Archives of Biochemistry and Biophysics 410 (2003) 83–88

ABB
www.elsevier.com/locate/yabbi

Thiol-activated serine proteinases from nymphal hemolymph of

the African migratory locust, Locusta migratoria migratorioides
Jacob Hanzon,a Patricia Smirnoff,a Shalom W. Applebaum,b Autar K. Mattoo,c
and Yehudith Birka,*
a
Institute of Biochemistry, Food Science and Nutrition, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel
b
Department of Entomology, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel
c
Vegetable Laboratory, Henry A. Wallace Beltsville Agricultural Research Center, Building 010A, Beltsville, MD 20705, USA
Received 8 July 2002, and in revised form 23 October 2002

Abstract

Two unique serine proteinase isoenzymes (LmHP-1 and LmHP-2) were isolated from the hemolymph of African migratory locust
(Locusta migratoria migratorioides) nymphs. Both have a molecular mass of about 23 kDa and are activated by thiol-reducing
agents. PMSF abolishes enzymes activity only after thiol activation, while the cysteine proteinase inhibitors E-64, iodoacetamide,
and heavy metals fail to inhibit the thiol-activated enzymes. The N-terminal sequence was determined for the more-abundant
LmHP-2 isoenzyme. It exhibits partial homology to that of other insect serine proteinases and similar substrate specificity and
inhibition by the synthetic and protein trypsin inhibitors pABA, TLCK, BBI, and STI. The locust trypsins LmHP-1 and LmHP-2
constitute a new category of serine proteases wherein the active site of the enzyme is exposed by thiol activation without cleavage of
peptide bonds.
Ó 2002 Elsevier Science (USA). All rights reserved.

Keywords: Insect trypsins; Thiol activation; Trypsin inhibitors; Locusta migratoria migratorioides

Proteinases often occur in the form of catalytically
inactive zymogen precursors. Enzyme activity is con-
trolled by the timing of activation and by subsequent in-
hibition by appropriate proteinase inhibitors. Classical
serine proteinase zymogens are activated by proteolysis
[1,2], while cysteine proteinase zymogens are activated by
proteolysis and thiol disulfide exchange [2,3,22]. In in-
sects, serine proteinase zymogens have been found in
Drosophila embryos [4], in the cocoonase family of pro-
teinases produced by the Bombyx mori [5], and in the
hemolymph of B. mori [6]. Prococoonase from A. pol-
phemus [23] has a molecular mass of 28 kDa, while the
active enzyme has a lower molecular mass of 24 kDa.
Trypsin-like and chymotrypsin-like enzymes have
been isolated from the digestive tract of Locusta mi-
gratoria in their active form only [7–9]. Cathepsin-like
enzymes activated in vitro by thiol-reducing agents have
been identified in the developing eggs of L. migratoria
[10]. Hemolymph serine proteinases may play vital roles
in various physiological processes in insects. A 29-kDa
hemocyte proteinase dissociates the fat body at meta-
morphosis of Sarcophaga peregrina [16]. Hemolymph
proteinases are involved in the activation of the pro-
phenoloxidase cascade in Blaberus craniifer [17]. Data
collected on hemolymph cysteine proteinase (CP1) from
Drosophila melanogaster suggest a role in immunity,
participating most likely in the degradation of inter-
nalized material in phagocytes [18].
We herein report on the purification and character-
ization of two novel thiol-activated serine proteinases
from L. migratoria hemolymph.
Materials and methods

Reagents. Solvents were from Merck (Darmstadt,

*
Corresponding author. Fax: +972-8-9473654. Germany). p-Nitroanilide chromogenic substrates, pro-
E-mail address: birk@agri.huji.ac.il (Y. Birk). teinase inhibitors, Sephadex G-75, and other reagents

