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Purification partial characterization and postembryonic levels of amylases from Sitophilus oryzae and Sitophilus granarius.

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Archives of Insect Biochemistry and Physiology 2:415-428 (1985)
Purification, Partial Characterization, and
Postembryonic Levels of Amylases From
Sitophilus oryzae and Sitophilus granarius
J.E. Baker and S.M. Woo
Stored Product Insects Research and Development Laboratory, Agricultural Research Sewice,
U.S.Department of Agricidture, Savannalz, Georgia
Amylases from adults of Sitophilus oryzae (L.) and S. granarius (L.) were
purified by using a sequential procedure of ammonium sulfate precipitation,
glycogen-complex formation, and ion exchange chromatography. Amylase of
S. oryaze was purified 47.4-fold to a specific activity of 478 unitdmg protein.
One amylase unit equals 1 mg maltose hydrate producedlmin at 3OOC.
Amylase of S. granarius was purified 85.4fold to a specific activity of 453
u n i t s h g protein. Amylase of S. oryzae had a Km of 0.173% for soluble starch
and consisted of two anionic isozyrnes with isoelectric points of pH 3.70 and
pH 3.76. Amylase of S. granarius had a Km of 0.078% for starch and was a
single protein with an isoelectric point of pH 3.76. Purified amylases of both
species had molecular weights of 56,000 estimated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis, were activated by chloride, and had
double energies of activation calculated from Arrhenius plots. Based o n fresh
weights of adults feeding on whole wheat through 10 weeks of age, S. oryzae
contained three-fold and eight-fold more amylase than S. granarius and S.
zeamais Motschulsky, respectively. High amylase levels in S. oryzae may
provide this species with an adaptive advantage when feeding o n cereals
containing naturally occurring amylase inhibitors.
Key words: Sitophilus, S. oryzae, S. granarius, S. zeamais, rice weevil, granary weevil,
maize weevil, amylase, purification, digestion, cereals, feeding, amylase
inhibitors, adaptive significance
INTRODUCTION
Amylases from larvae of the granary weevil, Sitophilus granarius (L,),and
the maize weevil, S. zeamais Motschulsky, are active in mildly acid buffers,
Mention of a proprietary product does not constitute a recommendation by the U.S. Department of Agriculture.
Received February 25,1985;accepted May 1,1985.
Address reprint requests to J.E. Baker, USDA-ARS, P.O. Box 22909,Savannah, GA 31403.
0 1985 Alan R. Liss,
Inc.
Baker and Woo
416
are activated by C1-, and are stabilized against thermal inactivation by Ca2+
[l].The amylases are of the endoamylase type (E.C.3.2.1.1) and hydrolyze
soluble starch and amylopectin with identical kinetic parameters. Electrophorectic analysis of larval midgut homogenates on polyacrylamide slab gels
indicated that S. zeurnais had two strongly anionic amylase isozymes, while a
single amylase was found in S. grunarius. Gel patterns of adult midgut
homogenates indicated that larvae and adults of a given species had identical
amylase isozymes [2]. Evidence also indicated that, in contrast to proteinases
that were secreted into the lumen by midgut epithelial cells, the origin of
amylase in adult S. grunurius was the salivary glands [3].
Adults of Sitophilus feeding on wheat live for extended periods and consume significantly more food than developing stages [4]. Since starch makes
up 55% of the wheat kernel and is the predominant nutrient in endosperm
[S], the relatively high levels of amylase present in Sitophilus [6] are important
in food utilization processes in these species. The role of amylase in starch
digestion by the weevils is complicated by the presence of potent a-amylase
inhibitors present in wheat [7]. These inhibitors interact with amylases from
Tenebrio rnolitor L. [6,8], Triboliurn custaneurn Herbst [9], as well as Sitophilus
[Z6/10]
To study the in vitro interactions of inhibitor with enzyme, purified amylases are required. A procedure based on ammonium sulfate precipitation,
formation of an insoluble glycogen-amylase complex, and ion exchange chromatography on DEAE-SephaceP was developed and used to purify the
amylases from adults of the rice weevil, S. oryzue (L.) and S. granarius. In
addition, several properties of the purified amylases as well as postembryonic levels of amylase activity in S. oryzue, S. grunurius, and S. zeurnais
were compared.
