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β-phosphorothioate analogs of nucleotide sugars are resistant to hydrolytic degradation and utilized efficiently by glycosyltransferases
ANALYTICAL
BIOCHEMISTRY
197,40-46
(1991)
,&Phosphorothioate Analogs of Nucleotide Sugars Are Resistant to Hydrolytic Degradation and Utilized Efficiently by Glycosyltransferases Richard B. Marchase,* Maureen F. Burkart,* Angel Barry D. Shur,? Gary T. Overmeyer,$ and D. Roger
A. Rivera,* Shaw$
Benjamin
L. Clarke,?
*Department of Cell Biology, The University of Alabama at Birmingham, Birmingham, Alabama and Tumor Institute, Houston, Texas; and $Dupont-NEN Products, Boston, Massachussetts
Received
January
35294; TMD Anderson
Hospital
l&l991
The (Y- and &phosphorothioate analogs of UDP-Gal and UDP-Glc, in which a sulfur is exchanged for a nonbridging oxygen at one of the phosphate groups, have been synthesized and tested for their resistance to enzymatic degradation and for their usefulness in glycosyltransferase reactions. The (Y analogs were found to be no more resistant to hydrolysis than the native nucleotide sugars, but as previously reported (R. B. Marchase et al. (1987) Biochim. Biophys. Acta 916: 157) the j3S analogs were approximately 10 times more resistant. The /IS analog and native UDP-Glc were found to have comparable Km’s when used in assays for glucosylphosphoryl dolichol synthase with rat liver and hen oviduct microsomes, although the apparent V,, of the reaction was about twofold higher for the analog, presumably due to its resistance to degradation. Partially purified 4&galactosyltransferase exhibited a V,, with (@S)UDP-Gal that was only slightly lower than that with UDP-Gal and a K,,, that was slightly increased. The effectiveness of the analog was especially apparent in assays for 4&galactosyltransferase on intact sperm and in rat liver homogenates, in which hydrolysis of the normal substrate was very rapid and net incorporation was at least 4 times greater with the @S analog in each 0 1991 Academic Press, Inc. system.
The assessment of glycosyltransferase activities is confounded in most tissue homogenates by the activities of enzymes such as nucleotide pyrophosphatases that rapidly hydrolyze the required sugar nucleotides. In certain tissues like gut (1,2) and liver (3), even millimolar levels of sugar nucleotide are degraded within minutes. In attempts to overcome this problem other
investigators have included metal chelators (4-6) or nucleotides or their derivatives (3) that do not significantly affect the transferase reaction of interest but inhibit or compete in the degradative reaction. An alternative approach to this problem would be to identify nucleotide sugar analogs that were utilized efficiently by glycosyltransferases but were resistant to degradation. The /3-phosphorothioate analogs of these compounds described here, in which a sulfur is exchanged for a nonbridging oxygen at the P-phosphate
group, appear to have these properties. Marchase et al. (7) first reported the synthesis of the P-phosphorothioate analog of UDP-Glc [ (@S)UDPGlc]. This synthesis came about as an outgrowth of studies of the UDP-glucose:glycoprotein glucose-l-phosphotransferase (Glc-phosphotransferase) and utilized 35S in the phosphorothioate to allow for the selective study of the incorporation of the sugar phosphate from the nucleotide sugar. Not only was the analog efficiently utilized by the Glc-phosphotransferase, but it proved to be at least 10 times more resistant to enzymatic degradation than the parent compound. Thus, much greater incorporation into product was observed than was seen with the natural substrate. This raised the possibility that other, more commonly studied glycosyltransferases could also be assayed using PS analog compounds containing an isotopically unlabeled phosphorothioate and a labeled sugar. We report here that the PS analog of UDP-Gal and UDP-Glc, but not the crS analogs, are resistant to enzymatic hydrolysis and appear to provide enhanced incorporation with impure sources of transferase in which hydrolysis of the nucleotide sugar is presently a significant limitation. The synthesis of (&S)UDP-Gal and (/3S)UDP-Glc has recently been independently reported by Singh et al. (8).
40 All
0003-269’7/91$3.00 Copyright 0 1991 by Academic Press, Inc. rights of reproduction in any form reserved.
ENZYME
MATERIALS
AND
ASSAY
OF
@-PHOSPHOROTHIOATE
METHODS
Materials Sprague-Dawley male rats and CD1 male mice were purchased from the Charles River Breeding Farm. White leghorn chicken eggs were obtained from a local hatchery and grown to Embryonic Day 10 in Sears Favorite egg incubators. Some initial preparations of the /3-phosphothioate analog of UDP-Glc were prepared by R.B.M. (as described in the next section), but all those used in the reported experiments were obtained commercially. (&S)UDP-[3H]Gal[NET-1014], (PS)UDPGal[NLP-0411, (/3S)UDP-[3H]G1~[NET-1015], and (PS)UDP-Glc[NLP-0421, as well as all other radioactive sugars and nucleotide sugars, were obtained from DuPont-NEN. Various other reagents and supplies were purchased from the following sources: phenylmethylsulfonyl fluoride (PMSF,l Boehringer-Mannheim; Triton X-100 (reagent grade), Research Product International; Aquasol-2, DuPont-NEN; and unlabeled sugar nucleotides, N-acetylglucosamine, ovalbumin, preparative enzymes, and 4P-galactosyltransferase, Sigma. Methods The Synthesis and degradation of nucleotide sugars. /3S analog of UDP-[3H]G1~ was synthesized in initial preparations essentially as previously described (7). Briefly, 100 nmol (@)ATP was combined with 80 nmol [l-3H]glucose (15 Ci/mmol) in the presence of 1.6 pmol MgCl,, 0.6 U hexokinase, and 50 mM Tris-HCl, pH 7.6, in a total volume of 200 ~1 and incubated for 18 h at 22’C; 20 pmol UTP, 40 pmol glucose 1,6-diphosphate, 1.6 U phosphoglucomutase, 25 U UDP-Glc pyrophosphorylase, and 15 U inorganic pyrophosphatase were then added in a total additional volume of 100 ~1 and the incubation was continued for 18 h. The reaction mixture was then applied to a Waters C,, reversed-phase highperformance liquid chromatography column (4.6 mm X 25 cm) and eluted in 5 mM tetrabutylammonium hydroxide and 30 mM phosphate in 12% methanol at 1 ml per minute. Retention times were determined by appropriate standards to be @S)UDP-Glc, 13.2 min; (cS)UDP-Glc, 18.1 min; UDP-Glc, 7.3 min; Glc-l(PO,S), 5.9 min; inorganic thiophosphate, 5.9 min. The aS analog of UDP-[3H]G1~ was synthesized from 5 nmol [3H]Glc-1-P (13 Ci/mmol) and 20 nmol (aS)UTP by the action of 25 U UDP-Glc pyrophosphorylase in the presence of 15 U inorganic pyrophosphate and 50 mM Tris-HCl, pH 7.6. Incubation was for 18 h at 22°C. The C,, purification described above was again utilized. However, all experiments reported in this con-
’ Abbreviations thase; PMSF, acid.
