From the Istituti di Biochimica e
§ Biologia e Genetica, Facoltà di Medicina e
Chirurgia, University of Ancona, via Ranieri,
60100 Ancona, Italy
Received for publication, September 22, 2000, and in revised form, October 4, 2000
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ABSTRACT |
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A 1329-base pair clone isolated from a human
placenta cDNA library contains a full-length 837-base pair coding
region for a 31.9-kDa protein whose deduced primary structure exhibits
high homology to consensus sequences found in other NMN
adenylyltransferases. Northern blotting detected a major 3.1-kilobase
mRNA transcript as well as a minor 4.1-kilobase transcript in all
human tissues examined. In several cancer cell lines, lower levels of
mRNA expression were clearly evident. The gene encoding the human
enzyme was mapped to chromosome band 1p32-35. High efficiency
bacterial expression yielded 1.5 mg of recombinant enzyme/liter of
culture medium. The molecular and kinetic properties of recombinant
human NMN adenylyltransferase provide new directions for investigating
metabolic pathways involving this enzyme.
Following genomic damage by various chemical or radiation
treatments, the extent of malignant transformation is thought to be
limited by enhanced DNA repair. Such repair is accomplished by
activation of different metabolic processes, some involving NAD+. This indicates that the pyridine coenzyme
NAD+ plays significant roles beyond its participation in
redox metabolism and in various types of ADP-ribosylation. The cellular
NAD+ concentration likewise appears to modulate expression
of stress response proteins, including the tumor suppressor protein
p53. In fact, high levels of pyridine dinucleotide reduce the time required for cellular rescue from DNA damage. Skin biopsy specimens from actinic keratoses and squamous cell carcinomas also exhibit an
inverse relationship between skin NAD+ content and the
severity of the malignant phenotype (1). In view of this role of
NAD+ in cellular rescue from DNA damage, a better
understanding of how human NAD+ biosynthesis is regulated
may be of vital importance in developing effective approaches for
preventing and treating cancer.
For many years, we have studied NMN adenylyltransferases
(NMNATs),1 which play central
roles in both de novo biosynthetic and salvage pathways for
nicotinamide nucleotides. These enzymes convert NMN (or nicotinic acid
mononucleotide) and ATP to NAD+ (or nicotinic acid
adenine dinucleotide) and inorganic pyrophosphate (2), and their
activity has been correlated with crucial cellular events such as
mitosis and DNA synthesis (3, 4). In prokaryotes, cell survival and
viability appear to require NMNAT activity (5). Because tumor cell
NMNAT concentrations are very low, the enzyme represents a potential
chemotherapeutic target (6, 7). This adenylyltransferase catalyzes the
essential last step in the metabolic conversion of the potent antitumor
agent tiazofurin from its prodrug form to tiazofurin adenine
dinucleotide. Low tumor levels of this enzyme activity are associated
with the development of drug resistance (8, 9).
Our laboratory has purified and characterized NMN
adenylyltransferases from yeast, bull testis, thermophilic
bacteria, and human placenta (10-13). We have also identified, cloned,
and expressed the gene for this enzyme from Methanococcus
jannaschii and Saccharomyces cerevisiae (12, 14), as
well as from Synechocystis sp. (slr0787). The bifunctional slr0787 protein is endowed with both nudix
hydrolase and NMNAT activities; and in Escherichia coli, the
NAD+ biosynthesis regulatory protein NadR possesses NMNAT
activity (15, 16). As part of an ongoing study on the role of this enzyme in regulating NAD+ levels, we successfully isolated
a full-length cDNA encoding NMNAT from a human placenta cDNA
library. In this report, we describe human NMNAT cDNA cloning and
its tissue-specific expression, levels in tumor cells, and chromosomal
location. We also describe the high efficiency expression and
purification of the protein in E. coli as well as its
kinetic properties and metal ion effects.
Materials
We purchased a human placenta cDNA library constructed in
Methods
Human placental NMNAT was purified (13) and subjected to tryptic
digestion (17). The resulting fragments were separated by reverse-phase
liquid chromatography on an ABI 173A Capillary LC/Microblotter system.
