Biochemical analysis of a papain like protease isolated from the latex of Asclepias curassavica L.

Acta Biochim Biophys Sin (2009): 154 – 162 | ª The Author 2009. Published by ABBS Editorial Office in association with Oxford University Press on behalf of
the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmn018.
Biochemical analysis of a papain-like protease isolated from the latex of Asclepias
curassavica L.
Constanza Liggieri1*, Walter Obrego´n1, Sebastia´n Trejo2, and Nora Priolo1
1Laboratorio de Investigacio´n de Prote?´nas Vegetales (LIPROVE), Departamento de Ciencias Biolo´gicas, Facultad de Ciencias Exactas,
Universidad Nacional de La Plata, C.C. 711, B1900AVW, La Plata, Argentina
2Institut de Biotecnologia i de Biomedicina ‘Vincent Villar i Palasi’, Universidad Auto`noma de Barcelona, 08193 Campus UAB, Bellaterra
(Cerdanyola del Valle`s), Barcelona, Spain
*Correspondence address. Tel: þ54-0221-423-0121; E-mail: cliggieri@biol.unlp.edu.ar
Most of the species belonging to Asclepiadaceae family
Received: October 23, 2008
Accepted: November 19, 2008
usually secrete an endogenous milk-like fluid in a network
of laticifer cells in which sub-cellular organelles intensively
synthesize proteins and secondary metabolites. A new
Introduction
papain-like endopeptidase (asclepain c-II) has been iso-
lated and characterized from the latex extracted from
Proteases are enzymes that catalyze the degradation of
petioles of Asclepias curassavica L. (Asclepiadaceae).
peptides and proteins, and occupy a significant role in
Asclepain c-II was the minor proteolytic component in the
numerous physiologic processes in the living beings, as
latex, but showed higher specific activity than asclepain
well as by their use in different industrial processes. It
c-I, the main active fraction previously studied. Both
has been verified that proteases direct specific and selec-
enzymes displayed quite distinct biochemical character-
tive modifications of proteins, such as the activation of
istics, confirming that they are different enzymes. Crude
proenzymes, sanguineous coagulation, digestion of fibrin
extract was purified by cation exchange chromatography
clots, secretory protein processing and transport through
(FPLC). Two active fractions, homogeneous by sodium
membranes, germination, senescense, defense against
dodecyl sulphate-polyacrylamide gel electrophoresis and
plant pathogens (especially fungi and insects), and acqui-
mass spectrometry, were isolated. Asclepain c-II displayed
sition of nutrients and apoptosis [1 – 7].
a molecular mass of 23,590 Da, a pI higher than 9.3,
In the world, the industries that apply enzymes
maximum proteolytic activity at pH 9.4–10.2, and showed
for their products invest annually near a trillion of
poor thermostability. The activity of asclepain c-II is inhib-
dollars in their commercialization. Of them, 75% belong
ited by cysteine proteases inhibitors like E-64, but not by
to hydrolytic enzymes and of this percentage, the pro-
any other protease inhibitors such as 1,10-phenantroline,
teases represent 60% of total of the world-wide sales [2].
phenylmethanesulfonyl fluoride, and pepstatine. The N-
The vast diversity of proteases, with its specification,
terminal sequence (LPSFVDWRQKGVVFPIRNQGQ
has attracted the attention of the scientists worldwide
CGSCWTFSA) showed a high similarity with those
that has taken them to explore its multiple physiologic
of other plant cysteine proteinases. When assayed on
and industrial applications. In relation to this last aspect,
N-a-CBZ-amino acid-p-nitrophenyl esters, the enzyme
the proteases develop to a protagonist role in the
exhibited higher preference for the glutamine derivative.
biotechnological processes because they maintain their
Determinations of kinetic parameters were performed
enzymatic activity within a wide range of pH and temp-
with N-a-CBZ-L-Gln-p-nitrophenyl ester as substrate:
erature [8].
