From the
Deoxyhypusine synthase is the first enzyme involved in the
post-translational formation of hypusine, a unique amino acid that
occurs at one position in a single cellular protein, eukaryotic
translation initiation factor 5A (eIF-5A). This NAD-dependent enzyme
catalyzes the formation of deoxyhypusine by transfer of the butylamine
portion of spermidine to the
The unusual amino acid hypusine
( N
Hypusine has been shown to be important for the
activity of eIF-5A in methionylpuromycin synthesis, an in vitro assay for protein synthesis initiation, yet the precise cellular
function of this putative initiation factor is unknown
(2, 3, 4, 5, 6) . Nonetheless,
the hypusine-containing protein, eIF-5A, which is ubiquitous and highly
conserved in eukaryotes, appears to be vital for cell proliferation
(2) . The synthesis of hypusine, and therefore of mature eIF-5A,
was correlated with proliferation in several eukaryotic cell types
(7, 8, 9, 10, 11) . Conversely,
inhibition of either of the two enzymes, deoxyhypusine synthase or
deoxyhypusine hydroxylase, was shown to arrest the growth of various
mammalian cells
(12, 13) . In yeast, the expression of
at least one of the two eIF-5A genes and modification of the eIF-5A
precursor protein to the hypusine form are essential for cell viability
(14, 15, 16) . In view of the implication of
hypusine in cell proliferation, its occurrence in a single protein, and
the specificities of the enzymes involved in its biosynthesis,
deoxyhypusine synthase was proposed as a target for antiproliferative
therapy
(2, 12) . Inhibitors of this enzyme that cause
effective suppression of hypusine formation in Chinese hamster ovary
cells
(17) were found to be antiproliferative
(12) ,
supporting the concept of a vital connection between hypusine and cell
growth and offering a potential means of defining the biological role
of hypusine and eIF-5A in cell growth.
Early recognition of the high
pH optimum
(18) and NAD dependence
(19) of
deoxyhypusine synthesis facilitated development of an in vitro assay for the enzyme and its preliminary characterization
(20, 21, 22) . The enzyme is unique in its
modification of a single lysine residue in one protein through a
multistep redox reaction (Fig. SI). Thus, while being the enzyme
responsible for deoxyhypusine synthesis, deoxyhypusine synthase can
also be viewed as a pro- R-specific NAD-dependent
dehydrogenase. In addition, because of its ability to cleave spermidine
between the secondary nitrogen and carbon 5 in either the presence or
absence of its eIF-5A precursor substrate (Fig. SI),
deoxyhypusine synthase may be considered a polyamine-metabolizing
enzyme. In early studies with partially purified enzyme
(20, 21) , it was not clear whether the reactions
leading to deoxyhypusine synthesis were catalyzed by a single enzyme, a
series of enzymes, or a multienzyme complex. We now report the
purification of deoxyhypusine synthase to homogeneity from rat testis
and provide evidence that this single enzyme indeed carries out the
overall synthesis of deoxyhypusine.
Materials ec-eIF-5A(Lys), purified from Escherichia coli lysates after
overexpression of the human eIF-5A cDNA as described
(23) , was
kindly supplied by Dr. Young Ae Joe of this laboratory. Frozen testes
from mature rats were purchased from Pel-Freez Biologicals;
[1,8-
The
enzyme activity of tissue homogenates was measured after ammonium
sulfate fractionation and dialysis of the precipitated proteins (see
Tables I and II). This was necessary in order to avoid dilution of
radiolabeled spermidine in the assay solution by nonradioactive
spermidine present in tissue extracts. Purification of Deoxyhypusine Synthase from Rat Testis Five hundred-gram batches of frozen rat testes were processed through
Step 3 (see below). The enzymatically active fractions from several of
these batches were combined for further purification. Chromatographic
separations were carried at ambient temperature, but the collected
fractions were immediately cooled in ice and stored frozen at -20
or -80 °C until used in the next step of purification. Other
operations were conducted at 4 °C.
Purification of Deoxyhypusine Synthase
The choice of rat testis as the source for purification of
deoxyhypusine synthase was based on a survey of the enzyme activity in
several rat tissues (). Of those tissues examined, the
testis was found to contain the greatest activity per milligram of
tissue as well as the highest specific activity of deoxyhypusine
synthase. It is interesting that the testis, an actively proliferating
tissue, is also the richest in hypusine content
(27) and in
deoxyhypusine hydroxylase
(28) .
