A Complement-Release Reaction; the Neutralization of the Anticomplementary Action of Sea-Urchin Fertilizin by AntifertilizinAuthor(s): Albert TylerSource: Proceedings of the National Academy of Sciences of the United States of America,Vol. 28, No. 10 (Oct. 15, 1942), pp. 391-395Published by: National Academy of SciencesStable URL: http://www.jstor.org/stable/87493 .

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PROCEEDINGS OF THE

NATIONAL ACADEMY OF SCIENCES Volume 28 October 15, 1942 Number 10

Copyright 1942 by the National Academy of Sciences

A COMPLEMENT-RELEASE REACTION; THE NEUTRALIZA- TION OF THE ANTICOMPLEMENTARY ACTION OF SEA-

URCHIN FERTILIZIN B Y ANTIFERTILIZIN*

BY ALBERT TYLER

WILLIAM G. KERCKHOFF LABORATORIES OF THE BIOLOGICAL SCIENCES, CALIFORNIA

INSTITUTE OF TECHNOLOGY

Communicated August 23, 1942

In previous articlesl"8 the interaction of fertilizin and antifertilizin has been considered analogous to that of an antigen and its specific antibody in the more usual serological reactions. Since man;y antigen-antibody re- actions exhibit complement-fixation, it was of interest to learn if this oc- curs in the union of fertilizin and antifertilizin. A. positive result would strengthen the analogy with serological reactions. However, it is by no means a necessary characteristic of antigen-antibody reactions that they fix complement in vitro, since there are a great many exceptions, such as all toxin-antitoxin reactions that have been examined,9 the reaction of horsel1 or humanll antipneumococcus antibodies with capsular carbohydrate, and most cases of the action of the normal iso-antibodies of the blood-groups.12 Also, in many instances antigen or antibody alone are too highly anticom- plementary to permit a determination of complement fixation by their in- teraction.

In complement-fixation tests with sea-urchin fertilizin-antifertilizin mixtures, the fertilizin was found to be highly anticomplementary. Dilu- tions below the anticomplementary limit gave with antifertilizin no complement-fixation. Above the anticomplementary limit a new phenom- enon was found; namely, that the addition of antifertilizin neutralized the anticomplementary action of fertilizin, or if the complement were first bound by fertilizin, antifertilizin released it. This phenomenon is there- fore designated a "Complement-Release Reaction." The data show it to be a quantitative measure of the interaction of fertilizin and anti-fertili- zin and to involve the fourth component (C'4) of complement.

Material and Methods.-The fertilizin and the antifertilizin solutions were partially purified preparations from the eggs and the sperm, respec- tively, of the sea-urchin Strongylocentrotus purpuratus, prepared according

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392 ZOOLOGY: A. TYLER PROC. N. A. S.

to previously described 48 t ;3~t~~ ~methods. Fertilizin titer 8t I o o o o o

is given as the reciprocal _________of the final dilution that

|O rn +4+ ++ +++4 ?-? + mgives microscopically per- + + + + + + + + + + ceptible agglutination with

zoPS + + + + + an equal volume of a 1%7 _ _________________- sperm suspension. Anti-

1 + + + + + + + + + + fertilizin titer is the recip- ~ g + + + + + + rocal of the dilution that z -+ +++++++++++

+ +?- + + + + + + + just neutralizes one unit of PQ --

..--- fertilizin. For use in the

8=Q +U +I + ++ -+ + hemolytic system fertilizin + + + + + + + + + + + =4 .'.- I-- --+ + + + + + + + + + and antifertilizin solutions g - I ++ + + + + + + + + were adjusted to 0.9%

|

o ~ .+++++salinity. Pooled fresh x a + +++++ +

'? o + + + + + + + guinea-pig serum was used

A,i U : +? + + + + + + as the source of comple- cd I z +++++++++ + *

8 ?+ ++ +?o +_____ _ment. The indicator con-

. a5 ^ + + + + sisted of a 2% suspension o i c + + + + + of sheep erythrocytes 0 h t .Q + + + + + +. z

+ + sensitized with three units m4 C , ( +++++++ool = C, B H - -

?_ - of amboceptor (rabbit U $ o + + antisheep cell serum) and

rt 0? ++++ C< , 4 + -+ + 0.5 ml. of this was used in

.

