PROTEIN
EXPRESSION
AND
PURIFICATION
2, 10-14
(19%)
Tyrosine Hydroxylase Purification from Rat PC1 2 Ceils
Laura G . Gahn and Robert Roskoski, Jr.
Department
of Biochemistry
Received January
and Molecular
Biology, Louisiana
Inc.
Tyrosine hydroxylase
(EC 1.14.16.2) catalyzes the
rate-limiting
step in catecholamine biosynthesis (1). It
catalyzes the hydroxylation
of tyrosine to form 3,4-dihydroxyphenylalanine
(Dopa).’ The activity of this enzyme is regulated by many factors, including phosphorylation (2), salts (3), andpolyanions
such as heparin (4).
Studies of purified tyrosine hydroxylase have been hindered by the difficulty in obtaining adequate amounts of
purified enzyme. Published purification
procedures report a yield of 0.2-2 mg of purified enzyme (5,6). Furthermore, purified tyrosine hydroxylase is reported to
be unstable and to lose substantial activity in a matter
of hours (7-9). The only large-scale purification of tyrosine hydroxylase reported to date is that of the bovine
adrenal enzyme (lo), in which 17 mg of tyrosine hydroxylase was obtained from 2.5 kg of bovine adrenal medulla. The rat pheochromocytoma
PC12 cell line established by Greene and Tischler (11) expresses high levels
* Abbreviations used: Dopa, 3,4-dihydroxyphenylalanine;
Pipes, piperazine-N,N’-bis(2-ethanesulfonic
acid); PMSF, phenylmethylsulfonyl fluoride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide
gel electrophoresis.
10
Medical Center, New Orleans, Louisiana
70119
25,199l
Tyrosine
hydroxylase
was purified
in high yield from
rat PC12 cells. This three-day
procedure
consisted
of
differential
ammonium
sulfate precipitation,
anion-exchange chromatography,
and heparin-Sepharose
affinity chromatography.
It yielded an average of I5 mg of
purified
protein
from 100 flasks of PC12 cells, with
greater than 4 0 % recovery
of tyrosine hydroxylase.
Sodium dodecyl sulfate-polyacrylamide
gel electrophoresis yielded
a single protein
band with
a molecular
weight of approximately
60,000. The protein had a specific activity
of 670 nmollminlmg
and had a K,,, for its
reducing .cofactor
tetrahydrobiopterin
of 1.8 mM. The
purified
protein
can be phosphorylated
and activated
o 1991 Academic
by cyclic AMP-dependent
protein kinase.
Press,
State University
of tyrosine hydroxylase and has been used to purify
small amounts (240-300 pug) of the enzyme (12,13). W e
report here a protocol for the purification
of about 15
mg of homogeneous, stable rat tyrosine hydroxylase in
high yield from PC12 cells in culture.
MATERIALS
AND METHODS
Materials
Catalase, tyrosine, piperazine-2\r,iV’-bis(2-ethanesulfonic acid) (Pipes), tris(hydroxymethyl)aminomethane,
KCl, and dithiothreitol
were obtained from Sigma
Chemical Co. (St. Louis, MO). Ammonium sulfate, potassium phosphate, NaCl, HCl, MgCl,, and decolorizing
carbon (Darco G-60) were obtained from J. T. Baker
(Jackson, TN). RPM1 1640 and gentamicin were from
GIBCO (Grand Island, NY). Fetal calf serum and heatinactivated horse serum were from Hazleton (Lenexa,
KS). Sucrose was obtained from Bio-Rad (Richmond,
CA). L-[3,5-3H]Tyrosine
was obtained from NEN Research Products (Boston, MA). Diethylaminoethyl-cellulose (DE 52) was obtained from Whatman Biosystems
(England)
and heparin-Sepharose
CL-6B was from
Pharmacia, Inc. (Piscataway, NJ). (6R)-5,6,7,8-Tetrahydro-L-biopterin
was obtained from Dr. B. Shircks
Laboratories (Switzerland).
