Hypusine formation on the eIF-5A (previously named eIF-4D)
precursor involves (i) the NAD
-dependent cleavage of
spermidine and the formation of deoxyhypusine (N
-(4-aminobutyl)lysine) (Chen and Dou, 1988)
and (ii) hydroxylation of the deoxyhypusine residue (Park et
al., 1984). The highly conserved nature of eIF-5A (Park et
al., 1993), the responsiveness to growth stimulation and the
specificity of hypusine formation (Cooper et al., 1982; Chen,
1983), together with the recognized importance of polyamines in growth
regulation (Cohen, 1971; Tabor and Tabor, 1984) suggest that hypusine
formation may have an important role in cell physiology. Disruption of
the two eIF-5A genes in yeast has been shown to be lethal (Schwelberger et al., 1993). Inhibition of deoxyhypusine synthase (
)in vivo affects the growth of Chinese hamster
ovary cells (Jakus et al., 1993). We have recently developed a
rapid assay method for deoxyhypusine synthase (Tao et al.,
1994). This assay has allowed us to explore various chromatographic
approaches. We found that deoxyhypusine synthase tenaciously binds to
1,12-diaminododecane-agarose and that the enzyme can be selectively
released by spermidine. This finding formed the basis for a simple
two-column procedure that enabled us to purify deoxyhypusine synthase
from Neurospora crassa to apparent homogeneity with good
yield.
EXPERIMENTAL PROCEDURES
Materials
Ni(II)-nitrilotriacetic
acid-agarose resins were purchased from Qiagen (Chatsworth, CA).
[1,8-
H]Spermidine (17.6 Ci/mmol) was obtained
from DuPont NEN. Nitrocellulose membrane (grade BA85) was purchased
from Schleicher & Schuell. NAD
, N-ethylmaleimide (NEM), (
)and iodoacetamide (IAM)
were obtained from Sigma. Except where noted, all chromatographic
matrices and non-ionic detergents were purchased from Sigma. All other
chemicals were of standard reagent grade. The polyhistidine-tagged Neurospora 21-kDa eIF-5A precursor, 6xHis-NC21K, was prepared
as described (Tao and Chen, 1994).
Growth of Cell Wall-less N. crassa
Mutant
The FGSC1118 strain cell wall-less N. crassa mutant cells (Scarborough, 1975) were grown in Vogel's N
medium supplemented with 2% (w/v) mannitol, 0.75% yeast extract, and
0.75% nutrient broth for 26-30 h at 30 °C with constant
shaking (150 rpm). Cells were harvested by centrifugation (4,000
g for 20 min).
Enzymatic Assay
The standard reaction
mixture, containing 1.2 µg of 6xHis-NC21K, 1 µCi of
[
H]spermidine (final concentration, 3
µM), 1 mM NAD
, and enzyme in 0.3 M glycine-NaOH buffer (pH 9.5) in a total volume of 30 µl,
was incubated at room temperature for various time periods as
indicated. The enzyme reaction was stopped by adding 60 µl of
phosphate buffer (0.5 M, pH 6.5) containing 10 mM spermidine. The labeled 6xHis-NC21K was absorbed onto the
Ni(II)-nitrilotriacetic acid-agarose and assayed with liquid
scintillation spectrometer (Tao et al., 1994).
Other Procedures
SDS-PAGE was carried out
as previously described (Dou and Chen, 1990). The native gel
electrophoresis was performed using precast mini-PROTEAN II ready gel
(4-20% gradient, Bio-Rad) under conditions as described in the
instruction manual. Silver staining was carried as described (Blum et al., 1987). Protein amount was determined by the Bradford
method(1976) using a Bio-Rad kit.
Purification of Deoxyhypusine
Synthase
Wall-less Neurospora cell pellets were
stored at -70 °C overnight, thawed, and suspended in buffer A
(10 mM phosphate, pH 7.0, 10% glycerol, 1 mM dithiothreitol, and 0.1 mM EDTA) containing 0.2
µM pepstatin A, 0.2 µM leupeptin, and 0.2
mM phenylmethylsulfonyl fluoride. All subsequent steps were
carried out on ice or at 4 °C. The mixture was centrifuged at 5,000
rpm for 25 min. The supernatant (
260 ml), containing about 5 g of
protein, was passed through one layer of cheesecloth and designated as
cell extracts. Ammonium sulfate was added to the cell extracts to 30%
saturation. The mixture was kept on ice for 1 h and then centrifuged at
10,000 rpm for 30 min. The supernatant was made to 60% saturation with
solid ammonium sulfate and was kept at 4 °C for 10 h. The mixture
was centrifuged at 10,000 rpm for 30 min. The pellet was dissolved in
buffer A for conventional column chromatography using hydroxyapatite
column, 1,8-diaminooctane-agarose column, Mono-Q, and Superose-6
columns.