+ + + 1 11

L0O + + + + + a+ o 0 a total reactant volume of

|c t 11-- o- + + :1.25 ml. All dilutions and

? + ++ C . v + Z 4 +' adjustments to total vol- Z + +

b o + + + + ume were made with a "sea I < z+ + + + -o o o o o water-Ringers" solution o + prepared by diluting three z K ~ + volumes of sea water (local

3 m . H+ + 4salinity = 3.3%) with 0 aU ++++?o000000 0 0

0s-4 ( +00 _____ _eight volumes of distilled 01 | ^ + water.

15 | X

+___ _++_o__o_O_o_o_Io Interaction of Fertilizin, Z

~ > ' - Antifertilizin and Comple- U C

8=8==8==8==8ment.-Solutions of S. pur- CU-4

q _ o o- "d C puratus fertilizin are found

U o to be very highly anticom- o o plementary and the extent

C r .+ cl'I 4 + of this action roughly g . . parallels the sperm-agglu- Cd p~~~

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VOL. 28, 1942 ZOOLOGY: A. TYLER 393

tinating titer. In the experiment listed in table 1 (last line) 0.25 ml. of a 32 unit fertilizin solution (I/16 dilution) inactivates 2 units of complement. In 13 other experiments the inactivation ratio ranged from 8:1 to 32:1 with an average of 20:1. On the other hand concentrated solutions of antifertilizin show no anticomplementary action (next to last column of table) nor do they have any hemolytic action on sensitized sheep cells (last column of table). The addition of antifertilizin overcomes the anticomplementary action of fertilizin and, as the data show, the amount required is directly propor- tional to the amount of fertilizin present. In these mixtures one might have expected to find complement fixation. Instead the presence of anti- fertilizin protects complement from inactivation by fertilizin. There is no sign of complement fixation with dilutions (such as '1/28 in the table) of fertilizin that are below the anticomplementary limit. From the data in the table it may be seen that one unit of antifertilizin neutralizes the anticomplementary action of 2 to 4 units of fertilizin and combined with 13 other less extensive experiments the ratio approximates 1:4.

If complement is allowed to react with fertilizin for some time before the addition of antifertilizin the results are the same as when all three are mixed immediately. If the sensitized sheep cells are added directly after the addition of antifertilizin, the same degree of hemolysis is ob- tained but there is a definite time lag when compared with a previously in- cubated mixture. Since antifertilizin has no hemolytic action on sensi- tized sheep cells the results mean that complement as a whole is not de- stroyed or irreversibly bound by fertilizin.

The interaction of fertilizin and complement is also manifested by an in- hibition of the sperm-agglutinating power of the former. For these tests the guinea-pig serum is adjusted to sea water salinity. In four experi- ments that were run one unit of complement neutralized the sperm-ag- glutinating action of 4 to 7 (av. 5) units of fertilizin. This is much less than might be expected from the anticomplementary ratio (20:1), but it is con- sistent with the ratios of fertilizin to antifertilizin that remove the anti- complementary action of the former. It may be interpreted as signifying a stronger interaction of fertilizin with antiferilizin (either in solution or on the sperm) than with complement or, in other words, a lower equilibrium constant for the former reaction than for the latter.

When guinea-pig serum is added to intact S. purpuratus eggs the latter

agglutinate and a precipitation membrane (4 fig. 1) forms on the gelatinous coat. Since the jelly coat is composed of fertilizin5'6 this action is, very likely, simply another manifestation of its interaction with complement.

Action of Univalent Fertilizin.-It has been shown6,13 that fertilizin can be converted into a non-agglutinating form, termed "univalent," that still combines with the species sperm or with antifertilizin. This, then, offered another means for testing for complement-fixation, if the anticomplemen-

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394 ZOOLOGY: A. TYLER PROC. N. A. S.

tariness of fertilizin were lost by the conversion. However, in three experi- ments that were run the univalent fertilizin was found to be just as anticom- plementary as the unaltered agent. In addition its effect on complement was likewise found to be neutralized by antifertilizin.

Component of Complement Involved.-Complement is known to be com- posed of two heat labile and two relatively heat stable components, now designated,14,15 respectively, C'l (mid-piece), C'2 (end-piece), C'3 (third component) and C'4 (fourth component). Guinea-pig serum that had been heated at 56?C. for 1/2 hour to inactivate C'l and C'2 was found to be capable of reactivating complement that had been inactivated by fertilizin. Also, such heated serum neutralized the sperm-agglutinating power of fertilizin and agglutinated the intact sea-urchin eggs. Serum absorbed with yeast (9 p. 16) to remove C'3 was likewise found to be capable of reac- tivating fertilizin-inactivated complement and agglutinating the eggs, while serum treated with ammonia16 to inactivate C'4 lacked the ability. These results are illustrated in table 2 which contains the data of one of four similar experiments. It is clear, then, that fertilizin combines with C'4.

