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Immunokinetic differences among human pancreatic, salivary and milk alpha-isoamylases
Immtmochemistry. Pergamon Press 1968. Vol. 5, pp. 135-137. Printed in Great Britain
C O M M U N I C A T I O N TO T H E E D I T O R S h n m t m o k i n e t l c differences a m o n g h u m a n pancreatic, salivary and milk alpha-isoamylases (Received 9 June 1967) THE INHmITION of human and animal a-amylases by antisera has been extensively investigated, especially by McGeachin and his co-workers [1, 2]. Using antisera prepared against hog pancreatic and human salivary amylases these workers measured the inhibition resulting from the incubation of equal volumes of amylase-containing solutions and diluted antisera. Interesting phylogenetic relationships could be drawn from the manner in which a particular antiserum inhibited the a-amylases of different species of animals. H u m a n pancreatic and salivary amylases were equally inhibited and McGeachin was of the opinion that they are possibly identical [2]. In assessing the above method of testing for immunological differences Samuels [3] felt that it was not sensitive enough. An excess of antiserum preincubated with the enzyme was likely to mask differences between cross-reacting proteins and he proposed the more sensitive method of titrating the enzyme [4]. The fundamental requirement that the antibody-enzyme complex should be inactive is fulfilled in case of human a-amylase where it is known that this obtains [5]. We have observed that human amylases precipitated with homologous antisera totally lose their enzymic activity. Samuels also proposed a method of graphing % inhibition of enzyme against % inhibition per ml antiserum used. The slope of the straight line obtained is roughly proportional to the equilibrium constant (K) of the reaction Ab + E = A b E
where Ab = antibody, E = enzyme and A b E =- antibody-enzyme complex. We wish to report the results of immuno-inhibition studies of purified human pancreatic, salivary and milk isoamylases. We have reported earlier the isoenzyme structure of these human amylases,[6] which have been subsequently purified on DEAE Sephadex and Sephadex G 75 [7]. Fairly monospecific antisera were prepared from rabbits to two pancreatic isoamylases, P1 and P2, and the salivary complex (S1 + $2). The rabbits, New Zealand Whites weighing 4-5 kg, were given 5 injections of enzyme protein, 10 mg on each occasion. The injections were spaced over 4 weeks. The first two were intradermal, the third intraperitoneal and the last two were given intravenously on consecutive days. The rabbits were bled from the ear vein 5 days after the last intravenous injection. The resultant antisera were checked for their specificity against the parent antigens by means ofimmunoelectrophoresis. They were coded AP1, AP2 and AS respectively. After preliminary studies the following dilutions were made: AP1 1:48, AP2 1:20 and AS 1:12. In each experiment four test-tubes were set up in duplicate, according to the following scheme : Antiserum
0.9% saline
Enzyme
(~l)
(~l)
(t,1)
!
0
2 3 4
10 20 30
30 20 10 0
100 100 100 I00
Tube No.
135
136
Communication to the Editors
Enzyme solutions were diluted purified isoamylases P1, P2 (S1 -+- $2), the milk group of isoenzymes (P2 q- $1 + $2), containing 0 - 4 - 0 . 5 Somogyi units of enzyme activity per ml, as estimated by the method described by Wootton [8]. Antisera and saline were incubated at 37°C separately from enzyme solution for 5 min. 100 F1 of enzyme solution was added to each set of tubes at one minute intervals. The remaining activity in each set of duplicate tubes was estimated exactly 1 hr later. The intrinsic amylolytic activities of the antisera were also estimated to enable the calculation of the true residual amylase activity. A series of experiments using control rabbit sera instead of antisera were also run. (b)
(o) I00
I00 --
o°~, °
PI
80 60
oo! 2C
0
80
"o3;~0 ° ~ o Api K / - [ "~,,.%'~1:~p2
KApi= 1.7 KAm=1"4 KAs =2"3 I
I
20
40
60
e~o,A
-
40 --
KApI=1.7 KAp2=14 KAS =4"5
20 --
60
I
I
80
S
20
I
40
I
60
I
80
& 0
~ iO0
(c)
PI+SI +$2
IO0-
(d) SI + $ 2
B0" ° "~°~°~~ ° ' "
AS
6C
60
4C
40
2C
KAPI=0"4 20 _K~2=1.3 KAs =0"7
K~=0.8 KAs =4.3 I
20
t
40
I
60
Percentage
l
80 inhibition
0
I
20
per ml antiserum
AS
I
[
40 60 x 10-2
I
BO
Fig. 1
The results are shown in Fig. 1. No inhibition of amylase activity was noted with the control rabbit sera. T h e pattern of inhibition of (S1 + $2), as compared with that of P1 and P2, by the same 3 antisera, show clear differences. It can be seen that the antisera AP1 and AS tend to inhibit P1 and P2 more than they do (S1 + $2). In the light of present understanding the mechanism of inhibition by antibody is attributed to steric hindrance. Steric hindrance is probably due to the position of antibody attachment with respect to the catalytic site. This was found to be so for ribonuclease [9]. The greater inhibition of pancreatic isoamylases than salivary by AS is then explainable on the assumption that the catalytic or binding sites of the former
