PHYTOCHEMICAL ANALYSIS Phytochem. Anal. 12, 363–365 (2001) DOI: 10.1002/pca.604 An HPLC Method for the Assay of UDP-glucose Pyrophosphorylase and UDP-glucose-4epimerase in Solieria chordalis (Rhodophyceae) F. Goulard,* M. Diouris, E. Deslandes and J. Y. Floc’h Laboratoire d’Ecophysiologie et de Biotechnologies des Algues Marines, IUEM-UBO, Technopole Brest-Iroise, place Nicolas Copernic, 29280 Plouzane, France An efficient method to assay both UDP-glucose pyrophosphorylase and UDP-glucose-4-epimerase in a crude extract of the red seaweed, Solieria chordalis is described. The method is based on the direct quantification by reverse-phase high-performance liquid chromatography of the UDP-sugars generated in the reaction mixture. UDP-glucose, UDP-galactose and UTP were detected by spectrophotometry at 254 nm and their recoveries ranged from 97 to 100%. In the course of the reaction, a correlation was observed between the reduction in the area of the substrate peak and the occurrence of product peak(s). This highly reproducible method for enzyme assay is fast since no intermediate reaction mixture is required. Copyright # 2001 John Wiley & Sons, Ltd. Keywords: High-performance liquid chromatography; UDP-glucose pyrophosphorylase; UDP-glucose-4-epimerase; Solieria chordalis. INTRODUCTION In plant tissues, nucleotide sugars (NDP-sugars) play a major role in polysaccharide biosynthesis. They are, indeed, the substrates for polysaccharide glycosyltransferases (see Gibeaut, 2000 for a review). The enzymes involved in NDP-sugar production are mainly pyrophosphorylases which catalyse the formation of NDP-sugars from NTP and a sugar-1-phosphate. Pyrophosphorylases are ubiquitous; the most active among them is UDPglucose pyrophosphorylase (EC 2.7.7.9), which catalyses the formation of UDP-glucose (Feingold and Avigad, 1980). This enzyme plays a major part in the distribution of glycosyl units to starch, sucrose and cell-wall polysaccharides in higher plants (Kleczkowski, 1994). Once UDP-glucose is formed, several enzymes act to provide the cell with most of the other NDP-sugars required in polysaccharide biosynthesis. In the first steps of metabolism, UDP-glucose can be converted into UDPgalactose by UDP-glucose-4-epimerase (EC 5.1.3.2), which catalyses the direct inter-conversion of UDPglucose and UDP-galactose (Leloir, 1951; Feingold and Avigad, 1980). In red algae, the production of UDPgalactose by this enzyme is required in the synthesis of both cell-wall components (agar, carrageenan and porphyran) and floridoside (Kremer and Kirst, 1981). HPLC is a fast and sensitive technique that provides the high resolution required for the detection and quantification of compounds in complex biological samples. This report describes a method for the separation and quantification of NDP-sugars by reverse-phase HPLC. Because of its capability to separate UDP-glucose, UDP* Correspondence to: F. Goulard, Laboratoire d’Ecophysiologie et de Biotechnologies des Algues Marines, IUEM-UBO, Technopole Brest-Iroise, place Nicolas Copernic, 29280 Plouzane, France. Email: F.Goulard@univ-brest.fr Contract/grant sponsor: Conseil Régional de Brétagne. Copyright # 2001 John Wiley & Sons, Ltd. galactose and UTP, this method is suitable for the assay of UDP-glucose pyrophosphorylase and UDP-glucose-4epimerase in crude extracts of the red seaweed Solieria chordalis (C. Agardh) J. Agardh (Rhodophyceae). EXPERIMENTAL Reagents. Glucose-1-phosphate (Glc-1-P), UTP, UDPglucose (UDP-glc) and UDP-galactose (UDP-gal) were purchased from Sigma (Poole, UK). HPLC analysis. A Waters (Bedford, MA, USA) HPLC system used was equipped with a model 626 pump, a Rheodyne injector and a model 486 variable wavelength detector set at 254 nm. Reverse-phase chromatography was performed on a Waters Nova-Pack column (25  4.6 cm i.d.; 4 mm) using 40 mm triethylamine phosphate (pH 6.5) as mobile phase at a flow rate of 1 mL/min. The injection volume was 50 mL, and all analyses were performed at room temperature. Peaks were identified by comparing their retention times with those of co-eluted standards, and quantification was performed by integration of peak areas and comparison with those produced by standards of known concentrations prepared in the same