From The Webb-Waring Antioxidant Research Institute and Department of Medicine, and § Department of Biochemistry, Biophysics, and Genetics, The University of Colorado Health Sciences Center, Denver, Colorado 80262
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
Molecular characterization of male and female rat
liver aldehyde oxidase is reported. As described for the mouse liver,
male and female rat liver expressed kinetically distinct forms of
aldehyde oxidase. Our data suggest that the two forms arise as a result of differences in redox state and are most simply explained by expression of a single gene encoding aldehyde oxidase in rats. In
support of this argument we have sequenced cDNAs from male and
female rat liver. We examined mRNA expression by Northern blot
analysis with RNA from males and females, from several tissues, and
following androgen induction. Purified rat liver enzyme from males or
females revealed a single 150-kDa species consistent with cDNA
sequence analysis. Both male and female forms were reactive to the same
carboxyl-terminal directed antisera.
Km(app) values obtained in crude
extracts of male or female rat liver and post-benzamidine-purified
aldehyde oxidase differed substantially from each other but could be
interconverted by chemical reduction with dithiothreitol or oxidation
with 4,4'-dithiodipyridine. Our data indicate that a single gene
is most likely expressed in male or female rat liver and that the
kinetic differences between male and female rat liver aldehyde oxidases
are sensitive to redox manipulation.
Aldehyde oxidase (AOX)1
is a member of the molybdenum cofactor containing enzymes. Native AOX
is routinely prepared as a homodimer of 300 kDa. Each 150-kDa subunit
contains two iron-sulfur centers, an FAD, and the molybdenum-pterin
cofactor (MoCo) (1-3). AOX (EC 1.2.3.1) catalyzes the oxidation of a
wide range of aldehydic compounds, purines, quinoliniums, and numerous
pharmacologic agents. While substrate specificity for AOX is very
broad, and wide species variation in substrate specificity exists, the
general catalytic reaction takes the form of hydroxyl transfer from
water to an aldehyde creating the cognate acid. For example, conversion
of benzaldehyde to benzoic acid is a very efficient reaction for AOX
from most species. Conversion of N-1-methyl nicotinamide
(NMN) to the 2- or -4-pyridone has been used as a standard definition of AOXs, although it is usually a kinetically less efficient reaction than benzaldehyde oxidation.
AOX is of interest both for its role in drug metabolism and as a source
of the reactive oxygen species (ROS), hydrogen peroxide, and the
superoxide anion, that have been related to numerous human pathologies.
The human gene encoding AOX has been linked to a rare form of
amyotrophic lateral sclerosis, although it is unknown if AOX encodes
the amyotrophic lateral sclerosis locus itself (4-6). ROS are
generated from AOX in an oxidative half-reaction following reduction of
the enzyme by substrate. The FeS, FAD, and MoCo cofactors comprise an
internal electron transfer chain in which electrons are passed from the
active site molybdenum center to the FeS centers and finally to FAD.
Partial reduction of oxygen at the the reduced FAD site produces ROS.
Unlike the related enzyme xanthine dehydrogenase (XDH), AOX does not
utilize NAD+ as a cofactor and therefore AOX does not
undergo the classical "D-form" to "O-form" conversion
characteristic of XDH (7).
Two distinct AOX activities have been reported for mouse liver (8).
Hepatic AOX from male and female mice differed in Km and Vmax for use of the substrate, benzaldehyde.
The male enzyme exhibited a Km of 40 µM while the female enzyme had a Km of
115.4 µM. The male Vmax was 423.7 nmol/min/mg of protein and the female enzyme was 203.0 nmol/min/mg of
protein. Furthermore, male and female mouse AOX enzymes were
dramatically regulated by testosterone. Treatment of female mice with
testosterone proprionate (TP) converted both Km and
Vmax values to the male pattern (8). Previous
work had shown that castration resulted in the loss of a male pattern
with conversion to a female pattern and TP supplementation restored the
male-specific pattern of AOX expression (9). This observation was
consistent with early reports of androgenic regulation of mouse hepatic
AOX (10, 11). More recently, regulation of mouse hepatic AOX by
testosterone was found to be mediated by growth hormone (12) where,
again, growth hormone supplementation was found to convert
Km and Vmax of the
female-specific pattern to a male-specific pattern. Nonetheless, when
male and female hepatic AOXs were purified to homogeneity they revealed
a single band of 150 kDa on SDS-PAGE analysis and a single active band
on native PAGE (8). How the differences between male and female hepatic
AOXs were generated remained unclear.
Two distinct AOX activities were also purified from rat liver and
identified as NMN oxidases I and II (13, 14). The two activities
exhibited different Km values for the oxidation of
NMN to either its 2- or 4-pyridone. These two activities also differed
by pH optima, heat stability, and inhibitor sensitivity. NMN oxidases I
and II were found to possess distinct kinetic parameters, Km and Vmax, for oxidation of
several different substrates, including benzaldehyde and NMN.
Furthermore, wide variation in Km has been observed
between different species and between individual rat strains (15-17).
As was found for mice, rat hepatic AOX could be purified to homogeneity
to yield a native enzyme of 300 kDa that resolved into two 150-kDa
subunits by SDS-PAGE (13, 14).
