Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Frank, D. W.
Right arrow Articles by Waechter, C. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Frank, D. W.
Right arrow Articles by Waechter, C. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

J Biol Chem, Vol. 273, Issue 19, 11791-11798, May 8, 1998


Purification and Characterization of a Polyisoprenyl Phosphate Phosphatase from Pig Brain
POSSIBLE DUAL SPECIFICITY*

David W. FrankDagger and Charles J. Waechter§

From the Department of Biochemistry, A. B. Chandler Medical Center, University of Kentucky College of Medicine, Lexington, Kentucky 40536

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Microsomal fractions from pig and calf brain catalyze the enzymatic dephosphorylation of endogenous and exogenous dolichyl monophosphate (Dol-P) (Sumbilla, C. A., and Waechter, C. J. (1985) Methods Enzymol. 111, 471-482). The Dol-P phosphatase (EC 3.1.3.51) has been solubilized by extracting pig brain microsomes with the nonionic detergent Nonidet P-40 and purified approximately 1,107-fold by a combination of anion exchange chromatography, polyethylene glycol fractionation, dye-ligand chromatography, and wheat germ agglutinin affinity chromatography. Treatment of the enzyme with neuraminidase prevented binding to wheat germ agglutinin-Sepharose, indicating the presence of one or more N-acetylneuraminyl residues per molecule of enzyme. When the highly purified polyisoprenyl phosphate phosphatase was analyzed by SDS-polyacrylamide gel electrophoresis, a major 33-kDa polypeptide was observed. Enzymatic dephosphorylation of Dol-P by the purified phosphatase was 1) optimal at pH 7; 2) potently inhibited by F-, orthovanadate, and Zn2+ > Co2+ > Mn2+ but unaffected by Mg2+; 3) exhibited an approximate Km for C95-Dol-P of 45 µM; and 4) was sensitive to N-ethylmaleimide, phenylglyoxal, and diethylpyrocarbonate. The pig brain phosphatase did not dephosphorylate glucose 6-phosphate, mannose 6-phosphate, 5'-AMP, or p-nitrophenylphosphate, but it dephosphorylated dioleoyl-phosphatidic acid at initial rates similar to those determined for Dol-P. Based on the virtually identical sensitivity of Dol-P and phosphatidic acid dephosphorylation by the highly purified enzyme to N-ethylmaleimide, F-, phenylglyoxal, and diethylpyrocarbonate, both substrates appear to be hydrolyzed by a single enzyme with an apparent dual specificity. This is the first report of the purification of a neutral Dol-P phosphatase from mammalian tissues. Although the enzyme is Mg2+-independent and capable of dephosphorylating Dol-P and PA, several enzymological properties distinguish this lipid phosphomonoesterase from PAP2.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Dolichyl monophosphate (Dol-P)1 plays an essential role as a glycosyl carrier lipid in the assembly of N-linked oligosaccharides in eukaryotes (1, 2). In view of the evidence that modulation of Dol-P levels in the ER are one factor regulating the rate of biosynthesis of dolichol-linked oligosaccharide intermediates and protein N-glycosylation (2-11), it will be important to understand the control of the enzymes involved in Dol-P metabolism. Recent studies on the biosynthesis of Dol-P indicate that the induction of the cis-isoprenyltransferase system, catalyzing the chain elongation stage in the de novo pathway, is a key regulatory event when the rates of lipid intermediate synthesis and protein N-glycosylation increase developmentally in embryonic rat brain (10) and proliferating B lymphocytes (11).

The presence of membrane-bound enzymes capable of phosphorylating dolichol and dephosphorylating Dol-P has been documented for a number of animal tissues (12). Reciprocal developmental changes in dolichol kinase and polyisoprenyl phosphate phosphatase activity have been reported for sea urchin embryos (13) and mammalian brain (14, 15). The metabolic balance in the phosphorylation/dephosphorylation of Dol-P could play a role in the "activation" of reserve pools of dolichol during developmental increases in protein N-glycosylation. Dol-P phosphatase (Dol-Pase) would provide a means of reducing the size of the Dol-P pool when the demand for N-linked glycoproteins decreases.

The developmental pattern for Dol-Pase described for estrogen-treated chick oviducts suggested a potential role for the enzyme in Dol-P biosynthesis (16). Since there is evidence that dolichol is formed by the enzymatic reduction of the double bond in the alpha -isoprene unit of the free long chain polyprenol (17), alpha -reduction would not occur until the fully unsaturated polyprenyl pyrophosphate intermediate was dephosphorylated by polyprenyl pyro- and monophosphatase activities. In this biosynthetic scheme, dolichol kinase would catalyze the terminal step in the de novo pathway.

Previous work from this laboratory has shown that crude microsomes from calf brain catalyze the dephosphorylation of endogenous and exogenous Dol-P (18). The brain polyisoprenyl phosphate phosphatase is enriched in light microsomes and axon plasma membranes (19). However, despite the potential importance of this class of phosphatases in the metabolism of Dol-P, the specificity and number of enzymes catalyzing the dephosphorylation reaction have not been determined.