After spotting recombinant human NMNAT on a polyvinylidene difluoride
membrane, Edman sequencing was performed on an Applied Biosystems
Procise Model 491 sequencer.
Identification and Isolation of Full-length cDNA for Human
NMNAT--
BLAST searches (18), conducted with NMNAT peptides
(YLVPDLVQEYIEK and NAGVILAPLQR) as query sequences, revealed a full
match with the predicted amino acid sequence of an expressed sequence tag cDNA (GenBankTM/EBI accession number AA307717).
This clone, containing a 342-bp cDNA insert, was used to design PCR
primers for the cloning of human NMNAT cDNA. We first employed PCR
using a human placenta cDNA 5'-stretch
The 5'-part of the human NMNAT cDNA was isolated by two consecutive
PCRs. A primary PCR was performed with the Construction of Expression Vector and Expression in E. coli--
To prepare a vector suitable for expression of recombinant
NMNAT in E. coli, we first generated an 840-bp DNA fragment
containing the coding sequence for NMNAT by PCR of the aforementioned
cDNA library using primers HF
(5'-CGGGGATTCATGGAAAATTCCGAGAAGACT-3') and HR
(5'-GCAGTCGACCTATGTCTTAGCTTCTGCAGT-3'). Primer HF contains a
BamHI site followed by the beginning of the open reading
frame; primer HR contains the end of the open reading frame followed by
a SalI cleavage site. PCR (1 min of denaturation at
95 °C, 1 min of annealing at 50 °C, and 1 min of elongation at
72 °C) was performed for 35 cycles with 20 pmol of each primer in a
final volume of 100 µl. After electrophoresis on a 1% agarose gel,
the amplified DNA, visible by ethidium bromide staining, was digested with BamHI and SalI and cloned into
BamHI-SalI-digested pT7-7 plasmid vector (19) to
obtain the construct pT7-7-HAT. The insert was sequenced to ascertain
that no mutations had been introduced during amplification. The
construct was used to transform E. coli TOP10 (Invitrogen)
for plasmid preparation and E. coli BL21(DE3) for protein expression.
Growth and Expression of Recombinant Human
NMNAT--
Transformed BL21(DE3) cells were grown at 37 °C to an
A600 of 0.6 in LB medium (50 ml) containing 100 µg/ml ampicillin. A 10-ml portion was used to inoculate 1 liter fresh
LB medium containing 100 µg/ml ampicillin, and the mixture was
incubated overnight at 120 rpm at 37 °C. Protein production was then
induced by 0.4 mM
isopropyl- Purification of Recombinant Human NMNAT--
All steps were
performed at 4 °C. The cell pellet was suspended in 60 ml of 100 mM Tris-HCl (pH 7.4) containing 0.5 mM EDTA, 1 mM MgCl2, and 1 mM dithiothreitol
(buffer A) and disrupted by sonication. The lysate was centrifuged at
15,000 × g for 30 min (crude extract). The crude
extract was applied to a Matrex Gel Green A chromatography
column, previously equilibrated with buffer A. The column was washed
with the same buffer containing 0.5 M NaCl and eluted with
a linear NaCl gradient (0.5-2.5 M) in the equilibration
buffer (Green A fraction). After adding NaCl to a final concentration
of 3 M, the Green A fraction was loaded onto a
phenyl-Sepharose column, equilibrated with buffer A containing 3 M NaCl. The column was washed with buffer A containing 2 M NaCl and then eluted with a linear NaCl gradient (2 to 0 M in buffer A). The active pool was concentrated through an
Amicon YM-30 membrane (phenyl-Sepharose fraction). This fraction
was injected onto a Superose 12 HR 10/30 fast protein liquid
chromatography column (Amersham Pharmacia Biotech), previously
equilibrated with 50 mM Tris-HCl (pH 7.4) containing 0.5 mM EDTA, 1 mM MgCl2, 1 mM dithiothreitol, and 0.5 M NaCl. Active
fractions were analyzed by SDS-polyacrylamide gel electrophoresis as
described below. Homogeneous fractions were pooled and used to
determine substrate specificity and other catalytic properties.
