It is important to determine the precise bio- chemical interactions involved in the action of
antifungal and anti-insect proteins, noting that such defensive proteins may have more than one
mode of action. The antifungal napins provide a good example of defensive proteins that have evi-
dent multiple functions and which have been shown to interact with more than one biochemical
entity. Thus napins are glutamine-rich storage proteins and can also be PIs [13,14], cause fungal
membrane changes and act synergistically with other membrane-altering antifungal proteins [18].
Napins and the napin-like proteins have a het- erodimeric subunit structure [15,16] and it has
been found that particular napin [16] and napin- like protein [14] small and large subunits are sub-
strates
for phosphorylation
by plant
Ca
2 +
-dependent protein kinase CDPK, although the biochemical significance of this has not been
established. In addition, some of these oxidized complexes and their constituent small and large
subunits can bind to calmodulin CaM, as de- tected
through inhibition
of CaM-dependent
myosin light chain kinase MLCK or through their
effect on
Ca
2 +
-dependent dansyl-CaM
fluorescence [16,19]. Some other plant antifungal proteins are also
substrates for plant CDPK [19], including various LTPs [20,21], soybean Bowman – Birk PI BBI-I
[22], potato tuber carboxypeptidase inhibitor protein [23] and plant defensins g-thionins [12].
Some plant defensins can interact with Ca
2 +
-CaM [12] in addition to being CDPK substrates and
antifungal proteins [4,5] and it is conceivable that further specific biochemical interactions may con-
tribute to the defensive function of this group of plant bioactive proteins.
PIs have a major role in anti-insect plant de- fence [1,17]. Inhibition of serine proteases by plant
protease inhibitory proteins or protease inhibitory secondary metabolites interferes with insect diges-
tion and can inhibit larval growth and develop- ment [1,17]. As part of an investigation into the
anti-insect components of various Indonesian Cas- sia species, the nature and structure of Cassia
fistula PI proteins have been investigated. The present paper describes the purification and se-
quencing of a PI protein from the seeds of C. fistula and its identification as a plant defensin. To
the authors’ knowledge this is the first demonstra- tion of a protease inhibitory function associated
with a representative of this major class of plant defensive proteins.
2. Materials and methods
2
.
1
. Materials Seeds of C. fistula were collected from the Jambi
forest in Sumatra, Indonesia. N-benzoyl-
D
,
L
- arginine-p-nitroanilide BAPNA and N-benzoyl-
L
-tyrosine-p-nitroanilide BYPNA were obtained from Sigma St. Louis, MO.
L
-1-Tosylamido-2- phenylethylchloromethyl-ketone TPCK-treated
bovine trypsin and Na-p-tosyl-
L
-lysine chloro- methyl ketone TLCK-treated chymotrypsin were
obtained from
Worthington USA.
Carbo- xymethylcellulose CM52 was obtained from
Whatman England. An Aquapore RP300 C8 RP-HPLC guard column 4.6 mm × 30 mm; 7 mm
particle size, 300 A , pore size was obtained from
Brownlee Laboratories USA and a Vydac C8 column 4.6 mm × 25 cm; 5 mm particle size, 300
A , pore size was obtained from Altech Associates
USA.
2
.
2
. Trypsin and chymotrypsin assays Trypsin or chymotrypsin inhibitory activity of
the isolated proteins was assayed in duplicate at 30°C in 96 well microtitre plates 100 ml well
volume containing 50 mM Tris Cl
−
, pH 8, 200 mM N-benzoyl-
D
,
L
-arginine-p-nitroanilide and 10 mgml trypsin or 200 mM N-benzoyl-
L
-tyrosine-p- nitroanilide and 10 mgml chymotrypsin in the
presence and absence of added test proteins. Protease activity was determined from the ab-
sorbance change at 405 nm, measured using a Spectra Max Pro 250 multiplate spectrophotome-
ter Molecular Devices. Alternatively, assays were conducted in duplicate in the same assay condi-
tions but with a final assay volume of 1 ml and the absorbance changes were measured at 405 nm
using an Hitachi 181 UV-VIS spectrophotometer. IC
50
values concentrations for 50 inhibition of proteins were determined using the microtitre
plate-based assay. Assays with increasing concen- trations of test protein were conducted in dupli-
cate and control assays no added inhibitor in sextuplet.
2
.
3
. Purification of C. fistula PIs Seeds of C. fistula 200 g were soaked overnight
in H
2
O at 20°C and were then homogenized in methanol 600 ml using an UltraTurrax blender
Janke Kunkel, Germany at full power for 10 min. The homogenate was passed through Mira-
cloth to recover the cell wall fraction. The cell wall fraction was washed with 1.5 l H
2
O until no more protein was detected in the eluate. The cell wall
fraction was then extracted by suspension in 200 ml 2 M NaCl in 10 mM glycine Na
+
, pH 9.5 the pH falling to 6 after addition of the cell wall
material. The suspension was filtered through Miracloth and the filtrate containing material ex-
tracted at high ionic strength from cell wall mate- rial was centrifuged at 15 000 × g for 15 min. The
supernatant was diluted 50-fold in 10 mM sodium phosphate pH 6.0 buffer and applied to 60 g
carboxymethylcellulose Whatman CM52 on a Bu¨chner funnel. The CM52 was washed with 200
ml 10 mM phosphate Na
+
, pH 6.0 and then the bound protein was eluted with 100 ml 2 M NaCl –
50 mM glycine Na
+
, pH 9.5. This chromato- graphic step was repeated with the CM52 being
eluted with a stepwise gradient of increasing NaCl concentration in 50 mM glycine Na
+
, pH 9.5, PI activity being detected in fractions containing
100 – 700 mM NaCl. The PI fractions were pooled, concentrated by pressure filtration Amicon YM 3
membrane and chromatographed on an Ultrogel AcA 44 column 7 cm
2
× 53 cm which was eluted
with 0.5 M NaCl in 50 mM Tris Cl
−
, pH 8.0. Low molecular weight fractions containing PI ac-
tivity peak 2, Fig. 1 were pooled and concen- trated
by pressure
filtration Amicon
YM3 membrane.