As an initial step toward the ultimate goal of cloning and sequencing polyisoprenyl phosphate phosphatase, and to learn more about its structure and specificity, we have purified the enzyme from pig brain microsomes. The brain enzyme was found to be an intrinsic membrane sialoglycoprotein, and specificity studies establish that although it is definitely not a nonspecific phosphomonoesterase, the highly purified enzyme is capable of actively dephosphorylating Dol-P and phosphatidic acid. The dual specificity of this novel membrane-bound Dol-Pase and the characteristics that distinguish it from PAP2 are discussed. Portions of this work have been presented in a preliminary report (20).

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- Dolichol (grade I), dolichyl phosphate (grade III), wheat germ agglutinin (WGA)-Sepharose 6MB, polyethylene glycol (average molecular weight = 8,000), Sephacryl S300, neuraminidase (from Arthrobacter ureafaciens and Clostridium perfringens), dioleoyl-phosphatidic acid, and 1,2-sn-dioleoylglycerol were obtained from Sigma. Individual polyprenols (C80-C100) and dolichols (C90-C100) were a generous gift from Dr. M. Mizuno, Kuraray Ltd. (Okayama, Japan). TSK-Gel Toyopearl-DEAE 650M and TSK-Gel Toyopearl-Blue 650M were purchased from Supelco. Phosphorus oxychloride was from Alfa. Carrier-free [32P]H3PO4 was supplied by ICN Biomedicals. Tetrabutylammonium hydroxide and trichloroacetonitrile were obtained from Aldrich, as was anhydrous acetonitrile. Lectin from the slug Limax flavus was a generous gift from Dr. Tom Oeltmann (Vanderbilt University). L. flavus agglutinin was conjugated to CNBr-activated Sepharose (Sigma) under conditions described by Miller et al. (21). Agarose-linked lectin from the peanut Arachis hypogaea (Arachis hypogaea agglutinin-agarose) and succinyl-WGA-agarose were obtained from EY Laboratories. Agarose-conjugated lectins from Ricinus communis (R. communis agglutinin-agarose), and Vicia villosa (V. villosa agglutinin-agarose) were purchased from Sigma.

Chemical Synthesis and Purification of Substrates-- [32P]Dol-P and [32P]polyprenyl phosphate were prepared as described by Adair and Keller (22) with an adaptation of the procedure of Danilov et al. (23). Briefly, 0.5-2 mCi of [32P]H3PO4, 1 µmol of H3PO4, and 1 µmol of tetra-n-butylammonium hydroxide were dissolved in 200 µl of anhydrous CH3CN. The solvent was evaporated under a stream of nitrogen. Dissolution in dry CH3CN and evaporation was repeated twice. The resulting tetra-n-butylammonium phosphate was dissolved in 50 µl of CHCl3 containing 1 mg (~0.8 µmol) dolichol or polyprenol and 5 µmol of trichloroacetonitrile. The reactants were mixed thoroughly with a vortex mixer and allowed to react for 30 min or longer at room temperature. The reaction was stopped by the addition of 4 ml of CHCl3/CH3OH (2:1) and 0.8 ml 0.9% NaCl. After 8-10 washings of the organic phase with 2 ml of CHCl3/CH3OH/0.9% NaCl (3:48:47) and once with CHCl3/CH3OH/H2O (3:48:47), the organic phase was then taken to dryness under a stream of nitrogen. The radiolabeled substrates were dissolved in 0.5 ml of CHCl3/CH3OH (2:1) and applied to a small column (1-ml bed volume in a Pasteur pipette) of TSK-Gel Toyopearl-DEAE 650M (acetate form) equilibrated in CHCl3/CH3OH (2:1). The column was washed with 5 ml each of CHCl3/CH3OH (2:1) and 1, 2, and 5 mM ammonium acetate in CHCl3/CH3OH (2:1). The phosphorylated alcohols eluted predominantly in the 2 mM ammonium acetate fraction and comigrated with authentic standards when subjected to thin layer chromatography on Silica Gel G 60 (Merck) developed with basic (CHCl3/CH3OH/H2O/NH4OH, 65:35:4:4), neutral (CHCl3/CH3OH/H2O, 75:25:4), and acidic (2,6-dimethyl-4-heptanone/acetic acid/H2O, 20:15:2) solvents. The method was also used to prepare [32P]phosphatidic acid from sn-dioleoylglycerol, and the product co-migrated with authentic phosphatidic acid in the same solvents.

A series of mono- and diphosphorylated dolichols and polyprenols of defined, homogeneous chain length were synthesized essentially by procedure B of the method of Danilov et al. (23), using chloroform as the solvent. Phosphorylated isoprenoid alcohols were identified following thin layer chromatography by spraying the plate with a phosphate-specific spray (24), allowing development of color, and then spraying with the p-anisaldehyde reagent described by McSweeney (25) for detection of isoprenoid compounds.

