1140 S.R. Marana et al. Insect Biochemistry and Molecular Biology 30 2000 1139–1146
even in insects from the same order. To understand the molecular basis and the physiological meaning of those
differences, it is necessary to characterise β
-glycosidases from different insects, describing their specificity, num-
ber of active sites and amino acid residues related to catalysis and substrate binding.
In this work, one of the major midgut β
-glycosidases from Spodoptera frugiperda larvae was purified and
shown to possess two active sites with different speci- ficities, resembling the mammalian dimeric lactase–
phlorizin hydrolase.
2. Materials and methods
2.1. Animals S. frugiperda Lepidoptera: Noctuidae were labora-
tory reared according to Parra 1986. The larvae were individually contained in glass vials with a diet based
on kidney bean Phaseolus vulgaris, wheat germ, yeast and agar and were maintained under a natural photoreg-
ime summer, 14L:10D; winter, 10L:14D at 25
° C.
Adults were fed a 10 honey solution. Fifth last instar larvae of both sexes were used in the experiments.
2.2. Enzyme samples Larvae were immobilised by placing them on ice, after
which they were rinsed in water and blotted with filter paper. Their guts were dissected in cold 125 mM NaCl,
and the midgut tissue was pulled apart. Midgut tissue, after being rinsed thoroughly with saline, was homogen-
ised in double distilled water, frozen-and-thawed three times and centrifuged at 25,000g for 30 min at 4
° C. The
resulting supernatant was stored at 220
° C until use.
2.3. Gel filtration Samples were applied to a Superose 12 HR 1030 col-
umn of a FPLC system Pharmacia-LKB Biotechnology, Sweden equilibrated and eluted with 20 mM triethanol-
amine–HCl buffer pH 7.5, or with this buffer plus 200 mM NaCl. Fractions of 0.4 ml were collected at a flow
rate of 0.4 mlmin. The active fractions were pooled and stored at
220 °
C until use. 2.4. Ion-exchange chromatography
An aliquot of the eluate from the Superose 12 column was applied to a column of Mono Q HR 55 FPLC-
system equilibrated with 20 mM triethanolamine–HCl buffer pH 7.5. The proteins were eluted with 20 ml of
a 300–500 mM NaCl gradient. The flow rate was 0.5 mlmin and 0.4 ml fractions were collected. Fractions
21–24 Q1 and 31–35 Q2 were pooled and stored at 220
° C.
2.5. Hydrophobic chromatography Sample Q1 or Q2, diluted in 50 mM phosphate buffer
pH 7.0, containing 2 M ammonium sulphate, were applied to a column of Alkyl Superose HR 55 FPLC
system. The proteins were eluted with 20 ml of 2–0.4 M ammonium sulphate gradient in the same buffer. The
flow rate was 0.5 mlmin and 0.4 ml fractions were col- lected. More active fractions from each chromatography
were pooled, stored at
220 °
C and used as an enzyme source.
2.6. Sodium dodecyl sulfate SDS-polyacrylamide gel electrophoresis PAGE
Samples containing approximately 4 µ
g protein were combined with sample buffer containing 60 mM Tris–
HCl buffer pH 6.8, 2.5 wv SDS, 0.36 mM β
-mer- captoethanol, 0.5 mM EDTA, 10 vv glycerol and
0.005 wv bromophenol blue. The samples were heated for 5 min at 95
° C in a water bath, before being
loaded onto a 7.5 wv polyacrylamide gel slab con- taining 0.1 SDS Laemmli, 1970. The gels were run
at a constant voltage of 200 V and stained for protein using a silver stain Blum et al., 1987. Mr values were
calculated according to Shapiro et al. 1967 using the following Mr standards: ovoalbumin Mr 45,000, bov-
ine serum albumin Mr 66,000, phosphorylase b Mr 97,400,
β -galactosidase Mr 116,250 and myosin Mr
200,000. 2.7. Microsequencing of purified
b-glycosidase Samples with 100
µ g of S. frugiperda purified
β -gly-
cosidase were concentrated in a vacuum desiccator Heto Lab Equipment, Denmark and then solubilised in 62.5
mM Tris–HCl buffer pH 6.75, containing 2 wv SDS, 5 vv
β -mercaptoethanol, 10 vv glycerol and
0.001 vv bromophenol blue. After a pre-run 10 mA, 30 min, with 0.1 M sodium thioglycolate in the running
buffer, the samples and pre-stained standards were loaded and electrophoretically resolved as described
above. The resolved peptides in the gel were electroblot- ted onto PVDF polyvinylidene difluoride membranes
according to Matsudaira 1987. The PVDF membranes were stained for proteins using 0.1 Coomassie Blue R-
250 in a 50 vv methanol solution, and destained with a 50 methanol solution. Dried PVDF membranes were
the source of peptides for microsequencing.
