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J Biol Chem, Vol. 273, Issue 52, 35381-35387, December 25, 1998
, andFrom the Department of Pharmacology, Joan and Sanford I. Weill Medical College of Cornell University, New York, New York 10021
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ABSTRACT |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Spodoptera frugiperda retinol
dehydratase catalyzes the conversion of retinol to the
retro-retinoid anhydroretinol. It shares sequence homology
with the family of mammalian cytosolic sulfotransferases and provides
the first link between sulfotransferases and retinol metabolism. In
this study the enzymatic properties of retinol dehydratase were
examined using bacterially expressed protein. We show that retinol
dehydratase can catalyze the transfer of the sulfonate moiety to small
phenolic compounds and exhibits many functional similarities to the
mammalian cytosolic sulfotransferases. The bisubstrate reaction that it
catalyzes between retinol and the universal sulfonate donor
3'-phosphoadenosine 5'-phosphosulfate seems to involve ternary complex
formation and to proceed via a Random Bi Bi mechanism. In addition to
the low nanomolar Km value for free retinol,
retinol dehydratase is strongly inhibited by retinol metabolites,
suggesting a preference for retinoids. Conversely, a number of tested
mammalian cytosolic sulfotransferases do not utilize retinol,
indicating that retinol is not a general substrate for sulfotransferases.
Vitamin A (retinol) regulates cellular function in multiple ways.
Since it is not known to have biological activity itself, it is thought
to serve as the parent compound for the biosynthesis of a number of
retinoid metabolites. 11-cis-Retinaldehyde, for example, has
been established as the essential chromophore for vision (1), and
all-trans-retinoic acid
(RA)1 has been widely studied
for its roles in cellular differentiation (2) and morphogenesis (3).
The two retro-retinoids,
14-hydroxy-4,14-retro-retinol (14-HRR) (4) and
anhydroretinol (AR) (5), are among the most recently characterized
bioactive retinoids. They are physiologically present in a number of
insect and mammalian cell types, and evidence to date suggests they
play a role in cell survival (reviewed in Ref. 6). 14-HRR appears to be
essential in lymphocyte and fibroblast activation and can prevent cell
death in retinol-dependent cell lines grown in serum-free
medium. AR can competitively inhibit the growth-supportive effects of
retinol and 14-HRR. These retro-retinoids seem to define a
novel class of messenger molecules.
Progress in the characterization of retinol metabolizing enzymes and
isolation of their genes has lagged behind the identification of the
various bioactive metabolites. Although most studies have focused on
alcohol and aldehyde dehydrogenases and their potential role in the
oxidation of retinol and retinaldehyde to RA (reviewed in Ref. 7),
other types of enzymes are required for the complete array of retinol
metabolism. The cloning of retinol dehydratase (8) raises the
possibility that sulfotransferases, a class of enzymes not previously
linked to vitamin A metabolism, may play a role in the biosynthesis of
certain retinol metabolites. Retinol dehydratase is the enzyme
responsible for the conversion of retinol to the
retro-retinoid AR (see Fig. 1)
and was cloned from the insect cell line Spodoptera
frugiperda (Sf)-21. It shares sequence homology to the family of
mammalian (reviewed in Refs. 9-11) and plant (reviewed in Ref. 12)
cytosolic sulfotransferases, and it represents the first insect
homologue of this growing family of enzymes. Like all
sulfotransferases, retinol dehydratase uses 3'-phosphoadenosine
5'-phosphosulfate (PAPS) as co-substrate (Fig. 1).
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (10K):
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Fig. 1.
Retinol dehydratase-catalyzed reaction.
* indicates the putative reaction intermediate, retinyl
sulfate.
Cytosolic sulfotransferases catalyze bisubstrate reactions where the
sulfonate moiety from the universal sulfonate donor PAPS is transferred
to small acceptor molecules, such as steroids and catecholamines. Many
of the putative substrates bind with high Km or
Ki values, and only a few physiological substrates
(i.e. endogenous substances that serve as substrates at
their physiological concentrations, e.g. 17
-estradiol)
have been described. A single enzymatic mechanism for the sulfonation of these compounds has not been established. A number of kinetic studies point toward a sequential mechanism (13-19), whereas the first
sulfotransferase crystal structure suggested that at least some
sulfotransferase reactions may follow a ping-pong mechanism (20).
