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Phenol ╬▓-glucosyltransferase and ╬▓-glucosidase activities in the tobacco hornworm larva Manduca sexta L.Properties and tissue localization

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Archives of Insect Biochemistry and Physiology 21 :207-224 (1992)
Phenol p-Glucosyltransferase and
p-Glucosidase Activities in the Tobacco
Hornworm Larva Manduca sexta (1.):
Properties and Tissue localization
Saad A. Ahmad and Theodore L. Hopkins
Department of Entomology, Kansas State Uniuersify, Manhattan, Kansas
Phenol p-glucosyltransferase (PGT; EC 2.4.1.35) was studied in feeding fifth
stadium larvae of the tobacco hornworm, Manduca sexta, using reversed-phase
HPLC with absorbance detection to separate and quantify both the model
substrate, p-nitrophenol (PNP), and the product, p-nitrophenyl f3-D-glucopyranoside (PNP-Clc). About 90% of total PGT activity in tissue homogenates was
associated with the particulate fraction (15,00(%),with the remainder in the
microsomal fraction. PGT activity was observed in all tissues, with highest
activities in the labial gland and fat body. Appreciable activity occurred in rnidgut
and hindgut tissue but none was found in hernolymph. PGT activity was also
observed in eggs and larval fat body at different times during development. Activity
was optimal at pH 7.5-9 and was highest with UDP-Glc as a glucose donor.
However, appreciable PCT activity was observed with dTDP-Clc or GDP-Glc in
place of UDP-Glc. The divalent cations Ca2+, Co2+, Mg", and Mn2' stimulated
activity, whereas Zn2+ and Hg2+, as well as pretreatment with the detergent Triton
X-100, were inhibitory. Endogenous P-glucosidase in the PGT-enrichedfractions,
especially from the midgut, antagonized the pglucosylation process, and interference was minimized with higher pH and the addition of D-gluconic acid
lactone to the incubation mixtures. The possible role of transglucosylation in the
detoxication of phenolic xenobiotics and biotransformation of endogenous phenolic compounds in insects i s discussed. Comparisons of PCT with some
glycosyltransferases involved in endogenous and xenobiotic conjugation in insects
and other organisms are reviewed. Q 1992 Wiley-Liss, Inc.
Key words: conjugation, glucosylation, p-glucosides, UDP-glucosyltransferase, insects
Acknowledgments: The authors thank Dr. Karl Kramer for supplying the experimental insects and
for helpful suggestions on the manuscript. This paper is contribution no. 92-543-Jfrom the Kansas
Agricultural Experiment Station, Manhattan, Kansas. This research was supported in part by KAES
Project 548.
Received April 29, 1992; accepted July 28, 1992.
Address reprint requests to T.L. Hopkins, Department of Entomology, Kansas State University,
Manhattan, KS 66506.
0 1992 Wiley-Liss, Inc.
208
Ahmad and Hopkins
INTRODUCTION
The ability of insects to synthesize p-glucoside conjugates of natural and
synthetic phenolic compounds is well established [1,2]. P-Glucosylation requires glucose donors in the form of specific high-energy glucosyl nucleotides
and is catalyzed by the enzyme phenol P-glucosyltransferase. Conjugation
with glucose is generally considered to be a mechanism of detoxification by
insects, resulting in increased water solubility and more rapid excretion of
xenobiotics and allelochemicals. 0-Glucosides are often less toxic than the
parent aglucones, probably because of rapid elimination and the masking of
functional groups [3]. Evidence is increasing that, in addition to detoxication,
P-glucoside formation in insects serves as a means to store and regulate levels
of endogenous phenolic metabolites [PB]and plant allelochemicals [9,10;
unpublished data] for physiological processes. Therefore, P-glucosylation
appears to be an important biochemical reaction in insects, allowing them to
cope with potentially harmful compounds in the diet, as well as accommodating metabolic needs during growth and development. However, relatively
little is known about the properties and functional role(s) of the glucosyltransferases that catalyze glucosylation reactions in insects [2]. In this
study, we have examined the localization and properties of PGT" in tissues of
feeding fifth instar tobacco hornworm larvae, Munducu sexfa (L.). A sensitive
method utilizing HPLC has been developed to measure PGT activity by
specific quantitation of product formation.
MATERIALS AND METHODS
Insects
Larvae of M . sextu reared at 27 f 1°C with a photoperiod of 16L:8D [ll]
were supplied by Dx. Karl J. Kramer, U.S. Grain Marketing Research Laboratory, Agricultural Research Service, U.S.D.A., Manhattan, KS.