The concentrated
fraction was
brought to 0.1 trifluoroacetic acid TFA, cen- trifuged at 10 000 × g for 5 min and filtered
through a 0.2 mm filter Phenomenex. The con- centrated acidified fraction was subjected to RP-
HPLC employing an Aquapore RP300 C8 RP-HPLC guard column 4.6 mm × 30 mm; 7 mm
particle size, 300 A
, pore size and a Vydac C8 column 4.6 mm × 25 cm; 5 mm particle size, 300
A , pore size which was eluted with a gradient of
increasing CH
3
CN concentration in 0.1 TFA 0 – 100 CH
3
CN over 100 min; flow rate 1 ml min; UV detection at 240 nm. A major protein
peak associated with trypsin inhibitory activity was resolved eluting at 33 CH
3
CN in 0.1 TFA Fig. 2A and was stored at − 30°C before
further analysis. A second procedure employed to purify the
defensin was essentially the same as that described above except that no overnight imbibing in H
2
O was employed seeds being frozen with liquid N
2
, ground in a mortar and pestle and then extracted
Fig. 1. Gel filtration of the basic, carboxymethylcellulose-binding fraction from C. fistula seeds on an Ultrogel AcA44 column. The gel filtration was conducted in 0.5 M NaCl – 50 mM Tris Cl
−
pH 8.0 with a flow rate of about 0.5 mlmin. Fractions of about 5 ml were collected. Protein was determined by the method of Sedmak and Grossberg [24]. Trypsin inhibition by 10
ml samples of each fraction was determined in the 100 ml final assay volume microtitre plate-based assay as described in Section 2. - , protein concentration; -, trypsin inhibition.
Fig. 2. Reversed phase HPLC RP-HPLC of the low molecular weight trypsin inhibitory basic protein fraction from C. fistula seeds. RP-HPLC and trypsin inhibition assays were conducted as described in Section 2. Continuous trace, absorbance at 240 nm
A or 220 nm B absorbance at 220 nm being used for greater measurement sensitivity; histogram ---, trypsin inhibition by 10 ml of the indicated fraction determined as described in the legend to Fig. 1. A, trypsin inhibitory material from procedure
1; B, trypsin inhibitory material from procedure 2.
as before in methanol, the cell wall fraction was not washed with H
2
O and the cell wall fraction was extracted with 2 M NaCl – 10 mM glycine
with the cell wall suspension in this solution having a final pH adjusted to 9.5 with NaOH. In
addition, the CM52 chromatographic step was conducted only once, with the PI fraction being
eluted batchwise in 2 M NaCl – 10 mM glycine pH 9.5. This second procedure yielded a major
peak of defensin on RP-HPLC that was associated with trypsin inhibitory activity peak 4, Fig. 2B
together with three further peaks of protein eluting just prior to the defensin peak peaks 1 – 3, Fig.
2B. All fractions were stored at − 30°C before further analysis. Protein was estimated from UV
absorbance or by the method of Sedmak and Grossberg 1977 [24] using crystalline bovine
serum albumin as a standard. SDS PAGE was conducted using 15 polyacrylamide BioRad
Minigels following the method of Laemmli [25].
2
.
4
. Amino acid sequencing N-terminal amino acid sequences were obtained
by sequential Edman degradation using a Hewlett- Packard G1005A automated protein sequencing
system calibrated
with phenylthiohydantoin
PTH-amino acid standards prior to each se- quencing run.
2
.
5
. Electrospray ionization mass spectrometry Prior to electrospray ionization mass spectrome-
try ESMS, protein fractions from RP-HPLC were concentrated under vacuum in a SpeedVac
concentrator and reconstituted with milli-Q H
2
O. ESMS was carried out using 1 – 100 pmol protein
in 2 – 4 ml of 50 CH
3
CN containing 0.1 formic acid. Samples were infused at a flow rate of 0.2
mlmin into a Perkin-Elmer Sciex API-300 triple quadrupole mass spectrometer fitted with a micro-
ionspray ion source and calibrated to an accuracy equivalent to
9 0.01 using singly charged
polypropylene glycol ions. Mass spectra, typi- cally 30 – 100 scans, were recorded in the first
quadropole Q1 scan mode over the mass-to- charge range mz 200 – 3000 Da per unit charge,
using a constant peak width at half peak height of 0.6 Da per unit charge. Mass-to-charge ratio
data were processed by signal-averaging, manual mass determination and transformation using PE-
Sciex Biomultiview software.
3. Results