S. frugiperda retinol dehydratase differs from the described
cytosolic sulfotransferases in several important ways: its molecular mass (41 kDa) is higher than that of the known cytosolic
sulfotransferases (30-36 kDa), and its end product, AR, is not
sulfonated. In addition, retinol dehydratase catalyzes the formation of
a putative signaling molecule, and its low Km value
for its apparent substrate, free retinol, indicates a specific
interaction between enzyme and substrate. We were interested in
examining whether retinol dehydratase functions as a sulfotransferase
and in establishing the specificity of interaction between retinol
dehydratase and retinoids. Our studies of the enzymatic properties of
retinol dehydratase are also important in understanding the mechanism of AR formation and the role of sulfotransferases in retinol metabolism.
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EXPERIMENTAL PROCEDURES |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Chemicals-- [3H]Retinol and [35S]PAPS were purchased from NEN Life Science Products. 14-Hydroxy-4,14-retro-retinol, 13,14-dihydroxyretinol, anhydroretinol, and 4-oxoretinol were kindly provided by Dr. Fadila Derguini (Sloan-Kettering Cancer Center, New York). All other chemicals were purchased from Sigma.
Expression and Purification of Retinol Dehydratase--
The
entire open reading frame of the cDNA coding for S. frugiperda retinol dehydratase was cloned in-frame into the
expression vector pET19b (Novagen, Madison, WI), transformed into
Escherichia coli strain BL21(DE3), and grown at 37 °C in
Luria Broth containing ampicillin (100 µg/ml) until the
A595 was 0.5. Isopropyl-1-thio--D-galactopyranoside was added (final
concentration of 1 mM), and the culture was grown for an
additional 3 h. Cells pelleted (4 °C, 10,000 × g, 10 min) from 4 liters of culture were lysed by sonication
in 50 ml of lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM dithiothreitol (DTT), 2 mM EDTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 100 µg/ml phenylmethylsulfonyl fluoride), and cellular debris was
removed by centrifugation (4 °C, 10,000 × g, 10 min). The supernatant was dialyzed at 4 °C against 20 mM
Tris-HCl, pH 7.4, 1 mM DTT (2 × 4 liters) and applied
to a DE52 column (Waters, Bedford, MA) (2.5 × 20 cm; flow rate,
1.5 ml/min 20 mM Tris-HCl, pH 7.4, 1 mM DTT;
linear gradient, 0-0.5 M NaCl over 280 min; fraction
volume, 9 ml). Fractions containing retinol dehydratase (eluting
between 0.18 and 0.23 M NaCl) were pooled, concentrated to
5 ml by Centricon (Amicon, Inc., Beverly, MA) ultrafiltration, and
applied to an Ultrogel AcA54 gel filtration column (Amersham Pharmacia
Biotech) (2 × 100 cm; flow rate, 1 ml/min 20 mM
Tris-HCl, pH 7.4, 1 mM DTT, 50 mM NaCl;
fraction volume, 8 ml). Fractions containing retinol dehydratase were
pooled and applied to a SourceQ column (Amersham Pharmacia Biotech)
(10-ml bed volume; flow rate, 0.65 ml/min 20 mM Tris-HCl,
pH 7.4, 1 mM DTT; linear gradient, 0-0.5 M
NaCl over 160 min; fraction volume, 2.5 ml). Three fractions (eluting
between 0.1 and 0.15 M NaCl) containing the majority of
retinol dehydratase were pooled. The protein purity was determined by
SDS-polyacrylamide gel electrophoresis followed by silver staining. The
protein concentration was determined to be 6 mg/ml by Bradford assay
referenced to bovine serum albumin (BSA) standard. Protein was stored
in 200-µl aliquots at 
70 °C and maintained stable enzymatic
activity for at least 6 months.
