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Immunochemical studies on pepsinogens A, C and D from the smooth dogfish, Mustelus canis
Immunochemistry'. Pergamon Press 1971. Vol.8, pp. 201-209. Printed in Great Britain
I M M U N O C H E M I C A L S T U D I E S O N P E P S I N O G E N S A, C AND D FROM T H E S M O O T H DOGFISH, M U S T E L U S CANIS* TERRENCE G. MERRETT,t LAWRENCE LEVINE and HELEN VAN VUNAKIS Graduate Department of Biochemistry, Brandeis University, Waltham, Mass. 02154, U.S.A.
(Received 27July 1970) Abstract-Rabbit antibodies to pepsinogens A, C and D (isolated from the gastric mucosae of the smooth dogfish) were characterized by immunodiffusion, immunoelectrophoresis, complement fixation and ability to inhibit the enzymic activity of the pepsin formed fi'om each zymogen. Similarities were detected only between pepsinogens A and D; pepsinogen C was unrelated to these proteins. Pepsin failed to precipitate or fix complement with the antisera directed toward the specific proenzyme from which it was derived. Therefi)re, some antigenic sites were either removed in the non-pepsin fragment and/or the pepsin moiety (which represents about 85 per cent of the pepsinogen molecule) underwent conformational changes during the conversion of the proenzyrne. However, pepsins A and D were still able to inhibit the pepsinogen A-antipepsinogen A system indicating some similarity to the proenzyme structure. The incubation of each pepsin with the antibody specific for its zymogen resulted in loss of enzymic activity indicating that the enzyme can bind to the antizymogen sera. The antigenic structure and potential enzymic activities of pepsinogens A and D were lost concurrently by first-order kinetics when these proenzymes were heated in aqueous solutions of neutral halide salts. The rate of thermal denaturation of these proenzymes was dependent upon the type and concentration of the anion present and increased in the following order I- > Br- > CI . INTRODUCTION T h e purification o f three pepsinogens isolated f r o m the gastric mucosae o f the s m o o t h dogfish with potential enzymic activity toward protein substrate h i s b e e n described [1]. Like the pepsinogens o f o t h e r species, dogfish pepsinogens A, C and D have molecular weights o f a p p r o x i m a t e l y 42,000 and contain a p r e d o m i n a n c e o f acidic over basic a m i n o acids. Dogfish p e p s i n o g e n s A and D have similar but not identical a m i n o acid compositions. Pepsinogen C contains half as m a n y basic a m i n o acid residues as pepsinogens A or D but it is unlikely that p e p s i n o g e n C represents a smaller molecule originating f r o m a p a r e n t p e p s i n o g e n A or D by limited hydrolysis since it contains greater a m o u n t s o f some o f the o t h e r a m i n o acids. Preliminary experiments indicate that the pepsins *Publication No. 749 from the Graduate Department of Biochemistry, Brandeis University, Waltham, Mass. 02154. Supported in part by research grants from the National Science Foundation (NSF GB4302) and tile National Aeronautics and Space Administration (NSG 375). L. L. is an American Cancer Society Professor of Biochemistry (Award No. PRP-21). H.V.V. is a recipient of a Puhlic Health Service Research Career Award (K6-AI-2372). tPresen! address : The Radiochemical Centre, Amersharn, Buckinghamshire, England. 201
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f o r m e d u p o n conversion of the p r o e n z y m e s account for approximately 85 per cent o f the p a r e n t molecule. With the intormation obtained from immunochemical techniques, some aspects o f the relationshi 1) o f the three precursors to each o t h e r and to their enzymes have been clarified. Although at least fimr different pepsinogens have been isolated from swine stomach mucosae [2-5], the published i m m u n o c h e m i cal studies deal with a single pepsinogen system [6-8]. Multiple pepsinogens and pepsins have also been detected in a n u m b e r o f o t h e r species. MATERIALS AND METHODS T h e three dogfish pepsinogens were p r e p a r e d fi'om the stomach mucosae o f smooth dogfish Mustelus canis stomachs and used to immunize New Zealand white rabbits [1[. Assays o f quantitative c o m p l e m e n t (C') fixation and the inhibition o f C' fixation assays were p e r f o r m e d according to the m e t h o d o f Wasserman and Levine[9]; double diffusion in agar by the m e t h o d o f Ouchterlony[10], and i m m u n o e l e c t r o p h o r e s i s hy the m e t h o d o f Grabar and Williams[l 1] except that 3 per cent agarose was substituted for agar. T h e d e n a t u r a t i o n curves were plotted f l o m a series of C' fixation curves obtained after exposing tim antigens to different conditions followed by dilution into chilled Tris buffer and immediately assaying for antigenic activity [121. Peptk" activities were d m e r m i n e