134 Biochimica et Biophysica Acta 872 (1986) 134-140

Elsevier

BBA32457

T h e amino acid sequence and reactive (inhibitory) site of the major trypsin
isoinhibitor (DE5) isolated from seeds of the Brazilian Carolina tree
( A denanthera pavonina L.)

M. R i c h a r d s o n a,,, F.A.P. C a m p o s b, j. X a v i e r - F i l h o b, M . L . R . M a c e d o b,
G . M . C . M a i a b a n d A. Y a r w o o d a
a Department of Botany, Unioersity of Durham, Science Laboratories, South Roaa~ Durham City Dill 3LE (U.K.) and
b Department of Biochemistry and Molecular Biology, Science Centre, Federal University of Cear,~,
60,000 Fortaleza (CE) (Brazil)

(Received February 17th, 1986)

Key words: Trypsin inhibitor; Amino acid sequence; Reactive site; Legume Kunitz inhibitor; (A. pavonina)

Eight iso-inhibitors of trypsin were isolated from seeds of the Brazifian Carolina tree (Adenantherapavonina
L.) by precipitation with ammonium sulphate, gel filtration, affinity chromato~tphy on enzymatically inert
anhydrotrypsin Sepharose 4B, and separated by ion-exchange chromatography on DEAE-Sepharose. The pl
values of the isoinhibitors 0DEI-DES) ranged from 5.10 to 4.40. Each isoinhibitor had an Mr of approx.
21000 and was composed of a large a chain (M r 16000) and a smaller fl chain (M r 5000) linked together by
a disulphide bond. The complete amino acid sequence of isoinhibitor DE5 (pl 4.75) was deduced by analysis
of peptides and fragments derived from the separated a and fl chains by digestion with trypsin, chyumtryp-
sin, pepsin, thermolysin, the Staphylococcus aureus V8 proteinase and iodosobenzoic acid. The sequence of
the Carolina DE5 isoinhibitor and the location of its reactive (trypsin-inhibitory) peptide bond showed clear
homology with the Kunitz-type proteinase inhibitors from soybean, winged bean and a number of other
legume seeds.

Introduction The partial sequences of the Albizzia [8,9], Acacia

[10.11], Erythrina [12-14] and Peltophorum [15]
Most of the protein inhibitors of serine pro- inhibitors are known, but the only complete
teinases which are of wide-spread occurrence in primary structures of the Kunitz family which
seeds of the family Leguminosae can be classified have been determined are those of the soybean
into two groups, the low ( = 8000) molecular weight (Glycine) [16-18] and the winged bean (Psopho-
cysteine-rich (Bowman-Birk) family, and the high carpus) [19].
( = 20000) molecular weight cysteine-poor Previous workers [20,21] have reported that
(Kunitz) family [1,2]. Although the amino acid seeds of Adenanthera pavonina contain very high
sequences of many examples of the Bowrnan-Birk levels of proteinase inhibitors which are 3-16-times
type have been determined [3-7], in this respect more abundant in this species than in the inhibi-
the Kunitz family has been relatively neglected. tor-rich seeds of soybean. We now report the
complete amino acid sequence of the major tryp-
sin isoinhibitor (DE5) isolated from seeds of the
Brazilian Carolina tree (A. pavonina L., Indian
* To whom correspondence should be addressed. red sandalwood or bead tree or coral tree), which
0167-4838/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
 135