used: GPDS, phenylmethylsulfonyl
glucosylphosphoryl fluoride; TCA,
dolichol syntrichloracetic
NUCLEOTIDE
SUGARS
41
tribution utilized reagents commercially prepared by DuPont-NEN. Degradation of nucleotide sugars as a function of time was assessed following incubations with rat liver homogenates utilizing descending paper chromatography. Sprague-Dawley male rats were decapitated, and the livers were dissected and placed in minimal essential medium. The livers were minced, washed three times with ice-cold 200 mM sodium cadodylate buffer (pH 6.25), and sonicated in about 1 tissue vol of this buffer containing 0.2 mM PMSF with a Heat Systems sonicator (output setting 6) for three l- to 2-s bursts. The resulting sonicate was kept on ice. The amount of protein in the sonicate was quantitated with Bio-Rad protein assay reagent. Liver sonicate containing 0.2-l mg of protein was added to a 1.5-ml Eppendorf tube containing 10 PCi UDP-[3H]G1~, (aS)UDP-[3H]G1~, (PS)UDP-[3H]G1~, UDP-[3H]Gal, or (&S)UDP-[3H]Gal (all at 11.5 Gil mmol), 5 pmol Na cacodylate (pH 7.4), and 0.5 pmol MnCl,, in a total volume of 100 ~1. The samples were incubated up to 240 min at 37°C. Ten-microliter aliquots of the assay mixtures were applied to Whatman No. 1 paper and chromatographed for 19 h using 95% ethanol:1 M ammonium acetate, pH 3.8 (5:2), as the solvent. The lanes were then cut into l-cm segments and counted in a Packard Minaxi liquid scintillation system. R, values for UDP-Glc (0.231, cuGlc-1-P (0.35), and Pi (0.82) were determined with appropriate radioactive standards. Initial rates of degradation were calculated from the differences in nucleotide sugar levels between 0 and 5 min. Glucosylphosphoryl dolichol synthase (GPDS) assays. The assay used for GPDS activity was a modification of the procedure of Jensen and Schutzbach (9) as described by Clarke et al. (10). Hen oviduct microsomes were prepared by the method of Welply et al. (11) and rat liver microsomes by the method of Dallner (12). Microsomes were suspended in medium consisting of 10 mM Tris, 250 mM sucrose, 10 mM NaCl, 154 mM KCl, and 10 mM MnCl,. Latency for the enzymes &glucuronidase and hexose-6-phosphatase was checked as described (11); values of 80-95% latency were routinely obtained. The assay mixture included 5 pg dolichyl phosphate solubilized in 1% Triton X-100 (final Triton X-100 concentration, 3.1 mM), 50 pmol MnCl,, and 60 pg microsomal protein. The assay was initiated by adding the sugar nucleotide substrate consisting of 5 nmol (PS)UDP-Glc or UDP-Glc (unless otherwise specified) and lo-20 PCi (PS)UDP-[3H]G1~ or UDP-[3H]Glc (resulting in a specific activity of 2-4 Ci/mmol). The final reaction volume was 50 ~1 in 7-ml scintillation vials; a mark was placed at the rim of the vial to facilitate reagent additions. Assays were incubated at room temperature for 10 min following the addition of radiolabeled
42
MARCHASE
sugar nucleotide. The assay was terminated by the addition of 450 ~1 H,O and 450 ~1 H,O-saturated butanol followed by 3.6 ml of a xylene-based scintillation fluid, and the vials were vigorously mixed. Then the vials were centrifuged at 3500 rpm in a table-top centrifuge for 3 min to separate phases. Synthesis of [3H]glucosylphosphoryl dolichol was determined by scintillation counting in a Packard Prias liquid scintillation counter (10). Zero time controls were included to correct for the small amount of labeled substrate that partitioned into the organic phase (less than 1% of the total). Each reported value represents the mean of triplicates with a standard deviation of less than 10% of the mean. In the experiments in which various concentrations of substrate were used, the specific activity was held constant throughout. Kinetic parameters were derived from Lineweaver-Burke analyses with linear correlation coefficients of 30.95. Assays for 4fl-galactosyltransferase in liver homogenates and with partially purified enzyme. Assays for 4fl-galactosyltransferase in rat liver homogenate employed the same constituents as those described for nucleotide sugar degradation with the addition of 0.1% Triton X-100 and 30 InM GlcNAc or 5 mg/ml ovalbumin as exogenous acceptor. The reactions with ovalbumin were terminated by the addition of 1 ml cold 10% TCA and the incorporation of [3H]galactose determined by precipitation onto glass fiber filters followed by scintillation counting. In assays utilizing GlcNAc as an acceptor, the incorporation of [3H]galactose into disaccharide was determined using high-voltage borate paper electrophoresis (13). Zero time controls were subtracted from all reported values. Assays utilizing Sigma’s 4P-galactosyltransferase preparation were carried out under the same conditions, except that the concentration of the nucleotide sugar (always at 11.5 Ci/mmol) was varied. These assays were incubated for 15 min and used 0.005 unit of enzyme per assay. Assays for 4@-galactosyltransferase on intact mouse sperm. Viable cauda epididymal sperm were collected from CD1 male mice (Charles River), filtered, and washed as described (14). Assays for 4/3-galactosyltransferase activity on intact mouse sperm followed the protocols outlined by Shur and Hall (13). Incubations were performed at 37°C with 6 X lo6 sperm in 300 ~1 of Hepes-buffered saline, pH 7.2, containing 1.05 nmol UDP-t3H]Ga1 or (&S)UDP-[3H]Gal (each at 11.5 Ci/ mmol), 3 pmol Mn2+, and 9 hmol GlcNAc. At the indicated times, 50-~1 aliquots were terminated with 10 ~1 of 0.2 M NaEDTA, pH 7.2, and subjected to high-voltage borate electrophoresis (3000 V, 280 mA, 42 min). RESULTS
Enzymatic Degradation of the Phosphorothioate of Nucleotide Sugars
Analogs
Preparations of the aS and /3S analogs of UDP-[3H]Glc were incubated with aliquots of rat liver homoge-
ET
AL.
0
20
40
60
Time
60
100
120
140
(mid
FIG. 1. Degradation of UDP-[3H]Glc and its phosphorothioate analogs by rat liver homogenate; 1.0 mg of protein was present in each loo-p1 assay. At the specified times lo-k1 aliquots were removed and subjected to paper chromatography. The percentage of cpm’s remaining as the nucleotide sugar is plotted. (0) UDP-[3H]Glc; (0) (@S)UDP-[3H]Glc; (m) (c&)UDP-[~H]G~C.
nate, and their degradation rates were compared to that of the parent compound (Fig. 1). As was seen previously (7) with the /335S analog, the initial rate of (@S)UDP[3H]Glc degradation was about 10 times less than the initial degradation rate of the parent nucleotide sugar. In contrast, the &-phosphorothioate analog displayed normal lability. Because of this, only the @S analog was investigated further. As might be expected, the same magnitude of resistance to degradation was seen when (/3S)UDP-[3H]Gal was compared to its parent compound.2 The rate of degradation seen in the experiments with UDP-Glc is much more rapid than the OH-- and Mn2+dependent decomposition of this nucleotide sugar that occurs via the formation of the 1,2-cyclic phosphate sugar (15). However, independent experiments performed in the absence of protein suggest that the P-phosphorothioate analogs of UDP-Gal and UDP-Glc are also more stable to this possible source of degradation as well (data not shown).