Substrate Elution Affinity
Chromatography
The post-ammonium sulfate (60%) fraction
(0.6 g of protein) was dissolved in 40 ml of buffer B (10 mM phosphate, pH 7.75, 10% glycerol, 1 mM dithiothreitol,
and 0.1 mM EDTA) containing 0.1% Tween 20, mixed with 17.2 ml
of 40% PEG8000 (in buffer B), and incubated at 4 °C for 10 h. The
mixture was centrifuged at 14,000 rpm for 25 min. The supernatant (53
ml) was mixed again with 40% PEG8000 to a final PEG concentration of
30%. The mixture was kept in ice for 2 h and then centrifuged at 16,000
rpm for 25 min. The precipitate was dissolved in 20 ml of buffer B and
designated as post-PEG fraction. The following two columns were then
used. (i) In the 1,12-diaminododecyl-agarose (C12) column, the resin
(2.0 ml) was washed with water and equilibrated with buffer B
containing 0.1% Tween 20. The post-PEG fraction was loaded onto the C12
column (1.6
8.5 cm). The column was eluted first with 100 ml of
buffer B and then with 50 ml of 1 M NaCl in buffer B, followed
by 10 mM spermidine in buffer B (50 ml) containing 1 M NaCl. The fraction eluted by spermidine was concentrated from 50
to 2 ml with Centricon P30. (ii) In the Mono-Q column, the post-C12
sample (2 ml) was diluted in buffer B to about 6 ml and applied onto
the Mono-Q column (HR5/5), and the chromatogram was developed with the
following program: 0-4 min, 20 mM phosphate buffer (pH
7.75), 0.5 ml/min; 4-24 min, 0.05 M NaCl in buffer B,
0.5 ml/min; 24-84 min, 0.05-0.15 M NaCl in buffer
B, 0.5 ml/min. Deoxyhypusine synthase activity always appeared at
retention time (68-72 min).
Microsequencing of Deoxyhypusine Synthase
Protein
About 4 µg of purified deoxyhypusine synthase
was electrophoresed on one-dimensional SDS-polyacrylamide gel. The
protein band was transferred from the gel to nitrocellulose membrane
(0.45 µ, Schleicher & Schuell) in a 25 mM Tris buffer
containing 192 mM glycine and 20% v/v methanol for 15 h at 30
V. The blot was briefly stained with 0.1% Ponceau S (Fluka, Buch,
Switzerland) solution in 1% acetic acid and washed in 1% acetic acid.
The band was cut with a razor and stored wet at -20 °C in
Milli Q water. Sequence determination of trypsin-digested peptide
fragments was carried out in the Microchemistry Core Facility at
Memorial Sloan-Kettering Cancer Center by a procedure previously
described (Tempst et al., 1990).
RESULTS
Initial Purification and Identification of
Deoxyhypusine Synthase
The results of an initial
purification using conventional approach were summarized in Table 1. The post-Mono-Q fraction gave a specific activity of
39,800 units/mg of protein and represented a 15,000-fold purification
from cell extracts. This fraction exhibited two major protein bands on
SDS-gel (Fig. 1A, lane2). The use of
Superose 6 column resolved these two protein bands (Fig. 1A, lanes3-16). The peak
fractions containing the highest amount of deoxyhypusine synthase
activity in the Superose 6 chromatogram (Fig. 1B, fractions 7-9) exhibited only the
40-kDa protein
band (Fig. 1A, lanes 7-9), suggesting
that the
40-kDa protein band was derived from deoxyhypusine
synthase.
Figure 1:
Purification of deoxyhypusine synthase
by Superose 6 column chromatography. Post-Mono-Q fraction in method I
was loaded onto a Superose 6 (HR10/30) column. Fractions collected
(0.125 ml/fraction at a flow rate of 0.25 ml/min) by Superose 6 column
chromatography were used for SDS-PAGE (12% gel) analysis (A)
and assay for deoxyhypusine synthase activity (B). A,
SDS-PAGE analysis. Each lane contained 10 µl of sample
from each fraction. The protein bands were detected by silver staining. Lane1, post-1,8-diaminooctane-agarose fraction; lane2, post-Mono-Q fraction; lanes3-16, samples from fractions 3-16 collected
between 61.5 and 68 min. Lanes indicated by M are
molecular size markers. B, Superose 6 column chromatogram.
