purification and characterization of a major esterase besb

日蚕雑 69 (2),121-130(2000)

J.Seric.Sci.JPn.

Purification and characterization of a major

esterase BesB from hemolymph of the silkworm, Bombyx mori

HITOSHI ARAI, TOSHIHISA OKIDO, HIROSHI FUJII and HIROSHI DOIRA

Institute of Genetic Resources, Faculty of Agriculture, Kyushu University

(Received May 31, 1999)

Total esterase activity in the hemolymph of Bombyx mori was measured using a-naphthyl acetate as a substrate. It was low during the early fifth instar, then sharply

increased at the spinning stage and stayed high throughout the pupal stage. One of the major hemolymph esterases of B. mori, previously named BesB (blood esterase B type), was

purified from day 1 pupae to homogeneity using procedures such as procainamide hydrochlor-ide affinity chromatography. BesB had an apparent molecular weight of 58,000 and an isoelectric point of 4.6. It was most active at pH 6-7, and was stable around pH 7 to 9. The sequence of N-terminal 28 amino acid residues was partially homologous to previously reported esterases isolated from the peach-potato aphid Myzus persicae. In kinetic parame-ters, there were no differences in Km values from four tested substrates, but there were differences in Vmax values. BesB had the highest sensitivity to paraoxon and diisopropyl f luorophosphate (DFP), and was moderately affected by eserin sulfate, phenylmethylsulf onyl fluoride (PMSF) and chloromercuribenzoic acid (pCMB). These results indicate that BesB is a carboxylesterase in hemolymph.

Key words: Bombyx mori, Bes, hemolymph purification carboxylesterase.

Introduction

Esterases (carboxylesterases; E. C 3. 1. 1.

1.) are ubiquitous in living organisms. Several

esterases have been isolated from various tissues

of plants and animals as well as microbes and

investigated for their biochemical properties

(BROGDON 1988; THOMAS et al., 1993). Also, esterases are widespread among insect species

and function in the digestion of nutritional mate-

rials (KAPIN and AHMAD, 1978), in detoxification

of xenobiotics (DAUTERMAN and HODGSON, 1978),

and in reproduction (RICHMOND et al., 1980).

Many esterases, however, do not show strict

substrate specificity, and it is difficult to reveal

their endogenous substrates except in the case of

cholinesterases.

In Bombyx mori, esterases have been stud-

ied from the standpoint of genetics. Polymor-

phism of esterase allozymes observed by their differential mobility on agar gel electrophoresis

was found in hemolymph, silk glands, fat bodies,

midguts, eggs and integuments (YOSHITAKE and

EGUCHI, 1965; YOSHITAKE and AKIYAMA, 1965;

EGUCHI et at., 1965; EGUCHI and YOSHITAKE,1967).

The hemolymph esterase of B. mori comprises

the three major allozymes BesA, BesB and BesC

(blood esterase), and their expression is controlled by the codominant alleles BesA, BesB and Besc,

respectively. The allele for BesO (absence of

Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, 6-10-1, Hakozaki, Higashi-ku, Fukuoka, 812-8581, Japan

-121-

122 The Journal of Sericultural Science of Japan Vol.69 No.2

Bes activity) is referred to as Bes° (YosHITAKE

and EGUCHI, 1965; EGUCHI et al., 1965). Later,

linkage analysis showed that the Bes genes are

located on the 11th chromosome (GAMO, 1978).

We have found minor esterases in hemolymph

other than Bes in BesA, BesB, BesC and BesO

strains (OKIDO et al., 1996) and they were present

in hemolymph from the larval to the pupal stage.

However, there are few studies on the physiologi-

cal function and biochemical properties of the

major hemolymph esterases, Bes, as well as the

other esterases. In order to elucidate the role of

esterase in hemolymph, we tried to isolate and

characterize Bes.