Km 5 0.1634 mM,
kcat 5 121.48 s21,
and
kcat/Km 5
At the moment several examples of the use of enzymes
7.4 3 105 s21/mM.
and of specifically proteases in different areas from the
industry can be mentioned: modified proteins for the food
Keywords
Asclepias
curassavica;
Asclepiadaceae;
industry, baking, beer elaboration, cheese production,
cysteine proteinase; latex; laticifers
detergent dust preparation, treatment of industrial effluents,
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Papain-like protease from latex Asclepias curassavica L.
textile industry, manufacture of leather, pharmaceutical
phosphate buffer ( pH 6.5) containing 5 mM ethylendia-
industry, cleaning of surgical supplies, and biomedicals
minetetraacetic acid (EDTA) and cysteine, in order to
[9–12].
avoid phenoloxidase activity and oxidation, respectively.
Species belonging to the Asclepiadaceae family
This suspension was first centrifuged at 16,000 g
usually contain proteolytic enzymes in latex. Latex is a
for 30 min at 48C to discard gums and other insoluble
milky fluid with a complex mixture of constituents, like
materials, and the supernatant was ultracentrifuged
proteins, vitamins, carbohydrates, lipids, terpenes, alka-
at 100,000 g for 1 h at 48C. This new supernatant
loids, and free amino acids. The presence of certain
containing soluble proteins (crude extract (CE), 12 ml),
enzymes like chitinases and proteases in latex vacuoles
was fractionated and conserved at 2208C for further
suggest that they may help plants for defense against
studies.
pathogens, parasites, and herbivores by attacking the
invader once the plant cell is lysed [13].
Purification of asclepain c-II
Our group has been largely devoted to screening new
The CE was applied in a column packed with
plant sources from the local flora, which could provide
SP-Sepharose Fast Flow (Pharmacia, Uppsala, Sweden)
new proteases, as plant proteases used in Argentina are
equilibrated with 0.05 M Tris – HCl buffer ( pH 8.25).
usually imported. Many members of the Asclepiadaceae
Cation exchange chromatography (FPLC, Pharmacia)
family have shown to contain very active proteases in the
was developed by adding the starting buffer (0.05 M
latex. The group has previously reported in the purification
Tris – HCl, pH 8.25), followed by a sodium chloride
and characterization of proteases present in lattices of
linear gradient (0 – 0.6 M) prepared in the same buffer.
several members of the Asclepiadaceae family [14– 22].
Cation exchange chromatography was monitored by
Asclepias curassavica L. is native of South America
measuring absorption at 280 nm. Caseinolytic activity
and grows from Mexico to Argentine but has become a
was tested in on the eluted fractions, and those showing
naturalized weed in tropical and subtropical areas through-
proteolytic activity were pooled and stored at 2208C for
out the world. The stems of this species exude large quan-
further studies (cf next section).
tities of latex when leaves are cut off, which has been used
in folk medicine as emetic, vermifuge, and antigonorrheic
Proteolytic activity assays
[23,24]. The main proteolytic component, named asclepain
Proteolytic assays were made using casein as substrate.
c-I, was isolated from the latex in a previous study [25]. In
The reaction mixture was prepared by mixing 0.1 ml of
this article the biochemical characteristics of a second pro-
the purified enzyme with 1.1 ml of 1% casein containing
tease is presented, which despite of being the minor pro-
12 mM cysteine in a 0.1 M Tris – HCl buffer ( pH 8.5).
teolytic component in the latex, it showed higher specific
The reaction was carried out at 428C and stopped 2 min
activity than asclepain c-I. Additionally, this new enzyme
later by the addition of 1.8 ml of 5% trichloroacetic acid
exhibited different properties that could be profitable when
(TCA). Each test tube was centrifuged at 3000 g for
used in industrial processes.
20 min and the absorbance of the supernatant measured
at 280 nm. An arbitrary enzyme unit (Ucas) was defined
Materials and Methods
as the amount of enzyme (g) that produces an increase
of one absorbance unit per minute in the assay
Plant material
conditions.
