Preparative Biochemistry
ISSN: 0032-7484 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/lpbb19
Stabilization and Purification of Tyrosine Aminotransferase from Rat Liver James L. Hargrove To cite this article: James L. Hargrove (1990) Stabilization and Purification of Tyrosine Aminotransferase from Rat Liver, Preparative Biochemistry, 20:1, 11-22, DOI: 10.1080/00327489008050174 To link to this article: http://dx.doi.org/10.1080/00327489008050174
Published online: 23 Oct 2006.
Submit your article to this journal
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Citing articles: 2 View citing articles
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Date: 08 November 2015, At: 20:54
PREPARATIVE BIOCHEMISTRY, 20(1), 11-22 (1990)
Downloaded by [NUS National University of Singapore] at 20:54 08 November 2015
STABILIZATION AND PURIFICATION OF TYROSINE AMINOTRANSFERASE FROM RAT LIVER James L. Hargrove Department of Foods and Nutrition Dawson Hall The University of Georgia Athens, GA 30602 Abbreviations: CM, carboxymethyl; DEAE, diethylaminoethyl; DTT, dithiothreitol; EDTA, Ethylenediamine tetraacetic acid; HEPPS, Hydroxyethylpiperazinepropanesulfonic acid; PLP, pyridoxal 5'-phosphate; SDS, sodium dodecyl sulfate. Purification of unmodified tyrosine aminotransferase from rat liver requires that the activity of cathepsin T be minimized, and that losses of enzyme due to dilution or oxidation be prevented. The enzyme was stabilized by pyridoxal 5'-phosphate, dithiothreitol, and potassium phosphate, but was destabilized by L-tyrosine or L-plutamate. A rapid, efficient method for purification of this enzyme included the following steps: twenty-fold induction with a high-casein diet plus dexamethasone phosphate administered in the drinking water; a heat step (65OC) followed by precipitation from 0.20 M_ sucrose at pH 5.0; and small-scale chromatography on DEAE-cellulose, hydroxyapatite and CM-Sephadex C50 at pH 6 . 0 . These steps yielded more than 10 mg of native enzyme from 35 rats, with a recovery of 68% of the initial activity
.
INTRODUCTION Recent studies suggest that tyrosine aminotransferase (E.C. 2.6.1.5)
contains structural features that are involved in the
rapid rate of degradation which characterizes this enzyme in vivo, and other features which underlie its ability to transaminate tyrosine (1-3). Thus, the enzyme contains PEST elements that are thought to participate in proteolytic degradation of soluble proteins (21, and features that may permit it to be taken up into lysosomes in a serum-dependent manner ( 3 ) .
11 Copyright 0 1990 by Marcel Dekker, Inc.
While it should be
12
HARGROVE
useful
to
characterize
the
features
causing
tyrosine
aminotransferase to interact with these degradative systems, the low abundance and instability of the enzyme in rat 1l.ver impede progress.
Downloaded by [NUS National University of Singapore] at 20:54 08 November 2015
In order to overcome these obstacles, the present work describes conditions permitting induction of the enzyme to high levels in rat liver, and rapid purification in high yield. method
involves
acidification
of
a
sucrose
homogenate
The to
precipitate the enzyme, which is stabilized in the presence of phosphate buffer, pyridoxal 5'-phosphate (PLP), and dithiothreitol (DTT). using
The resulting preparation can then be purified in one day small
columns
of
DEAE-cellulose, hydroxyapatite, and
CM-Sephadex C50 to avoid dilution and loss of activity. MATERIALS AND METHODS Sprague-Dawley rats were bred in the departmental vivarium, where they were given free access to Purina rat chow and water. To survey treatments that provide a high concentration of enzyme, the rats were either fed a high-protein diet (50% of calories as casein; ICN Biomedical Products) for three days, or were fasted overnight before being killed. Injections of hormones and other agents were given between 9 and 10 p.m., and animals were killed between 8 and 9 a.m. the next morning. Hydroxyapatite (Biogel HTP) was obtained from BioRad, and other media for chromatography were from Sigma.
Most chemicals
and reagents were obtained from standard suppliers.
Triamcinolone
acetonide was a gift from Lederle Laboratories (Pearl River, NY). Tyrosine aminotransferase was assayed by the procedure of Granner and Tomkins (41, with one unit of activity equal to formation of one micromole of product per minute at 37'C.
Protein
was measured by a dye binding assay using pre-mixed reagent from Pierce Chemical Company. Purity of the enzyme was assessed by SDS-polyacrylamide gel electrophoresis (5) using minigel apparatus from BioRad. RESULTS AND DISCUSSION Stabilization
of
Tyrosine
Aminotransferase.
Since
tyrosine
aminotransferase gradually loses activity during the time required
PURIFICATION OF TYROSINE AMINOTRANSFERASE
13
TABLE I: Stability of Tyrosine Aminotransferase in Vitro
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37", 4h
Addition
Minus
Plus
Glutamate
Glutamate
None
60", 15 min
84
18
63
106
84
91
a-Ketoglutarate, 2 mM
99
25
93
L-Tyrosine, 1 mM
36
N.D.~
18
Pyridoxal 5'-phosphate, 0.2 mM
E-Hydroxyphenylpyruvate, 1 mM
106
N.D.
N.D.
Dithiothreitol, 2 mM
113
23
110
108
100
N.D.