Preparation of Salt-washed Crude Microsomes from Pig Brain-- Fresh pig brains were obtained from 6-month-old pigs at the time of slaughter and used immediately or stored at -70 °C. Preparation of microsomes was performed at 4 °C unless otherwise indicated. Prior to homogenization, blood vessels and the meninges were removed from the brain. In a typical preparation, 7-10 brains (~1 kg, wet weight) were homogenized in 3 volumes of buffer A (0.1 M Tris-HCl, pH 7.5, 0.25 M sucrose, 10 mM EDTA, 1 mM 2-mercaptoethanol, 0.2 mM phenylmethylsulfonyl fluoride), for 1.5 min in a Waring blender. The homogenate was centrifuged at 10,000 × g for 15 min. The resulting pellet was diluted with 3 volumes of buffer A, rehomogenized, and centrifuged as before. The 10,000 × g supernatants were combined and centrifuged at 140,000 × gmax for 1 h in a Beckman type 35 rotor. Peripheral membrane proteins were removed by resuspending the microsomes (5 mg of membrane protein/ml) in buffer A containing 1 M NaCl. The suspension was stirred 1 h. The salt-washed membranes were sedimented by high speed centrifugation as above. The pellet was washed twice in 360 ml of buffer A and resuspended in buffer A at a protein concentration of 15-30 mg/ml. The membranes were stored at -20 °C.

Solubilization and Purification of Dol-Pase from Salt-washed Microsomes-- Salt-washed microsomes were resuspended in cold 50 mM Tris-HCl, pH 7.5, 10% (v/v) glycerol, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM EDTA and quantitatively transferred to a flask to obtain a final concentration of 2 mg of protein/ml. The flask was placed on ice, and 10% Nonidet P-40 was added to a final concentration of 1% Nonidet P-40. The mixture was stirred for 15 min, and insoluble material was removed by centrifugation at 140,000 × gmax for 1 h. The supernatant, containing Dol-Pase activity, was retained for further fractionation.

The detergent-solubilized enzyme was applied to a column of TSK-Gel Toyopearl-DEAE 650M (2.5 × 16 cm) equilibrated at 4 °C in buffer B (50 mM Tris-HCl, pH 7.5, 0.2% (w/v) Nonidet P-40, 10% (v/v) glycerol, 0.2 mM phenylmethylsulfonyl fluoride) at a flow rate of 8 ml/min. The column was washed with 2 column volumes of buffer B. Bound protein was eluted with a concave gradient of 0-0.75 M NaCl in buffer B and collected in 6-ml fractions.

Eluted fractions containing Dol-Pase activity were pooled and concentrated with an Amicon ultrafiltration cell fitted with a YM100 membrane (molecular weight cut-off of ~100 kDa). PEG (average molecular weight of 8,000) was added (150 mg of PEG/ml of protein solution) to the stirred solution in three equal portions over a 30-min period. Gentle stirring was continued for 1 h following dissolution of the PEG. The mixture was then centrifuged for 1 h at 140,000 × gmax. The supernatant solution, containing Dol-Pase activity, was saved.

Dol-Pase activity in the PEG supernatant was applied to a column (1 × 3 cm) of TSK-Gel Toyopearl-Blue 650M equilibrated in buffer B at 4 °C, and the column was washed with 20 ml of buffer B. Bound protein was eluted with a 50-ml linear gradient of 0-1 M NaCl in the same buffer at a flow rate of 1 ml/min.

Dol-Pase activity recovered from the dye column was pooled and concentrated to about 1 ml with an Amicon ultrafiltration cell fitted with a YM100 membrane then applied to WGA-Sepharose 6MB (0.75 × 25 cm) equilibrated in 50 mM Tris-HCl, pH 7.5, 0.1% (w/v) Nonidet P-40; 10% (v/v) glycerol, 0.15 M NaCl (buffer C). The enzyme solution was allowed to flow into the column bed under gravity, with a pressure head of 15 cm. After the sample had entered the bed, buffer C was layered over the gel, column flow was stopped, and the sample was equilibrated for 10-15 min with the lectin. The column was washed with 3 column volumes of buffer, and then bound glycoproteins were eluted with buffer C containing 0.1 M N-acetyl-D-glucosamine.

Fractions collected from each step of purification were assayed for Dol-Pase activity and protein concentration. Where indicated, NaCl concentration was estimated by comparison of conductivity of eluted fractions to standard solutions.

Assay for Dol-Pase Activity-- Microsomes, detergent extracts, and column fractions were assayed for Dol-Pase activity by the method of Sumbilla and Waechter (26). Reaction mixtures typically contained 50 µg of microsomal protein, 20 µg of Nonidet P-40-solubilized protein, or 5-10 µl from column fractions, 50 mM Tris-HCl, pH 7.2, 0.1% Nonidet P-40, 10 mM EDTA, and 0.2 mM [32P]Dol-P (500-2,000 cpm/nmol) in a total volume of 50 µl. Chemically phosphorylated dolichol from pig liver (C80-C105) was used routinely as substrate. Assays were performed at 37 °C for 5 or 10 min.