Materials And Methods

Insects and the collection of hemolymph

Two strains maintained at the Institute of

Genetic Resources at Kyushu University, the

strain n50 with the phenotype BesB and the strain

u10 with BesO (i, e., "null" with respect to Bes)

were used. Larvae were reared at 26•Ž with

fresh mulberry leaves. Hemolymph samples

were collected by cutting the larval or pupal

dorsal abdomen, centrifuged to remove hemocyte,

and stored with a small amount of phenylthiourea

at - 40•Ž until used.

Assay of esterase activity and determination of

protein concentrations

The total esterase activity was assayed

according to the method of SPARKS et al. (1979)

with slight modifications. When a-naphthyl ace-

tate (a-NA) or a-naphthyl propionate (a-NP) was

used as a substrate, the reaction mixture (1.1 ml)

containing 227 nM of substrate, 0.1 M sodium

phosphate buffer (pH 7.4) and 0.1 ml of diluted

hemolymph (1:10 in 0.1 M sodium phosphate

buffer, pH 7.4) was incubated at 30•Ž for 20 min.

The reaction was stopped through the addition of

0.5 ml 0.4% (w/v) o-dianisidine tetrazotized

containing 3.4% (w/v) sodium dodecyl sulfate

(SDS). The mixture was diluted with 3 ml of 0.05

M sodium phosphate buffer (pH 7.2) and mea-

sured for liberated 1-naphthol by monitoring the

absorbance at 600 nm. One unit of esterase

activity was defined as the amount of enzyme

which produces one n mole of 1-naphthol at 30•Ž

per min. When p-nitrophenyl acetate (p-NPA)

or p-nitrophenyl propionate (p-NPP) was used as

a substrate, the absorbance change at 405 mm

was recorded before and after reaction. The

reaction mixture (2 ml) was composed of enzyme

solution, 1 mM substrate and 0.5 M Tris-HCl

buffer (pH 8.0), at 30•Ž. This measurement was

done for the control without enzymes. The ester-

ase activity was calculated from the difference in

net increase between the two measurements using

the molar extinction coefficient (ƒÃ) of 4-

nitrophenol (16.4 mM-1 cm-1).

Protein concentrations were determined by

the method of LoWRY et al. (1951) with bovine

serum albumin as a standard.

Purification of BesB

Hemolymph specimens were fractionated

with ammonium sulfate, and the obtained precipi-

tate between 40 and 70% saturation was collected

and dialyzed against 20 mM sodium phosphate

buffer (pH 7.4) containing 1 mM dithiothreitol.

Then the solution was adjusted to 40% with

respect to ammonium sulfate and applied to a

column (2.2 x 30 cm) of Butyl-Toyopearl 650 M

(TOSOH Co., Tokyo, Japan) equilibrated with the

same buffer containing 40% ammonium sulfate.

The column was washed with the equilibration

buffer and eluted with a decreasing linear gradi-

ent of ammonium sulfate (from 40 to 0%) in the

same buffer. Five ml fractions were collected

and tested for esterase activity. Active fractions

were pooled and concentrated with Centricon (cut

off; 50,000, Amicon) to 2 ml. This chromatogra-

phy was performed twice. The active fractions

after the second Butyl-Toyopearl chromatogra-

phy were pooled and concentrated as described

above. After dialysis against 20 mM phosphate

buffer (pH 7.0), the sample was made as 0.1 M

with respect to NaCI and subjected to affinity

column chromatography using f ormyl-cellulof ine

(Seikagakukogyo, Tokyo, Japan). The f ormyl-

cellulofine had been previously bound to

procainamide hydrochloride as a ligand according

ARAI at al.: Purification of a major esterase from hemolymph 123

to the manufacture's instructions, and subsequent-

ly equilibrated with 20 mM sodium phosphate

buffer (pH 7.0), containing 0.1 M NaCI. After

sample loading, the column (1.0 x 10 cm) was

washed with the same buffer and eluted stepwise

using 0.1 and 0.5 M NaCI. One ml fractions were

collected and tested for the esterase activity.