although it has clear sequence homology with the gradient programmer using appropriately selected
single-chain Kunitz inhibitors from soybean and gradients of NaC1 (in the range 0.05-0.25 M) in
winged bean, differs in that it is composed of two 0.02 M Tris-HC1 (pH 7.6).
polypeptide chains linked by a disulphide bond. Assay of trypsin isoinhibitor activity. The inhibi-
tory activities of the native isoinhibitors against
Materials and Methods trypsin were determined by measuring the hydrol-
ysis of a-N-bcnzoyl-oL-ar~nine-p-nitroanilidc
Purification of Carolina isoinhibitors. Seeds of (Sigma) at pH 8 [25]. The concentration of active
A. pavonina were collected from trees growing in trypsin used was estimated by titration of the
Fortaleza in north-east Brazil. After removal of active site with p-nitrophcnyl-p'-guanidino-
the testas the cotyledons (50 g) were milled (20 benzoate HC1 as described in Rcf. 26.
mesh screen) and extracted three or four times Electrophoretic procedures. SDS-polyacrylamide
with 20 ml of acetone until the supernatant was gel electrophoresis in the presence and absence of
colourless. The dried meal was milled again (60 2-mcrcaptoethanol was carried out in 7.5% gels
mesh) and the flour was extracted with 0.1 M [27]. Isoclectric focusing was carried out on 5%
sodium phosphate buffer (pH 7.6) in 1% NaC1 acrylamide gels containing 4% crosslinker and a
(1 : 5, w/v) for 2 h. The extract was centrifuged at 2% (w/v) mixture of the pH 4-6, pH 6-8 and pH
10000 × g for 30 min at 4°C and the supernatant 3.5-10 ampholines ( 6 : 2 : 1 , v/v) as described in
was subjected to fractional precipitation with Rcf. 28.
(NH4)2SO 4. The 40-60% (5.7 g protein) precipi- Separation of two chains of isoinhibitor DE5.
tate was collected and dialyzed against distilled Isoinhibitor DE5 (40 mg) was reduced and S-
water and lyophilized. The protein (300 rag) was carboxymethylated as in Rcf. 29. The reaction
then applied to a column (95 × 2.5 cm) of Sep- mixture was applied directly to a column (1 × 150
hadex G-50 equilibrated with 0.1 M formic acid. cm) of Bio-Gcl P-60 (100-200 mesh) equilibrated
The single peak of anti-tryptic activity obtained in 0.1 M ammonium bicarbonate (pH 8.1) and
was recovered after dialysis against distilled water eluted in the dark. Pcptidcs were located by their
by lyophilization. This material was then applied absorbance at 280 nm and 230 nm.
to an affinity column (1.5 × 10 cm) of enzymati- Sequence determination. Samples (4-10 mg) of
cally inert anhydrotrypsin (dehydroalanine) the two polypcptide chains of the reduced and
[22-24] coupled to CNBr-activated Sepharos¢ 4B S-carboxymcthylated isoinhibitor DE5 were di-
(Pharmacia Ltd.) in 0.1 M phosphate (pH 7.6) gested separately with trypsin, chymotrypsin,
containing 0.3 M NaC1. After washing extensively pepsin, thermolysin and the S. aureus V8 pro-
with the same buffer the trypsin isoinhibitors were tcinasc, as described in Refs. 30 and 31. The
eluted with 0.1 M HC1, containing 0.3 M NaC1, mixtures of peptides produced by these methods
dialyzed against distilled water and lyophilized. were initially fractionated on columns (1 x 200
Separation of isoinhibitors. The mixture of isoin- cm) of Bio-Gcl P-6 in 0.05 M ammonium bi-
hibitors (130 mg) obtained from the anhydrotryp- carbonate as in Ref. 31. Further purification of
sin affinity column was applied to a column 1.5 × peak fractions was achieved by reverse-phase
50 cm) of DEAE-Sepharose CL-6B (Pharmacia HPLC as described in Ref. 32. Cleavage of the a
Ltd.) equilibrated in 0.05 M Tris-HCl (pH 7.6) chain of DE5 with iodosobenzoic acid and sep-
and eluted with a linear gradient of 0.05-0.25 M aration of the resulting fragments was carried out
NaCl in the same buffer (500 ml of each) at a flow as in Rcf. 32.
rate of 40 ml/h. This procedure yielded eight The intact a and fl chains of DE5 and the
major peaks of anti-trypsin activity (DE1-DE8). fragments or pcptidcs derived from them were
After dialysis the pooled material in each peak sequenced using the 4-N,N-dimcthylaminoazo-
was then subjected to rechromatography on the benzcnc-4'-isothiocyanatc (DABITC)/phenyhso-
same column or FPLC on a column (0.5 x 5 cm) thiocyanate double coupling method, the dansyl-
of Mono Q H R 5/5 (Pharmacia) with a Pharmacia Edman procedure, digestion with carboxypepti-
P-500 pumping system controlled by a GP-250 dase A and amino acid analyses all as in Ref. 31.
136