0003-9861/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved.
PII: S 0 0 0 3 - 9 8 6 1 ( 0 2 ) 0 0 6 5 7 - 4
84 J. Hanzon et al. / Archives of Biochemistry and Biophysics 410 (2003) 83–88

were from Sigma (St. Louis, MO). The ion exchangers
were from Whatman (England).
Insects. The African migratory locust (L. migratoria
migratorioides) was reared in gregarious phase on grass
and flaked oats and at temperature of 28–32 °C. Fifth
(last) instar nymphs were used in this study.
Enzyme assays. Specific proteinase activity was as-
sayed with various peptidyl-p-nitroanilide (pNA)1 sub-
strates. After a series of initial experiments to determine
suitable pH and constituents for optimal activity, assays
were routinely performed on the serine protease chro-
mogenic substrate Bz-Phe-Val-Arg-pNA. This was dis-
solved in dimethyl formamide (DMF) and diluted to a
final concentration of 2.5 mM in the reaction mixture,
which was buffered with 100 mM Tris–HCl, pH 8.0, and
supplemented with 2 mM dithiothreitol (DTT) and 2%
butanol. Proteolytic activity was assayed after preincu-
bation of the sample in assay buffer for 20 min. After
30 min incubation at 37 °C, proteolysis was terminated
with 30% acetic acid, and activity measured at 410 nm as
the release of pNA from Bz-Phe-Val-Arg-pNA. One unit
of activity was defined as A410 nm=30 min .
Inhibition of proteinase activity was assayed by
preincubation of the enzymes for 30 min at 37 °C in
2 mM DTT, which was later removed by dialysis, fol-
lowed by a second preincubation step of 15 min at 37 °C
with various inhibitors. Residual enzyme activity was
assayed with Bz-Phe-Val-Arg-pNA as detailed above.
Bowman–Birk inhibitor (BBI), soybean trypsin in-
hibitor (STI), phenylbutylamine (PBA), and p-chloro-
mercuribenzoic acid (pCMB) were dissolved in buffer.
Phenylmethysulfonyl fluoride (PMSF) was dissolved in
iso-propanol. N-a-tosyl-L -lysine chloromethyl ketone
(TLCK), N-a-tosyl-L -phenlalanine chloromethyl ketone
(TPCK), p-aminobenzamidine (pABA), and HgCl2 were
dissolved in ethanol. trans-Epoxy succinyl-L -leucylam-
ido-(4-guanidino)butane (E-64), was dissolved in di-
methyl sulfoxide (DMSO).
Polyacrylamide gel electrophoresis. PAGE was per-
formed according to Laemmli [19] in the presence of
SDS. Gels were run in a Hoeffer mini-gel apparatus at
120 V and 40 mA. Staining was performed with Coo-
massie blue R 250.
Proteolytic activity was detected in gels using 10%
polyacrylamide containing 0.1% gelatin as substrate.
Electrophoresis was performed according to Laemmli
[19] except that SDS was omitted. The gels were incu-
bated for 1 h at 37 °C in 100 mM Tris–HCl, pH 8.0,
supplemented with 10 mM DTT, and then stained with
Coomassie blue R 250.
Protein content. The protein content was measured by
the method of Bradford [20], using bovine serum albu-
min (BSA) as the standard protein, or estimated by
absorbance at 280 nm.
Sequence determination. The N-terminal amino acid
sequence was determined by Edman degradation using
an automated sequencer after reduction and alkylation
of the peptides. The respective PTH-amino acid deriv-
atives were identified by reverse-phase HPLC analysis.
Results
Purification of trypsin-like hemolymph proteinases
The sequence of steps leading to the purification of
the two L. migratoria trypsins (LmHP-1 and LmHP-2) is
presented in the form of a flowchart in Fig. 1.
Step 1. A total of 5 ml hemolymph was collected into
25 ml of 10 mM ammonium acetate, pH 6.5, from about
300 fifth instar locust nymphs. The dorsal integumental
membrane of each nymph, between the head capsule
and the pronotum, was delicately punctured and he-
molymph was transferred by capillary immediately into
ice-cold buffer. No darkening of hemolymph samples
was observed during this process. Diluted hemolymph
was centrifuged at 4 °C for 10 min at 15,000g.