solvent. Enzyme extraction. Fresh algae (ca. 10 g) was frozen in liquid nitrogen, ground to a fine powder in a mortar and thawed in 20 mL of 50 mM Tris-hydrochloride buffer (pH 8.5) containing 1 mM magnesium chloride, 0.5 M potassium chloride, 10 mM 2-mercaptoethanol, 0.5 mM phenylmethylsulphonyl fluoride (PMSF) and 0.1% Triton X-100. The cells contained in this suspension were disrupted with two 15 s bursts (50 W; 20 kHz) using a sonicator model Bioblock Scientific Vibra Cell (Bioblock Scientific, Illkrich, France). After a 20 min centrifugation Received 21 July 2000 Revised 26 December 2000 Accepted 2 January 2001 364 F. GOULARD ET AL. Figure 1. The HPLC separation of the standards UDP-gal, UDP-glc and UTP on a Nova-Pack reverse-phase column eluted with 40 mM triethylamine phosphate (pH 6.5; for chromatographic protocol see Experimental section). Key to peak identities: 1, UDP-gal; 2, UDP-glc; 3, UTP. (8500 g; 4°C) the supernatant was removed and is referred to as the “enzyme extract” in this report. Enzymes assay. For the assay of UDP-glucose pyrophosphorylase, the reaction mixture contained 0.4 mL of “enzyme extract”, 0.5 mL of 50 mM Tris-hydrochloride buffer (pH 8.5) containing 1 mM magnesium chloride, and 20 mL of inorganic pyrophosphatase (0.2 U; Sigma). The reaction was started by the addition of 0.1 mL of a mixture composed of 2.5 mM Glc-1-P and 1 mM UTP; after incubation at 20  1°C for 1 h the reaction was stopped by adding 50 mL of 1 M hydrochloric acid. Following centrifugation (10 min; 8500 g; 4°C) the supernatant was submitted to a solid-phase purification on an Oasis cartridge (Waters), then filtered through a Millipore (Bedford, MA, USA) cartridge (0.2 mm pore size) and assayed by HPLC. For the assay of UDP-glucose-4-epimerase, the reaction mixture contained 0.4 mL of “enzyme extract”, 0.5 mL of 50 mM Tris-hydrochloride buffer (pH 8.5) containing 1 mM magnesium chloride, and 0.1 mL 1 mM UDP-glc. The reaction was stopped by adding 50 mL of 1 M hydrochloric acid. After centrifugation, purification and filtration as described above, the supernatant was analysed by HPLC. Some extracts were heated in a boiling water bath to allow the comparison of enzymatic activity in a boiled extract (inactive enzyme) and a sample (active enzyme) during a 1 h incubation. Enzyme activity was determined through the integration of peak areas; protein concentration was measured using the method of Bradford (1976) using a BioRad (Hercules, CA, USA) reagent kit with bovine serum albumin as standard. Figure 2. HPLC pro®les of the UDP-glucose-4-epimerase reaction mixture containing ªenzyme extractº from Solieria chordalis [boiled extract in (A) and active sample in (B)] following a 1 h incubation. For key to peak identities see legend to Fig. 1. UDP-glc and UTP to the protein extraction buffer resulted in a recovery of nucleotides in the range 97–100%. Injections of 1–10 nmol of each of the three compounds above did not alter their chromatographic separation, and also demonstrated that the relationship between the amounts injected and the peak areas was linear: UDPgal (r = 0.998), UDP-glc (r = 0.994) and UTP (r = 0.996). Concentrations above 10 nmol were not employed. Figure 2 displays the elution pattern obtained from 50 mL of UDP-glucose-4-epimerase reaction mixture with samples containing inactivate enzyme (A) and active enzyme (B) after a 1 h incubation. These two chromatograms illustrate the enzymatic conversion of UDP-glc into UDP-gal; note the increase of UDP-gal peak [Fig. 2(B)]. RESULTS AND DISCUSSION Figure 1 shows the separation of the standards UDP-gal, UDP-glc and UTP on a Nova-Pack column; the standards were eluted with retention times of 8.7, 9.8 and 17.2 min, respectively. Addition of known amounts of UDP-gal, Copyright # 2001 John Wiley & Sons, Ltd. Figure 3. HPLC pro®les of the UDP-glucose pyrophosphorylase reaction mixture containing ªenzyme extractº from Solieria chordalis [boiled extract in (A) and active sample in (B)] following a 1 h incubation. For key to peak identities see legend to Fig. 1. Phytochem. Anal. 12: 363–365 (2001) ASSAY OF UDP-GLUCOSE PYROPHOSPHORYLASE AND 4-EPIMERASE Figure 3 shows the elution pattern obtained from 50 mL of UDP-glucose pyrophosphorylase reaction