A characterization of abrin a from the seeds of the Abrus precatorius plant

397

Biochimica et Biophysica Acta, 667 (1981) 397--410 © Elsevier/North-Holland Biomedical Press

BBA 38624

A CHARACTERIZATION OF ABRIN A FROM THE SEEDS OF THE A B R US PRECA TORIUS PLANT

MARY

S. H E R R M A N N

and W. D A V I D B E H N K E

Department of Biological Chemistry, University of Cincinnati Medical Center, 231 Bethesda Avenue, Cininnati, OH 45267 (U.S.A.) (Received October 29th, 1980)

Key words: A brin A; Carbohydrate content; Amino acid composition; Circular dichroism; (.4 brus precatorius)

Summary Abrin A was purified from the seeds of the Abrus precatorius plant and its physical and biological properties were studied. The biological properties of abrin A were found to be similar to the better studied Abrus protein, abrin C, in that it is toxic to cell-free protein synthesis and binds D-galactose. Abrin A contains carbohydrate moieties including both neutral and amine sugars but no metals, similar to the other two Abrus Pr0teins (abrin C and the Abrus agglutinin). Amino acid compositions of the subunits of abrin A indicated that it consists of two different subunits of comparable size. Furthermore, one of the subunits showed microheterogeneity suggesting that abrin A is a mixture of isolectins. A comparative study of abrin A and abrin C based on compositions and tryptic maps reveals them to be closely related. The evidence suggests that the two abrins may have the same mechanisms of toxic action. Far~ltraviolet circular dichroic studies of abrin A show it to contain 47% ~-pleated sheet and 10% a-helix, again similar to the other two Abrus proteins.

In~oducfion

The seeds of the Abrus precatorius plant contain the lectin abrin which is toxic towards animal cells by inhibiting protein synthesis [1,2]. The toxicity of abrin is higher for certain tumor cells than for normal cells and consequently it qualifies as an anti-tumor agent. Abrin suppresses the growth of solid Ehrlich

Abbreviation: SDS, sodium dodecyl sulfate.

398 ascites tumor and fibrosarcoma in mice and Yoshida sarcoma in rats [3,4]. More recently, it has been demonstrated that abrin has a strong inhibitory effect in nude mice bearing solid human cancers, including fibrosarcoma humori, ovarian carcinoma, and ovarian sarcoma [5]. In addition to abrin, other proteins from A. precatorius have been described. Another lectin, the Abrus agglutinin, is more typical of lectins in general in that it is non-toxic to animal cells and exhibits potent agglutinating activity towards erythrocytes [6,7]. In addition, Wei et al. [8] described two toxic proteins from the seed of A. precatorius. These two proteins, called abrin C and abrin A, have toxic activity comparable to the protein more commonly referred to as abrin. However, these two proteins differed from each other in other respects. Abrin C binds to a Sepharose column and to agglutinated sheep erythrocytes while abrin A did not display either function. In this communication, we examine the chemical and biological activities of abrin A, as little is known concerning this second toxic Abrus protein. Abrin A is then compared to the other two proteins, abrin C and the agglutinin, and possible structural relationships among the three are explored including their circular dichroic properties. Materials and Methods