Utilization Dolichol
of (&!3)UDP-[3H]Glc Synthase
by Glucosylphosphoryl
Previously, we had shown that net incorporation from (fi35S)UDP-Glc by the Glc-phosphotransferase in 2 Data may be found in DuPont-NEN Tech pp. 12-13,1989, and in subsequent corroborating
Update, data
Vol. (not
4, No. 4, shown).
ENZYME
ASSAY
OF
fl-PHOSPHOROTHIOATE
homogenized rat liver was about six times greater at 1 h than incorporation from the 32P-labeled parent compound (7). This result was partly due to the resistance of (@S)UDP-Glc to degradation and partly due to the resistance of the transferred cwGlc-1-P to degradation once on the acceptor glycoprotein ((7) and unpublished resuits) . In order to determine the generality of this increase, studies with the glucosyltransferase GPDS were initiated. These studies would not be confounded by differences in product stability, since only glucose is transferred to the lipid acceptor in this reaction (9,lO). GPDS activity utilizing (/3S)UDP-[3H]Gl~ was compared to that seen with UDP-[3H]Glc in previously frozen rat liver microsomes (Fig. 2A). Near-linear incorporation persisted for about twice as long when the analog was used in place of the parent compound and net incorporation after 1 h was 2.5 times as great. A LineweaverBurke plot of the apparent kinetics (assessed at 10 min) yielded nearly identical apparent K,,,‘s of about 2 pM but an apparent V,,, 3.6 times greater for the analog than for the normal substrate (Fig. 2B). In an independent experiment, the apparent kinetic parameters were assessed for GPDS from fresh preparations of both hen oviduct and rat liver microsomes. As can be seen in Table 1, the apparent Km’s for the analog and parent were again found to be about equal, while the apparent V,, s were greater for the analog by factors of 2.6 and 1.7, respectively. In an additional study the competitive effect of the unlabeled analog on incorporation from UDP-[3H]Glc by rat liver microsomes was determined. Apparent K,,,‘s were determined for UDP-Glc in the presence of 0,2.5, 10, and 25 PM (PS)UDP-Glc. Lineweaver-Burke analyses were consistent with a competitive mode of inhibition (16) and yielded, with a regression coefficient of 0.981, a Ki for @S)UDP-Glc of 5.0 PM (data not shown). This figure is, as expected, comparable to the Km found for @S)UDP-Glc in single-substrate experiments (Fig. 2B and Table 1). All of these data suggest that GPDS, like the Glcphosphotransferase (7), recognized @S)UDP-Glc and UDP-Glc with comparable affinity. The two- to threefold increase in net incorporation seen with GPDS is likely due to the increased rate of degradation of UDPGlc over that of the analog. Utilization
of (PS)UDP-Gal
by 4P-Galuctosyltransferase
The activity of 4@-galactosyltransferase in unfractionated rat liver homogenates, utilizing ovalbumin as acceptor, was assessed using (fiS)UDP-[3H]Gal and the parent UDP-[3H]Gal. Under one set of assay conditions used, the parent compound showed linear incorporation for less than 1 h, while the analog was still nearly linear after 2 h. Net incorporation was about four times greater with (/?S)UDP-[3H]Gal (Fig. 3). Comparable re-
NUCLEOTIDE 20
43
SUGARS
/A (@I
Time
UDP-t3H)Glc
(mid
0.01 d
0
1
2
3
4
-I
5
6
WI FIG. 2. (A) Synthesis of glucosylphosphoryl dolichol from UDP[3H]Glc or (&3)UDP-[3H]Glc by rat liver microsomes as a function of time. (0) UDP-[3H]Glc; (0) (@S)UDP-[aH]Glc. (B) LineweaverBurke analysis of apparent GPDS activity assessed following lo-min incubations. Units of velocity are fmol/min/mg protein. Units of substrate are lo- M. (0) UDP-[3H]Glc; (m) (@)UDP-[aH]Glc.
sults were found when 30 mM GlcNAc was employed as the acceptor.’ Since the inclusion of compounds that inhibit UDP-Gal breakdown results in higher net incorporation of Gal into its acceptors ((3-6) and data not shown), the most likely explanation for these data is that the resistance of the analog to hydrolysis is responsible for the observed increases in transfer. The advantage of the analog was also seen in experiments assaying cell surface 4P-galactosyltransferase on intact mouse sperm (Fig. 4). Increases in incorporation
44
MARCHASE TABLE
Utilization
of (@S)UDP-Glc Dolichol
ET
AL.
1
30
by the Glucosylphosphoryl Synthase
K,, (PM)
V,,
1 ?0
(fmol/min/mg) E & St (D
Rat microsomes UDP-glucose (@l)UDP-glucose
2.41 + 1.70 2.83 t 1.14
42.5 + 108.3 +
0 =E
19.1 30.5
10
8 Chicken UDP-glucose (@)UDP-glucose
2
microsomes
3.54 * 1.54 2.73 -t 1.36
3810 6360
Note. Glucosylphosphoryl dolichol synthase activity at various concentrations of either UDP-[3H]glucose [3H]glucose. The kinetic parameters were determined squares analysis fitted to a Lineweaver-Burke plot. listed as mean + SD with n = 3.
-t120 k940 was measured or (&S)UDPusing a leastEach value is
from UDP-[3H]Gal were not seen after 0.5 h, while incorporation from (PS)UDP-[3H]Gal continued to increase for 2 h. Net transfer at 90 min was nearly six times as great with the analog. These results thus confirm a preliminary report2 Since by 30 min hydrolysis of micromolar levels of UDP-Gal is nearly complete in these cell suspensions (data not shown), the increased transfer at later times is again attributable to the stability of the analog. Activity was also assessedusing a commercially available partially purified 4@-galactosyltransferase from milk. This preparation contains little nucleotide pyro-
20
I
:
UDP-QaI
o1/---0
30
60
90
120
time (min) FIG. 4. Synthesis of N-acetyllactosamine from 30 mM GlcNAc and UDP-[3H]Gal or (@S)UDP-[3H]Gal by intact mouse sperm. At the indicated times the formation of the product was determined by highvoltage borate electrophoresis. (0) UDP-[3H]Gal; (0) (@S)UDP[aH]Gal.
phosphatase activity (data not shown). With this preparation, in which nucleotide sugar hydrolysis was not a factor, a study of the formation of N-acetyllactosamine using 30 mM GlcNAc as acceptor was undertaken with both UDP-[3H]Ga1 and (pS)UDP-[3H]Gal. A Lineweaver-Burke analysis of the data, as shown in Fig. 5, determined that this enzyme preparation utilized the PS analog of UDP-Gal with a V,,, of 4.7 nmol/min/mg protein, 82% of the parent nucleotide sugar V,, of 5.7
0.4 -
(@S)UDP-(%)Gal
> F
Time (min) FIG. 3. Galactosylation of ovalbumin via transfer from UDP[3H]Gal or (&S)UDP-[3H]Gal by rat liver homogenate; 1.0 mg of protein was present in each lOO-~1 assay. Ten-microliter aliquots were assessed for macromolecular incorporation by TCA precipitation at the times indicated. (Q UDP-[‘HIGal; (+) (j3S)UDP-[SH]Gal.