Tyrosine Hydroxyluse
Activity
Assay
Tyrosine hydroxylase activity was assessed by measuring the release of 3H,0 in the conversion of L-[3,53H]tyrosine to Dopa by the method of Reinhard et al.
(14). Enzyme (5-15 fig/ml) was incubated at 37°C with
100 PM L-[3,5-3H]tyrosine
(0.5 &i per assay), 1 m M
(6R)-5,6,7,8-tetrahydro-L-biopterin,
1500 U/ml cataand 50 m M Pipes buffer (pH
lase, 5 m M dithiothreitol,
6.0) in a total volume of 30 ~1. Samples were incubated
for 10 min and the reaction was stopped by the addition
of 300 ~17.5% charcoal (Darco G-60) in 1 N HCl. Charcoal with adsorbed tyrosine and catechols was sedimented by centrifugation
at 13,000g for 2 min, aliquots
(100 ~1) of supernatant were removed, and 3H,0 radioac1046.5928/91
$3.00
Copyright 0 1991 by Academic Press, Inc.
All rights of reproduction in any form reserved.
TYROSINEHYDROXYLASEFROM PC12CELLS
tivity was determined by liquid scintillation
spectrometry. Samples were assayed in triplicate unless otherwise
noted. The blank radioactivity
(about 300 cpm) from
parallel samples containing
water in place of enzyme
was subtracted from the sample values to yield net activity.
Phosphorylation
of Tyrosine Hydroxylase by Cyclic
AMP-Dependent
Protein Kinase Catalytic Subunit
The catalytic subunit of cyclic AMP-dependent
protein kinase was purified by the method of Hart1 and
tyrosine hydroxyRoskoski (15). For phosphorylation,
lase (250-500 pg/ml) was incubated in the presence of 1
pM purified catalytic
subunit (40,000 Da), 100 pM ATP,
and 1 mM MgCl, in 50 m M Pipes buffer (pH 6.0) at 30°C
for 20 min. This procedure leads to the phosphorylation
and activation
of tyrosine hydroxylase
(16). Control
samples were incubated with MgCl, and buffer only.
The samples were diluted to provide a tyrosine hydroxylase concentration
of 15 pg/ml during the assay.
RESULTS
Purification
of Tyrosine Hydroxylase
Vulliet and co-workers (17) reported a procedure for
the purification
of 0.6 mg tyrosine hydroxylase from rat
pheochromocytoma.
We have used a modification
of
this procedure
to purify tyrosine
hydroxylase
from
PC12 cells. The cells were maintained
in RPM1 1640
medium with 10% horse serum (heat inactivated),
5%
fetal calf serum, and 50 pg/ml gentamicin in Nunc T175
flasks. Cells were kept at 37°C in a water-saturated,
5%
CO, atmosphere.
Greene et al. (18) reported that the
cells attach poorly unless the culture flasks are collagen-coated. We found, however, that PC12 cells adhered well to the Nunc flasks under these conditions.
Cultures were fed twice weekly by completely exchanging the medium. When confluent, the cells were either
subcultured
or harvested by trituration,
pelleted, and
frozen at -80°C until use for tyrosine hydroxylase purification. Cells were stored for 1 year with no loss of
tyrosine hydroxylase
activity. It is advisable to harvest
confluent or near confluent cells for purification,
since
cell-cell contact increases tyrosine hydroxylase expression in PC12 cells (19).