Materials. Mature seeds of the A. precatorius plant were obtained from Dr. Julia Morton of the Morton Collectanea, University of Miami in Coral Gables, FL. Bio-Gel and chemicals used for polyacrylamide gel electrophoresis were obtained from Bio-Rad Laboratories. Sepharose 4B was obtained from Pharmacia Fine Chemicals, DE52 and CM32 cellulose were purchased from Whatman. Monosaccharides, including glucose-free galactose, w e r e purchased from Sigma Chemical Co. as well as 5,5'
399 tein was chromatographed on CM-cellulose (2.5 X 10 c m column) as described by Wei et al. [ 8 ]. Isotation of subunits. The A and B chains of abrin C were purified according to the method of Olsnes and Pihl [9]. To obtain the subunits of abrin A and the agglutinin, the protein bands were extracted from SDS-polyacrylamide gels. Gels used were 13% in acrylmnide and were poured 3 mm thick and 10 cm high in a Bio-Rad vertical slab Gel apparatus. 300 /~g of protein contained in approx. 0.5 ml of sample buffer (see the following section) were loaded in a continuous band across the top of the gel. Each band was cut out with a razor blade and the excised band was cut into pieces about 1 mm s. The stain was removed from the protein within the gel and the protein eluted from the gel after the method of Gruenstein et al. [10]. The protein was then dialyzed against 30% ethanol followed by dialysis against distilled water and lyophilization. Gel electrophoresis. SDS-polyacrylamide gel electrophoresis was performed by a modification of the method of Laemmli [11]. Deviations from this method included 13% acrylamide in the separating gel and a current of 2 mA per gel. After electrophoresis the gels were stained overnight with 0.25% Coomassie brilliant blue in 9% acetic acid/41% methanol. Gels were then destained electrophoretically for 2 h with 7% acetic acid/5% methanol. Molecular weights were obtained by a comparison of mobilities to proteins of known molecular weights. Analytical disc gel electrophoresis was performed as described by Brewer et al. [12]. CeU-free system. The preparation of anemic rabbits and the purification of the lysate from the rabbit reticulocytes was performed as described by Lingrel [13]. The cell-free system used consisted of 1.0 ml of solution E (see Ref. 13) excluding leucine, 0.4 ml of reticulocyte lysate, 0.4 ml of distilled water and 0.1 ml of a leucine mixture/1.0 ml of cell-free system~ The leucine mixture contained [SH]leucine with a specific activity of 500 Citmol, unlabeUed leucine to a total leucine concentration of 1 mM and 50 mM Tris-HC1, pH 7.4. Routinely, 100 /~1 of the total lystate were added to the protein sample and the mixture incubated at 24°C for 30 min. The procedure used for recovery of acid-precipitable material and for counting of radioactivity incorporated was previously described [14]. Hemagglutination assay. Hemagglutination assays were performed according to the method of Sandvig et al. [15]. Ultracentrifuge studies. Meniscus depletion sedimentation equilibrium was performed using a Beckman Model E analytical ultracentifuge according to the method of Yphantis [16]. The buffer used was 30 mM NaCI, 10 mM Tris, pH 7.7. Sedimentation velocity experiments were also performed using a Beckman Model E ultracentrifuge. For these studies the temperature was maintained at 20°C and buffer used was 100 mM NaC1, 10 mM Tris, pH 7.7, for abrin C and the agglutinin and 100 mM NaC1, 10 mM sodium acetate, pH 6.0, for abrin A. Each protein was centrifuged at four different concentrations which were run concurrently in order to minimize experimental error. The sedimentation coefficients were calculated according to Schachman [17] and the observed s value

