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(Received for publication, October 28, 1994; and in revised form, December 9, 1994) From the
Retinoic acid, a hormone biosynthesized from retinol, controls
numerous biological systems by regulating eukaryotic gene expression
from conception through death. This work reports the cloning and
expression of a liver cDNA encoding a microsomal retinol dehydrogenase
(RoDH), which catalyzes the primary and rate-limiting step in retinoic
acid synthesis. The predicted amino acid sequence and biochemical data
obtained from the recombinant enzyme verify it as a short-chain alcohol
dehydrogenase. Like microsomal RoDH, the recombinant enzyme recognized
as substrate retinol bound to cellular retinol-binding protein, had
higher activity with NADP rather than NAD, was stimulated by ethanol or
phosphatidylcholine, was not inhibited by 4-methylpyrazole, was
inhibited by phenylarsine oxide and carbenoxolone and localized to
microsomes. RoDH recognized the physiological form of retinol,
holocellular retinol-binding protein, with a K
Retinol (vitamin A) undergoes metabolic activation by
dehydrogenation into retinal, followed by oxidation into the hormone
all-trans-retinoic acid (RA) Liver microsomal RoDH has been partially purified and its active
site has been associated with a 34-kDa polypeptide by covalent binding
with, and inactivation, by PAO and by chemical cross-linking with
holoCRBP. This work reports the cDNA cloning and expression of the 34-kDa
polypeptide from rat liver, provides new evidence that it is a
previously unknown SCAD, shows that it can catalyze the first step in
RA synthesis with holoCRBP as substrate, and reveals that it is
expressed tissue specifically.
Figure 1:
Diagram of the
sequencing strategy for rat liver RoDH cDNA. A 1.8-kilobase cDNA clone
included the complete coding region of the 34-kDa RoDH. The middle
line indicates the 1800 base pairs of the clone. The unlabeled
lines show the areas sequenced. Those above the middle
line were sequenced from left to right; the lines below the middle line were sequenced from right to left.
Figure 2:
Nucleotide and deduced amino acid sequence
of RoDH. Symbols identify the 25 amino acids conserved in at least 17
of the 24 SCAD; the 6 identical in all 24 SCAD are in bold
face(13, 14, 15, 16, 23) .
The amino acid sequences that had been determined by microsequencing
are underlined. Restriction enzyme cut sites are indicated in boldface.
A single open reading frame in p-DirextRo2 predicts
a polypeptide with a calculated molecular mass of 34.9 kDa, comparing
well to the value estimated by SDS-polyacrylamide gel electrophoresis
during the isolation of the 34-kDa RoDH. A total of 54 of the 317 amino
acids were verified by microsequencing. In addition to the four
internal oligopeptides sequenced, the polypeptide also had the expected
N-terminal amino acid sequence (Fig. 2). The predicted RoDH
is in the 25-35-kDa molecular mass range of SCAD and has amino
acid residues distinctive of
SCAD(13, 14, 15, 16, 17, 23) .
Six amino acid residues are identical in the 24 published SCAD
sequences, and all are conserved in RoDH. Nineteen other residues are
identical in at least 17 of the 24 SCAD; 16 of these are conserved in
RoDH, including the aforementioned sequence WXLVNNAG (at
Trp The closest amino acid similarity among RoDH and
other SCAD is between the hydroxybutyrate dehydrogenases and
17
SCAD generally have few cysteine residues: 14 of 24 SCAD have 2 or
fewer. Several mammalian SCAD, however, contain 4: human (R)-3-hydroxybutyrate dehydrogenase(16) ; rat
11
Figure 3:
Hydropathy plot and predicted secondary
structure of RoDH. Top panel, the hydropathy plot was
calculated as the average of the Kyte and Doolittle (28) values
with a 7-residue window. Hydrophobic areas are indicated by positive values. Bottom panel, secondary structure predictions
were made according to Garnier et
al.(29) .
