158 M.T. Cheeseman et al. Insect Biochemistry and Molecular Biology 31 2001 157–164
coln, 1976 and blackflies Wirtz, 1990 and tick phos- pholipase A
2
Bowman et al., 1997; Zhu et al., 1998, are not so clear because naturally occurring ester and
phospholipid substrates have not been identified. In this study we describe the heterogeneity and glyco-
sylation of cat flea salivary gland naphthyl esterases and document
salivary gland
platelet-activating factor
PAF-acetylhydrolase activity. The
potential anti-
inflammatory and anti-allergic role of salivary gland PAF-acetylhydrolase activity in fleas and other blood-
feeding arthropods is discussed.
2. Methods and materials
2.1. Animals Professor D. Jacobs of The Department of Pathology
and Infectious Diseases, The Royal Veterinary College, University of London, supplied the cat fleas. One to two
weeks old,
unfed male
and female
cat fleas
Ctenocephalides felis, strain RVC 003 were used in these experiments.
2.2. Chemicals Chemicals were purchased from Sigma unless other-
wise indicated. PAF 1-O-hexadecyl-2-O-acetyl-sn-gly- cero-3-phosphocholine was purchased from Bachem,
and [
3
H]PAF 1-O-hexadecyl-2-[
3
H]acetyl-sn-glyceryl- 3-phosphorylcholine, 13.5 Cimmol, was purchased
from NEN Life Sciences Inc. 2.3. Sample preparation
Cold immobilised fleas were dissected in ice-cold ster- ile phosphate-buffered saline PBS. Salivary glands
SG and the remainder of the carcass minus the salivary glands hereafter referred to as carcasses were each
transferred to an aliquot of appropriate ice-cold buffer ca. one salivary gland pair per
µ l; one carcass per 5 or
10 µ
l. Samples were stored frozen at 270 °
C for up to 8 weeks before thawing and homogenisation on ice. Sali-
vary gland preparations were adjusted to 0.5 Triton X-100 and homogenised with a micropestle in a 1.5 ml
polypropylene tube. Carcass samples were homogenised with short bursts of an electric dispersal probe Ultra-
Turrax, IKA Labotechnic. The homogenates were fil- tered through a 0.22
µ m microcentrifuge filter and the
filtrate stored on ice until use within 1 h. To determine the specific activity of whole body ester-
ases, groups of 80–100 frozen fleas were weighed, then homogenised in 1 ml of PBS with 0.5 Triton X-100.
Aliquots were assayed for protein Peterson, 1977 using bovine serum albumin BSA as a standard and for ester-
ase activity see below. 2.4. Esterase assay
Enzyme extracts were mixed with a 200 µ
l aliquot of substrate solution containing 0.83 mM
α -naphthyl acet-
ate α
NA or β
-naphthyl acetate β
NA in 0.1 M sodium phosphate buffer with 0.5 Triton X-100 pH 6.5 TP
buffer 4 vv 20 mM NA stock solution in ethanol diluted in TP buffer. In addition, a series of naphthyl
ester substrates — α
- and β
-naphthyl propionate, α
- and β
-naphthyl butyrate and α
-naphthyl caprylate — were used to assay partially purified esterase see below. The
mixture was incubated at 25 °
C for 15–120 min and the reaction stopped by the addition of 800
µ l of 0.19 mgml
Fast Garnet in TP buffer. The absorbance was read 10 min later at A
600
and A
490
for the α
- and β
-naphthol reaction products, respectively, and converted to nmol of
naphthol by interpolation with the appropriate standard curve. Values for enzyme reactions were corrected for
spontaneous breakdown of substrate by subtraction of control values from reactions in which buffer alone was
incubated with substrate solution. Boiling the enzyme preparation abolished activity. Esterase activities are
expressed in pmol of substratemin at 25
° C; assays per-
formed in triplicate have a standard error SE of 2–3 of the mean. Specific activity values for SG preparations
were calculated using an estimate of |21 ng protein per salivary gland pair data from Cheeseman, 1998.
Michaelis
constants K
m
were determined
with Lineweaver–Burk plots using 0.1 mM to 0.8 mM
α NA
and β
NA substrates. The acetylcholinesterase activity of salivary gland extracts was tested using the substrate
acetylthiocholine iodide Baker et al., 1998.
2.5. Detection of secreted esterase Unfed adult fleas, 1–2 week old, were allowed 15 min
to probe nitrocellulose membrane wetted with 2 mM adenosine triphosphate ATP solution. Nitrocellulose
membranes were air dried, rinsed with distilled water, and stained with
α NAFast Blue BB substratestain sol-
ution cytochemistry
kit from
Sigma Chemical
Company. This staining procedure was also employed for IEF, Native–PAGE and SDS–PAGE gels see
below.
2.6. Native–PAGE of esterases Samples were homogenised in Native–PAGE sample
buffer with 0.5 Triton X-100. Aliquots containing ca. 40 SG pairs or up two carcasses approximately equal
amounts of esterase activity were electrophoresed on a discontinuous 4 stacking and 4 resolving gel, using
Ornstein–Davis Tris–Glycine buffers, at 100 V for 2 h at 10
° C. The gel was stained as outlined above.