AOX genes are widely expressed phylogenetically and in some organisms
appear to arise from multigene families. Even the Archae express MoCo
enzymes related to AOX (18). Two different AOX cDNA sequences were
reported for corn plants that were themselves 83% identical (19).
Three cDNA sequences were reported for Arabidopsis thaliana (20), and tomato plants may also possess several AOX genes (21). Two AOX genes have been identified in Drosophila melanogaster, one encoding AOX and the other encoding the highly related pyridoxal oxidase (PO) which was identified as an AOX (22-24).
Sequences for AOX or PO in Drosophila have not been
reported. Multiplicity in AOX genes was also reported for the mouse
where independently segregating loci appeared to encode AOX enzymes with distinct electrophoretic mobilities and these were identified as
AOX1 and AOX2 (9). Importantly, the different isozymes appeared to
segregate as different AOX genetic loci under differential developmental and androgenic regulation. Human and bovine AOX sequences
have been published that are approximately 82% identical, and a small
fragment of a mouse AOX sequence was published (5, 25-27). However,
second copies of the vertebrate AOX genes have not been cloned or
sequenced. Therefore, while at least two or three AOX genes appear to
exist in plants and flys, they have not been confirmed by sequence
analysis in higher organisms. Furthermore, Southern blot analysis of
chromosomal DNA could be interpreted to suggest that only a single AOX
gene was present in humans (28).
Molecular characterization of AOX from the rat has not been reported.
Because both forms of AOX appeared to be expressed in rat liver, we
have examined expression of AOX genes from both male and female rat
livers. We have confirmed the existence of two kinetically different
forms of AOX in male and female rats. Sequence analysis of the
corresponding cDNAs indicates that a single AOX gene is most likely
activated in the liver in male and female rats. By Northern blot
analysis, male and female rat liver RNA contained a single mRNA
species that did not exhibit induction by TP. Purified rat liver AOX
from males and females revealed a single 150-kDa band on SDS-PAGE.
Present experiments suggest that a primary difference between male and
female forms of AOX may lie in their respective redox states.
RNA Purification and cDNA Synthesis--
RNA was prepared
from organs of freshly killed Sprague-Dawley rats by quick freezing the
tissue in liquid nitrogen followed by extraction in guanidine
isothionate and phenol:chloroform:isoamyl alcohol (24:24:1) (29).
Frozen tissues were stored at 3' and 5' RACE--
A region from the middle of AOX1 was
obtained by PCR amplification of reverse transcribed male rat liver
poly(A)+ RNA using synthetic oligonucleotides (Life
Technologies, Inc., Gaitherburg, MD) derived from a fragment of the
mouse liver AOX1 sequence (27). Nucleotides 1,682 through 2,217 were
amplified with the oligonucleotides MAO4RAT1 and MAO4RAT2 (Table
I) to produce a single 535-base pair
fragment that was gel purified, sequenced in its entirety from two
directions, and cloned as pMID. The resulting sequence was used to
derive the unique sequence oligonucleotides for rat AOX1, FORINRAO, and
REVINRAO. 3' RACE was performed as follows. Male rat liver
poly(A)+ RNA was reverse transcribed using the
oligonucleotide 3' RATRACE as a primer for reverse transcriptase. The
resulting single strand DNA was amplified by PCR using the
oligonucleotides MAO4RAT1 and 3' RATRACE. A second round of PCR
amplification was performed using the 5' nested oligonucleotide,
REVINRAO, and 3' RATRACE. A single product of 2,180 nt was obtained and
sequenced entirely from both directions. This fragment was cloned as
p3' RATRACE and showed 100% identity with the overlap region of pMID.
5' RACE was performed as follows. Male rat liver poly(A)+
RNA was reverse transcribed using random hexamer oligonucleotides. The
resulting single strand DNA was amplified by PCR using the oligonucleotides MAO4RAT2 and IVS22. A second round of PCR
amplification was performed using IVS22 and the nested oligonucleotide
FORINRAO. A single band of 1,930 nt was obtained, sequenced in its
entirety from both directions, and cloned as p5'RATRACE. This sequence revealed 100% identity in the region of overlap with pMID. The extreme
5' end and upstream region of the male rat AOX1 cDNA was obtained
by a modified 5' RACE as follows. Male rat liver poly(A)+
RNA was reverse transcribed using random hexamer oligonucleotides. RNA
was hydrolyzed in sodium hydroxide and the resulting single strand DNA
was subjected to two cycles of nested PCR, the first using an adapter
ligated oligonucleotide at the 5' end. Oligonucleotide 3'IVS11 was
phosphorylated with polynucleotide kinase in the presence of ATP.