Communication to the Editors
137
abut on the immunogenic sites more than they do in the latter. Attachment of antibody to the pancreatic isoenzymes will therefore hinder the approach of the substrate molecules to the catalytic sites to a greater degree than in the case of salivary isoenzymes. The three antisera evoke a striking response from the complex of milk isoenzymes (P2 + S1 + $2). AP1 and AP2 titrate it as they would salivary amylases, while AS titrates it in the same m a n n e r as it does P2. Another mechanism contributing to steric hindrance could be operative in the case of a-amylases that would be less significant for enzymes acting on smaller substrates. Aggregate formation between antibody and enzyme will produce a lattice-work and present an almost inpenetrable barrier to large polymer chains. This is possibly one reason why amylase-antibody complexes totally lose their enzymic activity. T h a t the h u m a n isoamylases bear a rather close resemblance immunologically is evident from the work of McGeachin and others. The method of titrating the isoenzymes is able to reveal that small differences do exist, at least around the binding and catalytic site or sites. S. E. Aw* Royal Postgraduate Medical School J . R . HOBBS Ducane Road, London W. 12 REFERENCES 1. McGEAcmN R. L., In Salivary Glands and their Secretions. (Edited by SRSEBNV L. M. and MSYER J.) p. 325. Pergamon Press, Oxford (1964). 2. MCGEACHINR. L., Ann. N.Y. Acad. Sci. 103, 1009 (1963). 3. SAMUELSA.J., Ann. N.Y. Acad. Sei. 94, 1001 (1961). 4. SAMUELSA. J., Biophys. J. 1, 437 (1961). 5. McGEAcmN R. L. and REVNOLDSJ. M., J. biol. Chem. 234, 1456 (1959). 6. Aw S. E. and HOBBSJ. R. Biochem. J. 99, 16P (1966). 7. Aw S. E., HOBBSJ. R. and WOOT'rONI. D. P., in preparation. 8. WOOTTONI. D. P., Microanalysis in Medical Biochemistry p. 106. J. & A. Churchill, London (1964). 9. CINADERB. and LAFFERTYK . J . Ann. N.Y. Acad. Sd. 103, 653 (1963). * Present address: Department of Biochemistry, Faculty of Medicine, Sepoy Lines, Singapore 3.
C O M M U N I C A T I O N TO T H E E D I T O R S h n m t m o k i n e t l c differences a m o n g h u m a n pancreatic, salivary and milk alpha-isoamylases (Received 9 June 1967) THE INHmITION of human and animal a-amylases by antisera has been extensively investigated, especially by McGeachin and his co-workers [1, 2]. Using antisera prepared against hog pancreatic and human salivary amylases these workers measured the inhibition resulting from the incubation of equal volumes of amylase-containing solutions and diluted antisera. Interesting phylogenetic relationships could be drawn from the manner in which a particular antiserum inhibited the a-amylases of different species of animals. H u m a n pancreatic and salivary amylases were equally inhibited and McGeachin was of the opinion that they are possibly identical [2]. In assessing the above method of testing for immunological differences Samuels [3] felt that it was not sensitive enough. An excess of antiserum preincubated with the enzyme was likely to mask differences between cross-reacting proteins and he proposed the more sensitive method of titrating the enzyme [4]. The fundamental requirement that the antibody-enzyme complex should be inactive is fulfilled in case of human a-amylase where it is known that this obtains [5]. We have observed that human amylases precipitated with homologous antisera totally lose their enzymic activity. Samuels also proposed a method of graphing % inhibition of enzyme against % inhibition per ml antiserum used. The slope of the straight line obtained is roughly proportional to the equilibrium constant (K) of the reaction Ab + E = A b E
where Ab = antibody, E = enzyme and A b E =- antibody-enzyme complex. We wish to report the results of immuno-inhibition studies of purified human pancreatic, salivary and milk isoamylases. We have reported earlier the isoenzyme structure of these human amylases,[6] which have been subsequently purified on DEAE Sephadex and Sephadex G 75 [7]. Fairly monospecific antisera were prepared from rabbits to two pancreatic isoamylases, P1 and P2, and the salivary complex (S1 + $2). The rabbits, New Zealand Whites weighing 4-5 kg, were given 5 injections of enzyme protein, 10 mg on each occasion. The injections were spaced over 4 weeks. The first two were intradermal, the third intraperitoneal and the last two were given intravenously on consecutive days. The rabbits were bled from the ear vein 5 days after the last intravenous injection. The resultant antisera were checked for their specificity against the parent antigens by means ofimmunoelectrophoresis. They were coded AP1, AP2 and AS respectively. After preliminary studies the following dilutions were made: AP1 1:48, AP2 1:20 and AS 1:12. In each experiment four test-tubes were set up in duplicate, according to the following scheme : Antiserum
0.9% saline
Enzyme
(~l)
(~l)
(t,1)
!