Electrophoresis

Polyacrylamide gel electrophoresis under

non-denaturing conditions (native PAGE) was

performed according to DAVIS (1964), and that in the presence of SDS (SDS-PAGE) according to

LAEMMLI (1970). The activity of esterase on the

gels after native PAGE was color-detected using the method of MARKERT and HUNTER (1959) using

a-NA as a substrate. After SDS-PAGE, the gels

were stained with Coomassie brilliant blue for the

detection of proteins. Isoelectric focusing was

performed in 5% polyacrylamide disc gels containing 2% (v/v) Ampholine (pH 3.5 to 10.0) at

200 V for 1 hr, then at 300 V for 15 hr and finally

at 500 V for 1 hr at 4°C (HORIO and YAMASHITA,

1981). After focusing, the gels were subjected to

the detection of esterase activity or cut into 0.5

cm-long pieces which were eluted in distilled

water to measure pH.

Gel filteation

Gel filtration was performed using a TSK

gel G3000SWxl column (7.8 mm x 30 cm) equili-brated with 0.3 M NaCI-20 mM sodium-potassium

phosphate buffer (pH 7.4), at a constant flow rate of 30 ml/hr. The molecular weight was calcu-

lated from the elution volume calibrated using the

standard curves made with molecular weight

markers.

Determination of optimum pH, pH stability and

thermostability

The optimum pH of BesB was determined

at 30°C for 20 min in a buffer of various pH

conditions, while the stability of BesB for pH was

determined at 4°C for 24 hr. The temperature

stability of BesB was studied by incubating the

enzyme at different temperatures between 0°C

and 100°C for 20 min followed by rapid cooling to 0°C. Esterase activity was assayed by the

spectrophotometric method described above for total esterase activity in the hemolymph.

N-terminus sequence analysis

Purified BesB was run on SDS-PAGE and was electrophoretically transferred to a

polyvinylidene dif luoride (PVDF) membrane (Bio Rad, CA, U. S. A.). The protein band was cut out and analyzed in a Model 476A gas phase peptide

sequencer (Applied Biosystem). Homology search was carried out with FASTA for esterase

sequences out of the pir database system (PEAR-SON and LIPMAN, 1988).

Western blot analysis

To make polyclonal antibodies against BesB, 0.2 mg of the purified enzyme preparation was emulsified with an equal volume of Freund's

complete adjuvant and the emulsified samples were injected into a mouse at multiple sites.

Second injection was done after 2 weeks and then a booster was given after 4 weeks. After inter-vals, the blood samples were collected and the

antibody was partially purified and diluted to 1:1, 000 dilution with the phosphate buffered saline

(FUJII and KAWAGUCHI, 1983). Proteins on the gel after native PAGE were electrophoretically transferred to a PVDF membrane. The mem-brane was first reacted with anti-BesB serum and then with digoxigenin-conjugated anti-mouse IgG

goat serum. The membrane was immersed to alkaline phosphatase labeled anti-digoxigenin

solution and was incubated in reaction mixture (0. 1M 2, 2'-iminodiethanol [diethanolamine] , 1mM

MgCl2, 0.02% sodium azide). After the reaction, the membrane was exposed to X-ray film.

Enzyme kinetic studies

To estimate apparent Michaelis constant

(Km) and maximal velocity ( Vmax) for BesB, ester-ase assay using purified BesB (40 ng) were perfor-med using four substrates, a-NA, a-NP, p-NPA

and p-NP. The concentration used as substrates for assay were 5 concentrations: 0.1-0.8 mM (a-

124 The Journal of Sericultural Science of Japan Vol.69 No-2

NA), 0.1-0.5 mM (a-NP), 0.05-0.25 mM (p-NPA)

and 0.1-0.5 mM (p-NP). Esterase activity was

measured in three times at 30•Ž. A Hanes-Woolf

plot ([S]/v vs [S]) was constructed for each

substrates and Km.and Vma. values for all sub-

strates were calculated.