FIG. 5. Lineweaver-Burke analysis of the synthesis of N-acetyllactosamine by a commercially available 4@-galactosyltransferase preparation from 30 mM GlcNAc and UDP-[3H]Gal or (fiS)UDP[aH]Gal as a function of increasing sugar nucleotide concentration. All assays were terminated at 15 min. Units of velocity are nmol/minl mg protein. Units of substrate are 10-s M. (0 UDP-[3H]Gal; (+) (@S)UDP-[SH]Gal.
ENZYME
I. -10
r. 0
r 10
.
[(QS)
r 20
ASSAY
.
"*Al]
I.
OF
I.
B-PHOSPHOROTHIOATE
14 50
(LM)
FIG. 6.
Apparent K,,,‘s of the partially purified 4@-galactosyltransferase for UDP-Gal in the presence of varying amounts of (@S)UDPGal. K,,,‘s were determined at four inhibitor concentrations from Lineweaver-Burke analyses. The n-intercept is equal to -Ki for @S)UDP-Gal (15).
nmol/min/mg protein. The K,,, with the analog was 23% larger than that with the parent, 13.9 as opposed to 11.4 PM. Comparable results were seen in experiments utilizing ovalbumin as acceptor (data not shown). Competition experiments indicated little significant difference in the potency of the unlabeled analog and the parent compound to dilute incorporated label from UDP-[3H]Ga1 (data not shown). In addition, apparent Km’s were determined for UDP-Gal in the presence of 0, 5,25, and 50 I.IM (PS)UDP-Gal. Lineweaver-Burke analyses, when plotted (16) according to the formula
were again consistent with a competitive mode of inhibition and yielded a Ki for (PS)UDP-Gal of 8.6 PM (Fig. 6). DISCUSSION
Nucleoside phosphorothioate analogs have previously proven to be useful in investigations of nucleic acid synthesis, protein phosphorylation, and certain regulatoryphenomena (for review, see (17)). The exchange of sulfur for a nonbridging oxygen at a phosphate group does not disrupt the utilization of the nucleotides by a host of enzymes, including most DNA and RNA polymerases and protein kinases. This modification was first useful to researchers interested in enzymatic mechanisms since the exchange can confer chirality on the substituted phosphorous, and a number of the resulting
NUCLEOTIDE
SUGARS
45
diastereomers have been employed to determine the stereochemical course of over 50 reactions (17). These compounds have also proven to be of pragmatic importance in aspects of molecular biology. (aS)dNTP precursors are efficiently utilized by almost all DNA polymerases (17,18), allowing the synthesis of phosphorothioate-containing DNA. The introduction of 35Sinto the LYanalogs creates a labeled substrate that has a longer half-life and affords better resolution in autoradiography than the s-32P-labeled parent compounds. This stability and resolution have led to the use of these analogs as standard substrates in DNA sequencing protocols (19). Another use of these analogs has arisen from the finding that a variety of phosphodiesterases and endo- and exonucleases are unable to cleave the phosphorothioate internucleotidic groups in the analog-containing DNA (20-22). This resistance has been exploited to study the consequences of gaps or mismatches in nucleotide sequences that cannot be repaired (23) and to channel endonuclease cleavage sites to a single, nonsubstituted strand (24). In addition, the y-phosphorothioate analog of ATP, ($S)ATP, is utilized efficiently by most protein kinases but the resulting thiophosphorylated proteins are poor substrates for most phosphatases (17,25). The metabolic stability that therefore characterizes thiophosphorylated proteins has been exploited as a means of perturbing various regulatory equilibria. The (yS)phosphorothioate analog of GTP has also proven useful in numerous studies of the signal-transducing guanine nucleotide binding proteins (26), which remain in an activated state due to the resistance of (+)GTP to enzymatic hydrolysis. Similar results have also made ($S)GTP a useful reagent in the study of monomeric GTP-binding proteins (27). Thus, in these instances, as well as in the results with the nucleotide sugars shown here, anabolic enzymes (DNA polymerases, kinases, glycosyltransferases) utilize the analogs relatively well, while many catabolic enzymes (phosphatases, ATPases, nucleases, pyrophosphatases) are significantly less efficient in the presence of the phosphorothioate. Our results demonstrate that the phosphorothioate analogs of nucleotide sugars are significantly more resistant to hydrolytic degradation in tissue homogenates than are the parent compounds. In addition, it appears that at least for 4P-galactosyltransferase and GPDS the phosphorothioate analogs are efficiently utilized in the transfer reaction. The combination of these two properties leads to increases in net incorporation when these transferases are assayed in impure preparations. Practically, it appears that approximately a two- to sixfold net increase in the product of interest can be generated from the same amount of precursor. It would thus appear that the phosphorothioate analogs of these nucleotide sugars may prove useful in studies of at least certain glycosyltransferases, especially in systems in which
46
MARCHASE
significant degradation of sugar nucleotides is a compromising variable. Singh et al. (8) have also reported the synthesis of @S)UDP-Glc and have tested the efficacy of this nucleotide sugar analog in the glycogen synthase reaction. The analog was found to be considerably less effective than the parent nucleotide sugar. However, Lomako et al. (28) have reported successful incorporation of glucose l-phosphate into glycogen from the @3 analog of UDP-Glc. This reaction appears to be catalyzed by an enzyme that is distinct from glycogen synthase. These results serve to point out the necessity of determining whether the phosphorothioate analog of the appropriate nucleotide sugar is efficaciously utilized in the particular glycosyltransferase reaction of interest. However, these analogs do provide an independent approach for the study of glycosyltransferases in crude systems where substrate degradation is a significant problem.
We are grateful to Peg White for superb secretarial assistance. This work was supported by USPHS EY 06714 and by an NSF Presidential Young Investigator Award to R.B.M., by USPHS HD 23479 to B.D.S., and by USPHS GM33184 to William J. Lennarz.
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ACKNOWLEDGMENTS
1. Carlson, Biophys.
ET
M., Regan,
25. Graces,
L., and Schimmel,
D., and Fischer,
mun. 58,960-967. 26. Gilman, A. G. (1987)
E. J. (1974)
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Biachem. Biaphys. Res. Com-
Annu. Rev. Biochem. 56,615-649.
P., Glick, B. S., Malhotra, V., Weidman, P. J., Sera27. Melancon, fini, T., Gleason, M. L., Orci, L., and Rothman, J. E. (1987) Cell 5 1,1053-1062. 28. Lomako, Marchase,
J., Lomako, W. M., Kirkman, B. R., Whelan, R. B. (1989) FASEB J. 3, A1254.