Figure 4:
Characteristics of RoDH transiently
expressed in P19 cells. RoDH activity was assayed with 5 µM holoCRBP/2 µM apoCRBP in the 10,000
A relatively high affinity interaction between
holoCRBP and the RoDH expressed in the P19 cell 10,000
Figure 5:
Affinity of transiently expressed RoDH
for holoCRBP. RoDH activity was measured from holoCRBP composed of
total CRBP/retinol in the ratio 1.4/1 with 180 µg of protein from
the 10,000
Figure 6:
Distribution of RoDH in rat tissues. A, RoDH mRNA. B, rat liver glyceraldehyde
dehydrogenase mRNA. C, RoDH activity of microsomes from rat
tissues determined with 5 µM holoCRBP/2 µM apoCRBP. Data are the means ± S.D. of six to eight
replicates. Controls of no cofactor or no microsomes produced no
retinal. D, immunoanalyses of rat tissue microsomal protein
with anti-34-kDa polypeptide or with preimmune serum: MW,
molecular weight marker; PI, preimmune serum; the lanes
between MW and PI are identified at the top of the figure.
Volume 270,
Number 8,
Issue of February 24, 1995 pp. 3900-3904
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A TISSUE-SPECIFIC, SHORT-CHAIN ALCOHOL DEHYDROGENASE (*)
of 0.9 µM, a value lower than the 
5
µM concentration of holocellular retinol binding protein
in liver. Northern and Western blot analyses revealed RoDH expression
only in rat liver, despite enzymatic activity in liver, brain, kidney,
lung, and testes. These data suggest that tissue-specific isozyme(s) of
short chain alcohol dehydrogenases catalyze the first step in retinoic
acid biogenesis and further strengthen the evidence that the
``cassette'' of retinol bound to cellular retinol-binding
protein serves as a physiological substrate.
. RA directs a variety
of biological responses by modulating gene expression during
development and postnatally, to control differentiation or entry into
apoptosis of numerous cell types in diverse
organs(1, 2, 3, 4) . Insight into RA
biosynthesis has been limited by a lack of data concerning enzymes
dedicated specifically to this pathway. The enzyme(s) that catalyze(s)
RA synthesis physiologically should recognize the predominant form of
retinol in vivo. Retinol in liver occurs bound in the
protected environment of CRBP: the concentration of CRBP exceeds that
of retinol, and CRBP envelops retinol in a high affinity (K
0.1 to 1 nM) binding
pocket(5, 6) . CRBP may confer specificity on RA
biosynthesis by restricting retinol access to enzymes capable of
recognizing the retinol/retinol binding-protein ``cassette.''
This would prevent opportunistic oxidation by dehydrogenases with broad
substrate tolerances, protect retinol from non-enzymatic oxidation and
protect cells from the membrane-disrupting potential of free retinol (7, 8, 9) . A pathway of RA synthesis
elucidated recently, consistent with this hypothesis, entails as the
first and rate-limiting step an NADP-dependent microsomal RoDH, that
recognizes holoCRBP as substrate (10, 11) . Retinal
generated in microsomes from holoCRBP by RoDH supports cytosolic RA
synthesis by an NAD-dependent retinal dehydrogenase(12) . 
The 34-kDa polypeptide has a subunit molecular
mass and other attributes typical of SCAD, including the conserved
sequence WXLVNNAG, Zn
independence,
inhibition by carbenoxolone (IC
= 55
µM), and insensitivity to inhibition by ethanol or
4-methylpyrazole. The 34-kDa polypeptide co-purified with a 54-kDa
polypeptide and RoDH activity was precipitated with either anti-34-kDa
or anti-54-kDa polypeptide antisera. It seems unlikely, however, that
RoDH exists as a heteromultimer between the 34- and 54-kDa
polypeptides, because known SCAD occur as
homomultimers(13, 14, 15, 16, 17) .