Chemicals
P-Glucosidase (almond) and all biochemicals were obtained from Sigma
Chemical Co., St. Louis, MO, except 7-methoxycoumarin, which was from
Aldrich Chemical Co., Milwaukee, WI. HPLC grade acetonitrileand methanol
were from Fisher Scientific Co., St. Louis, MO.
Tissue Preparation and Subcellular Fractions
Three-day-old fifth instars were immobilized in crushed ice, the anal horn
was cut, and hemolymph was collected in ice-cold 1.5 ml plastic centrifuge
vials containing a few crystals of diethyldithiocarbamic acid to inhibit phenoloxidases. The hemolymph was assayed immediately or stored at -20°C for
2-3 h. The bled larvae were dissected dorsally and tissues in situ were washed
with distilled water and then with 0.1 M sodium phosphate, pH 7.5, or with
*Abbreviations used: PGT = phenol P-glucosyltransferase; PIPES = piperazine-N,N-bis-(2-ethanesulfonic acid) monosodium; PNP = p-nitrophenol; PNP-Clc = pnitrophenyl-P-D-glucopyranoside.
Phenol 6-Glucosyltransferases in M. sexta
209
0.125 M KC1 (when the effect of pH was studied). The labial glands and fat
body were isolated. The mid- and hindguts (including Malpighian tubules)
were separated, cleaned of fat body, dissected longitudinally, and washed
with buffer or KC1 solution to remove the lumen contents, The skeletal muscle
and epidermis were cleaned of other tissues and pieces of epidermis-free
muscle were isolated. Epidermis with some muscle was collected by scraping
the inner surface of abdominal cuticle. Each tissue was kept in buffer or KCl
solution and homogenized in the proper medium in a glass tissue grinder for
1min. All steps were carried out at 0-5°C. The homogenates were centrifuged
at 15,0008 for 30 rnin at 5"C, and the supernatants were discarded unless
stated otherwise. The pellets were washed twice with medium without
disruption and then were resuspended for use as enzyme sources.
To study the subcellular distribution of PGT, the crude tissue hornogenate
in 60 mM phosphate buffer (pH7) was centrifuged at 600g for 15rnin to obtain
a pellet of nuclear and cell debris. The supernatant was centrifuged at 10,OOOg
for 20 min to obtain the mitochondria1 pellet, followed by centrifugation at
110,OOOg for 90 min to obtain a microsomal pellet and cytosol. Each pellet was
resuspended in buffer in a glass tissue grinder.
To study the activity of PGT during development, 15,000.g pellets and
supernatants were prepared from eggs (1-2 days before hatching) and fat
body from fourth and fifth instars (at ecdysis and 1 and 4 days old),
Enzyme Assays
All PGT assays were performed with same-day enzyme preparations, and
the typical incubation mixture contained 5 mM PNP, 5 mM UDP-Glc, 25 mM
MgC12,5mM D-gluconic acid lactone, and enzyme preparation (equivalent to
0.06-0.3 mg protein) in 0.1 M phosphate buffer (pH 7.5) in a final volume of
0.15 ml. The reaction was started by the addition of the enzyme source to the
incubation mixture at 30" C in a shaker water bath. The mixture was vortexed
and returned to the water bath. After 30 min, the reaction was stopped by the
addition of 75 PI of ice-cold 9% perchloric acid, vortexed, and held 15 min on
crushed ice. The mixtures were centrifuged to remove denatured protein, and
the supernatant was held at 6 5 ° C until analyzed for PNP-Glc by HPLC. In
control incubations in which UDP-Glc was omitted, no PGT activity was
observed. Antagonistic P-glucosidase activity, particularly in midgut tissues,
was suppressed by using the inhibitor, D-gluconic acid lactone, and an
alkaline buffer (see Results). The amount of PNP that disappeared was equal
to the amount of PNP-Glc formed in the PGT complete incubations, indicating
that PNP underwent P-glucosylation only. The identity of the products was
confirmed by comparing retention times with standards and by hydrolysis of
PNP-Glc formed by commercial P-glucosidase.
To study the effect of glucosyl nucleotides other than UDP-Glc, 5 mM of
ADP-Glc, CDP-Glc, GDP-Glc, dTDP-Glc, UDP-GlcNAc, or UDP-GlcUA replaced UDP-Glc in the incubations. When the effect of Triton X-100 was
studied, it was incubated with the enzyme source in the incubation mixture
for 15 min and the reaction was started by addition of UDP-Glc. To study the
effect of pH on PGT activity, PNP, UDP-Glc, and MgC12 were prepared in
210
Ahmad and Hopkins
appropriate buffers and added in incubation mixtures to yield 0.1 M final
buffer Concentration.
