Lectins in the hemolymph of Japanese horseshoe crab, Tachypleus tridentatus

71

Biochimica et Biophysica Acta, 500 (1977) 71--79 © Elsevier/North-Holland Biomedical Press

BBA 28357 LECTINS IN THE HEMOLYMPH OF JAPANESE HORSESHOE CRAB, TA CH Y PL E US TR I D E N T A TUS

S A T O R U SHIMIZU, MIE ITO and M A K O T O NIWA a

Laboratory o f Ultrastructure Research, Aichi Cancer Center Research Institute, Nagoya 464, and a Department o f Bacteriology, Osaka City University Medical School, Osaka 545 (Japan)

(Received April 5th, 1977)

Summary The h e m o l y m p h of the Japanese horseshoe crab, Tachypleus tridentatus contains lectins which agglutinate mammalian erythrocytes. Affinity chromatographic purification of the lectins using bovine submaxillary gland mucin-conjugated Sepharose resulted in the separation of the lectins into four fractions; one major and three minor lectins. Protein subunits revealed by polyacrylamide gel electrophoresis and the immunoprecipitin line of these lectins against antiserum to crude lectins were unique to each fraction. The activities of all the lectins were optimal at pH values between 6 and 8, and were destroyed by heating at 60 °C. Calcium chloride augumented the activities of three lectins, but the major lectin was not influenced by the salt. Bovine erythrocytes were not agglutinated by any of the lectins and comparative agglutination titers for other erythrocytes from various sources were different among these lectins. The activities of all the lectins were inhibited by N-acetylamino sugars. They were more effectively inhibited by glycoproteins which contain sialic acid.

Introduction The body fluid of many invertebrates has been shown to contain lectins that agglutinate erythrocytes from vertebrates, as was first reported by Noguchi in the h e m o l y m p h of the horseshoe crab, Lirnulus p o l y p h e m u s [1]. His crossabsorption study of the h e m o l y m p h with erythrocytes from various cold blooded animals resulted in the removing of a lectin reactive to one type of erythrocyte and the leaving of other lectins which agglutinate another type of

Abbreviations: BSM, bovine submaxillary gland mucin; PSM, porcine submaxillary gland mucin; OSM, ovine submaxillaxy gland mucin; SDS, sodium dodecyl sulphate.

72 e r y t h r o c y t e , suggesting t hat the h e m o l y m p h contained several kinds of lectins of different specificity for e r y t h r o c y t e s . Hall and Rowlands demonstrated biochemically the heterogeneity of lectins in the h e m o l y m p h of the American lobster by purifying two lectins of different specificity [2]. Recently lectins from plants which have strict binding specificity for sugar have been used as a tool in studies of the c a r b o h y d r a t e structure of glycoproteins and their distribution on cell surface [3]. It is of interest in this respect that some invertebrates have been reported to contain lectins reactive to sialic acid [2,4,5], and that a lectin of this specificity has not y e t been found in plants. The h e m o l y m p h of the horseshoe crab contains a lectin with this specificity [4]. Present work is intended to show the heterogeneity of lectins in the hemol y m p h o f the Japanese horseshoe crab, Tachypleus tridentatus and to compare the specificity o f these lectins for sugar with that of the Limulus lectins. Materials and Methods

ttemolymph Male and female T. tridentatus with shell diameter 20--30 cm were obtained from the T a m a n o Marine L a b o r a t o r y of O kayam a University, T am ano 706, Okayama Pref., Japan. Blood was collected from living crabs according to the m e t h o d described by Nakamura et at. [6]. After removing the h e m o c y t e s by centrifugation of 2500 rev./min for 10 min, the clear h e m o l y m p h was pooled and stored at --20°C until use.

