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Glycobiology Pages 1207-1213  

Topological studies on the enzymes catalyzing the biosynthesis of Glc-P-dolichol and the triglucosyl cap of Glc3Man9GlcNAc2-P-P-dolichol in microsomal vesicles from pig brain: use of the processing glucosidases I/II as latency markers
Introduction
Results
Discussion
Materials and methods
Acknowledgments
Abbreviations
References

Topological studies on the enzymes catalyzing the biosynthesis of Glc-P-dolichol and the triglucosyl cap of Glc<sub>3</sub>Man<sub>9</sub>GlcNAc<sub>2</sub>-P-P-dolichol in microsomal vesicles from pig brain: use of the processing glucosidases I/II as latency markers

Topological studies on the enzymes catalyzing the biosynthesis of Glc-P-dolichol and the triglucosyl cap of Glc3Man9GlcNAc2-P-P-dolichol in microsomal vesicles from pig brain: use of the processing glucosidases I/II as latency markers

Jeffrey S.Rush and Charles J.Waechter1

Department of Biochemistry, University of Kentucky College of Medicine, Lexington, KY 40536, USA

Received on March 19, 1998; revised on May 14, 1998; accepted on May 14, 1998

In the current model for Glc3Man9GlcNAc2-P-P-Dol assembly, Man5GlcNAc2-P-P-Dol, Man-P-Dol, and Glc-P-Dol are synthesized on the cytoplasmic face of the ER and diffuse transversely to the lumenal leaflet where the synthesis of the lipid-bound precursor oligosaccharide is completed. To establish the topological sites of Glc-P-Dol synthesis and the lipid-mediated glucosyltransfer reactions involved in Glc3Man9GlcNAc2-P-P-Dol synthesis in ER vesicles from pig brain, the trypsin-sensitivity of Glc-P-Dol synthase activity and the Glc-P-Dol:Glc0-2Man9GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) was examined in sealed microsomal vesicles. Since ER vesicles from brain do not contain glucose 6-phosphate (Glc 6-P) phosphatase activity, the latency of the lumenally oriented, processing glucosidase I/II activities was used to assess the intactness of the vesicle preparations. Comparative enzymatic studies with sealed ER vesicles from brain and kidney, a tissue that contains Glc 6-P phosphatase, demonstrate the reliability of using the processing glucosidase activities as latency markers for topological studies with microsomal vesicles from non-gluconeogenic tissues lacking Glc 6-P phosphatase. The results obtained from the trypsin-sensitivity assays with sealed microsomal vesicles from brain are consistent with a topological model in which Glc-P-Dol is synthesized on the cytoplasmic face of the ER, and subsequently utilized by the three Glc-P-Dol-mediated GlcTases after 'flip-flopping" to the lumenal monolayer.

Key words: ER/Glc-P-Dol synthesis/microsomal vesicles

Introduction

The precursor oligosaccharide for asparagine-linked carbohydrate chains of eukaryotic glycoproteins is synthesized as the dolichyl pyrophosphate-bound intermediate, Glc3Man9GlcNAc2-P-P-Dol (Kornfeld and Kornfeld, 1985; Hirschberg and Snider, 1987; Waechter, 1989; Cummings, 1992). It has been proposed that this assembly process is initiated on the cytosolic surface of the ER by the synthesis of Man5GlcNAc2-P-P-Dol, Man-P-Dol, andGlc-P-Dol with UDP-GlcNAc, GDP-Man, and UDP-Glc serving as the direct glycosyl donors. These three lipid intermediates then 'flip-flop" from the cytoplasmic leaflet to the lumenal monolayer, and the synthesis of Glc3Man9GlcNAc2-P-P-Dol is completed by the addition of four more [alpha]-linked mannosyl and 3 [alpha]-linked glucosyl residues donated by Man-P-Dol and Glc-P-Dol, respectively. The experimental evidence for this topological model has been rigorously reviewed (Hirschberg and Snider, 1987; Abeijon and Hirschberg, 1992). While most aspects of the model have been convincingly documented, protease-sensitivity studies on the topological sites of Glc-P-Dol synthesis and theGlc-P-Dol:Glc0-2Man9GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) have not provided clear-cut conclusions (Abeijon and Hirschberg, 1992).