PGlucosidase activity was measured with PNP-Glc as the substrate. The
incubation mixture contained, in a final volume of 0.15 ml, the enzyme
preparation (0.05-0.1 mg protein) in 0.125 M KCl and variable substrate
concentrations and buffers, as presented in Results. No PGT activity interfered with the P-glucosidase activity when UDP-Glc was omitted from the
incubations. Enzyme activity was determined by the amounts of PNP released
as quantified by HPLC.
Cytochrome P-450-dependent monooxygenase activity was measured with
7-methoxycoumarin as the substrate. The incubation mixture contained 1mM
NADPH, 50 p,M 7-methoxycoumarin, and midgut 15,0009 pellet or supernatant (equivalent to 0.1 mg protein) in 0.1 M phosphate buffer at pH 7.5. The
reaction was started by addition of the substrate in methanol, the final
concentration of which did not exceed 3% in the incubation mixture. After 1
h incubation at 30T, the reaction was stopped by addition of 4 volumes of
cold methanol. The mixture was vortexed, held 15min at - 20" C, centrifuged,
and the supernatant was analyzed for umbelliferone by HPLC,
HPLC
The separation and quantitation of PNP, PNP-Glc, 7-methoxycoumarin,
and umbelliferone were achieved using reversed-phase HPLC with a C18 5
pm spherical particle 4.6 x 250 mm column with a flow rate of 1 mVmin at
30°C and a variable wavelength absorbance detector. The mobile phase
contained either 20% methanol or 10%acetonitrile in 0.1 M sodium phosphate
buffer, pH 3, for PNP and PNP-Glc separation or 30% methanol in the same
buffer for separation of 7-methoxycoumarin and umbelliferone. The eluate
was monitored at 303 nm for PNP and PNP-Glc, and at 321 nm for 7methoxycoumarin and umbelliferone. Quantitation of products was determined by comparing their peak areas with those of known concentrations of
standard compounds.
RESULTS
SubcellularDistribution
PGT activity was found to be mainly associated with the particulate fraction,
with about 90% of total activity recovered in the 15,0008pellet from all tissues
(Fig. 1).Subcellular fractionation of the fat body by differential centrifugation
revealed that PGT activity was equally distributed between the nuclear (600g)
and mitochondria1 (10,OOOg)pellets (Fig. 1).The remaining 10% of activity in
the 15,0008supernatant was entirely microsomalbecause the cytosol obtained
from 110,OOOg centrifugation was devoid of activity. To determine if the PGT
in the 15,0009 pellet was a particulate enzyme and not due to early sedimentation of microsomes, we compared the subcellular distribution of PGT with
a microsomal marker enzyme, 7-methoxycoumarin 0-demethylase (a cytochrome P-450-dependent monooxygenase), from the midgut. Midgut incubations showed that 64.3% of total 7-methoxycoumarin demethylase activity
Phenol p-Glucosyltransferasesin M. sexta
100
A
x
1-
1 5 , 0 0 0 a supernatant
0Microsokmes'
80 - eZa 600g pellet
659 10,0009 pellet
EEd 15,OOOg pellet
v
x
4
._
.->
21 1
60 -
40
0 20
t
0
[ Fat body
1
Labia
_1
land
[ION
4k7-M€riOXYCOUMARIN
-I
0- DEMETHYLATION
Fraction
Fig. 1 . Subcellular distribution of PGP activity and cytochrome P-450-dependent monooxygenase
(7-methoxycoumarin 0-dernethylation) in tissues of fifth stadium larvae of Manduca sexta
was recovered in the 15,OOOg supernatant, whereas the remaining activity was
in the pellet (Fig. 1).In contrast, 87.4%of total PGT activity was found in the
pellet and the remainder was in the supernatant. Because of enrichment of
PGT in the particulate fraction, all further studies were carried out using the
15,OOOg pellet as the PGT source.
Tissue Distribution
All tissues except the hemolymph were active in the P-glucosylationof PNP
(Fig. 2). However, a marked difference was observed in specific activity of
PGT among tissues. The labial gland was the most active tissue, with a specific
activity of 12.2 nmol PNP-Glc formed/min/mg protein, followed by fat body,
6.6; midgut, 4; hindgut, 3.1; epidermis with muscle, 0.4; and muscle, 0.2.
Because of their higher activities, the labial gland and fat body were used in
subsequent studies.
Effect of pH
PGT activity in labial gland was detectable at pH 5.5, increased sharply at
pH 6, and reached a maximum at pH 7 . 5 9 (Fig. 3). Activity decreased steeply
at higher pHs and was undetectable above pH 10. PGT from the fat body
behaved similarly, except that it had a broader range of activity, with low
glucosylation rates detected at pH 4.5 and 10.5 (Fig. 4).