Previously, we reported the
partial purification (
Chromatofocusing, in which a gradient of decreasing pH is used to
separate proteins by virtue of their different pI values
(29) ,
was utilized for the final step of purification. A pH gradient range of
6 to 4 was chosen on the basis of the estimated pI of the enzyme (see
below). As shown in Fig. 1 A, a single sharp peak of
enzyme activity eluted at pH 4.5, coinciding with a peak of absorbance
at 280 nm. Upon SDS-PAGE (Fig. 1 B), the active
fraction(s) were found to contain one major protein of
Physical properties of rat testis
deoxyhypusine synthase are summarized in I. A molecular
mass of 40.8 ± 0.1 kDa was determined by matrix-assisted laser
desorption mass spectrometry of the pure enzyme. This value is in
agreement with the apparent molecular mass of 42 kDa estimated by
SDS-PAGE. However, gel filtration and ultracentrifugation indicated
that the predominant species in solution is not a monomer. Analysis of
the equilibrium sedimentation data of a 0.1 mg/ml sample of the pure
enzyme gave a weight average molecular mass of 144 kDa (with an assumed
partial specific volume of 0.73 cm
A recent study from this laboratory showed that a substantial
part of the primary structure of the eIF-5A precursor protein is
required for the enzymatic formation of deoxyhypusine
(23) .
This finding highlights the very stringent substrate specificity of
deoxyhypusine synthase and provides an explanation for the exclusive
occurrence of hypusine in eIF-5A. Two additional substrates, NAD and
spermidine, are involved in the enzymatic reaction leading to
deoxyhypusine. Cleavage of spermidine as the first step in this
reaction was postulated based on the observation that, in the absence
of the eIF-5A precursor protein, partially purified deoxyhypusine
synthase catalyzed another reaction, the conversion of spermidine to
1,3-diaminopropane and
The active form of
deoxyhypusine synthase catalyzing this complex reaction is probably a
tetramer of a protein with a monomer molecular mass of
This is the first purification to
homogeneity and physical characterization of mammalian deoxyhypusine
synthase, although a preliminary account of its purification from
Neurospora crassa from another laboratory has appeared
(34) . None of the partial amino acid sequences of peptides
isolated from the rat enzyme were found to correspond to any known
protein sequence. These peptide sequences and the antibody to the
purified rat enzyme were indispensable for the identification of yeast
genomic DNA encoding deoxyhypusine synthase
The enzyme activity was assayed as described under
``Experimental Procedures,'' and protein content was
determined by the bicinchoninic acid procedure (26). Approximately 90%
of the enzyme activity in tissues was precipitated between 40 and 50%
saturation with ammonium sulfate. This fraction was dialyzed versus Buffer A for 4 h before assay. Activity is expressed as units/mg
of tissue (original wet weight) and as units/mg of protein in the
40-50% ammonium sulfate fraction. One unit is the amount of
enzyme that catalyzes the formation of 1 pmol of deoxyhypusine/h
(equivalent to 1.67
The enzymatic assay and purification were carried
out as described under ``Experimental Procedures.'' Steps
1-6 show a representative preparation starting from 1121 g of rat
testes (two initial batches combined after Step 3). Approximately 50%
of the active material after Step 6 was used for Step 7, and the value
shown is adjusted accordingly.
The determination of enzymatic
activity after SDS-PAGE was as described in the legend to Fig. 3. Mass
spectrometry was performed by matrix-assisted laser desorption with a
Kratos Kompact Maldi III spectrometer on the pure enzyme (
Deoxyhypusine synthase, with rabbit
antiserum or with preimmune serum (each at 1:50 dilution in
phosphate-buffered saline) or without serum, was kept on ice for 18 h.
The mixture was added to Protein A liganded to agarose beads (Pierce),
shaken gently for 1 h at 23 °C, and centrifuged at 600
We are deeply indebted to Dan L. Sackett (NIDDK,
National Institutes of Health) for the ultracentrifugation analysis and
to Henry M. Fales and Edward A. Sokoloski (NHLBI, National Institutes
of Health) for the mass spectrometry.