W. J., and
BIOCHEMISTRY
197,40-46
(1991)
,&Phosphorothioate Analogs of Nucleotide Sugars Are Resistant to Hydrolytic Degradation and Utilized Efficiently by Glycosyltransferases Richard B. Marchase,* Maureen F. Burkart,* Angel Barry D. Shur,? Gary T. Overmeyer,$ and D. Roger
A. Rivera,* Shaw$
Benjamin
L. Clarke,?
*Department of Cell Biology, The University of Alabama at Birmingham, Birmingham, Alabama and Tumor Institute, Houston, Texas; and $Dupont-NEN Products, Boston, Massachussetts
Received
January
35294; TMD Anderson
Hospital
l&l991
The (Y- and &phosphorothioate analogs of UDP-Gal and UDP-Glc, in which a sulfur is exchanged for a nonbridging oxygen at one of the phosphate groups, have been synthesized and tested for their resistance to enzymatic degradation and for their usefulness in glycosyltransferase reactions. The (Y analogs were found to be no more resistant to hydrolysis than the native nucleotide sugars, but as previously reported (R. B. Marchase et al. (1987) Biochim. Biophys. Acta 916: 157) the j3S analogs were approximately 10 times more resistant. The /IS analog and native UDP-Glc were found to have comparable Km’s when used in assays for glucosylphosphoryl dolichol synthase with rat liver and hen oviduct microsomes, although the apparent V,, of the reaction was about twofold higher for the analog, presumably due to its resistance to degradation. Partially purified 4&galactosyltransferase exhibited a V,, with (@S)UDP-Gal that was only slightly lower than that with UDP-Gal and a K,,, that was slightly increased. The effectiveness of the analog was especially apparent in assays for 4&galactosyltransferase on intact sperm and in rat liver homogenates, in which hydrolysis of the normal substrate was very rapid and net incorporation was at least 4 times greater with the @S analog in each 0 1991 Academic Press, Inc. system.
The assessment of glycosyltransferase activities is confounded in most tissue homogenates by the activities of enzymes such as nucleotide pyrophosphatases that rapidly hydrolyze the required sugar nucleotides. In certain tissues like gut (1,2) and liver (3), even millimolar levels of sugar nucleotide are degraded within minutes. In attempts to overcome this problem other
investigators have included metal chelators (4-6) or nucleotides or their derivatives (3) that do not significantly affect the transferase reaction of interest but inhibit or compete in the degradative reaction. An alternative approach to this problem would be to identify nucleotide sugar analogs that were utilized efficiently by glycosyltransferases but were resistant to degradation. The /3-phosphorothioate analogs of these compounds described here, in which a sulfur is exchanged for a nonbridging oxygen at the P-phosphate
group, appear to have these properties. Marchase et al. (7) first reported the synthesis of the P-phosphorothioate analog of UDP-Glc [ (@S)UDPGlc]. This synthesis came about as an outgrowth of studies of the UDP-glucose:glycoprotein glucose-l-phosphotransferase (Glc-phosphotransferase) and utilized 35S in the phosphorothioate to allow for the selective study of the incorporation of the sugar phosphate from the nucleotide sugar. Not only was the analog efficiently utilized by the Glc-phosphotransferase, but it proved to be at least 10 times more resistant to enzymatic degradation than the parent compound. Thus, much greater incorporation into product was observed than was seen with the natural substrate. This raised the possibility that other, more commonly studied glycosyltransferases could also be assayed using PS analog compounds containing an isotopically unlabeled phosphorothioate and a labeled sugar. We report here that the PS analog of UDP-Gal and UDP-Glc, but not the crS analogs, are resistant to enzymatic hydrolysis and appear to provide enhanced incorporation with impure sources of transferase in which hydrolysis of the nucleotide sugar is presently a significant limitation. The synthesis of (&S)UDP-Gal and (/3S)UDP-Glc has recently been independently reported by Singh et al. (8).
40 All
0003-269’7/91$3.00 Copyright 0 1991 by Academic Press, Inc. rights of reproduction in any form reserved.
ENZYME
MATERIALS
AND
ASSAY
OF
@-PHOSPHOROTHIOATE
METHODS
Materials Sprague-Dawley male rats and CD1 male mice were purchased from the Charles River Breeding Farm. White leghorn chicken eggs were obtained from a local hatchery and grown to Embryonic Day 10 in Sears Favorite egg incubators. Some initial preparations of the /3-phosphothioate analog of UDP-Glc were prepared by R.B.M. (as described in the next section), but all those used in the reported experiments were obtained commercially. (&S)UDP-[3H]Gal[NET-1014], (PS)UDPGal[NLP-0411, (/3S)UDP-[3H]G1~[NET-1015], and (PS)UDP-Glc[NLP-0421, as well as all other radioactive sugars and nucleotide sugars, were obtained from DuPont-NEN. Various other reagents and supplies were purchased from the following sources: phenylmethylsulfonyl fluoride (PMSF,l Boehringer-Mannheim; Triton X-100 (reagent grade), Research Product International; Aquasol-2, DuPont-NEN; and unlabeled sugar nucleotides, N-acetylglucosamine, ovalbumin, preparative enzymes, and 4P-galactosyltransferase, Sigma. Methods The Synthesis and degradation of nucleotide sugars. /3S analog of UDP-[3H]G1~ was synthesized in initial preparations essentially as previously described (7). Briefly, 100 nmol (@)ATP was combined with 80 nmol [l-3H]glucose (15 Ci/mmol) in the presence of 1.6 pmol MgCl,, 0.6 U hexokinase, and 50 mM Tris-HCl, pH 7.6, in a total volume of 200 ~1 and incubated for 18 h at 22’C; 20 pmol UTP, 40 pmol glucose 1,6-diphosphate, 1.6 U phosphoglucomutase, 25 U UDP-Glc pyrophosphorylase, and 15 U inorganic pyrophosphatase were then added in a total additional volume of 100 ~1 and the incubation was continued for 18 h. The reaction mixture was then applied to a Waters C,, reversed-phase highperformance liquid chromatography column (4.6 mm X 25 cm) and eluted in 5 mM tetrabutylammonium hydroxide and 30 mM phosphate in 12% methanol at 1 ml per minute. Retention times were determined by appropriate standards to be @S)UDP-Glc, 13.2 min; (cS)UDP-Glc, 18.1 min; UDP-Glc, 7.3 min; Glc-l(PO,S), 5.9 min; inorganic thiophosphate, 5.9 min. The aS analog of UDP-[3H]G1~ was synthesized from 5 nmol [3H]Glc-1-P (13 Ci/mmol) and 20 nmol (aS)UTP by the action of 25 U UDP-Glc pyrophosphorylase in the presence of 15 U inorganic pyrophosphate and 50 mM Tris-HCl, pH 7.6. Incubation was for 18 h at 22°C. The C,, purification described above was again utilized. However, all experiments reported in this con-
’ Abbreviations thase; PMSF, acid.