1
MATERIALS AND METHODS
Insects
Adults of S. oryzue and S. granurius were removed from cultures maintained on soft, red winter wheat at 27°C and 50-609'0 RH with a 12:12 LD
photoperiod.
Assays
Amylase activity was measured with the DNS* procedure of Bernfeld [ll]
slightly modified from that of Baker El]. Activity at each step in the purification sequence was determined by adding 5 pl of sample to 1 ml 1%soluble
starch (potato, Lintner grade, Sigma Chemical Co., St. Louis, MO) in 20 mh4
acetate, pH 5.0, containing 20 mh4 NaCl and 0.1 mM CaC12. After 1 min at
30°C the reaction was stopped by adding 1 ml DNS, the tubes heated in
'Abbreviations: 3,5-dinitrosalicylic acid = DNS; energy of activation = Ea; molecular weight
= MW; polyacrylarnide gel electrophoresis = PAGE; mobility relative to brornophenol blue
= Rm; sodium dodecyl sulfate-polyacrylarnide gel electrophoresis = SDS-PAGE;
tris(hydroxymethy1)aminomethane = tris.
Purificationand Amylases
417
boiling water for 5 min, cooled, diluted with 2 ml H20, and read at 550 nm.
Reaction times for amylase from the final purification step were reduced to
30 sec and in later tests enzyme samples were diluted to maintain linearity.
Maltose was used as a standard. A unit of amylase activity was defined as
the amount of enzyme that produced 1mg of maltose hydrate per minute at
30°C. Specific activity was defined as units per milligram of protein. Protein
was estimated with the procedure of Lowry et a1 [12]. Column fractions were
monitored for amylase activity by measuring residual starch with a KI-iodine
procedure [13].
Purification Procedure
Adults of S. oryuze (20.0 g) and S. grunarius (16.3 g) were ground in 1%
NaCl (2 mllg) with a mortar and pestle, rinsed with 1%NaCl (1mllg) and
the total slurry centrifuged at 5,300g for 40 min. The supernatants were
decanted and recentrifuged to remove floating lipoidal material prior to
ammonium sulfate precipitation. Ammonium sulfate was stirred in at 0°C to
1.5 M and the precipitate that formed was removed by centrifugation. Additional ammonium sulfate was added to 4.0 M and the heavy precipitate that
formed after 60 min was collected by centrifugation for 40 min at 5,300g. The
supernatant was discarded and the pellet dissolved in standard buffer (20
mM acetate, pH 5.0, containing 20 mM NaCl and 0.01 mM CaC12).
Amylase was precipitated from the buffer solution by formation of an
alcohol-insoluble glycogen-amylase complex by using the procedure of Loyter and Schramm [14]. Because of the extreme activity of the amylases in
these species, glycogen was added at the rate of 1 mgl5 units of amylase
activity. The glycogen-amylase complex, insoluble in cold standard buffer
containing 40% ethanol, was collected by centrifugation and washed with
ethanolic buffer. Following centrifugation the pellet was dissolved in buffer
and incubated for 3 h at 37°C to remove glycogen by hydrolysis. Hydrolytic
products were separated from enzyme via ultrafiltration on YM-10 membranes on an Amicon stirred cell. When the ultrafiltrate no longer gave a
positive DNS reaction, the standard acetate buffer was exchanged on the
stirred cell for 20 mM tris-chloride, pH 7.5, containing 0.1 mM CaCl,.