Enzyme Activity Assays and Kinetic Characterization--
Enzyme
activity was measured continuously by a coupled spectrophotometric
assay (20) or by HPLC (20). Optimal reaction conditions were
established by varying divalent cation concentration and buffer pH (30 mM BisTris-HCl and 30 mM Tris-HCl adjusted to the desired pH). One enzyme unit is defined as the amount that catalyzes formation of 1 µmol of NAD+/min at
37 °C.
Chromosomal Mapping--
Human metaphase spreads were obtained
from phytohemagglutinin-stimulated peripheral lymphocytes of a
normal donor by standard procedures. Chromosome preparations were
hybridized in situ with biotinylated probes (full-length
human NMNAT cDNA) labeled by nick translation essentially as
described by Lichter et al. (21). Labeled probe (500 ng) was
used for fluorescence in situ hybridization at 37 °C in
2× SSC, 50% formamide, 10% dextran sulfate, 5 µg of COT1 DNA, and
3 µg of sonicated salmon sperm DNA (10-µl volume). Post-hybridization washing was carried out at 42 °C in 2× SSC and
50% formamide (three times), followed by three washes in 0.1× SSC at 60 °C. Biotin-labeled DNA was detected with Cy3-conjugated avidin. Chromosome identification was obtained by simultaneous 4,6-diamidino-2-phenylindole staining to produce the characteristic Q-banding patterns.
Northern Blot Analysis--
Blots containing 2 µg of
poly(A)+ RNA from different human tissues and cancer cell
lines were prehybridized at 68 °C for 1 h in
ExpressHybTM hybridization solution. The filters were then
hybridized with the 32P-labeled cDNA probe (nucleotides
89-928) at 68 °C for 16 h. After washing as recommended by the
manufacturer, blots were exposed to x-ray films at Southern Blot Analysis--
Human genomic DNA (10 µg) from
human peripheral white blood cells was digested with ApaI,
BamHI, EcoRI, PstI, or
PvuII. DNA fragments were resolved on a 1% agarose gel and
transferred to a nylon membrane by the method of Southern (22). A
118-bp cDNA fragment (nucleotides 800-917) was amplified with
primers HATF and HATR using the pT7-7-HAT construct as a template and
gel-purified. This fragment, radiolabeled with
[
Gel electrophoresis of recombinant human NMNAT was carried out by the
Laemmli method (23) on 15% polyacrylamide gel. Gel filtration of
recombinant human NMNAT was performed by fast protein liquid
chromatography on a Superose 12 HR 10/30 column equilibrated with 50 mM Tris-HCl (pH 7.4) containing 0.5 M NaCl, 1 mM dithiothreitol, 1 mM MgCl2, and
0.5 mM EDTA. Protein concentration was determined by the
Bradford method (24).
Isolation and Characterization of Human NMNAT cDNA--
Using
two tryptic peptide sequences from human placental NMNAT, we conducted
BLAST searches of the human expressed sequence tag data base. We
identified a cDNA clone (GenBankTM/EBI accession number
AA307717) encoding both sequences. The insert of this clone was too
short to account for the molecular mass of NMNAT (13), and we used the
clone to design specific primers for PCR experiments utilizing a human
placenta cDNA library. A scheme of our cloning strategy is shown in
Fig. 1. The composite sequence obtained
by assembling the products of PCR experiments resulted in a 1329-bp
transcript consisting of an 88-bp 5'-untranslated region, an 837-bp
coding region, and a 404-bp 3'-untranslated region with an incomplete
AATAA polyadenylation signal (Fig. 2, upper panel). The translational initiation site (ATG),
assigned to the first methionine codon (nucleotides 89-91), is in the
favorable initiation context ACCATGG, thus fulfilling Kozak's criteria
for initiation (25). An in-frame translational termination codon (TAG)
occurs after nucleotide 925. The open reading frame encodes a 279-amino
acid protein with a molecular mass of 31,900 Da, close to that
estimated by SDS-polyacrylamide gel electrophoresis (13). When the
sequences of both tryptic peptides were aligned with the deduced amino
acid sequence, there was a complete match (Fig. 2), confirming that the
cloned cDNA codes for NMNAT.