One technical problem that has limited studies on microsomal vesicles from tissues that do not contain glucose 6-P phosphatase (Glc 6-Pase), is the lack of a convenient assay for a lumenally oriented latency marker enzyme. The assay of Glc 6-Pase with mannose 6-P (Man 6-P) as an impermeant substrate has provided a simple way to assess the integrity of ER vesicles from liver for numerous topological studies of lipid-synthesizing enzymes (Bell et al., 1981). In this study we show that the integrity or 'intactness" of microsomal vesicles from pig brain can readily be assessed using the deoxnojirimycin-sensitive, processing glucosidase I/II activities as the latency marker enzyme. This approach has taken advantage of the previous observations that the glucosidases involved in the processing of the asparagine-bound precursor oligosaccharide, Glc3Man9GlcNAc2 are exposed on the lumenal side of the ER (Grinna and Robbins, 1979; Lucocq et al., 1986; Shailubhai et al., 1991), and that Glc3Man9GlcNAc2 does not enter a sealed ER vesicle (Grinna and Robbins, 1979). This assay is shown to be reliable with microsomal vesicles from rat kidney and pig brain, and should have applications for topological studies with nongluconeogenic tissues in addition to brain and cultured cells lacking Glc 6-Pase.

Using the processing glucosidase activities to assess the intactness of the microsomal vesicles, the topological orientation of the active sites of Glc-P-Dol synthase and the three Glc-P-Dol-mediated GlcTases in pig brain microsomal vesicles was investigated. The results indicate that Glc-P-Dol synthase has protease-sensitive sites, possibly the active site, exposed on the cytoplasmic surface of the ER vesicles. In contrast to this result, the Glc-P-Dol-mediated glucosyl transfer reactions are resistant to trypsin-inactivation in sealed ER vesicles, but the extent of inactivation of the GlcTases increases in parallel with the accessibility of [3H]Glc3Man9GlcNAc2 to the active sites of the processing glucosidases I/II when the vesicles are unsealed by increasing concentrations of Triton X-100. The latter results are consistent with a lumenal orientation of the Glc-P-Dol-mediated GlcTases.

The relationship of these studies to the previous reports on the topological orientation of Glc-P-Dol synthase and the submicrosomal location where the glucolipid intermediate is utilized as a glucosyl donor is discussed. A preliminary report of this work has been presented previously (Rush et al., 1997)

Results

Use of the lumenally oriented, processing glucosidase I/II activities to assess the intactness of microsomal vesicles from kidney and brain

One limitation for topological studies on microsomal vesicles from non-gluconeogenic tissues has been that brain and other tissues do not express the widely used microsomal integrity marker, Glc 6-Pase. To enable us to perform studies on the topological orientation of enzymes involved in the lipid intermediate pathway for N-glycosylation, we have evaluated the use of the lumenally-oriented processing glucosidase I/II activities as a means of assessing the intactness of pig brain microsomal vesicles. Brain microsomes were previously shown to contain processing exoglucosidase activities capable of sequentially cleaving the three glucose residues from free [3H]Glc3Man9GlcNAc2 (Scher and Waechter, 1979), and Grinna and Robbins (1979) have previously shown that the precursor oligosaccharide does not enter sealed liver microsomes. The experimental strategy for the latency assay used in this study is illustrated in Figure 1.


Figure 1. Experimental strategy for using the lumenally oriented, processing glucosidase I/II activities to assess the intactness of microsomal vesicles.

