-ml pool of saliva. Assuming an IgA concentration of 28 mg per i00 ml of saliva, as reported b y CHODIRKER AND TOMASI1, the yield is less than 5 %- For ascitic IgA the yield was 510% .

Samples of salivary IgA from three pools of saliva gave a single band on electrophoresis in starch gel, agar, cellulose acetate and polyacrylamide gel at p H 9.3 (Fig. IA). On polyacrylamide gel at p H 2.7, however, there was an additional pale band or trail towards the cathode (Fig. IH). Analytical ultracentrifugation of the three salivary IgA preparations showed a Biochim. Biophys. Acta, 181 (I969) 393-4o3

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large symmetrical peak and a much smaller peak with a higher sedimentation coefficient (Fig. 2). Upon immunoelectrophoresis at a o.2-1.o% protein concentration, each sample developed one precipitation line against anti-saliva, anti-normal human serum, antisalivary IgA and anti-ascitic IgA sera (Fig. 3A). At a 2 - 3 % protein concentration, two fine additional precipitation lines parallel to the IgA line were seen with anti-saliva serum. The samples did not show a precipitation line corresponding to lactoferrin at any concentration.

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E

Fig. i. Electrophoresis in p o l y a c r y l a m i d e gel. F r o m A to G electrophoresis was carried o u t at p H 9-3, f r o m H to L a t p H 2.7, in M a n d N a t p H 9-3 in 8 M urea. A a n d H : S a l i v a r y IgA. B a n d I: R e d u c e d a n d a l k y l a t e d s a l i v a r y IgA. C: P e a k I of reduced a n d a l k y l a t e d s a l i v a r y I g A p a s s e d t h r o u g h S e p h a d e x G-ioo. D a n d K : Ascitic IgA. E: R e d u c e d a n d a l k y l a t e d ascitic IgA. F: P e a k I of reduced a n d a l k y l a t e d ascitic I g A passed t h r o u g h S e p h a d e x G-ioo. G: P e a k II of r e d u c e d a n d a l k y l a t e d ascitic I g A p a s s e d t h r o u g h S e p h a d e x G-ioo. J: P e a k II of reduced a n d a l k y l a t e d s a l i v a r y I g A p a s s e d t h r o u g h S e p h a d e x G-ioo. L : Salivary I g A t r e a t e d w i t h guanidine. M : S a l i v a r y I g A t r e a t e d w i t h urea. N: Ascitic I g A t r e a t e d w i t h urea. Fig. 2. U l t r a c e n t r i f u g e p a t t e r n of s a l i v a r y IgA. S e d i m e n t a t i o n f r o m left to right. P h o t o g r a p h s t a k e n 4, 12 a n d 2o min. after r e a c h i n g full speed. T h e arrows indicate t h e m i n o r peak.

Ascitic IgA, on electrophoresis in starch gel, agar and on cellulose acetate, showed a band analogous to that of salivary IgA. In polyacrylamide at pH 9.3, the band was closer to the anode than was that of salivary IgA (Fig. IC). In polyacrylamide at pH 2.7 there were five bands : three dark bands were anodic and two pale bands were in the direction of the cathode (Fig. IK). Ascitie IgA sedimented in the analytical ultracentrifuge as a symmetrical peak. On immunoelectrophoresis, ascitic IgA at a protein concentration of I °/o , developed one precipitation line against the same anti-sera as were used to test salivary IgA, _Biochim. Biophys. Acta, 181 (I969) 393-403

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Fig. 3- Immunoelectrophoresis. A. Salivary IgA is revealed by anti-saliva and anti-salivary IgA sera. B. Reduced and alkylated salivary IgA is revealed by anti-salivary IgA, anti-ascitic IgA and anti-piece sera. C. Peak I of reduced and alkylated ascitic IgA passed through Sephadex G-ioo is revealed by anti-L-chain (anti L) and anti-a-chain (anti a) sera. D. Peak II of reduced and alkylated ascitic IgA passed through Sephadex G-ioo is revealed by anti-L-chain (anti L) and anti-a-chain (anti a) sera.