1
Abbreviations used: LmHP-1 and LmHP-2, Locusta migratoria
hemolymph proteases 1 and 2; PMSF, phenylmethylsulfonyl fluoride;
pABA, p-aminobenzamidine; TLCK, N-a-tosyl-L -lysine chloromethyl
ketone; BBI, Bowman–Birk soybean inhibitor; STI, soybean trypsin
inhibitor; pNA, p-nitroanilide; DMF, dimethyl formamide; DTT,
dithiothreitol; PBA, phenylbutylamine; pCMB, p-chloromercuriben-
zoic acid; TPCK, N-a-tosyl-L -phenylalanine chloromethyl ketone; E-
64, trans-epoxy succinyl-L -leucylamido-(4-guanidino)butane; DMSO,
dimethyl sulfoxide; PAGE, polyacrylamide gel electrophoresis; SDS,
sodium dodecyl sulfate; PTH, phenylthiohydantoin; HPLC, high-
performance liquid chromatography; EDTA, ethylenediaminetetra- Fig. 1. Flow chart of purification protocol for hemolymph trypsins
acetic acid. from L. migratoria.
 J. Hanzon et al. / Archives of Biochemistry and Biophysics 410 (2003) 83–88
Fig. 2. Enzymes activity in situ in gels detected as digestion of 0.1%
gelatin in native PAGE. Lane 1: Hemolymph proteolytic profile. Lane
2: Void volume fraction from DEAE-cellulose column. After electro-
phoresis the gel was incubated at 37 °C for 30 min with Tris–HCl, pH
8.0, containing 10 mM DTT and then was stained with Coomassie
brilliant blue.
Step 2. The supernatant from step 1 was loaded onto
a DE-52 anion-exchange column (1:4
25 cm) and
equilibrated with 10 mM ammonium acetate, pH 6.5.
The column was eluted at 4 °C with three stepwise
concentrations of NaCl (50, 200, and 500 mM) in the
same buffer at a flow rate of 20 ml/h. Four milliliter
fractions were collected (elution profile not shown). Two
proteinases eluted with the void volume while a minor
peak of proteolytic activity appeared after elution with
0.5 M NaCl. The void volume was collected, pooled, and
concentrated to a volume of 3 ml with an ultrafiltration
cell (Amicon 8010) MWCO-3000. This step separates
the two proteolytic activities, designated LmHP-1 and
LmHP-2, from other hemolymph proteinases, as shown
by gelatin-PAGE (Fig. 2). The major proteolytic activity
was found to be purified 17-fold with a yield of 55%
(Table 1).
Step 3. The void volume eluted in step 2 was sub-
jected to gel filtration on a Sephadex G-75 column
(1:4
110 cm), equilibrated with 50 mM ammonium
acetate buffer, pH 6.5, at a flow rate of 12 ml/h Fractions
Table 1
Purification of LmHP-1 and LmHP-2 from nymphal hemolymph of Locusta migratoria
Step Volume E280 nm
(ml)
Hemolymph 33 517
DEAE-cellulose 42 16.72
Sephadex G-75 35 0.18
Mono-S HP-1 2 0.006
HP-2 2 0.021
One unit of activity was defined as A410 nm=30 min .
85
of 2.5 ml each were collected. Both enzymes migrated as
a single peak (elution profile not shown). The peak
fractions containing the proteinases were pooled and
lyophilized. Specific activity increased 1550-fold with a
yield of 53%.
Step 4. The proteolytic activity that emerged from the
Sephadex G-75 column was applied to a cation-ex-
change Mono-S column (Fig. 3A). HPLC was per-
formed for 15 min with 10 mM ammonium acetate, pH
4.0, at a flow rate of 0.5 ml/min, followed by stepwise
elution with increasing buffer concentration after 15, 45,
and 75 min to 20, 50, and 100 mM, respectively. The
active fractions were pooled and lyophilized. This pro-
cedure separated the two isoenzymes, designated
LmHP-1 and LmHP-2. LmHP-1 and LmHP-2 were
homogeneous as shown by SDS–PAGE in the presence
of b-mercaptoethanol (Fig. 3B) with a recovery of 4.2
and 14.5%, respectively, and 3600-fold purification.
Characteristics of LmHP-1 and LmHP-2
The molecular mass of both LmHP-1 and LmHP-2 is
23 kDa as indicated by SDS–PAGE (Fig. 3B). Both are
activated by the thiol-reducing agents DTT, b-mercap-
toethanol, cysteine, and sulfite ions (Fig. 4). DTT was
routinely used to activate the two enzymes and subse-
quently removed by dialysis. Removal of the reducing
agent after activation did not affect enzyme activity. The
effect of proteinaceous and synthetic inhibitors on the
activity of LmHP-1 and LmHP-2 on Bz-Phe-Val-Arg-
pNA is given in Tables 2 and 3. The serine protease
inhibitor PMSF irreversibly inhibited the activated
LmHP-1 and LmHP-2 but did not inhibit if added be-
fore the DTT activation. All of the four inhibitors of
trypsin-like enzymes, TLCK, pABA, STI, and BBI, ef-
fectively inhibited the activation of LmHP-1 and
LmHP-2. These results indicate the presence of essential
serine and histidine residues at the active site. The spe-
cific cysteine proteinase inhibitor E-64 or the sulfydryl
reagents pCMB, HgCl2 ; AgNO3 , and iodoacetamide,
had no effect on LmHP-1 and LmHP-2 activity, either
prior or subsequent to thiol activation. These results
exclude the possibility of thiol being involved in the
active site of the enzymes. The metal chelators EDTA
Total activity Specific activity Yield Purification
(U) (U/E280 nm ) (%) factor
6000 11.6 100 1
3302 197 55 17
3150 18,010 53 1550
252 42,000 4.2 3620
870 41,430 14.5 3571
86 J. Hanzon et al. / Archives of Biochemistry and Biophysics 410 (2003) 83–88

