TMP 1 F28
TMP 1 F28
TMP 1 F28
ABB
www.elsevier.com/locate/yabbi
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 been identified in the developing eggs of L. migratoria
inactive zymogen precursors. Enzyme activity is con- [10]. Hemolymph serine proteinases may play vital roles
trolled by the timing of activation and by subsequent in- in various physiological processes in insects. A 29-kDa
hibition by appropriate proteinase inhibitors. Classical hemocyte proteinase dissociates the fat body at meta-
serine proteinase zymogens are activated by proteolysis morphosis of Sarcophaga peregrina [16]. Hemolymph
[1,2], while cysteine proteinase zymogens are activated by proteinases are involved in the activation of the pro-
proteolysis and thiol disulfide exchange [2,3,22]. In in- phenoloxidase cascade in Blaberus craniifer [17]. Data
sects, serine proteinase zymogens have been found in collected on hemolymph cysteine proteinase (CP1) from
Drosophila embryos [4], in the cocoonase family of pro- Drosophila melanogaster suggest a role in immunity,
teinases produced by the Bombyx mori [5], and in the participating most likely in the degradation of inter-
hemolymph of B. mori [6]. Prococoonase from A. pol- nalized material in phagocytes [18].
phemus [23] has a molecular mass of 28 kDa, while the We herein report on the purification and character-
active enzyme has a lower molecular mass of 24 kDa. ization of two novel thiol-activated serine proteinases
Trypsin-like and chymotrypsin-like enzymes have from L. migratoria hemolymph.
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 Materials and methods
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 Proteolytic activity was detected in gels using 10%
were from Whatman (England). polyacrylamide containing 0.1% gelatin as substrate.
Insects. The African migratory locust (L. migratoria Electrophoresis was performed according to Laemmli
migratorioides) was reared in gregarious phase on grass [19] except that SDS was omitted. The gels were incu-
and flaked oats and at temperature of 28–32 °C. Fifth bated for 1 h at 37 °C in 100 mM Tris–HCl, pH 8.0,
(last) instar nymphs were used in this study. supplemented with 10 mM DTT, and then stained with
Enzyme assays. Specific proteinase activity was as- Coomassie blue R 250.
sayed with various peptidyl-p-nitroanilide (pNA)1 sub- Protein content. The protein content was measured by
strates. After a series of initial experiments to determine the method of Bradford [20], using bovine serum albu-
suitable pH and constituents for optimal activity, assays min (BSA) as the standard protein, or estimated by
were routinely performed on the serine protease chro- absorbance at 280 nm.
mogenic substrate Bz-Phe-Val-Arg-pNA. This was dis- Sequence determination. The N-terminal amino acid
solved in dimethyl formamide (DMF) and diluted to a sequence was determined by Edman degradation using
final concentration of 2.5 mM in the reaction mixture, an automated sequencer after reduction and alkylation
which was buffered with 100 mM Tris–HCl, pH 8.0, and of the peptides. The respective PTH-amino acid deriv-
supplemented with 2 mM dithiothreitol (DTT) and 2% atives were identified by reverse-phase HPLC analysis.
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 Results
with 30% acetic acid, and activity measured at 410 nm as
the release of pNA from Bz-Phe-Val-Arg-pNA. One unit Purification of trypsin-like hemolymph proteinases
of activity was defined as A410 nm=30 min .
Inhibition of proteinase activity was assayed by The sequence of steps leading to the purification of
preincubation of the enzymes for 30 min at 37 °C in the two L. migratoria trypsins (LmHP-1 and LmHP-2) is
2 mM DTT, which was later removed by dialysis, fol- presented in the form of a flowchart in Fig. 1.
lowed by a second preincubation step of 15 min at 37 °C Step 1. A total of 5 ml hemolymph was collected into
with various inhibitors. Residual enzyme activity was 25 ml of 10 mM ammonium acetate, pH 6.5, from about
assayed with Bz-Phe-Val-Arg-pNA as detailed above. 300 fifth instar locust nymphs. The dorsal integumental
Bowman–Birk inhibitor (BBI), soybean trypsin in- membrane of each nymph, between the head capsule
hibitor (STI), phenylbutylamine (PBA), and p-chloro- and the pronotum, was delicately punctured and he-
mercuribenzoic acid (pCMB) were dissolved in buffer. molymph was transferred by capillary immediately into
Phenylmethysulfonyl fluoride (PMSF) was dissolved in ice-cold buffer. No darkening of hemolymph samples
iso-propanol. N-a-tosyl-L -lysine chloromethyl ketone was observed during this process. Diluted hemolymph
(TLCK), N-a-tosyl-L -phenlalanine chloromethyl ketone was centrifuged at 4 °C for 10 min at 15,000g.
(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.
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 85
Table 1
Purification of LmHP-1 and LmHP-2 from nymphal hemolymph of Locusta migratoria
Step Volume E280 nm Total activity Specific activity Yield Purification
(ml) (U) (U/E280 nm ) (%) factor
Hemolymph 33 517 6000 11.6 100 1
DEAE-cellulose 42 16.72 3302 197 55 17
Sephadex G-75 35 0.18 3150 18,010 53 1550
Mono-S HP-1 2 0.006 252 42,000 4.2 3620
HP-2 2 0.021 870 41,430 14.5 3571
One unit of activity was defined as A410 nm=30 min .
86 J. Hanzon et al. / Archives of Biochemistry and Biophysics 410 (2003) 83–88
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).
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