Amino Acid Sequence Analysis
The N-terminal
amino acid sequence of the 34-kDa polypeptide was determined to be
MWLYLLALVG. Four internal peptides were obtained by digestion in
situ with trypsin, isolated by high performance liquid
chromatography, and sequenced: LWGLVNNAGISVPV-PNE-M, ELTYFGVK,
VAIIEPGGFK, and YSPGWDAK.Cloning
Degenerate oligonucleotide primers for
reverse transcriptase PCR were synthesized based on two of the amino
acid sequences described above: sense from WGLVNNA,
5`-CTCGCTCGCCCATGGGGICTIGTIAA(C/T)AA(C/T)GC-3`; antisense from PGWDAK,
5`-CTGGTTCGGCCCATTIGC(G/A)TCCCAICCIGG-3`. Nucleotides used as linkers
for cloning into the vector p-Direct (Clontech) are underlined. The
cDNA template was prepared by allowing 1 µg of rat liver mRNA to
react with 0.5 µg of oligo(dT)
, 10 units of rRNase
ribonuclease inhibitor, and 15 units of avian myeloblastosis virus
reverse transcriptase in a total volume of 20 µl for 60 min at 42
°C. The reaction mixture was diluted one-tenth with water, and 4
µl of the diluted solution were added to a PCR reaction mixture
consisting of (final concentrations): 1 µM of each primer,
1.5 mM MgCl, 0.2 mM of each dNTP, and 2.5
units of Taq DNA polymerase (Promega) in 0.1 ml of 10 mM Tris-HCl, pH 9.0, 50 mM KCl, 0.1% Triton X-100. PCR was
done using 35 cycles of 2 min at 94 °C, 2 min at 52 °C, 3 min
at 72 °C, and 10 min at 72 °C after the final cycle. The
0.6-kilobase PCR product (nucleotides 612 through 1158 of the
final cDNA product) was gel-purified and cloned into p-Direct to
provide p-DRoDH1.Library Screening
A 323-base pair probe (probe A,
nucleotides 653-975 of the final cDNA product) was excised from
p-DirectRo1 with AvaII and labeled with 
P by the
random priming method(18) . Probe A was hybridized at 42 °C
to nitrocellulose filters containing 5 
10
plaques
from a 
gt11 rat liver cDNA library (Clontech). The final wash was
done at 65 °C with 0.1% SDS in 1 SCC. Twenty-five plaques
were identified after three rounds of screening. DNA from each plaque
was amplified by PCR and transferred to Nylon membranes for Southern
blot analyses with a 96-base pair probe (probe B, nucleotides
975-1071), also obtained by AvaII digestion of pDirectRo1.
Twenty-four of these PCR products hybridized at 42 °C to probe B
and were washed at 68 °C in 0.5% SDS in 0.1 SCC. The
longest, 1.8 kilobases, was cloned into p-Direct to provide
p-DirectRo2. The insert in p-DirectRo2 was sequenced in both directions
by dideoxy chain-termination with Taq DNA polymerase (Fig. 1).
Expression of RoDH
The coding region of RoDH in
p-DirectRo2 was amplified with the sense primer
5`-TGAGTCACGGCTGGGAGC-3`, nucleotides 179-196 and the antisense
primer 5`ATGAGTATGGTGAACAATGG-3`, nucleotides 1328-1309. To
produce pcDNA3/RoDH, the PCR product was ligated into pcDNA3
(Invitrogen), linearized with EcoRV. pcDNA3/RoDH was
transfected by calcium phosphate/DNA precipitation into semi-confluent
P19 cells, maintained in minimum essential medium with 10% fetal calf
serum. Mock transfections were done with pcDNA3 without insert.
Twenty-four h after transfection each group was harvested and used to
prepare a 10,000 g supernatant by differential
centrifugation(10, 11) .Rat Tissues and Microsomes
Male rats (250 g)
fed a Chow diet were sacrificed by cervical dislocation. Kidneys and
testes were decapsulated. Tissues were either used immediately for
preparation of RNA or were homogenized to prepare microsomes as
described elsewhere (10, 11) .Northern Blot Analysis
RNA was prepared by
guanidinium thiocyanate-phenol-chloroform extraction(19) . Five
µg of poly(A)
mRNA, isolated with the poly(A)Tract
mRNA isolation system (Promega), were fractionated on a 1.2% agarose
gel, transferred to nylon membranes, and cross-linked by UV
irradiation. The probe for RoDH was the 565-nucleotide NcoI/AvaI product from the 3`-untranslated region of
p-DirectRo2 (see Fig. 2). The blot was reprobed with
glyceraldehyde dehydrogenase cDNA, synthesized by PCR with rat liver
cDNA and the 35-60 and 994-1017-base pair fragments as the 5` and
3` primers, respectively. Probes were labeled by random priming to
specific activities of 10
cpm/µg with
[
-P]dCTP(18) . Hybridization was
done at 65 °C for 16 h in 7% SDS, 0.25 M sodium phosphate,
1 mM EDTA, and 1 mg/ml bovine serum albumin, pH 7. Membranes
were washed 2 in 5% SDS and 0.5 mg/ml bovine serum albumin in SE
(40 mM sodium phosphate, 1 mM EDTA, pH 6.8) at 65
°C for 15 min, and 4 for 15 min each in 1% SDS, SE, 65
°C. Autoradiography was with Kodak XAR films and two intensifying
screens at -70 °C.