Following extraction with phenol:chloroform:isoamyl alcohol and ethanol
precipitation the phosphorylated oligonucleotide was treated with
dideoxyadenosine triphosphate and terminal transferase from
bacteriophage T4 to block elongation from the 3' end. Blocked, phosphorylated oligonucleotide was then ligated to single strand DNA
using bacteriophage T4 RNA ligase in the presence of hexamine cobalt
chloride to produce adapter-ligated single strand DNA. Adapter-ligated
single strand-DNA was subjected to first round amplification using the
oligonucleotides 3' IVS11 and RAT5. The resulting PCR products were
subjected to a second round of amplification using 3' IVS11 and the
nested primer, RAT6. The resulting 160-base pair DNA was cloned
(p5'END-male), sequenced, and showed 100% identity with the overlap
region of p5'RATRACE. This sequence was inferred to contain the
translation initiation site and 49 nucleotides of 5'-untranslated
region because it showed excellent deduced amino acid sequence homology
with human and bovine AOX sequences, a single ATG was found to be
in-frame with the downstream sequence, and translational termination
sequences were observed upstream of this ATG and in the same reading
frame.
DNA Sequence Analysis--
Direct fluorescence sequence analysis
was performed on plasmid DNA or PCR products using oligonucleotide
primers designed to yield approximately 400 nt between primers and
approximately 100 nt of overlap. A list of sequencing oligonucleotides
and their sequences is available upon request. Prior to sequence
determination, plasmids were prepared by alkaline lysis (29). PCR
products were purified from low melting point agarose by phenol
extraction and precipitation in ethanol. Sequences were determined
using dideoxynucleotide chain terminating system from Perkin-Elmer
Applied Biosystems (Foster City, CA). Reactions used the ABI
PRISMTM Dye Terminator cycle sequencing ready reaction kits
(Perkin-Elmer). Sequence reactions were fractionated on an ABI PRISM
310 DNA sequencer equipped with a 43-cm microcapillary (Perkin-Elmer).
All sequences were determined from both directions and sequence data
were compiled manually.
Northern Blot Analysis and Quantitation--
Northern blots were
run using formaldehyde-agarose gels and 5 µg of
poly(A)+ RNA (6, 29). Hybridization probes were isolated
from the clones pMID, p3'RATRACE, and p5'RATRACE by PCR
amplification and agarose gel electrophoresis. Following isolation in
phenol, DNA fragments were labeled by random primed synthesis in the
presence of [32P]dATP for use as hybridization probes.
High stringency hybridization and washing were conducted as described
(6, 29). Following hybridization and autoradiography, each 4,500-nt
band was cut from the hybridization filter and counted by liquid
scintillation counting for quantitation. The remaining filter was
dissociated from residual 32P and rehybridized with a
Castration and Hormone Supplementation--
Castrated or sham
castrated male Sprague-Dawley rats were obtained from Charles Rivers
Laboratory (Wilmington, MA) following 1 week of recovery from surgery.
Surgery was performed when rats were 4 weeks of age. Rats were
maintained at Webb-Waring facilities for 1 additional week of
equilibration. Testosterone proprionate was administered at a dose of
50 mg/kg body weight in corn oil by daily subcutaneous injection.
Growth hormone was administered at a dose rate of 0.05 IU/100 g of body
weight in a buffer composed of 30 mM NaHCO3,
150 mM NaCl, pH 8.25, by subcutaneous injection twice
daily. Sham castrated and castrated controls received corn oil
injection. Following 10 days of treatment, rats were killed by sodium
pentabarbitol administration. Organs were harvested immediately and
dropped into liquid nitrogen for subsequent RNA preparation. RNA was
analyzed from individual organs with 4 rats in the sham controls, 4 rats in the castrated control group, 5 rats in the castrated and
testosterone supplemented group, and 5 rats in the castrated and growth
hormone supplemented group.
AOX Activity Assays--
AOX activity and initial rate data were
determined spectrophotometrically in a 1-ml reaction containing 50 mM potassium phosphate buffer, pH 8.0, NMN at 5 mM or as needed, 10% dimethyl sulfoxide, 250 international
units of CAT, 5-50 µM menadione as required, and
appropriate levels of purified or partially purified enzyme. Initial
rate data were obtained over a 5-min period. Formation of the pyridone
of NMN was monitored at 300 nm.
AOX Enzyme Purification and Characterization--
AOX enzyme
activity was purified from male and female Spraque-Dawley rat livers.
After removal of the liver, all procedures were performed at 4 °C or
on ice using ice-chilled buffers. Liver sections (10-20 g) were diced,
rinsed several times, homogenized, and dounced in 3 volumes of ice-cold
100 mM potassium phosphate, pH 7.5, containing 25 mM benzamidine hydrochloride, .2 mM
phenylmethylsulfonyl fluoride, .1 mM EDTA. Homogenates were
centrifuged for 1 h at 100,000 × g. The
supernatant was brought to 5 mM DTT and incubated for
1 h. MnCl2 was then added to the supernatant to a
final concentration of 10 mM. The solution was then
centrifuged for 5 min at 17,000 × g and the pellet was
discarded. Dry ammonium sulfate was added with stirring to achieve a
final concentration of 30%. The slurry was centrifuged and the pellet
discarded. The resulting supernatant was brought to 50% saturation
with ammonium sulfate, centrifuged, and the supernatant discarded. The
resulting pellet (50% pellet) was resuspended in 1/20 the original
volume of potassium phosphate buffer. Acetone fractionation was
subsequently achieved using acetone chilled with dry ice. Suspensions
were brought to 40% in chilled acetone, centrifuged, and the pellet
discarded. Supernatants were brought to 50% in acetone, centrifuged,
and the pellets collected. The 50% pellet was resuspended in the
original volume of buffer and dry ammonium sulfate was added to achieve
a 60% saturation. The pellet was recovered by centrifugation and
resuspended in 1/20 of the original volume in 100 mM
glycine, pH 9, containing 100 mM NaCl. After resuspension,
insoluble debris was removed by centrifugation and the solution was
desalted on a 5 × 15-cm Sephadex G-25 column in the above glycine
buffer. The desalted solution was loaded onto a 2.5 × 10-cm
benzamidine-Sepharose 6B (Pharmacia) column equilibrated in the same
buffer. The column was washed with 3 column volumes of buffer and
elution was achieved by flushing the column with 500 mM
benzamidine hydrochloride. Elution was monitored at 436 nM
and the single eluting peak was collected and precipitated with
ammonium sulfate at 60% saturation. The pellet was stored at 4 °C
for up to 1 day or was resuspended in a minimum volume of 100 mM potassium phosphate, pH 7.5, and dialyzed against the
same buffer overnight.