Agglutination assay Two-fold serial dilution of the samples was made in 25 pl of Tris-buffered saline (50 mM Tris • HC1, pH 7.0/150 mM NaC1/40 mM CaC12), and 25 pl of a 2% suspension o f hum an O e r y t h r o c y t e s washed 6 times with the saline were added, using a microtiter V-plate (Cooke Engineering Co). The plates were kept at r o o m t e m p e r a t u r e and the agglutination titer was read after I h. One unit o f agglutination activity per ml was expressed as titer -1, which was the reciprocal o f the highest dilution of sample which gave positive agglutination. In the experiments to test inhibition by sugars and glycoproteins, Tris-buffered saline containing an appropriate c o n c e n t r a t i o n of a inhibitor was used to dilute the samples, and a one-step reduction of titer was u n d e r s t o o d as indicating a 50% inhibition o f agglutination. In order to check the effect of divalent cation on agglutination, samples were first dialysed overnight against a large volume of Tris-buffered saline w i t h o u t CaC12, and then diluted with the buffer containing an appropriate c o n c e n t r a t i o n of cation. An increase of titer was taken as indicating the activation of the hemagglutinating activity of the samples. Protein was estimated by the m e t h o d of Lowry et al. [7] using bovine serum albumin as a standard.

Reagent Sugars were obtained from commercial sources at the highest purity. Bovine submaxillary gland mucin (BSM) and fetuin were purchased from Sigma and used w i t h o u t f u r the r purification. Porcine and ovine submaxillary gland mucins

73 (PSM and OSM) were prepared according to the m e t h o d of Katzman and Eylar [8] and Bhargava and Gottschalk [9], respectively. The sialic acid contents of fetuin, BSM, PSM and OSM, measured by the resorcinol method [10], were 0.32, 0.65, 1.31 and 1.34 #mol per mg protein, respectively.

Preparation o f BSM-Sepharose and GalNAc-Sepharose BSM-Sepharose and GalNAc-Sepharose used for purification of lectins were prepared by conjugating BSM to CNBr-activated Sepharose (5 mg per ml gel) and N-acetyl-D-galactosamine to epoxy-activated Sepharose (50 mg per ml gel), respectively, according to the instruction manual " A f f i n i t y C h r o m a t o g r a p h y " of Pharmacia Fine Chemicals.

Preparation of rabbit anti-lectin serum Anti-serum to crude lectins in rabbit was prepared as follows. Saline washed rabbit erythrocytes were incubated with the h e m o l y m p h for 1 h at room temperature. Erythrocytes that had adsorbed lectins were washed thoroughly with Tris-buffered saline to remove h e m o l y m p h , then mixed with Freund complete adjuvand (Difco). They were injected intradermally at 10-day intervals to the rabbit from which erythrocytes had been obtained, for a total of six injections. The rabbit was bled 2 weeks after the last injection. An immunodiffusion test was performed in 1.5% agar gel containing 20 mM N-acetyl-D-glucosamine.

Polyacrylamide gel electrophoresis Polyacrylamide gel electrophoresis in the presence and the absence of sodium dodecyl sulphate (SDS, B.D.H. Chemicals) and staining of proteins in gel with Coomassie blue were performed by the m e t h o d of Fairbanks et al. [11], except that the concentration of SDS was lowered to 0.1%. Results

Hemagglutinating activity o f the hemolymph and its inhibition by sugars Table I shows the agglutination titer of the h e m o l y m p h from T. tridentatus and its inhibition by several sugars. Hemolymph itself gave a titer of from 2048 to 8192 for human O erythrocytes, and its activity was inhibited by N-acetylneuraminic acid, N-acetyl-D-glucosamine and N-acetyl-D-galactosamine. Other sugars tested had no effect on the hemagglutinating activity of the hemolymph. TABLE I THE HEMAGGLUTINATING ACTIVITY FOR HUMAN O ERYTHROCYTES OF THE HEMOLYMPH F R O M T. T R I D E N T A T U S AND THE EFFECT OF SUGARS ON THE ACTIVITY F o r t i t e r - 1 d e f i n i t i o n see M a t e r i a l s a n d M e t h o d s . Sugar added

mM

T i t e r -1

-D-Glucose D-Galactose D-Mannose L-Fucose D-Glucosamine D-Galactosamine N- A c e t y l - D - g l u c o s a m i n e N- A c e t y l - D - g a l a c t o s a m i n e N-Acetylneuraminic acid