400 S0 was converted to standard conditions (20.w). Metal and carbohydrate analyses. Metal analyses were performed by Kettering Laboratories (Cincinnati, OH) using atomic absorption techniques. All three proteins were analyzed for the presence of 28 metals among which are those that most commonly occur in proteins. In order to determine neutral sugar content 1 mg of protein was placed in 0.5 rnl of 1 M HC1 in sealed tubes and hydrolyzed for 4 h at l l 0 ° C . Neutral sugar released was determined by the m e t h o d of Roe [18] using mannose as a standard. Total hexosamine was determined using the Elson-Morgan reaction. A protein sample of 10 mg was placed in 2 ml of 2 M HC1 and hydrolyzed for 15 h at 110°C. Hexosamine released was isolated on a Dowex 50 column as described elsewhere [19]. Hexosamine was qantitated using glucosamine as a standard as described by Boas [19]. Amino acid composition. A m i n o a c i d analyses were performed on a Durrum Model D-500 amino acid analyzer. Protein samples were hydrolyzed at 110°C in 6 M HC1 using sealed, evacuated ampules for 20 h. Values reported for serine and threonine are corrected for 10 and 5% destruction, respectively. Cysteine and methionine were determined as cysteic acid and methionine suifone, respectively, according to the m e t h o d of Spackman et al. [20]. Tryptophan content was determined by three different procedures. (1) Protein in 0.1 M acetic acid/8 M urea, pH 3.9, was titrated with N-bromosuccinimide and the amount of oxidized tryptophan determined spectroscopically [21]. (2) Protein was incubated for 30 rain in 6 M guanidine hyctrochloride, pH 7.0, containing 2-mercaptoethanol. The fluorescence emission of the resulting solution was then measured at 345 nm (excitation wavelength, 295 nm) using a Baird-Atomic spectrophotometer model SF-100 and tryptophan was quantitated as previously described [22]. (3) Protein was reacted with excess 2-hydroxy~5-nitrobenzyl bromide for 2 h [23] and modified residues were quantitated spectroscopically as previously described [24]. Total protein sulfhydryl content was determined using 5,5'~iithiobis(2-nitrobenzoic acid) as previously described [25]. Carbohydrate~stained gels. SDS-polyacrylamide gel electrophoresis was performed as before using 13% acrylamide. The gels were treated with Schiff's reagent as previously described [ 26 ]. Tryptic maps. Protein used for tryptic cleavage had been previously reduced and carhoxymethylated as described by Crestfield et al. [27]. The modified protein was added to 0.2 M NH4HCO3 and 2% (w/w) trypsin. Cleavage was allowed to proceed for 18 h at 37°C. The peptides were transferred to a cellulose chromagTam sheet (20 × 20 cm) and subjected to electrophoresis for 90 rain at 220 V using a buffer consisting of pyridine/acetic acid/water (25 : 1 : 225, v/v). This was followed by ascending chromatography in butanol/acetic acid/pyridine/water (15 : 3 : 10 : 12, v/v). The peptides were visualized by spraying the plate with fluorescamine (10 rag/100 ml acetone) and illuminating the fluorescent spots with an ultraviolet lamp. Isoeletric focusing gels. Isoeletric focusing gels were performed by a modification of the procedure of O'Farrell [28]. Each protein was run twice, once at each of the different pH ranges. To produce a pH profde of 5--7 in the gels,

401 80% of the ampholine solution was pH 5--7 ampholines a n d 20% was pH 3--10: to produce a pH profile of 6--8, 50% of the ampholines solution was pH 5--7 and 50% was pH 6--8. All samples were run twice, once using each combination of ampholines. This provided a pH range from 4.8 to 8.0. Following the prefocusing, the liquid on the top of the gel was removed and 20 ~1 (3/~g of protein) of sample was added. This was overlayered with 10/~1 of solution B (see Ref. 31) and then 0.02 M NaOH was added to fill the tube. The chamber was refilled and electrophoresis was carried out for 16 h at 400 V. To obtain a pH profile, three gels were cut every 0.5 cm and each section placed in a vial with 2 ml of deaerated, distilled water. The vials were capped and stirred overnight. The pH of the solution in each vial was measured resulting in a pH profile. The gels containing protein were fixed and stained as previously described [28]. Equilibrium dialysis. 1 ml of abrin A (5 mg/ml) dissolved in 0.01 M sodium acetate, pH 6.2, was placed in a dialysis bag. The experiment was performed with the bag in 25 ml of the same buffer and then adding varying amounts of [14C]gahctose. Dialysis was carried out for 18 h at 22°C. At the end of that time, aliquots were taken from the inside and outside of the bag for counting. The data were analyzed according to the m e t h o d of Scatchard [29]. Absorption spectra and extinction coefficients. Protein was routinely quantirated by measuring its absorption at 280 nm on a Cary Model 15 and using the appropriate extinction coefficient. The extinction coefficients (E~%cm,280 rim) used for the three Abrus proteins were 12.9 for abrin C [8], 14.6 for the agglutinin [30], and 11.8 for abrin A [8] as obtained elsewhere. Extinction coefficients used for the A and B chains of Abrin C were 7.87 and 18.2, respectively, as earlier described [30]. Circular dichroic studies. CD spectra were obtained at ambient temperature (22°C) with a Cary 61 spectropolarimeter. Standarization of the spectropolarimeter was accomplished using a 1 mg/ml aqueous solution of d-10-camphorsuifonic acid as specified by Varian Associates. All far-ultraviolet CD spectra were recorded in strain-free quartz cylindrical cells of 5 m m pathlength using concentrations of no more than 100/~g/ml. The CD spectra were analyzed for their content of the three structural components as previously described [31]. Results