The OD 280/450 ratio was between 5 and 7 for male or female
preparations. Specific activity for NMN hydroxylation to the pyridone was 100-250 nmol/min/mg for the female or male enzymes. Both enzyme preparations were inhibited to greater than 95% by inclusion of 50 µM menadione. We observed persistent aggregation of the
soluble protein fraction when preparations did not include treatment
with DTT early in the fractionation. Aggregation reduced the overall yield of AOX enzyme to less than 0.1% of the starting activity. Reduction of the crude lysate prior to MnCl2 treatment
improved the overall yield to 7% of the starting activity. SDS-PAGE
analysis of the aggregated proteins suggested no obvious bias for
specific aggregated proteins. Furthermore, reduction in 5 mM DTT was significantly more effective in preventing
aggregation than was 10 mM cysteine. Reduction of male or
female liver extracts permitted purification of both enzymes to homogeneity.
Preparation of Antibody to AOX--
The amino-terminal
decapeptide comprising the sequence NH2-DRASELLFYV-COOH and
the carboxyl-terminal decapeptide comprising the sequence
NH2-GSYVPWNIPV-COOH were synthesized by Dr. Hans-Richard Rackwitz (German Cancer Research Center, Heidelberg, Germany). Antibody
to the synthetic oligo peptides was produced in rabbits by intravenous
injection of 20 µg of peptide. Rabbits were boosted with peptide
every 2 weeks. The IgG fraction was prepared from serum by ammonium
sulfate precipitation following coagulation of the blood and
sedimentation. This produced two antisera preparations: AOX-NT
(amino-terminal antibody) and AOX-CT (carboxyl-terminal antibody).
Western Immunoblot Analysis--
Protein was electrophoresed on
SDS-PAGE and transferred to polyvinylidine difluoride membranes
(Bio-Rad). Filters were sliced for staining with Commassie Brilliant
Blue or processed for immunoblot analysis. For reaction with antisera,
filter strips were blocked with gelatin overnight prior to reaction
with preimmune sera, AOX-NT, or AOX-CT antisera. Antigen-antibody
complexes were detected by reaction with alkaline phosphatase
streptavidin kit (Bio-Rad).
Different Forms of AOX Exist in Male and Female Rat
Livers--
AOX enzyme activity was measured in crude extracts of male
and female rat livers. Apparent Km
(Km(app)) values were determined from
Lineweaver-Burk plots by measuring conversion of NMN to its pyridone.
Km(app) for male rat liver AOX was 538.8 µM and Km(app) for the
female was 1062.3 µM, consistent with previous reports
showing two forms of AOX in livers from rats and different forms of AOX
in livers from male and female mice. While no explanation for this
difference has been produced, the two AOX genes identified in mice,
AOX1 and AOX2, suggested the possibility that two different AOX genes may be expressed in rat liver.
cDNA Sequence Analysis of Male and Female Rat Liver
AOX1--
Fig. 1 illustrates the PCR
amplification strategy used to obtain segments of male rat liver AOX1.
DNA sequence of the three PCR products was assembled to produce the
male rAOX1 cDNA. RAOX1 comprised 4,304 nucleotides, including 30 nt
of polyadenylation, 210 nt of 3'-untranslated region, and 49 nt of
5'-untranslated region. A single open reading frame was identified that
encoded a protein of 1,333 amino acids and a deduced mass of 147,009 Da. The deduced male rAOX1 protein exhibited 82% sequence identity with human AOX1 and 81% sequence identity with bovine AOX1. Multiple sequence clustal analysis revealed conservation of co-factor domains corresponding to FeS I, FeS II, FAD, and five small domains within the
MoCo binding segment (Fig. 2).