Following concentration, samples were applied to a DEAE-SephaceP
(Pharmacia, Piscataway, NJ) column (1.6 x 13cm) equilibrated in 20 mh4 trischloride, pH 7.5, with 0.1 mM CaC12. Proteins were eluted with a linear
gradient of 250 m10.4 M NaCl in the tris buffer into 250 ml tris buffer. Initial
flow rate was 1 mllmin and 4-ml fractions were collected. Alternate tubes
were checked for absorbance at 280 nm and for amylase activity by using the
KI-iodine procedure for residual starch. Tubes containing amylase were combined and concentrated via ultrafiltration. Absorbance values of amylases in
distilled H20 were determined at 280 nm in 1-cm pathlength cuvettes.
Electrophoresis
Purified amylases were examined by PAGE on 7.5% gels according to
Davis [15] and Baker 1161. SDS-PAGE on 10% gels was performed according
418
Baker and Woo
to LKB manual 2001 except SDS was omitted from the gels and the SDS
concentration in the electrode buffer was reduced to 0.05%. PAGE and SDSPAGE gels were stained with Coomassie brilliant blue R-250. Protein standards were obtained from Sigma and BioRad (Richmond, CA). Plots of log
molecular weight versus mobility relative to bromophenol blue were prepared. Isoelectric points of the amylases were determined by isoelectric
focusing on polyacrylamide gels containing Pharmalyte 2.5-5.0 range ampholytes (Pharmacia) according to procedures previously described [lq,
Kinetic Parameters
Kinetic parameters for hydrolysis of starch were determined from duplicate anaylses of three protein concentrations of purified amylases from each
species reacting with eight concentrations of starch from 0.066 to 0.5% in 50
mM sodium acetate, pH 5.0, containing 20 mM NaCl and 0.1 mM CaC12for
2 min at 30°C. Protein concentrations used were 0.3, 0.45, and 0.6 p g for S.
o y z u e and 0.29, 0.39, and 0.48 p g for S. grunuuius. Each tube contained 1.0 ml
starch solution. Double reciprocal plots of velocity against starch concentration were prepared. Mean intercept values were obtained from plots using a
linear program analysis (Hewlett-Packard Model 97). Km (as percentage of
starch in reaction mixture) and Vmax were estimated for each species.
Effect of chloride on hydrolysis of starch was determined as above except
purified enzymes were diluted and washed with sodium acetate buffer (without chloride) on Centricon 10 Microconcentrator@tubes prior to assay. Starch
solutions were prepared in acetate buffer with and without 20 mM NaCl and
0.1 mM CaC12. Double reciprocal plots were prepared as above.
Effect of Temperature on Amylase Activity
Activity of purified amylases was determined at 5°C intervals from 10°C
to 50°C. Mean values obtained from two tests of amylases of each species
with triplicate analyses at each temperature were used to prepare Arrhenius
plots. Ea values were calculated from the equation: Ea = -2.303R X (slope
of 1I"K vs log v). Slopes of 1I"K ( X lo3)vs log v between given temperatures
were determined from a linear analysis program.
Comparative Levels of Amylase in Sitophilus
Amylase levels in whole body extracts of larvae and adults of Sitophilus
through 10 weeks of age were compared. Newly hatched larvae, 7-8-day-old
larvae, 15-18-day-old larvae, pupae, and newly eclosed adults of S. oryzae
and S. grunurius were obtained from groups reared on a casein-starch-based
meridic diet [18,19]. Groups of newly emerged adults of S. oyzue and S.
grunurius (from cultures reared on whole wheat) and S. zeumuis (from cultures
reared on whole corn) were placed on fresh whole wheat in 3.7-liter jars for
ad libitum feeding at 28°C and 50-60% RH in continuous darkness. At
weekly intervals for 10 weeks, three groups of ten weevils of each species
were removed, weighed, homogenized in 0.5 ml cold H20, the homogenates
centrifuged in 1.5-ml tubes, and triplicate 2 0 4 aliquots of supernatant analyzed for amylase and protein. Amylase assays for S. oryzue were conducted
Purificationand Amylases
419
for 1min at 30°C while assays for S. grunurius and S. zeurnuis were conducted
for 2 min. Amylase activity was expressed as milliunits per milligram of
protein or units per milligram fresh weight. Activity based on fresh weight
was determined by calculating total units of activity on a per midgut basis
and dividing by mean fresh weight of insects used in that particular replicate.