0
2 3 4
10 20 30
30 20 10 0
100 100 100 I00
Tube No.
135
136
Communication to the Editors
Enzyme solutions were diluted purified isoamylases P1, P2 (S1 -+- $2), the milk group of isoenzymes (P2 q- $1 + $2), containing 0 - 4 - 0 . 5 Somogyi units of enzyme activity per ml, as estimated by the method described by Wootton [8]. Antisera and saline were incubated at 37°C separately from enzyme solution for 5 min. 100 F1 of enzyme solution was added to each set of tubes at one minute intervals. The remaining activity in each set of duplicate tubes was estimated exactly 1 hr later. The intrinsic amylolytic activities of the antisera were also estimated to enable the calculation of the true residual amylase activity. A series of experiments using control rabbit sera instead of antisera were also run. (b)
(o) I00
I00 --
o°~, °
PI
80 60
oo! 2C
0
80
"o3;~0 ° ~ o Api K / - [ "~,,.%'~1:~p2
KApi= 1.7 KAm=1"4 KAs =2"3 I
I
20
40
60
e~o,A
-
40 --
KApI=1.7 KAp2=14 KAS =4"5
20 --
60
I
I
80
S
20
I
40
I
60
I
80
& 0
~ iO0
(c)
PI+SI +$2
IO0-
(d) SI + $ 2
B0" ° "~°~°~~ ° ' "
AS
6C
60
4C
40
2C
KAPI=0"4 20 _K~2=1.3 KAs =0"7
K~=0.8 KAs =4.3 I
20
t
40
I
60
Percentage
l
80 inhibition
0
I
20
per ml antiserum
AS
I
[
40 60 x 10-2
I
BO
Fig. 1
The results are shown in Fig. 1. No inhibition of amylase activity was noted with the control rabbit sera. T h e pattern of inhibition of (S1 + $2), as compared with that of P1 and P2, by the same 3 antisera, show clear differences. It can be seen that the antisera AP1 and AS tend to inhibit P1 and P2 more than they do (S1 + $2). In the light of present understanding the mechanism of inhibition by antibody is attributed to steric hindrance. Steric hindrance is probably due to the position of antibody attachment with respect to the catalytic site. This was found to be so for ribonuclease [9]. The greater inhibition of pancreatic isoamylases than salivary by AS is then explainable on the assumption that the catalytic or binding sites of the former
Communication to the Editors
137
abut on the immunogenic sites more than they do in the latter. Attachment of antibody to the pancreatic isoenzymes will therefore hinder the approach of the substrate molecules to the catalytic sites to a greater degree than in the case of salivary isoenzymes. The three antisera evoke a striking response from the complex of milk isoenzymes (P2 + S1 + $2). AP1 and AP2 titrate it as they would salivary amylases, while AS titrates it in the same m a n n e r as it does P2. Another mechanism contributing to steric hindrance could be operative in the case of a-amylases that would be less significant for enzymes acting on smaller substrates. Aggregate formation between antibody and enzyme will produce a lattice-work and present an almost inpenetrable barrier to large polymer chains. This is possibly one reason why amylase-antibody complexes totally lose their enzymic activity. T h a t the h u m a n isoamylases bear a rather close resemblance immunologically is evident from the work of McGeachin and others. The method of titrating the isoenzymes is able to reveal that small differences do exist, at least around the binding and catalytic site or sites. S. E. Aw* Royal Postgraduate Medical School J . R . HOBBS Ducane Road, London W. 12 REFERENCES 1. McGEAcmN R. L., In Salivary Glands and their Secretions. (Edited by SRSEBNV L. M. and MSYER J.) p. 325. Pergamon Press, Oxford (1964). 2. MCGEACHINR. L., Ann. N.Y. Acad. Sci. 103, 1009 (1963). 3. SAMUELSA.J., Ann. N.Y. Acad. Sei. 94, 1001 (1961). 4. SAMUELSA. J., Biophys. J. 1, 437 (1961). 5. McGEAcmN R. L. and REVNOLDSJ. M., J. biol. Chem. 234, 1456 (1959). 6. Aw S. E. and HOBBSJ. R. Biochem. J. 99, 16P (1966). 7. Aw S. E., HOBBSJ. R. and WOOT'rONI. D. P., in preparation. 8. WOOTTONI. D. P., Microanalysis in Medical Biochemistry p. 106. J. & A. Churchill, London (1964). 9. CINADERB. and LAFFERTYK . J . Ann. N.Y. Acad. Sd. 103, 653 (1963). * Present address: Department of Biochemistry, Faculty of Medicine, Sepoy Lines, Singapore 3.