Buffers showed a difference in their influence on PGT activity from both
the same and different tissues. PIPES and phosphate buffers gave maximum
PGT activity from labial glands, whereas PIPES was slightly superior to
212
Ahrnad and Hopkins
Tissue
Fig. 2. PGT activity in tissues of fifth stadium larvae of Manduca sexta (means
* SD, n = 3-6).
phosphate in the case of fat body PGT. Tris-HClwas the poorest in the optimal
pH range for both tissues. Highest activity in fat body was observed with
glycine-NaOH buffer at pH 8.75-9. Such an effect of glycine buffer was not
observed on PGT from labial glands; however, it was better than tris-HC1 at
2
4
a
6
10
12
PH
Effect of pH o n PGT activity in preparations from labial gland of fifth stadium larvae
(0)
universal, (@I citrate, (A 1 PIPES, (A)phosphate,
(0)
tris-HCI, (m) glycine-NaOH, and (4)borate.
Fig. 3.
of Manducasexta. Buffers (0.1 M) were
Phenol p-Clucosyltransferasesin M. sexfa
21 3
n
.-cat
Y
x Pa
4
.->
r
'In
.- -.-E
-P
0
Q
0
.+
.-
'c
E
D
E
P
0
0
1/)
0-
W GI
fl n
z
2
4
6
8
10
12
Fig. 4. Effect of pH on PCT activity in preparations from fat body of fifth stadium larvae of Manduca
sexta. Buffers (0.1 M)were (a)acetate, (A)PIPES, (A)phosphate, (0)tris-HCI, and (W)glycine-NaOH.
alkaline pH. Sodium phosphate buffer, pH 7.5, was used in further experiments because of its pKa2 of 7.2, which falls close to the optimal pH range of
PGT.
Interference by Endogenous P-Glucosidase
During investigation of the tissue site of tyrosine P-glucoside biosynthesis
in fifth stadium larvae of M. sexta, we observed that tyrosine glucoside in the
hemolymph was hydrolyzed when incubated with crude homogenates of
midgut at pH 7 (unpublished data). These homogenates also hydrolyzed
esculin, a natural p-glucoside of 6,7-dihydroxycournarin, as well as PNP-Glc,
PNP-a-glucoside, PNP phosphate, PNP sulfate, and PNP-N-acetyl glucosamine, but not PNP-glucuronide. Some inhibition of esculin hydrolysis was
achieved when 10 mM gluconic acid lactone was included in the incubation.
When a solution of esculin was perfused into the lumen of an isolated midgut
with both ends ligated and bathed in Manducu saline, samples of the saline
analyzed by HPLC 2 h later showed the presence of both esculin and its
aglucone, esculetin. Those preliminary results drew attention to the likely
presence of endogenous p-glucosidases, which may interfere with PGT assays
by hydrolyzing the PNP-Glc formed. PNP-Glc hydrolysis was also observed
with crude homogenates of labial gland and fat body, but to a lesser extent
than with homogenates of the midgut. In addition, both the 15,0009 pellet
and the supernatant of midgut homogenates hydrolyzed PNP-Glc. Washing
the pellet twice by resuspension in buffer, centrifugation, and discarding the
washes was unsuccessful in removing the hydrolytic activity. Two approaches
214
Ahmad and Hopkins
4
6
8
10
PH
Fig. 5. The influence of pH on PNP-Clc hydrolysis by endogenous P-glucosidase in the 15,OOOg
pellets from fat body and midgut of fifth stadium larvae of Manduca sexta. Buffers (0.1 M) were
acetate for pH 5.0, phosphate for pH 7.5, tris for pH 8.5, and glycine for pH 8.75-9.5. Enzyme
sources and substrate concentrations were midgut with (0)
3.33 mM PNP-Clc, ( A ) 0.25 mM
PNP-Clc, [A)0.25 mM PNP-Glc 10 rnM D-gtuconic acid lactone; fat body (El)with 0.25 rnM
PNP-CIC.
+
were studied to minimize P-glucosidase interference; (1)shifting the incubation buffer to a higher pH and (2) using the competitive inhibitor gluconic acid
lactone. The influence of pH on endogenous P-glucosidase activity in the
15,OOOg pellet from midgut and fat body tissue is depicted in Figure 5. As the
pH of incubation increased, the specific activity of p-glucosidase decreased.
At an initial concentration of 3.33 mM PNP-Glc, P-glucosidase activity in the
midgut preparation dropped from 3.54 nmol PNP-Glc hydrolyzed min/mg
protein at pH 5 to 3.08 at pH 7.5 and then sharply to 0.33 at pH 8.5. However,
glycine buffer at pH 9 was not effective in reducing hydrolytic activity (2.18
nmoVmin/mg).