Retinol Dehydratase Assay--
Recombinant retinol dehydratase
(6 µg/assay) was incubated with [3H]retinol (37.3 mCi/µmol) and PAPS in the presence of delipidated BSA (final BSA
concentration was 30 µM unless otherwise indicated) at
room temperature in 100 µl of buffer (20 mM Tris-HCl, pH
7.2, 1 mM DTT). The concentration of free retinol in each
assay was calculated based on the published value for the dissociation
constant of retinol for BSA (2 × 10
6 M)
(21). The reaction was allowed to proceed 5 min, and the reaction
mixtures were delipidated according to the procedure of McClean
et al. (22). The synthesis of [3H]AR was
determined by on-line liquid scintillation counting after separation by
high pressure liquid chromatography gradient elution (water/methanol/chloroform) from a C18 reverse-phase
201TP54 column (Vydac, Hesperia, CA) as described previously (5).
To examine the pH dependence of retinol dehydratase-catalyzed AR formation different buffers (50 mM Mes at pH 5.6, 6, 6.4; 50 mM Mops at pH 6.8, 7.2, 7.4; 50 mM Hepes at pH 7.2, 7.6, 8; 50 mM bis-tricine propane at pH 8, 8.4; 50 mM Ches at pH 8.6, 8.8, 9.2, 9.6, 9.8; 50 mM Caps at pH 10, 10.4, 10.8, 11.1) were substituted in the reaction mixture. To examine the effects of different cations, different salts (NaCl, KCl at final concentration 100 mM; MgCl2, MnCl2, CaCl2, CoCl2, NiCl2, ZnCl2 at final concentration 10 mM) were included in the assay.
Sulfonation Assay-- The method of Foldes and Meek (23) was used. Briefly, 50 µg of retinol dehydratase was incubated with 1 µl of [35S]PAPS (1.6 mCi/µmol) and 1 µl of substrate in 1 ml of buffer (20 mM Tris-HCl, pH 7.2) at room temperature for 0.5 h. The reaction was stopped by addition of 200 µl of 0.1 M Ba(OAc)2. Free [35S]PAPS was precipitated twice by addition of 100 µl of 0.1 M ZnSO4 and 100 µl 0.1 M Ba(OH)2. The precipitate was removed by centrifugation (5,000 × g, 5 min, room temperature). After the second precipitation 35S-sulfonated products in the supernatant were counted by liquid scintillation (Beckman Instruments, LS 6500). Assays were performed in triplicate.
Kinetic Studies--
Initial velocity measurements were made
under conditions where conversion of [3H]retinol did not
exceed 10%. Measurements of the kinetic constants for each substrate
were performed by varying the concentration of one substrate, while
keeping the other substrate at a fixed and near saturating
concentration. To determine the effect of an inhibitor, these
measurements were repeated at different fixed inhibitor concentrations.
The apparent Km, Vmax, and Ki values were determined using two computer
programs, k.cat (Biometallics, Inc., Princeton, NJ) and KinetAsyst II
(IntelliKinetics, State College, PA). Both programs use nonlinear
regression based on the algorithms by Cleland (24). Two-substrate
kinetic measurements were performed by varying the concentration of
retinol at several fixed concentrations of PAPS. The kinetic data were
fit to models of bisubstrate reactions (ping-pong, sequential, and
equilibrium ordered) using KinetAsyst II. For all-trans-RA,
17-estradiol, PAP, and AR the mode of inhibition was determined by
fitting the kinetic data to models of competitive, noncompetitive, and
uncompetitive inhibition using KinetAsyst II. The choice of fit was
based on a combination of visual examination and comparison of
parameter values and residuals for all models tested.
IC50 values were determined by measuring [3H]AR production at a fixed free retinol concentration (34 nM) in the presence of varying concentrations of inhibitor and plotting the log [inhibitor] versus vi/vo. The IC50 values calculated were based on duplicate measurements. In those cases, the Ki values were calculated from the IC50 values assuming competitive inhibition. The equation used to describe the relationship between Ki and IC50 for a competitive inhibitor was Ki = IC50/{1+([S]/Km)}.
Expression of Mammalian Sulfotransferases--
Oligonucleotide
primers were designed to specifically amplify the coding sequences of
phenol/aryl ST (25), hydroxysteroid ST-20 (26), estrogen ST (27), and
dopa/tyrosine ST (ST1B1) (28) and used for polymerase chain reaction
from rat liver first-strand cDNA. The products were cloned in-frame
into either the expression vector pET23a (estrogen ST and ST1B1) or the
expression vector pET28a (aryl ST and hydroxysteroid ST) and expressed
in E. coli strain BL21(DE3) similar to retinol dehydratase.