Asclepias
curassavica
L.,
‘scarlet
milkweed’,
(Asclepiadaceae) is an erect, evergreen perennial sub-
Protein determination
shrub with woody base. Like most milkweeds, it has
Proteins were measured according to Bradford’s method
opposite leaves and milky sap [26]. Latex was obtained
[27] using bovine albumin (Sigma Chemical Co.,
from plants grown in Rosario, Province of Santa Fe,
St. Louis, USA) as standard.
Argentina. A voucher specimen (UNR 1130) has been
deposited at the UNR herbarium (Faculty of Agricultural
pH profile
Sciences, University of Rosario, Argentina).
The effect of pH on enzyme activity was determined on
casein (pH range 6.0–10.5) at constant ionic strength using
Crude enzyme extract preparation
0.01 M sodium salts of the following buffers [28]:
Latex (2 ml), obtained by superficial incisions of
2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpho-
petioles, was collected on 13 ml of 0.1 M citric
lino) propanesulfonic acid (MOPS), N-tris(hydroxymethyl)
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Papain-like protease from latex Asclepias curassavica L.
methyl-3-aminopropanesulfonic acid (TAPS), 3-[(1,1-dime-
Controls were prepared by preincubating the enzyme
thyl-2-hydroxyethyl)amino]-2-hydroxy-propanesulfonic acid
with the appropriate solvent used to dissolve the inhibi-
(AMPSO), and 3-(ciclohexylamino)-1-propanesulfonic acid
tors [30]. A control assay of the enzyme activity was
(CAPS). At each pH a control assay was done without
done without inhibitors and the resulting activity was
enzyme and used as a blank.
taken as 100%.
pH stability
Electrophoresis
Two milliliter of the purified enzymatic preparation was
Purified samples of asclepain c-II were analyzed by
precipitated with three volumes of acetone, centrifuged
sodium dodecyl sulphate-polyacrylamide gel electrophor-
at 3000 g during 20 min, and then the precipitated was
esis (SDS-PAGE) with Tris – glycine cathodic buffer in
redissolved in the corresponding ‘Good’ buffer adjusted
10% polyacrylamide gels [31]. Voltage was kept con-
to pH 10.0. Samples (0.1 ml) were incubated at 208C for
stant at 40 mV for the stacking gel and at 150 mV for
1 – 3 h and the residual activity was assayed according to
the resolution gel. The gels were stained with coomassie
the method described above.
brilliant blue R-250 and the molecular weight of ascle-
pain c-II was estimated by using the Scion Image soft-
Thermal stability
ware. Protein molecular weight markers (SDS-PAGE
After incubating the enzyme solution (0.1 ml) for 30,
Molecular Weight Standards, Low Range; Bio Rad,
60, 90, and 120 min at 408C, 508C, 608C, and 708C, the
Hercules, USA) were used as standards to generate the
residual proteolytic activity was determined as indicated
calibration curve.
previously.
Isoelectric focusing and zymogram
Effect of inhibitors on proteolytic activity
Isoelectric focusing (IEF) was performed in a mini IEF
The effect of specific inhibitors on proteolytic activity
cell (Model III, Bio-Rad). The sample was concentrated
was determined by preincubating the protease prep-
and deionized with five volumes of cold acetone and
aration on casein or azocasein [29] with inhibitors and
further centrifugation at 3000 g for 15 min. The precipi-
then estimating the residual activity. The enzyme
tate obtained was redissolved with deionized water and
preparation (0.99 ml) was incubated with 0.01 ml of
the treatment repeated twice. Polyacrylamide gels con-
the one following inhibitors: 10 mM (2S,3S)-3-(N-
taining broad pH range ampholytes (3.0 – 10.0) were
f(S)-1-[N-(4-guanidinobutyl)carbamoyl]3-methylbutylg
used. Focusing was carried out under constant voltage
carbamoyl)oxirane-2-carboxylic acid (E-64), 100 mM
conditions in a stepped procedure: 100 V for 15 min,
phenylmethanesulfonyl
fluoride
(PMSF),
and
0.1 –
200 V for 15 min, and 450 V for 60 min. Gel was fixed
0.5 mM pepstatin A, during 30 and 60 min at 308C. The
and then stained with Coomassie brilliant blue R-250.