Addendum-While this manuscript was under review, a paper
describing the purification of deoxyhypusine synthase from N.
crassa was published (Tao, Y., and Chen, K. Y. (1995) J. Biol.
Chem.
270, 383-386).
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-amino group of a specific lysine
residue in the eIF-5A precursor. Its purification from rat testis was
accomplished by ammonium sulfate fractionation and successive
ion-exchange chromatographic steps, followed by chromatofocusing on a
hydrophilic resin (Mono P). A pI of 4.7 was determined by isoelectric
focusing. Amino acid sequences of five tryptic peptides of the pure
enzyme did not correspond to any sequences in the protein data banks.
The enzyme migrates as a single band on SDS-polyacrylamide gel
electrophoresis with an apparent monomer molecular mass of
42,000
Da. Matrix-assisted laser desorption mass spectrometry gave a monomer
mass of 40,800 Da. There is evidence, however, that the active enzyme
exists as a tetramer of this subunit. Rabbit polyclonal antibodies to
the 42-kDa protein precipitated deoxyhypusine synthase activity. The
enzyme shows a strict specificity for NAD. Purified deoxyhypusine
synthase catalyzes the overall synthesis of deoxyhypusine and, in the
absence of the eIF-5A precursor, catalyzes the cleavage of spermidine.
-(4-amino-2-hydroxybutyl)lysine) occurs in
only one cellular protein, eIF-5A,
(
)
and is
produced post-translationally in two successive enzyme-catalyzed
reactions: 1) deoxyhypusine synthesis and 2) deoxyhypusine
hydroxylation. The enzyme deoxyhypusine synthase mediates the
NAD-dependent transfer of the butylamine moiety of the polyamine
spermidine to the
-amino group of a single lysine residue in the
eIF-5A precursor protein (Lys
in the human precursor) to
form the deoxyhypusine
( N
-(4-aminobutyl)lysine) residue (Scheme I).
Hydroxylation of the deoxyhypusine residue by a specific hydroxylase
completes hypusine synthesis and the maturation of eIF-5A (for reviews,
see Refs. 1 and 2).
Figure SI:
Reactions
catalyzed by deoxyhypusine synthase. The product of the complete
reaction is eIF-5A(Dhp), the intermediate form of eIF-5A containing
deoxyhypusine ( Dhp). The dashed arrows indicate the reaction in the absence of the eIF-5A
precursor.
H]spermidine HCl (15 Ci/mmol) was from
DuPont NEN; NAD and NADH (Grade I; 100%) NADP (99%), NADPH (98%), FAD,
and FMN were from Boehringer Mannheim; 4-(2-aminoethyl)benzenesulfonyl
fluoride HCl, leupeptin, and aprotinin were from ICN Biochemicals;
Bistris and iminodiacetate
Na
H
O were
from Aldrich; CHES was from U. S. Biochemical Corp.; DEAE-cellulose
(DE23) was from Whatman; Q-Sepharose Fast Flow gel, Mono Q
HR 10/10 and Mono Q
HR 5/5 prepacked ion-exchange
columns, Mono P
HR 5/20 chromatofocusing columns,
Polybuffer
74, and protein standards for isoelectric
focusing were obtained from Pharmacia Biotech Inc.; precast
polyacrylamide gels and wide-range protein standards (Mark 12) were
from Novex; polyvinylidene difluoride ProBlott
membranes
were from Applied Biosystems; Immobilon-P membranes were from
Millipore; ImmunoPure (R) Plus Immobilized Protein A (6 mg of Protein
A/ml of gel, cross-linked, 6% beaded agarose) was from Pierce. Methods Assay of Enzyme Activity Deoxyhypusine synthase activity was measured as described previously
(21) typically in total volumes of 20 µl of 0.2
M
glycine/NaOH buffer, pH 9.5, containing 1 m
M dithiothreitol,
25 µg of bovine serum albumin, 0.5 m
M NAD, 7
µ
M (2 µCi) [1,8-
H]spermidine, 1
µ
M ec-eIF-5A, and enzyme. Incubations were at 37 °C
for 60 min. The radioactivity of [
H]deoxyhypusine
was measured after its ion-exchange chromatographic separation from the
hydrolyzed protein fraction, as described earlier
(21, 24) . One unit of activity is defined as the amount
of enzyme catalyzing the formation of 1 pmol of deoxyhypusine/h.