Potassium phosphate, pH 7 , 25 mM
aN.D., value not determined.
for its purification, means to optimize yield by stabilizing the enzyme were tested. A preparation of enzyme was incubated at 37°C in buffers from pH 6 to 8, in the presence of potassium phosphate, PLP,
dithiothreitol, and a-ketoglutarate.
The enzyme was most
stable in phosphate buffer at pH 6-7, and least stable at pH 8 in synthetic buffers. For the experiments shown in Table I, one hundred milliunits of partially purified tyrosine aminotransferase (30 unitslmg protein) was incubated for 4 h at 37" in 0.25 ml of
14
HARGROVE
0.1 M - Hepes, pH 7.5, with 1 mM EDTA and 0.5 mg of fatty acid-free
bovine serum albumin.
One set of samples included 1 mM glutamate,
which removes PLP from the enzyme.
Other agents were included at
the concentrations shown, and samples were taken for assay of
Downloaded by [NUS National University of Singapore] at 20:54 08 November 2015
enzyme activity before and after the incubation.
In a second
experiment, samples were heated to 60" for 15 min in 0 . 2 5 ml of 25 mM potassium phosphate, pH
7, containing 0 . 5 mg albumin.
Values
are expressed as percentages of the initial activity remaining after the incubations, and are means of duplicate determinations. In the presence of 1 mM L-glutamic acid (which resolves the coenzyme
[unpublished
observation]),
maximum
stabilization
occurred with 0 . 2 mM PLP or 25 mM potassium phosphate. During the heat step, the enzyme was also stablized by 1 mM a-ketoglutarate or 2 mM dithiothreitol.
Combinations of these agents totally
stabilized the enzyme; therefore, most of this work employed potassium phosphate buffer at pH 6 , containing 0 . 1 mM PLP, 2 mM a-ketoglutarate, and 2 mM dithiothreitol. The reducing agent was added to buffers immediately before use. However, the initial extract must be buffered
to pH 8 and heated to inactivate
lysosomal proteases that generate multiple
forms by
limited
proteolysis (6,7), for which reason hydroxyethylpiperazinepropanesulfonic acid (HEPPS) was chosen for that step. Induction of Tyrosine different
procedures
aminotransferase purification.
to
Aminotransferase have
high
been levels
used in
in Rat
Liver.
Many
to
induce
tyrosine
liver
before
starting
Since it i s essential to chose an efficent method
for induction, Table I1 summarizes results obtained in this laboratory
using
various
procedures.
The
concentration of
tyrosine aminotransferase is greater in male rats than in females both in the basal and induced states (8), so it is preferable to use males. However, as shown here, the enzyme can be increased to levels exceeding 250 units per liver in females.
This compares
well to maximum values observed in males, so that animals of either gender may be used. The procedure involves inducing the enzyme overnight
so
and purification.
that a full day may be used for the extraction Results of induction with combinations of
PURIFICATION OF TYROSINE AMINOTRANSFERASE
15
TABLE I1
Downloaded by [NUS National University of Singapore] at 20:54 08 November 2015
Induction of Tyrosine Aminotransferase in Rats
Sex
Treatment
Average
Tyrosine
Liver
Aminotransferase
Weight
Units/g Units /Rat
8.1
2.0
16.3
0 . 1 mg/100 g
8.2
17.4
142
10 mg/100 g
8.6
38.2
329
12.3
14.7
182
14.3
23.2
333
10.4
36.1
375
15.8
23.6
375
Males,
Fasted 20 h
fasted
Triamcinolone,
Males,
50X casein diet,
fed
3 days
Triamcinolone, 0.1 mg/100 g (fed) Triamcinolone, 0 . 1 mg/100 g (fed),
Cycloheximide, 10 d 1 0 0 g
Dexamethasone phosphate in drinking water, 1 mg/100 ml (fed)
Females, Triamcinolone, fasted
0 . 1 mg/100g, plus
Cycloheximide, 25 pg/100 g 12.4 23.9 296 Animals ( 5 per group) were injected with Triamcinolone and/or
cycloheximide at 9 p.m.
and killed between 8 and 9 a.m.
Enzyme
activity was determined in duplicate for 9,OOOxg supernatants of individual livers.
16
HARGROVE
Triamcinolone acetonide or dexamethasone phosphate, cycloheximide, and high-protein diets are shown in Table 11.
Lower levels result
from use of hydrocortisone, insulin, and theophylline (not shown). Although
10 ug of synthetic
steroid per
100 g body weight
Downloaded by [NUS National University of Singapore] at 20:54 08 November 2015
provides maximum induction in 5 h (9), overnight induction with steroid suspensions yields highest levels of enzyme at 10 mg per 100 g (10). Comparable effects can be achieved at 0.1-1 mg per 100 g
body
weight
by
dissolving
Triamcinolone
acetonide
in
dimethylsulfoxide and including 10-50 pg of cycloheximide/lOOg
(11).
Cycloheximide functions by elevating the mRNA for this
enzyme, which leads to increased enzyme synthesis when translation begins again (12). Induction to similar levels can be achieved without injecting the rats by adapting them to a diet containing 50% protein by weight for at least three days, and adding dexamethasone phosphate to the drinking water at a concentration of 10 ug/ml on the day prior to sacrifice. Preparation of Liver Homogenates. ether and decapitated between steroid was administered.
Rats were anesthetized with
8-9 a.m.
on the day after the
Rats were exsanguinated and the excised
livers were placed in a beaker of 0.154 M NaCl at
4OC.
