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Isolation and characterizatiopn of light chains from allotype suppressed b9 homozygous rabbits
Immunochemlstry,1973,Vol. 10,pp. 277-278. PergamonPress. Printedin Great Britain
COMMUNICATIONS ISOLATION AND FROM ALLOTYPE
TO T H E E D I T O R S
CHARACTERIZATION OF LIGHT SUPPRESSED b9 HOMOZYGOUS
CHAINS RABBITS
A L B E R T O CHERSI* and ROSE G. M A G E Laboratory of Immunology, National Institute of Health, Bethesda, Maryland, U.S.A.
(Received 17 August 1972) Abstract- Light chains have been isolated following sulfitolysis and gel filtration of immunoglobulin
G from allotype-suppressed b9 homozygous rabbits. Chemically and immunologically all light chains belong to the k type, exist only in the form with two disulfide infrachain bridges, and seem to be very similar to the b negative, h-type light chains which all allotype synthesize, in very low amount, under normal circumstances. As previously described, IgG from normal b9 rabbits, whose specificity is controlled at the b locus by a rather infrequent allele, has a relatively high proportion of light polypeptide chains lacking b locus allotypic specificity. (Carbonara, 1969). lgG from normal b9 rabbits can be fractionated by anion exchange chromatography so that the most basic IgG (fraction I, 15 per cent) contain essentially h-type molecules, and the most acidic IgG (fraction Ill, 11 per cent) k-type molecules. The major portion of IgG (fraction II, 74 per cent) contains both h-type and k-type molecules in the approximate ratio of 2 : 3 (Chersi, 1970). By oxidative sulfitolysis and gel filtration on Sephadex G 100 in 6 M urea, 0.05M formic acid, the first IgG fraction from normal b9 rabbits yields a light chain peak (Li; Kd = 0"38) composed only by k-type light chains, the second fraction two light chains peaks (Li and Ln, with Ka 0"35 and 0.48 respectively), the second of which is composed only of k-type light chains; the third IgG fraction only an Lit peak containing k-type light chains. All h-type light chains probably contain five half-cystine residues, while most k-type light chains contain seven half-cystine residues (Chersi, 1970). The production of a relatively high proportion of lambda light chains in normal b9 rabbits may be related to a low position of this gene in the 'pecking order' of phenotypic expression. It is possible that the 'pecking order' is not limited to the kappa chains genes, but that it also controls the expression of lambda chain genes. In the presence of a strong kappa chain gene, e.g. b4, the expression of the lambda genes may be less than in the presence of a weak kappa chain gene, i.e. b9. It might also be predicted that the suppression of the phenotypic expression in b9 rabbits should be easier than in b4 rabbits. The suppression o f k light chains in b9 rabbits, and their substitution with h-type light chains, might be expected to affect the distribution of IgG fractions in anion exchange chromatography, as well as elution and relative proportions of Li and Eli peaks in gel filtration of IgG fractions after sulfitolysis. In the present study we investigated also whether the h-type light chains in b9 *Present address: I stituto Regina Etena, Roma, Italy.
suppressed rabbits showed the same distribution of k chains as in normal b9 rabbits, or whether the suppression resulted in chains with chemical and physical properties different from those of normally expressed h-type light chains. If suppression altered relative expression of possible h-subtypes, this might be reflected in differences in number of half-cystine residues, aminoacid compositions and C- and N-terminal aminoacid residues. The suppression of the phenotypic expression in b9 rabbits was obtained by the procedure described for b5 rabbits (Dubiski, 1967). The rabbits were bled when the serum concentration of passively acquired b4 immunoglobulins decreased below the limit of detection, and when the serum concentration of b9 immunoglobulins was still below the limit of detection. We found that IgG from b9 suppressed rabbits was less acidic in nature than IgG from normal b9 rabbits: only two IgG fractions were obtained by chromatography on D E A E cellulose, under the standard pH and molarity conditions used for normal b9 IgG. The first peak of basic IgG, eluted with 0.0175M phosphate buffer pH 6.9, represented an amount (55 per cent) which is unusually high for normal b9 IgG. Peak II (45 per cent) was eluted with 0.035 M phosphate buffer pH 7.5. No peak III was eluted from the column with 0.05 M phosphate. Both Fraction I and II gave, after oxidative sulfitolysis and gel filtration, a similar pattern with one light chain peak with Ka values of 0-37 and 0.38, respectively. Both Li fractions were revealed to be of the h type by quantitative precipitation with goat anti-h antisera and by chemical analysis, which demonstrated only five half-cystine residues in each, no detectable N-terminal aminoacid by carbamylation, and serine as the major C-terminal residue, together with small amounts of glycine (L~ from Fraction I) and valine (Li from Fraction II). No striking differences were found between the two aminoacid compositions, and that of h light chains from normal b9 rabbits. Preliminary data presented here would suggest that the population of b negative light chains produced by b9 suppressed rabbits belong to the h-type, and exist only in the form with two disulfide bridges intrachain. The majority of them, if not all, have the usual C-terminal serine of the k chains and a blocked N-terminal residue. 277
278
Communications to the Editors
This k chain population is probably similar in structure and in chemical properties to the k-type light chains which the rabbits express under normal circumstances.
