Conservation of antigenic determinants among different seed lectins

ARCHIVES OF BIOCHEMISTKY AND BIOPHYSICS Vol. 192, No. 2, February, pp. 457-465, 1979

Conservation JOHN

of Antigenic

HOWARD, Department

Determinants

JUANITA

KINDINGER,

of Biochemistry, Received

among

May

IJniuersity

LELAND

AND

of California,

24, 1978; revised

Different

Riverside,

September

Seed Lectins’ M. SHANNON’

California

92521

1, 1978

Immunochemical techniques were employed to examine seed lectins for structural similarities. Antisera raised against eight homogeneous lectins were used to test for crossreacting material in crude seed extracts as well as highly purified lectins. The data provide immunochemical evidence that lectins isolated from different species may be structurally relat,ed proteins. As structurally related proteins, the cross-reacting lectins may also possess a similar function. In addition, antisera raised against 1JZe.xagglutinin I or Bandeiraea agglutinin, appeared to recognize an identical set, of determinants on those lectins showing cross-reactivity. This highly conserved region of the lectin molecule may be important for the proper functioning of these proteins.

antigenic lectins.

Lectins are multivalent carbohydrate binding proteins which are widely distributed in plants and are particularly abundant in the seeds of legumes. Very little is known about the structural properties of lectins and even less is known about their physiological role in plants (1, 2). Recently, six lectins from different leguminous plants were shown to possess extensive homology in primary structure near the N-terminus (3, 4). Other workers reported that wax bean lectin and PHA-P and PHA-M crossreact slightly with antiserum raised against lima bean lectin (5). These observations support the theory (3, 5) that during evolution, there was a conservation of a structural feature(s) among the different legume lectins. Conservation of structural features suggests that the conserved portion of the protein is performing a beneficial function, and that proteins possessing this conserved structural feature may have similar functions. Immunochemical techniques provide a sensitive method to detect structural similarities among lectins. Antibodies raised against a given lectin will react with a different lectin only when the two proteins share a common determinant(s). Using this approach we detected a conservation of ’ Supported dation (PCM ’ To whom

in part by the National Science 77-17612). inquiries should be addressed.

determinants MATERIALS

among AND

several

seed

METHODS

Seeds were obtained from the following sources: Virginia peanut, pea and soybean, were purchased from W. Atlee Burpee Co.; wheat germ, lens and rice were purchased from a local market; Robinia pseudocacia, Dolichos biflorus, Bauhinia purpurue alba,

Sophwa japonica, Cystisus scoparins, Ulex europeus, and Lotus tetragonolobus were obtained from Vector Laboratories (1479 Rollins Road, Burlingame, Calif. 94010); tobacco seeds were the kind gift of Mr. L. Oki, Department of Plant Science, University of California at Riverside. Purified lectins were obtained from the following sources: phytohemagglutinin was purchased from Sigma, peanut agglutinin, concanavalin A, Ricinus communis agglutinin 120, lens agglutinin, pea agglutinin, wheat germ agglutinin, Dolichos biflourus agglutinin, Bandeiraea agglutinin, Ulex agglutinin I, and Sophora agglutinin were obtained from Vector Laboratories. Lectinpurification. Seed flour (10 g) was suspended in 100 ml of phosphate-buffered saline (5 mM phosphate, pH 7.4, 150 mM NaCl). The mixt,ure was held at room temperature for 2 h, centrifuged at 10,OOOg for 10 min and the supernatant solution dialyzed against PBS.” The solution was then applied to an affinity ,’ Abbreviations used: PHA-E, erythrocyte phytohemagglutinin; PNA, peanut agglutinin; ConA, concanavalin A; RCA, Ricinus communis 120; WGA, wheat germ agglutinin; DBA, Dolichos biflorus agglutinin; SBA, soybean agglutinin; Ab, antibody; CRM, cross-reacting material; PBS, phosphate buffered saline; IgG, immunoglobulin G.

Foun-

457

0003-9861/79/020457-09$02.00/O Copyright Q 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

458

HOWARD,

KINDINGER,

chromatography column (1.5 cm x 5 cm). Soybean agglutinin and Bauhinia agglutinin were purified using N-acetylgalactosamine covalently attached to sepharose beads while Lotus agglutinin was purified using fucose covalently attached to sepharose beads. Affinity resins were obtained from Vector Laboratories. Bauhinia agglutinin and SBA were eluted with PBS containing 100 mM N-acetylgalactosamine while Lotus agglutinin was eluted with PBS containing 100 mM

fUCOSe.