At a protein concentration of 2%, an additional precipitation line corresponding to IgG was seen.

Sedimentation coefficients to salivary and ascitic IgA Two samples of salivary IgA were studied for their S°2o,w values. The first, examined at IO, 7.5, 5 and 2.5 mg protein per ml, had a value of lO. 9 S. The second, examined at 4.5, 2.25 and 1.125 mg protein per ml, gave a value of II.O S. One sample, examined at only 2. 9 mg protein per ml, had a coefficient of 9.9 S. A sample of ascitic IgA, examined at 12, 9, 6 and 3 mg protein per ml, gave a value for S°2o,,wof 6.28 S.

Diffusion coefficients of salivary and ascitic IgA IgA samples from three pools of saliva, analyzed at a protein concentration of 2.5 rag/m1, gave values for D20 of 2.73, 2.55 and 2.91. One sample of ascitic IgA, analyzed at 6 and 3 mg protein per ml, gave a D2o of 3.62, and another analyzed at 6 nag protein per ml, gave a D20 of 3-93.

Molecular weights of salivary and aseitic IgA Calculated from diffusion and sedimentation coefficients. The molecular weight for salivary IgA calculated according to SVEDBERG AND PEDERSONa5 was 385 ooo-~ Biochim. Biophys. Acta, 181 (I969) 393-403

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25 ooo, assuming a partial specific volume of o.74 (see ref. IO) and considering the different values obtained for D,zo. Assuming a S of 0.703 (see ref. 7), the molecular weight was 336 ooo :L 22 ooo. The molecular weight of ascitic IgA was found to be 167 ooo ~- 8 ooo, assuming a S of 0.74. Calculated from exclusion volume on Sephadex G-2oo. Exclusion volumes on Sephadex G-2oo for two different samples of salivary IgA were 194 and 192 ml and corresponded to molecular weights of 370 ooo and 380 ooo respectively (Fig. 4)The exclusion volume on Sephadex G-2oo for one sample of ascitic IgA was 236 ml and corresponded to a molecular weight of 18o ooo (Fig. 4).

Immunological comparison o[ ascitic and salivary IgA There was an identity reaction between salivary and ascitie IgA when they were tested against anti-normal human serum and anti-ascitic IgA sera by double diffusion

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Fig. 4. Calibration c u r v e on S e p h a d e x G-2oo. T h e p r o d u c t s used for c a l i b r a t i o n are i n d i c a t e d b y d a r k circles. T h e open circles, i n d i c a t e d b y arrows, correspond to elution v o l u m e s of ascitic I g A (circle in t h e middle of t h e curve) a n d of s a l i v a r y I g A (the t w o circles a t t h e b o t t o m of t h e curve). Fig. 5- Calibration c u r v e on S e p h a d e x G-ioo. T h e p r o d u c t s used for calibration are i n d i c a t e d b y d a r k circles. T h e open circles, i n d i c a t e d b y arrows, c o r r e s p o n d to elution v o l u m e s of P e a k s I a n d II of reduced a n d a l k y l a t e d s a l i v a r y I g A p a s s e d t h r o u g h S e p h a d e x G-ioo.

in agar (Fig. 7A). Against both anti-saliva and anti-salivary IgA sera, salivary IgA formed a spur over the precipitation line of ascitic IgA (Fig. 7B). With anti-piece serum, only salivary IgA formed a precipitation line (Fig. 7C).