To demonstrate that glucosidase I/II latency can be utilized to estimate the intactness of microsomal vesicles, glucosidase I/II latency and Man 6-Pase latency were compared in microsomes from rat kidney. As shown in Figure 2, very little glucosidase I/II or Man 6-Pase activities were detected in rat kidney microsomes in the absence of Triton X-100. Both enzyme activities increased in parallel with the addition of Triton X-100 between 0-2 mg/ml. Maximal enzymatic activities were detected in the presence of 5-10 mg/ml Triton X-100. These results indicate that similar estimates of microsomal integrity can be obtained using either glucosidase I/II (95%) or Man 6-Pase (98%) in kidney microsomes. Very similar results were reported by Grinna and Robbins (1979) for rat liver microsomes.


Figure 2. Comparison of latency of the processing glucosidase I/II activities with Man 6-Pase in intact rat kidney ER vesicles. Enzymatic reactions contained kidney microsomes (180 µg membrane protein), 0.25 M sucrose, 50 mM Tris-HCl (pH 7.4), the indicated concentration of Triton X-100, and either 1 mM Man 6-P (1000 c.p.m./nmol) (solid circles) or [3H]Glc1-3Man9GlcNAc2 (8000 c.p.m., open circles) in a total volume of 0.2 ml. Formation of free [3H]Man or [3H]Glc was determined as described in Materials and methods.

Based on glucosidase I/II latency, at least 98% of the pig brain microsomes used in the topological studies described below were sealed (Figure 3). As seen with rat kidney and liver (Grinna and Robbins, 1979) microsomes, glucosidase I/II latency was rapidly lost between 0-0.5 mg/ml Triton X-100. The [3H]Glc-labeled oligosaccharide was completely accessible to the active sites of glucosidase I/II at Triton X-100 concentrations between0.5-2 mg/ml. In our hands the brain vesicles generally unsealed at lower Triton X-100 concentrations than liver or kidney vesicles.


Figure 3. Latency of processing glucosidase I/II activities in intact pig brain microsomes. Glucosidase I/II reactions contained pig brain microsomes (126 µg membrane protein), 50 mM Tris-HCl (pH 7.4), 0.25 M sucrose, [3H]Glc1-3 Man9GlcNAc2 (8000 c.p.m.), and the indicated concentration of Triton X-100 in a total volume of 0.02 ml. Following incubation at 21°C for 5 min, formation of free [3H]glucose was determined as described in Materials and methods.

To confirm that the radiolabeled oligosaccharide substrate was cleaved by the lumenally-oriented, processing glucosidase, the sensitivity of [3H]glucose release to 1-deoxynojirimycin was tested. The brain glucosidase I/II activities were inhibited 50% in the presence of 10 µM deoxynojirimycin (Figure 4). This concentration is in close agreement with previously reported IC50 values determined for the processing glucosidase activities with 1-deoxynojirimycin (Hettkamp et al., 1982; Shailubhai et al., 1987; Bause et al., 1991).


Figure 4. Inhibition of pig brain microsomal glucosidase activity by 1-deoxynojirimycin. Glucosidase I/II reactions contained pig brain microsomes (100 µg membrane protein), 2 mg/ml Triton X-100, 50 mM Tris-HCl (pH 7.4), 0.25 M sucrose, [3H]Glc1-3Man9GlcNAc2 (8000 c.p.m.), and the indicated concentration of 1-deoxynojirimycin in a total volume of 0.02 ml. Following incubation at 21°C for 10 min, the release of free [3H]glucose was determined as described in Materials and methods.

Glc-P-Dol synthase is inactivated by treating intact microsomal vesicles from pig brain with trypsin

Establishing that the latency of the processing glucosidase I/II activities provided a reliable means to assess the intactness of microsomal vesicles, allowed us to determine whether the active site of Glc-P-Dol synthase was exposed on the cytoplasmic face of the ER in brain. From the results presented in Figure 5, it can be seen that there was time-dependent inactivation of Glc-P-Dol synthase when sealed microsomal vesicles were incubated with trypsin (80 µg/ml). Since Glc-P-Dol synthase was assayed in the presence of 0.1% Triton X-100, a concentration that would have exposed cryptic sites with a lumenal orientation, the loss of glucosyltransferase activity could not be due to trypsin-inactivation of the UDP-glucose transporter.