Identification of the reactive (trypsin inhibitory) 1 2 3

site. A sample (12 mg) of the native isoinhibitor
DE5 was subjected to limited hydrolysis with a
catalytic amount (0.3 mg) of trypsin in 0.2 M 3.55
acetic acid containing 0.04 M CaC12 and adjusted
to pH 2.5 with HC1 for 20 h at 37°C. After
reduction and S-carboxymethylation the modified 4.55 m
fragments which resulted were purified by column
-- D E S ( p l 4.75)
chromatography and HPLC as described above.

Results and Discussion 5.13

The trypsin inhibitors present in the extract'

after chromatography on Sephadex G-50 were
separated from the bulk of the seed proteins by
affinity chromatography on enzymatically inactive
anhydrotrypsin-Sepharose 4B. The trypsin inhibi-
tor fraction eluted at pH 2 contained approx. 20%
(w/w) of the seed protein extract. PAGE-IEF
showed the presence of at least eight isoinhibitors 8.30~
of trypsin whose pI values ranged from 5.10 to
4.40 (Fig. 1). Each of the isoinhibitors also in- Fig. 1. Polyacrylamide gel-isoelectric focusing of proteinase
hibited chymotrypsin when tested with a negative isoinhibitors from Carolina seeds. The focusing was carried out
on 5% acrylamide gels containing 4% crosslinker and a 2%
staining method using N-acetyl-DL-phenylalanine- (w/v) mixture of pH 4-6, pH 6-8 and pH 3.5-10 ampholines
naphthyl ester [33]. These proteinase isoinhibitors (6:2:1, v/v). Track 1, mixture of p l standard proteins;
were separated on DEAE-Sepharose CL-6B chro- amyloglucosidase (pl 3.55) soybean trypsin inhibitor (pl 4.55),
matography as shown in Fig. 2 and were des- lactoglobulin (pl 5.13), and sperm whale myoglobin (pl 8.30).
ignated DE1-DE8 according to their order of Track 2, mixture of Carolina isoinhibitors prepared by affinity
chromatography. Track 3, isoinhibitor DE5 purified by chro-
elution. Rechromatography of the major isoinhibi-
matography on DEAE-Sepharose CL-6B.
tor (peak DE5) on DEAE-Sepharose or FPLC on
Mono Q HR 5/5 yielded an essentially homoge-
neous product with a pI of 4.75 as revealed by
PAGE - IEF (Fig. 1). the expected two chains of approximate M r 16000
SDS-PAGE of each isoinhibitor without 2- (a chain) and 5000 (fl chain), respectively. The
mercaptoethanol yielded two very close bands with smaller fl chain lacked absorption at 280 nm and
an apparent M r of approx. 21000. In the presence was detected by its A230,m. The sum of the amino
of reducing agent, however, each of the isoinhibi- acid compositions of the a and fl chains of DE5
tots yielded a band of approximate M r 16 000 and was consistent with the composition of the whole
another of about Mr 5000, indicating that the DE5 isoinhibitor (Table I).
Carolina trypsin isoinhibitors are composed of The complete amino acid sequences .of the a
two polypeptide chains linked by a disulphide and fl chains of isoinhibitor DE5 are shown in
bridge. This two-chain structure of the isoinhibi- Fig. 3 together with the details of the overlapping
tots was not the result of their exposure to the peptides and fragments from which they were
immobilized anhydrotrypsin during affinity chro- deduced. The a chain of the protein contains 138
matography, as exactly the same pattern of SDS- amino acids, corresponding to an M, of 15 875
PAGE bands was obtained when the affinity pro- and the ~ chain 38 amino acids (M r 4883), both of
cedure was omitted from the scheme of purifica- which values are in excellent agreement with the
tion. Gel-filtration on Bio-Gel P-60 of the reduced estimates of M r made by SDS-PAGE. Each of the
and S-carboxymethylated isoinhibitor DE5 yielded other isoinhibitors had a similar sequence of amino
 137