-50 50 50 50 50 50 50 50 25

8192 16384 8192 8192 16384 8192 8192 256 256 64

74

Purification of lectins T h e lectins in the h e m o l y m p h were purified by affinity c h r o m a t o g r a p h y on BSM-Sepharose. 50 ml o f the h e m o l y m p h were added to 30 ml o f BSMS e p h a r o s e s u s p e n d e d in 50 ml o f T r i s - b u f f e r e d saline. T h e m i x t u r e was stirred in a cold r o o m for 30 rain. Over 90% of the activity was a d s o r b e d to BSMSepharose b y this t r e a t m e n t . T h e BSM-Sepharose was separated, washed five times with 100 ml o f T r i s - b u f f e r e d saline and p o u r e d into a c o l u m n (1.5 × 17 cm). Lectins were e l u t e d f r o m the c o l u m n at r o o m t e m p e r a t u r e , first with 0.1 M NaC1 in 0.1 M s o d i u m b o r a t e (pH 8.5), t h e n with 50 mM N-acetyl-Dglucosamine in Tris-buffered saline and finally with 1.0 M NaCI in 50 mM Tris • HC1 (pH 7.0) (Fig. 1). F o u r hemagglutinating activities were separated by these elution p r o c e d u r e s (M1, M2, M3 and M4). Each f r a c t i o n was pooled, concent r a t e d and dialysed against Tris-buffered saline. T h e lectin in the M3 fraction was f u r t h e r purified b y c h r o m a t o g r a p h y on a G a l N A c - S e p h a r o s e column. A l m o s t all t h e activity o f the M3 f r a c t i o n was a d s o r b e d o n t o the c o l u m n and e l u t e d with 50 mM N-acetyl-D-glucosamine (Fig. 2). A b o u t 1 2 0 0 - f o l d purification was o b t a i n e d in the M3 lectin in which 10--30% o f the activity in the h e m o l y m p h was r e c o v e r e d with the specific activity o f 37 000. Increase in the specific activity was also significant in the o t h e r lectins, b u t the yield o f the activity as a w h o l e was n o t so high. T y p i c a l result o f p u r i f i c a t i o n is shown in Table II. T h e p o l y a c r y l a m i d e gel e l e c t r o p h o r e t o g r a m o f intact lectins is shown in Fig. 3A. M1 gave several bands and M2 had one sharp band. M3 and M4 gave one and t w o bands, respectively. H o w e v e r , a large p o r t i o n o f the p r o t e i n s in M3 and M4 r e m a i n e d on top of the gel, and only a small p o r t i o n e n t e r e d into the gel, suggesting t h a t the large m o l e c u l a r weight c o m p o n e n t s were c o n t a i n e d in i n t a c t M3 and M4. The p r o t e i n s u b u n i t revealed by SDS p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s is u n i q u e to each fraction (Fig. 3B). M2 and M3 gave one broad band, b u t M1 and M4 had several bands. T h e m o l e c u l a r weight o f the p r o t e i n s u b u n i t o f M2 and M3 e s t i m a t e d f r o m their m o b i l i t y in the gel was

3000

!!M3

M'I

2OOO,

0.6

i

E c o

?