Figure 5. Glc-P-Dol synthase is inactivated by treating sealed microsomal vesicles with trypsin. Trypsin digestion mixtures contained pig brain microsomes (1 mg membrane protein), 5 mM MgCl2, 50 mM Tris-HCl (pH 8), 0.25 M sucrose, and 80 µg/ml trypsin in a final volume of 0.08 ml. Following incubation in the presence of trypsin for the indicated periods of time, 0.8 mg trypsin inhibitor (soy bean) was added and aliquots were removed and analyzed for either Glc-P-Dol synthase (solid circles) or glucosidase I/II (open circles). Glc-P-Dol synthase reaction mixtures contained trypsin-treated pig brain microsomes (0.9 mg membrane protein), 8.5 mM MgCl2, 0.1% Triton X-100, 1 mM AMP, and 2.5 µM UDP-Glc (440 c.p.m./pmol) in a total volume of 0.1 ml. Following incubation at 37°C for 10 min, transfer of [3H]glucose from UDP-[3H]glucose to Glc-P-Dol was determined as described in Materials and methods. Glucosidase I/II assay mixtures contained trypsin-treated pig brain microsomes (0.13 mg membrane protein), 0.2% Triton X-100, 0.25 M sucrose, 25 mM Tris-HCl (pH 8), and [3H]Glc1-3Man9GlcNAc2 (8,000 c.p.m.) in a total volume of 0.02 ml. Following incubation at 20°C for 15 min the release of free [3H]glucose was determined as described in Materials and methods.

Under the same conditions, glucosidase I/II activities were unaffected, confirming that the vesicles remained sealed during the incubation period. The data depicted in Figure 6 (upper panel, solid circles) show that pig brain glucosidases I/II are substantially inactivated when unsealed microsomal vesicles are exposed to trypsin for 5-20 min (25 µg/ml). These results are consistent with the active site of Glc-P-Dol synthase being exposed on the cytoplasmic face of the ER in brain.


Figure 6. Glc-P-Dol:Glc0-2Man9GlcNAc2-P-P-Dol GlcTases are not inactivated by treating sealed microsomal vesicles from pig brain with trypsin. Trypsin digestion mixtures contained pig brain microsomes (1 mg protein), 50 mM Tris-HCl (pH 8), 0.25 M sucrose, and 25 µg/ml trypsin in a final volume of 0.2 ml. Following incubation at 37°C for the indicated periods of time in either the presence (solid circles) or the absence (open circles) of Triton X-100 (2 mg/ml), 0.2 mg trypsin inhibitor (soy bean) was added and aliquots were removed and analyzed for either glucosidase I/II (upper panel) or Glc-P-Dol:Glc0-2Man9GlcNAc2-P-P-Dol GlcTase (lower panel). Glucosidase I/II and GlcTase activities were assayed as described in Materials and methods.

Topological orientation of the active sites of the Glc-P-Dol:Glc0-2Man9GlcNAc2-P-P-Dol glucosyltransferases in pig brain microsomes

While the Glc-P-Dol mediated GlcTases have previously been shown to be highly enriched in the rough ER in brain (Scher et al., 1984), the topological orientation of the enzymes has not been rigorously established. To investigate the orientation of the active site(s) of the Glc-P-Dol-mediated GlcTases, the effect of trypsin treatment on the membrane-bound enzymes was examined in intact and unsealed pig brain microsomes. The results in Figure 6 show that both glucosidase I/II (Figure 6, upper panel) and the GlcTases (Figure 6, lower panel) were resistant to trypsin when sealed microsomal vesicles were incubated with the protease (25 µg/ml) for at least 20 min. However, if the vesicles were unsealed by the addition of Triton X-100 (2 mg/ml), GlcTase and glucosidase I/II activities were inactivated within 5 min. Furthermore, the data in Figure 7 show that GlcTase (solid circles) and glucosidase I/II (open circles) activities become sensitive to trypsin-inactivation in close parallel when intact pig brain vesicles are unsealed by the addition of increasing concentrations of Triton X-100.