d using d e n a t u r e d hemoglobin as substrate al p H 2[13] a n d / o r by using a modification o f the milk clot test at p H 5.4114]. In tile latter assay, the substrale was p r e p a r e d by a d d i n g 170 ml o f 0'022 M acetate buffer (pH 4'6) to a 13 oz can o f Borden's Ew~porated Milk with continuous stirring. For potential enzynfic activity, the pepsinogens were activated to pepsin by e x p o s u r e to pH 2 for 3 rain at 37 ° prior to e n z y m e assay. T h e pepsins were p r e p a r e d by exposing the purified p r e c u r s o r to pH 2 at 37 ° for 3 rain followed by dialysis against 0-1 M sodium acetate at pH 3 for 24 hr at 4 °. This yielded products with m o l e c u h n weight o f approximately 34,000 a c ( o r d i n g to high speed sedimentation to equilibrium [15]. Such products were t b u n d to be at least !15 l)er cent pepsin by the milk clot assay, RESI;I~TS hnmunochemical characlerizalim~ o/the antipepsinown.~ All antisera were examined fi)r immunochenfical h o m o g e n e i t y by double diffusion in agar. With highly purified antigens, only a single band o f precipitation was observed in each case (Fig. I). In addition, c r u d e extracts o f the stomach mucosae yielded single bands o f precipitation with these antisera. T h e serological reactions of pepsinogens A and D with antipepsinogens A and D w e r e indistinguishable; there was no sign o f s p u r r i n g between the h o m o l o g o u s and heterologous systems. Pepsinogen C did nol react with either of' the heterologous antibodies and pepsinogens A and D thiled m react wifll antipcpsinogen C. Single lines o f precipitation were obtained (Fig. 2) on i m m u n o e l e c t r o p h o r e s i s o f lhese three antibodies with Iheir amisera. A pattern superimposable on tile one shown was obtained if antipepsinogen D and antipepsinogen A were interchanged in the wells. In the (Y fixation assay (Fig. 3), pepsinogens A and D fixed C' with either
Fig. 1. Immunodiffusion analyses of pepsinogens A, D and C and antipepsinogens A, D and C (a-A. a-D and a-C).
(Faciytg page 202)
I mmunochemistry of Dogfish Pepsinogens
a-C
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Immunochemical relatio~zships of the pepsi~zs Io the pepsinogens T h e pepsin obtained f r o m each purified pepsinogen failed to react with the antibody to the p r e c u r s o r in any o f the immunological assays, i.e. precipitation or direct ('/ fixation. T h e results shown in Fig. 4 using antipepsinogen A with all three pepsins are typical o f those obtained with the o t h e r two antisera in tile ('/ fixation assay. Each o f tlae pepsins was t h e r e f o r e tested for its ability to inhibit the pepsinogen A - a n t i p e p s i n o g e n A system. As shown in Table 1, pepsin A and pepsin D gave 50 per cent inhibition at about equivalent concentrations but no inhibition was obtained with either pepsin C or pepsinogen C even when concentrations o f up to 17/xg were used. T h e antibodies directed toward the dogfish zymogens thiled to react with swine and chicken pepsinogens or pepsins, while the antibodies specific t o t the zymogens o f these species failed to react with the chicken zymogens or enzymes. T h e ability o f the antibodies to neutralize enzymic activity was tested bv incubating each pepsin with the antibody specific for its respective zymogen [6i. As controls, an antiserum directed toward h u m a n y-globulin was also incubated with the enzymes and the enzymes were incubated in tile absence o f any antisera. T h e residual activity after the incubation was d e t e r m i n e d by the milk clot test at pH 5"4, since the conditions o f the hemoglobin assay (pH 2) would cause dissociation o f the a n t i g e n - a n t i b o d y complex. In each case, 50 per cent neutralization was obtained with approximately 0-1 ml o f a n t i s e r u m and the enzymes were fully active in the control experiments. While such inhibition shows that an a n t i g e n - a n t i b o d y reaction has taken place, it does not necessarily prove that the antibody is directed towards the enzymatically active site. Similar results may be obtained if the large substrate sterically hinders the tormation o f the an tibody-substrate complex [ 16].
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Fig. 3. C' fixation of pepsinogens A (A), D (O) and C(X) with top) antipepsinogen A, middle) antipepsinogen D and bottom) antipepsinogen C.
Immunochemistry of Dogfish Pepsinogens
205
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Fig. 4. C' fixation of pepsinogen A (O), pepsin A (Q), pepsin D(A) and pepsin C(X) with antipepsinogen A. Table 1. Inhibition of pepsinogen Aantipepsinogen A immune system* Amount required for (50~) inhibition 0*g)
I nhibitor Pepsin A Pepsin A Pepsin C Pepsinogen C
0-3 0"2 t t
*Pepsinogen A(0"I p,g/ml); antipepsinogen A (304 C-2) diluted 1/4000 [9]. ?No inhibition at a concentration of 17'0/zg.
