380 H.K. Lehman et al. Insect Biochemistry and Molecular Biology 30 2000 377–386
developmental stage was quantified by HPLC and scin- tillation counting.
3. Results
3.1. Tyramine- b-hydroxylase assay
We developed an assay for T β
H activity as a first step in an analysis of mechanisms that are responsible for the
increases in the levels of OA in the CNS during adult development. Incubation of [ring-
3
H]tyramine with crude brain extracts resulted in the formation of two
radiolabeled products that were distinguished by reverse- phase HPLC Fig. 1. The total amount of radioactivity
collected from these two fractions was less than 5 of that in the [ring-
3
H]tyramine used as substrate. The identity of the compound that eluted in HPLC fraction
7 was determined by comparing its elution time to that of unlabeled OA Figs. 1 and 2. In addition, the elution
of the methylated fraction 7 was compared to the elution of N-methyl OA synephrine on HPLC, and the
mobility of fraction 7 treated with sodium periodate was compared to the mobility of p-hydroxybenzaldehyde on
TLC data not shown. In each instance the product reco- vered from HPLC fraction 7 was indistinguishable from
synthetic OA.
The conversion of tyramine to OA by brain homogen- ates depended upon incubation time, protein concen-
tration, and pH Fig. 3. Production of [
3
H]OA was linear for at least 20 min Fig. 3A. Little [
3
H]OA synthesis was detected at protein concentrations below 0.2
µ g
µ l,
but at higher protein concentrations 0.5–2.0 µ
g µ
l, a linear rate of synthesis of [
3
H]OA was observed. At pro- tein concentrations greater than 2.0
µ g
µ l, additional pro-
duction of [
3
H]OA was not detected, and the enzymatic
Fig. 1. Reverse-phase HPLC radiochromatograms of the enzymatic
products from incubation of M. sexta CNS extracts with [ring-
3
H]tyra- mine. Arrows indicate the elution of synthetic OA and tyramine
detected by UV absorbance 223 nm. I: Radiochromatogram of the enzymatic products from untreated nervous system extracts. s: Com-
pounds recovered from boiled enzyme reaction. Fig. 2.
Identification of OA as a product of a standard tyramine- β
- hydroxylase assay. Following a standard tyramine-
β -hydroxylase
assay, 10 µ
l of 10
25
M synthetic OA was added to the reaction pro- ducts and the mixture was separated by reverse-phase HPLC. Each
fraction was tested for the presence of OA using PNMT and [methyl-
3
H]SAM as described in the text. I: Radiochromatogram of the enzy- matic products from untreated CNS extracts. s: Radiochromatogram
of the enzymatic products from PNMT and [methyl-
3
H]SAM.
reaction appeared to be substrate-limited Fig. 3B. The optimum pH for the conversion of tyramine to OA was
7.0 Fig. 3C. T β
H activity also depended on the pres- ence of copper, catalase, and ascorbate Table 1. Copper
concentrations greater than 0.001 mM were required for activity, and maximal activity was achieved with 0.05
mM cupric sulfate. Metal-complexing agents, including 1.0
µ M diethyldithiocarbamate and 0.4 mM KCN,
reduced enzyme activity 95 data not shown. Catal- ase, at concentrations in the range 0.0001–0.1 mg
µ l,
was required for T β
H activity; the optimal concentration was 0.01 mg
µ l. Finally, maximal T
β H activity was
measured in enzyme incubation mixtures containing 5.0 mM ascorbic acid; higher concentrations appeared to
be saturating. In addition to OA, one other enzymatic product was
routinely detected in the T β
H assay. This unidentified product, which eluted in HPLC fractions 11 and 12, was
re-incubated with 1.0 µ
g µ
l of brain homogenate for 15 min at room temperature, and the reaction mixture was
analyzed by HPLC to determine if its radioactivity could contribute to the radioactivity co-eluting with OA. The
retention times of the metabolites of fractions 11 and 12 were compared to the retention times of OA and tyram-
ine. Under these conditions, the unidentified product did not co-elute with OA. In similar experiments, [
3
H]OA produced in our assay was not converted to the unidenti-
fied product. Furthermore, inhibitors of N-acetylation and amine uptake, including nomifensine 0.5 mM, xyl-
amine 0.5 mM, and bupropion 0.5 mM had no effect on the formation of this unidentified product data not
shown.
381 H.K. Lehman et al. Insect Biochemistry and Molecular Biology 30 2000 377–386
Fig. 3. Effects of incubation time, protein concentration, and pH on activity of M. sexta tyramine-
β -hydroxylase. The reaction mixture contained
5.0 µ
Ci [ring-
3
H]tyramine, 1.0 mg catalase, 0.1 mM N-ethylmaleimide, 0.05 mM CuSO
4
, 5.0 mM disodium fumarate, and 5.0 mM ascorbic acid. Hydroxylase activity was estimated by the standard assay procedure as described in Materials and methods except for the parameter under study.
A Incubation time varied from 0 to 20 min. B Protein concentration varied from 25 to 500 µ
g100 µ
l. C Constant-ionic-strength sodium phosphate buffer was used over the pH range 4–8.