Chemical and biological properties The size and purity of abrin A were examined by several different techniques. (1) Polyacrylamide electrophoresis was performed both at pH 4.3 and pH 8.3 (data n o t shown). At t h e low pH a single, narrow band resulted while one broad band resulted at the high pH. (2) Polyacrylamide gel electrophoresis with 1% SDS results appear in Fig. 1. Abrin A without 2-mercaptoethanol revealed one band at 70 000 daltons. Inclusion of 2-mercaptoethanol resulted in three bands which decreased in intensity with increasing mobility. The Mr of these subunits were found to be 35 500, 32 500 and 31 000. SDS-polyacryl-

402

I 2 5 4 56 7 8 Fig. 1. SDS-polyacrylamide gels of purified abrus proteins. SDS-polyacrylamide gel electrophoresls, using 139b acrylamide, was performed as described in Materials and Methods. Gel 1 contained three proteins of k n o w n molecular weight: bovine serum albumin (67000), ovalbumin ( 4 5 0 0 0 ) a n d carbonic anhydrase (S0 000). Abrin A without 2-marcaptoethanol in the sample buffer is shown in gel 2. Gel 8 is abrin C with 2-mercaptoethanol, while gels 4 and 5 are purified the B and A chain of abrin C0 respectively. Agglutinin without and with 2-mel~'.aptoethanol is s h o w n in gels 6 and 7, respectively. Gel 8 is abrin A with 2-mercaptoethanol. Tracking dye is indicated by a thin line.

amide gel electrophoresis of the two other Abrus proteins (abrin C and the agglutinin) are included in Fig. 1 for comparison. (3) The Mr of native abrin A as determined by high,speed sedimentation equilibrium was 63 500. A plot of the logarithm of concentration vs. r 2 was linear, demonstrating the presence of a single, homogeneous, sedimenting species. (4) The sedimentation rate of abrin A was determined through a series of velocity experiments at varying concentrations and extrapolation to zero concentration. The s°0, w value thus determined was 4.88 and the photographs showed single, symmetric peaks indicating homogeneity. A summary of these data appears in Table I along with comparable data for abrin C and the abrus agglutinin.

Biological activities The toxicity of abrin A was determined by measuring inhibition of protein synthesis in a cell-free system. The index of toxicity was arbitrarily chosen as the least amount of protein which resulted in 75% inhibition of protein synthesis. This level was i.0 /~g/ml for abrin A as compared to 0.5/~g/ml for abrin C. A b r i n A showed no agglutinating ability for human red blood cells. The sugar binding of abrin C and the agglutinin has been shown to be specific for v~galactose [6]. The aff~mity of abrin A for D~alactose was deter-

403

TABLE I SIZE OF THE ABRUS PROTEINS Protein

SDS-polyac~ylamide gel e l e c t r o p h o r e s i s

Sedimentation equilibrium (daltons)

sOo,w

Abrin C A chain B chain

70 000 29 0 0 0 36 0 0 0

65 500

4.76

Abrin A Subunit TI T2 T3

70 000

62 500

4.88

Agglutinin Dimer 1 2 Subunit A1 A2 A3

--

134 900

7.25

35 500 32 500 31 00O

72000 7 0 O00 39 000 37 0 0 0 32 000

mined using equilibrium d~lysis. An initial experiment showed that abrin A did indeed bind galactose, and therefore equilibrium dialysis was performed at several different abrin A concentrations and the data were analyzed according to Scatchard [29]. The resulting Scatchard plot is shown in Fig. 2. The data result in a straight line with a slope equal to 7900 M -1. From the data, it was also deduced that abrin A has 0.45 galactose binding sites per molecule. Carbohydrate content Previous reports [30] indicate that abrin C and the agglutinin are glycopro4.0