The livers
were rinsed several times with the cold saline solution, blotted on paper towels, and weighed.
Samples weighing one gram were used
to obtain the values shown in Table 11, and the rest of the livers were pooled and used to purify the enzyme as shown in Table 111. They were minced with scissors and homogenized in four volumes of 0.2 M sucrose containg 0.05 M HEPPS, pH 8.2, dithiothreitol, and 0.2 mM PLP.
1 mM EDTA, 2 mM
Disruption of tissue was achieved
with a Tekmar tissue homogenizer in 1 minute at 70% maximum power. The extract was immediately centrifuged at 9,OOOxg for 20 minutes in a preparative centrifuge, and the resulting supernatant solution was decanted through a double layer of cheesecloth. The supernatant was divided into two portions of about 600 ml each, and was heated to 65OC in a water bath maintained at 70°C
.
After five minutes, the sample was placed into an ice bath and cooled to 23°C.
The cooled extract was then centrifuged for ten
PURIFICATION OF TYROSINE AMINOTRANSFERASE
17
TABLE III Purification of Tyrosine Aminotransferase from 35 Rats (Liver wet weight, 370 g)
Downloaded by [NUS National University of Singapore] at 20:54 08 November 2015
Volume (ml)
Enzyme
Protein
(Units/ml)
(mg/ml)
Total
Specific
Units
Activity
(Units/mg protein)
12,000 xg 1290
5.4
21.6
6960
0.25
1090
5.6
5.7
6120
1.0
precipitation
180
34
6.0
6180
5.7
DEAE Cellulose
45
110
1.4
4980
78
Hydroxyapatite
9
492
3.1
4780
158
CM Sephadex C50
10.9
436
0.85
4760
510
Supernatant Heat to 65" for 5 m i n Isoelectric
Liver supernatants from the hormone-treated groups of male and female rats shown in Table XI were pooled and purified as described in the text.
minutes at
9,OOOxg
in a rotor at 4OC.
The volume of the
supernatant fluid was recorded, and the solution was acidified to pH 5.0 by dropwise addition of 10% acetic acid.
This causes
tyrosine aminotransferase to precipitate; after stirring the fluid for fifteen minutes at 4OC, the solution was centrifuged for five minutes at 2,700xg.
The supernatant fluid was decanted and
HARGROVE
18
discarded after verifying that the tyrosine aminotransferase had precipitated quantitatively.
The pellet was resuspended using a
Dounce-type tissue grinder in 50 mM potassium phosphate, pH 7.0, containing 0 . 1 M KC1, 2 mM dithiothreitol, 0.1 mM PLP, and 0.5 m M
Downloaded by [NUS National University of Singapore] at 20:54 08 November 2015
EDTA,
using
one-fifth
of
the
volume
recorded
prior
to
acidification. The suspended material was then centrifuged for 30 minutes
material
and
nucleoproteins that impede chromatography on DEAE cellulose.
at
100,OOOxg
to
remove
undissolved
The
precipitation yields a five-fold purification of the enzyme and a five-fold reduction in volume to permit more rapid chromatographic steps. Chromatography of Tyrosine Aminotransferase. next
series
of
steps
is
that
the
The basis for the
enzyme
DEAE-cellulose and CM-Sephadex C50 at pH 6.0,
binds
to
both
and that it can be
concentrated from solutions containing KC1 without dialysis using a small column of hydroxyapatite. Therefore, the redissolved enzyme was loaded onto a column of DEAE-cellulose equilibrated 50
with
mM
potassium
phosphate,
pH
7.0.
One
gram
of
DEAE-cellulose is used per rat, and the column should be 2 . 5 cm o r more
in diameter to permit application at a rate of about 100 ml
per hour without using a pump.
The column used for DEAE
-
cellulose chromatography was made from a plastic, 50 ml syringe, and
the ones used
for chromatography on hydroxyapatite and
CM-Sephadex C50 were made from 10 ml syringes.
After the enzyme
has been applied, the column is washed with 100 ml of column buffer (25 mM potassium phosphate, pH 6 . 0 , 2 mM dithiothreitol, 2 a-ketoglutaric acid, and 0.5 mM EDTA) containing 0.1 M KC1. PLP binds to DEAE cellulose and i s omitted during this step. A
mM
400 ml gradient of 0.1 to 0.5 M KC1 in column buffer is then
applied.
The enzyme elutes between 0.2-0.3 M KC1 (not sham).
The eluate from the DEAE-cellulose column is applied to column
containing
10
ml
of
hydroxyapatite;
a
tyrosine
aminotransferase binds without removal of potassium chloride. The column is washed with 20 ml of column buffer minus EDTA (EDTA is omitted because it chelates calcium to some extent); a yellow band should now be visible on the column, corresponding to bound
PURIFICATION OF TYROSINE AMINOTRANSFERASE tyrosine aminotransferase.
19
The enzyme is then eluted by washing
with 10 ml each of 0.2 M and 0.3 M potassium phosphate, pH 6.0, containing the same concentrations of dithiothreitol, PLP, and a-ketoglutaric acid as used in the earlier steps.
Elution can be
Downloaded by [NUS National University of Singapore] at 20:54 08 November 2015
monitored by watching movement of the yellow band.
The enzyme
should now be concentrated in less than 10 ml of buffer,
so
that
loss of activity due to dilution can be avoided. The optimum conditions for binding aminotransferase to CM-Sephadex C50 are
of 25-50
native mM
tyrosine
sodium or
potassium phosphate at pH 6.0 or less (unpublished observation). Therefore, the eluate from the hydroxyapatite may either be dialyzed or simply diluted to equilibrate it with the appropriate buffer.