Results

Developmental changes in esterase activity

In previous study, it was revealed that the

BesB was found in plasma, but not in hemocytes,

as well as in BesA and BesC. The esterase

activity of the plasma specimens collected during

the fifth instar and the pupal stage of the strain

n50, which possesses BesB, was assayed with

a-NA as a substrate (Fig. 1A). The activity was

low, between 186 and 486 units/ml, both in males

and females for the first 3 days of the fifth instar.

Thereafter, it increased sharply until the onset of

spinning and remained high throughout the pupal

stage. In the banding pattern of activity-

staining, BesB was detected on day 0 of the 5th

instar. The bands became intense during pupal

ecdysis, and then fairly constant during the pupal

period (Fig. 1B). However, other minor bands of

Est-2, Est-3, Est-4 and Est-5 were recognized from

the 5th instar to the pupal stage. Because this

result showed that BesB was a major plasma

esterase of n50 strain, we started the purification

of BesB.

Purification of BesB

The plasma collected from day-1 pupae of

the n50 strain was used for purification of BesB.

The elution profile of hydrophobic chromatogra-

phy on Butyl-Toyopearl showed BesB was eluted

at 9% ammonium sulfate (Fig. 2A). Affinity chro-

matography on procainamide-cellulofine of the

active fractions of the 2nd Butyl-Toyopearl showed

that BesB could bind to resin, but no other pro-

teins could (Fig. 2B). Then BesB was eluted as a

single peak at 0.5M NaCl. Table. 1 summarized

the results of purification of BesB. Starting

from 5 ml hemolymph, 10 ug (1,200 units) of

purified esterase was obtained with a purification

factor of about 2,000-fold and a yield of about 9%.

(A)

(B)

Fig. 1. (A) Developmental changes of ester-

ase activity in the hemolymph of Bombyx

mori n50 strain during the fifth larval instar

and pupal stage. (A) Each plot represents the

mean of 6 independent determination. Verti-

cal bars show standard deviations. -•œ-:

females, -•›-: males. (B) Pattern of ester-

ase activity band of gel electrophoresis under

non-denaturing condition from the 5th instar

to pupal period. The numbers along left

margin represent esterase a components:

BesB; Est-2; Est-3; Est-4, Est-5.

Characterization of purified BesB

The final esterase preparation showed one

protein band on native PAGE followed by activity staining (Fig. 3A) and SDS-PAGE followed by

protein staining (Fig. 3B). By SDS-PAGE, BesB showed a molecular weight of about 58,000.

Estimation by gel filtration showed the molecular

weight of 62,000 (data not shown). The


(A)

(B)

Fig. 2. (A) Second Butyl-Toyopearl column

chromatography of 1st Butyl-Toyopearl frac-

tions. The fractionated and then concen-

trated protein (780 tcg) was applied on to the

column (2.2 x 30cm) equilibrated with 20 mM

sodium phosphate buffer (pH 7.4) containing

40% ammonium sulfate. The column was

washed with 3 volumes of the equilibration

buffer. Elution was performed in a linear

gradient of ammonium sulfate (from 40 to

0%) made up to the equilibration buffer at a

flow rate of 30 ml/ hr. Fractions of 5 ml

were collected and assayed for esterase activ-

ity (-•œ-). Protein concentration was

monitored by the absorbance at 280 nm (-•›-

). (B) Procainamide hydrochloride bound

formyl-cellulofine affinity chromatography

of the second Butyl-Toyopearl fractions.

The fractionated and then concentrated pro-

tein (220 ug) was applied onto the column (1.

0 x 10 cm) equilibrated with 20 mM sodium

phosphate buffer (pH 7.4) containing 0.1 M

NaCl. The column was washed with 3 vol-

umes of equilibration buffer. Elution was

carried out using 0.5 M NaCI in the equilibra-

tion buffer at a flow rate of 10 ml/ hr.

Fractions of 1 ml were collected and assayed

for esterase activity (-•œ-). Protein con-

centration was monitored by absorbance at

280 nm (-•›-).