used: GPDS, phenylmethylsulfonyl
glucosylphosphoryl fluoride; TCA,
dolichol syntrichloracetic
NUCLEOTIDE
SUGARS
41
tribution utilized reagents commercially prepared by DuPont-NEN. Degradation of nucleotide sugars as a function of time was assessed following incubations with rat liver homogenates utilizing descending paper chromatography. Sprague-Dawley male rats were decapitated, and the livers were dissected and placed in minimal essential medium. The livers were minced, washed three times with ice-cold 200 mM sodium cadodylate buffer (pH 6.25), and sonicated in about 1 tissue vol of this buffer containing 0.2 mM PMSF with a Heat Systems sonicator (output setting 6) for three l- to 2-s bursts. The resulting sonicate was kept on ice. The amount of protein in the sonicate was quantitated with Bio-Rad protein assay reagent. Liver sonicate containing 0.2-l mg of protein was added to a 1.5-ml Eppendorf tube containing 10 PCi UDP-[3H]G1~, (aS)UDP-[3H]G1~, (PS)UDP-[3H]G1~, UDP-[3H]Gal, or (&S)UDP-[3H]Gal (all at 11.5 Gil mmol), 5 pmol Na cacodylate (pH 7.4), and 0.5 pmol MnCl,, in a total volume of 100 ~1. The samples were incubated up to 240 min at 37°C. Ten-microliter aliquots of the assay mixtures were applied to Whatman No. 1 paper and chromatographed for 19 h using 95% ethanol:1 M ammonium acetate, pH 3.8 (5:2), as the solvent. The lanes were then cut into l-cm segments and counted in a Packard Minaxi liquid scintillation system. R, values for UDP-Glc (0.231, cuGlc-1-P (0.35), and Pi (0.82) were determined with appropriate radioactive standards. Initial rates of degradation were calculated from the differences in nucleotide sugar levels between 0 and 5 min. Glucosylphosphoryl dolichol synthase (GPDS) assays. The assay used for GPDS activity was a modification of the procedure of Jensen and Schutzbach (9) as described by Clarke et al. (10). Hen oviduct microsomes were prepared by the method of Welply et al. (11) and rat liver microsomes by the method of Dallner (12). Microsomes were suspended in medium consisting of 10 mM Tris, 250 mM sucrose, 10 mM NaCl, 154 mM KCl, and 10 mM MnCl,. Latency for the enzymes &glucuronidase and hexose-6-phosphatase was checked as described (11); values of 80-95% latency were routinely obtained. The assay mixture included 5 pg dolichyl phosphate solubilized in 1% Triton X-100 (final Triton X-100 concentration, 3.1 mM), 50 pmol MnCl,, and 60 pg microsomal protein. The assay was initiated by adding the sugar nucleotide substrate consisting of 5 nmol (PS)UDP-Glc or UDP-Glc (unless otherwise specified) and lo-20 PCi (PS)UDP-[3H]G1~ or UDP-[3H]Glc (resulting in a specific activity of 2-4 Ci/mmol). The final reaction volume was 50 ~1 in 7-ml scintillation vials; a mark was placed at the rim of the vial to facilitate reagent additions. Assays were incubated at room temperature for 10 min following the addition of radiolabeled
42
MARCHASE
sugar nucleotide. The assay was terminated by the addition of 450 ~1 H,O and 450 ~1 H,O-saturated butanol followed by 3.6 ml of a xylene-based scintillation fluid, and the vials were vigorously mixed. Then the vials were centrifuged at 3500 rpm in a table-top centrifuge for 3 min to separate phases. Synthesis of [3H]glucosylphosphoryl dolichol was determined by scintillation counting in a Packard Prias liquid scintillation counter (10). Zero time controls were included to correct for the small amount of labeled substrate that partitioned into the organic phase (less than 1% of the total). Each reported value represents the mean of triplicates with a standard deviation of less than 10% of the mean. In the experiments in which various concentrations of substrate were used, the specific activity was held constant throughout. Kinetic parameters were derived from Lineweaver-Burke analyses with linear correlation coefficients of 30.95. Assays for 4fl-galactosyltransferase in liver homogenates and with partially purified enzyme. Assays for 4fl-galactosyltransferase in rat liver homogenate employed the same constituents as those described for nucleotide sugar degradation with the addition of 0.1% Triton X-100 and 30 InM GlcNAc or 5 mg/ml ovalbumin as exogenous acceptor. The reactions with ovalbumin were terminated by the addition of 1 ml cold 10% TCA and the incorporation of [3H]galactose determined by precipitation onto glass fiber filters followed by scintillation counting. In assays utilizing GlcNAc as an acceptor, the incorporation of [3H]galactose into disaccharide was determined using high-voltage borate paper electrophoresis (13). Zero time controls were subtracted from all reported values. Assays utilizing Sigma’s 4P-galactosyltransferase preparation were carried out under the same conditions, except that the concentration of the nucleotide sugar (always at 11.5 Ci/mmol) was varied. These assays were incubated for 15 min and used 0.005 unit of enzyme per assay. Assays for 4@-galactosyltransferase on intact mouse sperm. Viable cauda epididymal sperm were collected from CD1 male mice (Charles River), filtered, and washed as described (14). Assays for 4/3-galactosyltransferase activity on intact mouse sperm followed the protocols outlined by Shur and Hall (13). Incubations were performed at 37°C with 6 X lo6 sperm in 300 ~1 of Hepes-buffered saline, pH 7.2, containing 1.05 nmol UDP-t3H]Ga1 or (&S)UDP-[3H]Gal (each at 11.5 Ci/ mmol), 3 pmol Mn2+, and 9 hmol GlcNAc. At the indicated times, 50-~1 aliquots were terminated with 10 ~1 of 0.2 M NaEDTA, pH 7.2, and subjected to high-voltage borate electrophoresis (3000 V, 280 mA, 42 min). RESULTS
Enzymatic Degradation of the Phosphorothioate of Nucleotide Sugars
Analogs
Preparations of the aS and /3S analogs of UDP-[3H]Glc were incubated with aliquots of rat liver homoge-
ET
AL.
0
20
40
60
Time
60
100
120
140
(mid
FIG. 1. Degradation of UDP-[3H]Glc and its phosphorothioate analogs by rat liver homogenate; 1.0 mg of protein was present in each loo-p1 assay. At the specified times lo-k1 aliquots were removed and subjected to paper chromatography. The percentage of cpm’s remaining as the nucleotide sugar is plotted. (0) UDP-[3H]Glc; (0) (@S)UDP-[3H]Glc; (m) (c&)UDP-[~H]G~C.
nate, and their degradation rates were compared to that of the parent compound (Fig. 1). As was seen previously (7) with the /335S analog, the initial rate of (@S)UDP[3H]Glc degradation was about 10 times less than the initial degradation rate of the parent nucleotide sugar. In contrast, the &-phosphorothioate analog displayed normal lability. Because of this, only the @S analog was investigated further. As might be expected, the same magnitude of resistance to degradation was seen when (/3S)UDP-[3H]Gal was compared to its parent compound.2 The rate of degradation seen in the experiments with UDP-Glc is much more rapid than the OH-- and Mn2+dependent decomposition of this nucleotide sugar that occurs via the formation of the 1,2-cyclic phosphate sugar (15). However, independent experiments performed in the absence of protein suggest that the P-phosphorothioate analogs of UDP-Gal and UDP-Glc are also more stable to this possible source of degradation as well (data not shown).