Fig. 4. Effect of various thiols on LmHP-2 activity. Purified proteinase

was assayed at 37 °C in 100 mM Tris–HCl, pH 8.0, 2% butanol, and a
thiol-reducing agent. Activities are expressed as A410 nm per 30 min.
DTT ðsÞ, cysteine ðDÞ, Na2 SO4 ðdÞ, b-mercaptoethanol ðÞ.

Table 2
Activation mechanism of LmHP-1 and LmHP-2 by thiol reducing
agent (DTT)
Preincubation Enzyme activity
with
In the absence of DTT In the presence of DTT
LmHP-1 LmHP-2 LmHP-1 LmHP-2
Control (none) 0 0 100 100
DTT 2 mM 107 108 101 98
PMSF 10 mM 0 0 100 102
DTT 2 mM + PMSF 7 4 6 5
10 mM
LmHP-1 and LmHP-2 were preincubated in 100 mM Tris–HCl, pH
8.0, at 37 °C for 20 min, with different combinations of 10 mM PMSF
and 2 mM DTT. After the preicubation step, the enzymes were dia-
lyzed at 4 °C and assayed in 100 mM Tris–HCl, pH 8.0, containing 2%
butanol, with or without 2 mM DTT at 37 °C for 30 min. These values
are expressed as percentages relative to control and are given as means
(N ¼ 3).