Crude protein lysates were prepared as described above for retinol
dehydratase. Crude protein lysates were also prepared from cells where
sulfotransferase expression was not induced with
isopropyl-1-thio--D-galactopyranoside and from cells
that were transformed with vector alone (either pET23a or pET28a) and
were used as controls.
Activity Assays for Rat Sulfotransferases--
Sulfonation of
p-nitrophenol (1 mM) and
all-trans-retinol (52 µM) was determined using
100 µl of crude protein lysates as described above. To test for AR
production, crude protein lysates were incubated with
[3H]retinol, and reaction mixtures were analyzed as
described above. The activity assays for mammalian sulfotransferases
were performed at 37 °C.
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RESULTS |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Expression of Recombinant Retinol Dehydratase-- Recombinant S. frugiperda retinol dehydratase was overexpressed in E. coli and purified to homogeneity (Fig. 2A) by sequential column chromatography. Enzyme activity was measured by quantitating the reaction product, [3H]AR, after separation by C18 reverse-phase high pressure liquid chromatography. The apparent Km and Vmax values were 3.9 nM and 494 pmol AR/min·mg enzyme for free all-trans-retinol, and 0.95 µM and 375 pmol of AR/min·mg enzyme for PAPS (Fig. 2, B and C). These values are consistent with what was previously reported for the enzyme purified from Sf-21 cells (8) and indicated the recombinant enzyme was representative of the native form.
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Effects of pH and Divalent Cations-- Enzyme activity was assayed in different buffers covering pH values ranging from 5.6 to 11. Maximal activity was obtained at two distinct pH values, pH 7.4 and pH 9.2 (data not shown). Cations have been reported to differentially regulate the activity of mammalian cytosolic sulfotransferases and have been proposed as tools for distinguishing between various sulfotransferases (29). For this reason we tested the effects of different divalent cations on retinol dehydratase activity and found that whereas Mg2+, Mn2+, Ba2+, and Ca2+ had no effect, Zn2+, Ni2+, and Co2+ had a significant inhibitory effect (40-60% inhibition at 10 mM concentration) (data not shown). In addition, the monovalent cations NaCl and KCl had no effect on enzyme activity.
Retinol Dehydratase Is a Sulfotransferase-- Retinol dehydratase was originally identified as a sulfotransferase based on sequence homology (8), but its function as a sulfotransferase had not been directly demonstrated. We tested its ability to sulfonate a variety of compounds that have been extensively studied as substrates for mammalian cytosolic sulfotransferases (e.g. p-nitrophenol). Under the conditions used, several compounds, including p-nitrophenol, vanillin, and serotonin, were sulfonated by retinol dehydratase (Table I). The highest level of activity was obtained with vanillin. Surprisingly, we observed ethanol sulfonation with as low as 0.1% ethanol concentration (corresponding to 17 mM).
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Substrate Specificity of Retinol Dehydratase--
To study the
substrate specificity of retinol dehydratase, structurally different
compounds were tested for their ability to inhibit AR production (Table
II). The mode of inhibition
versus retinol was established for two compounds,
all-trans-RA and 17-estradiol. For both these compounds
the best data fit was to a model of competitive inhibition with
Ki values in the range of 0.24 µM for all-trans-RA (Fig.
3A) and 17.6 µM
for 17--estradiol (Fig. 3B). For the other compounds
listed in Table II, including retinoids and steroids, IC50
values were determined at a fixed retinol concentration (34 nM), and Ki values were calculated from
the measured IC50 assuming competitive inhibition. The
retinoid compounds tested were strong inhibitors of retinol dehydratase
with Ki values in the low micromolar range similar
to all-trans-RA. Certain steroids were also found to be
potent inhibitors of retinol dehydratase activity, in particular
hydrocortisone and androsterone, even though these compounds were not
sulfonated when presented as substrates (Table I). In contrast, the
ecdysteroids tested, which play an important role in the post-embryonic
development and metamorphosis of insects (30), did not seem to have
significant affinity for retinol dehydratase. Other compounds that
serve as substrates for mammalian sulfotransferases, including
p-nitrophenol, minoxidil, and several catecholamines, were
weak inhibitors of retinol dehydratase activity at millimolar
concentrations. As might be expected from the sulfonation assays,
ethanol inhibited retinol dehydratase activity at ethanol
concentrations above 0.1% (17 mM).