Here, the enzyme was diluted with five parts of deionized
water containing 2 mM dithiothreitol and 0.2 mM PLP.
The pH was
adjusted with a few drops of 10% acetic acid, and the conductivity was checked to be sure it was very close to that of 50 mM potassium phosphate. The diluted enzyme was then applied to a column containing ten ml of CM-Sephadex C50 equilibrated with 50 mM potassium phosphate, pH 6.0, containing 2 mM DTT, 2 mM a-ketoglutarate, and 0.2 mM PLP.
The column was washed with 20 ml of the same buffer,
and the enzyme was eluted
stepwise with 10 mls each of buffer
containing 0.1, 0.2 and 0.4 M KC1.
The enzyme was recovered in
nine ml of buffer, was dialyzed against four liters of 25
mM
ammonium bicarbonate and then lyophilized.
A summary of the procedure is shown in Table 111, and a photograph of an electrophoretic gel containing enzyme purified by this method is shown in Figure 1.
The undegraded enzyme migrates
with an apparent molecular weight of 52,000,
indicating that
cathepsin T activity has been suppressed. Otherwise, a band would be present at 48,000 Daltons (6,7). Comments on the Procedure.
Purified tyrosine aminotransferase is
extremely stable, so long as it is protected against oxidation and dilution (unpublished observations). than at pH 6-7,
It is less stable at pH 8
but must be extracted from liver under alkaline
conditions to minimize the activity of cathepsin T, the lysosomal
Downloaded by [NUS National University of Singapore] at 20:54 08 November 2015
20
HARGROV E
FIGURE 1 Analysis of Purified Tyrosine Aminotransferase by SDS-PAGE Tyrosine aminotransferase samples resulting from chromatography on DEAE cellulose , hydroxyapatite (HAP) and Carboxymethyl Sephadex C50 (CM) were analyzed on a polyacrylamide gel containing 12% total acrylamide monomer, in the following amounts:
(lanes 1 and 2),
and 16 pg of protein; CM (lanes 1 and 2), Molecular
DEAE
7 and 14 pg of protein; HAP (lanes 1 and 2 1 , 8
weight
standards
are
5 and 10 pg of protein.
bovine
serum
albumin,
immunoglobulin G, and ovalbumin.
protease that rapidly cleaves an N-terminal segment from tyrosine aminotransferase (6,7).
Since cathepsin T is inactivated by
heating, the chromatographic steps may be performed safely at pH 6 after the initial heat step, Conditions that effectively stabilize tyrosine aminotransferase in vitro are shown in Table I.
The cofactor, PLP, protects
against inactivation, as does phosphate
ion.
Dithiothreitol
protects against losses due to oxidation, mechanical agitation, freezing and thawing, or heating, with an optimum concentration of
PURIFICATION OF TYROSINE AMINOTRANSFERASE about 20 mM.
21
These three protective agents are very effective
when combined in the concentrations used in this protocol.
By
minimizing dilution and the time required for preparation, the yield observed in the present work was 6 8 % . as compared to 15-43%
Downloaded by [NUS National University of Singapore] at 20:54 08 November 2015
in previous reports.
Glycerol at concentrations of 25-50% (v:v>
protects against inactivation due to freezing and thawing, and may be added during storage.
Enzyme stored at -20" in 40% glycerol
retains its primary structure but loses activity with a half-time of about 6 months; an inert atmosphere such as nitrogen or argon is beneficial.
This rapid procedure for purifying tyrosine aminotransferase
relies upon precipitation with acetic acid to reduce the volume of the initial extract, which must be done after the heat step because cathepsin T has an acidic pH optimum. Precipitation works reliably only in the absence of KC1, which solubilizes the enzyme. Therefore, sucrose i s included to lessen release of cathepsin T from lysosomes due to osmotic lysis.
Using ammonium sulfate,
polyethylene glycol, or ethanol as precipitating agents either gave less purification, lower recovery, or introduced salts that must be removed prior to chromatography. Small volumes of chromatographic media are used to minimize dilution during
application
contaminating proteins.
of
gradients
and
adsorption of
To provide for moderate rates of flow,
columns may conveniently be made from 50-ml or 10-ml plastic syringes, with sample being applied by way of a second syringe (minus plunger) connected to the column with plastic tubing. Together, these steps minimize dilution and permit a high degree of purification to be achieved in one or two days.
Since several
milligrams of enzyme may be prepared quickly from a small number of rats, it should now be possible to characterize functional regions in tyrosine aminotransferase with comparative ease.
ACKNOWLEDGEMENT This work was supported by U . S . P . H . S .
grant number DK39329.
HARGROVE
22
REFERENCES 1.
J. L. Hargrove, H. A. Scoble, W. R. Mathews, B. R. Baumstark, and K. Biemann, J. Biol. Chem.
2.
S. Rogers, R.
3.
J. F. Dice and H.
4.
D. K. Granner and G. M.
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(1986)
.
(1989)
264,
45-53
(1989).
Wells, and M. Rechsteiner, Science
Chiang, Biochem.
L.
SOC.
234, 364-368
Symp.
. Tomkins, Meth. Enzymol.
55,
45-55
E , 633-637
(1970). 5.
U. K. Laemmli, Nature
6.