and 1,10-phenanthroline did not affect the two enzymes,

Fig. 3. (A) Cation-exchange HPLC of the proteolytic peak from the
Sephadex G-75 column on a column of Mono-S. Elution was per-
formed for 15 min with 10 mM ammonium acetate, pH 4.0, at a flow
rate of 0.5 ml/min, followed by stepwise elution with 20, 50, and
100 mM ammonium acetate, pH 4.0, after 15, 45, and 75 min, respec-
tively. The upper graph is the protein elution profile monitored at
280 nm. The lower graph represents the plotted levels of pNA released
by proteolytic activity of each individual fraction, as described under
Materials and methods. (B) SDS–PAGE of the purified proteinases
LmHP-1 and LmHP-2 obtained after Mono-S column chromatogra-
phy. Lane 1: purified LmHP-2. Lane 2: purified LmHP-1. Lane 3:
Molecular weight markers.
indicating that the enzymes are not metalloproteinases.
The optimal pH of the proteinases for the hydrolysis
of Bz-Phe-Val-Arg-pNA was in the range of 7.5–8.5
(data not shown). The enzymes were not influenced by
either of the cations, tested Naþ ; Kþ ; Mgþ2 , or Caþ2 .
Short aliphatic alcohols, on the other hand, increased
the activity; 70% activation was obtained by the addi-
tion of 2% butanol to the reaction mixture.
Substrate specificity
The specificity of purified LmHP-1 and LmHP-2 was
investigated using a number of pNA substrates. The
preferred substrate was Bz-Val-Leu-Arg-pNA (Table 4).
Both enzymes preferred substrates with arginine at the
P1 position. Substituting lysine for arginine at P1 re-
duced activity by 49 and 39% for LmHP-1 and LmHP-2,
 J. Hanzon et al. / Archives of Biochemistry and Biophysics 410 (2003) 83–88 87

Table 3 LmHP-1 and LmHP-2, respectively, compared to -Val-

Effect of proteinase inhibitors on amidolytic activity of purified Leu-. Substituting Gly for Leu at the P2 position re-
LmHP-1 and LmHP-2
duced activity by 60 and 40% for LmHP-1 and LmHP-2,
Inhibitor Concentration % Activity remaining respectively. No activity was found when substrates for
HP-2 HP-1 chymotrypsin and elastase were used.
None 100 100
Serine proteinase inhibitors Discussion
PMSF 10 mM 4 5
TLCK 1 mM 50 33
TPCK 1 mM 91 90 Two L. migratoria larval hemolymph isoenzyme
PABA 10 mM 2 3 proteinases, Lm HP-1 and LmHP-2, have been purified
PBA 10 mM 77 71 to homogeneity and characterized. Both enzymes are
STI 2 lM 30 6 activated by thiol or sulfite ions, which is characteristic
BBI 2 lM 4 5
of cysteine proteinases [3] but are not inhibited by E-64,
Cysteine proteinase inhibitors a specific inhibitor of cysteine proteinases, nor by the