Failure of Antifertilizin to Act as C'4.-A reasonable interpretation of the fact that antifertilizin and C'4 can displace one another from com- bination with fertilizin is that both possess very similar combining groups. On this basis one would expect antifertilizin to be capable of acting as a substitute for C'4 in the hemolytic system. Attempts were therefore made to reactivate ammonia-treated guinea-pig serum by the addition of anti- fertilizin. The results (table 2) were consistently negative, however. This might mean that while antifertilizin resembles C'4 in some respects

TABLE 2

REACTIVATION OF FERTILIZIN-INACTIVATED COMPLEMENT AND TEST OF ANTIFERTILIZIN

FOR C'4 ACTIVITY

DILUTIONS OF 0.25 ML. OF:

YEAST-

NORMAL ABSORBED NH40H-TREATED FERTILIZIN GUINEA-PIG GUINEA-PIG GUINEA-PIG ANTIFERTII,IZIN DEGREE OF

(TITER = 256) SERUM SERUM SERUM (TITER = 128) HEMOLYSES

... 1/25 . ........ .... ++++ 1/16 1/25 ..... ..... 0

1/32 1/25 .... ........ + 1/64 1/25 ... .... + +

1/128 1/26 ..... .... ....+ + + 1/2 ..... .... 0

/2 .... 0

... . 1/2o /20 0 .... + + + + 1/16 1/2 /20 ..... .... + + + +

1/16 /2 1/5 .... 0

.. . .. .. 1/2 and less 1 and less 0

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VOL. 28, 1942 ZOOLOGY: A. TYLER 395

it differs in properties that enable C'4 to collaborate with the other three

components and the sensitized cells in the hemolytic system. Another

interpretation is that the mutual displacement is due, not to similarity of

combining groups of antifertilizin and C'4, but to sufficiently close prox- imity of combining sites on the fertilizin molecule so that union with one of them inhibits union with the other.

Summary.-The fertilizin obtained from sea-urchin eggs is found to be

highly anticomplementary. There is no evidence for complement-fixation in its interaction with antifertilizin from the species sperm, but instead there appears a new type of phenomenon which is termed "Complement Release." This consists in the liberation by antifertilizin of complement that has been bound by fertilizin or in the neutralization by antifertilizin of the anticomplementary action of fertilizin. The effect is a quantitative measure of the interaction of fertilizin and antifertilizin. Only the fourth

component (C'4) of complement is involved and the results may imply chemical similarity between C'4 and antifertilizin. However, antifertilizin cannot replace C'4 in the hemolytic system.

I am indebted to Professor Sterling Emerson for his generous coopera- tion and advice in this work.

* This investigation was aided by a grant from the Penrose Fund of the American Philosophical Society.

1 Tyler, A., Proc. Nat. Acad. Sci., 25, 317 (1939). 2 Tyler, A., and Fox, S. W., Science, 90, 516 (1939). 3 Tyler, A., Biol. Bull., 78, 159 (1940). 4 Tyler, A., Proc. Nat. Acad. Sci., 26, 249 (1940). 6 Tyler, A., and Fox, S. W., Biol. Bull., 79, 153 (1940). 6 Tyler, A., Ibid., 81, 190 (1941). 7 Tyler, A., and O'Melveny, K., Ibid., 81, 364 (1941). 8 Tyler, A., West. Jour. Surg., Obstet. & Gynec., 50, 126 (1942). 9 Osborn, T. W. B., Complement or Alexin, Oxford Univ. Press, 1937, p. 36.

10 Zinsser, H., and Parker, J. J., Jour. Exp. Med., 49, 25: (1923). 11 Stats, D., and Bulowa, J. G. M., Jour. Immunol., 44, 41 (1942). 12 Wiener, A. S., Blood Groups and Blood Transfusion, Charles C. Thomas, Baltimore,

1939, p. 25. 13 Metz, C. B., Biol. Bull., 82,446 (1942). 14 Pillemer, L., and Ecker, E. E., Science, 94, 437 (1941); Ann. N. Y. Acad. Sci., 43,

63 (1942). 15 Heidelberger, M., Jour. Exp. Med., 73, 681 (1941). 16 Whitehead, H. R., Gordon, J., and Wormall, A., Biochem. Jour., 19, 618 (1925).

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