Phytochemistry, Vol. 30, No.9, pp. 2865-2867, 1991 0031-9422/91 $3.00 +0.00 Printed in Great Britain. Pergamon Press pic

CHARACTERIZATION OF MAIZE ENDOSPERM UDP-: DOLICHOL- GLUCOSYLTRANSFERASE

JAN A. MIERNYK and WALTER E. RIEDELL* Seed Biosynthesis Research Unit, USDA, Agricultural Research Service, National Center for Agricultural Utilization Research, Peoria, IL 61604, U.S.A.

(Received ill revised form 12 February 1991)

Key Word Index-Zea mays; Gramineae; maize; endosperm; tissue culture; dolichol cycle; glycolipid; glycosyl­ transferase.

Abstract-Uridine-diphosphate-glucose:dolichol-phosphate glucosyltransferase [EC 2.4.1.1l7J was solubilized from maize endosperm microsomes by treatment with 0.25% Triton X-IOO. The solubilized enzyme was partially purified by chromatography on columns of hydroxylapatite, diethylaminoethyl-Sephacel, concanavalin A-Sepharose, and uridine-diphosphate-hexanolamine-agarose. The purified transferase had no activity unless a -acceptor was included in the assays. K m values for uridine-diphosphate-glucose, dolichol-phosphate, and magnesium were 0.5 /lM, 1 1.2 /lg ml- , and 0.7 mM, respectively. The K j values for uridine-diphosphate, uridine-monophosphate and uridine­ diphosphate-glucuronic acid were 150, 650 and 7.6/lM, respectively. The in vitro activity of the transferase was inhibited 50% by 40 /lg ml- 1 of tunicamycin.

INTRODUCTION were less efficient in terms of either solubilization or stabilization of the enzyme (data not presented). The We have previously presented the results of detailed results of a typical purification are presented in Table 1. studies on the localization of the dolichol-cycle enzymes The degree of purification ranged from 80- to 100-fold from maize endosperm cells [1]. During the preliminary with recoveries from 7 to 10%. The final preparations characterization of the it was ob­ were not homogeneous, and when examined by SDS­ served that uridine-5'-diphosphate-glucose (UDP-Glc): PAGE there were three major and three minor bands lipid Glc-transferase activity in vitro was not dependent (data not presented). We were not able to determine upon added dolichol-phosphate (dolichol-P) acceptor. At which band(s) corresponded to the Glc-transferase. Re­ that time we speculated that the activity assayed might be cently, it was reported that the subunit mass of the yeast a combination of two UDP-Glc:lipid Glc-transferases, UDP-Glc:dolichol-P Glc-transferase is 35000 [3J; how­ one using endogenous sterols [e.g. 2J as the lipid acceptor ever none of the bands in the purified maize transferase and the other using endogenous dolichol-phosphate [1]. corresponded to this M value. There has previously been The solubilization and separation of the Glc-transferases r difficulty in purifying the enzymes of the dolichol cycle was undertaken in order to characterize the dolichol­ because ofinstability after solubilization [e.g. 4]. We were cycle enzyme without complications in interpretation of able to achieve some degree of stabilization by the the results due to competing reactions. inclusion of protease inhibitors plus UDP-Glc in the isolation and solubilization buffers. RESULTS AND DISCUSSION Anion-exchange chromatography resolved solubilized Glc-transferase activity into two peaks (Fig. 1). The Maize UDP-Glc:dolichol-P Glc-transferase was solu­ activity in the first peak was relatively high even without a bilized from washed microsomes with 0.25% Triton lipid-acceptor included in the assay mixtures and was X-lOO, as previously described [lJ, and purified by increased less than two-fold by addition of dolichol-P. sequential hydroxylapatite-, anion-exchange-, immob­ The second peak, eluting at a conductivity of ca ilized lectin- and affinity-chromatography. The maize 7 mMho, had very low activity when assayed without a enzyme was not solubilized by treatment of the micro­ lipid-acceptor and was stimulated more than lOO-fold somes with 1.0 M KCI or alkaline sodium carbonate. when dolichol-P was added to an assay previously lack­ Other detergents, including Tween-20, Brij 35, Nonidet ing acceptor (data not presented). The later eluting peak P-40, and the tensides octyl-glucoside and dodecyl­ of Glc-transferase activity from the anion-exchange col­ maltoside, at concentrations ranging from 0.1 to 1%, umn is the dolichol-cycle enzyme, and further purification of this activity was undertaken. After two additional chromatography steps (Table 1) there was no detectable *Present address: USDA, Agricultural Research Service, Glc-transferase activity without addition ofa ­ Northern Grain Insects Research Laboratory, RR No.3, P acceptor to the enzyme assays and there was zero Brookings, SD 57006, U.S.A. activity when 10 /lg ml- 1 of soybean p-sitosterol was

2865 2866 1. A. MIERNYK and W. E. RIEDELL

Table I. Partial purification of UDP-glucose:dolichol-phosphate glucosyltransferase from maize endosperm cultures

Total Specific Units activity Fold- Yield I Fraction (nkat) (mg) (units mg- ) purification (%J

Solubilized microsomes 116 750 0.15 1 100 Hydroxyapatite 102 283 0.36 2.4 88 DEAE-Sephacel 27 6.8 3.97 26.4 23 Con A-Sepharose 21 4.8 4.37 29.1 18 UDP-hexanolamine-agarose 8 0.6 13.11 87.3 7

Starting material was 1.5 kg of seven-day-old cells.