Utilization Dolichol
of (&!3)UDP-[3H]Glc Synthase
by Glucosylphosphoryl
Previously, we had shown that net incorporation from (fi35S)UDP-Glc by the Glc-phosphotransferase in 2 Data may be found in DuPont-NEN Tech pp. 12-13,1989, and in subsequent corroborating
Update, data
Vol. (not
4, No. 4, shown).
ENZYME
ASSAY
OF
fl-PHOSPHOROTHIOATE
homogenized rat liver was about six times greater at 1 h than incorporation from the 32P-labeled parent compound (7). This result was partly due to the resistance of (@S)UDP-Glc to degradation and partly due to the resistance of the transferred cwGlc-1-P to degradation once on the acceptor glycoprotein ((7) and unpublished resuits) . In order to determine the generality of this increase, studies with the glucosyltransferase GPDS were initiated. These studies would not be confounded by differences in product stability, since only glucose is transferred to the lipid acceptor in this reaction (9,lO). GPDS activity utilizing (/3S)UDP-[3H]Gl~ was compared to that seen with UDP-[3H]Glc in previously frozen rat liver microsomes (Fig. 2A). Near-linear incorporation persisted for about twice as long when the analog was used in place of the parent compound and net incorporation after 1 h was 2.5 times as great. A LineweaverBurke plot of the apparent kinetics (assessed at 10 min) yielded nearly identical apparent K,,,‘s of about 2 pM but an apparent V,,, 3.6 times greater for the analog than for the normal substrate (Fig. 2B). In an independent experiment, the apparent kinetic parameters were assessed for GPDS from fresh preparations of both hen oviduct and rat liver microsomes. As can be seen in Table 1, the apparent Km’s for the analog and parent were again found to be about equal, while the apparent V,, s were greater for the analog by factors of 2.6 and 1.7, respectively. In an additional study the competitive effect of the unlabeled analog on incorporation from UDP-[3H]Glc by rat liver microsomes was determined. Apparent K,,,‘s were determined for UDP-Glc in the presence of 0,2.5, 10, and 25 PM (PS)UDP-Glc. Lineweaver-Burke analyses were consistent with a competitive mode of inhibition (16) and yielded, with a regression coefficient of 0.981, a Ki for @S)UDP-Glc of 5.0 PM (data not shown). This figure is, as expected, comparable to the Km found for @S)UDP-Glc in single-substrate experiments (Fig. 2B and Table 1). All of these data suggest that GPDS, like the Glcphosphotransferase (7), recognized @S)UDP-Glc and UDP-Glc with comparable affinity. The two- to threefold increase in net incorporation seen with GPDS is likely due to the increased rate of degradation of UDPGlc over that of the analog. Utilization
of (PS)UDP-Gal
by 4P-Galuctosyltransferase
The activity of 4@-galactosyltransferase in unfractionated rat liver homogenates, utilizing ovalbumin as acceptor, was assessed using (fiS)UDP-[3H]Gal and the parent UDP-[3H]Gal. Under one set of assay conditions used, the parent compound showed linear incorporation for less than 1 h, while the analog was still nearly linear after 2 h. Net incorporation was about four times greater with (/?S)UDP-[3H]Gal (Fig. 3). Comparable re-
NUCLEOTIDE 20
43
SUGARS
/A (@I
Time
UDP-t3H)Glc
(mid
0.01 d
0
1
2
3
4
-I
5
6
WI FIG. 2. (A) Synthesis of glucosylphosphoryl dolichol from UDP[3H]Glc or (&3)UDP-[3H]Glc by rat liver microsomes as a function of time. (0) UDP-[3H]Glc; (0) (@S)UDP-[aH]Glc. (B) LineweaverBurke analysis of apparent GPDS activity assessed following lo-min incubations. Units of velocity are fmol/min/mg protein. Units of substrate are lo- M. (0) UDP-[3H]Glc; (m) (@)UDP-[aH]Glc.
sults were found when 30 mM GlcNAc was employed as the acceptor.’ Since the inclusion of compounds that inhibit UDP-Gal breakdown results in higher net incorporation of Gal into its acceptors ((3-6) and data not shown), the most likely explanation for these data is that the resistance of the analog to hydrolysis is responsible for the observed increases in transfer. The advantage of the analog was also seen in experiments assaying cell surface 4P-galactosyltransferase on intact mouse sperm (Fig. 4). Increases in incorporation
44
MARCHASE TABLE
Utilization
of (@S)UDP-Glc Dolichol
ET
AL.
1
30
by the Glucosylphosphoryl Synthase
K,, (PM)
V,,
1 ?0
(fmol/min/mg) E & St (D
Rat microsomes UDP-glucose (@l)UDP-glucose
2.41 + 1.70 2.83 t 1.14
42.5 + 108.3 +
0 =E
19.1 30.5
10
8 Chicken UDP-glucose (@)UDP-glucose
2
microsomes
3.54 * 1.54 2.73 -t 1.36
3810 6360
Note. Glucosylphosphoryl dolichol synthase activity at various concentrations of either UDP-[3H]glucose [3H]glucose. The kinetic parameters were determined squares analysis fitted to a Lineweaver-Burke plot. listed as mean + SD with n = 3.
-t120 k940 was measured or (&S)UDPusing a leastEach value is
from UDP-[3H]Gal were not seen after 0.5 h, while incorporation from (PS)UDP-[3H]Gal continued to increase for 2 h. Net transfer at 90 min was nearly six times as great with the analog. These results thus confirm a preliminary report2 Since by 30 min hydrolysis of micromolar levels of UDP-Gal is nearly complete in these cell suspensions (data not shown), the increased transfer at later times is again attributable to the stability of the analog. Activity was also assessedusing a commercially available partially purified 4@-galactosyltransferase from milk. This preparation contains little nucleotide pyro-
20
I
:
UDP-QaI
o1/---0
30
60
90
120
time (min) FIG. 4. Synthesis of N-acetyllactosamine from 30 mM GlcNAc and UDP-[3H]Gal or (@S)UDP-[3H]Gal by intact mouse sperm. At the indicated times the formation of the product was determined by highvoltage borate electrophoresis. (0) UDP-[3H]Gal; (0) (@S)UDP[aH]Gal.
phosphatase activity (data not shown). With this preparation, in which nucleotide sugar hydrolysis was not a factor, a study of the formation of N-acetyllactosamine using 30 mM GlcNAc as acceptor was undertaken with both UDP-[3H]Ga1 and (pS)UDP-[3H]Gal. A Lineweaver-Burke analysis of the data, as shown in Fig. 5, determined that this enzyme preparation utilized the PS analog of UDP-Gal with a V,,, of 4.7 nmol/min/mg protein, 82% of the parent nucleotide sugar V,, of 5.7
0.4 -
(@S)UDP-(%)Gal
> F
Time (min) FIG. 3. Galactosylation of ovalbumin via transfer from UDP[3H]Gal or (&S)UDP-[3H]Gal by rat liver homogenate; 1.0 mg of protein was present in each lOO-~1 assay. Ten-microliter aliquots were assessed for macromolecular incorporation by TCA precipitation at the times indicated. (Q UDP-[‘HIGal; (+) (j3S)UDP-[SH]Gal.