Figure 7. The active sites of the three Glc-P-Dol-mediated GlcTases catalyzing the addition of the triglucosyl cap to Glc3Man9GlcNAc2-P-P-Dol are lumenally-oriented. Trypsin digestion mixtures contained pig brain microsomes (1 mg membrane protein), 50 mM Tris-HCl (pH 8), 0.25 M sucrose, 25 µg/ml trypsin, and the indicated amount of Triton X-100 in a final volume of 0.2 ml. Following incubation at 37°C for the indicated periods of time, 0.2 mg trypsin inhibitor (soy bean) was added and aliquots were removed and analyzed for either glucosidase I/II (open circles) or Glc-P-Dol:Glc0-2Man9GlcNAc2P-P-Dol GlcTases (solid circles). Glucosidase I/II and GlcTase activities were assayed as described in Materials and methods.

Similar results were obtained when the same protease-sensitivity study was conducted with subtilisin instead of trypsin. All of these results represent solid evidence that the processing glucosidases I/II and the lipid-mediated GlcTases contain protease-sensitive domains, possibly the active sites of the enzymes, exposed in the lumenal compartment of ER vesicles from pig brain.

Discussion

In this article we outline an experimental strategy for reliably assessing the intactness of ER vesicles from nongluconeogenic tissues lacking Man 6-Pase, the latency marker enzyme routinely used for topological studies in microsomal vesicles from liver. As illustrated in Figure 1, the principle of this approach is similar to the use of Man 6-Pase in ER vesicles from liver. Since [3H]Glc3Man9GlcNAc2 does not enter sealed vesicles, the enzymatic release of [3H]Glc residues is only observed when the vesicles are unsealed and the active sites of glucosidase I/II activities are accessible. It should be possible to use the same experimental approach used in these studies on brain microsomes, for topological studies on ER vesicles from other non-gluconeogenic tissues and cultured mammalian cells.

Based on glucosidase I/II latency, at least 98% of the microsomal vesicle preparations from pig brain used for these studies are sealed. These ER vesicle preparations have allowed us to evaluate the topological orientation of Glc-P-Dol synthase and the three GlcTases (Runge et al., 1984; Runge and Robbins, 1986; D'Souza-Schorey and Elbein, 1993; Stagljar et al., 1994; Zufferey et al., 1995) utilizing the glucolipid intermediate as a glucosyl donor for Glc3Man9GlcNAc2-P-P-Dol biosynthesis in brain.

This investigation was undertaken because although there is good evidence to support most details of the model described by Hirschberg and Snider, (1987), the studies on the topological orientation of the active sites of Glc-P-Dol synthase and the Glc-P-Dol-mediated GlcTases have yielded inconclusive results. For example, Hanover and Lennarz (1978) found that Glc-P-Dol synthase was inactivated by 40% when sealed hen oviduct vesicles were incubated with trypsin, and the extent of inactivation increased to 70% when the vesicles were unsealed. Snider et al. (1980) found that 70% of the Glc-P-Dol-mediated GlcTase transfer reactions were sensitive to pronase inactivation in sealed microsomal vesicles from liver. Thus, these studies did not provide conclusive answers regarding the orientation ofGlc-P-Dol synthase or the lipid-mediated GlcTases. The sensitivity of Glc-P-Dol synthase in ER vesicles from thyroid to DIDS and trypsin indicated a cytoplasmic orientation (Spiro and Spiro, 1985). In a related study, Drake et al. (1992) found that a polypeptide with the molecular size expected for Glc-P-Dol synthase (37 kDa) could be photolabeled with [32P]5-azido-UDP-glucose in intact rat liver microsomes, presumably at the active site of the enzyme. The sensitivity of the photolabeled polypeptide to trypsin digestion in intact microsomal vesicles is also consistent with a cytoplasmic orientation of the glucosyltransferase. The use of the processing glucosidases (I/II) as latency markers enabled us to examine the topology of Glc-P-Dol synthesis and its function as a glucosyl donor in ER vesicles from brain.