The enzymatic dephosphorylation of [3H]glucose 6-phosphate, [2-3H]mannose 6-phosphate, and 5'-[3H]AMP by the purified phosphatase was assayed as described previously for mannose-6-phosphatase (27). The dephosphorylation of [3H]farnesyl phosphate was determined by analyzing an aliquot of the phosphatase reaction mixture by thin layer chromatography on Silica Gel G 60 and developing with CHCl3/CH3OH/NH4OH/H2O (65:35:4:4). The reaction products were located with a Bioscan Imaging Scanner System 200-IBM and subsequently scraped from the plate and quantitated by scintillation counting.

To locate polyprenyl phosphate phosphatase activity in nondenaturing gels, individual gel lanes were sliced with a razor blade. The gel slices were macerated in a 1.5-ml centrifuge tube and incubated with 50 mM Tris-HCl, pH 7.2, 0.1% Nonidet P-40, 12.5 mM EDTA, and 0.2 mM [32P]Dol-P (500-1,000 cpm/nmol) in a total volume of 0.2 ml at 37 °C for 1 h with periodic mixing. A 50-µl aliquot was removed, and released 32Pi was extracted and quantitated.

Denaturing and Nondenaturing Polyacrylamide Gel Electrophoresis-- Denaturing gel electrophoresis was performed in SDS-polyacrylamide minigels according to Laemmli (28). Gels were fixed and silver-stained (29).

Electrophoresis of Dol-Pase under nondenaturing conditions was performed in gradient gels of 5-20% (2.7% cross-linker) acrylamide, buffered with 0.375 M Tris-HCl, pH 8.8, 0.1% Nonidet P-40. No stacking gel was employed. The tank buffer was 25 mM Tris, 0.192 M glycine, 0.1% Nonidet P-40, pH 8.3. Gels were run for 4-18 h at an initial current of 10 mA (voltage limited to 500 V) at 4 °C. Individual gel lanes were separated, and the resulting strips were used for silver staining of protein or location of Dol-Pase activity (see above).

Isoelectric Focusing of Dol-Pase-- Purified Dol-Pase was subjected to solution isoelectric focusing in a Bio-Rad Rotofor apparatus. The focusing chamber contained 50 ml of 1% Pharmalyte 4-6.5 (Amersham Pharmacia Biotech), 2% Nonidet P-40, 15% glycerol and was prefocused for 2 h at 12 watts, 4 °C. The anolyte was 0.1 M acetic acid, and the catholyte was 0.1 M NaOH. After the pH gradient was established, Dol-Pase in 1-3 ml 50 mM Tris-HCl, pH 7.5, 0.1% Nonidet P-40 was added. Power was applied at 12 watts for 3 h. Temperature was maintained by a recirculating ice water bath. At the end of the isoelectric focusing period, fractions were collected and analyzed for phosphatase activity and pH.

Analytical Lectin Affinity Chromatography of Dol-Pase-- Purified Dol-Pase was analyzed by lectin affinity chromatography using 0.5-1 ml of gel. The columns were equilibrated, and the sample was applied in 50 mM Tris-HCl, pH 7.5, 0.1% (w/v) Nonidet P-40, 10% (v/v) glycerol, 0.15 M NaCl at room temperature and equilibrated for 10 min. The column was washed with 10 column volumes of buffer, and bound protein was eluted with the appropriate monosaccharide or disaccharide specific for the lectin. Fractions of 0.5-1 ml were collected and assayed for Dol-Pase activity.

Treatment of Purified Dol-Pase with Neuraminidase-- Purified Dol-Pase (25 µg in 50 µl or 105 dpm of 125I-Dol-Pase prepared using Iodogen (Pierce) according to the manufacturer's instructions) was incubated with 10 milliunits of A. ureafaciens neuraminidase or 7.5 milliunits of neuraminidase from C. perfringens at room temperature (~24 °C) for 4 h, in 50 mM Tris-HCl, pH 7.5, 0.1% (w/v) Nonidet P-40, 10% (v/v) glycerol, 1 mM EDTA. Treated Dol-Pase was then analyzed by SDS-PAGE or analytical lectin affinity chromatography as described elsewhere.

Treatment of Purified Dol-Pase with Alkylating Reagents-- Purified Dol-Pase (25 nmol/min of activity in 9 µl) was incubated with 0-10 mM diethylpyrocarbonate (DEPC), 0-25 mM N-ethylmaleimide (NEM), or 0-25 mM phenylglyoxal for 30 min at 37 °C in 50 mM Tris-HCl, pH 7.2, 0.095% Nonidet P-40, 0.95 mM EDTA in a total volume of 10 µl. Fresh stock solutions of each alkylating reagent in absolute ethanol were prepared just prior to use, and 1 µl of each reagent stock solution at the appropriate concentration was added to Dol-Pase to initiate the reaction. Following the incubation period, each reaction mixture was diluted with 40 µl of cold 50 mM Tris-HCl, pH 7.2, 0.1% (w/v) Nonidet P-40, 25 mM EDTA. In some experiments, treatment of Dol-Pase with DEPC was followed by the addition of 2 µl of 1 M NH2OH (final concentration, 170 mM). Tubes receiving NH2OH were incubated an additional 30 min at 37 °C and then diluted to 50 µl with cold buffer, as described above. After dilution of each sample to 50 µl, one-tenth of that volume was removed and assayed for Dol-Pase activity as described above.