Fractions from 3 to 23 were collected for enzyme assay. Samples from
fraction 3 to 16 were also analyzed by SDS-PAGE. Sample in fractions
7-9 exhibited a single band at the 40-kDa position on
SDS-polyacrylamide gel (A, lanes7-9)
and contained the highest enzyme activity. The specific activity in
these fractions was about 80 units/µg of
protein.
Binding Properties of Deoxyhypusine
Synthase
We have examined the binding of the enzyme to 12
different sorbents, including various diaminohydrocarbons.
Deoxyhypusine synthase bound tightly to diaminodecane-agarose (C10) and
diaminododecane-agarose (C12) at a low salt concentration (data not
shown), suggesting that the amino group of C12 may also be involved in
the binding with deoxyhypusine synthase. The finding prompted us to
examine whether the enzyme can be released specifically from the C12
column by polyamines or diaminohydrocarbons. We found that spermidine
(10 mM), but not putrescine or spermine, specifically eluted a
40-kDa protein band as shown by SDS-PAGE. Although diaminodecane or
diaminododecane (10 mM each) could release a 40-kDa protein
band, they also eluted other major proteins (data not shown).
Purification of Deoxyhypusine Synthase by Affinity
Chromatography
The major protein eluted by spermidine from
the C12 column appeared to be the deoxyhypusine synthase. Fig. 2A shows the recovery of deoxyhypusine synthase
activity in the post-C12 fraction by employing the Mono-Q column. Fig. 2B indicates that the fractions containing high
deoxyhypusine synthase activity exhibited a single protein band with an
apparent molecular mass of 40 kDa on SDS-polyacrylamide gel. The
specific activity of the purified enzyme was about 130 units/µg of
protein, representing a 64,000-fold purification from cell extracts.
The purified enzyme exhibited a single protein band on a native gel (Fig. 3, lane3). All of the deoxyhypusine
synthase activity was found to be associated with this band, indicating
that this protein band was indeed deoxyhypusine synthase. We therefore
concluded that a combination of two columns, namely, the C12 and
Mono-Q, has led to the purification of deoxyhypusine synthase to an
apparent homogeneity. The results of a representative purification are
summarized in Table 2.
Figure 2:
A,
purification of deoxyhypusine synthase by C12 and Mono-Q column
chromatography. The post-C12 fraction eluted by spermidine was
concentrated to
2 ml (
40 µg/ml) and then loaded onto the
Mono-Q column. The chromatogram was developed by a program as described
under ``Experimental Procedures.'' The fractions collected
between 68 and 72 min contained pure deoxyhypusine synthase. B, SDS-PAGE analysis of purified Neurospora deoxyhypusine synthase. LaneM, protein standard
(68, 45, and 30 kDa as indicated); lane1, 1 µg
of post-Mono-Q fraction; lane2, 4 µg of
post-Mono-Q fraction; lane3, 0.5 µg of ovalbumin (OV); lane 4, 2 µg of ovalbumin; lane5, 4 µg of ovalbumin. The gel was stained by 0.1%
Ponceau S.
Figure 3:
Native gel electrophoresis of pure
deoxyhypusine synthase. The native gel was treated by silver staining. Lane1, protein standard (240, 67, and 45 kDa as
indicated); lane2, 240-kDa protein; lane3, post-Mono-Q fraction (0.5 µg of protein). An
identical post-Mono-Q sample was run on another lane for enzymatic
assay. The gellane was sliced (4 mm/slice) and
minced in the assay buffer. The enzyme activity in each slice was
measured using SDS-PAGE and fluorography as described before (Dou and
Chen, 1990).
Estimate of the Molecular Mass of the Native
Enzyme
Size exclusion chromatography using Superose 6
HR10/30 indicated that the native deoxyhypusine synthase had an
apparent molecular mass of
180 kDa (data not shown). However,
SDS-PAGE analysis of purified deoxyhypusine synthase revealed a single
band at 40 kDa (Fig. 2B). These results suggest that Neurospora deoxyhypusine synthase is likely a homotetramer.
Since a single protein was found to be associated with deoxyhypusine
synthase activity, we concluded that deoxyhypusine synthase is a single
multifunctional enzyme that is capable of catalyzing both the
spermidine dehydrogenation and the transfer of aminobutyl group to the
eIF-5A precursor protein.