FIG. 5. Lineweaver-Burke analysis of the synthesis of N-acetyllactosamine by a commercially available 4@-galactosyltransferase preparation from 30 mM GlcNAc and UDP-[3H]Gal or (fiS)UDP[aH]Gal as a function of increasing sugar nucleotide concentration. All assays were terminated at 15 min. Units of velocity are nmol/minl mg protein. Units of substrate are 10-s M. (0 UDP-[3H]Gal; (+) (@S)UDP-[SH]Gal.
ENZYME
I. -10
r. 0
r 10
.
[(QS)
r 20
ASSAY
.
"*Al]
I.
OF
I.
B-PHOSPHOROTHIOATE
14 50
(LM)
FIG. 6.
Apparent K,,,‘s of the partially purified 4@-galactosyltransferase for UDP-Gal in the presence of varying amounts of (@S)UDPGal. K,,,‘s were determined at four inhibitor concentrations from Lineweaver-Burke analyses. The n-intercept is equal to -Ki for @S)UDP-Gal (15).
nmol/min/mg protein. The K,,, with the analog was 23% larger than that with the parent, 13.9 as opposed to 11.4 PM. Comparable results were seen in experiments utilizing ovalbumin as acceptor (data not shown). Competition experiments indicated little significant difference in the potency of the unlabeled analog and the parent compound to dilute incorporated label from UDP-[3H]Ga1 (data not shown). In addition, apparent Km’s were determined for UDP-Gal in the presence of 0, 5,25, and 50 I.IM (PS)UDP-Gal. Lineweaver-Burke analyses, when plotted (16) according to the formula
were again consistent with a competitive mode of inhibition and yielded a Ki for (PS)UDP-Gal of 8.6 PM (Fig. 6). DISCUSSION
Nucleoside phosphorothioate analogs have previously proven to be useful in investigations of nucleic acid synthesis, protein phosphorylation, and certain regulatoryphenomena (for review, see (17)). The exchange of sulfur for a nonbridging oxygen at a phosphate group does not disrupt the utilization of the nucleotides by a host of enzymes, including most DNA and RNA polymerases and protein kinases. This modification was first useful to researchers interested in enzymatic mechanisms since the exchange can confer chirality on the substituted phosphorous, and a number of the resulting
NUCLEOTIDE
SUGARS
45
diastereomers have been employed to determine the stereochemical course of over 50 reactions (17). These compounds have also proven to be of pragmatic importance in aspects of molecular biology. (aS)dNTP precursors are efficiently utilized by almost all DNA polymerases (17,18), allowing the synthesis of phosphorothioate-containing DNA. The introduction of 35Sinto the LYanalogs creates a labeled substrate that has a longer half-life and affords better resolution in autoradiography than the s-32P-labeled parent compounds. This stability and resolution have led to the use of these analogs as standard substrates in DNA sequencing protocols (19). Another use of these analogs has arisen from the finding that a variety of phosphodiesterases and endo- and exonucleases are unable to cleave the phosphorothioate internucleotidic groups in the analog-containing DNA (20-22). This resistance has been exploited to study the consequences of gaps or mismatches in nucleotide sequences that cannot be repaired (23) and to channel endonuclease cleavage sites to a single, nonsubstituted strand (24). In addition, the y-phosphorothioate analog of ATP, ($S)ATP, is utilized efficiently by most protein kinases but the resulting thiophosphorylated proteins are poor substrates for most phosphatases (17,25). The metabolic stability that therefore characterizes thiophosphorylated proteins has been exploited as a means of perturbing various regulatory equilibria. The (yS)phosphorothioate analog of GTP has also proven useful in numerous studies of the signal-transducing guanine nucleotide binding proteins (26), which remain in an activated state due to the resistance of (+)GTP to enzymatic hydrolysis. Similar results have also made ($S)GTP a useful reagent in the study of monomeric GTP-binding proteins (27). Thus, in these instances, as well as in the results with the nucleotide sugars shown here, anabolic enzymes (DNA polymerases, kinases, glycosyltransferases) utilize the analogs relatively well, while many catabolic enzymes (phosphatases, ATPases, nucleases, pyrophosphatases) are significantly less efficient in the presence of the phosphorothioate. Our results demonstrate that the phosphorothioate analogs of nucleotide sugars are significantly more resistant to hydrolytic degradation in tissue homogenates than are the parent compounds. In addition, it appears that at least for 4P-galactosyltransferase and GPDS the phosphorothioate analogs are efficiently utilized in the transfer reaction. The combination of these two properties leads to increases in net incorporation when these transferases are assayed in impure preparations. Practically, it appears that approximately a two- to sixfold net increase in the product of interest can be generated from the same amount of precursor. It would thus appear that the phosphorothioate analogs of these nucleotide sugars may prove useful in studies of at least certain glycosyltransferases, especially in systems in which
46
MARCHASE
significant degradation of sugar nucleotides is a compromising variable. Singh et al. (8) have also reported the synthesis of @S)UDP-Glc and have tested the efficacy of this nucleotide sugar analog in the glycogen synthase reaction. The analog was found to be considerably less effective than the parent nucleotide sugar. However, Lomako et al. (28) have reported successful incorporation of glucose l-phosphate into glycogen from the @3 analog of UDP-Glc. This reaction appears to be catalyzed by an enzyme that is distinct from glycogen synthase. These results serve to point out the necessity of determining whether the phosphorothioate analog of the appropriate nucleotide sugar is efficaciously utilized in the particular glycosyltransferase reaction of interest. However, these analogs do provide an independent approach for the study of glycosyltransferases in crude systems where substrate degradation is a significant problem.
We are grateful to Peg White for superb secretarial assistance. This work was supported by USPHS EY 06714 and by an NSF Presidential Young Investigator Award to R.B.M., by USPHS HD 23479 to B.D.S., and by USPHS GM33184 to William J. Lennarz.
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7. Marchase, R. B., Saunders, A. (1987) B&him. Biophys. Acta. 8. Singh, A. N., Newborn, J. S., Chem. 16,206-214. 9. Jensen, J. W., and Schutzbach, 41-48. 10. Clarke,
B. L., Naylor,
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4. Lau, J. T. Y., and Carlson, D. M. (1981) J. Biol. Chem. 266, 7142-7145. 5. Faltynek, C. R., Silbert, J. E., and Hof, L. (1981) J. Biol. Chem. 256, 7139-7141. 6. Kean, E. L., and Bighouse, K. J. (1974) J. Biol. Chem. 249,78137823.
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21. Brody, R. S., and Frey, P. A. (1981) Biochemistry 20,1245-1252. 22. Romaniuk, P. J., and Eckstein, F. (1982) J. Bial. Chem. 257, 7684-7688. D., Grisafi, P., Benkovic, S. J., and Botstein, D. (1982) 23. Shortle,
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ACKNOWLEDGMENTS
1. Carlson, Biophys.
ET
M., Regan,
25. Graces,
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