The N-terminal sequence analysis was performed at the Macromolecular Structure Analysis Facility at the
University of Kentucky, Lexington, KY, USA.
1141 S.R. Marana et al. Insect Biochemistry and Molecular Biology 30 2000 1139–1146
2.8. Protein determination and hydrolase assays Protein was determined according to Bradford 1976
using ovoalbumin as a standard. β
-glycosidase activity was determined by measuring the release of p-nitrophenolate Terra et al., 1979 from
NP β
Glu p-nitrophenyl-
β -d-glucopiranoside;
and NP
β Gal p-nitrophenyl-
β -d-galactopiranoside, reducing
groups Noelting and Bernfeld, 1948 from laminarin and
CMC carboxymethyl
cellulose or
glucose Dahlqvist, 1968 from different alkyl
β -glucosides, cel-
lobiose, cellotriose, cellopentaose, gentiobiose, prunasin, amygdalin, phlorizin, lactose, laminaribiose and glucos-
ylceramide. In the last case, the solubilization was achi- eved according to Dinur et al. 1984.
All substrates were assayed in 50 mM citrate–sodium phosphate pH 6.0 at 30
° C under conditions such that
activity was proportional to protein concentration and to time. Controls without enzyme or without substrate were
included. One unit of enzyme U is defined as the amount that hydrolyses 1
µ mol of substratemin.
2.9. Chemical modification studies Purified
β -glycosidase was incubated at 30
° C with 6
mM EDC
1-ethyl-3-3-dimethylaminopropyl carbodiimide, 40 mM glycine ethyl ester and 100 mM
TemedN,N,N 9,N9-tetramethyl-ethylenediamineHCl buffer
pH 5.2 or 6.0. When the substrate used to follow the inactivation was cellobiose, the incubation with EDC
was done with or without 25 mM NP β
Gal or 14 mM cellobiose. When the substrate was NP
β Gal, the incu-
bation with EDC was done with or without 14 mM cello- biose. Samples were collected at different periods of
time and the reaction was stopped by a two-fold dilution with 400 mM citrate–sodium phosphate buffer pH 6.0.
When the substrate used to follow the inactivation was cellobiose, after this initial dilution, samples were sub-
mitted to two cycles of five-fold dilution followed by concentration in Microcon centrifuge filters YM-10
Amicon. This procedure is necessary to avoid cello- biose hydrolysis inhibition by NP
β Gal present in the
reaction media. 2.10. Kinetic studies
The effect of substrate concentration on purified β
- glycosidase was determined using at least 10 different
substrate concentrations. K
m
and V
m
values mean and SEM were determined by linear regression using the
software Enzfitter Elsevier, Biosoft. When the inhibition of the hydrolysis of one substrate
NP β
Glu or NP β
Gal by another substrate NP β
Glu, NP
β Gal or cellobiose was studied,
β -glycosidase was
incubated with at least five different concentrations of substrate in each of at least five different concentrations
of the substrate used as inhibitor. In these studies, two samples were taken from each reaction medium at the
end of incubation. In one sample, p-nitrophenolate was determined to calculate the amount of NP
β Glu or
NP β
Gal that was hydrolysed; in the other sample, glu- cose was measured according to Dahlqvist 1968, with
the final addition of sulfuric acid to change p-nitrophen- olate into the colourless p-nitrophenol. In the medium
with NP β
Glu, after allowance for the amount of glucose originating from it, it is possible to calculate the activity
on cellobiose or NP β
Gal. K
i
values were determined from replots of slopes of Linewaver–Burk plots against
inhibitor concentration Segel, 1975, using the software Enzfitter Elsevier, Biosoft.
3. Results