Step 1: Homogenization
Frozen rat testes were minced and
homogenized in a Waring Blendor in 60-g portions at a ratio of 1 g
(wet weight)/4.2 ml of ice-cold Buffer A (0.05
M Tris acetate,
pH 6.7, containing 1 m
M dithiothreitol and 0.1 m
M
EDTA) to which were added the protease inhibitors aprotinin (2
µg/ml) and leupeptin (0.5 µg/ml). The combined homogenate was
centrifuged at 30,000
g for 60 min, and the
supernatant solution was used for Step 2.
Step 2: Batch Adsorption on DE23
The enzyme
activity in the homogenate supernatant solution from 500 g of rat
testes was adsorbed in a batchwise fashion on
150-170 g
(damp weight) of DEAE-cellulose (Whatman DE23) that had been
equilibrated with Buffer A. The enzyme activity was eluted with
0.5-1.5 liters of 0.5
M KCl in Buffer A after two
stepwise washes with 2 liters of Buffer A and up to 1.3 liters of
Buffer A containing 0.1-0.2
M KCl. Portions of
100
ml were collected, and those with enzymatic activity were pooled.
Step 3: Ammonium Sulfate Fractionation
To the
combined active fractions from Step 2 was added 230 g/liter ammonium
sulfate (to 38% saturation). The precipitate obtained was discarded,
and ammonium sulfate at a level of 121 g/liter (to 55% saturation) was
added to the supernatant. The precipitated proteins were collected,
dissolved in 10-15 ml of Buffer A, and dialyzed against the same
buffer before assay.
Step 4: Ion-exchange Chromatography on
Q-Sepharose
The dialyzed fractions from two to five batches
after Step 3 were applied to a 2.5 14.4-cm (71 ml) column of
Q-Sepharose gel that had been equilibrated with Buffer A. The enzyme
activity was eluted with a 600-ml linear gradient of 0.1-0.6
M KCl in Buffer A, and the enzyme in the active fractions was
concentrated by ammonium sulfate precipitation by the addition of 351
g/liter ammonium sulfate and dialyzed versus Buffer A.
Step 5: Ion Exchange on Mono Q (Large Scale)
The
partially purified enzyme from Step 4 was applied to a Mono Q HR 10/10
column and eluted at 2 ml/min with a 120-ml linear gradient of
0.2-0.6
M KCl in Buffer A, pH 6.5, after a wash with 20
ml of Buffer A and 20 ml of 0.2
M KCl in Buffer A.
Step 6: Ion Exchange on Mono Q (Small Scale)
The
fractions containing enzyme activity from Step 5 were combined,
concentrated, and dialyzed as described above; applied to a Mono Q HR
5/5 column equilibrated with Buffer A, pH 6.5, 1 m
M EDTA; and
eluted with a 20-ml linear gradient of 0.1-0.6
M KCl in
Buffer A at a flow rate of 0.5 ml/min. Fractions of 0.5 ml were
collected. The fractions with the peak of enzyme activity (eluted in
0.25-0.3
M KCl) showed three to five prominent bands
upon SDS-PAGE. The enzyme purity was estimated as 25% in the peak
fractions.
Step 7: Chromatofocusing
Chromatofocusing was
carried out on a Mono P HR 5/20 column equilibrated with starting
buffer (0.025
M Bistris adjusted to pH 6.3 with iminodiacetic
acid). The pH gradient for elution was generated with Polybuffer 74,
diluted 10-fold according to the manufacturer's recommendations
(35) and adjusted to pH 3.8 with iminodiacetic acid. After
injection of the sample and washing with 5 ml of starting buffer,
elution was initiated with 100% elution buffer at a flow rate of 1
ml/min. Absorbance at 280 nm was monitored continuously; the pH was
determined on individual fractions. PAGE Small samples of the fractions from each of the chromatographic
procedures were analyzed by SDS-PAGE
(25) to monitor the
progress of the purification. Protein bands were visualized by staining
with Coomassie Blue R-250. Amino Acid Analysis and Sequence Determinations Protein (156 µg, 25% enzyme protein) from peak fractions after
Step 6 was subjected to electrophoresis in the presence of SDS on a
precast gel (10% in acrylamide), and the proteins were transferred to a
polyvinylidene difluoride (ProBlott) membrane by electrophoresis at 300
mA and 100 V for 1 h at 6 °C in CAPS. The membrane was stained
briefly with Coomassie Blue, and the band at
42 kDa was excised.
