©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Deoxyhypusine Synthase from Rat Testis: Purification and Characterization (*)

Edith C. Wolff (§) , Young Bok Lee , Soo Il Chung , J. E. Folk , Myung Hee Park

From the (1) Enzyme Chemistry Section, Laboratory of Cellular Development and Oncology, National Institute of Dental Research, NIH, Bethesda, Maryland 20892-4330

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 -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.


INTRODUCTION

The unusual amino acid hypusine ( N-(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 (Lysin 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).

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.


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.




EXPERIMENTAL PROCEDURES

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-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 iminodiacetateNaHO were from Aldrich; CHES was from U. S. Biochemical Corp.; DEAE-cellulose (DE23) was from Whatman; Q-Sepharose Fast Flow gel, Mono QHR 10/10 and Mono QHR 5/5 prepacked ion-exchange columns, Mono PHR 5/20 chromatofocusing columns, Polybuffer74, 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 ProBlottmembranes 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.

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.

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.


RESULTS

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 (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.

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 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.

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/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.


DISCUSSION

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 -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.

The active form of deoxyhypusine synthase catalyzing this complex reaction is probably a tetramer of a protein with a monomer molecular mass of 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.

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() 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

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 10µmol/min).


  
Table: Purification of deoxyhypusine synthase from rat testis

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.


  
Table: Physical properties of deoxyhypusine synthase from rat testis

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 (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

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 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.



FOOTNOTES

*
A preliminary account of a portion of this work has been presented at the Satellite Meeting on Protein Structure, Function, and Engineering at the Bose Institute, Calcutta, India on September 17, 1994 as part of the XVIth Congress of the International Union of Biochemistry and Molecular Biology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: NIDR, NIH, Bldg. 30, Rm. 211, 30 Convent Dr., MSC 4330, Bethesda, MD 20892-4330. Tel.: 301-496-5056; Fax: 301-402-0823.

The abbreviations used are: eIF-5A, eukaryotic translation initiation factor 5A; ec-eIF-5A, the precursor of eIF-5A (containing lysine in place of hypusine) expressed in E. coli from a human cDNA; Bistris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; CHES, 2-(cyclohexylamino)ethanesulfonic acid; CAPS, 3-(cyclohexylamino)-1-propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis.

E. C. Wolff, unpublished observations.

Preliminary experiment carried out by Curt Pendergrass in the laboratory of Dr. Boyd Haley (University of Kentucky).

K. R. Kang, E. C. Wolff, M. H. Park, J. E. Folk, and S. I. Chung, submitted for publication.

Y. A. Joe, E. C. Wolff, and M. H. Park, manuscript in preparation.


ACKNOWLEDGEMENTS

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).


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