The results presented here using intact microsomal vesicles from pig brain provide corroborative evidence for a cytoplasmic orientation of the active site of Glc-P-Dol synthase, and indicate that the active sites of the GlcTases, catalyzing the formation of the triglucosyl cap of Glc3Man9GlcNAc2-P-P-Dol, are lumenally-oriented. These results are consistent with the model proposed by Hirschberg and Snider, (1987), and the results of studies by Snider and Robbins (1982) using Con A as a nonpenetrating probe for the orientation of Glc3Man9GlcNAc2-P-P-Dol.

This model raises interesting questions about the mechanisms by which the hydrophilic, polar headgroup of Glc-P-Dol, as well as Man-P-Dol and Man5GlcNAc2-P-P-Dol, are translocated through the hydrophobic core of the bilayer in the ER. The possibility that the transverse diffusion of these intermediates is mediated by ER proteins ('flippases"), as proposed for membrane glycerophospholipids, glucosylceramide, and GPI-anchor intermediates (Bishop and Bell, 1985; Zachowski, 1993; Trotter and Voelker, 1994; Menon, 1995) is addressed in the accompanying article (Rush et al., 1998).

Materials and methods

Materials

UDP-glucose, baker's yeast hexokinase (H-4502), chicken muscle phosphoglucomutase (P-6156), baker's yeast pyrophosphatase (I-1643), UDP-glucose pyrophosphorylase (U-8501), trypsin, soybean trypsin inhibitor, DEAE cellulose, and Concanavalin A Sepharose were purchased from Sigma Chemical Co. (St. Louis, MO). [1-3H]Glucose (15.5 Ci/mmol) and [2-3H]mannose (15 Ci/mmol) were obtained from American Radiolabeled Chemicals (St. Louis, MO). [2-3H]Man 6-P was synthesized enzymatically and purified as described previously (Rush and Waechter, 1992). [3H]Glc-P-Dol (500 c.p.m./pmol) was prepared enzymatically as described by Waechter and Scher (1978, 1981). [3H]Glc3Man9GlcNAc2 was prepared as described by Scher and Waechter (1979). Dolichyl phosphate (C95) was obtained from Dr. Taduesz Chojnacki, Warsaw, Poland. Econosafe counting cocktail is a product of Research Products International Corp. (Mount Prospect, IL). All other chemicals and reagents were obtained from standard commercial sources.

Enzymatic synthesis of UDP-[3H]glucose

Initially [3H]Glc 6-P was synthesized by incubating 1 U of yeast hexokinase, 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 1 mM ATP, and 1 mCi [3H]glucose in a total volume of 0.25 ml. Following incubation at 21°C for 1 h, the reaction mixture was transferred to a DEAE-cellulose column (5 ml) equilibrated in distilled water. After elution with 5 column volumes of distilled water, [3H]Glc 6-P was eluted with a 50 ml gradient of NH4HCO3 (0-1 M). Fractions containing [3H]Glc 6-P were pooled and concentrated to 1 ml by rotary evaporation under reduced pressure at 30°C and desalted by gel permeation chromatography on a Sephadex G-10 column (1.5 × 20 cm) equilibrated in distilled water. The column was eluted with H2O and fractions containing [3H]Glc 6-P were pooled and concentrated to 1 ml by rotary evaporation. Purified [3H]Glc 6-P was stored at -20°C until used for the enzymatic synthesis of UDP-Glc.