E-64 0.2 mM 100 101 sulphydryl reagents iodoacetamide, PCMB, HgCl2 , or
Iodoacetamide 100 mM 100 100
HgCl2 1 mM 114 110 AgNO3 . LmHP-1 and LmHP-2 are unaffected by PMSF
AgNO3 1 mM 100 100 prior to thiol activation, but are completely inhibited by
pCMB 1 mM 100 102 PMSF after thiol activation; PMSF inhibition of papain
Metalloproteinase inhibitors
is reversed by reducing agents [21], but the inhibition of
1,10-Phenantroline 10 mM 101 100 LmHP-1 and Lm HP-2 by PMSF is not reversed by
EDTA 10 mM 100 102 DTT. These results suggest the presence of a serine
The purified enzymes were preincubated in 100 mM Tris–HCl, pH residue at the reactive site as the nucleophilic agent and
8.0, containing 2 mM DTT, and 2% butanol for 20 min at 37 °C (en- exclude the possibility of a cysteine residue being the
zyme activation). After dialysis to remove DTT, a second incubation nucleophilic agent. Classification of the enzymes as
step was performed with the inhibitor for 15 min. The activity was
serine type proteinases is also supported by the simi-
measured at 37 °C with Bz-Phe-Val-Arg-pNA as substrate in 100 mM
Tris–HCl, pH 8.0, containing 2% butanol. These values are expressed larity of LmHP-2 N-terminal sequence to those of other
as percentages relative to control and are given as means  SD, N ¼ 3. trypsin-like and chymotrypsin-like enzymes (Fig. 5).
From the effect of the inhibitors pABA, SBTI,
TLCK, and BBI (Table 3), and substrate specificity
Table 4 (Table 4), both LmHP-1 and LmHP-2 can be classified
Substrate specificity of purified LmHP-1 and LmHP-2 as trypsin-like enzymes. We conclude that these enzymes
Substrate Relative activity (%) are serine proteinase zymogens activated by the opening
HP-1 HP-2 of disulfide bridge(s). Thiol-activated LmHP-1 and
LmHP-2 are unrelated to the clip-domain family of
Val-Leu-Arg-pNA 100 100
Val-Leu-Lys-pNA 51 61 serine proteinases [22].
Bz-Phe-Val-Arg-pNA 75 87 Different from the hemolymph proteinases LmHP-1
Bz-Val-Gly-Arg-pNA 40 60 and LmHP-2, no inactive zymogen have been identified
Boc-Leu-Ser-Thr-Arg-pNA 50 64 for the trypsin- and chymotrypsin-like enzymes purified
Bz-Pro-Phe-Arg-pNA 39 46
from L. migratoria digestive tract [7,8]. Trypsin zymo-
Bz-Arg-pNA 3 4
Ac-Ala-Ala-Ala-pNA 0 0 gens in the digestive tract of insects have been previously
Bz-Glu-Phe-Leu-pNA 0 0
Ac-Tyr-pNA 0 0
LmHP-1 and LmHP-2 were assayed at 37 °C in 100 mM Tris–HCl,
pH 8.0, 2% butanol, and 2 mM DTT. Activities are expressed as a
percentages of the maximal activity. Values are given as means (N ¼ 3).
Abbreviations: Bz, N-a-benzoyl; Boc, N-tert-butoxy-carbonyl; Ac,
acetyl.