The hydrophobicity profile (26) of the predicted protein (Fig. 2,
lower panel) suggests the existence of several potential transmembrane regions, in agreement with the observation that NMNAT is
located at the inner nuclear membrane level (27). As previously
reported (28), Reinhardt's method (34) for cytoplasmic/nuclear discrimination also suggests a nuclear localization. When the deduced
amino acid sequence of NMNAT was examined for a number of structural
motifs, we identified a single potential Asn-linked glycosylation site
(36NGTG39) in the N-terminal region. To provide
additional evidence that the isolated cDNA encodes NMNAT, we
compared the deduced amino acid sequence with sequences of other known
NMNATs. The human enzyme was 40% identical to the yeast enzyme and
34 and 33% identical to the two putative Caenorhabditis
elegans homologs, respectively (14). There was very little
similarity with respect to the prokaryotic counterparts (12, 15, 16).
In addition, we used the ClustalW program to obtain an optimized
multiple sequence alignment of known NMNATs, including the NadR protein
recently demonstrated to exhibit NMNAT activity (16). All proteins
possess the sequence (H/T)XXH (Fig.
3), which is very similar to the motif
implicated in the Expression of Human NMNAT cDNA in E. coli and Purification of
the Recombinant Protein--
To confirm that the isolated cDNA
encodes NMNAT, we developed a bacterial expression system. An 840-bp
fragment containing the complete coding sequence was PCR-amplified and
cloned in the polylinker region of the expression vector pT7-7 (see
"Experimental Procedures"). After nucleotide sequencing of the
coding insert, the resulting plasmid pT7-7-HAT was used to transform
E. coli BL21(DE3). Transformed bacteria were induced by
treatment with isopropyl-
NMNAT was isolated by three chromatographic steps (Fig. 4, lanes
4-6; and Table I). The
purified recombinant protein appeared as a single band with a molecular
mass of 33 kDa, agreeing with the value calculated from the deduced
sequence (Fig. 4, lane 6). The N-terminal sequence of
the first 18 residues of the recombinant protein exactly matched the
deduced sequence. The native molecular mass of the active recombinant
enzyme was determined by gel filtration to be 139 kDa. These data
indicate that the recombinant enzyme is an oligomer of four identical
subunits, agreeing with previous experiments on the wild-type placental
enzyme (13).
Chromosomal Localization of the Human NMNAT Gene--
We were
interested in investigating the chromosomal localization of the NMNAT
gene to ascertain if it corresponded to the position of any known
disease locus and to obtain insights regarding the genomic organization
of the NMNAT locus. A fluorescent cDNA probe was used for in
situ hybridization on metaphase chromosome spreads. The analysis
showed that the NMNAT gene is located on chromosome 1p32-35 (Fig.
5). The gene for the closely related enzyme poly(ADP-ribose) polymerase is located on the same chromosome, albeit in a different region (1q41-42) (30). Genes for other enzymes
in the NAD+ metabolic pathway map to different chromosomes
(31, 32).
Expression of NMNAT in Human Tissues and Cancer Cell Lines--
To
evaluate the distribution of NMNAT mRNA, Northern blot analyses
were performed with mRNAs from various human tissues. Two messages
of ~3.1 and 4.1 kilobases, respectively, were detected with variable
intensity in all examined tissues. The 3.1-kilobase mRNA was
considerably more abundant (Fig.
6A). The major sites of NMNAT
expression were skeletal muscle, heart, liver, and kidney, whereas
organs such as thymus and spleen showed a very weak signal. The
widespread distribution of NMNAT in human tissues is consistent with
its essential role in cellular metabolism; with no alternative biosynthetic routes for NAD synthesis, this enzyme is vitally important. However, the wide variability of NMNAT expression suggests that, in addition to its housekeeping role in pyridine metabolism, NMNAT may play a more specific role in those tissues, such as skeletal
muscle, where its level is substantially higher.
Further analysis of NMNAT mRNA expression in a panel of cancer cell
lines indicated that, with the exception of Burkitt's lymphoma and
chronic myelogenous leukemia, the enzyme is expressed at relatively low
quantities in tumor cells (Fig. 6B). This result agrees with
a previous report concerning reduced NMNAT activity in cells after
malignant transformation (5).