Enzymatic reactions for the synthesis of UDP-[3H]Glc contained 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 1 U baker's yeast pyrophosphatase , 0.1 U chicken muscle phosphoglucomutase, 0.1 U yeast UDP-Glc pyrophosphorylase, 1 mM UTP, 10 µM Glc 1,6-di-P and 220 µCi [3H]Glc 6-P in a total volume of 0.1 ml. Following incubation at 20°C for 3 h, UDP-[3H]glucose was purified as described above for [3H]Glc 6-P. UDP-[3H]glucose was stored at -20°C in 50% ethanol until used for the synthesis of either [3H]Glc3Man9GlcNAc2-P-P-Dol or [3H]Glc-P-Dol.

Preparation of hen oviduct microsomes

Hen oviduct microsomes were prepared from fresh oviduct magnum sections of mature laying hens immediately after slaughter exactly as described by Pless and Lennarz (1975) and stored at -80°C. Prior to utilization for the synthesis of Glc-P-Dol or [3H]Glc1-3Man9GlcNAc2-P-P-Dol, the microsomes were thawed, resuspended in 2 vol ice-cold 10 mM Tris-HCl (pH 7.4), 0.25 M sucrose, 5 mM EDTA and sedimented at 100,000 × g, 10 min in a TL100 ultracentrifuge. The EDTA-treated microsomes were resuspended in 10 mM Tris-HCl, (pH 7.4), 0.25 M sucrose (buffer A) and resedimented two times. The microsomes were suspended in buffer A to a final protein concentration of 15-20 mg/ml and used for the enzymatic synthesis of Glc-P-Dol or [3H]Glc1-3Man9GlcNAc2-P-P-Dol.

Preparation of microsomes from rat kidney and pig brain gray matter

Microsomes were prepared from fresh rat kidneys as described for rat liver by Coleman and Bell (1978). Fresh pig brains were obtained at the time of slaughter and immediately placed on ice. ER-enriched, myelin-free microsomes were prepared exactly as described previously (Waechter and Harford, 1977) except that 10 mM HEPES (pH 7.4) was used instead of 0.1 M Tris-HCl (pH 7.4).

Assessment of the integrity of microsomal vesicles

The integrity of microsomal vesicles from rat kidney was determined by measuring Man 6-Pase latency using [3H]Man 6-P as substrate (Rush and Waechter, 1992). To assess the integrity of pig brain microsomal vesicles, the processing, deoxynojirimycin-sensitive glucosidase I/II activities were measured using [3H]Glc1-3Man9GlcNAc2 as substrate before and after complete disruption of the microsomal vesicles with Triton X-100 (2 mg/ml). Glucosidase I/II assay mixtures contained 50 mM Tris-HCl (pH 7.4), 0.25 M sucrose, 50-200 µg of pig brain microsomal protein, the indicated amount of Triton X-100, and [3H]Glc1-3Man9GlcNAc2 (8000 c.p.m.) prepared as described by Scher and Waechter (1979) in a total volume of 0.02 ml. Following incubation at 20°C for 5-15 min the enzymatic reaction was stopped by the addition of 0.08 ml of 95% ethanol. The precipitated protein was sedimented by centrifugation (1 min) in a Costar microcentrifuge, and 0.08 ml of the supernatant was transferred into 0.5 ml of 10 mM Tris-HCl (pH 7.4), 0.15 M NaCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2 (Con A buffer). The sample was transferred to a column (0.5 ml) of Con A-Sepharose. The column was eluted directly into a 20 ml scintillation vial with an additional 1.5 ml of Con A buffer. The amount of free [3H]glucose released by glucosidase I/II activity was measured by liquid scintillation spectrometry in a Packard Tri-Carb 2100 TR liquid scintillation spectrometer. Vesicular integrity is calculated by comparison of glucosidase I/II activity detected before and after disruption of the permeability barrier with Triton X-100 (2 mg/ml).