The thermal denaturation of pepsinogens A and D T h e effect o f heat on the immunological activities o f pepsinogens A and D are c o m p a r e d in Fig. 5. T h e thermal transition curve o f pepsinogen A has a slight plateau, less defined than that f o u n d for swine pepsinogen[12]. T h e r e was no difference in first-order rates o f thermal d e n a t u r a t i o n if pepsinogens A and D were h e a t e d in Tris buffer at concentrations r a n g i n g f r o m 0-015 M-1.5 M (Fig. 6). I f neutral halide salts were present d u r i n g the heating (Figs. 7 and 8), the rates varied d e p e n d i n g u p o n the type and concentration o f the specific anion. T h e time r e q u i r e d for 50 per cent inactivation o f antigenic and potential enzymic activities o f pepsinogens A and D are shown in Table 2. DISCUSSION C o m p l e m e n t fixation has p r o v e n itself to be a m o n g the most sensitive tech-
206
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Fig. 5. Loss of antigenic activities of pepsinogen A (©) and pepsinoge. D (O) after exposure to varying temperatures [6]. Pepsin0gen
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Fig. 6. Loss of' antigenic activity of pepsinogen A and pepsinogen D after heating for various periods of time in 0"015 M (O), 0-15 M (Q) and 1"5 M (X) Tris buffer (pH 7-5). niques in detecting structural differences in proteins[17, 18]. Since only slight differences in reactivity were f o u n d between the i m m u n e systerns of pepsinogens A and D, these proteins must possess similar conformations. T h e differences which do exist, e.g. in the thermal denaturation profiles, in chromatographic behavior and in electrophoretic mobilities, are evidently not sufficient to alter the gross topographical structure o f these proteins. T h e chromatographic properties which make their separation possible as well as their migration on
2O7
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Fig. 7. Effect on the antigenic and potential enzymic activities o f pepsinogen A heated at 65 ° in solutions o f neutral halide salts in 0-1 M T r i s buffer at p H 7"5. Pepsinogen
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Fig. 8. Effect on the antigenic and potential enzymic activities o f pepsinogen D heated at 65 ° in solutions o f neutral halide salts in 0"1 M Tris buffer at p H 7'5.
T. G. MERRETT
208
et al.
Table 2. Loss of antigenic and potential enzymic activities of pepsiongens A and D in neutral halide salt solutions
Halide solutions 1-5 M NaC1 1'5 M NaBr I-5M NaI 0.15M NaCI 0"I5M NaBr 0' 15 M Nal
Time required for 50% loss of activities Pepsinogen A Pepsinogen D Antigenic Peptic Antigenic Peptic 63 rain .q <1 12 9 6
70 rain !) <1 18 9 9
60 8 <1 15 8 6
69 10 --~1 15 10 5
Samples were heated at 65° at a protein concentration of 10/,g/ml in solutions containing the halide salt in 0" 1 M Tris buffer and maintained at pH 7"5. disc gel electrophoresis would suggest that pepsinogen A is m o r e acidic than pepsinogen D[1]. Swine pepsinogen D appears to be a d e p h o s p h o r y l a t e d pepsinogen A [5], but we do not yet have sufficiently good data to make a statem e n t about the p h o s p h a t e content o [ the dogfish proenzymes. T h e lack of immunological reaction o f pepsinogen C with antipepsinogen A or D would indicate that this protein has a different c o n f o r m a t i o n . Preliminary e x p e r i m e n t s indicate that the dogfish pepsinogens (42,000 mol. wt.) lose fragments o f 5000-8000 daltons d u r i n g their autocatalytic conversion into the active enzyme[151. A f r a g m e n t o f the same o r d e r , i.e. containing 41 amino acid residues, is lost d u r i n g the conversion of" swine pepsinogen to pepsin[19, 20]. T h e dogfish pepsins, which comprise some 85 per cent of the p a r e n t molecule, fail to react directly with the antisera specific tk)r the proenzyme. Pepsins A and D, however, can inhibit the pepsinogen A - a n t i p e p s i n o g e n A system to approximately the same extent. T h e s e two enzymes, therefore. resemble each o t h e r and also have some antigenic determinants in c o m m o n with the pepsinogen A. Using the same technique, analogous resuhs were obtained with the swine p e p s i n o g e n - p e p s i n system [6] However, swine pepsin was d e n a t u r e d at the assay conditions o f the c o m p l e m e n t fixation assay[61. Dogfish pepsin is stable [15] and still does not possess sufficient antigenic determinants to fix c o m p l e m e n t directly. We are still not in a position to decide w h e t h e r the loss o f the peptide f r a g m e n t a n d / o r contormational