Female rat liver AOX1 cDNA sequence was obtained
using a similar strategy with the exception that a unique sequence
oligonucleotide derived from the male sequence, 3' RATUTR, was used to
obtain the 3' RACE product. Thus, 47 nucleotides of 3'-untranslated
region was obtained for the female and this does not include the
polyadenylation site (Fig. 1). The assembled cDNA sequence for
female rat liver AOX1 encoded a deduced protein of 1,333 amino acids
and 146,919 Da. The female cDNA sequence was 99.8% identical to
the male sequence and the deduced protein sequence was 99.6% identical
to the male sequence. Of the 10 nucleotide differences detected between
the male and female rat liver AOX1 cDNA sequences described here, five resulted in changes to the deduced amino acid sequences (Table II). However, while nucleotides 405 and
408 differ between male and female sequences reported here and to the
GenBank data base, these variations were also found between individual
male clones and therefore do not represent gender differences but
differences between individual rats. The full extent of individual
variation was not determined and it remains possible that all of the
differences observed between the two clones described may be attributed
this cause alone.
Expression of Rat AOX1 mRNA--
Fig.
3A shows Northern blot analysis of
poly(A)+ RNA from male rat liver. Hybridization probes were
derived from each of the three male clones, pMID, p5'RATRACE, and
p3'RATRACE. The region between nucleotides 1,682 and 2,217 of the male
cDNA, corresponding to the pMID hybridization probe, produced
hybridization signals at approximately 4,500 and 2,500 nt.
Hybridization probes derived from both the p5'RATRACE and p3'RATRACE
clones produced predominantly a single band at 4,500 nt with weak
hybridization to the band at 2,500 nt. We conclude that the predominant
mRNA for rAOX1 detected by Northern blot analysis in males is
approximately 4,500 nt, consistent with the cDNA sequence assembled
for rAOX1. The unexpected signal at 2,500 nt may represent a
cross-reactive species largely localized to the pMID region.
Hybridization probes derived from the p5'RATRACE produced predominantly
a single band from both male and female RNA (Fig. 3B). This
RNA was also estimated to be 4,500 nucleotides in size, and no
difference in size or number of hybridizing bands was detected between
males and females.
Northern blot analysis of poly(A)+ RNA from several
different tissues showed expression of a single 4,500-nt RNA for all
tissues examined (Fig. 3C). Different tissues did not show
variation in either the size or multiplicity of AOX RNAs. Variation in
the
RNA from male rats that had been sham castrated, castrated, castrated
and treated with TP, or castrated and treated with growth hormone was
analyzed by Northern blot. Fig. 4 shows
that steady state RNA levels were only slightly affected by any of
these treatments. When hybridization signals were quantitated and
normalized to either OD 280 or to AOX Enzyme Purification and Characterization--
AOX enzyme
activity was purified to homogeneity from male rat livers (Fig.
5A). These preparations
produced a single band of 150 kDa by SDS-PAGE analysis. We were unable
to sequence this protein by direct Edman degradation suggesting that
its amino terminus was blocked, as was found for both the rabbit liver
and bovine liver AOXs (5, 26). Western immunoblot analysis of partially
purified AOX from male and female rat livers revealed excellent
reactivity of each enzyme preparation to this synthetic peptide-derived
antibody (Fig. 5B). Furthermore, reactive proteins from male
and female revealed a predominant polypeptide of approximately 150 kDa
with no evident difference in size between genders. (Fig. 5C).
Km(app) values for oxidation of NMN to
its pyridone were determined by analysis of Lineweaver-Burk plots. Fig.
6 and Table
IV show these results. As noted above,
crude liver extracts from males produced
Km(app) of 538.8 and 1062.3 µM for females. Reduction of the crude liver extract with
5 mM DTT shifted Km(app) for
males and females. Reduced male rat liver produced
Km(app) of 359.9 µM and
for the reduced female enzyme Km(app)
was 354.5 µM (Fig. 6B). Thus, male and female
forms of AOX can be converted to a form with indistinguishable Km(app) values by chemical reduction in
a crude lysate. Oxidation of the reduced female preparation with
4,4'-DTDP converted Km(app) back to a
form similar to that obtained from the untreated female preparations
(Fig. 6B). Furthermore, AOX purified from female rat liver
through the post-benzamidine stage could be reduced with DTT to yield
an enzyme with Km(app) of 261 µM. Reoxidation of the reduced enzyme with 4,4'-DTDP
converted the Km(app) to 1673 µM (Fig. 6C). Thus, the capacity to modulate
Km(app) of rat liver AOX was maintained
through purification of the enzyme and may therefore reflect an
intrinsic property of the enzyme.
In the present work have assembled full-length sequences for AOX
cDNAs from male and female rat liver. We examined expression of AOX
by Northern blot analysis from males and females, from several tissues,
and have examined the effect of androgen regulation. We purified AOX
from rat livers of males and females and obtained a single 150-kDa band
by SDS-PAGE analysis consistent with the deduced amino acid sequences
from males or females of 1,333 amino acids and 147 kDa. Antisera raised
against synthetic decapeptides reacted with AOX from males or females.
Furthermore, Km(app) values for crude
extracts of male or female rat liver and post-benzamidine purified AOX
differed substantially but could be interconverted by chemical
reduction with DTT or oxidation with 4,4'-DTDP.