residual proteolytic activity was determined on casein as
In order to ascertain if the protein fraction had proteo-
indicated above.
lytic activity, the unstained gel was contacted for 10 min
Due to 1,10-phenantroline exhibits high absorbance at
at 508C with an agarose gel imbibed for 20 min in 1%
280 nm, for this assay casein was changed by azocasein.
casein solution (0.1 M Tris – HCl buffer, pH 8.3) with
The enzyme preparation (0.99 ml) was incubated with
12 mM cysteine and washed twice with distilled water.
0.01 ml of 100 mM 1,10-phenanthroline and then the
Then, the agarose gel was stained by Coomassie brilliant
residual activity was determined as follows: 0.25 ml of
blue R-250. Clear bands on the stained agarose gels evi-
2% azocasein in 0.1 M glycine – NaOH buffer ( pH 9.5)
dence the presence of proteolytic activity [32].
containing 20 mM cysteine was added to 0.15 ml of
enzyme sample and incubated at 458C for 30 min. The
Mass spectrometry
reaction was stopped by adding 1 ml of 10% TCA. After
The molecular weight and purity of asclepain c-II were
centrifugation at 4000 g for 15 min, 0.9 ml of the
determined by MALDI-MS/TOF. Mass spectrum was
supernatant obtained was added to 1 ml of 1 M NaOH,
acquired on a Bruker Daltonicsw model Ultraflex spec-
and the absorbance was measured at 440 nm. In this
trometer equipped with a pulsed nitrogen laser (337 nm),
case, one unit of proteolytic activity (Uazocas) was
in a linear positive ion mode, using a 20 kV acceleration
defined as the amount of enzyme (g) that produced an
voltage. Samples were prepared by mixing equal
absorbance increase of one unit per minute under the
volumes of a saturated solution of the matrix (sinapic
assay conditions.
acid) in 0.1% TFA (aq.): acetonitrile (2:1), and protein
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Papain-like protease from latex Asclepias curassavica L.
solution. From this mixture, 1 ml was spotted on the
Kinetic parameters
sample slide and allowed to evaporate to dryness.
N-a-CBZ-L-Gln-p-nitrophenyl ester was used to deter-
Bovine trypsinogen was used for internal calibration.
mine Vm, Km, kcat, and kcat/Km of asclepain c-II.
Estimation of kinetic parameters was performed follow-
N-terminal sequence
ing the method described previously for this substrate
The N-terminal sequence was determined by Edman’s
[34]. Substrate final concentration ranged from 125 Â
automated degradation using a Beckman LF3000 protein
1026 to 2 Â 1023. Km, kcat, Vm, and kcat/Km values were
sequencer equipped with a System Gold (Beckman,
calculated by hyperbolic regression analysis using the
Fullerton, USA) PTH-amino acid analyzer. The Basic
nonlinearized form of the Michaelis – Menten equation.
Local Alignment Search Tool (BLAST) network service
[33] was used to perform protein homology studies but
Results
considering only those specific residues that are identical
(‘identities’).
Purification
Cation exchange chromatography allowed the separation
of three fractions (Fig. 1). No proteolytic activity was
Measurement of endoesterolytic activity
found in the former fraction eluted, but the two other
These assays were carried out by the Silverstein’s [34]
fractions eluted with the linear salt gradient used were
method
modified
to
reach
optimal
conditions
of
active. The active fractions were named asclepain c-I and
the
enzyme.