changes in the pepsin moiety are responsible for the loss in the ability o f pepsin to react directly with the antisera to the p r o e n z y m e . In the chicken pepsinogen system, a m u c h smaller f r a g m e n t o f the p r o e n z y m e is lost d u r i n g the conversion to yield a stable pepsin which reacts directly with the antisera [21]. T h e antigenic and potential enzymic activities o f pepsinogens A and D are lost at approximately the same rate (first-order kinetics) when these proteins are heated in aqueous solutions o f neutral halide salts. T h e rates are affected by the type o f anion as well as by its concentration. Generally, it has been observed that those ions which are particularly effective in 'salting in' proteins a p p e a r to be effective destabilizers of the native structure, while those ions which
Immunochemistry of Dogfish Pepsinogens
209
t e n d to 'salt o u t ' proteins h e l p p r e s e r v e the 'native' state o f the proteins [22, 23]. T h e relative effectiveness o f C1- > B r - > I in stabilizing the 'native' f o r m o f p e p s i n o g e n s A a n d D is similar to the r a n k i n g that these anions have in the H o f f m e i s t e r series [22, 24] a n d similar to that f o u n d with o t h e r proteins [23]. Dogfish p e p s i n o g e n s A a n d D are m o r e stable in 1 "5 M NaCI t h a n in 0" 15 M NaCI but are m o r e labile in 1-5 M N a I c o m p a r e d to 0" 15 M NaI. T h i s effectivehess o f c o n c e n t r a t i o n oi the individual anions is similar to that f o u n d with the p o l y p e p t i d e model, acetyl tetraglycylethylester (ATGEE), i.e. the solubility o f A T G E E at 25 ° d e c r e a s e d with increasing molarity o f NaC1 (salted out) while it increased (salted in) with increasing molarity o f N a I a n d r e m a i n e d virtually the same with N a B r ( f r o m 0 to 2"0 M) [22]° T h e antigenic a n d potential enzymic activities o f swine p e p s i n o g e n are lost m o r e rapidly at 1-5 M NaCI t h a n at 0-15 M NaCI, a situation opposite to that f o u n d for dogfish p e p s i n o g e n . Since the effects o f neutral salts on m a c r o m o l e c u l a r s t r u c t u r e are c o m p l e x and not yel u n d e r stood [23], we can now only stress the similarities which exist between dogfish t)epsinogens A a n d D a n d their difference f r o m swine p e p s i n o g e n . T h e s e observations may be i n t e r p r e t e d , as knowledge a b o u t the s t r u c t u r e o f proteins is a c c u m u l a t e d and the exact n a t u r e o f the electrostatic and lyotrot)ic effects o f salts on m a c r o m o l e c u l e s is defined.
1. 2. 3. 4. 5. 6. 7. 8. 9. l(l. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
REFERENCES Merrett T. G., Bar-Eli E. and Van Vunakis H., Biochemist~y 8, 3696 (1969). Herriott R. M.,J. gen. Physiol. 21,575 (1938). Ryle A. P., Biochem.J. 96, 6 (1965). Ryle A. P. and Hamihon M. P., Biochem.J. 101,176 (1966). Lee D. and Ryle A. P., Biochem.J. 104, 735 (1967). Van Vunakis H., Lehrer H. I., Allison W. S. and Levine L., J. gen. Physiol. 46, 589 (1963). Arnon R. and Perlmann C,. E.,J. biol. Chem. 238, 963 (1963). Schlamowitz M., Varandani P. T. and Wissler F. C., Biochemist~7 2,238 11963) Wasserman E. and Levine L.,J. Immun. 87, 290 (1961). Ouchterlony, O., Actapath. microbiol, stand. 26, 507 (1949). (;rabar P. and Williams C. A., Biochim. biophys. Acta 10, 193 (1953). (;erstein J. F., Van Vunakis H. and Levine L., Biochemistry, 2,964 (1963). Anson M. L.,J. gen. Physiol. 22, 79 (1938). Herriott R. M.,J. gen. Pbysiol. 24, 325 (1941). Merrett T. G. and Bar-Eli E., (Unpublished data). Cinader B., Antibodies to Biologically, Active Molecules. Pergamon Press, Oxfl)rd (1967). Levine L., Fedn Proc. 21,711 (1962). von Fellenberg R., Bethell M. R.,.Jones M. E. and Levine L.. Biochemistry: 7, 4322 (1968). Koehn t'. V. and Perlmann G. E.,J. biol. Chem. 243, 6099 (1968). Ong E. B. and Perlmann G. E.,J. biol. Chem. 243, 6104 (1968). Donta S. and Van Vunakis H., Biochemistry 9, 2798 (1970). Robinson D. R. andJencks W. P.,J. Am. chem. Soc. 87, 2470 (1965). Von Hippel P. H. and Schleich T., in Structure and Stability oJBiolo~cal Macromolecule~ (Edited by Timasheff S. N. and Fasman G. D.). Marcel Dekker, New York (1969). Hofinesiter F., Arch. expt. Pathol. Pharmakol. 24, 247 (1888).
IMM VoL 8 . 2 - - F
I M M U N O C H E M I C A L S T U D I E S O N P E P S I N O G E N S A, C AND D FROM T H E S M O O T H DOGFISH, M U S T E L U S CANIS* TERRENCE G. MERRETT,t LAWRENCE LEVINE and HELEN VAN VUNAKIS Graduate Department of Biochemistry, Brandeis University, Waltham, Mass. 02154, U.S.A.