1000

0.4

<

M2

20

40 Fraction

B |

; ",

60 Number

C

/

60

M4

100

Fig. 1. P u r i f i c a t i o n o f Tachyple~ts l e c t i n s o n a B S M - S e p h a r o s e c o l u m n . F r o m B S M - S e p h a r o s e t h a t w a s t r e a t e d w i t h t h e h c m o l y m p h (see t h e t e x t ) , l e c t i n s w e r e c l u t e d b y t h r e e b u f f e r s y s t e m s (A, B a n d C). C h a n g e s i n e l u e n t are i n d i c a t e d b y t h e a r r o w s . 2 - m l f r a c t i o n s w e r e c o l l e c t e d . A l l f r a c t i o n s w e r e m o n i t o r e d for absorbance at 280 nm ( ) and agglutinating activity ( ........ ,), B u f f e r s y s t e m A , 0 . 1 M N a C l i n 0 . 1 M s o d i u m b o r a t e ( p H 8 . 5 ) ; B, 5 0 m M N - a c e t y l - D - g l u c o s a m i n e i n T r i s - b u f f e r e d s a l i n e ; C, 1 . 0 M NaC1 i n 5 0 m M T r i s - HC1 ( p H 7 . 0 ) .

75

0.6

'~

5000

""

3000 ~,,

0.4

T

0.2

ooo

10 20 Fraction

30 40 Number

F i g . 2. Purification of M 3 lectin on a G a l N A c - S e p h a r o s e c o l u m n . column of GalNAc-Sepharose (0.7 X 12 cm) equilibrated with mM N-acetyl-D-glucosamine in t h e b u f f e r . A c h a n g e in e l u e n t were collected. All f r a c t i o n s w e r e m o n i t o r e d for a b s o r b a n c e activity (: ....... ).

0 . 2 m g o f M 3 ]ectin was passed through a Tris-buffered saline, and eluted with 50 is i n d i c a t e d b y t h e a r r o w . 1 - m l f r a c t i o n s at 2 8 0 n m ('~ ) and agglutinating

20 000 and 23 0 0 0 , respectively. Each fraction gave its own precipitin line against antiserum to lectins in immunodiffusion gel, except M2 which had no precipitin line (Fig. 4). M1 gave two broad lines, and both M3 and M4 had one sharp line. These precipitin lines did not fuse with each other, indicating that the lectins in each fraction had different antigenicities.

Properties of lectins M1 and M2 had optimal pH of hemagglutination at 7, M3 at between 6 and 8, M4 at between 6 and 9. Over 80% of the activities was lost when the fractions were heated at 60°C for 10 min. The rate of inactivation was not the same among these fractions: M1 was the most stable; no inactivation was observed in this fraction for 5 min. Heating at 50°C for 20 min did not inactivate any of the lectins. Calcium chloride activated the lectins in M1, M2 and M4 fractions, but did not effect the activity of the M3 lectin. The concentration of CaC12 resulting in maximum activation was from 20 to 100 mM for M2, and from 40 to 100 mM for M1 and M4. Magnesium chloride had n o effect on the activities of any of the lectins. Table III shows the comparative agglutination titers of each lectin for erythrocytes from various mammalian species plotted against the titer for human O erythrocytes, taken as 256. Comparative activity TABLE

|I

RESULT

OF PURIFICATION

Fraction

Hemolymph M1 M2 M3 M4

(50 ml)

Protein (mg)

Units

Specific activity (XIO -3 )

Yield (%)

3678 3.35 0.24 0.46 0.31

102 1 3 16

0.03 0.53 16.4 36.7 1.7

100 1.7 3.8 16.5 0.5

400 776 936 869 524

76

B

A M1

M2

M3 M4

M2 M3

M1

M4

i

il

Fig. 3. P o l y a e r y l a m i d e gel e l e c t r o p h o r e s i s o f "I'achypleas l e e t i n s . A, 1 5 p g o f e a c h l e c t i n w e r e l o a d e d o n a 5 . 6 % p o l y a c r y l a m i d e gel ( 0 . 6 X 11 era). E l e e t r o p h o r e s i s w a s p e r f o r m e d a t p i t 8.3 i n t h e a b s e n c e o f S D S . B. E a c h l e c t i n w a s i n c u b a t e d w i t h 4 0 m M d i t h i o t h r e i t o l a n d 0 . 2 5 % S D S a t 37~'C f o r 1 h , a n d l o a d e d o n a 5 . 6 % gel p r e p a r e d a c c o r d i n g t o t h e m e t h o d o f F a i r b a n k s e t al. [ 1 1 ] . E l e c t r o p h o r e s i s w a s p e r f o r m e d at p H 7.4 i n t h e p r e s e n c e o f 0 . 1 % S D S .