Whole body extracts of larvae were prepared as above except only five
mature larvae (15-18 days old) were used per replicate. Adults of S. zurnuis
feeding on wheat were analyzed at biweekly intervals after four weeks of
age. Weevils of all three species feeding on wheat were removed at 3-week
intervals and placed on clean wheat to prevent mixing of newly emerged
weevils with the original weevils of known age.
RESULTS
Purification of Amylases
Results of purification procedures for amylase from adults of S. oryzue are
presented in Table 1. Amylase complexed with glycogen on a nearly quantitative basis, resulting in a 12-fold purification over the ammonium sulfate
step and a 20.8-fold purification relative to the crude homogenate. Amylase
from S. oryme eluted from the DEAE-SephaceP column in a single, symetrical
peak with 0.3 M NaCl in buffer at pH 7.5 (Fig. 1).Tubes 94 to 102 were
combined and concentrated. Specific activity after column chromatography
was 478.6 unitslmg protein, which represented a 47.4-fold increase over that
of the original homogenate. Final recovery was 59% of original activity.
Absorbance coefficient (Alyo, 280 nm) of the purified amylase of S. oqzue was
13.3.
Amylase from S. gvunurius was isolated and purified by using the same
sequence of procedures used for S. oryzue. The glycogen-complex step resulted in a 28-fold increase in specific activity over the ammonium sulfate
step and a 45-fold increase in activity over the original homogenate (Table 2).
A single, symetrical peak of amylase activity was eluted from the DEAESephaceP column with 0.32 M NaCl in buffer at pH 7.5. Tubes 104 to 110
were combined and concentrated. Although specific activity of the original
homogenate of S. grunurius was only half that of S. or-yme, (5.3 unitslmg
protein compared to 10.1 unitslmg protein, respectively), final activity following chromatography was 452.8 unitslmg protein, nearly identical to that of
TABLE 1. Purification Sequence for Amylase From the Rice Weevil, Sitophilus oryzae
Step
Crude homogenate
(Nh)2S04
Glycogen complex
DEAE-SephaceF
Volume
(ml)
Total
proteir t
(mg)
Amylase”
(units)
Recoveryb
(%)
Specific
activity‘
Fold
purificationd
46.0
44.0
23.2
7.8
947.6
431.2
31.5
11.7
9,531
7,453
6,625
5,598
100
78
70
59
10.1
17.3
209.9
478.6
1.0
1.7
20.8
47.4
aOne unit of activity equals 1.0 mg maltose hydrate produced per minute at 30°C.
bunits of activity at a given step divided by units in crude homogenate.
“Units per milligram of protein.
dSpecific activity at a given step divided by specific activity of crude homogenate.
Baker and Woo
420
0 81
1.21
0 4
03
I
02
:z:
01
0
3
TUBE
Fig. 1. Elution of glycogen-precipitated protein (A28o)and amylase (Aa) of S. oryzae from a
DEAE-Sephacele ion exchange colum (1.6 x 13 crn) with a linear gradient of 0.4 M NaCl in 20
rnM tris-chloride, pH 7.5. Amylase was monitored by KI-iodine detection of residual starch.
Tubes 94 to 102 were combined.