Miscellaneous Procedures-- Estimations of protein concentration utilized the BCA protein assay (Pierce) essentially as described by Smith et al. (30). The reference protein for all assays was bovine serum albumin. The concentration of Dol-P and other lipids in solution or dispersion was determined by the modified Bartlett procedure described by Kates (31). Radioactivity was determined by liquid scintillation counting in a Packard Tri-Carb liquid scintillation spectrometer. Dried samples were dissolved in 0.8 ml of 1% SDS, and 9.2 ml of liquid scintillation fluid (3a70b Complete Counting Mixture or Econo-Safe; Research Products International, Inc.) was added. All data are average values obtained by measuring the amount of radioactivity in each sample for 10 min.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Purification of Polyisoprenyl Phosphate Phosphatase from Pig Brain-- Solubilization trials indicated that the most efficient conditions for extraction of Dol-Pase activity from salt-washed pig brain microsomes required 1% Nonidet P-40 at a detergent/protein ratio of 5-10 mg of detergent/mg of protein (Fig. 1). The yield of solubilized activity varied with different microsomal fractions and the amount of membrane protein used for the preparation.


View larger version (20K):
[in this window]
[in a new window]
  		Fig. 1.   Solubilization of polyisoprenyl phosphate phosphatase activity from microsomal membranes with Nonidet P-40. Salt-washed microsomes were mixed with the indicated concentration of Nonidet P-40. Following extraction and centrifugation, aliquots of the soluble and particulate fractions were assayed to determine the percentage of Dol-Pase activity (bullet ) and protein (open circle ) that was solubilized as described under "Experimental Procedures."

Triton X-100 was nearly as effective, but the polyisoprenyl phosphate phosphatase activity was inhibited by Brij 35, Lubrol PX, CHAPS, sodium deoxycholate, sodium taurocholate, and cetylpyridinium chloride in the same concentration range. Extraction of the enzyme with 1% Nonidet P-40 at neutral pH resulted in solubilization of 60-80% of the activity, with a slight (2-3-fold) increase in specific activity (Table I).

                              
View this table:
[in this window]
[in a new window]
 
Table I
Purification of polyisoprenyl phosphate phosphatase from pig brain microsomes
The details of the purification protocol are described under "Experimental Procedures."

When the detergent-solubilized activity was applied to a TSK-Gel Toyopearl-DEAE 650M column, Dol-Pase was completely retained and was eluted in two narrowly resolved peaks with 0.05-0.1 M NaCl, using a concave 0-0.75 M NaCl gradient (Fig. 2A). The two closely eluted peaks from the anion-exchange column could be due to differential amounts of sialyl residues in a single enzyme, since no evidence for multiple Dol-Pases was seen in the subsequent purification steps (see below).


View larger version (22K):
[in this window]
[in a new window]
  		Fig. 2.   Chromatographic steps in the purification of detergent-solubilized polyisoprenyl phosphate phosphatase activity. Salt-washed microsomes were extracted with 1% Nonidet P-40, and the soluble protein was applied to a column of TSK-Gel Toyopearl-DEAE 650M. The elution profiles of polyisoprenyl phosphate phosphatase (bullet ) and protein (open circle ) are illustrated in A. The supernatant fraction from PEG fractionation of polyisoprenyl phosphate phosphatase activity was subjected to chromatography on TSK-Gel Toyopearl-Blue 650M (B), and fractions from the TSK-Gel Toyopearl-Blue 650M column containing polyisoprenyl phosphate phosphatase activity (bullet ) were pooled and applied to a WGA-Sepharose 6MB column. Aliquots of the indicated fractions were assayed for Dol-Pase activity (bullet ) and protein concentration (open circle ). The details of the chromatographic procedures and assays are described under "Experimental Procedures."

Typically, 60-70% of the phosphatase activity was recovered, resulting in a 10-12-fold purification. Dol-Pase activity in peak fractions was not stimulated by the addition of aliquots from the flow-through fraction or other included fractions, suggesting that the modest loss of activity was due to partial denaturation of the enzyme and not to separation of the enzyme from an unidentified cofactor or regulatory protein during this chromatographic step.

Pooled fractions from the narrowly resolved peaks of polyisoprenyl phosphate phosphatase activity were separated from 80% or more of the other proteins by polyethylene glycol fractionation (Table I). Following high speed centrifugation, the phosphatase was found predominantly in the supernatant fraction, while the bulk of the protein was precipitated.

The 15% PEG supernatant solution containing Dol-Pase was then further purified by adsorption to a column of TSK-Gel Toyopearl-Blue 650M and eluted with a gradient of 0-1 M NaCl (Fig. 2B). The enzyme activity eluted in a single peak centered at 0.75 M NaCl. Most of the applied protein was not retained by the column. The purification resulting from this chromatographic step was generally at least 3-5-fold.