The deduced amino acid sequences for male and female rat liver AOX are
81 and 82% identical to human and bovine AOXs and are themselves
99.6% identical. They show excellent conservation in the domains
attributed to iron, FAD, and MoCo binding. By this criterion, the rat
AOXs clearly belong to the molybdenum iron-sulfur flavoproteins that
include AOX, XDH, and XO. Furthermore, the rat AOXs conform to the
domain models proposed for XDHs (7, 30, 31). Amino acids critical for
catalysis of this class of enzymes are conserved in the rat AOX
sequences as they are in most other AOXs and XDHs. In particular,
Glu-869 of the Mop enzyme is critical for catalysis and is conserved in
nearly all XDHs and AOXs (see Ref. 21 for alignment of several
sequences), including the two rat sequences reported here where it is
found at amino acid 1265 in MoCo domain 5 (Fig. 2).
We observed that the male and female cDNA sequences were not
identical. While several arguments could be advanced to explain the
differences, we suggest that the least tenable argument is for the
expression of two different AOX genes in the livers of male and female
rats. Nucleotide differences between males and females could arise
during PCR amplification, cloning, or sequence analysis since this is
also PCR based. The small number of differences observed between male
and female sequences could also arise from allelic differences between
males and females or simply from individual differences between rats.
Sequence analysis of only one additional male cDNA uncovered two of
the changes (nt 405 and 408) found between the male and female clones.
The minimal number of differences found between males and females
coupled with the individual variation already observed suggests that
the differences are unlikely to represent expression of alternative
genes. Furthermore, sequence polymorphism is a well described
phenomenon in Drosophila XDH (32-34).
Multiplicity of AOX genes has been established for some organisms
(19-24), and in plants these data are supported by multiple cDNA
sequences of approximately 80% identity (19, 20). Thus, the
observation that mice appear to express two AOX genes in the liver was
not surprising (9). However, genetic data derived in the mouse have not
been supported yet by corresponding molecular data and few efforts have
been made to establish AOX gene copy number in vertebrates. Southern
blot analysis of chromosomal DNA failed to reveal second AOX genes in
several vertebrates under conditions in which at least 80% identity
would have produced hybridization (28).
Northern blot analysis of AOX mRNA from male or female rats, and
from several different tissues, demonstrated expression of a single
4,500 nt RNA. We did not observe variation in size or multiplicity of
AOX mRNAs in the liver from males or females where kinetically
distinct forms of AOX were identified. Furthermore, castration and/or
testosterone supplementation resulted in no significant alteration in
AOX mRNA abundance and we infer that testosterone does not appear
to exert significant regulation of AOX mRNA abundance or form in
the rat liver. We did observe surprisingly strong hybridization to an
RNA of 2,500 nt that could be localized to the region from +1,682 to
+2,217. While the identity of this RNA is unknown, it is too small to
encode an AOX of 150 kDa. Thus, RNA analysis supports expression of a
single AOX gene in the liver where post-translational events may be
important for determining the differences between male and female
kinetic variants.
Interestingly, AOX-3 from A. thaliana encoded a protein of
only 568 amino acids truncated at the amino terminus (20). It must
differ from a true AOX because it cannot encode a protein capable of
binding the full set of co-factors. AOX-3 from A. thaliana showed striking homology to AOX-1 and AOX-2 in the MoCo-binding region,
confirming that it is indeed a member of the MH family. Confirmation
that vertebrates encode such a protein would be of great interest since
it may represent the RNA detected at 2,500 nt.
Since our observations suggested that rats express a single AOX gene in
the liver, we examined the possibility that redox status might underlie
the differences in male and female variants. We found that partially
purified crude extracts of male and female rat liver did indeed reveal
different Km(app) variants of AOX.
Reduction of crude extracts from males or females with DTT resulted in
conversion to a single form. Subsequent reoxidation of the reduced AOX
with 4,4'-DTDP resulted in conversion to a more female like
Km(app). Reduction or oxidation of
post-benzamidine purified AOX also resulted in interconversion between
the two extremes of Km(app) suggesting
that the variants differed by their intrinsic oxidation state. Since
DTT directly reduces protein disulfides to thiols while 4,4'-DTDP forms
disulfides from thiols (35-38), we infer that manipulation of thiol
oxidation state can interconvert kinetic variants of AOX.
Redox effects on XDH are well known. Conversion between the
NAD+ dependent, D-form, and the oxygen dependent, O-form,
is a redox-dependent process reversible by chemical
reduction (7, 39, 40). Furthermore, redox effects on XDH have dramatic
effects on the kinetic parameters of the enzyme (41). AOX does not have
an NAD+-dependent form of the enzyme, and
therefore conversion between D-form and O-form is irrelevant. While
cysteine residues critical for D-form to O-form conversion in XDH were
not conserved in AOX (42), most of the 41 cysteine residues found in
rat liver AOX are conserved with vertebrate XDHs. These cysteine
residues would be expected to take part in the same biochemical
reactivities as those found in XDH. Since we observed that redox
effects on Km(app) were preserved from
crude extracts through post-benzamidine-purified enzyme and that these
effects alone were sufficient to explain the differences between male
and female variants, we infer that the different
Km(app) variants result not from
expression of alternative genes but from redox effects that may act
either on AOX itself or on a closely associated protein capable of
modulating Km(app) for AOX.