The excised portion was extensively destained in 50% MeOH and washed
with water. Portions of this washed membrane were used for ( a)
amino acid analysis after acid hydrolysis (kindly carried out by Lyuben
Marekov, NIAMS, National Institutes of Health), ( b) sequence
analysis of the intact protein (carried out at the University of
California Protein Structure Laboratory, Davis, CA), and ( c)
preparation and sequence analysis of tryptic peptides (carried out at
the Macromolecular Structure Facility, Department of Biochemistry,
Michigan State University, East Lansing, MI). Antibody Production Rabbit polyclonal antibodies to deoxyhypusine synthase were generated
from the 42-kDa protein isolated by SDS-PAGE of enzyme from a peak
fraction (
25% pure) after Step 6. The prominent stained band at 42
kDa was excised; the gel strip was washed in two changes of 50%
methanol, one change of 10% methanol, and equilibrated with water.
Protein electroeluted from the minced gel was used for immunization
(Assay Research, Inc., College Park, MD). The antibody produced was
titered against the enzyme by enzyme-linked immunosorbent assay, dot
blotting, and Western blotting techniques.
760-fold, to
1% purity) of
deoxyhypusine synthase from rat testis by ammonium sulfate
precipitation, ion-exchange chromatography, and gel filtration
(21) . In the present study, the protocol was modified to
include a rapid ion-exchange chromatographic step (on DE23) prior to
the ammonium sulfate fractionation in order to process a larger scale
preparation. Since size-exclusion chromatography on Sephadex G-200 gave
only a small enrichment
(21) , this step was omitted. Instead,
exploiting the strong affinity of the enzyme protein for
cation-exchange resins, we resorted to successive ion-exchange
chromatographic steps (Table II, Steps 4-6). At Step 6, the
synthase activity was eluted from a Mono Q HR 5/5 column in a single,
slightly asymmetric peak. In one preparation, the two peak fractions
showed specific activities of
200,000 units/mg of protein,
respectively, representing
20,000-fold enrichment and 25% purity.
Isoelectric focusing of this enzyme from Step 6 showed separation of
the enzyme from contaminating proteins (see below) and suggested a
means of further purification based on pI differences.
42 kDa.
Proteins that eluted at other points in the pH gradient were found to
be devoid of enzyme activity. In this chromatogram and in the preceding
step, it was apparent that the intensity of staining of the 42-kDa band
corresponded closely to the level of enzyme activity. Furthermore, the
two peak fractions from chromatofocusing, Fractions 42 and 43,
displayed a constant specific activity of
800,000 units/mg of
protein. The yield at this final step was low, probably due in part to
the inevitable losses incurred in processing a small amount of protein
and possibly to a partial loss of activity due to the low pH at the
elution point. Overall, a purification of
80,000-fold was achieved
with a recovery of
4%. From the data of , it can be
estimated that the enzyme represents only 0.0012% (w/w) of the total
protein of testis. Properties of Deoxyhypusine Synthase
Figure 1:
Chromatofocusing of deoxyhypusine synthase
from rat testis on Mono P HR 5/20. Approximately 240,000 units of
activity from Step 6 (see Table II) was applied in starting buffer
(0.025
M Bistris/iminoacetate, pH 6.26). The pH gradient was
established as described under ``Experimental Procedures''
with Polybuffer 74/iminoacetate, pH 3.78, at a flow rate of 1 ml/min.
Fractions of 1 ml were collected for 24 min, then of 0.5 ml for 10.5
min, and then of 1 ml for the final fractions. Total elution time was
60 min. A, comparison of enzymatic activity (),
absorbance at 280 nm (--), and the pH of individual
fractions (- - -). B, SDS-PAGE on a precast
gel (10% in acrylamide). Lane 1, molecular mass
standards; lane 2, starting material; lane
3, Fraction 40; lane 4, Fraction 42; lane 5,
Fraction 44; lane 6, Fraction 46; lane 7, Fraction
48; lane 8, Fraction 50; lane 9, Fraction 52;
lane 10, Fraction 54 (15 µl from each fraction). The
arrowhead marks the position of the 42-kDa
protein.