Reduction and alkylation Salivary IgA. On polyacrylamide-gel electrophoresis at pH 2.7, reduced and alkylated salivary IgA formed two bands both of which had faster mobilities than native salivary IgA (Fig. II). In polyacrylamide (pH 9-3), this material formed a single broad band of faster mobility than that of native salivary IgA (Fig. IB). The reduced and alkylated salivary IgA was analyzed by immunodiffusion. With anti-ascitic IgA serum, one precipitation line identical with the line of ascitic IgA Biochim. Biophys. Acta, 181 (1969) 393-4o3

A

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399

was formed (Fig. 7A). With anti-salivary IgA serum, two precipitation lines were formed; one was identical with the line of ascitic IgA, the other with the additional antigenic determinants of salivary IgA and with the free piece (Figs. 7B and 8A). On immunoelectrophoresis, anti-salivary IgA serum developed two precipitation lines, one of/3 mobility and the other of)~ mobility. Anti-ascitic IgA serum developed only the precipitation arc of)J mobility while anti-piece serum developed only the precipitation arc of/3 mobility (Fig. 3B). Reduced and alkylated salivary IgA was applied to a Sephadex G-Ioo column, and two peaks were obtained (Fig. 6). The first corresponded to a component with a molecular weight of 17o ooo, the second to a component with a molecular weight of

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Fig. 6. Gel-filtration c u r v e of reduced a n d a l k y l a t e d s a l i v a r y I g A on S e p h a d e x G-ioo. T h e S e p h a d e x G - i o o c o l u m n (2.4 c m × 8 5 crn) was equilibrated w i t h p h o s p h a t e - b u f f e r e d saline. Flow r a t e 12 ml/h, 3 ml per tube. T h e p e a k s labeled I a n d II were a n a l y z e d b y double diffusion in a g a r a n d b y p o l y a c r y l a m i d e - g e l electrophoresis.

54 ooo (Fig. 5)- Peak I had a sedimentation coefficient of 5.8 S at a protein concentration of 3 mg/ml. This value corresponded to that of ascitic IgA at the same protein concentration. Immunodiffusion showed Peak I to be identical with ascitic IgA. On polyacrylamide-gel electrophoresis at pH 2. 7 and 9.3 (Fig. IC), Peak I formed bands similar to that of ascitic IgA. On immunodiffusion, Peak II was shown to be identical with the free piece (Fig. 8A). Peak II contained no antigenic determinants common to ascitic IgA, IgG, Bence Jones protein I or II. In polyacrylamide-gel electrophoresis at pH 2. 7, Peak II formed one band with the same mobility as the faster band of reduced and alkylated salivary IgA (Fig. I J). Reduced and alkylated salivary IgA obtained from another pool of saliva gave the same elution pattern on Sephadex G-Ioo. Immunological analysis showed that Peak I from each pool was similar; Peak II from the second pool, however, differed from Peak II of the first in that it contained not only antigenic determinants of the secretory piece but also those of ascitic IgA and IgG. Ascitic IgA. Reduced and alkylated ascitic IgA on polyacrylamide gel at pH 9-3 showed one band broader than the band of native ascitic IgA (Fig. IE). In polyacrylamide at pH 2.7, there was a broad band with three sharp cathodic bands. On immunoelectrophoresis, nonabsorbed anti-salivary IgA revealed a splitting of the precipitation line in its anodic region. Filtration on Sephadex G-Ioo of the reduced and alkylated ascitic IgA gave two peaks. On polyacrylamide-gel electrophoresis (pH 9.3), Peak I Biochim. Biophys. Acta, 181 (1969) 393-4o3

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Fig. 7. Double diffusion in agar. Ascitic IgA (1), salivary IgA (2), reduced and alkylated salivary IgA (3) are revealed by (A) anti-ascitic IgA serum (4), by (B) anti-salivary IgA serum (5), and (C) by anti-piece serum (6). Fig. 8. Double diffusion in agar and immunoelectrophoretic analysis. A. Salivary IgA (2), secretory piece obtained by reduction and alkylation of salivary IgA (3) and free piece (4) are revealed b y anti-salivary IgA serum (i). B. Salivary IgA (2), salivary IgA treated with guanidine (6) and ascitic IgA (7) are revealed by anti-salivary IgA serum (I). C. Salivary IgA treated with guanidine is revealed by immunoelectrophoresis with three antisera: anti-piece serum, anti-L-chain serum and anti-a-chain serum.