TABLE 2. Purification Sequence for Amylase From the Granary Weevil, Sitophilus
granarius
Step
Crude homogenate
(NH4)2S04
Glycogen complex
DEAE-SephaceF
Volume
(ml)
Total
protein
(mg)
Amylase"
(units)
Recoveryb
38.5
31.5
11.8
3.5
469.7
270.9
8.8
3.4
2,500
2,223
2,095
1,521
100
89
84
61
(YO)
Specific
activity'
Fold
purificationd
5.3
8.2
239.9
452.8
1.0
1.5
45.3
85.4
"One unit of activity equals 1.0 mg maltose hydrate produced per minute at 30°C.
bunits of activity at a given step divided by units in crude homogenate.
'Units per milligram of protein.
dSpecific activity at a given step divided by specific activity of crude homogenate.
the purified amylase from S. oryzae. Final activity represented an %-fold
purification with 61% of the original activity recovered. Absorbance coefficient (Al%,280 nm) of the purified amylase of S . granarius was 12.5.
Electrophoresis
Purified amylase from adults of S. oyzue consisted of two highly anionic
isozymes that traveled with Rm values of 0.90 and 0.91 in a 7.5%polyacrylamide gel and 0.74 and 0.75 in a 10% gel, respectively. The amylases from S.
o y m e were electrophoretically pure. Isoelectric points of the two amylase
isozymes of S. oryzae were at pH 3.76 and 3.70.
Purified amylase from adults of S. grunurius consisted of a single protein
with R, 0.91 on the 7.5% gel and 0.75 on the 10% gel, identical to the fastest
moving isozyme from S. otyzue. Except for a faint, diffuse, blue-staining band
Purification and Amylases
421
at R
, 0.85, the amylase sample from S. grunarius was electrophoretically
clean. Isoelectric point of the amylase from S. grunurius was at pH 3.76.
Both isozymes of S. oryme and the amylase of S. granarius had identical
mobilities on the SDS-PAGE system (Fig. 2). Molecular weights of the enzymes were estimated to be about 56,000 for both species. The similar relative
mobilities of the two isozymes of S. oryme on the anionic gels of both 7.5%
and 10% concentration and the single band obtained with SDS-PAGE indicates that the amylase isozymes are charge isomers rather than size isomers.
Kinetic Parameters
Mean values (kSE) of Km with soluble starch as substrate were 0.173 k
0.005% for amylases from S. oryme and 0.078 f 0.003% for S. grunarius. Mean
Vmax values for purified amylases at 30°C were 621 + 3 unitslmg protein for
S. oryme and 592 f 11unitslmg protein for S. granarius. Expressed in terms
of pmol maltoselminlmg protein, values were 1,730 pmollminlmg protein for
amylases from S. oryzae and 1,640 pmollminlmg protein for amylase from
s. grunurius.
Amylases from both S. oryzue and S. granurius were activated by chloride
(Fig. 3). Vmax for amylase increased 7.6-fold in S. oryzae and 6.5-fold for S.
granarius in the presence of 20 mM NaC1. Presence of chloride did not affect
the Km of S. oyzue but shifted the Km of S. grunarius slightly from about 0.09
to 0.07% starch.
Fig. 2. Comparison of mobilities of SDS-treated amylases from S. oryzae and S. granarius
with porcine pancreatic m-amylase and molecular weight standards. Lanes 1, 10, a-amylase,
15 pg; lanes 2, 3, Sigma standards, 5 and 10 &band; lanes 4, 5, amylase from S. oryzae, 5 and
10 pg; lanes 6, 7, amylase from S. granarius, 5 and 10 pg; lanes 8 , 9 , BioRad standards, 5 and 10
&band. Estimated M W of Sitophilus amylases was 56,000.
422
Baker and Woo
Effect of Temperature
Arrhenius plots of effect of temperature indicate that double energies of
activation occur in purified amylases from both S. oyme and S. granarius (Fig.
4). Slopes of temperature effect between 10°C and 25°C for both species
were identical with apparent Ea values 9.2 kcallmol. Between 25°C and 50°C
activity slopes of the two species were not parallel and apparent Ea values
were 6.3 and 7.5 kcal/mol for S. oyme and S. granarius, respectively.