Table. 1. The summary of purification steps

of Bes-B

*One unit corresponds to hydrolyzing one n

mol of a-naphthyl acetate per min at 30•Ž

(A) (B)

Fig.3. Polyacrylamide gel electrophoresis of purified esterase. (A) Under the non-denaturing conditions of 7.5% acrylamide gel. lane 1: activity staining for esterase, lane 2: Coomassie brilliant blue staining for proteins.

(B) In the presence of SDS of 12.5% acrylamide gel. The numbers along the right margin represent molecular weights;

phosphorylase a (94 kDa), bovine albumin (67 kDa), ovalbumin (43 kDa), carbonic anhy-drase (30 kDa), soybean trypsin inhibitor (20 kDa) and a-lactalbumin (14 kDa).

isoelectric point of this enzyme, as determined by

electrofocusing using 5% polyacrylamide gel, was

4.6. (data not shown)

The highest activity of the enzyme occur-

red at a pH of 7.0 (Fig. 4A). Treatment of the

enzyme at different pHs at 4°C for 24 hr or at

different temperatures for 20 min at pH 7.0

showed that it was most stable between pH 7 and

9, and lost activity below pH 4 and over pH 10

(Fig. 4B), and that the activity declined gradually as the temperature increased, becoming 80% of


(A)

(B)

(C)

Fig. 4. (A) Optimal pH of BesB. The pur-

ified BesB was incubated at various pHs at

30•Ž for 30 min. •›: citrate, š˜: phosphate, •¢

: Tris-HC1, •Þ: glycine-NaOH. (B) pH sta-

bility of BesB. The purified BesB was in-

cubated at various pHs at 4•Ž for 24 hr. •œ:

citrate, •¡•: phosphate, •£: Tris-HC1, •¥:

glycine-NaOH. (C) Thermostability of

BesB. BesB was incubated at various tem-

perature for 10 min, and then placed on ice.

Fig. 5. N-terminal amino acid sequence of

BesB and its alignment with reported ester-

ases. MPESTRFE4: Myzus persicae esterase

FE4, MPESTRE4: Myzus persicae esterase E4.

The numbers indicate the residues. Shad-

owed box indicates identical amino acids.

the original level at 30•Ž and 0% at 60•Ž (Fig. 4C).

The sequence of the first 28 residues of

N-terminal amino acids was determined (Fig. 5).

A homology search with 291 previously registered

esterases showed that the sequence was 30%

homologous to those of the N-terminal amino

acids of esterases FE4 and E4 from the peach-

potato aphid Myzus persicae (FIELD et al., 1993).

Western blot analysis

The mouse polyclonal antibody raised

against the purified BesB reacted with the pur-

(A) (B)

Fig. 6. Western blot analysis of purified

esterase and hemolymph. After native PAGE, the gels were blotted to PVDF mem-

branes and were immunostained with the antibody raised against purified BesB. (A),

purified BesB (Lane 1); hemolymph of the strain n50 (Lane 2); activity staining of pur-ified BesB (Lane 3). (B), Several strains homologous to respective types of Bes all-ozymes. n51 for BesA (Lane 1), n50 for BesB

(Lane 2), w31 for BesC (Lane 3) and u10 for BesO (Lane 4). purified BesB (Lane 5).


ified preparation specifically, producing a single

band that co-migrated with the purified prepara-

tion stained for the esterase activity (Fig. 6A).

Figure 6B also showed that the antibody was

reactive to other Bes allozymes, BesA and BesC

from different strains of the silkworm. This

finding is compatible with the notion that BesA, B

and C are the products of codominant alleles.

The antibody produced no band with the speci-

(A)

(B)

Fig. 7. Hanes-Woolf plot of [S] vs [S] /v

for BesB hydrolyzing a-NA and a-NP (A),

and p-NPA and p-NPP (B). The assay was

performed using 40 ng of purified BesB at

30•Ž. Each point is based on the mean of

three determinations (each with triplicate

incubations, n=3). Regression coefficients

(r) >0.98.

mens from the BesO strain (Lane 4).