Lower P-glucosidase activities were obtained when midgut and fat body
enzyme preparations were incubated with a 0.25 mM initial PNP-Glc concentration (equals 5% of initial PNP concentration in PGT assays and presumably
equals the concentration of PNP-Glc formed when 0.1 mg labial gland pellet
is incubated for 30 min in the complete PGT assays). P-Glucosidase specific
activities decreased from 1nmol PNP-Glc hydrolyzed/midmg protein at pH
5 to 0.04 nmol PNP-Glc hydrolyzed/min/mg at pH 8.5. Glycine buffer at a
higher pH range was also ineffective compared to the other buffers in
suppressing p-glucosidase activity. The addition of 10 mM D-gluconic acid
lactone to the midgut incubation mixture at pH 7.5 reduced P-glucosidase
activity by about one half.
D-Gluconicacid lactone inhibition of P-glucosidase from the midgut 15,0009
pellet and supernatant is shown in Figure 6. Percent inhibition was linear with
Phenol p-Glucosyltransferasesin M. sexfa
215
100
15,OOOg pellet
o 15,OOOg supernatant
801
.+
.n
D-Gluconic Acid Lactone
[ mM ]
Fig. 6 , The inhibitory effect of D-gluconic acid lactone on PNP-Glc hydrolysis by endogenous
P-glucosidase in midgut 15,OOOgfractions of (0)
pellet and (0)supernatant from fifth stadium larvae
of Manduca sexta. The initial PNP-Glc concentration was 3.33 mM. Buffer was 0.1 M sodium
phosphate, pH 7.5.
the concentration (mM) of the inhibitor. Fifty percent inhibition occurred at
7.58 and 9.92 mM for the supernatant and pellet, respectively. Addition of a
high concentration of the inhibitor resulted in lowering the pH of the incubation mixture. Therefore, 5 mM D-gluconic acid lactone was added in the PGT
assays at pH 7.5, effectively suppressing P-glucosidase activity to less than 50
pmol PNP-Glc hydrolyzed/min/rng protein.
Effect of Divalent Cations
The effect of various divalent cations on PGT activity is presented in Figure
7. Zn2+ was the most inhibitory, with PGT activities being 11.1,0.1, and 0%
of the control value, whereas H$+ was the next most inhibitory, with
activities 69, 7.1, and 0% of control at concentrations of 1, 5, and 25 mM,
respectively. Co2+ and Mn enhanced activity at 1 and 5 mM, but the stimulatory effect of Co2+ decreased, whereas Mn2+ was inhibitory at 25 mM.
Mg2+ and Ca2+ steadily increased PGT activity as the concentration was
increased from 1to 25 mM with Ca2+ slightly more effective at all concentrations. No difference occurred in the pattern of stimulation between the sulfate
and chloride salts of Mg. The Mg effect reached a plateau at concentrations
between 15 and 45 mM, but PGT activity decreased slowly at higher concentrations (data not shown).
Effect of Triton X-100
The non-ionic detergent, Triton X-100, was studied in regard to its effect on
PGT activity (Fig. 8). It inhibited PGT at a final concentration as low as 0.07
216
Ahrnad and Hopkins
250
I
01 mM
IEEl 5 mM
L
0
150
W
I
1004
n
HZ'
' :Z
Co2+
Mn2+
Cation
Fig. 7. The influence of divalent cations on PNP glucosylation by PGT from labial glands of fifth
stadium larvae of Manduca sexta.
Ratio ( w/w ) Triton X-1 OO/Protein
0.0
0.0
0.4
0.2
1.2
0.8
0.4
0.6
1.6
0.8
1.0
2.0
1.2
2.4
1.4
Triton X-100 Conc. ( mg/ml )
Fig. 8. The effect of Triton X-100 on PNP glucosylation by PGT from labial glands of fifth stadium
larvae of Manduca sexta.
Phenol P-Glucosyltransferases in M. sexfa
217
7GDP-Glc
&481 dTDP-Glc
Hindgut
Midgut Fat body Labial gland
Tissue
Fig. 9. Utilization of glucosyl nucleotides as glucose donors by PGT from tissues of fifth stadium
larvae of Manduca sexta.
mg/ml (or a ratio of detergent/protein of 0.113wiw). PGT activity was strongly
inhibited at concentrations higher than 0.2 mg/ml.