_ .3o

It.o

I~

.Lo,,..,oo

•

i.o

I 0,1

I 0.2

I 0.3

0.4

f

Fig. 2. S c a t e h a r d p l o t o f g a l a c t o ~ b i n d i n g t o a b r i n A . V a r y i n g m o u n t s o f [ 1 4 C ] g a l a e t o s e w e r e r e a c t e d w i t h a b r i n A u s i n g e q u l l t b r i u m dialys£s a n d a S c a t c h a r d p l o t p n e r a t e d (see M a t e r i a l s a n d M e t h o d s ) . r, t h e a m o u n t o f p r o t e i n w i t h s u ~ b o u n d d i v i d e d b y t h e a m o u n t o f u n b o u n d p r o t e i n . T h e b u f f e r u s e d was 0 . 0 1 M s o d i u m a c e t a t e , p H 6.2. T h e a s s o c i a t i o n c o n s t a n t d e t e r m i n e d f o r m t h e s l o p e was 7 9 0 0 M - I .

404

I0

2 b ...........

3b

Fig. 3. SDS-polyaczy/am/de gel electzophoresls of the subun/ts of the Abrus proteins stained for carbohydrate. SDS-polyacrylamide gel eleetrophoresls of the Abrus prot e i ns was p e r f o r m e d as described in Fig. 1 with 2-mercaptoethanol included in the sample buffer. Gels m a r k e d (a) were stained for prot e i n as described in Materials and Methods (Gel electrophoresls section) and the gels m a r k e d (b) were stained for car bohydrate as described in Mater/a~ and Methods ( C e r b o h y d z a t e - s t a ~ e d gels section). Gels I , abrin ~ ; gels 2, abrin A, an d geld 3, agglutinin. Tracking dye is indicat e d by a t hi n line.

teins. Therefore, the amount of total neutral sugar and total amine sugar in abrin A was determined and found to be 5 and 0.5%, respectively. In order to ascertain which subunits contain t h e carbohydrate, abrin A was rerun on SDS-polyacrylamide gels as before, but this time stained for carbohydrate rather than protein. The results, shown in Fig. 3, indicate that abrin A contains carbohydrate in all three subunits. Also shown is the agglutinin which was also positive for carbohydrate in all three subunits and abrin C which contained carbohydrate in the B chain only. Metal c o n t e n t

No metals were found in any of the three Abms proteins except calcium which waspresent in less than one molar equivalent (0.17 mol/mol of abrins C and A and 0.33 mol/mol of agglutinin) and as such, t h e activity was unaffected by EDTA.

405

Amino acid compositions The amino acid composition of abrin A is given inTable II. The values are given in residues per protein molecule whose molecular weight has been adjusted to exclude carbohydrate. No free sulfhydryl group was found in abrin A and, therefore, all the cysteine residues are involved in disulfide bonds. For comparison the compositions of abrin C and the .agglutinin are also given in Table II. The composition of abrin A was almost equal to that of abrin C if considered on the basis o f equivalent molecular weights. Furhermore, for most amino acids, the agglutinin contains approximately twice as many residues as either o f the abrins. The similarity of the amino acid compositions raises the possibility that one or more of the subunits in abrin A may be common with those within the other two Abrus proteins. To examine this possibility, the amino acid compositions of the subunits were determined. The subunits o f abrin A and the agglutinin were purified by extracting the bands from SDS-polyacrylamide slab gels as described in Materials and Methods. This isolation procedure allowed for the quantitation of most of the amino acids within the subunits. The subunits of abrin C were isolated as previously described [9]. Amino acid compositions of the abrin A subunits are given in Table III. The upper band, as it appears in the gel, is referred to as subunit T1, the middle band as subunit T2, and the bottom band as subunit T3 (T is used to indicate toxin as opposed to A for agglutinin, see below). The corn-

