378 H.K. Lehman et al. Insect Biochemistry and Molecular Biology 30 2000 377–386
levels of neurotransmitters such as OA. In order to understand the mechanisms controlling the levels of this
amine, we have investigated its biosynthesis in the sphinx moth, Manduca sexta.
OA is biosynthesized from tyrosine in the insect ner- vous system, and the studies of Livingstone and Temple
1983 suggested that OA biosynthesis requires two enzymatic steps: decarboxylation of tyrosine by tyrosine
decarboxylase to form tyramine, followed by hydroxyl- ation of tyramine by tyramine-
β -hydroxylase T
β H to
produce OA. Little information is available about either of these enzymes in insects. Tyrosine decarboxylase
activity has not been characterized or identified in any arthropod, but it appears to be distinct from DOPA
decarboxylase, a well-studied enzyme required for the conversion of l-DOPA to dopamine in the insect ner-
vous system and cuticle Livingstone and Temple, 1983; Hirsh, 1989. In the only biochemical characterization
of T
β H, Wallace 1976 showed that extracts of nervous
tissue from the American lobster, Homarus americanus, can hydroxlyate tyramine to produce OA, and that this
conversion is dependent upon pH, ascorbic acid, copper, and catalase. These properties suggested that T
β H is
similar to dopamine- β
-hydroxylase D β
H, which in mammals is required for the hydroxylation of dopamine
to form norepinephrine. Indeed, Wallace 1976 demon- strated that purified lobster T
β H can produce norepi-
nephrine via the β
-hydroxlyation of dopamine. More- over, D
β H exhibits relatively broad substrate specificity
and can hydroxylate tyramine to form OA Creveling et al., 1962; Goldstein and Contrera, 1962; reviewed by
Kaufman and Friedman, 1965. These results indicate that lobster T
β H is functionally related to mammalian
D β
H. In this study we focused on T
β H because it is the last
and putatively rate-limiting enzyme in the biosynthetic pathway leading to OA. We document that, as in Mames-
tra configurata Bodnaryk, 1980, levels of OA increase during metamorphosis in M. sexta. To determine if this
OA increase is associated with an increased rate of syn- thesis, we characterized T
β H activity in extracts of M.
sexta CNS tissue and showed that during metamor- phosis, there is a direct correlation between increased
levels of T β
H activity and levels of OA. Based upon apparent T
β H kinetic constants measured in extracts of
late-larval and adult CNS tissue, we conclude that increased levels of T
β H protein account for the increase
in T β
H activity during metamorphosis. The possible cellular and molecular mechanisms that may be respon-
sible for a long-term increase in T β
H are discussed.
2. Materials and methods
2.1. Experimental animals Larvae of M. sexta Lepidoptera: Sphingidae were
reared on an artificial diet modified from that of Bell and Joachim, 1976 and maintained on a long-day photo-
period regimen 17L:7D at 25–26 °
C and 50–60 rela- tive humidity. Pharate adults were staged according to
previously published criteria Sanes and Hildebrand, 1976; Tolbert et al., 1983, with adult development
occurring in 18 stages and completed about 21 days after pupation.
2.2. Reagents Radiochemicals, [ring-
3
H]tyramine and S-[methyl-
3
H]adenosyl-l-methionine SAM, were purchased from New England Nuclear Boston, MA, and OA and N-
ethylmaleimide were obtained from Research Biochemi- cals Inc. Natick, MA. Unless otherwise noted, all other
chemicals were reagent grade, and those products as well as catalase and phenylethanolamine-N-methyl transfer-
ase PNMT used in assays were purchased from Sigma Chemical Co. St. Louis, MO.
2.3. Protein assay The amount of soluble protein in samples was esti-
mated using a bicinchoninic acid kit obtained from Pierce Chemical Co. Rockford, IL. The kit was used
according to the manufacturer’s instructions, and bovine serum albumin was used to prepare the standard curves.
2.4. Octopamine assay The assay used to estimate OA concentrations in sol-
ution was modified from the procedures of Molinoff et al., 1979. Brains comprising both the supraoesophageal
ganglion, including optic lobes, and the suboesophageal ganglion and abdominal ganglia AG; third, fourth,
fifth, and terminal abdominal ganglia were dissected at various stages of adult development and immersed in
80 ice-cold acetone. Each tissue sample was homogen- ized, centrifuged 10 min, 10,000 rpm, and re-extracted
with aqueous acetone. The supernatant solution was con- centrated to dryness in a vacuum centrifuge Savant
Industries, Farmingdale, NY and resuspended in 100
µ l
of 0.1 M Tris buffer pH 8.6. PNMT 25 unitsmg; 0.2 units in 0.1 M Tris was added to each tube along with
5.0 µ
Ci of [methyl-
3
H]SAM 55–85 Cimmol. The tubes were incubated for 2 h at 37
° C. The reaction was
stopped by the addition of 200 µ
l of 0.5 M sodium borate pH 10. The N-[
3
H]methyl OA i.e. [
3
H]synephrine produced by the enzymatic reaction was then isolated
from the reaction mixture by three extractions, each with 2 ml of a mixture of toluene and isoamyl alcohol 3:2
v:v. The toluene–isoamyl alcohol extracts were then pooled for each sample, combined with 10 ml of scintil-
lation cocktail ScintiVerse LC, Fisher Scientific, and subjected to liquid scintillation counting. The radioen-
zymatic products were confirmed by HPLC using con-
379 H.K. Lehman et al. Insect Biochemistry and Molecular Biology 30 2000 377–386
ditions described below. More than 90 of the radioac- tivity extracted with toluene–isoamyl alcohol co-eluted
with synephrine. Unknowns were compared to a stan- dard curve produced with OA standards. The curve was
linear r
= 0.95 over the range 0.1–2.5 pmol.