J. L. Hargrove, M.
227,
680-685
(1970).
D i e s t e r h a f t , T. Noguchi, and D.
Granner
J. Biol. Chem 255, 71-77 (1980). 7.
Godha and H.
E.
C.
Pitot,
J.
Biol.
Chem.
255,
7371-7379
235,
E374-E380
(1980). 8.
J. Voigt and C.
E. S e k e r i s , h e r . J. Physiol.
(1978). 9.
T. H i r o t a , K. H i r o t a , Y. Sanno, and T. Tanaka, Endocrinology
117, 10.
1788-1795 (1985).
F A. V a l e r i o t e , F. Auricchio, G . M. Tomkins, and D. R i l e y , J. Biol. Chem. 244, 3618-3624 (1969).
11.
F.
M.
Baccino,
T e s s i t o r e , and M.
G.
Cecchini,
F.
Palmucci,
V.
Z u r e t t i , Biochim. Biophys. Acta
Sverko,
L.
9. 91-97
(1977). 12.
M. J. Ernest, L. DeLap, and P. Feigelson, J. Biol. Chem. 2895-2897 (1978).
253,
ISSN: 0032-7484 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/lpbb19
Stabilization and Purification of Tyrosine Aminotransferase from Rat Liver James L. Hargrove To cite this article: James L. Hargrove (1990) Stabilization and Purification of Tyrosine Aminotransferase from Rat Liver, Preparative Biochemistry, 20:1, 11-22, DOI: 10.1080/00327489008050174 To link to this article: http://dx.doi.org/10.1080/00327489008050174
Published online: 23 Oct 2006.
Submit your article to this journal
Article views: 9
View related articles
Citing articles: 2 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=lpbb20 Download by: [NUS National University of Singapore]
Date: 08 November 2015, At: 20:54
PREPARATIVE BIOCHEMISTRY, 20(1), 11-22 (1990)
Downloaded by [NUS National University of Singapore] at 20:54 08 November 2015
STABILIZATION AND PURIFICATION OF TYROSINE AMINOTRANSFERASE FROM RAT LIVER James L. Hargrove Department of Foods and Nutrition Dawson Hall The University of Georgia Athens, GA 30602 Abbreviations: CM, carboxymethyl; DEAE, diethylaminoethyl; DTT, dithiothreitol; EDTA, Ethylenediamine tetraacetic acid; HEPPS, Hydroxyethylpiperazinepropanesulfonic acid; PLP, pyridoxal 5'-phosphate; SDS, sodium dodecyl sulfate. Purification of unmodified tyrosine aminotransferase from rat liver requires that the activity of cathepsin T be minimized, and that losses of enzyme due to dilution or oxidation be prevented. The enzyme was stabilized by pyridoxal 5'-phosphate, dithiothreitol, and potassium phosphate, but was destabilized by L-tyrosine or L-plutamate. A rapid, efficient method for purification of this enzyme included the following steps: twenty-fold induction with a high-casein diet plus dexamethasone phosphate administered in the drinking water; a heat step (65OC) followed by precipitation from 0.20 M_ sucrose at pH 5.0; and small-scale chromatography on DEAE-cellulose, hydroxyapatite and CM-Sephadex C50 at pH 6 . 0 . These steps yielded more than 10 mg of native enzyme from 35 rats, with a recovery of 68% of the initial activity
.
INTRODUCTION Recent studies suggest that tyrosine aminotransferase (E.C. 2.6.1.5)
contains structural features that are involved in the
rapid rate of degradation which characterizes this enzyme in vivo, and other features which underlie its ability to transaminate tyrosine (1-3). Thus, the enzyme contains PEST elements that are thought to participate in proteolytic degradation of soluble proteins (21, and features that may permit it to be taken up into lysosomes in a serum-dependent manner ( 3 ) .
11 Copyright 0 1990 by Marcel Dekker, Inc.
While it should be
12
HARGROVE
useful
to
characterize
the
features
causing
tyrosine
aminotransferase to interact with these degradative systems, the low abundance and instability of the enzyme in rat 1l.ver impede progress.
Downloaded by [NUS National University of Singapore] at 20:54 08 November 2015
In order to overcome these obstacles, the present work describes conditions permitting induction of the enzyme to high levels in rat liver, and rapid purification in high yield. method
involves
acidification
of
a
sucrose
homogenate
The to
precipitate the enzyme, which is stabilized in the presence of phosphate buffer, pyridoxal 5'-phosphate (PLP), and dithiothreitol (DTT). using
The resulting preparation can then be purified in one day small
columns
of
DEAE-cellulose, hydroxyapatite, and
CM-Sephadex C50 to avoid dilution and loss of activity. MATERIALS AND METHODS Sprague-Dawley rats were bred in the departmental vivarium, where they were given free access to Purina rat chow and water. To survey treatments that provide a high concentration of enzyme, the rats were either fed a high-protein diet (50% of calories as casein; ICN Biomedical Products) for three days, or were fasted overnight before being killed. Injections of hormones and other agents were given between 9 and 10 p.m., and animals were killed between 8 and 9 a.m. the next morning. Hydroxyapatite (Biogel HTP) was obtained from BioRad, and other media for chromatography were from Sigma.
Most chemicals
and reagents were obtained from standard suppliers.
Triamcinolone
acetonide was a gift from Lederle Laboratories (Pearl River, NY). Tyrosine aminotransferase was assayed by the procedure of Granner and Tomkins (41, with one unit of activity equal to formation of one micromole of product per minute at 37'C.