As reported above, two messages of different size were detected in all
examined tissues. The library screening and the chromosomal localization suggest that both signals arise from the NMNAT locus since
there were no data indicating the existence of a highly related gene
that (cross-)hybridizes with the NMNAT probe. Currently, it is not
known whether the bands of different size are alternatively spliced
mRNAs from the NMNAT locus or if the larger transcript represents a
mRNA retaining intron sequences.
Southern Blot Analysis--
To estimate the copy number of the
NMNAT gene, genomic DNA isolated from human peripheral white blood
cells was digested by one of five restriction enzymes with 6-base
recognition sequences: ApaI, BamHI,
EcoRI, PstI, or PvuII. The genomic
Southern blot prepared from these digests shows a single band in all
lines (Fig. 7), suggesting that the NMNAT
gene exists as a single copy/haploid in humans. This result agrees with
the results of fluorescence in situ hybridization
experiments, indicating that there are no other closely related loci in
the human genome.
Catalytic Properties of Recombinant Human NMNAT--
Because the
functional properties of recombinant human NMNAT are of fundamental
importance, we performed a detailed kinetic characterization. The
activity profile of recombinant NMNAT at different pH values using 30 mM BisTris-HCl and 30 mM Tris-HCl buffer
mixtures was determined. Like other NMNATs (10-13), recombinant human
NMNAT exhibited a broad pH optimum, ranging from pH 6.0 to 8.0. As
observed with the enzyme purified from other sources (10-12, 15),
recombinant human NMNAT absolutely required divalent cations. Optimal
activity occurred in the presence of 12 mM
Mg2+, but other metal ions can replace magnesium (Table
II). Noteworthy is the very low
Kapp value of the enzyme for Zn2+.
The results reported above seem to indicate a similarity, with respect
to the cation requirement, between human NMNAT and its thermophilic
counterpart, whereas the yeast recombinant enzyme exhibits maximal
activity with Ni2+ (14, 33). As described previously for
the native enzyme (13), the activity of recombinant human NMNAT was
very depressed by several heavy metal ions, including Hg2+,
Cd2+, Cu2+, and Cr3+, when present
in the assay mixture at a concentration of 250 µM.
Recombinant human NMNAT exhibited linear kinetics with respect to NMN
and ATP; the Michaelis constants for these reactants, reported in Table
III, as well as the substrate
inhibition exerted by ATP, especially at low levels of NMN, are in good
agreement with the catalytic properties described previously for the
native enzyme (13). When deamido-NMN was used as the substrate, the reaction occurred at a comparable rate with respect to that measured in
the presence of NMN; however, the higher Km value calculated for nicotinic acid mononucleotide, with respect to NMN, suggests that, in human as well as in other eukaryotes, the amido
pathway is predominant (10). Table III shows that the human enzyme can
replace ATP with its deoxy form, even though with a considerably lower
reaction rate, as described previously for the yeast enzyme (14).
The results reported above clearly indicate that the recombinant enzyme
possesses a kinetic behavior indistinguishable from that of the native
form. Therefore, the availability of the human cDNA encoding NMNAT
should allow more detailed studies on the function and structure of
this enzyme. In this regard, a further investigation on the specificity
exhibited by recombinant human NMNAT will be helpful to expand our view
of metabolic pathways that could involve NMNAT and to evaluate its
functional role in human cellular homeostasis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
gt11 as well as blots containing poly(A)+ RNAs from
human tissues and cancer cell lines from CLONTECH
(Palo Alto, CA). Restriction endonucleases and other cloning reagents were purchased from New England Biolabs, Inc. (Beverly, MA) or Promega
(Madison, WI). Double-stranded DNA probes were radiolabeled with
[
-32P]dCTP (3000 Ci/mmol) from Amersham Pharmacia
Biotech (Buckinghamshire, United Kingdom) using a commercial random
priming kit (Amersham Pharmacia Biotech, Uppsala, Sweden). All other
reagent-grade chemicals were obtained from standard suppliers.
gt11 library and primers
based on the expressed sequence tag sequence (HATF,
5'-TACTTGGTACCAGATCTTGTCC-3'; and HATR, 5'-CTTCTGCAGTGTTTCTCTGCAA-3').