of each lectin was distinct. M1 had a relatively high activity for human A and B, and rabbit erythrocytes, and M4 gave a high titer for rat, mouse and horse erythrocytes. M3 agglutinated human erythrocytes more completely, while it did not agglutinate porcine erythrocytes which were agglutinated by the other lectins. Bovine erythrocytes were not agglutinated at all by any of the Tachypleus lectins.

Fig. 4. l m m u n o d i f f u s i o n t e s t o f Tach?,,pleus l e e t i n s . A, r a b i t a n t i - l e c t i n s e r u m ; B, w h o l e h e m o l y m p h ; M1 l e c t i n ; D, M 2 l e c t i n ; E, M3 l e c t i n ; F, M 4 l e c t i n .

C,

77 T A B L E III COMPARATIVE

AGGLUTINATION

TESTS OF TACHYPLEUS

LECTINS

Agglutination titers were assayed under the standard condition using erythrocytes T h e y are p l o t t e d a g a i n s t t h e t i t e r f o r h u m a n 0 e r y t h r o c y t e s , t a k e n as 2 5 6 . Erythxocytes

Human O A B Mouse Rat Rabbit Horse Sheep Pig Cattle

from various sources.

T i t e r -1 M1

M2

M3

M4

256 2048 1024 128 128 512 64 32 256 0

256 512 512 128 128 32 64 32 32 0

256 128 256 64 32 4 16 4 0 0

256 512 256 1024 512 64 512 128 256 0

Inhibition by sugars and glycoproteins The hemagglutinating activities of these lectins were inhibited by the following N-acetylamino sugars and glycoproteins: N-acetylneuraminic acid, N-acetylD-glucosamine, N-acetyl-D-galactosamine, N-acetyl-D-mannosamine, N-acetylmuramic acid, fetuin, BSM, PSM and OSM. The concentration of N-acetylneuraminic acid resulting in the 50% inhibition of agglutination was the same among M1, M2 and M3 (1 mM). A much higher concentration of N-acetylneuraminic acid (30 mM) was required for M4. The other N-acetylamino sugars listed above had a similar inhibiting effect; the concentration required for 50% inhibition was 1--5 mM for M1, M2 and M3 lectins, and 30--50 mM for M4 lectin. D-Glucosamine, D-galactosamine, D-glucose, D-galactose, D-mannose, Lfucose and D-glucuronic acid had no inhibiting effect. BSM was a potent inhibitor of these lectins. In this case, M2, M3 and M4 were inhibited in much the same way, while M1 required a relatively high concentration of BSM for the 50% inhibition. The concentration of BSM resulting in the 50% inhibition was 1 pg per ml for M2, M3 and M4, and 10 pg for M1. Fetuin, PSM and OSM had very much the same effect as BSM upon these lectins. Discussion

The importance of sialic acid on cell surface in the agglutination of erythrocytes by Limulus lectins was first suggested by Cohen who reported the reduction in the agglutinability of human erythrocytes treated with neuraminidase [12], and this was confirmed by Pardoe et al. [4]. Following the isolation of Limulus lectin by Marchalonis and Edelman [13], using starch gel electrophoresis, a variety of procedures were applied to purify the lectin: adsorption and elution from formalin-fixed erythrocytes by Nowak and Barondes [14], DEAE-Sephadex chromatography by Roche and Monsigny [15], and BSMSepharose chromatography by Oppenheim et al. [16] and Roche et al. [17]. These procedures provided highly purified lectin from the h e m o l y m p h of L. polyphemus in good yield, but as to the specificity of the lectin for sugar,