Effect of Glucose Donors
Five glucosylated nucleotides were compared in relation to their preference
as glucose donors in PGT mediated reactions (Fig. 9). UDP-Glc was the
preferred glucose donor in all tissues. dTDP-Glc (a synthetic p-glucosyl
nucleotide derivative of dTDP, unlike the other nucleotides that are glucosylated with a-linkages) was next in preference by PGT from all tissues,
followed by GDP-Glc. Very low rates of PNP glucosylation in incubation
mixtures of fat body (85and 13 pmollmidmg protein) and labial gland (40 and
51 pmollmidmg protein) were detected with ADP-Glc and CDP-Glc, respectively. When UDP-GlcUA was substituted for glucose donors in the labial
gland incubations, the observed activity was 29 pmol PNP-glucuronide/min/mg protein. When the glucosyl nucleotides were individually included with UDP-Glc in the incubations, PGT activity was not affected
compared with mixtures containing only UDP-Glc, except that UDP-GlcUA
caused 13.6%inhibition.
PGT Kinetics
A plot of PGT specific activity from labial glands and fat body vs. specific
activity/PNPconcentration (mM)is depicted in Figure 10. Apparent Kms were
0.14 and 0.1 mM PNP and Vmaxs were 15.2 and 7.9 nmol PNP-Glc
formed/min/mg protein for the labial gland and fat body, respectively. PGT
from both tissues obeyed simple Machaelis-Mentin kinetics, in that the K
,
did not change throughout the range of substrate concentration examined.
218
Ahmad and Hopkins
1
c1
c
.W
+
e4
-' m1
-'.-Ec
E
0
B
I
a
z
a
0
20
40
60
80
100
120
Fig. 10. Determination of kinetic parameters and specific activity (inset) of PGT towards PNP in
the 15,OOOg pellets of (0)
fat body and (a) labial gland of fifth stadium larvae of Manduca sexta.
No inhibitory effect of the substrate on PGT was observed as the reaction
approached zero-order kinetics at 1 mM PNP (Fig. 10, inset). To ensure a
steady-state reaction, a final concentration of 5 mM PNP was employed in
most experiments. Kinetic constants for UDP-Glc were not determined.
PGT Activity During Larval Development
Specific activities of PGT in the 15,OOOg pellet and supernatant of egg (1-2
days before hatching) homogenates were 2.5 and 0.3 nmol PNP-Glc
formedmidmg protein, respectively. Activities in the 15,0009 pellet of fat
body homogenate from fourth stadium feeding larvae and 0-, 1-, and 4-dayold fifth instars were 7.5,7.4,6.9, and 8.5 nmoYmidmg protein, respectively.
DISCUSSION
The subcellular distribution of PGT activity in different tissues of M . sexta
showed that heavy cellular particles are the main source of the enzyme. About
half of the enzyme activity was associated with the nuclear fraction, and most
of the remaining activity in the supernatant sedimented with the mitochondria, Golgi apparatus, and lysosomes in fat body. About 10% of the total
activity was present in the microsomes, whereas the soluble fraction (cytosol)
was devoid of activity. The particulate fractions were also found to be the
major or sole source of PGT activity in adult cockroaches, P e r ~ ~americana
~ ~ ~ e ~ a
[12], and the locust, Schistocerca cancellafa [13]. Our results differ from those
of Mehendale and Dorough [14], who found that 1-naphthol glucosylation
activity was contained in the 105,OOOg soluble fraction of an M . sexta larvae
Phenol p-Clucosyltransferases in M, sexfa
21 9
whole body homogenate and that little activity was present in the 15,0009
pellet. However, in adult houseflies and P. americana, the microsomal fractions
appear to have the highest PGT sources (14-17). Our studies of the distribution
of PGT and 7-methoxycoumarin 0-demethylase, a microsomal marker enzyme, confirmed that PGT is indeed associated with the heavy cellular
particles and is not a result of early sedimentation of microsomes. The
0-demethylation reaction was found to be a quantitative measure of microsoma1 contents in the centrifugal fractions of homogenates from the larval
midgut of M . sexfa [ 181. Mannosyltransferases involved in the glycosylation
of phospholipids and proteins have also been found in a high percentage in
the particulate fractions from pupae of the stable fly, Stomoxys culcifrans [19].
Although this conjugation reaction differs from p-glucosylation, the coexistence of both types of glycosyltransferasein cell structures and their stimulation by M g suggests a possible interrelationship between these enzymes.
In mammals, a glucosyltransferase specificfor endogenous lipid acceptors has
been localized in the plasma membrane and was stimulated by divalent
cations [ZO].