Comparative Amylase Levels in Sitophilus
Specific activities of amylases were higher in larvae than in adults of S.
oryzae and S. granarius. Activities in first-stage larvae (prior to feeding) were
10.7 f 1.5 and 7.3 & 1.1 unitslmg protein for S. oryzae and S. granarius,
respectively. Activities in 7-8-day-old larvae were 24.3 & 0.8 and 14.6 k 2.2
+
unitslmg protein, respectively. Highest amylase activity of 25.7 1.1units/
mg protein was found in fourth-instar larvae of S. oryzae. Amylase activities
in larvae of S. oryzae at a given age were always significantly higher than
those of larvae of S. grunarius. Only trace amounts of activity (< 0.1 unit/mg
protein) were detected in pupae of mixed ages of either species. Activity in
whole body extracts of newly eclosed adults (obtained from the artificial diet)
was also low, 2.8 unitslmg protein for S. oyzue.
Activities in whole body extracts of adults of S. oryzae, S. grunarius, and S.
zeamais reared on whole wheat through 10 weeks of age are given in Figure
5. In S. oryzue, there was a slight trend toward higher specific activities as the
weevils became older; however, amylase levels in all three species were
variable from week to week. Significant differences in activity levels were
80 -
0
5. gronoriur
5. oryzoe
50
N o Chloride
s
*
40-
Fig. 3. Effect of chloride (20 m M NaCI) on hydrolysis of soluble starch in 20 m M acetate, pH
5.0, by purified amylases from A) S. oryzae (7.6-fold increase in Vmax) and B) S. granarius (6.5fold increase in Vrnax).
Purification and Amylases
423
3.0
Ea kcal/rnole
2.8
Temp
5. oryzae
--
5. gronariur
10'-25"
25"-50"
9.2 f 1 . 1
6 . 3 f 0 .4
9.2 f 0 - 9
7.5 f 0.5
2.6
>
m
2
2.4
2.2
2 .o
3.1
3.2
3.3
'/K
3.4
3.5
3.6
(~103)
Fig. 4. Arrhenius plots of effect of temperature on activity of purified amylases from S.
oryzae and S. granarius. Double energies of activation were demonstrated for each species.
5. gronariut
U
4
w
4
4-
a
2-
I
n
z
5. zeomair
$
04
0
1
2
3
4
5
6
7
8
9
1
0
ADULT AGE (Weeks)
Fig. 5. Amylase activities of S. oryzae, S. granarius, and S. zeamais feeding on whole, soft
red winter wheat at 28OC and 60% RH. Each point at each age interval is the mean of three
replicates of ten weevils each for each species.
424
Baker and Woo
found among species. Mean specific activities, excluding the initial (0 time)
readings, for all values within the 10-week interval were 11.8 k 0.3, 4.3 k
2.0, and 1.5 k 0.2 unitslmg protein for S. o y m e , S. granarius, and S. zearnais,
respectively. Based on protein, S. oyzue contained a mean of 2.9-fold and
7.8-fold more amylase activity than S. granarius and S. zearnais, respectively.
S. grunarius contained 2.9 fold more amylase than S. zearnais. Mean activities
of whole body extracts expressed on a fresh weight basis were 725 k 15, 237
& 9, and 90.2 k 8.5 milliunitslmg fresh weight for S. oryzae, S. granarius, and
S. zeurnais, respectively. Thus, based on fresh weight, S. o y m e contained
threefold and eightfold more amylase activity than S. granurius and S . zearnais,
respectively. S. granarius contained 2.6-fold more amylase than S. zearnais.
Adults of S. zearnais were largest (mean fresh weight of about 3.8 mg compared to 2.8 mg for S. granarius and 2.2 mg for S. oyzue throughout the 10week period) but contained extremely low amylase levels relative to the two
other species.