Protein
was measured by a dye binding assay using pre-mixed reagent from Pierce Chemical Company. Purity of the enzyme was assessed by SDS-polyacrylamide gel electrophoresis (5) using minigel apparatus from BioRad. RESULTS AND DISCUSSION Stabilization
of
Tyrosine
Aminotransferase.
Since
tyrosine
aminotransferase gradually loses activity during the time required
PURIFICATION OF TYROSINE AMINOTRANSFERASE
13
TABLE I: Stability of Tyrosine Aminotransferase in Vitro
Downloaded by [NUS National University of Singapore] at 20:54 08 November 2015
37", 4h
Addition
Minus
Plus
Glutamate
Glutamate
None
60", 15 min
84
18
63
106
84
91
a-Ketoglutarate, 2 mM
99
25
93
L-Tyrosine, 1 mM
36
N.D.~
18
Pyridoxal 5'-phosphate, 0.2 mM
E-Hydroxyphenylpyruvate, 1 mM
106
N.D.
N.D.
Dithiothreitol, 2 mM
113
23
110
108
100
N.D.
Potassium phosphate, pH 7 , 25 mM
aN.D., value not determined.
for its purification, means to optimize yield by stabilizing the enzyme were tested. A preparation of enzyme was incubated at 37°C in buffers from pH 6 to 8, in the presence of potassium phosphate, PLP,
dithiothreitol, and a-ketoglutarate.
The enzyme was most
stable in phosphate buffer at pH 6-7, and least stable at pH 8 in synthetic buffers. For the experiments shown in Table I, one hundred milliunits of partially purified tyrosine aminotransferase (30 unitslmg protein) was incubated for 4 h at 37" in 0.25 ml of
14
HARGROVE
0.1 M - Hepes, pH 7.5, with 1 mM EDTA and 0.5 mg of fatty acid-free
bovine serum albumin.
One set of samples included 1 mM glutamate,
which removes PLP from the enzyme.
Other agents were included at
the concentrations shown, and samples were taken for assay of
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enzyme activity before and after the incubation.
In a second
experiment, samples were heated to 60" for 15 min in 0 . 2 5 ml of 25 mM potassium phosphate, pH
7, containing 0 . 5 mg albumin.
Values
are expressed as percentages of the initial activity remaining after the incubations, and are means of duplicate determinations. In the presence of 1 mM L-glutamic acid (which resolves the coenzyme
[unpublished
observation]),
maximum
stabilization
occurred with 0 . 2 mM PLP or 25 mM potassium phosphate. During the heat step, the enzyme was also stablized by 1 mM a-ketoglutarate or 2 mM dithiothreitol.
Combinations of these agents totally
stabilized the enzyme; therefore, most of this work employed potassium phosphate buffer at pH 6 , containing 0 . 1 mM PLP, 2 mM a-ketoglutarate, and 2 mM dithiothreitol. The reducing agent was added to buffers immediately before use. However, the initial extract must be buffered
to pH 8 and heated to inactivate
lysosomal proteases that generate multiple
forms by
limited
proteolysis (6,7), for which reason hydroxyethylpiperazinepropanesulfonic acid (HEPPS) was chosen for that step. Induction of Tyrosine different
procedures
aminotransferase purification.
to
Aminotransferase have
high
been levels
used in
in Rat
Liver.
Many
to
induce
tyrosine
liver
before
starting
Since it i s essential to chose an efficent method
for induction, Table I1 summarizes results obtained in this laboratory
using
various
procedures.
The
concentration of
tyrosine aminotransferase is greater in male rats than in females both in the basal and induced states (8), so it is preferable to use males. However, as shown here, the enzyme can be increased to levels exceeding 250 units per liver in females.
This compares
well to maximum values observed in males, so that animals of either gender may be used. The procedure involves inducing the enzyme overnight
so
and purification.
that a full day may be used for the extraction Results of induction with combinations of
PURIFICATION OF TYROSINE AMINOTRANSFERASE
15
TABLE I1
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Induction of Tyrosine Aminotransferase in Rats
Sex
Treatment
Average
Tyrosine
Liver
Aminotransferase
Weight
Units/g Units /Rat
8.1
2.0
16.3
0 . 1 mg/100 g
8.2
17.4
142
10 mg/100 g
8.6
38.2
329
12.3
14.7
182
14.3
23.2
333
10.4
36.1
375
15.8
23.6
375
Males,
Fasted 20 h
fasted
Triamcinolone,
Males,
50X casein diet,
fed
3 days
Triamcinolone, 0.1 mg/100 g (fed) Triamcinolone, 0 . 1 mg/100 g (fed),
Cycloheximide, 10 d 1 0 0 g
Dexamethasone phosphate in drinking water, 1 mg/100 ml (fed)
Females, Triamcinolone, fasted
0 . 1 mg/100g, plus
Cycloheximide, 25 pg/100 g 12.4 23.9 296 Animals ( 5 per group) were injected with Triamcinolone and/or
cycloheximide at 9 p.m.
and killed between 8 and 9 a.m.
Enzyme
activity was determined in duplicate for 9,OOOxg supernatants of individual livers.
16
HARGROVE
Triamcinolone acetonide or dexamethasone phosphate, cycloheximide, and high-protein diets are shown in Table 11.
