160 M.T. Cheeseman et al. Insect Biochemistry and Molecular Biology 31 2001 157–164
3. Results
Homogenates of salivary glands had esterase activity against
α NA
|210 pmolmingland pair,
10.0 µ
molminmg specific
activity and
β NA
|110 pmolmingland pair, 5.2 µ
molminmg specific activity. The K
m
for SG homogenates are 59 ±
6 µ
M α
NA mean ±
SE, n =
2 and 132 ±
12 µ
M β
NA. When fleas were allowed to probe nitrocellulose membranes wetted
with a phagostimulant 2 mM ATP solution, they deposited clusters of naphthyl-esterase-positive saliva
spots that were approximately the diameter of the mouthparts Fig. 1. Control membranes that had been
exposed to flea probing but developed in solution omit- ting
α NA did not give a positive reaction. Fleas weigh
on average |0.5 mg and whole flea homogenates yielded 26
± 2
µ g
of soluble
protein per
flea n
= 4
and 3.9
± 0.7 nmolmin of
α NA esterase activity per flea
|0.15 ±
0.01 µ
molminmg esterase specific activity. Native–PAGE resolves SG esterases into three major
bands of low relative mobility; one band is retained in the stacking gel. The fastest migrating band has a similar
mobility to the slowest migrating of the seven carcass esterase bands some faint carcass esterase bands do not
photograph well enough to be seen in Fig. 2. The same patterns of SG and carcass esterases occur when
β NA
is used as the substrate data not shown. IEF of SG esterases resolves four sharply focused bands of pI|6.4,
|6.2, |5.9 and |5.7, and two less distinct bands of pI|8.3 and |7.5 average of two separate experiments.
Carcass esterases are resolved into a separate pattern of four bands at pI|5.4, |5.2, |5.1 and |4.9 Fig. 3. Rena-
turation of enzyme activity after SDS–PAGE gave sharp, closely grouped |56 kDa, |57 kDa and |58 kDa naph-
thyl-esterase-positive bands Fig. 4.
Concanavalin A affinity chromatography of SG
Fig. 1. Esterase activity against
α -naphthyl acetate in blots of
secreted saliva ×
250. Unfed adult fleas, 1–2 week old, were allowed 15 min to probe nitrocellulose membrane wetted with 2 mM ATP sol-
ution. Membranes were air dried, rinsed with distilled water and stained with
α -naphthyl acetateFast Blue BB, substratestain solution.
See text for details. Fig. 2.
Native–PAGE of cat flea esterases. Salivary glands have three major esterase bands with low relative mobility and one retained in
the stacking gel, compared with bands with higher relative mobility found in the remainder of the carcass. Lane 1, two carcasses; lane 2,
one carcass; lane 3, 40 salivary gland pairs. Extracts were electrophor- esed on a discontinuous native gel the boundary between the 4
stacking and 4 resolving gels is marked with an arrow and stained with
α -naphthyl acetateFast Blue BB. See text for details.
Fig. 3. IEF of cat flea esterases. Salivary glands have six esterase
bands of pI|8.3, |7.5, |6.4, |6.2, |5.9 and |5.7 lane 2, compared with four carcass esterases of pI|5.4, |5.2, |5.1 and |4.9 lane 1.
Extracts two salivary glands; 0.33 carcasses were focused on a 5 polyacrylamide gel with pH 3–10 ampholytes alongside coloured stan-
dards, and focused gel stained for esterase activity. Positions of indi- vidual esterases are marked and the lowest and highest pI values are
indicated. See text for details.
161 M.T. Cheeseman et al. Insect Biochemistry and Molecular Biology 31 2001 157–164
Fig. 4. Renaturation of cat flea salivary gland esterase activity after
SDS–PAGE resolves closely grouped |56 kDa, |57 kDa and |58 kDa naphthyl-esterase-positive bands. Extract 25
µ l was electrophoresed
on a discontinuous SDS–PAGE 4 stacking and 12 resolving gel and, after renaturation, stained for esterase activity. Molecular-weight
markers in kDa are shown in the right-hand column. See text for details.
extracts gave two peaks of esterase activity. The first unbound smaller peak eluted in the preliminary wash;
the second, accounting for |77 of recovered activity, eluted at |150 mM competing sugar Fig. 5. Treatment
of SG extracts with 0.65 U of glycopeptidase F to enzy- matically remove oligosaccharide moieties produced a
Fig. 5. Concanavilin A affinity chromatography of salivary gland
esterases gives two peaks of activity; the first is eluted in the prelimi- nary wash, the second is eluted at 150 mM competing sugar. An extract
of 100 salivary gland pairs was loaded on to a 0.5 ml Concanavilin agarose gel equilibrated with 20 mM Tris pH 7.0, 50 mM NaCl, 1 mM
CaCl
2
and 0.1 Triton X-100, washed at 4.8 mlh and 0.4 ml fractions collected. After five fractions, an 8 ml gradient of 0–300 mM
α -d-
methyl mannopyranoside was applied. Fractions were assayed for activity against
α -naphthyl acetate. See text for details.
new, fast-migrating esterase band, while treatment with 0.13 U produced a smaller amount of this entity Fig. 6.
Salivary gland homogenates had PAF-acetylhydrolase activity
in calcium-free
buffer systems
[5.0 ±
0.6 pmolmingland pair
n =
4 separate
experiments; |0.24 µ
molminmg specific activity]. Production of [
3
H]acetate was linear up to 30–60 mins with up to |1.5 gland pairs and is curvilinear thereafter
Fig. 7. On gel filtration, salivary gland naphthyl esterase and
PAF-acetylhydrolase co-elute with an apparent molecu- lar weight of |59 kDa Fig. 8. Fractions containing this
material were pooled and 100 µ
l aliquots tested for activity against a series of substrates:
α -naphthyl propi-
onate 500
± 1 pmolmin,
β -naphthyl
propionate 413
± 3 pmolmin,
β -naphthyl acetate 311
± 6 pmolmin,
α -naphthyl acetate 280
± 1 pmolmin,
α -naphthyl butyr-
ate 134 ±
1 pmolmin, α
-naphthyl caprylate 102 ±
1 pmolmin and
β -naphthyl butyrate 76
± 1 pmolmin
n =
3 for all determinations. Salivary gland extracts did not have detectable acetyl-
cholinesterase activity against the substrate acetylthioch- oline iodide.
4. Discussion