Immunoblots
Microsomal protein (7-10 µg)
from 10% SDS-PAGE was transferred to poly(vinylidene fluoride)
(Bio-Rad) for immunoblotting with a rabbit anti-34-kDa polypeptide or
preimmune serum, 1/500 dilution each. The signal was
developed with the Bio-Rad alkaline phosphatase kit.RoDH Assay
Assays for retinal biosynthesis were
done with retinol bound to excess CRBP for 30 min at 37 °C in 0.5
ml of 10 mM HEPES, 150 mM KCl, and 2 mM EDTA, 2 mM NADP, pH 8, with 2 mM egg yolk L--phosphatidylcholine (added in 2 µl of ethanol).
Reactions were quenched and the retinal generated was quantified by
high performance liquid chromatography as previously
described(20, 21) .Preparation of CRBP
CRBP saturated with
all-trans-retinol was prepared and purified from CRBP
generated in Escherichia coli with the vector
pMONCRBP(22) , as described elsewhere(10) . ApoCRBP was
prepared and purified as was holoCRBP, except for saturation with
retinol. The concentration of functional apoCRBP was determined by
saturating an aliquot with retinol, separating free and bound retinol
by size-exclusion chromatography and determining the A
/A
ratio. The ratio A/A ratio of holoCRBP was
not affected in the presence of the phosphatidylcholine concentration
used in the RoDH assays, indicating that the integrity of the protein
was not affected.
cDNA and Amino Acid Sequence
The active site of
rat liver RoDH was identified in a 34-kDa polypeptide by covalent
binding and inactivation with PAO, and by chemical cross-linking with
holoCRBP. PCR amplification of rat liver cDNA, with primers
based on two internal amino acid sequences of this polypeptide, yielded
a 546-base pair product that encoded four internal amino acid sequences
determined by microsequencing (nucleotides 613 through 1158 of the
final product, Fig. 2). Two probes, obtained by AvaII
digestion of this PCR product, were then used to screen a rat liver
gt11 cDNA library through three rounds, followed by Southern blot
of the clones. The longest of the clones identified by these procedures
was subcloned to give p-DirectRo2 and was sequenced in both directions (Fig. 1).
), the putative SCAD cofactor binding site
G(X)
GXG (at Gly
), and the
putative SCAD active site Y(X)K (at
Tyr
). As for other SCAD, and in contrast to the
medium-chain (classical) alcohol dehydrogenases, the cofactor binding
site lies N-terminal to the active site. One of the three nonidentical
residues is a conservative substitution, V159I; the others are not,
R104G and W107D.
-hydroxysteroid dehydrogenase, type II (15, 16, 25) (Table 1). There is less
similarity among RoDH, 11-hydroxysteroid dehydrogenases,
17-hydroxysteroid, type I, and 15-hydroxyprostaglandin
dehydrogenase (17, 24, 26, 27) .
-hydroxysteroid dehydrogenase(24) ; human
17-hydroxysteroid dehydrogenase, type I(17) ; human
15-hydroxyprostaglandin dehydrogenase(26) . RoDH has 6 cysteine
residues, more than others except the rat liver D--hydroxybutyrate dehydrogenase, which also has
6(15) .Secondary Structure Predictions
The first 18
N-terminal amino acid residues of RoDH have an average hydropathy of
1.6, calculated with Kyte and Doolittle (28) values. This
span, consisting mostly of helix, determined according to Garnier et al.(29) , is sufficiently long and hydrophobic for
membrane-anchoring (Fig. 3). A short stretch of the most
hydrophilic amino acids, RERK, flanks this hydrophobic span, a typical
feature at membrane junctures, presumably to aid in precise positioning
in the membrane. One other area, the 22 amino acids from residues 131
through 152, also has an average hydropathy of 1.6, but consists
of helix-sheet-helix. This may be a hydrophobic CRBP-interaction site:
it lies close to the active site, centered about residues
176-180. The active site itself lies in a hydrophobic pocket,
bounded by two very hydrophilic sections. There are no other areas
sufficiently hydrophobic to be unequivocally associated with spanning a
lipid bilayer. The scarcity of transmembrane helical domains in the
membrane-associated 34-kDa RoDH is similar to the secondary structure
of human heart (R)-3-hydroxybutyrate dehydrogenase, an inner
mitochondrial membrane SCAD which lacks any transmembrane
helices(16) .