(Received 27July 1970) Abstract-Rabbit antibodies to pepsinogens A, C and D (isolated from the gastric mucosae of the smooth dogfish) were characterized by immunodiffusion, immunoelectrophoresis, complement fixation and ability to inhibit the enzymic activity of the pepsin formed fi'om each zymogen. Similarities were detected only between pepsinogens A and D; pepsinogen C was unrelated to these proteins. Pepsin failed to precipitate or fix complement with the antisera directed toward the specific proenzyme from which it was derived. Therefi)re, some antigenic sites were either removed in the non-pepsin fragment and/or the pepsin moiety (which represents about 85 per cent of the pepsinogen molecule) underwent conformational changes during the conversion of the proenzyrne. However, pepsins A and D were still able to inhibit the pepsinogen A-antipepsinogen A system indicating some similarity to the proenzyme structure. The incubation of each pepsin with the antibody specific for its zymogen resulted in loss of enzymic activity indicating that the enzyme can bind to the antizymogen sera. The antigenic structure and potential enzymic activities of pepsinogens A and D were lost concurrently by first-order kinetics when these proenzymes were heated in aqueous solutions of neutral halide salts. The rate of thermal denaturation of these proenzymes was dependent upon the type and concentration of the anion present and increased in the following order I- > Br- > CI . INTRODUCTION T h e purification o f three pepsinogens isolated f r o m the gastric mucosae o f the s m o o t h dogfish with potential enzymic activity toward protein substrate h i s b e e n described [1]. Like the pepsinogens o f o t h e r species, dogfish pepsinogens A, C and D have molecular weights o f a p p r o x i m a t e l y 42,000 and contain a p r e d o m i n a n c e o f acidic over basic a m i n o acids. Dogfish p e p s i n o g e n s A and D have similar but not identical a m i n o acid compositions. Pepsinogen C contains half as m a n y basic a m i n o acid residues as pepsinogens A or D but it is unlikely that p e p s i n o g e n C represents a smaller molecule originating f r o m a p a r e n t p e p s i n o g e n A or D by limited hydrolysis since it contains greater a m o u n t s o f some o f the o t h e r a m i n o acids. Preliminary experiments indicate that the pepsins *Publication No. 749 from the Graduate Department of Biochemistry, Brandeis University, Waltham, Mass. 02154. Supported in part by research grants from the National Science Foundation (NSF GB4302) and tile National Aeronautics and Space Administration (NSG 375). L. L. is an American Cancer Society Professor of Biochemistry (Award No. PRP-21). H.V.V. is a recipient of a Puhlic Health Service Research Career Award (K6-AI-2372). tPresen! address : The Radiochemical Centre, Amersharn, Buckinghamshire, England. 201
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f o r m e d u p o n conversion of the p r o e n z y m e s account for approximately 85 per cent o f the p a r e n t molecule. With the intormation obtained from immunochemical techniques, some aspects o f the relationshi 1) o f the three precursors to each o t h e r and to their enzymes have been clarified. Although at least fimr different pepsinogens have been isolated from swine stomach mucosae [2-5], the published i m m u n o c h e m i cal studies deal with a single pepsinogen system [6-8]. Multiple pepsinogens and pepsins have also been detected in a n u m b e r o f o t h e r species. MATERIALS AND METHODS T h e three dogfish pepsinogens were p r e p a r e d fi'om the stomach mucosae o f smooth dogfish Mustelus canis stomachs and used to immunize New Zealand white rabbits [1[. Assays o f quantitative c o m p l e m e n t (C') fixation and the inhibition o f C' fixation assays were p e r f o r m e d according to the m e t h o d o f Wasserman and Levine[9]; double diffusion in agar by the m e t h o d o f Ouchterlony[10], and i m m u n o e l e c t r o p h o r e s i s hy the m e t h o d o f Grabar and Williams[l 1] except that 3 per cent agarose was substituted for agar. T h e d e n a t u r a t i o n curves were plotted f l o m a series of C' fixation curves obtained after exposing tim antigens to different conditions followed by dilution into chilled Tris buffer and immediately assaying for antigenic activity [121. Peptk" activities were d m e r m i n e d using d e n a t u r e d hemoglobin as substrate al p H 2[13] a n d / o r by using a modification o f the milk clot test at p H 5.4114]. In tile latter assay, the substrale was p r e p a r e d by a d d i n g 170 ml o f 0'022 M acetate buffer (pH 4'6) to a 13 oz can o f Borden's Ew~porated Milk with continuous stirring. For potential enzynfic activity, the pepsinogens were activated to pepsin by e x p o s u r e to pH 2 for 3 rain at 37 ° prior to e n z y m e assay. T h e pepsins were p r e p a r e d by exposing the purified p r e c u r s o r to pH 2 at 37 ° for 3 rain followed by dialysis against 0-1 M sodium acetate at pH 3 for 24 hr at 4 °. This yielded products with m o l e c u h n weight o f approximately 34,000 a c ( o r d i n g to high speed sedimentation to equilibrium [15]. Such products were t b u n d to be at least !15 l)er cent pepsin by the milk clot assay, RESI;I~TS hnmunochemical characlerizalim~ o/the antipepsinown.~ All antisera were examined fi)r immunochenfical h o m o g e n e i t y by double diffusion in agar. With highly purified antigens, only a single band o f precipitation was observed in each case (Fig. I). In addition, c r u d e extracts o f the stomach mucosae yielded single bands o f precipitation with these antisera. T h e serological reactions of pepsinogens A and D with antipepsinogens A and D w e r e indistinguishable; there was no sign o f s p u r r i n g between the h o m o l o g o u s and heterologous systems. Pepsinogen C did nol react with either of' the heterologous antibodies and pepsinogens A and D thiled m react wifll antipcpsinogen C. Single lines o f precipitation were obtained (Fig. 2) on i m m u n o e l e c t r o p h o r e s i s o f lhese three antibodies with Iheir amisera. A pattern superimposable on tile one shown was obtained if antipepsinogen D and antipepsinogen A were interchanged in the wells. In the (Y fixation assay (Fig. 3), pepsinogens A and D fixed C' with either
Fig. 1. Immunodiffusion analyses of pepsinogens A, D and C and antipepsinogens A, D and C (a-A. a-D and a-C).