Lower levels result
from use of hydrocortisone, insulin, and theophylline (not shown). Although
10 ug of synthetic
steroid per
100 g body weight
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provides maximum induction in 5 h (9), overnight induction with steroid suspensions yields highest levels of enzyme at 10 mg per 100 g (10). Comparable effects can be achieved at 0.1-1 mg per 100 g
body
weight
by
dissolving
Triamcinolone
acetonide
in
dimethylsulfoxide and including 10-50 pg of cycloheximide/lOOg
(11).
Cycloheximide functions by elevating the mRNA for this
enzyme, which leads to increased enzyme synthesis when translation begins again (12). Induction to similar levels can be achieved without injecting the rats by adapting them to a diet containing 50% protein by weight for at least three days, and adding dexamethasone phosphate to the drinking water at a concentration of 10 ug/ml on the day prior to sacrifice. Preparation of Liver Homogenates. ether and decapitated between steroid was administered.
Rats were anesthetized with
8-9 a.m.
on the day after the
Rats were exsanguinated and the excised
livers were placed in a beaker of 0.154 M NaCl at
4OC.
The livers
were rinsed several times with the cold saline solution, blotted on paper towels, and weighed.
Samples weighing one gram were used
to obtain the values shown in Table 11, and the rest of the livers were pooled and used to purify the enzyme as shown in Table 111. They were minced with scissors and homogenized in four volumes of 0.2 M sucrose containg 0.05 M HEPPS, pH 8.2, dithiothreitol, and 0.2 mM PLP.
1 mM EDTA, 2 mM
Disruption of tissue was achieved
with a Tekmar tissue homogenizer in 1 minute at 70% maximum power. The extract was immediately centrifuged at 9,OOOxg for 20 minutes in a preparative centrifuge, and the resulting supernatant solution was decanted through a double layer of cheesecloth. The supernatant was divided into two portions of about 600 ml each, and was heated to 65OC in a water bath maintained at 70°C
.
After five minutes, the sample was placed into an ice bath and cooled to 23°C.
The cooled extract was then centrifuged for ten
PURIFICATION OF TYROSINE AMINOTRANSFERASE
17
TABLE III Purification of Tyrosine Aminotransferase from 35 Rats (Liver wet weight, 370 g)
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Volume (ml)
Enzyme
Protein
(Units/ml)
(mg/ml)
Total
Specific
Units
Activity
(Units/mg protein)
12,000 xg 1290
5.4
21.6
6960
0.25
1090
5.6
5.7
6120
1.0
precipitation
180
34
6.0
6180
5.7
DEAE Cellulose
45
110
1.4
4980
78
Hydroxyapatite
9
492
3.1
4780
158
CM Sephadex C50
10.9
436
0.85
4760
510
Supernatant Heat to 65" for 5 m i n Isoelectric
Liver supernatants from the hormone-treated groups of male and female rats shown in Table XI were pooled and purified as described in the text.
minutes at
9,OOOxg
in a rotor at 4OC.
The volume of the
supernatant fluid was recorded, and the solution was acidified to pH 5.0 by dropwise addition of 10% acetic acid.
This causes
tyrosine aminotransferase to precipitate; after stirring the fluid for fifteen minutes at 4OC, the solution was centrifuged for five minutes at 2,700xg.
The supernatant fluid was decanted and
HARGROVE
18
discarded after verifying that the tyrosine aminotransferase had precipitated quantitatively.
The pellet was resuspended using a
Dounce-type tissue grinder in 50 mM potassium phosphate, pH 7.0, containing 0 . 1 M KC1, 2 mM dithiothreitol, 0.1 mM PLP, and 0.5 m M
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EDTA,
using
one-fifth
of
the
volume
recorded
prior
to
acidification. The suspended material was then centrifuged for 30 minutes
material
and
nucleoproteins that impede chromatography on DEAE cellulose.
at
100,OOOxg
to
remove
undissolved
The
precipitation yields a five-fold purification of the enzyme and a five-fold reduction in volume to permit more rapid chromatographic steps. Chromatography of Tyrosine Aminotransferase. next
series
of
steps
is
that
the
The basis for the
enzyme
DEAE-cellulose and CM-Sephadex C50 at pH 6.0,
binds
to
both
and that it can be
concentrated from solutions containing KC1 without dialysis using a small column of hydroxyapatite. Therefore, the redissolved enzyme was loaded onto a column of DEAE-cellulose equilibrated 50
with
mM
potassium
phosphate,
pH
7.0.
One
gram
of
DEAE-cellulose is used per rat, and the column should be 2 . 5 cm o r more
in diameter to permit application at a rate of about 100 ml
per hour without using a pump.
The column used for DEAE
-
cellulose chromatography was made from a plastic, 50 ml syringe, and
the ones used
for chromatography on hydroxyapatite and
CM-Sephadex C50 were made from 10 ml syringes.
After the enzyme
has been applied, the column is washed with 100 ml of column buffer (25 mM potassium phosphate, pH 6 . 0 , 2 mM dithiothreitol, 2 a-ketoglutaric acid, and 0.5 mM EDTA) containing 0.1 M KC1. PLP binds to DEAE cellulose and i s omitted during this step. A
mM
400 ml gradient of 0.1 to 0.5 M KC1 in column buffer is then
applied.
The enzyme elutes between 0.2-0.3 M KC1 (not sham).