Kinetic parameters

The substrate specificity of BesB was

investigated at various concentrations using four

substrates. Figure 7 shows Hanes-Woolf plots

for BesB hydrolyzing for a-NA, a-NP (A), p-

NPA and p-NPP (B). Km and Vmax values were

1.73 X 10-4 M and 1.15 X 10-6 (M • min-1) for a-NA,

1.68 x 10-4 M and 1.10 x 10-6 (M • min-1) for a-NP,

1.10 X 10-4 M and 1.09 X 10-4 (M • min-1) for p-NPA

and 3.14><10 _4 M and 1.07><10 _4 (M • min-1) for

p-NPP. BesB showed 100-times higher values for Vmax to a-NA and a-NP than to p-NPA and

p-NPP. BesB was unable to hydrolyze acetylth-iocholin (ATCh).

Inhibition assay for BesB

The effects of the following inhibitors on

the activity of BesB were investigated: paraoxon,

diisopropyl f luorophosphate (DFP), eserin sulfate,

p-chloromercuribenzoic acid (pCMB), ethylen-diamine tetraacetic acid disodium salt (EDTA

2Na), and phenylmethylsulfonyl fluoride (PMSF)

(Table 2). The 150 value (the concentration required to inhibit 50% of esterase activity) of

paraoxon was the lowest among them, and that of EDTA was the highest. Paraoxon was the stron-

gest inhibitor among those tested for BesB.

Discussion

The major hemolymph esterase of B. mori

is composed of the allozymes BesA, BesB or BesC

(or a pair), depending on the strain. Among Bes allozymes of hemolymph, we found that the BesO

strain is hardly distinguishable from the strains of

Table. 2. Effect of inhibiotrs on activity of

purified Bes-B.


BesA, BesB or BesC phenotypes in respect to

their apparent characters; morphology, viability,

growth, fecundity and others. A similar phenom-

enon has been recognized with amylase in diges-

tivejuice. Two types of amylase activity, weak

and strong, are observed among B. mori strains,

and they are controlled by the ae and + ae gene,

respectively (MATSUMURA, 1938). The strain

with weak amylase activity was as healthy as

those with strong activity, although the reason is

also be unknown.

The banding pattern of esterases in

hemolymph of the n50 strain during the 5th instar

to pupal stage were consistent with reports on the

BesA strain OKIDO et al. (1996), although the

pattern of minor esterases bands on the n50 strain were much stronger than that on the BesA (n51)

strain. Judging from the intensity of the BesB

band, BesB activity in total esterase might be

higher in the n50 strain than that in the examined

strain of our laboratory. The developmental

pattern of hemolymph esterase activity in B. mori resembled that of esterase-6 in Drosophila

melanogaster (SHEEHAN et al., 1979) and of a-

naphthyl esterase in hemolymph of Manduca sexta

(JESUDASON et al., 1990). Since there were ester-ases which resembled the pattern of activity of

esterase during development in other insects,

BesB was always a major esterase component

and its activity was higher during pupal periods

than during the larval period, suggesting that

BesB in particular might play role after the late

5th instar.

In our purification procedure, more than

half of total esterase activity was discarded after

the ammonium sulfate precipitation step so that it

would be easy to remove contaminant proteins

through the subsequent column chromatography

steps. BesB with an apparent molecular weight

of 58,000, was similar in size to the other insect

esterases such as the brown rice leafhopper (62,

000 and 64,000) (CHEN and SUN, 1994) and midgut

carboxylesterase of a mosquito (59,000) (WHYARD

et al., 1994), as well as hepatic and renal carbox-

ylesterase of the guinea pig (56,000) (ECOBICHON, 1969). In gel filtration, BesB gave one peak at

62,000, suggesting that BesB is a monomeric

protein. Previously known esterases were repor-ted to be monomeric except for pig liver and

kidney aliesterases, which might be in a tetramer-

ic aggregate (CHOW and ECOBICHON, 1973). The

isoelectric point of BesB (pI 4.6) was also similar

to those of the above mentioned insect esterases.