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Two-substrate Kinetics-- The crystal structure of mouse estrogen sulfotransferase showed similarities between protein kinases and sulfotransferases and suggested that sulfonation may proceed via a ping-pong mechanism (20). In order to examine whether a ping-pong mechanism is operative in retinol dehydratase, [3H]AR formation was measured as a function of retinol at different fixed concentrations of PAPS. The kinetic data was fit to several models of bisubstrate reactions, and the best fit was obtained in the case of a sequential model. As shown in Fig. 4, the double-reciprocal plots of 1/v versus 1/[retinol] at different fixed PAPS concentrations resulted in an intercepting family of lines, which suggests the reaction proceeds via ternary complex formation.
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Product Inhibition-- We tested the effects of PAP and AR on retinol dehydratase activity in order to see whether there is product inhibition and to get more information about the reaction mechanism. PAP was found to be a very potent inhibitor of retinol dehydratase similar to what has been reported for other mammalian (e.g. Refs. 14-16) and plant (e.g. Ref. 17) sulfotransferases. It inhibited PAPS binding competitively (Fig. 5A) with a very low Ki value (0.38 µM ± 0.16), whereas it showed a noncompetitive mode of inhibition versus retinol (Fig. 5B) (Ki = 9.9 µM ± 1.83). AR inhibited retinol binding competitively (Fig. 5C), whereas inhibition of PAPS binding appeared to be noncompetitive (Fig. 5D). These data taken together suggest that the retinol dehydratase-catalyzed reaction between all-trans-retinol and PAPS follows a Random Bi Bi mechanism.
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AR Is Not Produced by Known Mammalian Cytosolic Sulfotransferases-- Despite the fact that the substrate specificities of mammalian cytosolic sulfotransferases have been extensively studied, there are no reports indicating utilization of retinol as substrate. To see whether mammalian sulfotransferases could utilize retinol, we expressed four rat sulfotransferases representing the known sulfotransferase subfamilies (shown in Fig. 6; expressed STs are highlighted) and tested them for their ability to produce AR and/or sulfonate retinol. The activity of the sulfotransferases was assayed in crude bacterial lysates, and in the case of phenol ST and dopa/tyrosine ST the results were confirmed with purified protein. All of the sulfotransferases were active in sulfonating p-nitrophenol, confirming we were expressing enzymatically active proteins (data not shown). When retinol was tested as a substrate (at total concentrations as high as 52 µM), none of the expressed mammalian sulfotransferases were able to produce AR or sulfonate retinol (data not shown), indicating that retinol is not a general substrate for cytosolic sulfotransferases.
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DISCUSSION |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Our results show that retinol dehydratase catalyzes the transfer of the sulfonate moiety from PAPS to a variety of acceptor molecules and that it has many other functional similarities to known cytosolic sulfotransferases. Like them, it exhibits product inhibition, has a low micromolar Km value for PAPS, and low micromolar Ki value for PAP. Additionally, dual pH optima and inhibition by various metals have been reported for a number of sulfotransferases (e.g. Refs. 17 and 29).
Mammalian cytosolic sulfotransferases are believed to have broad
substrate specificities (9), whereas plant sulfotransferases exhibit
strict substrate specificity (12). The low nanomolar Km value of retinol dehydratase for free retinol and the low micromolar Ki values for other retinoids
suggest a specific interaction between the insect sulfotransferase and retinoids. Among the wide variety of other compounds tested,
hydrocortisone and androsterone, and to a smaller extent
17-estradiol, seemed to bind tightly to retinol dehydratase. However
these compounds do not appear to be substrates. We could not detect
sulfonated products, and based on their chemical structures, it is not
likely that the sulfonated forms of these steroids are converted to
more stable products lacking the sulfonate moiety. It is worth noting the effect of ethanol on retinol dehydratase. As far as we know, sulfonation of ethanol has not been reported, even though many putative
substrates for sulfotransferases are often dissolved in ethanol or
other alcohols. Our results indicate that any analysis of the
sulfonation of such compounds needs to be revisited to take into
account the effect of the solvent.