A c k n o w l e d g e m e n t - W e thank Dr. S. Dubiski, The Toronto Western Hospital, Canada, for his generous gift of sera from b9 suppressed rabbits.
lmmunochemistry, 1973, VoL 10, pp. 278-280. Pergamon Press.
REFERENCES
Carbonara A., Tosi R., Mancini G. and Luzzati A. (1969) Progr. lmmunobiol. Standard, Vol. 4. Karger, Basel. Chersi A., Mage R., Rejnek J. and Reisfeld R. A. (1970) J. lmmun. 104, 1205. Dubiski S. (1967) Nature, Lond. 214, 1365.
Printed in Great Britain
I N H I B I T I O N OF H O R S E R A D I S H P E R O X I D A S E BY S P E C I F I C A N T I S E R A A. A. M A R U C C I Department of Microbiology, Upstate Medical Center, State University of New York, Syracuse, New York 13214 U.S.A.
(First Received 3 July 1972; in revised form i 5 September 1972) Abstract-Antiperoxidase has been prepared in the rabbit, cat, mouse, hamster and chicken. All of these antisera inhibit peroxidase activity. INTRODUCTION
During the past 5 yr the enzyme peroxidase has become an increasingly useful tool in various immunological investigations, viz. site of antibody formation (Straus, 1968), as an antibody label by chemical coupling (Nakane and Pierce, 1967) as an antibody label by immunological coupling (Dougherty et al., 1972; Sternberger et al. 1970) and recently to study localization of antigen in the inflammatory response (Graham and Shannon, 1972). There are contradictory reports in the literature on the effect of the antibody-peroxidase reaction on the enzyme activity. Straus (1968) working with rabbit antiperoxidase demonstrated that the enzyme could be efficiently inhibited by specific antibody. Sternberger et al. (1970) have also reported inhibition by rabbit antisera. Most recently Hess et al. (1971) have stated that there is no inhibition of peroxidase by pooled mouse antiperoxidase or by specific rabbit antiserum. In the present communication the results of inhibition reactions between peroxidase and specific antisera are presented. Antisera from five different species of animals have been assayed. M A T E R I A L S AND METHODS
Enzyme. Horseradish peroxidase (PO), Type VI, was purchased from the Sigma Chemical Co., St. Louis, Mo. Antisera: Rabbits were injected intramuscularly on days 1 and 15 with 1 and 4mg PO in complete Freund's adjuvant (CFA) and on day 29 with 2.5mg in saline. They were bled on day 35. The second course included 0.8 and 1.6 mg in CFA on days 49 and 63. They were then bled on day 77. Cats were inoculated with 4mg in CFA on days 1 and 15, 2 mg on day 29, 1 mg on days 48 and 57 and finally I mg in saline on day 69. The cats were bled on day 78. Mice were inoculated intraperitoneally with 0.13mg in C F A on days 1, 15, and 24.
They were bled on day 29 and all the sera were pooled. The preparation of chicken and hamster antiperoxidase has been described (Dougherty et al., 1972). All of the antisera gave only one precipitin line in agar when reacted against 200/.rg PO nitrogen. Quantitative precipitin analyses were done as described in Kabat and Mayer (1961). Enzyme assay. Peroxidase activity was measured by following the rate of decomposition of hydrogen peroxide with 3-3' dimethoxybenzidine (o-dianisidine) as hydrogen donor. To 16× 100mm test tubes which contained the enzyme or enzyme-antiserum mixture was added sufficient 0.1 M citrate buffer, pH 5.3, to bring the total volume up to 4.5 ml. The o-dianisidine (0-04 ml of a 1% solution) was added and the contents of the tube were mixed on a vortex mixer. The reaction was started by addition of 0.5 ml of H~O~ contained 340/~g H2OJml in citrate buffer. The tube was immediately thoroughly mixed then a portion was poured into a 1 cm cuvette. The color development was followed in a Beckman DK recording spectrophotometer at 460 rim. The chart speed was set at 3 in/min. From the recording of the straight line the change in optical density for 20 sec (AO.D./20 sec) was obtained. All assays were done at room temperature. Under these conditions the reaction kinetics were zero order. RESULTS
(1) Inhibition assays. A constant volume of varying dilutions of the antiserum under study was added to each tube followed by an appropriate enzyme dilution which gave a AO.D./20 sec of 0.250-0-300. The reactions were set up and remained in a crushed ice bath for at least 1 hr. This was sufficient time for maximal inhibition by all antisera studied. The tubes were then warmed to room temperature and the enzyme assay was per-
COMMUNICATIONS ISOLATION AND FROM ALLOTYPE
TO T H E E D I T O R S
CHARACTERIZATION OF LIGHT SUPPRESSED b9 HOMOZYGOUS
CHAINS RABBITS
A L B E R T O CHERSI* and ROSE G. M A G E Laboratory of Immunology, National Institute of Health, Bethesda, Maryland, U.S.A.