Lectin homogeneity. Electrophoresis was performed with 50 pg of lectin on 7% acrylamide gels using the procedure of Davis (6). Gels were stained with 1% Coomassie blue in 7.5% acetic acid-50% methanol and destained in the same solution without dye. Following this procedure, all purified lectins showed a single major band. Lectins, for which antiserum was available, showed a single precipitin band following immunoelectrophoresis. Seed extracts. Seed flour (5 g) was suspended in 10 ml PBS and held at room temperature for 2 h. The mixture was centrifuged at 10,OOOgfor 10 min and the supernatant solutions collected. Antibody preparation. Antibodies were raised against purified SBA, DBA, WGA, ConA, lens, pea, Ulex I, and Bandeiraea lectins utilizing methods described earlier (7). The immunoglobulins were purified from each antiserum by precipitation with 37% saturated (NH4)2S04 followed by DEAE chromatography (8). Purified IgG was stored in 50% saturated (NH4)2S04 and dialyzed against PBS prior to each experiment. Zmmunochemical techniques. Immunoelectrophoresis was performed using Davis gels (6) following procedures outlined previously (8). Double diffusion assays were performed according to the procedure of Ouchterlony (9) using 0.01 ml antiserum and 0.01 ml of sample, and incubation at 37°C. Precipitin bands were observed over a 24-h period. “Sugar plates” were made by dissolving agar in PBS containing 10 mM galactose, glucose, fucose, N-acetylgalactosamine, and N-acetylglucosamine. Agglutination assays. A 2% suspension of red blood cells in PBS (0.01 ml) was incubated with an equal volume of lectin in PBS for 30 min on a Petri dish at room temperature. Cells were examined for agglutination using a dissecting microscope. The titer of a particular lectin sample was determined by making serial two-fold dilutions of the lectin. The reciprocal of the last dilution capable of agglutination was recorded as the titer for the sample. All agglutination assays were performed with trypsinized rabbit red blood cells unless noted otherwise. RESULTS

CRM in seed extracts. The results in Table I show that each antiserum raised against a purified lectin reacted with the

AND SHANNON

seed extract containing that lectin (homologous reaction). A positive reaction between a seed extract and antiserum raised against a purified lectin from another species (heterologous reaction) denoted the presence of cross-reacting material. The predominate heterologous reactions involved Ulex I-Ab. CRM was observed between each seed extract and Ulex I-Ab. CRM inpurified lectins. Using the Ouchterlony double diffusion assay, 14 purified lectins were tested with antisera raised against 8 lectins. The results of these experiments (Table II) show that the 8 lectins reacted with their corresponding homologous antiserum. In addition, 11 of the 14 purified lectins reacted with heterologous antisera. Table II also shows the minimum concentration of each lectin needed to elicit a reaction with a particular antiserum. A low concentration of lectin is indicative of a strong reaction. The predominant crossreaction with purified lectins involved Ulex I-Ab, however, lens-Ab, pea-Ab, and Bandeiraea-Ab also detected CRM in purified lectins. Purified RCA and ConA yielded precipitin bands with preimmune serum, suggesting that these lectins interacted with a carbohydrate component in preimmune serum. To prevent the reaction with preimmune serum, RCA, and ConA binding sites were saturated by mixing respectively, 100 InM N-acetylgalactosamine and cu-methylmannoside, with the lectin in the Ouchterlony well. In addition, whole antiserum was replaced with purified IgG. The final preventive measure involved replacing standard Ouchterlony plates with sugar Ouchterlony plates. These precautions eliminated precipitin bands between normal serum and ConA and RCA. Using preventive measures, ConA reacted only with ConAAb, and RCA reacted only with Ulex I-Ab (RCA-Ab was not available). Other purified lectins did not react with preimmune serum. The following experiments were performed to ensure that the precipitin bands which formed between lectins and whole antisera were specific antibody reactions rather than reactions with carbohydrate components in the antisera. Each lectin

CONSERVATION

OF DETERMINANTS TABLE

AMONG

459

LECTINS

I

CRM OBSF.RVED IN OUCHTERLONY

DOUBLE DIFFUSION ASSAYS WHEN SEED EXTRACTS ANTISERA RAISED AGAINST PURIFIED LECTINS”

WERE

TESTED

Antisera

Seed extracts DBA Dolichos Lens Pea Soybean Ulen Wheat Germ Bauhinirc Cystisus Lotus Peanut Rice Sophora Tobacco

Lens

Pea

+ * +

+ /

SBA .~

+

*

WGA

Ulea I ~~~~ ~~~~ + + + + * + + + + + + + +

Bandeiraeu

+ + +

+

” Formation of precipitin bands are denoted as + for heterologous reactions and * for homologous Neither pre-immune serum, nor antiserum to Con A, reacted with the seed extracts.