T A B L E II AMINO ACID COMPOSITIONS OF THE ABRUS PROTEINS

A m i n o acid Asp Thr Set Glu Pro CIy

Ala Val lie Leu TyT Phe His Lys Arg Cys * Met Trp-Nbs * * Fluor * * Hnbb ** M r based o n

Abzin A

Aulutinin

74.0 44.1 57.1 58.1 23.0 42.9 32.9 28.6

69,5 38.7 51.4 53.8 21.2 37.8 86.5 29.9

31.6

29.4

41.8 20.4

19.6

130.8 78.5 107.3 117.8 45.5 68.1 76.5 71.5 54.9 88.8 39.4 42.5 12.9

Abrin C

40.9

16.6 9.8 21.1 30.8

8,7 18.5 28.8

9.8

10.9

16.4

38.1 49.2 21.7

13.0

11.6

29.0

10.8 10.2

10.5 11.0

27.5 26.7 27.4

63 800

59 4 0 0

124 200

* Cys, half-cystine. ** These abx~eviations indicate three different procedures used for the d e t e r m i n a t i o n of t : y p t o p h a n (see Materials and Methods): Nbs, N - b r o m o s u c c i n i m i d e ; Finor, fluorescence, and Hnbb, 2 - h y d r o x y - 5 - n i t z o -

b e n z y l bromide.

406 T A B L E IIl AMINO ACID COMPOSITIONS OF ABRINS C AND A SUBUNITS Amino ac/d

Abrin C A chain

Asp Thr Set Glu Pro GIy *

Abr/n A subunlt B chaln

Val

31.2 19.7 22.5 33.1 15.1 15.6 18.2 13.5

14.3

He

15.6

16.9

Leu Tyr Phe His LFs Ax'g C y s ** Met T r p - N b s * ** F l u o r ***

21.3 8.7 11.0 6.0 2.1 17.4 1.0 2.0 2.09 1.96

20.9 13.2 4.5 3.4 16.6 11.0 9.0 12.0 8.75 9.23

Ala

Mr based on

29 000

T1

T2

T3

42.9 23.3 30.5 28.7 11.0 26.5

29.8 14.7 22.6 24.1 9.4 --

--

--

15.5

15.7

19.9

13.5

20.3 14.8

14.3

-I 2 . 4

13.6

13.0

18.8 9.8 5.1 4.8 14.2 11.7 -7.7

19.9 8.8 8.9 3.6 5.7 16.8 -4.0

19.6 8.6 9.7 3.8 5.1 16.9 -3.5

34 800

25 000

24.6 15.2 21.3 27.7 9.4

25 O00

24.7 14.9 20.9 28.8 10.4

25 000

* V a l u e s f o u n d f o r g l y c i n e w e r e c o n s i s t e n t l y h i g h f o r t h e s u b u n i t s e x t r a c t e d f r o m gels d u e t o c o n t a m ination from the electrophoresis buffer. ** C y s , h a l f - c y s t i n e . *** See f o o t n o t e s t o T a b l e If.

position of subunit T1 is very different from those of T2 and T3 and also from the parent molecule. Since some of the amino acids are higher in subunits T2 and T3 (Ala, Phe, Arg) than in the parent molecule, it is very improbable that subunits T2 and T3 are proteolytic degradation products of subunit T1, as was previously suggested [8]. Furthermore, the compositions of T2 and T3 are virtually the same. It appears that each abrin A molecule contains one subunit T1 and one of either T2 or T3. This implies that there are two different abrin A molecules varying by about 1000 in molecular weight. The amino acid compositions of the abrin A subunits proved interesting from another aspect. The composition of subunit T1 was similar to that of the B chain of abrin C, while subunits T2 and T3 had compositions similar to the A chain of abrin C (see Table II). This is a further indication that there are one or two common subunits within these two toxins. Amino acid compositions of the agglutinin subunits appear in Table IV. Here, the upper band is referred t o a s subunit A1, and the middle as subunit A2 and the bottom as subunit A3. In this case, subunits A1 and A2 are the same and are different from subunit A3. Again, there appear to be two different subunits based on compositions. The compositions of the two aggiutinin dimers were also determined after their extraction from gels, and these are given in Table IV. The two compositions were found to be virtually equal to each other. In addition, they are intermediate between the two different subunit compositions and are very similar to that of the whole protein. This suggests that there