(Received 17 August 1972) Abstract- Light chains have been isolated following sulfitolysis and gel filtration of immunoglobulin
G from allotype-suppressed b9 homozygous rabbits. Chemically and immunologically all light chains belong to the k type, exist only in the form with two disulfide infrachain bridges, and seem to be very similar to the b negative, h-type light chains which all allotype synthesize, in very low amount, under normal circumstances. As previously described, IgG from normal b9 rabbits, whose specificity is controlled at the b locus by a rather infrequent allele, has a relatively high proportion of light polypeptide chains lacking b locus allotypic specificity. (Carbonara, 1969). lgG from normal b9 rabbits can be fractionated by anion exchange chromatography so that the most basic IgG (fraction I, 15 per cent) contain essentially h-type molecules, and the most acidic IgG (fraction Ill, 11 per cent) k-type molecules. The major portion of IgG (fraction II, 74 per cent) contains both h-type and k-type molecules in the approximate ratio of 2 : 3 (Chersi, 1970). By oxidative sulfitolysis and gel filtration on Sephadex G 100 in 6 M urea, 0.05M formic acid, the first IgG fraction from normal b9 rabbits yields a light chain peak (Li; Kd = 0"38) composed only by k-type light chains, the second fraction two light chains peaks (Li and Ln, with Ka 0"35 and 0.48 respectively), the second of which is composed only of k-type light chains; the third IgG fraction only an Lit peak containing k-type light chains. All h-type light chains probably contain five half-cystine residues, while most k-type light chains contain seven half-cystine residues (Chersi, 1970). The production of a relatively high proportion of lambda light chains in normal b9 rabbits may be related to a low position of this gene in the 'pecking order' of phenotypic expression. It is possible that the 'pecking order' is not limited to the kappa chains genes, but that it also controls the expression of lambda chain genes. In the presence of a strong kappa chain gene, e.g. b4, the expression of the lambda genes may be less than in the presence of a weak kappa chain gene, i.e. b9. It might also be predicted that the suppression of the phenotypic expression in b9 rabbits should be easier than in b4 rabbits. The suppression o f k light chains in b9 rabbits, and their substitution with h-type light chains, might be expected to affect the distribution of IgG fractions in anion exchange chromatography, as well as elution and relative proportions of Li and Eli peaks in gel filtration of IgG fractions after sulfitolysis. In the present study we investigated also whether the h-type light chains in b9 *Present address: I stituto Regina Etena, Roma, Italy.
suppressed rabbits showed the same distribution of k chains as in normal b9 rabbits, or whether the suppression resulted in chains with chemical and physical properties different from those of normally expressed h-type light chains. If suppression altered relative expression of possible h-subtypes, this might be reflected in differences in number of half-cystine residues, aminoacid compositions and C- and N-terminal aminoacid residues. The suppression of the phenotypic expression in b9 rabbits was obtained by the procedure described for b5 rabbits (Dubiski, 1967). The rabbits were bled when the serum concentration of passively acquired b4 immunoglobulins decreased below the limit of detection, and when the serum concentration of b9 immunoglobulins was still below the limit of detection. We found that IgG from b9 suppressed rabbits was less acidic in nature than IgG from normal b9 rabbits: only two IgG fractions were obtained by chromatography on D E A E cellulose, under the standard pH and molarity conditions used for normal b9 IgG. The first peak of basic IgG, eluted with 0.0175M phosphate buffer pH 6.9, represented an amount (55 per cent) which is unusually high for normal b9 IgG. Peak II (45 per cent) was eluted with 0.035 M phosphate buffer pH 7.5. No peak III was eluted from the column with 0.05 M phosphate. Both Fraction I and II gave, after oxidative sulfitolysis and gel filtration, a similar pattern with one light chain peak with Ka values of 0-37 and 0.38, respectively. Both Li fractions were revealed to be of the h type by quantitative precipitation with goat anti-h antisera and by chemical analysis, which demonstrated only five half-cystine residues in each, no detectable N-terminal aminoacid by carbamylation, and serine as the major C-terminal residue, together with small amounts of glycine (L~ from Fraction I) and valine (Li from Fraction II). No striking differences were found between the two aminoacid compositions, and that of h light chains from normal b9 rabbits. Preliminary data presented here would suggest that the population of b negative light chains produced by b9 suppressed rabbits belong to the h-type, and exist only in the form with two disulfide bridges intrachain. The majority of them, if not all, have the usual C-terminal serine of the k chains and a blocked N-terminal residue. 277
278
Communications to the Editors
This k chain population is probably similar in structure and in chemical properties to the k-type light chains which the rabbits express under normal circumstances.