DISCUSSION
Properties of Amylases
Procedures used to purify amylases from insects include glycogen precipitation followed by ion exchange chromatography [ZO],coprecipitation with
amylopectin [21], and affinity gel chromatography [22]. The glycogen precipitation and anionic ion exchange chromatography purification sequence used
for S. oryzae and S. granarius resulted in extremely active preparations with
good yields. Comparisons of activities of purified amylases from insects are
complicated since definitions of "units" of activity vary in each case. Activity
of amylase from larvae of Callosobruchus chinensis (L.) after a glycogen precipitation step was 590 unitslmg protein, 1 unit equivalent to 1 mg maltosell0
min at 37°C [20]. Disregarding temperature, this activity corresponds to 59
"Sitophilus" units, which is 3.5- and 4-fold lower than activity of amylases
from S. oryzae and S . grunarius, respectively, at the equivalent step in the
purification procedure. Activity of electrophoretically pure amylase from
Tenebrio molitor L. was 859 unitslmg protein, 1 unit equivalent to 1 pmol
maltoselmin at 37°C [23]. If activity units for amylases from S. oyzue and S .
granarius are expressed in pmol maltoselmin, final activities at 30°C were
1,300 and 1,250 unitslmg protein, respectively. Thus, the purified amylases
from Sitophilus are apparently much more active than that from C. chinensis
and are also more active than the purified amylase from T. rnolitor.
Molecular weights of mammalian and bacterial amylases range from about
40,000 to 59,000 [13]. Molecular weights of insect amylases are comparable.
Amylase from T. rnolitor was a single polypeptide chain with an SDS-PAGE
estimated molecular weight of 68,000 [22]. Estimates of the molecular weight
of purified amylase from Bornbyx mori were 57,000 by SDS-PAGE and 47,000
by guanidine column chromatography on Sepharose [21]. Molecular weight
estimates of 56,000 for amylases of both S. oryzae and S . grunarius by SDSPAGE are comparable to these purified enzymes. Amino acid analyses of the
Sifophilus amylases have not been determined. Absorbance coefficients of aamylases at 280 nm are relatively high because of high concentrations of
Purification and Amylases
425
tryptophan and tyrosine [13]. However, A*%values of 13.3 and 12.5 for S.
oy m e and S. granarius, respectively, were about half those calculated for
porcine pancreatic amylase and human salivary amylase.
Isoelectric points of pH 3.70 and 3.76 for the isozymes of S. oryme and pH
3.76 for the single amylase of S. grunurius were slightly lower than that of T.
molifor (pH 4.0) [22] and generally lower than isoelectric points (range pH 4.2
to 5.7) for most other amylases [13]. The highly anionic nature of the Sitophilus amylases at neutral to slightly alkaline pH apparently accounts for their
fast mobility on the polyacrylamide gels.
Amylases from larvae of S. grunuritds and S. zearnuis were activated by 0.1
mM NaCl in buffered starch solution [l]. Presence of C1- also resulted in
substantial increases in Vmax for the purified enzymes from adult Sitophilus.
Chloride generally activates insect amylases [22,24] but C1- inhibited amylase
from C. chinensis [20]. Ca2+ stabilized the larval amylases of S . grunarius and
S. zeamais against thermal inactivation [l]. The role of Ca2+ in the purified
amylases from adults has not been determined but presumably this divalent
cation has a similar function.
Purified amylases from both S. oryzue and S. grunarius have double energies
of activation, 9.2 Kcallmol between 15°C and 25°C and 6.3 and 7.5 Kcallmol
between 25°C and 50"C, respectively. Most mammalian and bacterial amylases have single Ea values [13]. However, double energies of activation were
shown for the amylase from the bacterium Pseudornonas saccharophilu, 14.4
Kcallmol between 0°C and 15°C and 8.5 Kcallmol between 15°C and 40°C
[25]. Although there have been only a few such studies with insects, insect
amylases with both single and double energies of activation have been
demonstrated. Single Ea values were shown for C. chinensis, 7.3 Kcallmol
between 10°C and 60°C [20] and Rhynchosciara umericanu, 4.8 Kcal/mol between 12°C and 35°C [24]. Double Ea values were calculated for the purified
amylase from B. mod, 17 Kcallmol between 0°C and 25"C, and 3.8 Kcallmol
between 25°C and 40°C [21].