In immunological experiments, anti- BesB

antibodies cross-reacted with hemolymph speci-

mens from day 1 pupae of BesA, BesB and BesC

strains, while no band was detected in the BesO

hemolymph specimen. This result is compatible

with the notion that BesA, BesB and BesC are the

product of codominant alleles (allozymes), and suggests that no cross reactive protein of compa-

rable size has in the hemolymph of BesO strain.

Enzymatic characterization showed that

BesB had a broad range of affinity for substrates.

The Vmax values of BesB for p-NPA and p-NPP

were three orders of magnitude larger than for

a-NA and a-NP. In this respect, the BesB

roughly resembled other insect esterases such as

esterase-6 of D. melanogaster (MANE et al., 1983),

midgut carboxylesterase TE-I and TE-II from the

termite, Odentotermes horni (SREERAMA and

VEERABHADRAPPA, 1991), carboxylesterase of the

brown rice planthopper, Nilaparvata lugens (CHEN

and SUN, 1994) and salivary-gland esterase of the

mosquito, Aedes aegypti (ARGENTINE and JAMES,

1995). It was also confirmed that BesB does not

belong to acetylcholinesterase in hemolymph.

The activity of BesB was strongly inhibited

by organophosphorus (OPs) such as paraoxon and

DFP, and moderately by PMSF. It has been

reported that the activity of several esterases in

insects is strongly inhibited by organophosphorus

(CHEN and SUN, 1994: SHU and CLARK, 1994: ARGENTINE and JAMES, 1995). It also is known

that the amino acid residue in the active centesr

of some esterases inhibited by organophosphorus

in insects is serine (RUSSELL et al., 1996). The

present result suggests that the amino acid resi-due in the active center of BesB may also be

serine. The active center of BesB may also be

similar to that of serine protease because of

PMSF inhibition, although a detailed amino acid


sequence attributing to the active center has not

been described. Esterases can be divided into

four classes on the basis of their sensitivities to

three groups of inhibitors, sulfhydryl regents

(such as PCMB), OPs (such as paraoxon), and eserin sulfate (HOLMES and MASTERS, 1967).

Carboxylesterases are the most inhibited by OPs,

while cholinesterases are inhibited by eserin sul-

fate, and arylesterases are inhibited by sulfhydryl

reagents. The result of inhibitory experiment

revealed that BesB would be carboxylesterase in

hemolymph.

Acknowledgments:

We thank Prof. K. KOGA, Kyushu Univer-

sity, for a critical reading of the manuscript and

suggestions, and Prof. S. KUHARA, Kyushu Uni-

versity, for his helpful advice on amino acid

homology searches. This work was supported in

part by two Grant-in-Aid for Scientific Research

(A12306005) and (09045069) from the Ministry of Education, Science and Culture of Japan.

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新井仁・大城戸利久・藤井博・土井良宏:カイコ体液の主要エステラーゼの精製とその

性質

カイコ体液の全エステラーゼ活性を基質として α-ナフチル酢酸を用いて測定した。その

結果,5齢初期は低かったが,吐糸期以降急激に上昇し,蛹期は高い状態であった。以前BesB

と名付けられていたカイコ体液の主要エステラーゼを塩化プロカインアミドをリガンドにし

たアフィニティークロマトグラフィーを用いた方法で蛹1日の体液から精製した。BesBの

分子量は58,000,等電点は4.6であった。活性はpH7.0の時が最も高く,また,pH7～9の

範囲で安定であった。N末端28残基のアミノ酸配列はモモアカアブラムシから精製されたエ

ステラーゼと相同性が見られた。kinetic解析では用いた4種の基質に対するKm.値に差異は

見られなかったが,Vmax値には違いが認められた。BeSBはparaoxonとdiisopropyl fluoro-

phosphate(DFP)に感受性が高く,硫酸エゼリン,phemylmethylsulfonyl fluoride(PMSF),

p-chloromercuribenzoic acid(pCMB)にも阻害効果が見られた。これらの結果から,BesBは体液中のカルボキシルエステラーゼあると思われた。

purification and characterization of a major esterase besb

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