Molecular Mass: Identification of the 42-kDa Protein as a
Subunit of Deoxyhypusine Synthase
The observation that a protein
with an apparent molecular mass of 42 kDa on SDS-PAGE increased in
prominence in Steps 5-7 of the purification procedure in parallel
with the increase in enzyme activity was the first indication of
association of the enzyme with this specific protein. Further evidence
of this association was obtained by isoelectric focusing (Fig. 2).
Coomassie Blue staining of an isoelectric focusing gel of the enzyme
from Step 6 revealed several proteins (Fig. 2, lane 2), only one of which was found to possess deoxyhypusine
synthase activity. This enzymatically active protein of pI 4.7 migrated
as a 42-kDa protein upon subsequent SDS-PAGE (data not shown).
Figure 2:
Isoelectric focusing of deoxyhypusine
synthase. A, Coomassie Blue staining of standard proteins
(Pharmacia pI calibration kit, pI 3.50-8.15) ( lane 1)
and deoxyhypusine synthase (25% pure) ( lane 2); B,
enzyme activity () and pH (+) of individual gel slices after
elution. The top of the gel is at the left. Samples were mixed with an
equal volume of 2
sample buffer (80 m
M lysine, 30%
glycerol; Novex) and applied to a precast isoelectric focusing gel, pH
3-7; and the electrofocusing was conducted at 4 °C according
to the manufacturer's recommendations, using a cathode buffer of
40 m
M lysine and an anode buffer of 10 m
M phosphoric
acid, increasing the voltage in a stepwise fashion (100, 200, and 500
V) for a total time of 2 h, 40 min. Different amounts of deoxyhypusine
synthase (peak fraction from Mono Q column, Step 6) were run in
parallel lanes. Slices from one lane were extracted with Buffer A
containing 0.2
M KCl and bovine serum albumin (1 mg/ml) for
enzyme activity assay, while the other set was treated with SDS sample
buffer prior to SDS-PAGE. A blank lane was sliced into 0.5-cm pieces
and extracted in 10 m
M KCl (1 ml for 6 h at 25 °C) for pH
determination. Lanes 1 and 2 were fixed in a solution
containing 17.3 g of sulfosalicylic acid, 57.3 g of trichloroacetic
acid in 500 ml prior to staining with Coomassie
Blue.
In an
effort to identify this 42-kDa protein as a component of deoxyhypusine
synthase, a sample from the peak fraction from the Mono Q column (Step
6) was subjected to SDS-PAGE, and eluates of gel slices were assayed
for activity before and after a renaturation procedure (Fig. 3,
open and closed bars, respectively)
(30) .
Although no enzymatic activity was seen in eluates from the region of
the 42-kDa band before renaturation, a measurable enzymatic activity
was observed after renaturation of the protein from urea (Fig. 3,
open bars)
(30) . Surprisingly, even without
the renaturation procedure, a small, but clearly measurable, activity
was detected in extracts of gel slices from the region at 84 kDa
(Fig. 3, closed bars), even though there was no
visible protein staining at this position.
Figure 3:
Determination of the apparent molecular
mass of the subunit of deoxyhypusine synthase. A, shown is a
schematic diagram of the SDS-PAGE pattern of the migration of proteins
of known molecular mass (Novex Mark 12 wide-range standards) and the
three major bands of the partially purified enzyme preparation used.
B, the enzyme activity measured in eluates of gel slices
( before renaturation) is shown as closed
bars, and the activity of eluted samples of gel slices from a
separate lane after removal of SDS and renaturation from urea
( after renaturation) is shown as open bars.
The enzyme from Step 6 (25% pure) was mixed with an equal amount of 2
sample buffer (containing 2% SDS, but no reducing agents)
without heating and applied to a 10% precast gel. After
electrophoresis in Tris/glycine buffer containing 0.1% SDS, the gel was
rinsed with Buffer A; the lane of interest was cut into 2.5-mm slices;
and each crushed slice was eluted with 0.2
M KCl in Buffer A
(100 µl) for 16 h at 4 °C. Portions were taken for assay of
enzymatic activity, which represented a further dilution of SDS to
<0.02% ( closed bars). The extraction and renaturation of
proteins from gel slices from a separate lane were according to the
procedure of Weber and Kuter (30). The renatured samples were dialyzed
versus Buffer A, pH 7.22, to remove residual urea and assayed
for enzyme activity ( open bars). The recovery was 1.2
and 0.7% ( closed and open bars,
respectively) before and after the renaturation
procedure.