(Faciytg page 202)
I mmunochemistry of Dogfish Pepsinogens
a-C
L,
~
~
203 I
XC a-D
i
I
XD a-C
i
i
XA a-A
i
l
Fig. 2. lmmunoelectrophoresis of pepsinogens A, D and C with antipepsinogens A. D and C (a-A, a-D and a-C). antipepsin A or D but failed to react with antipepsinogen C. T h e r e was no reaction between pepsinogen C and antipepsinogen A or D. It was not possible to get a complete fixation curve in the region o f antigen excess in the pepsinogen C system since the antigen was a n t i c o m p l e m e n t a r y at high concentrations.
Immunochemical relatio~zships of the pepsi~zs Io the pepsinogens T h e pepsin obtained f r o m each purified pepsinogen failed to react with the antibody to the p r e c u r s o r in any o f the immunological assays, i.e. precipitation or direct ('/ fixation. T h e results shown in Fig. 4 using antipepsinogen A with all three pepsins are typical o f those obtained with the o t h e r two antisera in tile ('/ fixation assay. Each o f tlae pepsins was t h e r e f o r e tested for its ability to inhibit the pepsinogen A - a n t i p e p s i n o g e n A system. As shown in Table 1, pepsin A and pepsin D gave 50 per cent inhibition at about equivalent concentrations but no inhibition was obtained with either pepsin C or pepsinogen C even when concentrations o f up to 17/xg were used. T h e antibodies directed toward the dogfish zymogens thiled to react with swine and chicken pepsinogens or pepsins, while the antibodies specific t o t the zymogens o f these species failed to react with the chicken zymogens or enzymes. T h e ability o f the antibodies to neutralize enzymic activity was tested bv incubating each pepsin with the antibody specific for its respective zymogen [6i. As controls, an antiserum directed toward h u m a n y-globulin was also incubated with the enzymes and the enzymes were incubated in tile absence o f any antisera. T h e residual activity after the incubation was d e t e r m i n e d by the milk clot test at pH 5"4, since the conditions o f the hemoglobin assay (pH 2) would cause dissociation o f the a n t i g e n - a n t i b o d y complex. In each case, 50 per cent neutralization was obtained with approximately 0-1 ml o f a n t i s e r u m and the enzymes were fully active in the control experiments. While such inhibition shows that an a n t i g e n - a n t i b o d y reaction has taken place, it does not necessarily prove that the antibody is directed towards the enzymatically active site. Similar results may be obtained if the large substrate sterically hinders the tormation o f the an tibody-substrate complex [ 16].
204
T . G . MERRETT et al.
a) Antipepsinogen
A
I00
80
60
40
20
o
~'
b) Antipepsinogen D I00
80
--
60
u_ 40
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c) Antipepsinogen C I00
80
60
/
40
20 x
/
/
X-~--'-X ~ x
S I
0.01
0.1
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I0.0
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/~g Pepsinogen
Fig. 3. C' fixation of pepsinogens A (A), D (O) and C(X) with top) antipepsinogen A, middle) antipepsinogen D and bottom) antipepsinogen C.
Immunochemistry of Dogfish Pepsinogens
205
I00
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"o 40
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/J.g P e p s i n o g e n
Fig. 4. C' fixation of pepsinogen A (O), pepsin A (Q), pepsin D(A) and pepsin C(X) with antipepsinogen A. Table 1. Inhibition of pepsinogen Aantipepsinogen A immune system* Amount required for (50~) inhibition 0*g)
I nhibitor Pepsin A Pepsin A Pepsin C Pepsinogen C
0-3 0"2 t t
*Pepsinogen A(0"I p,g/ml); antipepsinogen A (304 C-2) diluted 1/4000 [9]. ?No inhibition at a concentration of 17'0/zg.