A23O
I I I I I I = , [NcICI] T h e o n l y m a j o r h e t e r o g e n e i t y o b s e r v e d in iso-
i n h i b i t o r D E 5 o c c u r r e d at the N - t e r m i n u s of the fl
c h a i n where b o t h p r o l i n e a n d leucine were ob-
served in a p p r o x i m a t e l y equal a m o u n t s . M i n o r
" 0.8 L (less than 10%) e x a m p l e s o f heterogeneity were
0.6
/
./ / A - 0.1 M d e t e c t e d in the a chain in p o s i t i o n s 4 (Phe r e p l a c -
/
04 / t /
ing Leu), 6 ( A l a for Val), 11 (Phe for Leu), 22 (Val
/ for Ala), 49 (Ser for Ala), 52 ( G l n for Ser), 67 (Phe
0.2
_ ^ /~ ~ ~ ~_~ ~7 for Tyr), 89 ( G l y for Asp), 97 ( A s p for Glu), 108
40 80 120 160 ( G l u for Lys), 112 ( A r g for G l n ) , 113 (His for
FRACTION NO. Leu) a n d 120 ( G l n for Lys), a n d in the fl c h a i n at
Fig. 2. Separation of Carolina isoinhibitors on a column (1.5 x 22 (Lys for Glu). T h e existence of traces o f such
50 cm) of DEAE-Sepharose CL-6B. The mixture of isoinhibi- p o l y m o r p h i s m is n o t surprising in view o f the
tors (130 mg) obtained by affinity chromatography was applied m u l t i p l i c i t y of t r y p s i n isoinhibitors p r e s e n t in
in 0.05 M Tris-HCl (pH 7.6) to the column equilibrated in the C a r o l i n a seeds.
same buffer. The column was developed with a linear gradient
of NaCI concentration (0.05-0.25 M, 500 ml of each) at a flow Fig. 4 shows the clear sequence h o m o l o g y which
rate of 40 ml/h. The fractions (4 hal) indicated by bars were exists b e t w e e n the Adenanthera D E 5 i n h i b i t o r a n d
pooled. the Glycine and Psophocarpus K u n i t z - t y p e inhibi-
tors for which c o m p l e t e sequences are available.
a c i d s at their respective N - t e r m i n i , a n d their a m i n o C o n s i d e r a t i o n o f the three c o m p l e t e K u n i t z t y p e
a c i d c o m p o s i t i o n s d e a r l y i n d i c a t e d their close ho- sequences in Fig. 4 reveals that 47 residues are
m o l o g y with i s o i n h i b i t o r D E 5 ( u n p u b l i s h e d re- invariant, b u t this n u m b e r falls to 43 when the
suits). i n c o m p l e t e sequences o f the Acacia, Albizzia,

TABLEI
THE AMINO ACID COMPOSITIONS OF THE TRYPSIN ISOINHIBITOR DE5 FROM SEEDS OF CAROLINA
(ADENANTHERA PAVONINA) AND ITS CONSTITUENT a AND fl CHAINS
n.d., not determined.

Amino acid a chain fl chain Isoinhibitor DE5

analysis sequence analysis sequence analysis sequence
Asp 8.4 8 10.4 10 18.2 18
Thr 4.8 5 0 0 4.8 5
Ser 8.3 9 0.9 1 10.5 10
Gin 24.0 24 3.7 4 26.8 28.
Pro 10.7 11 1.4 1.5 = 12.6 12.5 ~
Gly 13.6 13 3.2 3 16.3 16
Ala 5.9 6 2.2 2 8.3 8
½Cys 3.2 3 1.0 1 3.8 4
Val 6.6 7 4.3 4 11.6 11
Met 0 0 0 0 0 0
lie 7.2 7 1.8 2 9.4 9
Leu 11.9 12 3.6 3.5 i 16.6 15.5 a
Tyr 6.3 7 0 0 6.9 7
Phe 6.3 6 1.1 1 7.7 7
Trp n.d. 1 n.d. 0 n.d. 1
His 0 0 0 0 0.1 0
Lys 7.1 7 2.3 2 9.2 9
Arg 11.8 12 2.9 3 14.9 15
Total 138 38 176
a Both Pro and Leu are present in equal amounts at the N-terminus of the fl-chain.
 138