TABLE MINIMUM

CONCENTRATION

Antisera Bandeiraea

ConA

DBA

reactions.

II

OF PURIFIED LECTIN REQUIRED TO ELICIT AGAINST PURIFIED LECTIN~

Purified lectins Bandeirnea ConA” DBA Lens Pea SBA C1e.r I WGA Bauhinia Lotus PNA PHA-E RCA” Sophora

WITH

CRM WITH ANTISF.RA

RAISED

(pg/ml)

Lens

Pea

SBA

Ulex I

WGA 60

60 30 30

1000 15 15

500 30 30 30

1000

30

500 500

120 120

60

1000

1000 1000

250 120

60

30

-. ” Double diffusion assays were performed using 0.01 ml antiserum and 0.01 ml lectin at the concentration noted. The absence of a value means that no reaction occurred at 1 mg/ml lectin. ’ Tested in the presence of 100 mM n-methylmannoside (ConA) or galactose (RCA) on sugar Ouchterlony plates.

showing CRM was tested using the preventive measures outlined for Con A and RCA. The results using the modified procedure were the same as those using whole antiserum. Furthermore, lectin solutions (1 mg/ml in PBS containing 1% deoxycholate and 1% Triton X-100) were submerged in a

boiling water bath for 10 min. All lectin hemagglutination activity was destroyed. The boiled solutions, however, retained their ability to react with antisera on Ouchterlony double diffusion plates. These observations indicate that the precipitin bands resulted from antibody specific re-

460

HOWARD,

KINDINGER,

actions and were not the result of lectins reacting with carbohydrate components in the antisera. Immunoelectrophoresis. While the above experiments confirm that the precipitin bands were the result of antibody reactions, they do not distinguish whether the antibody reacted specifically with the lectin or a protein contaminant. To show that lectins were involved in the antibody reactions, each purified lectin was first subjected to gel electrophoresis and the protein detected using Coomassie blue. Each lectin showed a single major band. A parallel unstained gel was placed in a double diffusion plate and tested with homologous antiserum. In each case a single precipitin band formed which corresponded with that position of the gel stained with Coomassie blue. A second series of unstained gels was simultaneously tested with heterologous antisera. One example is shown in Fig. 1A. A single precipitin band formed between Bandeiraea lectin and Ulex I-Ab. No precipitin band occurred between the lectin and the preimmune serum. Comparison of the precipitin and protein band positions for Bandeiraea lectin can be seen by superimposing the immunoelectrophoretic gel over the gel stained with Coomassie blue. Figure 1B reveals that the position on the gel responsible for the CRM superimposes with the position on the gel which stained with Coomassie blue. Rf values for the precipitin band and the protein band of several lectins are summarized in Table III. In each case, the Rf value obtained with Coomassie stained gels corresponded with the Rf value of the precipitin band. These observations suggest that the lectin molecules were responsible for the CRM. Adsorption experiments. The following experiments were undertaken to confirm that the lectins were responsible for the CRM. Lectin samples (100 pg in 0.1 ml) were incubated with cross-reacting antiserum (0.1 ml) for 30 min at room temperature. In each instance a precipitate formed and was removed by centrifugation. The supernatant solution was then tested for lectin activity using the red blood cell agglutination assay. Table IV summarizes the results. In each instance the cross-reacting