Table 2. Some characteristics of partially purified maize endosperm UDP-glucose: dolichol-phosphate glucosyltransfer­ ase

Optimum [Triton X-I00] (%) 0.Q15 pH optimum 7.5

K m UDP-glucose (tIM) 0.5 1 K m dolichol-P (pgml- )* 1.2 K m MgCl z (mM) 0.7 K i UDP (pM) 150.0 K i UMP(pM) 650.0

K i UDP-glucuronic acid (pM) 7.6 Fraction Number 10 .5 tunicamycin (pg ml- 1) 40.0

Fig. 1. Separation of the maize endosperm UDP-glucose: lipid *The kinetic constants for dolichol-P and tunicamycin are glucosyltransferase by anion-exchange chromatography. both presented as mass volume - 1 values rather than as molar solubilized from microsomal membranes by treatment concentrations because both compounds are mixtures of mo­ with 0.25% Triton X-IOO were chromatographed on a 1.6 x 22 lecular species. column of DEAE-Sephacel previously equilibrated with 10 mM Kinetic constants were derived from initial-rate studies ana­ TES pH 7.5 containing 0.25% Triton X-lOO. After washing, lysed by iterative curve-fitting using nonlinear regression [11]. bound proteins were eluted with a linear gradient of 0 to 0.5 M KCl in equilibration buffer. Maximum activity was 2.4 nkat fraction -I. Recovery of activity in fractions 12-34 was 69% of that loaded onto the column. Tunicamycin is a potent inhibitor of UDP­ GlcNAc:dolichol-P GlcNAc-transferase activity both in vivo and in vitro [5]. It is thought that tunicamycin

resembles a transition-state reaction intermediate. The K i substituted for dolichol-P. At equal substrate concentra­ value for the chick embryo GlcNAc-transferase is 5 nM tions, activity with decaprenol-P was 43% of that with [5J, and we previously reported that the K i for the maize dolichol-P (data not presented). Preparations purified endosperm GlcNAc-transferase is 14 ng ml-! [1]. While through the affinity-chromatography step were used for our observation that tunicamycin also inhibited maize in vitro catalytic and kinetic analyses. endosperm UDP-Glc: dolichol-P Glc-transferase was un­ In vitro catalytic activity of the purified dolichol-cycle expected, the previous results were verified with the transferase was maximal at a Triton X-100 concentration purified enzyme (Table 2). Because the kinetic mechanism of 0.015%, and pH value of 7.5 (Table 2). This pH of inhibition of the maize enzyme is unknown, we are optimum is significantly more alkaline than the pre­ presenting an 10 .5 value instead of a K i value. It was viously reported value [lJ which was the combination of previously reported that relatively high concentrations of dolichol and sterol transferase activities. Additionally, the tunicamycin nonspecifically inhibited protein synthesis,