78 results were different among these preparations. The lectin reported by Roche et al. [17] was inhibited by a low c onc e nt r at i on of glycoproteins that contain sialic acid, but free N-acetylneuraminic acid inhibited only with much higher concentration. A high c onc e nt r at i on of N-acetyl-D-glucosamine also inhibited the activity. The lectin preparation by Nowak and Barondes [14] was inhibited by a low co n cent r a t i on of both N-acetylneuraminic acid and D-glucuronic acid. Sela et al. [18] recently reported a simple procedure to isolate lectin from various sources. The lectin obtained from L. polyphemus agglutinated only horse er y th r o cy te s . These results suggest that the h e m o l y m p h of L. polyphemus might contain several kinds of lectins with slightly different specificity. Our results on anot her species of horseshoe crab, Tachypleus tridentatus show that the lectins in the h e m o l y m p h are heterogeneous. Tachypleus lectins, which are inhibited by N-acetylamino sugars, were successfully separated into four fractions by c h r o m a t o g r a p h y on BSM-Sepharose. The first step of elution by sodium borate, which is the application of the m e t h o d used in the purification of neuraminidase by Geisow [19], resulted in the fractionation of two lectins (M1 and M2), and the second step, eluted by N-acetyl-D-glucosamine, fractionated the major lectin of the h e m o l y m p h (M3). The third step of elution was the same as the procedure described by Roche et al. in the purification of Limulus lectin [171. 10--30% of the activity was recovered in the M3 fraction, b u t the overall yield of the activity was not so high, suggesting that some lectins still remained on the BSM-Sepharose column. The protein subunit revealed by polyacrylamide gel electrophoresis and the antigenic properties of each lectin suggest that the proteins in these fraction are unique. These lectins are also unique in their biological properties. M1, M2 and M4 were activated by CaC12, but M3 was n o t influenced by the salt. Relative titers of agglutination for e r y t h r o c y t e s from various sources were different between these lectins. In spite of these biochemical and biological properties of the lectins, an inhibition study by sugars revealed a c o m m o n p r o p e r t y of them. All lectins are not absolutely specific for N-acetylneuraminic acid, but are inhibited by the sugars with the N-acetylamino group and by glycoproteins that contain sialic acid in their c a r b o h y d r a t e chains, although susceptibility of each lectin to these inhibitors varies from lectin to lectin. The concent rat i on of sialic acid bound to the glycoproteins that produced a 50% inhibition of agglutination was very low c o mp ar ed to that of free sialic acid, indicating that the binding site of Tachypleus lectins have a stronger affinity for sugars bound to the proteins than for the monosaccharides. The specificity of M4 lectin which required a high concentration of the monosaccharide for inhibition resembles that of the Limulus lectin reported by Roche et al. [17]. The other three Tachypleus lectins have a completely different specificity for sugar from any of the Limulus lectins reported. A lectin which is specific for N-acetylamino sugars has been reported in the h e m o l y m p h of the American lobster by Hall and Rowlands [2]. It is of interest that these lectins of similar specificity gave a relatively different agglutination titers for e r y t h r o c y t e s from various sources. This might be some reflection of a slight difference in the specificity among these lectins and an elucidation of the difference might prove useful for the structural study of c a r b o h y d r a t e chains in glycoproteins. Bovine e r y t h r o c y t e s that are not agglutinated by any of the Tachypleus lectins and porcine e r y t h r o c y t e s that have no

79 receptor site for M3 lectin might possess some unique structure of N-acetylamino sugars in the carbohydrate chains of cell surface glycoproteins. Further purification and characterization of these lectins is now in progress. Acknowledgments

The authors wish to express their thanks to Drs. M. Yoshida, Tamano Marine Laboratory of Okayama University, and H. Nishii for having assisted in the bleeding of horseshoe crabs, and to Messrs. K. Iki and H. Ohiwa, Nagoya City Meat Inspection Center, for having supplied fresh blood from horses, cattle and pigs, and porcine submaxillary gland. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