mixture with samples containing inactivate enzyme (A) and active enzyme (B) after a 1 h. Comparison of the profiles reveals a reduction of the UTP peak concomitant with an increase of UDP-glc and the occurrence of UDP-gal. As UDP-glc was formed from Glc-1-P and UTP, it was partly transformed into UDP-gal by UDP-glucose-4epimerase, an enzyme contained in the “enzyme extract”. Thus, in order to quantify UDP-glucose pyrophosphorylase activity, the occurrence of UDP-gal must be taken into account. Using this method, the enzymatic activities of the extract were determined as 10 nmol UDP-glc formed per min per mg protein for UDP-glucose pyrophosphorylase, and 4 nmol UDP-gal formed per min per mg protein for UDP-glucose-4-epimerase. Several methods for the determination of UDP-glucose pyrophosphorylase activity have been described in the literature, and some of these are based on enzymatic assays. Thus, the occurrence of UDP-glc in the course of the reaction has been determined by UDP-glucose dehydrogenase, the NADH produced being followed spectrophotometrically at 340 nm (Dillard et al., 1983; Elling, 1995). The same method has been applied to detect UDP-glucose-4-epimerase (Fan and Feingold, 1969; Thomson et al., 1984), but this procedure supposes that all of the UDP-glc that appears or disappears is related to the activity of the pyrophosphorylase or the epimerase since only the reaction product is quantified. When, as illustrated in Fig. 3, the UDP-glc produced by the UDP-glucose pyrophosphorylase is partly converted into UDP-gal by UDP-glucose-4-epimerase, it is not detected when using the enzymatic assay mentioned above. Furthermore, such enzymatic procedures require tedious and time-consuming intermediate reactions. Chemical assays have also been performed in order to determine the amount of inorganic pyrophosphate produced by UDP-glucose pyrophosphorylase (Hondo et al., 1983). Inorganic pyrophosphate was converted into inorganic phosphate by inorganic pyrophosphatase, and the product was measured chemically. However, this method implies that all of the pyrophosphate and 365 phosphate produced must result from UDP-glucose pyrophosphorylase activity. Pyrophosphorylase activities measured in this way are typically much greater than those measured using a radioactive assay. However, the transformation of inorganic pyrophosphate into inorganic phosphate may result in the channelling of the UDPglucose pyrophosphorylase reactants toward UDP-glucose synthesis This is why, in our experiment, inorganic pyrophosphatase was added to the reaction mixture in order to shift the equilibrium constant and mimic what happens in plant tissues that contain pyrophosphatases. The best procedure to ensure that any product measured results from enzyme activity is, of course, through the use of radioactive labelling. Radioactive assays have used either [14C]-Glc-1-P to measure the synthesis of [14C]-UDP-glucose (Ghosh and Preiss, 1966) or [32P]-inorganic pyrophosphate to measure the [32P]UTP formed in the pyrophosphorolysis reaction (Shen and Preiss, 1964). Unfortunately, such techniques, although precise and very sensitive, are very expensive and, owing to the need to avoid risk of contamination, require specific infrastructure and equipment. It is for these reasons that we have developed a fast procedure for the separation of UDP-gal, UDP-glc and UTP in order to detect and quantify UDP-glucose pyrophosphorylase and UDP-glucose-4-epimerase activities in cell-free extracts of S. chordalis. The HPLC procedure described in this paper permits the separation of the first three compounds with a good recovery and reproducibility. The method has proved to be a reliable and easy-to-use technique to measure enzyme activities in S. chordalis, and would also be useful to detect these enzymes in samples from various origins. Compared with chemical procedures, HPLC is reproducible and does not require any intermediate, which is of a very high interest. Acknowledgements This work was supported by the Conseil Régional de Brétagne with a fellowship to F. G. The authors wish to thank M. P. Friocourt for the correction of the English manuscript. REFERENCES Bradford MM. 1976. 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