When the fractions from the Toyopearl-Blue column containing Dol-Pase were pooled and applied to a column of WGA-Sepharose 6MB, most of the enzyme was bound by the lectin and could be eluted as a single peak of activity with 0.1 M GlcNAc (Fig. 2C), consistent with the presence of sialylated oligosaccharide chains. Lectin affinity chromatography produced an enzyme preparation that was purified approximately 1,107-fold relative to salt-washed brain microsomes (Table I).

A small portion (5-15%) of the activity did not bind to the column, and repeated applications of the flow-through fraction resulted in only minor additional binding of Dol-Pase to the lectin. This result may indicate heterogeneity in the oligosaccharide chains.

Analysis of the Purified Polyisoprenyl Phosphate Phosphatase by Gel Electrophoresis and Isoelectric Focusing-- SDS-PAGE analysis of purified Dol-Pase revealed a major silver-stained polypeptide of Mr 33,000 (Fig. 3A). To confirm that the major polypeptide observed by silver staining the SDS-gel was the phosphatase assayed using Dol-P as substrate, the purified enzyme was run on a nondenaturing gel by substituting Nonidet P-40 for SDS in the Tris-glycine buffer reported by Laemmli (28). After allowing electrophoresis to proceed for 18 h, with constant cooling of the gel, silver staining revealed a well defined band of protein migrating 1.0-1.5 cm, which corresponds to the gel slices containing Dol-Pase activity (Fig. 3B).


View larger version (27K):
[in this window]
[in a new window]
  		Fig. 3.   Gel electrophoresis of purified polyisoprenyl phosphate phosphatase. Fractions of purified polyisoprenyl phosphate phosphatase eluted from WGA-Sepharose 6MB were pooled, and an aliquot (1 µg; 5 nmol/min enzyme activity) was subjected to SDS-PAGE (A). The gel was a gradient of 8-20% acrylamide, and following electrophoresis the gel was stained with silver (29). B, purified Dol-Pase (1 µg) was subjected to nondenaturing gel electrophoresis through a 5-20% acrylamide gradient as described under "Experimental Procedures." The calibration markers were bovine serum albumin (66 kDa), ovalbumin (45 kDa), glyceraldehyde-3-phosphate dehydrogenase (36 kDa), carbonic anhydrase (29 kDa), trypsinogen (24 kDa), trypsin in hibitor (20 kDa), and lactalbumin (14 kDa). The gel was sliced longitudinally between lanes, and appropriate lanes were silver-stained. A lane adjacent to the one shown above was sliced into 0.5-cm sections and assayed for Dol-Pase activity as a control.

The isoelectric point of Dol-Pase was examined by focusing the purified enzyme in detergent solution. The sharp peak obtained in the plot of phosphatase activity against the corresponding pH values indicates the presence of a single purified polyisoprenyl phosphate phosphatase with an acidic isoelectric point of 5.2 (Fig. 4).


View larger version (17K):
[in this window]
[in a new window]
  		Fig. 4.   Isoelectric point of purified polyisoprenyl phosphate phosphatase. Purified polyisoprenyl phosphate phosphatase was subjected to isoelectric focusing in a Bio-Rad Rotofor apparatus. Fractions were assayed for Dol-Pase activity, and the pH of each fraction was determined as described under "Experimental Procedures."

Presence of Sialyl Residues in Purified Polyisoprenyl Phosphate Phosphatase-- The retention of polyisoprenyl phosphate phosphatase by WGA (Fig. 2C) and the isoelectric point of 5.2 were consistent with the enzyme containing sialooligosaccharide chains. To strengthen the evidence for the presence of sialyl residues, the effect of treating the purified enzyme with neuraminidase on its ability to bind to WGA and other lectins was examined.

As seen in Fig. 2C, over 90% of the untreated enzyme binds to WGA-Sepharose 6MB, but following incubation with neuraminidase the phosphatase eluted directly through the column, indicating the presence of one or more sialyl residues (Table II). Consistent with this result, the purified enzyme also bound to L. flavus agglutinin-agarose, a lectin that is specific for sialic acid (21). Succinylated WGA, a chemically modified form of WGA that eliminates binding to sialyl residues (32) but not GlcNAc moieties, did not bind the purified enzyme (Table II). Also consistent with the presence of sialic acid, treatment of the phosphatase with neuraminidase resulted in an increase in electrophoretic mobility of the treated enzyme relative to the untreated enzyme when compared by SDS-PAGE (Fig. 5).

                              
View this table:
[in this window]
[in a new window]
 
Table II
Lectin binding properties of polyisoprenyl phosphate phosphatase before and after neuraminidase treatment
Purified polyisoprenyl phosphate phosphatase was analyzed by lectin chromatography before and after treatment with neuraminidase (A. ureafaciens) as desribed under "Experimental Procedures."