AND SHANNON

antiserum removed at least 95% of the agglutination activity from solution. We next inquired if the CRM could bind to red blood cells, a common feature among lectins. Samples of purified lectins (10 pg in 0.1 ml, except Bandeiraea which contained 100 pg in 0.1 ml) were incubated with rabbit red blood cells for 10 min to adsorb the lectin. The cells were collected by centrifugation and the supernatant solution was tested for CRM using the Ouchterlony double diffusion assay. The results are presented in Table V. In each instance the CRM was adsorbed by the red blood cells and removed from solution. Examination of spur formation. Spur formation in Ouchterlony double diffusion assays can distinguish if two proteins are antigenically similar or distinct. The formation of a single spur is the result of two different antigens which share some common determinant(s) (10). Lectins which showed precipitin bands with heterologous antisera were examined for their ability to form spurs. In the first example, Bandeiraea and Ulex I lectins were tested with Ulex I-Ab. The results (Fig. 2) show the formation of a single spur, indicating that these lectins have some determinant(s) in common. RCA, Lotus, Bauhinia, Sophora, DBA and PHA-E were each assayed in a similar manner with Ulex I lectin and Ulex I-Ab. In each instance a single spur was produced similar to that shown in Fig. 2. These observations indicate that each of the above lectins has some determinants in common with Ulex I lectin and that Uler I lectin possesses some distinct determinants. Since lens and pea lectins reacted with each other’s antiserum (Table II), these lectins were examined for their ability to form spurs with either lens-Ab or pea-Ab. The results in Fig. 3 show that lens and pea lectin form a line of identity with lens-Ab. When lens-Ab was replaced with pea-Ab, a precipitation pattern, indistinguishable from that shown in Fig. 3, was obtained. These observations indicate that the lectins isolated from lens and pea are antigenically identical proteins. Examination for common antigenic determinant(s). The unusually high frequency of cross-reactivity with Ulex I-Ab led us to

CONSERVATION

OF DETERMINANTS

AMONG

LECTINS

461

FIG. 1. (A) Immunoelectrophoretic pattern of Bandeiraea lectin. Left trough contains preimmune serum; right trough contains Ulex I antiserum. (B) Diagram of Bandeiraea lectin immunoelectrophoretic pattern superimposed on a gel stained with Coomassie blue. Left trough contains preimmune serum; right trough contains Ulex I-antiserum.

inquire if Ulex I-Ab recognized the same set of determinants with each of the crossreacting lectins. Recognition of identical determinants would suggest that a portion(s) of each of the lectins had been conserved. This possibility was tested by examining the ability of Ulex I-Ab to discriminate between two different cross-reacting lectins. In the first example Bandeiraea and Bauhinia lectins were challenged with Ulex I-Ab and examined for the formation of spurs. The results (Figure 4) show that a line of identity was formed, indicating that the determinants recognized by Ulex I-Ab are identical for these two lectins. This was repeated by testing Ulex I-Ab with the lectins, Bandeiraea and RCA, RCA and

Lotus, and Bauhinia and Lotus. In each case a line of identity was observed similar to that shown in Figure 4. In addition, Bandeiraea-Ab was tested with lectins from Bauhinia and Sophora, Ulex I and Sophora, and Ulex I and Bauhinia lectins. Again, in each case, a line of identity was formed similar to that shown in Fig. 4. This indicates that Bandeiraea-Ab, like Ulex IAb, recognizes an identical set of determinants on each of its cross-reacting lectins. DISCUSSION

We began our study by raising antiserum to each of 8 different homogeneous lectins. Each antiserum raised against a purified lectin reacted with the seed extract contain-

462

HOWARD.

KINDINGER.

ing that lectin and with the highly purified lectin. We then tested 14 seed extracts with each of the 8 antisera. We found that each seed extract showed CRM when challenged with Ulex I-Ab. Some of the seed extracts also showed CRM when challenged with lens-Ab, pea-Ab, DBA-Ab and Bandeiraea-Ab. We then tested highly purified lectins for CRM and found that several of the purified lectins were capable of reacting with heterologous antisera. Additional experiments were performed to confirm that the lectins were responsible for the CRM. First, we found that the CRM and lectin had similar migration patterns on gel electrophoresis. Second, we observed that the CRM, like the lectin, could be adsorbed on

TABLE K, VAI,UES

AND SHANNON

red blood cells. We also found that crossreacting antisera were capable of precipitating lectin activity, indicating that the antibody recognized the lectin molecule. These observations suggest that the lectin molecules were responsible for the precipitin bands when tested with heterologous antisera. Since lectins are multivalent carbohydrate binding proteins, it was conceivable that the lectins could bind to a carbohydrate component in the antiserum, thereby causing precipitin bands similar to those obtained in antibody reactions. This possibility was ruled out in the following manner. First, carbohydrate binding was inhibited by saturating the lectin binding sites with sugars. Second, the lectins were tested with purified IgG rather than whole antiserum. These precautions failed to di-