Marked differences occurred among specificactivities of PGT from different
tissues of M . sexta larvae. Activities from fat body and midgut supernatants
were reported to be the same magnitude with 1-naphthol as a substrate
[14,21].We have found that the fat body fraction with the major PGT activity
was over 1.5 times as active as the midgut fraction in PNP glucosylation. The
labial gland, however, had the highest PGT activity, whereas the epidermis
and muscle were lowest. The specific activities of PGT in labial gland are the
highest thus far reported for insects. It is not uncommon that some detoxication enzymes are present at varying levels of activity in the tissues of different
insects, indicating the potential role of each tissue in the detoxication of
harmful xenobiotics [2]. The major site of P-450 monooxygenase was the fat
body, midgut, or Malpighian tubules, depending upon the insect species. The
common occurrence of PGT at different activity levels in M . sexta illustrates
similarities among detoxication systems regarding tissue distribution and
importance. The high activity of PGT compared to that of P-450 monooxygenase, 1.34 nmol/min/midgut [18], as well as its presence in early larval
development stage, may reflect the role P-glucosylation plays in coping with
phenolic xenobiotics.
The role of PGT in the labial gland is uncertain. In saturniid moths, the silk
gland secretes 0-glucosides of 3- and 5-hydroxyanthranilic acid or gentisic
acid for silk tanning [22]. In M. sexfa, the labial gland does not produce silk,
except during the first larval stadium, but does produce a proteinaceous
secretion [23]. Because the midgut has high P-glucosidase activity as well as
PGT, it could hydrolyze ingested plant glucosides and liberate the more toxic
aglucones. We can speculate that the labial gland in this species actively
participates in the detoxication of plant phenolics during the feeding stage.
PGT from the labial gland accepted over 30 plant phenolics as substrates.
However, only the fat body contained P-glucosyltransferases active towards
endogenous phenolic precursors for cuticle tanning as well as towards plant
phenolics (unpublished data).
'
220
Ahmad and Hopkins
The effect of pH on PGT activity in the labial glands and fat body from M .
sexta is in close agreement with results obtained for o-arninophenolglucosylation by P. americana [12] and generally agrees with other studies on insects in
regard to a broad optimum pH range [14,15]. Phosphate and PIPES were
generally the best buffers for optimal activity of PGT from salivary gland and
fat body, although the glycine buffer also gave high activity for the latter.
Tris-HCI gave lower PGT activity in the optimum pH range from both tissues,
in contrast to studies of PGT activity from the midgut of M . sexta [14] and the
hepatopancreas from the crustacean Homarus americanus [24]. High activities
of microsomal PGT with glycine buffers were also observed in the housefly,
Musca domestica [15], and in P. americana [17].
A similar pattern of effects of divalent cations on glucosyltransferases
appears to be present in such distantly related organisms as plants, insects,
and mammals. The stirnulatory effect of Mg2', Ca2+, and Mn2+ on PGT
activity in this study is comparable with the activation of a specific glucosyltransferase from mouse liver plasma membranes [20] and PGT from papaya
fruit [25]. UDP-glucuronyltransferase from mammals, on the other hand,
although stimulated by M$+, is inhibited by Ca2+ and MI-?-+ [26]. Co2+
stimulated PGT activity in this study, whereas Mehendale and Dorough [14]
found it inhibitory to glucosylation of 1-napthol by the insect species. Hg2+
and Zn2+ were inhibitory to glucosyltransferases from insects and plants and
were mixed inhibitors (affectingboth K, and V,,,) to PGT from sugar beets [27].
Detergents generally activate membrane-bound glycosylation enzymes,
such as microsomal glucosyltransferase from houseflies [161; glucuronyltransferase from rat liver nuclear membrane [28], and rat intestinal and hepatic
microsomes [29]; and UDP-G1c:sterol glucosyltransferasefrom white mustard
seedlings [30]. The activation is suggested to be due to membrane structure
modification, exposure of otherwise nonfunctioning active sites, increased
permeability to UDP-Glc, or solubilization of the enzyme, The non-ionic
detergent, Triton X-100, however, inhibited PGT in this study. Detergents
probably activate the enzyme from membrane preparations because glucuronyltransferase from intact, whole nuclei of rat liver was inhibited by digitonin but was activated in the nuclear membrane preparations [28]. In this
study PGT could have been in the membranes of intact cell organelles obtained
by differential fractionation. Support for this suggestion comes from the work
on PGT from white mustard seedlings [30]. PGT is a membrane-bound
enzyme occurring mainly in cellular structure sedimented at 3,000-15,OOOg.
When the crude membrane preparations of the sediment were delipidated,
Triton X-100 solubilized and activated the enzyme. Further studies on the
exact localization of PGT in insects would enhance efforts for purification and
characterization of the enzyme.