The
activity
was
studied
using
c-II, according to nomenclature recommendations for
N-a-Cbz-L-Gln-p-nitrophenyl esters of some L-amino
proteases
obtained
from
latex
of
species
of
the
acids (Ala, Asn, Gln, Gly, Ile, Leu, Trp, Pro, and Val)
Asclepiadaceae family [36,37].
as substrates. Assays were made at 378C in 0.1 M Tris –
Asclepain c-I was the main fraction [25]. Asclepain
HCl buffer ( pH 8.0) containing 2 mM EDTA, 25 mM
c-II was purified with 12.1-fold, the percent recovery in
cysteine and 1 mM of each substrate in the reaction
terms of total activity was 9.8% and the specific activity
mixture. Liberation of p-nitrophenol was followed spec-
was 12.8 Ucas/mg (Table 1).
trophotometrically at 405 nm in an Agilent 8453 E
UV-visible spectroscopy system (Santa Clara, CA, USA)
Homogeneity of the enzyme
equipped with a chamber termostatized at 378C. An arbi-
Purity of asclepain c-II was checked both by SDS-PAGE
trary enzyme activity unit (Ucbz) was defined as the
and mass spectrometry: 23,500 and 23,590 Da, respect-
amount of protease (g) that released one micromol of
ively (Figs. 2 and 3). The presence of a single band both
p-nitrophenolate per min in the assay conditions. To
in SDS-PAGE and IEF (Fig. 4) show that the enzyme is
determine the micromoles of p-nitrophenolate produced
homogenous, like asclepain c-I.
during the reaction, a standard curve with p-nitrophenol
As it has already been mentioned before, IEF and
(5 – 50 mM) in 0.1 M Tris – HCl buffer pH 8.0 containing
zymogram confirmed the presence of a unique band
5% acetonitrile was carried out.
with proteolytic activity, corresponding to a basic
Measurement of amidolytic activity
Amidolytic activity was determined by hydrolysis of
L-pyroglutamil-L-phenylalanyl-L-leucine-p-nitroanilide
(PFLNA) according to Filippova et al. [35]. This assay
was performed using a solution of 1 mM PFLNA in
dimethyl sulfoxide (DMSO). The reaction mixture con-
tained 1.5 ml of 0.1 M phosphate buffer, pH 6.5, 0.3 M
KCl, 1024 M EDTA, 0.003 M dithiothreitol (DTT),
0.18 ml
substrate,
and
0.12 ml
enzyme.
The
p-nitroaniline released at 378C was detected spectropho-
tometrically at 410 nm. An arbitrary enzyme activity unit
Fig. 1 Cation exchange chromatography
Elution buffer: 0.05 M
(UPFLNA) was defined as the amount of protease (g) that
Tris – HCl ( pH 8.25). Gradient: sodium chloride 0 – 0.6 M. Flow rate:
released one micromol of p-nitroaniline per min in the
0.75 ml/min. Fraction volume: 2.0 ml. SP-Sepharose Fast Flow,
assay conditions.
column Pharmacia Biotech XK 16.
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Papain-like protease from latex Asclepias curassavica L.
Table 1 Purification of the proteolytic components present in the latex of Asclepias curassavica L
Sample
Volume
Protein
Total
Arbitrary enzyme unit
Total
Specific activity
Purification
Yield
(ml)
(mg/ml)
proteins
Ucas (ml21)
Ucas
(Ucas/mg)
(fold)
(%)
CE
1.5
0.9333
1.399
0.9880
1.480
1.0586
1
100
Asclepain c-I
7.5
0.0243
0.182
0.2649
1.980
10.877
10.27
26.80
Asclepain c-II 10.5
0.0075
0.078
0.0968
1.016
12.836
12.125
9.80
An arbitrary enzyme unit (Ucas) was defined as the amount of enzyme (g) that produces an increase of one absorbance unit per minute in the
assay conditions.
Fig. 2 SDS-PAGE
M, Bio Rad molecular weight standards:
phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa),
ovoalbumin (45.0 kDa), carbonicanhydrase bovine (31.0 kDa), soybean
trypsin inhibitor (21.5 kDa), and lysozyme (14.4 kDa); 1, crude
extract; 2, asclepain c-I; 3, asclepain c-II.