PCR was carried out in a GeneAmp PCR system 2400 (PerkinElmer Life
Sciences) for 35 cycles of denaturation (95 °C, 30 s annealing
(60 °C, 1 min), and extension (72 °C, 1 min). The resulting
118-bp fragment was sequenced to confirm the presence of NMNAT cDNA
(see Fig. 1, sequence B, nucleotides 800-917).
gt11-specific primer V1
(5'-GAGCTCACACCAGACCAACTGGTAATG-3') in combination with primer HATR
directed upstream of sequence B (5'-CTTCTGCAGTGTTTCTCTGCAA-3'). To increase the specificity, the product of the first round of PCR was
diluted 1:10 in water, and 1 µl was used as a template in a second
round of PCR using the nested
gt11-specific primer V2
(5'-CAACTGGTAATGGTAGCG-3') and the nested specific primer HATRN (5'-GGACAAGATCTGGTACCAAGTA-3'). This final combination yielded an
821-bp DNA fragment, which was cloned into the pGEM-T vector by T-A
ligation for transformation of E. coli JM109 and sequenced on both strands (see Fig. 1, sequence A, nucleotides
1-821). Primers HATF and HATFN (5'-TTGCAGAGAAACACTGCAGAAG-3'),
in combination with the
gt11-specific primers V1 and V2, were used
to isolate the 3'-part of the cDNA sequence. The product of the
second round of PCR was a 434-bp fragment, which was cloned into the
pGEM vector and sequenced (see Fig. 1, sequence C,
nucleotides 896-1329).
-D-thiogalactopyranoside. After a 3-h
induction at 37 °C, cells were harvested by centrifugation
(10,000 × g for 10 min at 4 °C) and either used for
purification or stored at
80 °C.
80 °C with an
intensifying screen for 4 days. RNA integrity and loading were assessed
with an actin probe.
-32P]dCTP (3000 Ci/mmol) using the commercial
random priming kit, was used as a probe. Hybridization was performed
overnight at 45 °C in ULTRAhyb buffer (Ambion Inc.). The filter was
then washed and exposed for 48 h to x-ray film at
80 °C.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Schematic representation of the cloning and
sequencing strategy. The composite sequence of the human NMNAT
cDNA is shown schematically at the top. Sequences A-C
represent PCR-amplified fragments corresponding to different parts of
the full-length sequence. The PCR primers and their positions in the
full-length cDNA are indicated. See "Experimental Procedures"
for details.
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Fig. 2.
Nucleotide and deduced amino acid sequences
of cDNA encoding human NMNAT. In the upper panel,
the numbers on the right refer to nucleotide and amino acid
positions. The sequences determined for tryptic fragments of NMNAT from
human placenta are underlined. The potential Asn-linked
glycosylation site is marked with an asterisk. In the
lower panel, the hydrophobicity profile for the translation
of the cDNA sequence is shown. The analysis was performed according
to Kyte and Doolittle (26). The hydropathicity value of each amino acid
residue is plotted against its position in the polypeptide
(x axis), starting with the amino terminus.
/
-phosphodiesterase activity of
nucleotidyltransferases (29). Furthermore, this motif appears to be a
part of the conserved sequence
GXFXPX(T/H)XXH, which may
represent a novel NMNAT motif. This region may constitute part of the
enzyme's active center, but site-directed mutagenesis is needed to
evaluate the role of conserved sequences in NMNAT catalysis. These
efforts will be augmented by ongoing x-ray diffraction studies to
obtain the enzyme's three-dimensional structure.
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Fig. 3.
Comparison of the amino acid sequence of
human NMNAT with those of other homologous proteins. Shown is an
optimized multiple sequence alignment created using the ClustalW
program. Protein sequences included in the comparison are the
translation of the human NMNAT cDNA (Homo sapiens), the
two putative C. elegans homologs, the S. cerevisiae and M. jannaschii homologs, the
Synechocystis sp. slr0787 bifunctional protein,
and E. coli NadR. Asterisks indicate identical
amino acids; double and single dots indicate
strong and weak conserved amino acid substitutions, respectively. Gaps
introduced to optimize the alignment are indicated by
dashes. Boxed residues represent the novel
putative consensus sequence.