Our observations can be explained by the activity of the hepatic
microsomal monooxygenase. The cystamylating flavin monooxygenase catalyzes oxidation of cysteamine, a thiol, to cystamine, a disulfide, and this reaction provides a significant source of disulfide
responsible for maintaining the intracellular thiol:disulfide potential
(43, 44). The thiol:disulfide potential is thought to reflect two ratios: the GSH:GSSG ratio and the cysteamine:cystamine ratio. Protein
oxidation state depends on overall thiol:disulfide potential. Higher
levels of monooxygenase lead to a more oxidizing environment, while
reduced monooxygenase levels result in a more reducing environment. Significantly, monooxygenase from mice or rats is regulated by testosterone in precisely the fashion that kinetic variants of AOX are
regulated (43). Male rat hepatic monooxygenase levels are lower than
female levels. Castration of male rats elevates monooxygenase and leads
to elevated cystamine and a more oxidizing cytosol. Testosterone
reverses this effect and restores the reducing environment.
Furthermore, testosterone treatment of females lowers monooxygenase and
elevates cysteamine levels. These observations may explain both the
effect of testosterone and the ability to purify kinetically distinct
forms of AOX from male and female rats since neither thioltransferase
nor glutathione reductase, the two other enzymes establishing
thiol:disulfide potential, are regulated by testosterone (43). We posit
that AOX is sensitive to the thiol:disulfide potential and that kinetic
variants of AOX reflect the intracellular thiol:disulfide potential
through thiol modification of AOX. Activity of the monooxygenase could thereby regulate AOX Km(app) by
"setting" the thiol:disulfide potential leading to a more oxidized
form of AOX in the female or a more reduced form of AOX in the male.
This may have direct consequences for ROS generation from AOX since the
kinetically less efficient enzyme may bias ROS generation for
superoxide anion. This argument does not require expression of a second
AOX gene and can account for the failure of testosterone to regulate
AOX gene expression in rats despite finding kinetically distinct forms in males and females. AOX thiol modification could also explain both
novel electrophoretic variants (9) and the widely variant kinetic
characteristics of AOX from different organs and different species (16,
45-47).
INTRODUCTION
Top
Abstract
Introduction
References
MATERIALS AND METHODS
70 °C until use.
Poly(A)+ RNA was prepared by fractionation on
oligo(dT)-cellulose (Stratagene, La Jolla, CA). cDNA was prepared
by reverse transcription in a final volume of 20 µl as follows. 1.0 µg of poly(A)+ RNA was mixed with diethyl
pyrocarbonate-treated water, 1.0 µl of primer oligonucleotide at 20 µM, 1.0 µl of 10 mM deoxyribonucleoside triphosphates (ACGT), 0.5 µl of RNase inhibitor, 1.0 µl of
recombinant Moloney murine leukemia virus reverse transcriptase
(CLONTECH Laboratories, Palo Alto, CA), and 4.0 µl of 5 × buffer (final conditions: 50 mM Tris-HCl,
pH 8.3, 75 mM KCl, 3 mM MgCl2).
Reactions were incubated at 42 °C for 60 min and then heated to
94 °C for 5 min to inactivate reverse transcriptase. Prior to use,
reactions were diluted to 100 µl and 5 µl was used for PCR
amplification. Reverse transcriptase reactions were stored at
70 °C.
Oligonucleotides used for amplification of male and female rat AOX1
-actin specific probe. Actin hybridization was also quantitated by
excising the bands from the filter and liquid scintillation counting.
Each AOX1 hybridization signal was normalized to the corresponding
signal for
-actin after correction for background hybridization.
RESULTS
View larger version (14K):
[in a new window]
Fig. 1.
PCR amplification, cloning, and sequence
strategy for male and female rat AOX1. The upper bar
shows the deduced cDNA structure for both male and female rat liver
AOX1. PCR amplified and cloned fragments are shown below.
Note that the 3' RACE product for the female has been truncated within
the untranslated region and does not comprise the entire untranslated
region. Each cloned PCR fragment was subjected to DNA sequence analysis
using a battery of oligonucleotides designed to encompass approximately
400 nucleotides between oligonucleotide primers. Each fragment was
sequenced entirely from both directions. A list of sequence analysis
primers and their sequences is available upon request. The assembled
cDNA sequences for both male and female rat liver AOX have been
deposited in the NCBI gene bank data base.
View larger version (55K):
[in a new window]
View larger version (71K):
[in a new window]
Fig. 2.
Alignment of deduced amino acid
sequences. Amino acid sequences for the four vertebrate AOXs have
been aligned by multiple Clustal analysis. Identical amino acids are boxed in
black and biochemically conserved differences are shown in
gray. Regions thought to mediate cofactor binding are
indicated by the overline. Five sites within the large
MoCo-binding domain have been identified and are shown individually.
The 5 amino acid differences between male and female rat liver AOXs
have been indicated with an asterisk. The programs ClustalW,
Boxshade, and Paint were used to create the figure.
Sequence differences in male and female rat AOX1 clones described
here
View larger version (25K):
[in a new window]
Fig. 3.
Northern blot analysis of rat AOX1
expression. A, each of the three clones used to
assemble male rat liver AOX1 was used as a hybridization probe for
independent Northern blots of male rat liver poly(A)+ RNA.