formed one band analogous to that of ascitic IgA and was shown to be identical with ascitic IgA by immunoelectrophoresis (Fig. 3C) Peak I I eluted later corresponded to material of a molecular weight of 6o ooo; that in polyacrylamide gel at p H 9.3 formed a band of faster mobility than that of ascitic IgA (Fig. IG). This material, analyzed b y immunoelectrophoresis, was shown to be a mixture of molecules, some of faster mobility having only a-chain determinants and others of slower mobility having only L-chain determinants (Fig. 3D).

Treatment with guanidine Salivary IgA dialyzed against 5 M guanidine. HC1 in o.I M Tris buffer (pH 8.o) was applied to a Sephadex G-Ioo column eqailibrated with the same buffer. Only one peak was obtained which was then dialyzed against distilled water and lyophilized. This material was analyzed by immunodiffusion. With anti-salivary IgA serum, two precipitation lines were formed. One, closer to the antiserum well, had some antigenic determinants common and some supplementary to ascitic IgA. The other is antigenically identical with ascitic IgA (Fig. 8B). This same material was analyzed by immunoelectrophoresis. Two precipitation arcs were formed; one of/5 mobility had Biochim. Biophys. Acta, i81 (1969) 393-4o3

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the antigenic determinants of a chains and of piece while the other of y mobility had the antigenic determinants of a chains and of L chains (Fig. 8C). On polyacrylamide-gel electrophoresis at p H 9.3, the material of the Sephadex G-ioo peak gave a broad band. In polyacrylamide gel at p H 2.7, there was a broad band and two sharp dark bands (Fig. IL). The most cathodic had the same mobility as the slower band of reduced and alkylated salivary IgA. Treatment

with urea

Salivary IgA dialyzed against 8 M urea in glycine buffer was analyzed by polyacrylamide-gel electrophoresis in 8 M urea at p H 9-3 and three bands were seen (Fig. IM). The most prominent band was of intermediate mobility and had the same position as the band of ascitic IgA treated with urea (Fig. IN). DISCUSSION The isolated salivary and ascitic IgA were not pure. However, less than 5 % impurity was detected with the analytical methods used, and this did not interfere with our results. The immunological study confirms other data3,4, 36 which indicate that salivary IgA possesses antigenic determinants not present in ascitic IgA. We found a sedimentation coefficient of i i . o S which is similar to that reported for secretory IgA in man3, n, 12,14 and rabbit 9. The molecular weight of salivary IgA calculated both by exclusion on Sephadex and by the diffusion and sedimentation coefficients is 370 ooo ~ 33 ooo; this result is in accordance with that of CEBRA AND SMALL2 for rabbit colostrum IgA and of NEWCOMB et al. 37 for human colostrum IgA. Taking into account the molecular weights of ascitic and salivary IgA, it appears that salivary IgA consists of a m a x i m u m of two ascitic type IgA molecules; this corresponds to the model of CEBRA AND SMALL7. In addition, there is a fragment with antigenic determinants specific for secretions. I f the molecular weight of ascitic IgA is subtracted from that of salivary IgA, the fragment could have a molecular weight of up to 60 ooo. This fragment isolated b y reduction, alkylation and filtration on Sephadex has a molecular weight of 54 ooo which agrees with the values found b y HONG et al. 1° and HANSON"AND JOHANSSON 12 (see NEWCOMB et al.37). From our data it is not possible to determine whether this fragment actually has a molecular weight of 54 ooo or is a polymer of smaller fragments analogous to the T chains of CEBRA AND SMALL2. The secretory piece is covalently bound by disulfide bonds to ascitic-type IgA molecules as shown b y reduction and alkylation. Noncovalent bonds were not found. Treatment with guanidine failed to separate secretory piece from the salivary IgA. These results are in disagreement with those of other investigators 1°,12. The bonds which bind secretory piece to IgA are probably similar to those which connect the polymers of serum IgA 36and b y which IgA molecules are attached to various proteins as. In particular, it was shown that 5 M guanidine fails to dissociate bonds between albumin and IgA which are split only b y reduction with 2-mercaptoethanoP '~. However, salivary IgA treated with guanidine was dissociated into fragments of identical molecular weights as judged by exclusion volume on Sephadex. Some fragments are probably formed by two heavy chains bound to one secretory piece, while others are formed b y two heavy and two light chains bound as in a molecule of Biochim. Biophys. Acta, 181 (1969) 393-4o3