Transient Transfection in P19 Cells
RoDH was
expressed transiently in P19 cells to determine its biochemical
characteristics. The 10,000 g supernatant of
homogenates prepared from mock-transfected cells did not convert
CRBP-bound retinol into retinal in four separate experiments. In
contrast, the 10,000 g supernatant of cells
transfected with the expression vector pcDNA3/RoDH produced retinal
from holoCRBP (Fig. 4). The characteristics of the RoDH
expressed were consistent with those of the rat liver microsomal
enzyme, using microsomes or semipurified
RoDH(10, 11) . Recombinant RoDH was not
inhibited by the alcohol dehydrogenase inhibitor 4-methylpyrazole, had
3-fold higher activity with NADP compared to NAD, was stimulated by
ethanol and phosphatidylcholine, and was inhibited by PAO and
carbenoxolone. PAO has been considered an active-site inhibitor that
forms covalent heterocyclic adducts between spatially proximal
sulfhydryl groups (30, 31, 32) . One of the 6
cysteine residues of RoDH, C37, occurs in the putative cofactor binding
site and another, C177, occurs in the putative active site. Binding of
PAO to either one or both of these and another cysteine residue nearby
in the secondary or tertiary structure may realize the requirement for
inhibition. Carbenoxolone, the steroidal aglycone of licorice-derived
glycyrrhizin, inhibits other SCAD, such as 11-hydroxysteroid
dehydrogenase(33, 34) . Finally, centrifugation
(100,000 g for 1 h) of the transfected P19 cell 10,000
g supernatant localized retinal synthesis, supported
by 5 µM holoCRBP/2 µM apoCRBP, to the
microsomal fraction (33 and 35 pmol of retinal/100 µg of protein),
with no detectable activity in the cytosol (duplicates/180 µg of
protein each).
g supernatant from P19 cells transfected on two separate occasions. A, 200 µg of protein; B, 125 µg of protein). 1, pcDNA3 (mock transfected); 2-8, pcDNA3/RoDH.
Assays were done with the complete assay medium, 1 and 2 or with alterations: 3, + 500 µM carbenoxolone; 4, +1 mM ethanol; 5, -NADP, + 2 mM NAD; 6,
-phosphatidylcholine; 7, +1 mM PAO; 8, +500 mM 4-methylpyrazole. B2 is the
mean ± S.D. of four replicates. The others are averages of
duplicates, each within 2 pmol of its mean.
g supernatant was indicated by an average K
value of 0.9 µM (mean of two separate transfections,
0.5 ± 0.1, 1.2 ± 0.5, ± S.E., determined by
fitting data with the non-linear regression program Enzfitter(35) ) (Fig. 5). This K value compares well with the previously determined values for
holoCRBP of 1.6 µM for rat liver microsomes and 0.6
µM for partially purified
RoDH(10, 11) . The >5 µM of holoCRBP in normal rat liver exceeds the K value for the expressed recombinant RoDH considerably, consistent
with a physiological role for RoDH in RA
biogenesis(7, 8, 9) .
g supernatant of P19 cells transfected with
pcDNA3-RoDH. The Michaelis-Menten data were fit with the nonlinear
regression program Enzfitter(35) . The curve shown is
one of two experiments, each done with a separate transfection. In both
experiments, cells mock-transfected at the same time had no RoDH
activity in their 10,000 g supernatants.
Tissue Distribution of RoDH mRNA
Many tissues and
cell types convert retinol into RA (7, 9, 10, 36, 37) and CRBP
has widespread tissue and cellular
distribution(38, 39, 40, 41) .