N o g u c h i , H. ( 1 9 0 3 ) Z e n t r a l b l . B a k t e r i o l . P a r a s i t e n k d . I n f e k t i o n s k r . H y g . A b t . Orig. 3 4 , 2 8 6 - - 2 8 8 Hall, J . L . a n d R o w l a n d s , J r . , D.T. ( 1 9 7 4 ) B i o c h e m i s t r y 1 3 , 8 2 1 - - 8 2 7 N i c o l s o n , G . L . a n d S i n g e r , S.J. ( 1 9 7 1 ) P r o c . Natl. A c a d . Sci. U.S. 6 8 , 9 4 2 - - 9 4 5 P a r d o e , G . I . , U h l e n b r u c k , G. a n d B i r d , G . W . G . ( 1 9 7 1 ) I m m u n o l o g y 18, 7 3 - - 8 3 K h a l a p , S., T h o m p s o n , T.E. a n d G o l d , E . R . ( 1 9 7 0 ) V o x S a n g . 1 8 , 5 0 1 - - 5 2 6 N a k a m u r a , S., I w a n a g a , S., H a r a d a , T. a n d N i w a , M. ( 1 9 7 6 ) J. B i o c h e m . 8 0 , 1 0 1 1 - - 1 0 2 1 L o w r y , O . H . , R o s e b r o u g h , N . J . , F a r r , A . L . a n d R a n d a l l , R . J . ( 1 9 5 1 ) ,I. Biol. C h e m . 1 9 3 , 2 6 5 - - 2 7 5 Katzman, R.L. and Eylar, E.H. (1966) Arch. Biochcm. Biophys. 117, 623~637 B h a x g a v a , A.S. a n d G o t t s c h a l k , A. ( 1 9 6 6 ) B i o c h i m . B i o p h y s . A c t a 1 2 7 , 2 2 3 - - 2 3 1 S v e n n e r h o l m , L. ( 1 9 5 7 ) B i o c h i m . B i o p h y s . A c t a 2 4 , 6 0 4 - - 6 1 1 F a i r b a n k s , G., S t e c k , T . L . a n d W a l l a c h , D . F . H . ( 1 9 7 1 ) B i o c h e m i s t r y 1 0 , 2 6 0 6 - - 2 6 1 7 C o h e n , E. ( 1 9 6 8 ) T r a n s . N.Y. A c a d . Sci. Ser. II 3 0 , 4 2 7 - - 4 4 3 M a r c h a l o n i s , J.J. a n d E d e l m a n , G.M. ( 1 9 6 8 ) J. Mol. Biol. 3 2 , 4 5 3 - - 4 6 5 N o w a k , T.P. a n d B a r o n d e s , S.H. ( 1 9 7 5 ) B i o c h i m . B i o p h y s . A c t a 3 9 3 , 1 1 5 - - 1 2 3 R o c h e , A.C. a n d M o n s i g n y , M. ( 1 9 7 4 ) B i o c h i m . B i o p h y s . A c t a 3 7 1 , 2 4 2 - - 2 5 4 O p p c n h e i m , J . D . , N a c h b a r , M.S., S a l t o n , M . R . J . a n d Aull, F. ( 1 9 7 4 ) B i o c h e m . B i o p h y s . Res. C o m mun. 58, 1127--1134 17 R o c h e , A.C., S c h a u e r , R. a n d M o n s i g n y , M. ( 1 9 7 5 ) F E B S L e t t . 5 7 , 2 4 5 - - 2 4 9 18 Sela, B . A . , W a n g , J . L . a n d E d e l m a n , G.M. ( 1 9 7 5 ) J. Biol. C h e m . 2 5 0 , 7 5 3 5 - - 7 5 3 8 19 G e i s o w , M.J. ( 1 9 7 5 ) B i o c h e m . J. 1 5 1 , 1 8 1 - - 1 8 3