A c k n o w l e d g e m e n t - W e thank Dr. S. Dubiski, The Toronto Western Hospital, Canada, for his generous gift of sera from b9 suppressed rabbits.
lmmunochemistry, 1973, VoL 10, pp. 278-280. Pergamon Press.
REFERENCES
Carbonara A., Tosi R., Mancini G. and Luzzati A. (1969) Progr. lmmunobiol. Standard, Vol. 4. Karger, Basel. Chersi A., Mage R., Rejnek J. and Reisfeld R. A. (1970) J. lmmun. 104, 1205. Dubiski S. (1967) Nature, Lond. 214, 1365.
Printed in Great Britain
I N H I B I T I O N OF H O R S E R A D I S H P E R O X I D A S E BY S P E C I F I C A N T I S E R A A. A. M A R U C C I Department of Microbiology, Upstate Medical Center, State University of New York, Syracuse, New York 13214 U.S.A.
(First Received 3 July 1972; in revised form i 5 September 1972) Abstract-Antiperoxidase has been prepared in the rabbit, cat, mouse, hamster and chicken. All of these antisera inhibit peroxidase activity. INTRODUCTION
During the past 5 yr the enzyme peroxidase has become an increasingly useful tool in various immunological investigations, viz. site of antibody formation (Straus, 1968), as an antibody label by chemical coupling (Nakane and Pierce, 1967) as an antibody label by immunological coupling (Dougherty et al., 1972; Sternberger et al. 1970) and recently to study localization of antigen in the inflammatory response (Graham and Shannon, 1972). There are contradictory reports in the literature on the effect of the antibody-peroxidase reaction on the enzyme activity. Straus (1968) working with rabbit antiperoxidase demonstrated that the enzyme could be efficiently inhibited by specific antibody. Sternberger et al. (1970) have also reported inhibition by rabbit antisera. Most recently Hess et al. (1971) have stated that there is no inhibition of peroxidase by pooled mouse antiperoxidase or by specific rabbit antiserum. In the present communication the results of inhibition reactions between peroxidase and specific antisera are presented. Antisera from five different species of animals have been assayed. M A T E R I A L S AND METHODS
Enzyme. Horseradish peroxidase (PO), Type VI, was purchased from the Sigma Chemical Co., St. Louis, Mo. Antisera: Rabbits were injected intramuscularly on days 1 and 15 with 1 and 4mg PO in complete Freund's adjuvant (CFA) and on day 29 with 2.5mg in saline. They were bled on day 35. The second course included 0.8 and 1.6 mg in CFA on days 49 and 63. They were then bled on day 77. Cats were inoculated with 4mg in CFA on days 1 and 15, 2 mg on day 29, 1 mg on days 48 and 57 and finally I mg in saline on day 69. The cats were bled on day 78. Mice were inoculated intraperitoneally with 0.13mg in C F A on days 1, 15, and 24.
They were bled on day 29 and all the sera were pooled. The preparation of chicken and hamster antiperoxidase has been described (Dougherty et al., 1972). All of the antisera gave only one precipitin line in agar when reacted against 200/.rg PO nitrogen. Quantitative precipitin analyses were done as described in Kabat and Mayer (1961). Enzyme assay. Peroxidase activity was measured by following the rate of decomposition of hydrogen peroxide with 3-3' dimethoxybenzidine (o-dianisidine) as hydrogen donor. To 16× 100mm test tubes which contained the enzyme or enzyme-antiserum mixture was added sufficient 0.1 M citrate buffer, pH 5.3, to bring the total volume up to 4.5 ml. The o-dianisidine (0-04 ml of a 1% solution) was added and the contents of the tube were mixed on a vortex mixer. The reaction was started by addition of 0.5 ml of H~O~ contained 340/~g H2OJml in citrate buffer. The tube was immediately thoroughly mixed then a portion was poured into a 1 cm cuvette. The color development was followed in a Beckman DK recording spectrophotometer at 460 rim. The chart speed was set at 3 in/min. From the recording of the straight line the change in optical density for 20 sec (AO.D./20 sec) was obtained. All assays were done at room temperature. Under these conditions the reaction kinetics were zero order. RESULTS
(1) Inhibition assays. A constant volume of varying dilutions of the antiserum under study was added to each tube followed by an appropriate enzyme dilution which gave a AO.D./20 sec of 0.250-0-300. The reactions were set up and remained in a crushed ice bath for at least 1 hr. This was sufficient time for maximal inhibition by all antisera studied. The tubes were then warmed to room temperature and the enzyme assay was per-