III

OF PURIFIED LFXTINS FOIJ~OWIN~:

IMMU~.OEI.F.CTROPH~KF.SIS” Lectin Protein Band&am Bauhinin Lens Lens Lotus Pea Pea PHA-E RCA L~les I

TABLE Antisera

4 Precipitin

.20 .I8 .I9 .19 .%Y .27 .27 .I7 ,026 30

.21 .21 23 .21 32 .29 32 .17 ,022” 22

l&x L7ex Lens Pea lTlex Pea Lens Ulex 1%x I.Tlex

I I

Bandeiraea Lens Pea PHA-E 1.~1e.rI

I

I I I

TABLE

Lectin Bandeiraea Lens Pea PHA-E Ulex 1

ACTIVITY

Initial

OF LECTINS

agglutination ter 260,000 65,000 16,000 32,000 128”

ti-

Ulex I

0

>97

4 8 4 4

Pea Lens Ulex I Clex I

0 0 1 0

95 X3; 75 >75

IV

BEFORE AND AFTER SERA”

Antisera

32

” Purified lectin samples (10 pg in 0.1 ml PBS, except Bandeiraea which contained 100 pg in 0.1 ml PBS) were incubated with fresh rabbit red blood cells (0.2 ml of 21, suspension) for 10 minutes. The cells were then removed by centrifugation and the supernatant tested for CRM by the double diffusion assay.

” The Rl values of protein and precipitin bands were calculated following immunoelectrophoretic procedures described in Fig. 1. ” Immunoelectrophoresis using sugar plate as described in Materials and Methods.

HEMAGGLUTINATION

V

R~MOVAI~ OF CRM FROM PURIFIED LECTINS BY ADSORPTION WITH RABBIT RED BLOOD CFJJS” Lectin Initial Antisera Ouchterlony ‘I lnhihiouchtertested titer after lion Ion? titer RRC treat ment

tested

Ulex I Pea Lens 1:1ex I Ulex I

PRECIPITATION

WITH CROSS-REACTING

Agglutination titer after incubation

% Decrease in agglutination titer

0 2000 1000 0

>99 97 94 >99

1

” Lectins (100 pg in 0.1 ml PBS) were incubated 30 min with antisera (0.1 ml). the precipitate by centrifugation, and the agglutination activity measured in the supernatant solution. ” Human 0 positive cells were used instead of trypsinized rabbit red blood cells.

>99

was removed

CONSERVATION

OF DETERMINANTS

AMONG

LECTINS

463

FIG. 2. Ouchterlony double diffusion pattern of Ulex I antiserum (0.01 ml) [I] with Ulex I lectin (IO ,~g in 0.01 ml PBS) [2] and B~~C/EWWW lectin (10 p’g in 0.01 ml PBS) [3] showing the presence of a single spur.

minish the ability of the antisera to form precipitin bands with the lectins. Third, heat treatment of the lectin abolished its ability to agglutinate red blood cells, but failed t,o diminish its ability to form precipitin bands with antiserum. If the precipitin bands resulted from lectin interactions with the bands carbohydrate components, should not appear when lectin agglutination activity is destroyed. Since the precipitin bands were not diminished by heat treatment of the lectins, we again concluded that the precipitin bands resulted from antibody specific reactions. Purified ConA, SBA, and WGA failed to react with any heterologous antibody. In these instances the purified lectin was not responsible for CRM detected in the crude seed extracts. For example, purified SBA failed to react with any heterologous antisera, while crude soybean seed extracts showed CRM with both Ulex I-Ab and DBA-Ab. These observations suggest that soybean seeds possess a protein that is im-

munologically related to other seed lectins, but distinct from SBA, a point which is presently under investigation. Among the purified lectins that did show cross-reactivity, lens and pea were unique in that they were antigenically identical. Lens and pea lectins also share similar molecular weights, metal ion requirements, amino acid and carbohydrate compositions (11, 12). Lens and pea lectins are also unique in that they possess much greater sequence homologies with each other than with other lectins (3,4). These observations suggest that lens and pea lectins possess very similar structures. This is not too surprising since, botanically, lens and pea are closely related. The other cross-reacting lectins showed a typical spur formation when assayed with Ulex I lectin and Ulex I-Ab. These cross-reacting lectins appear to possess some structural properties in common with the UZex I lectin. Perhaps the most significant result was that both Ulex I-Ab and Bandeiraea-Ab

464

HOWARD,

KINDINGER,

AND SHANNON

FIG. 3. Ouchterlony double diffusion pattern of lens antiserum (0.01 ml) [l] with lens lectin (10 ag in 0.01 ml PBS) [2] and pea lectin (10 pg in 0.01 ml PBS) [3] showing a line of identity.