View larger version (33K):
[in this window]
[in a new window]
  		Fig. 5.   Effect of neuraminidase treatment on mobility of purified polyisoprenyl phosphate phosphatase in SDS-PAGE. Purified Dol-Pase was radiolabeled with 125I and analyzed by SDS-PAGE before (lane 1) and after (lane 2) neuraminidase treatment. Details of the iodination reaction, SDS-PAGE analysis, and preparation of the autoradiogram are described under "Experimental Procedures." The arrows indicate electrophoretic positions of the center of the protein band before and after neuraminidase treatment.

Attempts to identify the glycosyl unit that sialic acid was attached to by examining the binding of the neuraminidase-treated enzyme to lectins specific for beta -Gal/GalNAc (R. communis agglutinin), alpha -GalNAc (V. villosa agglutinin), and Gal(beta -1,4)GalNAc (A. hypogaea agglutinin) were unsuccessful, since the internal glycosyl unit exposed by neuraminidase treatment did not bind to any of the other lectins tested (Table II). All of these results indicate that the anionic Dol-Pase contains at least one sialylated oligosaccharide chain, but further studies will be required to determine the length and complete composition of the carbohydrate unit(s).

Enzymatic Properties of Purified Dol-Pase-- In agreement with previous findings with microsomal Dol-Pase activity (18), the purified enzyme was found to be a neutral phosphatase that exhibited maximum activity at pH 7. The purified Dol-Pase required detergent and was optimally active at Nonidet P-40 concentrations of 0.1-0.2% (not shown).

As seen in Fig. 6A, purified Dol-Pase was inactivated in the presence of F- in a concentration-dependent manner. At 20 mM NaF enzymatic activity was reduced to about 10% of the control level. Orthovanadate, a phosphatase transition state analog, inhibited Dol-Pase by 90% when added at 10 mM. Purified Dol-Pase was inhibited by Zn2+ and Mn2+ but essentially unaffected by the presence of Mg2+ in the assay, up to a concentration of 10 mM (Fig. 6B). Zn2+ was the most potent inhibitor of Dol-Pase of those studied, providing complete inactivation of the enzyme at a concentration of 1 mM ZnCl2. This observation is interesting in view of the fact that Zn2+ ions stimulate brain dolichol kinase (33, 34), suggesting a potential regulatory role for the divalent cation influencing the balance of the phosphorylation/dephosphorylation scheme.


View larger version (16K):
[in this window]
[in a new window]
  		Fig. 6.   Inhibition of purified polyisoprenyl phosphate phosphatase by fluoride, orthovanadate, and divalent cations. A, purified Dol-Pase was dialyzed against 50 mM Tris-HCl, pH 7.5, 0.1% Nonidet P-40 and then assayed for activity in the presence of the indicated concentrations of NaF (open circle ) or Na3VO4 (black-diamond ). B, Dol-Pase activity was assayed in the presence of the indicated concentrations of MgCl2 (bullet ), MnCl2 (down-triangle), or ZnCl2 (diamond ) included in the assay of the dialyzed enzyme, and EDTA was omitted from the reaction mixture. A control assay with NaCl is also shown in A. Other details of the assay procedure were as described under "Experimental Procedures."

Evaluation of Various Phosphomonoesters as Substrates for Purified Dol-Pase-- To examine the substrate specificity of the phosphatase purified by following Dol-Pase activity, several structurally related and unrelated phosphoesters were tested as substrates. There were only very modest differences in the rate of dephosphorylation of Dol-P homologues of varying chain length from C55 to C100 (Table III). The rate of dephosphorylation of the water-soluble analogue of Dol-P, citronellyl phosphate (C10-Dol-P), was approximately 50% of the rate observed with C95-Dol-P. Although there was not an absolute requirement for the presence of the saturated alpha -isoprene unit in the dolichyl moiety, the purified polyisoprenyl phosphate phosphatase did exhibit a preference for Dol-P over the fully unsaturated C95-polyprenyl phosphate with the same chain length.

                              
View this table:
[in this window]
[in a new window]
 
Table III
Substrate specificity of purified Dol-Pase
Purified Dol-Pase was incubated under the standard assay conditions in the presence of each indicated substrate added at a concentration of 200 µM, and the rate of dephosphorylation was assayed as described under "Experimental Proceures."

Kinetic analyses of purified enzyme activity toward defined isoprenologues of Dol-P were done to estimate the relative affinities and maximal initial rates of the enzyme with each substrate. Double reciprocal plots of the data obtained from saturation curves revealed similar approximate Km values (46-52 µM) for C90-, C95-, and C100-Dol-P (Fig. 7A).


View larger version (22K):
[in this window]
[in a new window]
  		Fig. 7.   Double-reciprocal plots of initial rates of dephosphorylation of C90-100-Dol-P and dioleoyl-PA by purified polyisoprenyl phosphate phosphatase at varying concentrations of each lipid phosphomonoester. Purified polyisoprenyl phosphate phosphatase was assayed as described with C90-Dol-P (open circle ), C95-Dol-P (bullet ), and C100-Dol-P (down-triangle) as substrate (A). C95-Dol-P (bullet ) and dioleoyl-PA (black-square) were compared in B. All substrates were assayed in the concentration range of 10-250 µM.