K m value for UDP-Glc was 10-times lower than the mRNA synthesis, and even the transport of UDP-galac­ previously reported value [lJ, which also was the combi­ tose across Golgi membrane vesicles [5J, and it could be nation of the two enzyme activities. Other kinetic con­ that the effect upon the maize endosperm Glc-transferase stants (Table 2) for the purified dolichol transferase are is this type of nonspecific inhibition. similar to those previously reported. UDP-glucuronic During purification, maize endosperm Glc-transferase acid was a potent inhibitor of the maize dolichol trans­ was adsorbed to immobilized concanavalin A and then ferase (Table 2). Inhibition was competitive with respect specifically eluted with C(-methyl-D-mannoside. This to UDP-Glc (data not presented) and the K i value was lectin has a binding selectivity for high- type 7.6 tiM. UDP-glucuronic acid is also a potent inhibitor of glycans [6]. The enzymes in plant cells that convert high­ the mammalian GIc-transferase, with Shailubhai et al. [4J mannose type glycans to the complex-type are localized reporting 10 .5 of 15 tlM. within the Golgi apparatus [7]. The occurrence of Maize endosperm glucosyltransferase 2867 high-mannose glycans on maize endosperm UDP­ directly onto a 1.6 x 20 cm column of con A-Sepharose pre­ Glc:dolichol-P Glc-transferase is consistent with the viously equilibrated with 10 mM TES, pH 7.5, containing 0.25% localization of this enzyme as a resident protein of the Triton X-IOO, 100 mM KCl, 1 mM MgCI 2 , 1 mM CaCl2 and rough-ER [1]. It has been reported that the plant UDP­ 1 mM MnCI2 . After washing with equilibration buffer the bound Glc:sterol Glc-transferase is localized in the Golgi appar­ proteins were specifically eluted with 250 mM x-methyl-D­ atus and plasma membrane [8]. If the sterol-transferase is mannoside in equilibration buffer. The combined Glc-trans­ a then it might be expected that the glycans ferase containing frs were dialysed overnight against 10 mM would have complex-type modifications and would not TES, pH 7.5, containing 100 mM KCl and 20% ethylene glycol, bind to Con A. then applied to a 0.9 x 14 cm column of UDP-hexanolamine­ agarose previously equilibrated with dialysis buffer. After was­ hing with equilibration buffer, bound proteins were eluted with EXPERIMENTAL 2 mM UDP-glucose in equilibration buffer. The final enzyme Chemicals. Hydroxylapatite (Bio-Gel HT) was from BioRad. preparations were either used immediately or stored for up to 4 Con A-Sepharose and DEAE-Sephacel were from Pharmacia. weeks at -70'. The sources of other materials were previously described [1]. Other analytical methods. Protein concentrations were deter­ Unless otherwise noted, all other reagents were from Sigma. mined by the dye-binding method of ref. [10J, using BSA as the Enzyme assay and purification. Enzyme activity and product standard. Kinetic data were analysed by iterative curve-fitting analysis were conducted exactly as previously described [1]. using nonlinear regression as described in [11]. When determin­ Maize endosperm cells were harvested by filtration 7 days after ing inhibition constants, at least 3 concentrations of inhibitor transfer [9]. Washed microsomes were prepared as previously were used spanning a range from 0.1 to 10-times the pre­ described [1]. The microsomal pellets were frozen in liquid N 2 liminarily determined K, value. and stored at - 70' until used for enzyme purification. UDP­ Glc: dolichol-P Glc-transferase was solubilized as previously AcknolVledgement-The excellent technical assistance of C described [lJ except that the solubilization buffer contained in Kessler is gratefully acknowledged. addition, lOI1M leupeptin, 20l1M E64, 2 mM benzamidine, 2 mM EDTA, 200l1M UDP-Glc, and 20l1M (+)-l-deoxy­ nojirimycin. The additives were included in all solutions through REFERENCES the DEAE-Sephacel column step in enzyme purification, after which only the UDP-Glc was included. The UDP-GIc was 1. Riedell, W. E. and Miernyk, J. A. (1988) Plant Physiol. 87, omitted from solns used immediately preceding affinity chro­ 420. matography. 2. Durr, M., Bailey, D. S. and Maclachlan, G. (1979) Eur J. After solubilization, glucosyltransferase activity was adsorbed Biochem. 97, 445. to a 1.6 x 20 cm column of hydroxylapaptite previously equilib­ 3. Palamarczyl, G., Drake, R. R., Haley, B. E. and Lennarz, rated with 5 mM K-Pi buffer. After washing with equilibration W. J. (1990) Proc. Natl Acad. Sci. U.S.A. 87, 2666. buffer, bound proteins were eluted with a linear gradient from 5 4. Shailubhai, K., Illeperuma, C, Tayal, M. and Vijay, 1. K. to 500 mM K-Pi, pH 7.1. A single symmetrical peak of GIc­ (1990) J. Bioi. Oem. 265, 14105. transferase activity eluted at - 280 mM K-Pi (data not pre­ 5. Elbein, A. D. (1987) Annu. Rev. Biochem. 56, 497. sented). The GIc-transferase-containing frs from the hydroxy­ 6. Slodki, M. E., Ward, R. M. and Boundy, R. M. (1973) lapatite column were combined, diluted 1: 5 with deionized H 2 0, Biochim. Biophys. Acta 304, 449. and loaded onto a 1.6 x 22 cm column of DEAE-Sephacel 7. Sturm, A., Johnson, K. D., Szumilo, T., Elbein, A. D. and previously equilibrated with 10 mM TES, pH 7.5, containing Chrispeels, M. J. (1987) Plant Physiol. 85, 741. 0.25% Triton X-l00. After washing with equilibration buffer, 8. Duperon, R. and Duperon, P. (1987) C. R. Acad. Sc. Paris t. bound proteins were eluted with a linear gradient of0 to 500 mM 304, serie III, 235. KCl in equilibration buffer. Product analysis revealed that the 9. Miernyk, J. A. (1987) J. Plant Physiol. 129, 19. second peak of GIc-transferase activity eluted from the ion­ 10. Bradford, M. M. (1976) Analyt. Biochem. 72, 248. exchanger was the dolichol-cycle enzyme (data not presented). 11. Garland, W. H. and Dennis, D. T. (1977) Arch. Biochem. The combined peak frs from the anion-exchanger were loaded Biophys. 182, 614.

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