159 M.T. Cheeseman et al. Insect Biochemistry and Molecular Biology 31 2001 157–164
2.7. IEF Samples were prepared in 50 vv glycerol with 0.5
Triton X-100. Aliquots 2 µ
l of extract containing ca. two SG pairs or |0.33 carcass equivalents were focused
on a 5 acrylamide gel with pH 3–10 ampholytes according to the manufacturer’s instructions Mini IEF
cell, Biorad alongside coloured IEF markers Sigma. The pI of the stained esterase bands was calculated by
interpolation with a standard curve.
2.8. Renaturation of esterase activity after SDS–PAGE A sample of 75 SG pairs in 75
µ l SDS–PAGE sample
buffer with 50 mM dithiothreitol was heated at 37 °
C for 60 min without homogenisation. Aliquots 25
µ l of the
filtrate were electrophoresed on a discontinuous SDS– PAGE 4 stacking and 12 resolving gel alongside
coloured molecular-weight markers Biorad. The ester- ase activity was renatured by incubating the gel for
60 min at 37
° C in 2.5 Triton X-100, rinsing three times
with distilled water, and then incubating for 30 min in 0.5 M Tris–HCl pH 7.6, 0.5 M NaCl, 0.375 M CaCl
2
and 0.67 Brij 35. The gel was stained for esterase activity
as outlined above. The molecular weight of esterase bands was interpolated by reference to the standard
curve of marker proteins.
2.9. Concanavalin A lectin chromatography of salivary gland esterases
A sample of 100 SG pairs prepared in 20 mM Tris pH 7.0, 50 mM NaCl, 1 mM CaCl
2
and 0.1 Triton X- 100 was layered on to a 0.5 ml Concanavalin A agarose
gel Vector Laboratories equilibrated in the same buffer and allowed to stand for 10 min. The column was
washed with four bed volumes at 4.8 mlh and 0.4 ml fractions collected. Bound material was eluted with an
8 ml gradient containing 0–300 mM
α -d-methyl manno-
pyranoside in the same buffer. Chromatography was per- formed at room temperature. Fractions were collected on
ice and then assayed for esterase activity against α
NA. 2.10. Glycopeptidase F deglycosylation of salivary
esterases Aliquots 11
µ l of PBS extract containing |40 SG
pairs were incubated at 25 °
C for 2 h with a 1 µ
l aliquot of glycopeptidase F containing either 0.65 U or 0.13 U
of activity Sigma. Controls reactions included omission of glycopeptidase F or SG extract. At the end of the
incubation period each reaction mixture was mixed with 24
µ l of 2
× -concentrated Native–PAGE buffer with 1
Triton X-100, electrophoresed on a native gel and stained as above.
2.11. Gel filtration Samples of 175 to 275 SG pairs were prepared in
50 mM Tris pH 7.0, 300 mM NaCl and 0.01 Triton X- 100, and passed through a 120 cm
× 1 cm diameter col-
umn packed with Ultrogel ACA 44 Sigma. The column was eluted at 4 mlh and 1 ml fractions collected. Frac-
tions were assayed for esterase activity with α
NA and PAF-acetylhydrolase see below. Chromatography was
performed at 5 °
C, and the column calibrated with mol- ecular-weight markers from Sigma alcohol dehydrogen-
ase, 150 kDa; albumin, 66 kDa; carbonic anhydrase, 29 kDa; cytochrome C, 12.4 kDa; and the void volume
determined with Blue Dextran, 2000 kDa. The molecu- lar weights of enzymes were determined by interpolation
with a standard curve of V
e
V against log molecular
weight. A pool of fractions containing esterase activity was frozen at 270
° C until assayed for activity against
a series of α
- and β
-naphthyl esters. 2.12. PAF-acetylhydrolase assay
PAF-acetylhydrolase activity in whole salivary gland extracts or gel filtration fractions was assayed using
[
3
H]PAF as described by Tselepis et al. 1991 and Kitsi- ouli et al. 1999. Aliquots of salivary extract prepared
in 1 mgml BSA in PBS pH 7.4, or gel filtration fractions diluted with an equal volume of 2 mgml BSA in PBS,
were added to 150 µ
l of substrate solution containing 40
µ M PAF and 148 nM [
3
H]PAF |0.3 µ
Ci per assay in 1 mgml BSAPBS and incubated at 37
° C for 30 min
or 60 min. The reaction was stopped by adding an equal volume of ice-cold 20 trichloroacetic acid TCA. The
mixture was held on ice for 5 min and then centrifuged at 14,000g for 4 min. An aliquot of supernatant contain-
ing the [
3
H]acetate reaction product was mixed with an equal volume of Microscint 40 Packard and counted on
a Packard Topcount Microscintillation counter. Assays performed in duplicate have an SE of 7 of the mean.
Control reactions included incubation of column buffer or boiled enzyme preparation with the substrate solution.
The total amount of non-isotopically labelled PAF hydrolysed in each reaction was extrapolated from the
proportion of [
3
H]acetate released in each reaction from the total [
3
H]PAF added calculated from counting an aliquot of [
3
H]PAF substrate solution. After TCA pre- cipitation, less than 0.15 of [
3
H] appeared in the super- natant of control reactions incubated with buffer alone,
boiling abolished enzyme activity and release of super- natant [
3
H] in control reactions without enzyme was ,0.15 in 60 mins. Triton X-100 in gel filtration frac-
tions 50 µ
l aliquots containing 0.01 detergent did not interfere with TCA precipitation of unhydrolysed
[
3
H]PAF with the BSA carrier.
160 M.T. Cheeseman et al. Insect Biochemistry and Molecular Biology 31 2001 157–164
3. Results