The eluate from the DEAE-cellulose column is applied to column
containing
10
ml
of
hydroxyapatite;
a
tyrosine
aminotransferase binds without removal of potassium chloride. The column is washed with 20 ml of column buffer minus EDTA (EDTA is omitted because it chelates calcium to some extent); a yellow band should now be visible on the column, corresponding to bound
PURIFICATION OF TYROSINE AMINOTRANSFERASE tyrosine aminotransferase.
19
The enzyme is then eluted by washing
with 10 ml each of 0.2 M and 0.3 M potassium phosphate, pH 6.0, containing the same concentrations of dithiothreitol, PLP, and a-ketoglutaric acid as used in the earlier steps.
Elution can be
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monitored by watching movement of the yellow band.
The enzyme
should now be concentrated in less than 10 ml of buffer,
so
that
loss of activity due to dilution can be avoided. The optimum conditions for binding aminotransferase to CM-Sephadex C50 are
of 25-50
native mM
tyrosine
sodium or
potassium phosphate at pH 6.0 or less (unpublished observation). Therefore, the eluate from the hydroxyapatite may either be dialyzed or simply diluted to equilibrate it with the appropriate buffer.
Here, the enzyme was diluted with five parts of deionized
water containing 2 mM dithiothreitol and 0.2 mM PLP.
The pH was
adjusted with a few drops of 10% acetic acid, and the conductivity was checked to be sure it was very close to that of 50 mM potassium phosphate. The diluted enzyme was then applied to a column containing ten ml of CM-Sephadex C50 equilibrated with 50 mM potassium phosphate, pH 6.0, containing 2 mM DTT, 2 mM a-ketoglutarate, and 0.2 mM PLP.
The column was washed with 20 ml of the same buffer,
and the enzyme was eluted
stepwise with 10 mls each of buffer
containing 0.1, 0.2 and 0.4 M KC1.
The enzyme was recovered in
nine ml of buffer, was dialyzed against four liters of 25
mM
ammonium bicarbonate and then lyophilized.
A summary of the procedure is shown in Table 111, and a photograph of an electrophoretic gel containing enzyme purified by this method is shown in Figure 1.
The undegraded enzyme migrates
with an apparent molecular weight of 52,000,
indicating that
cathepsin T activity has been suppressed. Otherwise, a band would be present at 48,000 Daltons (6,7). Comments on the Procedure.
Purified tyrosine aminotransferase is
extremely stable, so long as it is protected against oxidation and dilution (unpublished observations). than at pH 6-7,
It is less stable at pH 8
but must be extracted from liver under alkaline
conditions to minimize the activity of cathepsin T, the lysosomal
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20
HARGROV E
FIGURE 1 Analysis of Purified Tyrosine Aminotransferase by SDS-PAGE Tyrosine aminotransferase samples resulting from chromatography on DEAE cellulose , hydroxyapatite (HAP) and Carboxymethyl Sephadex C50 (CM) were analyzed on a polyacrylamide gel containing 12% total acrylamide monomer, in the following amounts:
(lanes 1 and 2),
and 16 pg of protein; CM (lanes 1 and 2), Molecular
DEAE
7 and 14 pg of protein; HAP (lanes 1 and 2 1 , 8
weight
standards
are
5 and 10 pg of protein.
bovine
serum
albumin,
immunoglobulin G, and ovalbumin.
protease that rapidly cleaves an N-terminal segment from tyrosine aminotransferase (6,7).
Since cathepsin T is inactivated by
heating, the chromatographic steps may be performed safely at pH 6 after the initial heat step, Conditions that effectively stabilize tyrosine aminotransferase in vitro are shown in Table I.
The cofactor, PLP, protects
against inactivation, as does phosphate
ion.
Dithiothreitol
protects against losses due to oxidation, mechanical agitation, freezing and thawing, or heating, with an optimum concentration of
PURIFICATION OF TYROSINE AMINOTRANSFERASE about 20 mM.
21
These three protective agents are very effective
when combined in the concentrations used in this protocol.
By
minimizing dilution and the time required for preparation, the yield observed in the present work was 6 8 % . as compared to 15-43%
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in previous reports.
Glycerol at concentrations of 25-50% (v:v>
protects against inactivation due to freezing and thawing, and may be added during storage.
Enzyme stored at -20" in 40% glycerol
retains its primary structure but loses activity with a half-time of about 6 months; an inert atmosphere such as nitrogen or argon is beneficial.
This rapid procedure for purifying tyrosine aminotransferase
relies upon precipitation with acetic acid to reduce the volume of the initial extract, which must be done after the heat step because cathepsin T has an acidic pH optimum. Precipitation works reliably only in the absence of KC1, which solubilizes the enzyme. Therefore, sucrose i s included to lessen release of cathepsin T from lysosomes due to osmotic lysis.
Using ammonium sulfate,
polyethylene glycol, or ethanol as precipitating agents either gave less purification, lower recovery, or introduced salts that must be removed prior to chromatography. Small volumes of chromatographic media are used to minimize dilution during
application
contaminating proteins.
of
gradients
and
adsorption of
To provide for moderate rates of flow,
columns may conveniently be made from 50-ml or 10-ml plastic syringes, with sample being applied by way of a second syringe (minus plunger) connected to the column with plastic tubing. Together, these steps minimize dilution and permit a high degree of purification to be achieved in one or two days.
Since several
milligrams of enzyme may be prepared quickly from a small number of rats, it should now be possible to characterize functional regions in tyrosine aminotransferase with comparative ease.
ACKNOWLEDGEMENT This work was supported by U . S . P . H . S .
grant number DK39329.
HARGROVE
22
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