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ascitic IgA. These data could be in part explained by the results reported by GREY et al. ~°. These investigators have shown that secretory IgA consists principally of 7A2-type proteins in which the heavy and light chains are not linked to each other by disulfide bonds. Treatment with guanidine releases light chains from ?JA2 protein. In our experiments, L chains were not found as dimers but rather were bound to heavy chains. Therefore it seems necessary to consider disulfide interchange reactions in order to explain this phenomenon. The secretory piece obtained by reduction and alkylation of salivary IgA has a/5 mobility on electrophoresis in agar that is similar to that of free piece from a patient lacking serum IgA. This is in agreement with the data of SOUTH et al. 4 and HANSON AND JOHANSSON 1~. The piece has a fast mobility on polyacrylamide-gel electrophoresis at pH 2.7, as already reported by CEBRA AND SMALL 7, which probably explains the fast band found when L chains of ?~-globulins from human eolostrum are analyzed on starch-gel electrophoresis in 8 M urea41m. The secretory piece isolated by reduction and alkylation has tile same antigenic determinants as the free piece. It possesses only specific determinants a n d no determinants common to heavy or light chains of IgA or IgG. The fact that determinants common to L and a chains were found with secretory piece in one pool of saliva did not indicate that secretory piece possesses determinants common to L chains (see HONG et al. ~°) and to a chains, but that secretory piece is contaminated by a- and L chains. These chains are released from serum-type IgA during the reduction and alkylation of salivary IgA. Further evidence is seen when ascitic IgA is reduced and alkylated (pH 8.0) and is dissociated into fragments of a molecular weight of 60 ooo consisting of heavy chains and dimers of L chains. Dissociation of serum-type IgA b y reduction and alkylation at a neutral pH, is known. ISHIZAKA4a showed that IgA monomer isoagglutinins lose 80% of their antigen-combining activity b y reduction and alkylation (pH 8.0). VAERMAN et al. 44 noticed that, in presence of o.I M 2-mercaptoethanol (pH 7.o), preparations of 7- and IO.5-S serum IgA show an additional 3.5-S peak not seen in the absence of 2-mercaptoethanol. I f it is accepted that secretory piece does not possess determinants common to the chains of immunoglobulins and that it is bound to IgA by disulfide bonds, further investigation is necessary to explain the mechanism in vivo of this attachmenO 5. It is not known whether different cells exist for the synthesis of piece and IgA a or if there is only one cell synthesizing secretory piece and IgA 46. The mechanism of formation of the whole salivary IgA clearly depends upon which of these theories is correct. ACKNOWLEDGEMENTS

The authors wish to thank Drs. J. M. McKenzie and H. Jaquet for their suggestions. The cooperation of Mrs. H. Darling in the preparation of the manuscript is gratefully acknowledged. We wish to thank Dr. Ph. Frei, Clinique Universitaire de M6decine, Lausanne, for providing the saliva from a patient without IgA. We wish so thank Dr. Ts. Susuki for providing Bence Jones protein I and II. This work was supported by the Swiss National Foundation for Scientific Research. REFERENCES I W. B. CHODIRKER AND T. B. TOMASI, Science, 142 (1963) lO8O.

Biochim. Biophys. Acta, 181 (1969) 393-403

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