~chain
I0 20 30 40 50 60
RELLDVDGNFLRNGGSYYIVPAFRGKGGGLELARTGSETCPRTVVQAPAEQSRCLPARLS

TI ~ l l T2 - - 4 1 T3 II T4 Jl T5 IF---.T6-~
I T3a I
cl ~ 1 c2 I ~ - c 3 - ~ i ..... c4 II c5 II c6 :~..

I vl ~1 v2 I~---...--v3 II v4

P1 II P2 II P3 : I P4 II

70 80 90 100 110 120

TPPRI RYI CPEFYLTI EFEEQKPP SCLRDSNLQWKVEEESQ I VKIA SKEEEQLFGSFQIK

- T 7 - 1 ~ ~T8~ I T9 -~ ~-----TI0 II ?11 II T12 _ _

I TI2VI__
mC7 IF.--.¢8..._.t1_.c9_._11 rlO t ~ ---on II c12 _ _ IF¢13~i_.c14_
tP---v5----II-v6 "41 v7 II v8 II v9 I
~ P 5 ip.--P6.---II P7 ~lt-.~S -'-II P9 4~EIL~_pII.

I HI~I~H2-----~ I IOB 1 , -

chain
130 I0 20 30
PYRDDYKLVYCEPQQGGR --r
~ E~C-K.D~L-GrI- v ~ ~ ~S ~I ~D'D,D N~N RRLAVKEGDPLVVQFVNADREGN

- - - t-T13~ I ~14 - - ' 1 ~--¢1~ I ~2 - - t ) T3 . . . . . . . . . "~

a ~3a ---II T3b ,j
.-.~F-Cl5.~I C16 I l---cl -----41 C2 ~ 1 1 c3 i~_c4._41 c5 I

I Vl0 I ~---VI "~1 V2 II V3 - - I I v4 - -

V4a II V4b I
--Pll I~ P12 I
-I
Fig. 3. The amino acid sequences of the a and ~8 chains of the proteinasc isoinhibitor DE5 from seeds of Carolina (A. pavonina). T,
tryptic pectides C, chymotryptic pectides; V, pectides from digestion with S. aureus V8 protcinasc; P, pectic pectides; H,
thermolysin pectides; IOB, fragment obtained after cleavage with iodosobenzoic acid. Solid lines indicate regions of pectides
sequenced by the DABITC method, dashed lines indicate residues which were not sequenced or yielded unsatisfactory results. - . ,
residues determined by the DABITC method applied to the intact a and ~ chains; . - , residue determined by digestion with
carboxypectidasc A.

Erythrina and Peltophorum inhibitors are included proteins [12-19]. The precise physiological signifi-
in the comparison. Glycine and Psophocarpus, cance of the proteolytic processing of a single-
which are both members of the Papilionoideae chain precursor which is presumed [8] to give rise
(Phascoleae) show approx. 45~ homology between to the two-chained inhibitors of the Mimosoideae
their sequences, but Adenanthera which is classi- is unknown [10], as is the reason why. this cleavage
fied separately in the Mimosoideae (Aden- of a susceptible bond appears to be restricted to
anthereae) exhibits only 33-35~ homology with this sub-family.
either of these. Previous workers [21] have shown that the
The Adenanthera inhibitor is composed of two Adenanther~ inhibitor forms a 1 : 1 molar complex
disulphide-linked polypeptide chains and is simi- with trypsin, and this observation was confirmed
lar in this respect to the inhibitors in the other during this work by titration of the native inhibi-
representatives (Acacia, Albizzia) of the Mimo- tor with trypsin. The reactive (inhibitory) peptide
soideae sub-family which have been examined bond was identified in the usual way by limited
[8-11], whereas the Kunitz inhibitors found in hydrolysis of the native isoinhibitor DE5 with a
Glycine, Erythrina and Psophocarpus belonging to catalytic amount of trypsin at p H 2.5 for 20 h
the sub-family Papilionoideae and in Peltophorum which led to hydrolysis of the Arg-64-Ilc-65
(Caesalpinoideae) all appear to be single-chained peptide bond. This bond is in an exactly homolo-
 139