During the purification,
all steps were performed at
0-4°C. PMSF and leupeptin, protease inhibitors,
were
added to all solutions just before use. PC12 cells from
100 T175 flasks (30 ml of partially thawed, packed cells
containing
1$2 g of protein, approximately
1 X lOlo
cells) were homogenized with a polytron in 250 ml of 50
m M potassium
phosphate
(pH 7.0), 7.5 m M mercaptoethanol, 1 mM EDTA, 5 pg/ml leupeptin, and 10 pM
phenylmethylsulfonyl
fluoride (PMSF) (Buffer A) with
0.27 M sucrose.
The resulting solution was centrifuged
11
at 40,OOOg for 60 min. To the supernatant,
solid ammonium sulfate (30% saturation)
was added over 20 min
with gentle stirring and the solution was stirred an additional 20 min. After centrifugation
at 15,000g for 15 min,
the supernatant
was collected. Additional
ammonium
sulfate was added to 42% saturation as described above
and the precipitate
was collected. The precipitate
was
then dissolved in buffer A and dialyzed against 100 vol
of the buffer overnight with one change of solution. The
dialysate was then applied to a DEAE-cellulose
column
(1.5 X 18 cm) equilibrated
with Buffer A. The column
was washed with 1 column vol of Buffer A, and tyrosine
hydroxylase was eluted with a linear gradient of 40-300
m M NaCl in Buffer A (Fig. 1). The enzyme eluted at
about 100 mM NaCl. The tyrosine hydroxylase-containing fractions were then pooled, ammonium sulfate was
added to 44% saturation,
and the precipitate
was collected as described above.
This precipitate
was dissolved in 20 m M potassium
phosphate (pH 7.4), 1 m M dithiothreitol,
5 pg/ml leupeptin, and 10 pM PMSF (Buffer B). Following overnight dialysis against 100 vol of Buffer B with one
change of solution, the dialysate was applied to a heparin-Sepharose
column (1 X 18 cm) equilibrated
with
Buffer B. The column was then washed with Buffer B,
350 m M Tris-HCl in Buffer B, and Buffer B again. Tyrosine hydroxylase
was not eluted from the column by
Tris-HCl
while other proteins were, so that the TrisHCl wash enhanced purification
at this step (5). Tyrosine hydroxylase was eluted from the column with a linear gradient of 100-500 m M KC1 in Buffer B (Fig. l),
with the enzyme eluting at approximately
250 m M KCl.
The tyrosine
hydroxylase-containing
fractions
were
then pooled and concentrated
by pressure dialysis
(Amicon stirred cell with Diaflo PM10 ultrafilter).
For
PC12 enzyme preparations,
the sample was stored in
0.5-ml aliquots of 0.4-2.5 mg/ml protein at -80°C in 5
m M Tris-acetate
(pH 8.2), 2 m M dithiothreitol,
and 10%
(w/v) sucrose until use. The resulting enzyme was stable
under these conditions for more than 2 years. Enzyme
was thawed prior to use and could be stored up to 6
weeks at 2°C without appreciable loss of activity. There
was no loss of activity with up to four freeze-thaws, but
additional
freeze-thaws
were avoided. These preparations appear to be homogeneous as judged by visualization on sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE)
with Coomassie blue staining
(Fig. 2).
Results from a typical purification
cells are summarized in Table 1. Total protein was assessed by the
method of Bradford
(20) with bovine y-globulin
as a
standard.
Relative amounts of tyrosine hydroxylase
were determined immunochemically
by the method of
Haycock (21), using affinity-purified
polyclonal
rabbit
antibodies to rat tyrosine hydroxylase
and the purified
enzyme as the standard. We used the immunochemical
12
GAHN
AND
ROSKOSKI
I
i
0
0
10
20
30
40
50
60
0
Fraction
FIG. 1.
measured
20
40
60
Fraction
Tyrosine hydroxylase
elution profiles from (A) DEAE-cellulose
and (B) heparin-Sepharose.
Tyrosine hydroxylase
in fractions as described under Materials and Methods, and protein was measured by the method of Bradford (20).
quantification
of tyrosine hydroxylase
to determine the
amount of tyrosine hydroxylase in the starting material,
which, in the purification
shown in Table 1, was 3% of
the total protein in the high-speed
supernatant.
This
quantification
indicated that the homospecific
activity
(activity per milligram tyrosine hydroxylase)
decreased
somewhat during the purification
but increased in the
final step. The basis for this change is unknown.
The
yield based on total activity of the dialyzed fractions and
immunoreactivity
agreed within 10%.