Assay of Glc-P-Dol synthase activity in pig brain microsomes

Enzymatic reactions for the assay of Glc-P-Dol synthase contained 5 mM MgCl2, 50 mM Tris-HCl (pH 8), Triton X-100 (1 mg/ml), 0.125 M sucrose, 1 mM AMP, 2.5 µM UDP-[3H]glucose (440 c.p.m./pmol), and pig brain microsomes (0.9 mg protein) in a final volume of 0.1 ml. After incubating for 10 min at 37°C, the enzymatic reaction was stopped by the addition of 20 vol CHCl3/CH3OH (2:1). The reaction mixture was centrifuged and the organic extract freed of water-soluble reactants as described previously (Waechter and Scher, 1978, 1981). The organic layer was dried under a stream of N2 and hydrolyzed in 50% isopropanol containing 0.1 M HCl, 50°C, 45 min. The reaction was then neutralized, diluted with 10 vol CHCl3/CH3OH (2:1), and partitioned with 1/5 vol of water. The mild acid stable product, [3H]Glc-ceramide remained in the lower (organic) phase. The aqueous layer, containing [3H]glucose released from the mild acid-labile product, [3H]Glc-P-Dol, was dried under a stream of air and the amount of [3H]glucose was measured by scintillation spectrometry in a Packard Tri-Carb 2100TR liquid scintillation spectrometer.

Assay of Glc-P-Dol:Glc0-2Man9GlcNAc2-P-P-Dol GlcTase activity

Reaction mixtures for the assay of Glc-P-Dol:Glc0-2Man9-GlcNAc2-P-P-Dol GlcTase activity contained 50 mM Tris-HCl (pH 8), 0.2 M sucrose, 5 mM EDTA, 2 mg/ml Triton X-100, 0.75 mg of pig brain microsomal protein and 0.3 µM [3H]Glc-P-Dol (500 c.p.m./pmol) in a total volume of 0.1 ml. Following incubation for 2 min at 37°C the incorporation of [3H]glucose into Glc1-3Man9GlcNAc2-P-P-Dol was determined by a multiple extraction procedure (Waechter and Scher, 1978).

Proteolytic digestion of pig brain microsomes with trypsin

Protease mixtures contained 50 mM HEPES (pH 8.0), 0.25 M sucrose, trypsin (0.025-1 mg/ml), pig brain microsomes (5 mg/ml membrane protein), and the indicated concentration of Triton X-100. After the indicated periods of time at 37°C, further proteolysis was blocked by the addition of soy bean trypsin inhibitor at a 10:1 (mg/mg) ratio of inhibitor/trypsin. The reactions were then assayed for either Glc-P-Dol synthase, Glc-P-Dol:Glc0-2Man9GlcNAc2-P-P-Dol GlcTase, or processing glucosidase I/II as described above. For each enzymatic reaction, the effects of the preincubation in the presence or absence of trypsin-inhibitor were assessed.

Analytical methods

Protein concentrations were determined by the method of Rodriquez-Vico et al. (1989) using a protein assay reagent (BCA, Pierce, Rockford, IL). Lipid-phosphorus was determined by the method of Bartlett (1959). Samples were analyzed for radioactivity by scintillation spectrometry in a Packard Tri-Carb 2100TR liquid scintillation spectrometer following the addition of Econosafe Liquid Scintillation Counting Cocktail.

Acknowledgments

We thank Austin Cantor, Mike Ford, and Jim May (Department of Animal Science, University of Kentucky) for providing the hen oviducts and pig brains used in these studies. This work was supported by NIH Grant GM36065 awarded to C.J.W.

Abbreviations

Glc-P-Dol, glucosylphosphoryldolichol; Man-P-Dol, mannosylphosphoryldolichol; GlcTases, Glc-P-Dol:Glc0-2Man9GlcNAc2-P-P-Dol glucosyltransferases, Glc 6-P, glucose 6-phosphate; Man 6-P, mannose 6-phosphate; DIDS, 4,4[prime]-diisothiocyano-2,2[prime]-stilbenedisulfonate.

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