LlO

b) IEPLLI]
i
s z l ~ zz~. v R s G
A P ^I~]Q s R

c) ~FVL~ TIY Y1,21~ S D T T A , - ~ I '1~ AIAI P TL~I NURI~ LIT V VIQ $ R N EL~ D K
d) ~ K E L L ~ A DIG ~1~ L L N G
e) ¢ g I K G L L r
~10
f) ' ~ * L L r ADIGDLL*SG
g) ILLI c ~lc ~ v ~ z kc
h) 1DFVL[ A El G KI~ L - N G

a) L P A RLSTP R Y I G PEFYLTI EFEE-QKPP S L RD SN LQ W Kr~IE~ OQ I - - R

b) PLRI6~ SQ oF I PTg- - Y- S~VRI GFA s~PPK AP SP-Wg~TV D Q P Q Q [~OS
c) ~T[ I S6~ P R F I A ET~F P L ~ L g F D S8~ A V I 8 L V C 19~ T Z W S V U D LI~0E G r AU
d) NINTPS ASLTPA YLN*EF

~ r'~ d120 1
S K E E1 | [ 120
b) S E L g S T X r D Y L[FIK F E g V T S - K F S SlY K L[K g ] q A g R
c) GENKDA~-DGW SDDEFNNYKLVF QQ.A

b)
o)
1o1 "1°1Z IDIII¢ 0Y R I°1Q
• ~ ~ ~IcIGi~i~i~|~ s ~i.|. v ~ ~
~ 6 V T
,,. g- [ 180
T l ~ l ~ U V M S K . ~1 P L v vIQ~jQ ~ ~ V ~lqS L
d) ' K D D H iclXioi Llor oS I ioI D D E N
~1 I I I I lezo I I
~) v n s SlClZPir.iGi I s HDI- v E S
/31 I I I i~101 I I
f) G w WE *IClQIDILIGII * VL~D " E

Fig. 4. Homology of the Carolina (Adenantkera) isoinhibitor DE5 (a), with the Kunitz inl'fibitors from (b) Psopkocarpus [19], (c)
Glycine [34], (d) Albizzia [8,9], (c) Acacia elata [10], (f) Acacia sieberana [11], (g) Erythrina latissima [12], and (h) Peltophorum [15].
Homologous residues are shown in boxes. The reactive (inhibitory) peptide bonds for trypsin are indicated by a vertical arrow. *,
unidentified residues. - , gaps inserted to facilitate comparisons.

gous position to the reactive sites identified in the References

soybean inhibitor at Arg-63-Ile-64 [16] in Albizzia
at Arg-66-I1e-67 [9] and in Psophocarpus at Arg- 1 Liener, I.E. and Kakade, M.L. (1980) in Toxic constituents
of Plant Foodstuffs (Liener, I.E., ed.), 2nd Edn., pp. 7-71,
64-Ser-65 [19] (Fig. 4).
Academic Press Inc., New York
2 Ryan, C.A. (1981) in The Biochemistry of Plants (Marcus,
Acknowledgements I., cd.), Vol. 6, pp. 351-370, Academic Press, New York
3 Richardson, M. (1980) Food Chem. 6, 235-253
The authors wish to thank the Science and 4 Norioka, S. and Ikenaka, T. (1983) J. Biochem. 94, 589-599
5 Wilson, K.A. and Chen, J.C. (1983) Plant Physiol. 71,
Engineering Research Council, the CNPq
341-349
(Brazilian National Council for Scientific and 6 Shimokawa, T., Kuromizu, K., Araki, T., Ohata, J. and
Technological Development), the British Council Abe, O. (1984) Eur. J. Biochem. 143, 677-684
and the Smith Kline Foundation for financial ? Ishikawa, C., Watanabe, K., Sakata, N., Nakagaki, C.,
support. We also thank Mr. J. Gilroy and Mr. P. Nakanura, S. and Takahashi, K. (1985) J. Biochem. 97,
55-70
Miller for valuable technical assistance, Professor
8 0 d a n i , S., Odani, S., Ono, T. and lkenaka, T. (1979) J.
P. Roug6 (University Paul Sabatier, Toulouse) for Biochem. 86, 1795-1805
amino acid analysis and Professor D. Boulter for 9 0 d a n i , S., Ono, T. and Ikenaka, T. (1980) J. Biochem. 88,
the provision of certain facilities. 297-301
140