Table 1 Requirements for tyramine
β -hydroxylase activity
Compound Final concentration activity
Cupric sulfate 0.0 mM
19.3 0.001 mM
64.2 0.005 mM
84.7 0.01 mM
95.6 0.05 mM
100.0 0.1 mM
74.2 Catalase
0.0 mg µ
l 8.3
0.0001 mg µ
l 46.3
0.001 mg µ
l 55.8
0.01 mg µ
l 100.0
0.10 mg µ
l 83.7
Ascorbate 0.05 mM
46.6 0.10 mM
33.5 0.50 mM
74.2 1.00 mM
56.1 5.00 mM
92.3 10.00 mM
100.0 50.00 mM
86.2
3.2. Tyramine- b-hydroxylase kinetics
The rate of synthesis of [
3
H]OA was a function of the concentration of tyramine in the incubation mixture. The
apparent K
M
for tyramine, calculated from reaction mix- tures of stage-18 brains and abdominal ganglia, was
0.22 ±
0.04 mM, and the calculated V
max
was 17.86 ±
1.85 pmolminmg protein Fig. 4A. The apparent K
M
for ascorbate was 2.75 mM. With reaction mixtures contain-
ing stage-P0 CNS homogenates, the apparent K
M
for tyr- amine was 0.30
± 0.05 mM, and the calculated V
max
was 2.05
± 0.14 pmolminmg protein Fig. 4B. Although the
apparent V
max
of the stage-P18 homogenates was sig- nificantly greater than that of stage-P0 homogenates, the
apparent tyramine K
M
values estimated with the two homogenates were not significantly different Student’s
t-test, P ,0.05.
3.3. Developmental changes in tyramine- b-hydroxylase
activity The levels of T
β H activity in the brain and abdominal
ganglia varied with the developmental stage of the moth. Minimal T
β H activity was measured in brain extracts
from animals early in adult development stage P2, 149.6
± 29.3; stage P6, 246.0
± 34.4; stage P10, 352.6
± 75.4
cpm, whereas later in adult development OA synthesis was elevated stage P14, 1051.6
± 197.6; stage P18,
1672.6 ±
238.0 Fig. 5A. Significant differences in T β
H activity were detected between stage P2 and stages P14
and P18, although no significant differences in T β
H activity were measured between stage P2 and stages P6
and P10 ANOVA, P ,0.05. A similar increase in T
β H
activity occurred in the abdominal ganglia. Relatively little production of [
3
H]OA was detected with homogen- ates of abdominal ganglia taken from pupae at stages
P2, P6, and P10 61.0 ±
8.9, 89.6 ±
41.3, 116.6 ±
38.3 cpm, respectively. Later in adult development much greater
[
3
H]OA synthesis
occurred 351.0
± 103.9
and 547.0
± 46.2 cpm from stages P14 and P18, respectively
Fig. 5B. Whereas no significant differences in T β
H activity were observed between stage P2 and stages P6
and P10, significant differences in T β
H activity were detected between stage P2 and stages P14 and P18
ANOVA, P ,0.05.
3.4. Developmental changes in octopamine levels Coincident with the rise in T
β H activity in the brain
and abdominal ganglia were increases in the levels of OA Fig. 5. In the first third of adult development, from
stage P2 following apolysis through stage P6, the level of OA in the brain remained stable stage P2: 0.40
± 0.07
pmolbrain; stage P6: 0.46 ±
0.01 pmolbrain. Beginning at stage P10, however, OA levels rose from 0.78
± 0.01
to 6.00 ±
2.0 pmolbrain by stage P18, the final stage of adult development Fig. 5A. The changes in brain levels
382 H.K. Lehman et al. Insect Biochemistry and Molecular Biology 30 2000 377–386
Fig. 4. Effect of varying concentrations of tyramine on tyramine-
β -
hydroxylase activity from CNS homogenates obtained from stage-P18 and P0 animals. A Kinetic constants estimated from CNS homogen-
ates obtained from stage-P18 animals. Assay was performed under standard conditions except that the concentration of unlabeled tyramine
was varied from 0.007 to 2.4 mM. Inset: double reciprocal plot; abscissa
= 1S mM, ordinate
= 1V pmolminmg. Apparent kinetic
constants were: V
max
, 17.86 ±
1.85 pmolminmg; K
M
, 0.22 ±
0.04 mM n
= 3, mean
± SEM. B Kinetic constants estimated from CNS homo-
genates obtained from stage-P0 animals. Assay was performed under standard conditions except that the concentration of unlabeled tyramine
was varied from 0.007 to 1.0 mM. Inset: Double reciprocal plot; abscissa
= 1S mM, ordinate
= 1V pmolminmg. Apparent kinetic
constants were: V
max
, 2.05 ±
0.14 pmolminmg; K
M
, 0.30 ±
0.05 mM n
= 3, mean
± SEM.
of OA through stage P14 were not statistically signifi- cant, but the levels measured at stages 2 and 18 were
significantly different ANOVA, P ,0.05. The level of
OA in the abdominal ganglia also increased. In P2 ani- mals, OA levels were 0.89
± 0.22 pmolAG. Beginning at
stage P10, the level of OA increased from 1.03 ±
0.25 pmolAG to 2.71
± 0.57 pmolAG by stage P18 Fig. 5B.
No significant differences in OA levels were observed at stages P2, P6, and P10, but the differences between
stage P2 and stages P14 and P18 were significant ANOVA, P
,0.05.
Fig. 5. Tyramine-
β -hydroxylase activity and octopamine levels in the
brain and abdominal ganglia AG of M. sexta during adult develop- ment. A Tyramine-
β -hydroxylase activity and OA levels detected
from brain extracts at 5 different stages of adult development. I: T β
H activity. s: OA levels. Each point represents the mean of 3
replicates ±
SEM. B Tyramine- β
-hydroxylase activity and OA levels detected in extracts of abdominal ganglia at five different stages of
adult development. I: T β
H activity. s: OA levels. Each point rep- resents the mean of 3 replicates
± SEM.
4. Discussion