Partial Amino Acid Sequences of Deoxyhypusine
Synthase
Four internal peptide fragments derived from the Neurospora deoxyhypusine synthase though in situ trypsin digestion were sequenced. The partial amino acid sequences
of these peptides are HVSLIVTTAGGIEEDSIK, NGAESAVYINTAQEFD,
NDIPVFCPALTDGWLGDMLK, and IGNLVVPNSNYCAFEDWVVPI. If deoxyhypusine
synthase subunit has a molecular mass of 40 kDa, the 75 amino acid
residues in these peptides represent approximately 25% total sequence
of the enzyme. The GenBank search indicated that these sequences did
not share any homology with other known proteins.
Hydrophobicity and Stability of Deoxyhypusine
Synthase
Deoxyhypusine synthase bound tightly to
phenyl-Sepharose column in the presence of high salt concentration (1 M Na
SO
in buffer B), suggesting the
presence of hydrophobic patches at the surface of the enzyme (data not
shown). Freeze-thawing of pure or partially purified enzyme, even in
the presence of 10% glycerol, caused a rapid loss of enzyme activity.
Non-ionic detergent such as Tween 20 was effective in maintaining the
enzyme activity against multiple freeze-thawing (data not shown). The
hydrophobic nature and the multisubunit structure of the protein may
contribute to the instability of the enzyme.
Protection Against the Inhibition of Sulfhydryl
Reagents by NAD
Fig. 4illustrates
the inhibitory effects of both NEM and IAM on purified deoxyhypusine
synthase. The results indicate a direct inactivation of the enzyme by
NEM and IAM. Fig. 4also shows that the inactivation could be
partially blocked by pre-incubation of the enzyme with
NAD
, suggesting the presence of cysteine residues at
or near NAD
binding site. Alternatively,
NAD
may induce global conformational change that
renders exposed cysteine group(s) cryptic.
Figure 4:
Protection of deoxyhypusine synthase from
the inhibition by sulfhydryl reagents. Deoxyhypusine synthase
(Post-Mono-Q fraction from method I) was mixed without or with
different concentrations of NAD
for 5 min, followed by
an incubation without (control) or with 10 mM of IAM
or 1 mM of NEM for another 30 min at room temperature. Enzyme
assays were then carried out as described under ``Experimental
Procedures.''
DISCUSSION
We described here for the first time the purification of
deoxyhypusine synthase from N. crassa to apparent homogeneity
as judged by SDS-PAGE. The initial purification using the conventional
chromatographic approach resulted in a 31,000-fold purification but
with only 0.25% yield (Table 1). In contrast, the two-columns
method is rapid and simple, and the recovery is good (Table 2).
From the degree of purification ( Table 1and Table 2), we
estimated the abundance of deoxyhypusine synthase in wall-less Neurospora mutant cells to be about 0.001% or less. The
abundance of 21-kDa eIF-5A, the modified substrate protein, in
wall-less mutants was estimated to be about 0.1% by immunostaining. (
)Gel filtration and SDS-PAGE analysis of purified
deoxyhypusine synthase suggest that the enzyme is a homotetrameric
protein consisting of four identical subunits.
The binding affinity
of deoxyhypusine synthase to diaminododecane resembles that of
putrescine oxidase from Micrococcus rubens and spermidine
dehydrogenase from Serratia marcescens (Okada et al.,
1979), suggesting that there may exist certain structural similarities
among these enzymes. Whether the hydrophobic nature of the enzyme may
affect its stability is not clear. We noticed, however, that during
purification the enzyme became less stable as the degree of
purification increased. The finding that non-ionic detergent such as
Tween 20 stabilizes the enzyme activity may suggest the intracellular
environment of the enzyme is also hydrophobic in nature. It is
noteworthy that spermidine dehydrogenase in Citrobacter freundii has been found in membranous fractions (Hisano et al.,
1992). The finding that deoxyhypusine synthase is sensitive to
sulfhydryl reagents (Fig. 4) indicates that cysteine residues
are required for enzymatic action. It also suggests the possibility
that the enzyme can be subjected to redox regulation. The finding that
NAD
can protect the enzyme from the inhibition
strongly suggests that the cofactor NAD
may cause some
conformational change of the enzyme. This is consistent with the
finding that the enzyme binds to its substrate protein only in the
presence of NAD
(Tao and Chen, 1994).