Electrophoresis of the
same material from Step 6 on a 6% acrylamide gel under nondenaturing
conditions showed that the enzyme activity migrated as a single band
close to the position of -galactosidase (molecular mass of 116.5
kDa). However, the protein eluted from this region of the nondenaturing
gel, when subjected to electrophoresis under denaturing conditions
(SDS-PAGE), moved to a 42-kDa position. Thus, the
42-kDa protein
appears to be a subunit of a larger enzyme. In this regard,
size-exclusion chromatography data obtained with earlier preparations
(21)
(
)
had indicated a molecular size
(160-180 kDa) for the active enzyme considerably greater than
that of the putative subunit.
/g) (I).
Assuming a monomer of 41,000 Da, the data fitted well to an equation
describing monomer to tetramer association with an association constant
of
10
, indicating a predominance of the tetramer
form.
Recognition of the Synthase by Polyclonal
Antibodies
Rabbit polyclonal antibodies raised against the
42-kDa protein were found to recognize purified deoxyhypusine synthase
by enzyme-linked immunosorbent assay and by Western blotting. Antisera
at 1:1000 dilution showed a positive response with 1 ng of the synthase
(data not shown). When allowed to react with the enzyme at 0-4
°C, the antibodies inhibited the enzyme activity by only 30%, but
did immunoprecipitate it in the presence of Protein A-liganded beads.
As shown in , the activity lost from the supernatant
solution was associated with the Protein A-liganded beads.
Nucleotide Specificity
The nucleotide specificity
and requirements of deoxyhypusine synthase were re-evaluated using the
purified enzyme (Fig. 4). Unlike a crude preparation of the
enzyme
(20) or Neurospora lysates
(32) , the
pure enzyme showed a strict specificity for NAD and was not stimulated
by NADP. At 100 µ
M or less NAD, some inhibition by added
NADH, FAD, or FMN could be demonstrated (data not shown). However, no
inhibition by NADP or NADPH was observed.
Figure 4:
Nucleotide requirement of pure
deoxyhypusine synthase. The assay was conducted as described under
``Experimental Procedures,'' except that NAD was replaced as
indicated. The pure enzyme (20 units of combined concentrated
Fractions 41-45; Fig. 1) was used.
pH Optimum
The pH optimum for the purified enzyme
of pH 9.5-9.6, determined in glycine/NaOH or CHES/NaOH buffer, is
essentially the same as that reported earlier
(18, 20, 21) .
Partial Reaction
The NAD-dependent cleavage of
spermidine by a partially purified deoxyhypusine synthase preparation
(1% purity) in the absence of the eIF-5A precursor protein was
reported earlier
(21) . Two products, 1,3-diaminopropane and
-pyrroline, were identified. Incubation of the pure
enzyme with spermidine and NAD in the absence of the eIF-5A precursor
protein also yielded the same two products, 1,3-diaminopropane and
-pyrroline. As observed earlier
(21) , the
partial reaction was completely dependent on the presence of NAD and
proceeded at a slower rate than that of the full reaction (data not
shown).
Amino Acid Composition and Partial Sequence
An
amino acid composition (with the exception of tryptophan and cysteine)
was estimated based on a Mof 40,000 as
Ala
, Arg
, Asx
, Gly
,
Glx
, His
, Ile
, Leu
,
Lys
, Met
, Phe
, Pro
,
Ser
, Thr
, Tyr
, and
Val
, for a total of 381 amino acids. Attempted Edman
degradation of the intact protein yielded no sequence data, consistent
with a blocked N-terminal amino acid. Internal tryptic peptides were
isolated, however, and the sequences obtained for five of these are as
follows: P1, Gly-Val-Asp-Tyr-His-Ala-Leu-Leu-Glu-Ala-Tyr-Gly-Thr; P2,
Glu-Ile-Asn-Asn-Pro-Glu-Ser-Val-Tyr-Tyr-Tyr-Ala-His; P3,
Asn-His-Ile-Pro-Val; P4, Asn-Pro-Gly-Leu-Val-Leu-Asp-Ile-Val-Glu-Glu;
and P5, Asn-Gly-Ala-Asp-Tyr-Ala-Val-Tyr-Ile-Asn-Thr-Ala-Gln-Glu-Gly.