The thermal denaturation of pepsinogens A and D T h e effect o f heat on the immunological activities o f pepsinogens A and D are c o m p a r e d in Fig. 5. T h e thermal transition curve o f pepsinogen A has a slight plateau, less defined than that f o u n d for swine pepsinogen[12]. T h e r e was no difference in first-order rates o f thermal d e n a t u r a t i o n if pepsinogens A and D were h e a t e d in Tris buffer at concentrations r a n g i n g f r o m 0-015 M-1.5 M (Fig. 6). I f neutral halide salts were present d u r i n g the heating (Figs. 7 and 8), the rates varied d e p e n d i n g u p o n the type and concentration o f the specific anion. T h e time r e q u i r e d for 50 per cent inactivation o f antigenic and potential enzymic activities o f pepsinogens A and D are shown in Table 2. DISCUSSION C o m p l e m e n t fixation has p r o v e n itself to be a m o n g the most sensitive tech-
206
T . (;. M E R R E T T
et al.
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I
,
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,
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Fig. 5. Loss of antigenic activities of pepsinogen A (©) and pepsinoge. D (O) after exposure to varying temperatures [6]. Pepsin0gen
A
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E
Pepsinogen D
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Fig. 6. Loss of' antigenic activity of pepsinogen A and pepsinogen D after heating for various periods of time in 0"015 M (O), 0-15 M (Q) and 1"5 M (X) Tris buffer (pH 7-5). niques in detecting structural differences in proteins[17, 18]. Since only slight differences in reactivity were f o u n d between the i m m u n e systerns of pepsinogens A and D, these proteins must possess similar conformations. T h e differences which do exist, e.g. in the thermal denaturation profiles, in chromatographic behavior and in electrophoretic mobilities, are evidently not sufficient to alter the gross topographical structure o f these proteins. T h e chromatographic properties which make their separation possible as well as their migration on
2O7
l m m u n o c h e m i s t r y of Dogfish Pepsinogens Pepsinogen A
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:
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0
"
0
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Time
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Fig. 7. Effect on the antigenic and potential enzymic activities o f pepsinogen A heated at 65 ° in solutions o f neutral halide salts in 0-1 M T r i s buffer at p H 7"5. Pepsinogen
=
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Time (Minutes)
Fig. 8. Effect on the antigenic and potential enzymic activities o f pepsinogen D heated at 65 ° in solutions o f neutral halide salts in 0"1 M Tris buffer at p H 7'5.
T. G. MERRETT
208
et al.
Table 2. Loss of antigenic and potential enzymic activities of pepsiongens A and D in neutral halide salt solutions
Halide solutions 1-5 M NaC1 1'5 M NaBr I-5M NaI 0.15M NaCI 0"I5M NaBr 0' 15 M Nal
Time required for 50% loss of activities Pepsinogen A Pepsinogen D Antigenic Peptic Antigenic Peptic 63 rain .q <1 12 9 6
70 rain !) <1 18 9 9
60 8 <1 15 8 6
69 10 --~1 15 10 5
Samples were heated at 65° at a protein concentration of 10/,g/ml in solutions containing the halide salt in 0" 1 M Tris buffer and maintained at pH 7"5. disc gel electrophoresis would suggest that pepsinogen A is m o r e acidic than pepsinogen D[1]. Swine pepsinogen D appears to be a d e p h o s p h o r y l a t e d pepsinogen A [5], but we do not yet have sufficiently good data to make a statem e n t about the p h o s p h a t e content o [ the dogfish proenzymes. T h e lack of immunological reaction o f pepsinogen C with antipepsinogen A or D would indicate that this protein has a different c o n f o r m a t i o n . Preliminary e x p e r i m e n t s indicate that the dogfish pepsinogens (42,000 mol. wt.) lose fragments o f 5000-8000 daltons d u r i n g their autocatalytic conversion into the active enzyme[151. A f r a g m e n t o f the same o r d e r , i.e. containing 41 amino acid residues, is lost d u r i n g the conversion of" swine pepsinogen to pepsin[19, 20]. T h e dogfish pepsins, which comprise some 85 per cent of the p a r e n t molecule, fail to react directly with the antisera specific tk)r the proenzyme. Pepsins A and D, however, can inhibit the pepsinogen A - a n t i p e p s i n o g e n A system to approximately the same extent. T h e s e two enzymes, therefore. resemble each o t h e r and also have some antigenic determinants in c o m m o n with the pepsinogen A. Using the same technique, analogous resuhs were obtained with the swine p e p s i n o g e n - p e p s i n system [6] However, swine pepsin was d e n a t u r e d at the assay conditions o f the c o m p l e m e n t fixation assay[61. Dogfish pepsin is stable [15] and still does not possess sufficient antigenic determinants to fix c o m p l e m e n t directly. We are still not in a position to decide w h e t h e r the loss o f the peptide f r a g m e n t a n d / o r contormational changes in the pepsin moiety are responsible for the loss in the ability o f pepsin to react directly with the antisera to the p r o e n z y m e . In the chicken pepsinogen system, a m u c h smaller f r a g m e n t o f the p r o e n z y m e is lost d u r i n g the conversion to yield a stable pepsin which reacts directly with the antisera [21]. T h e antigenic and potential enzymic activities o f pepsinogens A and D are lost at approximately the same rate (first-order kinetics) when these proteins are heated in aqueous solutions o f neutral halide salts. T h e rates are affected by the type o f anion as well as by its concentration. Generally, it has been observed that those ions which are particularly effective in 'salting in' proteins a p p e a r to be effective destabilizers of the native structure, while those ions which
Immunochemistry of Dogfish Pepsinogens
209
t e n d to 'salt o u t ' proteins h e l p p r e s e r v e the 'native' state o f the proteins [22, 23]. T h e relative effectiveness o f C1- > B r - > I in stabilizing the 'native' f o r m o f p e p s i n o g e n s A a n d D is similar to the r a n k i n g that these anions have in the H o f f m e i s t e r series [22, 24] a n d similar to that f o u n d with o t h e r proteins [23]. Dogfish p e p s i n o g e n s A a n d D are m o r e stable in 1 "5 M NaCI t h a n in 0" 15 M NaCI but are m o r e labile in 1-5 M N a I c o m p a r e d to 0" 15 M NaI. T h i s effectivehess o f c o n c e n t r a t i o n oi the individual anions is similar to that f o u n d with the p o l y p e p t i d e model, acetyl tetraglycylethylester (ATGEE), i.e. the solubility o f A T G E E at 25 ° d e c r e a s e d with increasing molarity o f NaC1 (salted out) while it increased (salted in) with increasing molarity o f N a I a n d r e m a i n e d virtually the same with N a B r ( f r o m 0 to 2"0 M) [22]° T h e antigenic a n d potential enzymic activities o f swine p e p s i n o g e n are lost m o r e rapidly at 1-5 M NaCI t h a n at 0-15 M NaCI, a situation opposite to that f o u n d for dogfish p e p s i n o g e n . Since the effects o f neutral salts on m a c r o m o l e c u l a r s t r u c t u r e are c o m p l e x and not yel u n d e r stood [23], we can now only stress the similarities which exist between dogfish t)epsinogens A a n d D a n d their difference f r o m swine p e p s i n o g e n . T h e s e observations may be i n t e r p r e t e d , as knowledge a b o u t the s t r u c t u r e o f proteins is a c c u m u l a t e d and the exact n a t u r e o f the electrostatic and lyotrot)ic effects o f salts on m a c r o m o l e c u l e s is defined.
1. 2. 3. 4. 5. 6. 7. 8. 9. l(l. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
REFERENCES Merrett T. G., Bar-Eli E. and Van Vunakis H., Biochemist~y 8, 3696 (1969). Herriott R. M.,J. gen. Physiol. 21,575 (1938). Ryle A. P., Biochem.J. 96, 6 (1965). Ryle A. P. and Hamihon M. P., Biochem.J. 101,176 (1966). Lee D. and Ryle A. P., Biochem.J. 104, 735 (1967). Van Vunakis H., Lehrer H. I., Allison W. S. and Levine L., J. gen. Physiol. 46, 589 (1963). Arnon R. and Perlmann C,. E.,J. biol. Chem. 238, 963 (1963). Schlamowitz M., Varandani P. T. and Wissler F. C., Biochemist~7 2,238 11963) Wasserman E. and Levine L.,J. Immun. 87, 290 (1961). Ouchterlony, O., Actapath. microbiol, stand. 26, 507 (1949). (;rabar P. and Williams C. A., Biochim. biophys. Acta 10, 193 (1953). (;erstein J. F., Van Vunakis H. and Levine L., Biochemistry, 2,964 (1963). Anson M. L.,J. gen. Physiol. 22, 79 (1938). Herriott R. M.,J. gen. Pbysiol. 24, 325 (1941). Merrett T. G. and Bar-Eli E., (Unpublished data). Cinader B., Antibodies to Biologically, Active Molecules. Pergamon Press, Oxfl)rd (1967). Levine L., Fedn Proc. 21,711 (1962). von Fellenberg R., Bethell M. R.,.Jones M. E. and Levine L.. Biochemistry: 7, 4322 (1968). Koehn t'. V. and Perlmann G. E.,J. biol. Chem. 243, 6099 (1968). Ong E. B. and Perlmann G. E.,J. biol. Chem. 243, 6104 (1968). Donta S. and Van Vunakis H., Biochemistry 9, 2798 (1970). Robinson D. R. andJencks W. P.,J. Am. chem. Soc. 87, 2470 (1965). Von Hippel P. H. and Schleich T., in Structure and Stability oJBiolo~cal Macromolecule~ (Edited by Timasheff S. N. and Fasman G. D.). Marcel Dekker, New York (1969). Hofinesiter F., Arch. expt. Pathol. Pharmakol. 24, 247 (1888).
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