The specificityof PGT toward the glucosylnucleotidesubstrate is apparently
less restricted in M . sexfa than in other organisms studied so far. Nucleotides
of the pyrimidines, uracil and thymine, and of the purine, guanine, served as
glucose donors in this study and trace activity was observed with ADP-Glc
and CDP-Glc. Although UDP-Glc was preferred, activity with dTDP-Glc was
appreciable. The utilization of GDP-GLc as a glucose donor by PGT has not
previously been reported [24,31]. For binding of the nucleotide to the enzyme,
Phenol p-Glucosyltransferases in M. sexfa
221
the bases appear to need a carbonyl group on carbon 4 of the pyrimidine ring
or on carbon 6 of the purine ring, in addition to a reduced nitrogen atom on
carbon 3 of the pyrimidine and carbon 1of the purine. The same enzyme may
transfer glucose from UDP-Glc, dTDP-Glc, and GDP-Glc because activity was
not additive when glucose donating nucleotides were combined in the incubation mixtures. The specificity towards the sugar moiety was higher, however, because UDP-GlcUA and UDP-Glc NAc did not serve as donors and
UDP-GlcUA inhibited PGT activity, probably due to competitive binding of
the uridine moiety. Such inhibition was not observed in the analogous
vertebrate glucuronyltransferase [26].It is interesting that PGT of insects was
able to transfer glucose whether in the a- or p-form in the donor, and the
product in both cases was a P-glucoside. Only the a-form of UDP-GlcUA
served as donor in UDP-glucuronyltransferase reactions [26].
Unlike crustacean PGT [24] and rat liver glucuronyltransferase [28], which
deviated from simple Michaelis-Menten kinetics and had 2 apparent K, values,
the M. sexta PGT preparation had one K, value for PNP. This could be due to
the presence of only one enzyme that catalyzed the conjugation of PNP.
The presence of p-glucosidases in the digestive system of insects is well
documented [32]. P-Glucosidases seem to have broad substrate specificityand
are involved in the digestive hydrolysis of carbohydrates, as well as the
hydrolysis of plant allelochemical glucosides. Aglucones often are more toxic
than their glucosides, and their release catalyzed by P-glucosidase may be
harmful to insects. Lindroth [33] concluded that lower p-glucosidase activity
in the eastern tiger swallowtail subspecies, Papilio glaucus canadensis, relative
to that in P. g. glaucus, contributed to the adaptation of the former subspecies
to members of the Salicaceae that produce a variety of phenolic glucosides in
substantial quantities. In M. sexta, p-glucosidase in the labial gland might be
secreted in the saliva for hydrolysis of phenolic glucosides during feeding.
We have also found that the labial gland of the black cutworm, Agrotis ipsilon,
was active in PNP-Glc hydrolysis (unpublished data). The activity of pglucosidase from M. sexfa larval rnidgut was of a comparable magnitude to
that from other lepidopteran species [3], but was much lower than that from
larvae of the processionary moth Thaurnetopoca pityocampa [32]. The 50%
inhibition value of the P-glucosidase inhibitor D-gluconic acid lactone was
more than 10 times higher for the M. sexta enzyme than for the fall armyworm
enzyme [3]. The antagonist enzymes, PGT and p-glucosidase, have not been
studied simultaneously to determine more definitively the consequences of
deglucosylation-glucosylation in conjunction with the net capacity of phytophagous insects to tolerate and adapt to host plants with defensive arsenals
of phenolic glucosides.
In this study, we have demonstrated the presence of both PGT and
P-glucosidase in the same tissues and subcellular fractions. The coexistence
of related antagonistic enzymes has been shown in other organisms. Rat liver
microsomal preparations contained both P-glucuronidase and UDP-glucuronyltransferase [34]. Glycosyltransferases and glycosidases active on the
same type of secondary compounds have been demonstrated in the same
plant tissues [35]. UDP-G1c:sterol glucosyltransferase and steryl p-D-glucoside hydrolase were found to be of similar subcellular distribution in white
222
Ahmad and Hopkins
mustard seedlings [30]. Why these antagonistic enzymes are present in the
same tissues or cellular membranes is not understood, but probably they
regulate endogenous compound levels through glycosylation and hydrolysis.
If so, then the enzymes themselves must be under tight regulation. Herbivorous insects are suitable models for exploring the regulation of phenolic
conjugation as influenced by natural and modified diets. Regulation of endogenous glucosyl conjugates of tyrosine and catecholamines is important for
cuticle tanning in insects [4,6], and the timing of glucosylation of tyrosine and
hydrolysis of tyrosine glucoside is under hormonal control [36,37].
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properties, glucosyltransferase, tobacco, larvae, localization, tissue, phenols, hornworm, activities, sexta, manduca, glucosidase
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