Amylases from larvae of S. grunurius and S. zeurnuis were endoamylases of
the a-amylase type [l]. Km values for soluble starch of larval amylases were
0.077% for S. grunurius and 0.13% for S. zeumais. The Km value for starch for
purified amylase from adult S. granarius was 0.078%, nearly identical to that
of the larval amylase. Single isozymes were found in both larvae and adults
of S. granurius. Km for starch for adults S. oryme was 0.173%, somewhat
higher than the Km determined for larvae of S . zeurnuis. Both S . o y m e and S.
zeamais have two amylase isozymes that are resolved on polyacrylamide gels.
Amylase from Sitophilus have a higher affinity for starch than the amylase
from C. chinensis (Km of 0.23%) [20] but the amylase of S . oryme has a Km
similar to that of T. rnolifor (Km of 0.18%) [22].
Adaptive Significance of Amylase Levels in Sitophilus
Although amylase of S. oryzae and S . grunurius were purified to the same
specific activity, total amylase levels in S. oqzue were significantly higher
than those of S. grunurius in developing and adults stages. These high
amylase levels may provide a selective advantage to S. oryme even though
426
Baker and WOO
the Km for starch for S. oyme (0.173O/0) was much higher than that of S.
granarius (0.078%).
When reared on wheat, the capacity for increase of S. o y m e is greater than
that of both S. zeumuis [26] and S. grunurius [27,28]. In addition, S. oryzue is
more efficient than S. grunurius in utilizing whole wheat [4,29] or a starchbased meridic diet [30] as food sources and out-competes S. granarius in
competition experiments [31]. A combination of behavioral and physiological
responses, including the differential presence of symbiotic microorganisms
in these species [32], probably accounts for faster population development
by S. oryzae. Nevertheless, the presence of significantly higher levels of aamylase in s. otyzae may also confer an adaptive advantage for this species
by negating the effect of a-amylase inhibitors when feeding on wheat.
Amylase levels demonstrated in adults of S. oyzue, S. granarius, and S.
zeurnais do not always correlate to development profiles of these species on
different cereals or the relative susceptibility of different cereals to weevil
attack. Although S. mmuis has relatively low levels of amylase, this species
develops faster than S. grunurius on triticale, wheat, barley, and maize [33].
However, barley and maize do not contain the a-amylase inhibitors [34] so
insect amylase levels may not confer any advantage or disadvantage when
Sitophilus feed on these cereals. On the other hand, triticale does contain an
amylase inhibitor [35] but softness of the endosperm rather than biochemical
composition of this cross between wheat and rye may be more important in
its susceptibility to weevil attack [33].
A number of the low molecular weight, water soluble albumins found in
wheat inhibit amylases [36]. The albumins form reversible complexes with
the amylase from T. rnolifor [37] and are resistant to proteolytic degradation
by midgut proteinases from this species [8]. Since midgut proteinase levels
in Sitophilus are very low [16], detoxification of ingested amylase inhibitor by
proteolytic degradation also seems unlikely in these species. Thus, the relatively high amylase levels in Sitophilus and the significantly higher levels in
S. ovyzae may be an adaptation not only to high dietary levels of starch but
also to counter the effect of ingested inhibitors from cereals by actually
flooding the inhibitors with enzyme to thereby allow normal digestive mechanisms to proceed.
LITERATURE CITED
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34. Kneen E, Sandstedt RM: Distribution and general properties of an amylase inhibitor in
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