Northern blot analysis of liver RoDH expression in rat tissues revealed
a 1.8-kilobase mRNA in liver. Unexpectedly, no mRNA was apparent in
brain, kidney, lung or testis (Fig. 6). Immunoblot analysis
detected this RoDH protein only in liver. Assayed with holoCRBP,
microsomes from rat brain, kidney, lung and testis had RoDH activity
20-30% of that in rat liver microsomes, verifying that the
holoCRBP-supported path of RA biosynthesis occurs in each, as noted
previously(10) . These data indicate extra-hepatic tissues must
contain isozyme(s) of hepatic RoDH which recognize holoCRBP as
substrate, i.e. tissue-specific RoDH isozyme(s) occur.
Concluding Summary
This work identifies the liver
microsomal NADP-dependent RoDH, an enzyme that catalyzes the first and
rate-limiting step in RA biosynthesis, as a heretofore unknown SCAD and
indicates that tissue-specific isozyme(s) may be important to RA
biogenesis. Retinol activation by members of a larger family of lipid
metabolizing enzymes expands and complements insight developing into
retinoid biology. The six ligand-activated receptors that mediate RA
action and the four cellular retinol/RA binding-proteins that function
in retinoid metabolism belong to superfamilies of sterol/lipid-hormone
receptors (42, 43, 44) and
sterol/lipid-binding proteins(45, 46) , respectively,
which are expressed tissue specifically. It is also notable that the
maize sex differentiation gene Tasselseed2 encodes an
SCAD(23) . Perhaps SCAD enjoy widespread involvement in
activating hormones.
))
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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 T. C. Roos, F. K. Jugert, H. F. Merk, and D. R. Bickers Retinoid Metabolism in the Skin Pharmacol. Rev., June 1, 1998; 50(2): 315 - 333. [Abstract] [Full Text] [PDF]
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X. Chai, Y. Zhai, and J. L. Napoli cDNA Cloning and Characterization of a cis-Retinol/3alpha -Hydroxysterol Short-chain Dehydrogenase J. Biol. Chem., December 26, 1997; 272(52): 33125 - 33131. [Abstract] [Full Text] [PDF]
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R. J. Haselbeck, H. L. Ang, L. Deltour, and G. Duester Retinoic Acid and Alcohol/Retinol Dehydrogenase in the Mouse Adrenal Gland: A Potential Endocrine Source of Retinoic Acid during Development Endocrinology, July 1, 1997; 138(7): 3035 - 3041. [Abstract] [Full Text] [PDF]
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M. G. Biswas and D. W. Russell Expression Cloning and Characterization of Oxidative 17beta - and 3alpha -Hydroxysteroid Dehydrogenases from Rat and Human Prostate J. Biol. Chem., June 20, 1997; 272(25): 15959 - 15966. [Abstract] [Full Text] [PDF]
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F. Grun, N. Noy, U. Hammerling, and J. Buck Purification, Cloning, and Bacterial Expression of Retinol Dehydratase from Spodoptera frugiperda J. Biol. Chem., July 5, 1996; 271(27): 16135 - 16138. [Abstract] [Full Text] [PDF]
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X. Wang, P. Penzes, and J. L. Napoli Cloning of a cDNA Encoding an Aldehyde Dehydrogenase and Its Expression in Escherichia coli. RECOGNITION OF RETINAL AS SUBSTRATE J. Biol. Chem., July 5, 1996; 271(27): 16288 - 16293. [Abstract] [Full Text] [PDF]
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H. L. Ang, L. Deltour, T. F. Hayamizu, M. &cjs2657;gombic-Knight, and G. Duester Retinoic Acid Synthesis in Mouse Embryos during Gastrulation and Craniofacial Development Linked to Class IV Alcohol Dehydrogenase Gene Expression J. Biol. Chem., April 19, 1996; 271(16): 9526 - 9534. [Abstract] [Full Text] [PDF]
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M. H. E. M. Boerman and J. L. Napoli Cellular Retinol-binding Protein-supported Retinoic Acid Synthesis J. Biol. Chem., March 8, 1996; 271(10): 5610 - 5616. [Abstract] [Full Text] [PDF]
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 B U Lee, K Lee, J Mendez, and L J Shimkets A tactile sensory system of Myxococcus xanthus involves an extracellular NAD(P)(+)-containing protein. Genes & Dev., December 1, 1995; 9(23): 2964 - 2973. [Abstract] |
ABSTRACT