respectively. The minimal substrate, Bz-Arg-pNA, was

hydrolyzed at a very slow rate. The secondary specificity
of the enzymes was examined by substituting groups at
P2 ; P3 , and P4 . Replacement of the sequence -Val-Leu-
with -Phe-Val-, -Pro-Phe-, or -Leu-Ser-Thr- resulted in Fig. 5. N-terminal sequence of LmHP-2, and its comparison with
reduced activity. The least favored substitution -Pro- N-terminal sequences of other trypsin-like and chymotrypsin-like
Phe- resulted in 61 and 54% activity reduction for enzymes from insects.
88 J. Hanzon et al. / Archives of Biochemistry and Biophysics 410 (2003) 83–88
deduced on the basis of isolation of cDNA from
Manduxa sexta [24] and Aedes aegypti [25] midgut.
Preliminary evidence [26] indicates that the activity
profile of LmHP-1 and LmHP-2 in the hemolymph
during the fifth larval instar is coincident with apolysis
and the molting process. This supports the notion that
these hemolymph proteinases may be transferred at the
appropriate time into the integumental molting fluid and
might participate in cuticle degradation.
Chitinases of the silkworm (B. mori) and the tobacco
hornworm (M. sexta) are presumably synthesized dur-
ing the larval to pupal transition as inactive zymogens,
which are apparently activated by limited proteolysis
[27]. During the progress of cuticle degradation pre-
ceding eclosion of the tobacco hornworm to the adult
form, the developmental profiles of two molting fluid
proteases are in correlation to progression of cuticle
degradation whereas chitin-degradative enzymes did not
parallel weight loss from the old cuticle. Protease ac-
tivity is a proposed to be prerequisite to chitin degra-
dation by chitinase [28].
References
[1] L. Polg
ar, in: A. Neuberger, K. Brocklehurst (Eds.), Hydrolytic
Enzymes, Elsevier, Amsterdam, 1987, pp. 159–170.
[2] A.R. Khan, M.N.G. James, Protein Sci. 7 (1998) 815–836.
[3] K. Brocklehurst, F. Willenbrock, E. Salih, in: A. Neuberger, K.
Brocklehurst (Eds.), Hydrolytic Enzymes, Elsevier, Amsterdam,
1987, pp. 39–158.
[4] C.L. Smith, R. DeLotto, Nature 368 (1994) 548–551.
[5] F.C. Kafatos, C.M. Williams, Science 146 (1964) 538–540.
[6] Y. Katsumi, H. Kihara, M. Ochiai, M. Ashida, Eur. J. Biochem.
228 (1995) 870–877.
[7] E. Sakal, S.W. Applebaum, Y. Birk, Int. J. Pept. Protein Res. 32
(1988) 590–598.
[8] E. Sakal, S.W. Applebaum, Y. Birk, Int. J. Pept. Protein Res. 34
(1989) 98–505.
[9] W. Lam, G.M. Coast, R.C. Rayne, Insect Biochem. Mol. Biol. 27
(1999) 653–660.
[10] S. Kuk-Meiri, N. Lichtenstein, A. Shulov, M.P. Pener, Comp.
Biochem. Physiol. 18 (1966) 783–795.
[11] C. Kellenberger, C. Boudier, I. Bermudez, J.G. Bieth, B. Luu, H.
Hietter, J. Biol. Chem. 270 (1995) 25514–25519.
[12] N. Nakakura, H. Hietter, A. Van Dorsselaer, B. Luu, Eur. J.
Biochem. 204 (1992) 147–153.
[13] E. Kromer, N. Nakakura, M. Lagueux, Insect Biochem. Mol.
Biol. 24 (1994) 329–331.
[14] A. Hamdaoui, S. Wataleb, B. Devreese, S.J. Chiou, J. Vanden
Broeck, J. Van Beeumen, A. DeLoof, L. Schoofs, FEBS Lett. 422
(1998) 74–78.
[15] Z. Malik, S. Amir, J. P
al, Z. Buz
as, E. V
arallyay, J. Antal, Z.
Szil
agyi, K. Vek
ey, B. Asb
oth, A. Patthy, L. Gr
af, Biochim.
Biophys. Acta 1434 (1999) 143–150.
[16] S. Kurata, H. Saito, S. Natori, Dev. Biol. 153 (1992) 115–
121.
[17] C. Leonard, K. S€ oderh€all, N.A. Ratcliffe, Insect Biochem. 15
(1985) 803–810.
[18] Y. Tryselius, D. Hultmark, Insect Mol. Biol . 6 (1997) 173–181.
[19] U.K. Laemmli, Nature 227 (1970) 680–685.
[20] M.M. Bradford, Anal. Biochem. 72 (1976) 248–254.
[21] J.R. Whitaker, J. Perez-Villase~ nor, Arch. Biochem. Biophys. 124
(1968) 70–78.
[22] H. Jiang, M.R. Kanost, Insect Biochem. Mol. Biol. 30 (2000) 95–
105.
[23] J.H. Law, P.E. Dunn, K.J. Kramer, Insect proteases and
peptidases, in: A. Meister (Ed.), Adv. Enzymol., vol. 35, Wiley,
New York, 1977, pp. 389–425.
[24] A.M. Peterson, C.V. Barillas-Mury, M.A. Wells, Insect Biochem.
Mol. Biol. 24 (1994) 463–471.
[25] C. Barillas-Mury, R. Graf, H.H. Hagedorn, M.A. Wells, Insect
Biochem. 21 (1991) 825–831.
[26] J. Hanzon, Ph.D thesis, The Hebrew University of Jerusalem,
1997.
[27] D. Koga, T. Funakoshi, K. Mizuki, A. Ide, K.J. Kramer, K.C.
Zen, H. Choi, S. Muthukrishnan, Insect Biochem. Mol. Biol. 22
(1992) 305–311.
[28] R.I. Samuels, S.E. Reynolds, Arch. Insect Biochem. Physiol. 24
(1993) 33–44.