None of these corresponded to any known protein sequence in the data
banks.
-pyrroline in conjunction with
the reduction of NAD (Fig. SI, dashed line)
(21) . Although both reactions appeared to have a similar
dependence on NAD and high pH, it was not clear whether spermidine
cleavage in the absence of the eIF-5A precursor was catalyzed by
deoxyhypusine synthase as an abortive partial reaction or whether it
was the result of the action of a different enzyme that copurified with
the synthase. Speculation that catalysis of the two reactions was
mediated through a common enzyme-spermidine intermediate
(Fig. SI) required the assumption that a single enzyme was
involved. The present findings strongly support the proposal that the
two reactions are catalyzed by a single enzyme through a common
intermediate, as shown in Fig. SI.
41,000 Da
and a pI of 4.7. Since no other protein bands of similar intensity were
detected after the purification (Fig. 1), it is unlikely that the
enzyme consists of different subunits. Several lines of evidence
support the association of deoxyhypusine synthase activity with a
protein composed of
42-kDa subunits. These include the correlation
of intensity of the 42-kDa protein band on SDS gels and enzyme activity
through steps of ion-exchange chromatography and chromatofocusing and
the detection of a 42-kDa monomer upon SDS-PAGE of enzymatically active
fractions after nondenaturing gel electrophoresis and after isoelectric
focusing. Consistent with the identity of the 42-kDa protein as a
subunit of deoxyhypusine synthase was the result of an
experiment
(
)
with 2-azido-NAD
(33) , a
photoaffinity probe for NAD-binding sites. Incubation of the pure
enzyme (Fraction 43; Fig. 1) with the
P-labeled
probe produced labeling of the 42-kDa band on an SDS gel, as expected
for the subunit of this NAD-binding protein. The most convincing
evidence, however, is the recovery of deoxyhypusine synthase activity
upon removal of SDS and renaturation of the protein extracted from the
42-kDa region of an SDS gel (Fig. 3) and the immunoprecipitation
of enzyme activity by rabbit antibodies raised against the 42-kDa
protein (). The detection of a small amount of enzyme
activity in the extract of a gel slice from the position of a
84
kDa protein after SDS-PAGE might be due to the activity of a dimer
per se or to its reassociation to a tetramer under the assay
conditions. Whether the dimer has intrinsic activity or whether it must
reassociate to a tetramer before it becomes functional cannot be
determined from the present data. The size-exclusion chromatography
suggests a tetrameric structure, and the molecular mass determined by
equilibrium centrifugation confirms the predominance of the tetramer
form of the native enzyme.
(
)
and
for cloning the human cDNA.
(
)
The availability of
pure recombinant enzyme should lead to a better understanding of its
complex reaction mechanism and of the physical structure of the binding
sites for its three substrates and to the design and development of
structure-based inhibitors of this unique modification reaction.
Table: Deoxyhypusine synthase activity in
rat tissues
10
µmol/min).
Table: Purification of deoxyhypusine synthase from
rat testis
Table: Physical properties of deoxyhypusine
synthase from rat testis
0.6
µg) after chromatofocusing. Ultracentrifugation of the pure enzyme
(
0.1 mg/ml) was conducted in 0.05
M Tris acetate, pH 6.3,
0.1 m
M EDTA, 0.2
M KCl (0.2-cm column height) at
15,000 rpm and 20 °C in a Beckman Model XLA ultracentrifuge. The
centrifugation was monitored by absorbance at 230 nm and was continued
to equilibrium as determined by a difference method (31).
Size-exclusion chromatography was carried out on three different
preparations (protein concentration in the peak area of enzyme activity
varied from 0.08 to 0.4 mg/ml (Ref. 21; E. C. Wolff, unpublished
observations)).
Table: Reactivity of enzyme protein with rabbit
polyclonal antibodies
g for 2 min. The supernatant solution was removed, and the
pelleted beads were washed twice with phosphate-buffered saline and
then resuspended in the enzyme assay mixture. Portions of the
enzyme/serum mixtures after preincubation and of the supernatant
solutions after Protein A precipitation were also assayed for enzyme
activity.
and PBE
, Rahms i Lund, Uppsala
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.