Fig. 4 isoelectric focusing and corresponding zymogram
M, pI
markers, amyloglucosidase ( pI 3.50), trypsin inhibitor ( pI 4.55),
b-lactoglobulin a ( pI 5.20), carbonic anhydrase II ( pI 5.85), carbonic
anhydrase I ( pI 6.55), myoglobin ( pI 6.85, 7.15, and 7.35), lectins from
Lens culinaris ( pI 8.15, 8.45, and 8.65), and trypsinogen ( pI 9.30); 1,
crude extract; 2, asclepain c-I; 3, asclepain c-II; 4, zymogram.
Dependence of enzyme activity on pH
Asclepain c-II showed maximum activity within the
range of pH 9.4 and 10.2 (casein), which is higher than
that exhibited by the CE of Asclepias curassavica and
asclepain c-I [25], and similar to that of funastrain c-II
[22]. On the other hand, asclepain c-II showed a high
stability at pH 10 retaining about 95% of the residual
activity after 1 h (Fig. 5).
Fig. 3 Mass spectroscopy of asclepain c-II
MALDI-MS/TOF was
Dependence of enzyme activity on temperature
used for the determination of purity degree as well as of molecular
Asclepain c-II exhibited poor thermostability, as only
weight of asclepain c-II. Sample was mixed with sinapinic acid
30% of its activity was retained after 2 h of incubation at
(matrix) dissolved in 0.1% trifluoroacetic acid. Bovine trypsinogen was
508C (Fig. 6). At 60 – 708C the lost of activity is rapid.
used for internal calibration.
protein, which focused at pI higher than 9.3 (Fig. 4).
Effect of inhibitors on proteolytic activity
The pI value of asclepain c-II is comparable with those
Different inhibitors specific for distinctive classes of pro-
of other plant proteases from the Asclepiadaceae family
teases were used to determine the class to which the pur-
(Table 2).
ified protease belongs (Fig. 7). The enzyme was
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Papain-like protease from latex Asclepias curassavica L.
Table
2
Comparison
of
biochemical
characteristics
of
phytoproteases belonging to Asclepiadaceae family
Phytoproteases Molecular
pI
pH
References
mass (Da)
optima
Asclepain c-II
23,590
9.3 8.5
This study
Asclepain c-I
23,200
9.3 8.5
[25]
Araujiain a-I
23,464
9.3 8.00
Our previous
work
Araujiain a-II
23,528
9.3 8.25
Our previous
work
Araujiain a-III
23,488
9.3 9.25
Our previous
work
Fig. 6 Thermal stability on the proteolytic activity of the asclepain c-II
Araujiain h-I
24,031
9.3 8.75
[19]
at 408C, 508C, 608C, and 708C following 30, 60, 90, and 120 min
incubation
Araujiain h-II
23,718
8.9 8.5
[19]
Araujiain h-III 23,446
10.5 8.5
[19]
Asclepain f
23,652
9.3 9.5
[21]
Funastrain c-II 23,636
9.3 9.5
[22]
Morrenain b-I
23,205
9.3 8.70
[15]
Morrenain b-II 26,000
9.3 8.25
[17]
Morrenain o-I
27,000
9.3 8.5
[17]
Morrenain o-II 25,800
9.3 8.5
[17]
Fig. 7 Effect of inhibitors on the proteolytic activity of asclepain c-II
incubated on each inhibitor solution during 30 and 60 min at 308C
immediately and irreversibly inhibited by 10 mM E-64
(cysteine proteases inhibitor). Incubation with 100 mM
PMSF (serine proteases inhibitor) for 30 min decreased
activity to 63% of the initial value, but inhibition
was partially reverted when 12 mM cysteine was
added to the reaction mixture (data not shown). As
1,10-phenantroline
(metaloproteases
inhibitor)
and
pepstatine (aspartic proteases inhibitor) do not affect the
activity of asclepain c-II.