This purification
procedure yielded 9-25 mg (an average of 15 mg from four preparations)
of tyrosine hydroxylase from PC12 cells. The recovery was consistently
4050%. The variability
in the quantity of purified protein obtained from the PC12 cells was related to variable expression of enzyme (22).
kDa
’
*
116+
was
We purified tyrosine hydroxylase
from a radiationinduced rat pheochromocytoma
by the same procedure
with the following additional
manipulations
(16). After
frozen tumor (30 g) extract was carried through the heparin-Sepharose
step, tyrosine hydroxylase-containing
fractions were pooled and concentrated
by pressure dialysis to about 1 mg protein/ml.
The sample was then
subjected to 52% saturation ammonium sulfate precipitation as described above and resolubilized
in 5 mM
Tris-acetate
(pH 8.2), 2 mM dithiothreitol.
The sample
(about 25 mg protein) was centrifuged
at 100,OOOg in a
Beckman ultracentrifuge
(SW27 rotor) for 40 h in a 35
ml linear 5-20% sucrose gradient in 5 mM Tris-acetate
(pH 8.2) and 2 mM dithiothreitol.
Tyrosine
hydroxylase-containing
fractions
were pooled and stored at
-80°C as previously described. This procedure yielded
about 5 mg purified protein. The enzyme from these two
sources was indistinguishable
with the following exception. The enzyme prepared from the transplantable
tumor migrated as a doublet in SDS-PAGE
(23) while the
enzyme prepared from the PC12 cells migrated as a single band (Fig. 2). The doublet was identified
in SDS
extracts of the tumor following Western blot immunochemical localization
(not shown). Only one band was
observed following SDS extraction of the PC12 cells.
Characterization
FIG. 2. SDS-PAGE
of fractions from the purification
of tyrosine
hydroxylase. Equal amounts of total protein (10 rg) from lane 1, the
high-speed supernatant,
and lane 2, the final purified protein, were
subjected to electrophoresis
on an 8% acrylamide gel.
activity
of Purified
Tyrosine
Hydroxylase
As noted above, the purified PC12 cell tyrosine hydroxylase appeared to be homogeneous,
with a subunit
molecular
weight of approximately
60,000 by SDSPAGE. Reports of the specific activity of tyrosine hydroxylase vary depending on assay conditions, but the
specific activity of this enzyme (0.67 pmol/min/mg
at
37°C 1 mM tetrahydrobiopterin,
pH 6.0) was similar to
that reported by others for purified rat tyrosine hydroxylase (5,12,17,23). The enzyme had a K,,, of 1.8 + 0.5 mM
(n = 2) for tetrahydrobiopterin
and a V,,, of 860 & 70
nmol/min/mg
(50 mM Pipes, pH 6,100 pM tyrosine).
TYROSINE
HYDROXYLASE
TABLE
Purification
Step
High-speed supernatant
Applied to DEAE
Applied to heparin-Sepharose
Purified product
Protein
(md
645
218
25.1
9.17
FROM
1
of Tyrosine Hydroxylase
Total
activity
(nmol/min)
from PC12 Cells
Specific
activity
(nmol/min/mg)
14,500
10,300
6,300
6,440
13
PC12 CELLS
Immunoreactive
TH
(md
22.5
47.5
250
664
19.7
15.7
10.6
9.17
Homospecific
activity
(nmol/min/mg
TH)
736
656
594
702
Yield
(SD)
100
80
54
46
at pH 6.0 as described under Materials
Note. Enzyme activity was assessed in the presence of 100 pM tryosine and 1 mM tetrahydrobiopterin
and Methods. All samples, which were stored at -8O”C, were dialyzed for 4 h against 10 mM phosphate buffer (pH 6.8) prior to assay to avoid
salt effects on activity (3). Specific activity refers to activity per milligram of total protein while homospecific activity refers to activity per
milligram of immunoreactive
tyrosine hydroxylase. Yield is based upon relative amount of tyrosine hydroxylase immunoreactive
material.
Similar results were obtained in five other preparations
with the yield ranging from 9 to 25 mg.