407

TABLE IV AMINO ACID COMPOSITIONS OF AGGLUTININ

SUBUNITS AND DIMERS

G l y e i n e is n o t r e p o r t e d b e c a u s e t h e s e v a l u e s w e r e consistently high due to contnmtnmtion f r o m the electrophore~ buffer. Amino acid

Subunlt A

Asp Thr Set Glu Pro Ala Val Met Ile Leu Tyr Phe His Lys Arg Mr based on

Dimer A2

29.1 13.7 23.7 30.6 12.1 16.6 14.3 6.3 13.0 19.6 S.7 6.6 2.3 11.7 9.0 25 000

A3 28.0 13.3 24.0 32.5 13.2 17.9 12.1 5.4 12.4 19.6 7.8 6.8 2.7 12.2 9.0

25 000

1 30.9 1 S.1 30.0 22.3 13.9 22.9 17.5 2.5 14.7 24.0 7.9 II.8 5.0 10.6 10.7

25 000

2 55.2 30.8 49.6 48.9 19.5 36.4 33.5 13.4 24.2 38.6 18.0 16.4 6.7 19.9 23.5

50 000

56.0 32.2 46.3 46.4 19.1 36.4 35.0 14.7 25.0 39.6 17.9 16.2 5.8 18.9 24.7 50 000

are two agglutinin dimers, each held together by disulfide bonds, one which contains subunits A1 and A3, and one with subunits A2 and A3. If this is so, then the agglutinin tetramer probably contains one of each dimer.

Tryptic maps ~ In ol~lerto furthercompare the primary structureof the three proteins,they were digested with trypsin and the peptides mapped by two
408

with a minimum occurring at 210 nm. This type of spectrum is typical of proteins whose secondary structure is primarily ~-pleated sheet [32]. This was confirmed and quantitated using the data compiled by Chen et al. [32]. The spectrum of abrin A was applied to their standard protein data using a best-fit computer program and the computer-fitted curve was in good agreement with the experimental values. This revealed 47% B-pleated sheet and 10% a-helix. The CD spectrum of abrin A was compared to that of the other two Abrus proteins. The spectrum of the Abrus agglutinin previously reported [31] indicated the same secondary structure (48% B-pleated sheet and 10% a-helix). That of abrin C was also very similar (data not shown). In this spectrum there is a small maximum at 233 nm and a minimum occurring at 205 nm. The secondary structure content of abrin C was found to be 40% ~-pleated sheet and 10% a-helix. Because the A chain o f abrin C can be purified in an active conformation, possessing extremely high toxicity [30], its far-ultraviolet CD spectrum was also studied. The far-ultraviolet CD spectrum of the A chain is somewhat like that of abrin C except from 245 to 230 nm where the A chain spectrum is negative instead of positive. The best-fit curve is an excellent fit down to 215 nm and revealed 25% ~-pleated sheet and 21% a-helix. This means that all the a-helix present in the parent molecule is contained within the A chain, unless the A chain undergoes a dramatic conformational change when removed from the B chain. Also, the A chain contains approx, one-third of the B-pleated sheet in abrin C, the rest coming from the B chain. Discussion This work has greatly expanded the information on an almost unknown prorein, abrin A, and has compared it to and differentiated it from the other two more c o m m o n Abrus proteins, abrin C and the Abrus agglutinin. There was good agreement with the only previous report on abrin A [8] on molecular weight, amino acid composition and subunit pattern on SDS-polyacrylamide gel electrophoresis. However, the somewhat unusual subunit pattern was interpreted differently in this earlierwork [8]. It was suggested that the two smaller components are due to proteolytic degradation occurring during the preparations and that abrin A is actually a dimer of one subunit type. In contrast, our results suggest that abrin A is a mixture of isolectinshaving subunit structures ab, a'b, and a"b where a subunit m a y differ from each other only in carbohydrate content. Abrin A has a similar sugar-binding site and m a y further indicate that abrin A has the same mechanism of toxic action as abrin C [35]. The finding of 0.45 galactose binding sites per molecule is unusual. It m a y be that one or two of the abrin A isolectins hck sugar-binding activity perhaps due to additional carbohydrate moieties.. These could interfere sterically or cause a conformational change in the protein. If abrin A has the same mechanism of action as abrin C, then any molecular species lacking sugar binding would also lack toxicity. Abrin A showed one-half the toxicity of abrin C in a cell-free system. The toxicity of abrin A in a cell-free system had not been previously reported, but Wei et al. [8] studying LDs0 values in mice also found it to be slightlyless toxic