2.5. Enzyme extracts CNS tissue from M. sexta was dissected under ice-
cold normal saline solution 150 mM NaCl, 3 mM KCl, 3 mM CaCl
2
, 20 mM MgCl
2
, and 10 mM N-tris[hyd- roxymethyl]methyl-2-aminoethanesulfonic acid TES,
pH 6.9; Christensen et al., 1991, frozen on dry ice, and stored at
280 °
C. This material was kept for up to 2 weeks without loss of enzyme activity. Tissue was
thawed immediately prior to use, placed in a ground- glass homogenizer containing normal saline solution
with catalase 19,000 unitsmg; 10.0 mgml, and homo- genized with 20 strokes by hand. The homogenate was
centrifuged 10,000 rpm, 10 min at 4
° C, and the
resulting supernatant fraction was used as the crude enzyme source.
2.6. Tyramine- b-hydroxylase assay
The assay we developed to measure T β
H activity was based upon the hydroxylation of [ring-
3
H]tyramine to form [ring-
3
H]OA. First, [ring-
3
H]tyramine 20–40 Cimmol was pre-purified on a C-18 HPLC column
Nova-Pak, Waters Assoc., Milford, MA by isocratic elution with a solution of 10 methanol, 0.1 M potass-
ium phosphate, and 5.0 mM octanesulfonic acid pH 3.8. Radioactivity co-eluting with tyramine was col-
lected, desalted with a Sep-Pak Waters Assoc., Milford, MA, and concentrated on a vacuum concentrator. Then
5.0
µ Ci of [ring-
3
H]tyramine was added to 90 µ
l of an enzyme
reaction mixture
containing in
final concentrations: 0.1 M potassium phosphate pH 6.9,
1.0 mg catalase, 0.1 mM N-ethylmaleimide, 0.05 mM CuSO
4
, 5.0 mM disodium fumarate, and 5.0 mM ascor- bic acid. The concentration of tyramine used in most
assays was lower than that required to saturate the enzyme, but these levels of [ring-
3
H]tyramine offered the highest sensitivity and therefore were used in assays
necessary to characterize the enzyme. Greater concen- trations of tyramine 3.5 times the K
M
were used to esti- mate the levels of T
β H during development see below.
The reaction was initiated by the addition of 100 µ
g to give a final protein concentration of 1
µ g
µ l of the crude
enzyme tissue homogenate, and then the mixture was incubated at room temperature ca. 22
° C for 15 min
without shaking. Background radioactivity was determ- ined by using an enzyme mixture containing heat-inacti-
vated enzyme. The reaction was stopped by boiling the incubation mixture for 5 min. It then was centrifuged
for 5 min at 14,000 rpm, the supernatant solution was collected, and the reaction products were separated and
identified by C-18 reverse-phase HPLC as described above. The solvent was pumped at a rate of 1 mlmin,
and 1 min fractions were collected. Each fraction was combined with 5 ml of scintillation fluid ScintiVerse
LC, Fisher Scientific, Pittsburgh, PA, and radioactivity was quantified by scintillation counting.
The identity of [
3
H]OA produced in the T β
H assay was verified by three methods. First, the HPLC elution
times of radioactive enzyme products were compared to the elution time of synthetic OA added to the enzyme
reaction mixture immediately prior to termination of the reaction; detection was by UV absorbance 223 nm.
Second, [
3
H]OA produced in the T β
H assay was methyl- ated by incubating the purified, desalted [
3
H]OA with 0.2 units of PNMT and 5.0
µ Ci of [methyl-
3
H]SAM for 2 h at 37
° C. The reaction was stopped by boiling, and
the elution time of the reaction product was then com- pared to that of synthetic N-methyl OA synephrine by
HPLC using the column and solvents described above. Third, [
3
H]OA produced in the T β
H assay was oxidized with NaIO
4
to form p-hydroxybenzaldehyde, which then was detected with sulfanilic acid Touchstone and Dob-
bins, 1983 as follows: the [
3
H]OA was collected from HPLC, Sep-Pak purified, dried, resuspended in 20
µ l of
H
2
O, and incubated with 20 µ
l of 2 NaIO
4
for 30 min at room temperature; the reaction was stopped by
addition of 20 µ
l of 10 sodium metabisulfite, silica TLC plates LK-6, Whatman were spotted with 20
µ l
of the reaction mixture, and finally the TLC plates were developed in n-butanolacetic acidwater 4:1:2, dried,
scraped into scintillation vials, combined with scintil- lation fluid, and assayed by scintillation counting. The
R
f
values of radioactivity produced by treatment with NaIO
4
were compared to the mobilities of synthetic OA, tyramine, and p-hydroxybenzaldehyde detected with sul-
fanilic acid. Kinetic parameters of the crude enzyme for tyramine
were determined from the data in Fig. 5. The data were displayed as double-reciprocal plots, and estimates of
V
max
and K
M
were obtained from the slopes and inter- cepts of the straight lines generated in these plots.
2.7. Developmental studies Levels of T
β H in the developing brain and abdominal
ganglia were estimated by assaying enzyme activity in homogenates of CNS tissue at various stages of adult
development. Brains and abdominal ganglia were dis- sected separately from developing adults at stages P2,
P6, P10, P14, and P18 and subjected to the T
β H assay
procedure. Homogenates were processed as described above, 1
µ g
µ l of protein was added to the T
β H reaction
mixture containing 0.76 mM [ring-
3
H]tyramine 0.007 Cimmol, and the mixture was incubated at room tem-
perature for 15 min. The amount of OA produced at each
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