-D-thiogalactopyranoside to
produce the recombinant enzyme. Protein extracts were prepared from the
induced bacteria and assayed for NMNAT activity. Even without
isopropyl-
-D-thiogalactopyranoside, high NMNAT activity
could be detected in BL21 cells transformed with the recombinant
plasmid, whereas BL21 cells containing unmodified plasmid showed no
detectable enzyme activity. SDS-polyacrylamide gel electrophoresis
demonstrated that bacteria transformed with the recombinant plasmid
contained a polypeptide of the expected size, whereas crude extracts of
bacteria lacking human NMNAT-coding sequence did not (Fig.
4).
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Fig. 4.
Expression and purification of recombinant
human NMNAT. A 15% polyacrylamide gel containing 0.1% SDS was
stained with Coomassie Blue. Lane 1, reference proteins;
lanes 2 and 3, E. coli BL21 containing
the expression vector (10 µg) without and with the coding sequence,
respectively; lane 4, Green A fraction (10 µg); lane
5, phenyl-Sepharose fraction (10 µg); lane 6, 4 µg
of purified recombinant NMNAT.
Purification of recombinant human NMNAT
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Fig. 5.
Chromosomal localization of the human NMNAT
gene by fluorescence in situ hybridization. Shown
is a photograph of human metaphase chromosomes counterstained with
4,6-diamidino-2-phenylindole. Arrows point to the site of
hybridization of the biotin-labeled human cDNA probe on both copies
of chromosome 1 at p32-35.
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Fig. 6.
Expression of the NMNAT gene in human tissues
and cancer cell lines. A, 2 µg of
poly(A)+ RNA prepared from the indicated tissues were
analyzed by Northern blot hybridization with a radiolabeled probe
corresponding to the coding region of human NMNAT cDNA. The
positions of RNA markers are shown. Filters were subsequently
hybridized with a human actin probe to ascertain the differences in RNA
loading among the different samples. B, 2 µg of
poly(A)+ RNA prepared from the indicated tumor cells were
hybridized with the probe described for A. Filters were
finally hybridized with a human actin probe. kb,
kilobases.
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Fig. 7.
Southern blot analysis. Human genomic
DNA (10 µg) was digested with ApaI, BamHI,
EcoRI, PstI, or PvuII. The sizes (in
base pairs) of DNA markers are indicated. The fragments were separated
on a 1% agarose gel, blotted, and hybridized with a 118-bp
radiolabeled cDNA probe (nucleotides 800-917).
Requirement of divalent cations for NMNAT activity
Kinetic parameters of purified recombinant NMNAT
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge Prof. Mariano Rocchi (Institute of Genetics, University of Bari, Bari, Italy) for help with the chromosomal localization and Dr. Marta Menegazzi (Department of Neuroscience and Vision, Laboratory of Biological Chemistry, University of Verona, Verona, Italy) for valuable assistance in Northern blot experiments.
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FOOTNOTES |
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* This work was supported in part by Consiglio Nazionale delle Ricerche Target Project "Biotechnology" and by Cofinanziamento Ministero dell'Università e della Ricerca Scientifica e Tecnologica "Nucleotidi e Nucleosidi: Segnali Chimici, Regolatori Metabolici e Potenziali Farmaci."The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF312734.
¶ To whom correspondence should be addressed. Tel.: 390-71-2204678; Fax: 390-71-2802117; E-mail: magnig@popcsi.unian.it.
Published, JBC Papers in Press, October 10, 2000, DOI 10.1074/jbc.M008700200
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ABBREVIATIONS |
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The abbreviations used are: NMNAT, NMN adenylyltransferase; bp, base pair; PCR, polymerase chain reaction; HPLC, high performance liquid chromatography; BisTris, 2-[bis(2-hydroxyethyl)amino]- 2-(hydroxymethyl)propane-1,3-diol.
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REFERENCES |
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