The structure of mouse estrogen sulfotransferase suggested that a sulfotransferase-catalyzed reaction may proceed via the formation of a sulfonated enzyme intermediate, and for this reason, we tested whether this is the case for retinol dehydratase. As shown in Fig. 4, the retinol dehydratase-catalyzed reaction appears to proceed via ternary complex formation, rather than a ping-pong mechanism, although it is not possible to exclude a mechanism with an unstable enzyme intermediate. Previous kinetic studies with mammalian and plant sulfotransferases (13-19) have provided evidence for several types of sequential mechanisms. For example, studies of human phenol sulfotransferases (15, 16) and flavonol (17) sulfotransferases suggested an ordered Bi Bi mechanism where PAPS binds first, whereas sulfonation of cortisol was reported to follow an ordered mechanism where the steroid binds first (18). In the case of retinol dehydratase, the competitive modes of inhibition of all-trans-RA (Fig. 3A) and AR (Fig. 5C) versus retinol and of PAP versus PAPS (Fig. 5A), as well as the noncompetitive mode of inhibition of PAP versus retinol (Fig. 5B) and AR versus PAPS (Fig. 5D) are consistent with a Random Bi Bi reaction mechanism.
Based on the functional similarities between retinol dehydratase and mammalian cytosolic sulfotransferases, it is reasonable to believe that AR formation proceeds via the formation of retinyl sulfate (see Fig. 1). Our inability to detect retinyl sulfate in our sulfonation assay is presumably due to the fact that retinyl sulfate is an unstable and short-lived intermediate. This would also support the assumption inherent in our interpretation of the kinetic data that the formation of retinyl sulfate is the rate-limiting step in AR production. A more rigorous and detailed determination of the mechanism of AR formation awaits the chemical synthesis of retinyl sulfate which has not been reported to date.
Anhydroretinol is present in significant amounts in mammalian liver and
in smaller amounts in mammalian lung (5). By analogy to the insect
enzyme, we speculate that the mammalian enzyme responsible for the
production of AR will also be a sulfotransferase. None of the rat
sulfotransferases tested (Fig. 6) were able to produce AR, indicating
that retinol is not a general substrate for sulfotransferases. Based on
this finding we speculate that an as yet unidentified mammalian
sulfotransferase will have retinol dehydratase activity. We are
currently pursuing the cloning and characterization of additional
mammalian sulfotransferases to identify the mammalian retinol
dehydratase in the hope that it will provide important information
about the metabolism of vitamin A and the synthesis of
retro-retinoids.
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ACKNOWLEDGEMENTS |
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We thank Dr. Fadila Derguini (Sloan-Kettering Cancer, New York) for kindly providing us with 14-HRR, 13,14-dihydroxyretinol, AR, and 4-oxoretinol; Dr. Philip Cole (Rockefeller University, New York) for help in using the computer program KinetAsyst II and analyzing our data; Dr. Lonny Levin (Joan and Sanford I. Weill Medical College of Cornell University, New York) for critical reading of the manuscript; and an anonymous reviewer for their helpful insights.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant DK48022.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.
Supported by National Eye Institute Training Grant NRSA T32EY07138.
§ To whom correspondence should be addressed: Dept. of Pharmacology, Joan and Sanford I. Weill Medical College of Cornell University, 1300 York Ave., New York, NY 10021. Tel.: 212-746-6274; Fax: 212-746-8835; E-mail: jobuck{at}mail.med.cornell.edu.
The abbreviations used are: RA, retinoic acid; 14-HRR, 14-hydroxy-retro-retinol; AR, anhydroretinol; PAPS, 3'-phosphoadenosine 5'-phosphosulfate; PAP, 3'-phosphoadenosine 5'-phosphate; BSA, bovine serum albumin; ST, sulfotransferase; DTT, dithiothreitol; Mops, 4-morpholinepropanesulfonic acid; Ches, 2-(cyclohexylamino)ethanesulfonic acid; Caps, 3-(cyclohexylamino)propanesulfonic acid; Mes, 4-morpholineethanesulfonic acid.
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REFERENCES |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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