409 than abrin C. The fact that abrin A inhibited cellular protein synthesis further supports the possibility that abrin A has the same mechanism of toxic action as abrin C. In addition to the c o m m o n biological properties between abrin A and abrin C, abrin A shares many physical properties with the other two Abrus proteins. All three were shown to be glycoproteins but not metalloproteins. They have similar subunit sizes, secondary structures and also similar near-ultraviolet circular dichroic spectral properties [36]. Furthermore, the amino acid compositions were closely related, especially with respect to the two abrins. These similarities bring up the possibility that one or more of the proteins have comm o n subunits. The existence of c o m m o n subunits between the agglutinin and either of the abrins is extremely unlikely. First, the amino acid composition of the abrin A subunits as well as the A chain of abrin C differed significantly from that of any of the agglutinin subunits. Secondly, the results of tryptic digestion and mapping of the three proteins demonstrated only a few c o m m o n fragments between the agglutinin and the other two proteins. On the other hand, there is good evidence that the two abrins share at least one subunit. The N-terminal sequence of the A chain of abrin C appears to be also present in abrin A (data n o t presented). In addition, both subunits in the two proteins had very similar amino acid compositions and tryptic maps. The data support the possibility that the two abrins have identical polypeptide chains, the only difference in the two being additional carbohydrate moieties added to the 'A chain' of abrin A. The extra sugar could explain the increased isoelectric point, the decreased sugar affinity, and the decreased toxicity of abrin A as compared to abrin C. Many other plants also contain more than one lectin having almost identical properties, including lectins from Ricinus communis (discussed earlier), soy. bean [37], garden pea [38], wheat germ [39]. Lotus tetragonolobus [40], lentil [41], and kidney bean [42]. This p h e n o m e n o n is n o t limited to glycoproteins as the pea, lentil and wheat germ agglutinins do not contain carbohydrate moieties. Circular dichroic studies of the three Abrus proteins in the far-ultraviolet indicated very similar secondary structures, all containing primarily ~-pleated sheet. All the a-helix of the parent molecule was present in the A chain assuming there is n o t a gross conformational change when the subunits are separated. This may indicate that a-helix is required for the toxic action of the A chain. Based on their far-ultraviolet CD, lectins have been grouped into three classes [31]. The spectrum of abrin A would place it in class II along with the other two A b m s proteins and the Ricinus agglutinin. Thus far, all lectins with toxic activity whose far-ultraviolet CD spectra are known are grouped in this class with a exclusion of all non-toxic lectins. Perhaps this type of spectrum is indicative of a lectin with toxic activity. References 1 IAn, J.Y., Kao, W.Y., Tserng, K.Y., C h e n , C.C. and T u n g , T.C. (1970) Cancer Res. 30, 2 4 3 1 - - 2 4 3 3 2 Benson, S., Ohmes, S., Pibl, A., Skorve, J. a n d A b r a h a m , A.K. (1975) Ettr. J. Biochem. 5 9 , 5 7 3 - - 5 8 0

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