N-terminal protein sequence
The N-terminus sequence of asclepain c-II (30 amino
Fig. 5 Effect of pH
(A) Proteolytic activity of asclepain c-II
acids) was analyzed with the BLAST (http://www.ncbi.
measured on casein (range pH 6.0 – 11.0) using 0.01 M sodium salts of
nlm.nih.gov/BLAST/). This sequence showed higher
the following buffers: MES, MOPS, TAPS, AMPSO, and CAPS. (B)
identity (86% and 80%, respectively) with proteases iso-
Stability at pH optimum on the proteolytic activity of asclepain c-II,
using buffers adjusted to pH 10.0 following 1 – 3 h incubation at 208C.
lated from species of Asclepias and Funastrum genera
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Papain-like protease from latex Asclepias curassavica L.
(Table 3). Both, asclepain c-I and c-II, shared the motifs
ester. The obtained results were: K ¼
¼
m
0.1634 mM; Vm
surrounding the catalytic cysteine (CGS and WTFS), that
0.000974 mM/s; k
¼
¼
cat
121.48 s21; and kcat/Km
7.4 Â
also occur in most of the sequences compared. The
105 s21/mM. The kcat/Km values within a range from
DWR and QG motifs are notably conserved and are
105–106 s21/mM revealed a high affinity of the enzyme for
present in all the cases, as well as the proline residue
this substrate.
located in position 2, to which is attributed a protective
anti-aminopeptidase role [38].
Discussion
Measurement of endoesterolytic activity
When the enzyme was assayed using N-a-Cbz-L-amino
Although asclepain c-II is the minor fraction, it showed
acid-p-nitrophenyl esters, the highest relative esterolytic
higher specific activity (12.8 Ucas/mg) than asclepain c-I
activity was obtained for the glutamine derivative
(10.8 Ucas/mg). The molecular mass of this new enzyme
(Table 4), as happened with asclepain c-I [25].
is very close to that of asclepain c-I [25] and similar to
those obtained for other species of the Asclepias genus,
Measurement of amidolytic activity
like Asclepias syriaca, that displays proteases of molecu-
Asclepain c-II exhibited very low activity on PFLNA, a
lar weight 21,000 and 23,000 Da [39,40], Asclepias
typical substrate for cysteine endopeptidases (data not
glaucescens, with a single protease of molecular weight
shown), unlike asclepain c-I, which showed an important
23,000 Da [36,37] and Asclepias fruticosa, also contain-
activity on this substrate [25].
ing a single protease of molecular weight 23,652 Da
[21], as well as those from other genera of the same
Kinetic parameters
family, within the interval from 20 to 35 kDa [16,18,22].
Km, kcat, and kcat/Km ratio of asclepain c-II were determined
The important loss of proteolytic activity at 60 – 708C
with the substrate for which the CE showed the greatest
of asclepain c-II denotes a different behavior from other
preference, that is N-a-Cbz-L-amino acid-p-nitrophenyl
proteases
studied
in
our
laboratory
[15,16,18,19].
Table 3 Comparison of N-terminal amino acid sequences of asclepain c-II and other cysteine plant endopeptidases
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Papain-like protease from latex Asclepias curassavica L.
Table
4
Asclepain
c-II’s
endoesterolytic
activity
using
As most cysteine proteases obtained from other plant
N-a-Cbz-L-Gln p-nitrophenyl esters of some L-amino acids as
latex sources, asclepain c-II, the new protease isolated
substrates
and purified from the latex of A. curasavica, could be
useful in pharmaceutical and biotechnology industries
N-a-Cbz-L-amino acid p-nitrophenyl ester
Ucbz
due to their wide ranges of activity over temperature and
Gln
75.35
pH [43,44].
Asn
130.00
Ala
181.28
Funding
Gly
226.06
Tyr
361.88
This work was supported by the grants from ANPCyT
Asp
904.26
(PICT
9-9916),
CONICET
(PIP
2813)
and
the
Val
0
University of La Plata, Argentina, as well as for CYTED
Leu
0
IV.22. C.L. is member of the CIC Support Professional
Ile
0
Career Program. W.D.O. is CONICET fellowship holder.
Lys
0
An arbitrary enzyme activity unit (Ucbz) was defined as the amount
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