Tyrosine
hydroxylase
is phosphorylated
and activated by cyclic AMP-dependent
protein kinase in purified rat pheochromocytoma
tyrosine hydroxylase
(17)
and in intact PC12 cells (24). Our purified tyrosine hydroxylase was phosphorylated
by the kinase, with a stoichiometry of 0.7 mol phosphate/m01
subunit and activated about 20-fold (at pH 7.2 and 125 PM tetrahydrobiopterin).
As observed
with tyrosine
hydroxylase
purified from rat pheochromocytoma
(16), the stoichiometry varied from preparation to preparation presumably due to variation in the endogenous phosphate content in the different preparations
of the enzyme.
DISCUSSION
We developed a procedure for the purification
of rat
tyrosine hydroxylase
from cultured PC12 cells which
yielded 9-25 mg of purified protein from 30 ml of packed
cells. Previously
reported purifications
of rat tyrosine
hydroxylase result in less enzyme, typically 0.2 mg from
360 rat brains (5) to 2 mg from 20 g of rat pheochromocytoma (6). Our greater yield compared to that from
other PC12 cell preparations
(12,13) is probably due to
two factors. First, our PC12 cell cultures express a high
level of tyrosine hydroxylase, typically 35% of the total
cellular protein. Second, the high purification
afforded
by the heparin-Sepharose
chromatography
(with the
Tris wash) allowed us to minimize purification
steps
and maximize yield. The transplantable
tumor used in
our procedure yielded 5 mg of tyrosine hydroxylase
from 30 g of tumor (30 rats). Use of the transplantable
rat pheochromocytoma
necessitates maintenance
of an
inbred New England Deaconess rat colony for tumor
transplantation
and growth. We found maintenance
of
the colony to be costly, time-consuming,
and labor-intensive. Purification
from PC12 cells required approximately 100 T175 (cm2) flasks of PC12 cells and yielded
greater quantities of the enzyme. Our procedure can be
scaled up or down readily. Although the cost of materials for the transplantable
tumor and PC12 cells in cul-
ture is similar, the preparation
of PC12 cells was much
less labor-intensive.
Moreover, both of these sources
were less costly than the 360 rats required for preparing
200 pg from rat brain (5). Haavik and co-workers (10)
have purified bovine adrenal tyrosine hydroxylase with
a yield of 17 mg enzyme from 2.5 kg of adrenal medulla
(this corresponds to about 500 bovine adrenal glands).
The procedure of Haavik et al. takes 6 days to complete,
as compared to the 3-day procedure reported here.
Another major problem frequently encountered with
tyrosine hydroxylase is the extreme lability of the purified enzyme. For example, Togari and co-workers
(8)
purified tyrosine hydroxylase
from rat adrenal and rat
striatum, but they reported that the enzyme lost 50% of
its activity in 5 h at 4°C. Similar problems were reported
for purified bovine and human tyrosine hydroxylase
(7,9). The enzyme purified by our procedure, from either tumor or PC12 cells, is quite stable and can be
stored at -80°C for over 2 years with no loss of activity.
Richtand et al. (5) emphasized the importance of including protease inhibitors
to increase the stability of
rat brain tyrosine hydroxylase
during purification
and
storage. We point out, furthermore,
that tyrosine hydroxylase is unstable during storage at pH 6.0 and is
much more stable at higher pH. We store our enzyme at
pH 8.2 as noted previously. Storage at 2°C overnight at
pH 6.0 leads to a complete loss of activity, while at pH
8.2 the enzyme can be stored for up to 6 weeks at 2°C
with little or no decrease in activity.
ACKNOWLEDGMENTS
The authors thank Ms. Josephine Roussell for maintenance of the
PC12 cells, Mr. Jeffrey Kubinek for purification
of the catalytic subunit of cyclic AMP-dependent
protein kinase, Dr. Harvey Wilgus for
rat pheochromocytoma
samples, and Dr. John W. Haycock for providing reagents for the tyrosine hydroxylase immunoassay. This work
was supported by USPHS Grant NS 15994.
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