The major band at 4,500 nt corresponds to the AOX1 mRNA. The band
at 2,500 nt that has greater localization to the pMID region of AOX is
assumed to represent a cross-reactive species. Although it has not been
excluded that this may represent a breakdown product of the larger RNA,
it is too small to encode a full-length AOX. B,
poly(A)+ RNA from male and female rat liver has been
analyzed by Northern blot using the p5'RATRACE clone as a hybridization
probe. No attempt is made here to indicate a difference in abundance of
the AOX mRNA between males and females. C,
poly(A)+ RNA from several different male rat tissues has
been analyzed by Northern blot using the AOX1 insert from p5'RATRACE as
a hybridization probe.
-actin control precludes drawing firm conclusions at this point concerning relative levels of expression between tissues.
-actin hybridization signal (Table
III), we found no statistically
significant difference between groups in AOX1 mRNA abundance. These
data do not support significant regulation of rat AOX1 mRNA
abundance by TP.
View larger version (39K):
[in a new window]
Fig. 4.
Testosterone does not regulate steady state
levels of rat hepatic AOX. Poly(A)+ RNA from
individual male rat livers has been analyzed by Northern blot.
Individuals from the following four groups have been used: castrated,
sham castrated, castrated and testosterone supplemented, castrated and
growth hormone supplemented. -Actin was used as a control to reveal
uniform loading of RNA. Blots were first probed with the AOX probe, the
corresponding region cut from the filter for counting, and the
remaining filter was dissociated of all 32P and
rehybridized with the
-actin probe.
Quantitation of RNA hybridization
-actin signal derived from the same
lane, and the normalized, background subtracted counts were multiplied
by 100. The number of animals in each group is shown by the
n. The mean and standard error of the mean (SE) were
calculated for each group. No statistical significance could be
established between group means.
View larger version (28K):
[in a new window]
Fig. 5.
Analysis of rat liver AOX1 protein.
A, male rat liver AOX was purified to homogeneity from
initially reduced extracts as described under "Materials and
Methods." Purified AOX was analyzed by SDS-PAGE and stained with
Ponceau S prior to subjecting the enzyme to amino-terminal sequence
analysis. B, antibody was raised against synthetic
decapeptides from the amino (AOX-NT) and carboxy
(AOX-CT) termini. AOX was partially purified to retain
numerous unrelated proteins, analyzed by SDS-PAGE (Crude
AOX), and reacted to each antibody as described under "Materials
and Methods." Preimmune, AOX-NT, and AOX-CT antisera were used at a
1:500 dilution. C, male and female rat liver AOX was
purified to homogeneity from initially reduced preparations. Enzymes
were analyzed by SDS-PAGE and subjected to Western immunoblot analysis
using the AOX-CT antisera. Both male and female preparations migrated
with apparent size of 150 kDa and both preparations reacted to the
AOX-CT antisera.
View larger version (15K):
[in a new window]
Fig. 6.
Chemical reduction or oxidation abolishes
differences in Km(app) between
male and female rat liver AOX. AOX activity was purified from
unreduced male and female rat livers through
(NH4)2SO4 fractionation and either
treated with 5 mM DTT or not to produce the crude native or
crude reduced preparations. A separate preparation was reduced
initially and purified through benzamidine fractionation to produce the
post-benzamidine preparation. A, Lineweaver-Burk plots were
determined for male (squares) and female
(diamonds) preparations using NMN as the oxidizing
substrate. B, Lineweaver-Burk plots were determined for
initially reduced male and female crude extracts. In addition, the
reduced female extract was then treated with 1 mM 4,4'-DTDP
to reoxidize it (open squares) and subjected to
Lineweaver-Burk analysis. C, female rat liver AOX was
purified through benzamidine fractionation from an initially reduced
extract. Enzyme was treated with 5 mM DTT or 1 mM 4,4'-DTDP to reduce or oxidize the enzyme.
Lineweaver-Burk analysis was then conducted on the reduced
(diamonds) or oxidized (squares)
preparations.
Effect of reduction and oxidation on Km(app) for crude
and purified rat liver AOX
DISCUSSION
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Enrico Garrattini and Mineko Terao (Milan, Italy) for discussion of these and other data.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants HL52509 and HL45582, The Muscular Dystrophy Association, and the Robert and Helen Kleberg Foundation.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) AF110477 and AF110478.
To whom correspondence should be addressed. The Webb-Waring
Antioxidant Research Institute and The University of Colorado Health
Sciences Center, Dept. of Medicine, 4200 East 9th Ave., Denver, CO
80262. Tel.: 303-315-4593; Fax: 303-315-3776; E-mail: richard.m.wright{at}uchsc.edu.
The abbreviations used are: AOX, aldehyde oxidase; XDH, xanthine dehydrogenase; NMN, N-methyl nicotinamide; DTT, dithiothreitol; 4, 4'-DTDP, 4,4'-dithiodipyridine; PAGE, polyacrylamide gel electrophoresis; ROS, reactive oxygen species; AOX-NT, anti-AOX antisera for the amino-terminal decapeptide; AOX-CT, anti-AOX antisera for the carboxyl-terminal decapeptide; MoCo, molybdenum-pterin cofactor; PCR, polymerase chain reaction; TP, testosterone proprionate; nt, nucleotide; RACE, rapid amplification of cDNA.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|