FIG. 4. Ouchterlony double diffusion pattern of Ulex I antiserum (0.01 ml) [l] with Bauhinia lectin (10 pg in 0.01 ml PBS) [2] and Bandeiraea lectin (10 ag in 0.01 ml PBS) [3] showing a line of identity.

CONSERVATION

OF DETERMINANTS

recognized an identical set of determinants for all of the lectins which cross-reacted with them. This suggests that RCA, DBA, PHA-E, Ulex I, Bandeiraea, Bauhinia, Sophora, and Lotus share common structural features which have been conserved throughout their evolutionary process. These lectins are all glycoproteins containing mannose and glucosamine (13-20). Their amino acid compositions are also similar in that they are rich in acidic and hydroxyl amino acids and low in sulfhydryl amino acids, This raises the question of whether their protein or carbohydrate domains account for the conserved portion of the lectins, or if it is a combination of both. Whatever the case, this highly conserved feature may be required for proper functioning of these proteins and may serve a crucial function for the plant, since every seed extract tested showed CRM. ACKNOWLEDGMENTS We would like to thank Ms. Elena de1 Campillo, Dr. Charles Hankins and Mr. dames Koehn for their helpful discussions. REFERENCES H., AND SHARON, N. (1973) Ann. Rw. Biothem. 42, 541-574. 2. LIENER, I. (1976) Ann. Rez,. Plant. Ph,wiol. 27, 291-319. 1. LIS,

3. FORIERS, A., WCILMART, STROSBERG, D. (1977)

Commun. 75, 980-986.

C., SHARON, N., AND Biochem. Biophys. I&.

AMONG

4. ETZLER,

465

LECTINS

M. E., TALBOT,

C. F., ANI) ZIAYA,

I’. R.

(1977) FEBS Lett. 82, 39-41. 5. GALBRAITH,

W., AND GOLDSTEIN,

I. .J. 11972) Bio-

chemistry 11, 3975-3984. 6. DAVIS, G. J. (19ti4) Ann. h’.Y. Acad. 404-427. 7. HOWARD,

J., AND SHANNON,

L. (1975)

Sci. 121, Anal. Bio-

them. 79 234-239. 8. SHANNON, L., ASD MILLS, E. (1976) Eur. J. Riothem. 63,563-568. 9. OUCHTERLONY. 0. (1948) Acta Pathol. Microhol. Stand. 25, 186. 10. OUCHTERLONY, O., AND Nr~ssos. L. (1973) In Handbook of Experimental Immunology (Weir. D. M., ed.), pp. 19.17-19.20, Blackwell Scientific Publications, Oxford. 11. HOWARD, I., AND SAGE. H. (1969) Biochemistry 8,

2436-2441. 1%. TROWBHIDGE. I. (1974) J. Biol. Chem. 249, 6004-6012. 13. NICOLSON, G., BLAUSTEIN, .J., AND ETZLEK, M. 13, 196-204. (1974) Biochemistry 14. ETZLF.R, M.. AND KARAT, E. (1970) Biochemistry

9,869-87;. 15. MILLER, J. B., Hsv, K., HEINRIKSOX, R., ANI) YACHNIN, S. (1975) Proc. Nat. Acad. Sri. L’S’A 72, 1388-1391. 16. MATSUMOTO, I.. AND OSAWA, T. (1969) Biochim.

Biophys. Acta 194, 180-189. I. (1974) J. Biol. 17. HAYES, C., AND GOLDSTEIN, Chem. 18. IHIMCRA.

249,

1904-1914.

T.. AND OSAWA, T. (1972) Arch. Biothem. Biophys. 151, 4i5-482. 19. PFXEIRA. M., AND KABAT, E. (19i4) Biochemistry 13, 3184-3192. 20. PORETZ, R., RISS, H.. TIMBF.RLAKE, J., AND CHIEh.. W. (1974) Biochemistry 13, 250-256.