10 Kortt, A.A. and Jermyn, M.A. (1981) Eur. J. Biochem. 115, 24 Xavier-Filho, J. and Campos, F.A.P. (1985) 13th Int. Congt
551-557 Biochem., Abstract TV-182, Amsterdam
11 Joubert, F.J. (1983) Phytochemistry 22, 53-57 25 Erlanger, B.F., Kokowski, N. and Kohen, W. (1961) Arch
12 Joubert, F.J., Carlsson, F.H.H. and Haylett, T. (1981) Biochem. Biophys. 95, 271-278
Hoppe-Seyler's Z. Physiol. Chem. 363, 531-538 26 Chase, T.J. and Shaw, E. (1967) Biochem. Biophys. Res.
13 Joubert, F.J. (1982) Phytochemistry 21, 1213-1217 Commun. 29, 508-514
14 Joubert, F.J. (1982) Int. J. Biochem. 14, 187-193 27 Chase, T.J. and Shaw, E. (1967) Biochem. Biopbys. Res.
15 Joubert, F.J. (1981) Hoppe-Seyler's Z. Physiol. Chem. 362, Commun. 29, 508-514
1515 27 Reisfeld, R.A., Lewis, U.J. and Williams, D.E. (1962) Na-
16 Koide, T. and lkenaka, T. (1973) Eur. J. Biocliem. 32, ture 195, 281-283
401-407 28 Sigma Technical Bulletin (1984) No. IEF-100
17 Koide, T., Tsunasawa, S. and Ikenaka, T. (1973) Eur. J. 29 Crestfield, A.M., Moore, S. and Stein, W.H. (1983) J. Biol.
Biochem. 32, 408-416 Chem. 238, 622-627
18 Koide, T. and Ikenaka, T. (1973) Eur. J. Biochem. 32, 30 Richardson, M. (1974) Biochem. J. 137, 101-112
417-431 31 Richardson, M., Campos, F.A.P., Moreira, R.A., Ainouz,
19 Yamamoto, M., Hara, S. and Ikenaka, T. (1983) J. Bio- I.L., Begbie, R., Watt, W.B. and Pusztai, A. (1984) Eur. J
chem. 94, 849-863 Biochem. 144, 101-111
20 Xavier-Filho, J., Juca, M.B. and Lopes, A.L.S. (1976) 32 Yarwood, A., Richardson, M., Sousa-Cavada, B. and Rou86,
Ciencia Cult. 28, 494-497 P. (1985) FEBS Lett. 184, 104-109
21 Prahbhu, K.S. and Pattabiraman, T.N. (1980) J. Sci. Food 33 Uriel, J. and Berges, J. (1968) Nature 218, 578-580
Agric. 31, 967-980 34 Kim, S.H., Hara, S., Hase, S., Ikenaka, T., Toda ,H.,
22 Ako, H., Foster, R.J. and Ryan, C.A. (1974) Biochemistry Kitamura, K. and Kaizuma, H. (1985) J. Biochem. 98,
13, 132-139 435-448
23 Xavier-Filho, J. and Campos, F.A.P. (1983) Braz. J. Med.
Biol. Res. 16, 11-15