Phosphatidate has been shown to be a potent inhibitor of Dol-Pase in some tissues (16, 18, 35-37). Direct assay for enzymatic activity with dioleoyl-PA as substrate revealed that the phospholipid was efficiently hydrolyzed by the purified enzyme (Table III), suggesting that the enzyme may possess a dual specificity. A kinetic comparison of the enzymatic dephosphorylation of C95-Dol-P and dioleoyl-PA revealed similar approximate Km values and maximum initial rates for both lipophilic phosphomonoesters (Fig. 7B). Consistent with a common substrate binding site, Dol-P competitively inhibited dephosphorylation of PA (data not shown).

Although the purified enzyme was capable of dephosphorylating the two structurally diverse lipophilic substrates, Dol-P and PA, it is clearly not a general, nonspecific phosphatase, since no detectable dephosphorylation of farnesyl monophosphate, glucose 6-phosphate, mannose 6-phosphate, p-nitrophenyl phosphate, or 5'-AMP was observed (Table III).

The Enzymatic Dephosphorylation of Dol-P and PA by Purified Polyisoprenyl Phosphate Phosphatase Is Identically Sensitive to Several Alkylating Reagents-- Specific alkylating reagents were tested in an attempt to identify essential aminoacyl residues in purified polyisoprenyl phosphate phosphatase and to see if the enzymatic dephosphorylation of Dol-P and PA could be distinguished on the basis of differential sensitivity to the chemical reagents.

First, DEPC totally inhibited enzymatic activity with both substrates at 10 mM (Fig. 8A), suggesting the presence of a critical histidinyl residue (38). The sensitivities of the enzymatic dephosphorylation of both phospholipid substrates to NEM were virtually identical, implicating a sulfhydryl residue in the active site (Fig. 8B). Phenylglyoxal (25 mM), an arginine-specific reagent, also reduced the rate of Dol-P and PA hydrolysis in close parallel (Fig. 8C). Thus, an arginyl residue may also be critical for Dol-Pase activity acting on both substrates.


View larger version (27K):
[in this window]
[in a new window]
  		Fig. 8.   Inactivation of the enzymatic dephosphorylation of Dol-P and PA by NEM, phenylglyoxal, and diethylpyrocarbonate. Purified polyisoprenyl phosphatase was incubated with the indicated concentrations of DEPC (A), NEM (B), or phenylglyoxal (C) and then assayed for the enzymatic dephosphorylation of Dol-P (bullet ) or PA (open circle ) as described under "Experimental Procedures."

Overall, the results in Fig. 8 show that treatment of the purified enzyme with all three of the chemical reagents inactivated Dol-P and PA dephosphorylation in close parallel. These data strongly suggest that a single phosphatase has been purified and that it is capable of dephosphorylating Dol-P and PA. Although the possibility that two separate phosphatases have been co-purified in this study cannot be completely eliminated, if this were true both enzymes must contain sialyl residues and have virtually identical molecular weights, isoelectric points, and enzymatic properties.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Although it has been known for many years that crude microsomal fractions from brain and numerous other mammalian tissues catalyze the dephosphorylation of Dol-P (18, 35, 36, 39-41), the exact number and specificity of the membrane-bound phosphatase(s) have not been established. This paper describes the isolation and properties of a highly purified polyisoprenyl phosphate phosphatase from pig brain. The pig brain enzyme was solubilized from salt-washed microsomes with the nonionic detergent Nonidet P-40 and purified 1,107-fold by a protocol including anion exchange chromatography, PEG-fractionation, dye column chromatography, and lectin affinity chromatography. The purified enzyme has been shown to be a 33-kDa polypeptide, containing sialylated oligosaccharide chains, and appears to be an integral membrane glycoprotein, since it could not be extracted from microsomes with high ionic strength buffer solutions. In preliminary experiments, the sialoglycoprotein was not sensitive to N-glycanase, but the detailed structure of the carbohydrate moiety remains to be elucidated. The presence of a sialylated carbohydrate chain is consistent with a light microsomal/plasma membrane location previously reported for the membrane-bound activity (19, 35).

The enzymological properties of the purified enzyme are very similar to the neutral phosphatase activity associated with calf brain microsomes (18). Based on the activity profiles for each step in the purification procedure, this enzyme appears to be responsible for the majority of the "Dol-P" phosphatase activity detected in microsomal fractions. Moreover, the observation that calf brain microsomes dephosphorylate endogenous, prelabeled Dol-P in its native membrane environment (18) strongly suggests that the in vitro dephosphorylation reaction is not just an artifact of presenting the exogenous substrate in an unnatural detergent dispersion.

A Dol-Pase activity could play important roles in the catabolism and biosynthesis of the glycosyl carrier lipid. Since Dol-P is synthesized via a fully unsaturated, long chain polyprenyl pyrophosphate (42-46) and most cellular dolichol is present as the free polyisoprenol (12), it is quite likely that mammalian cells contain a phosphatase responsible for the dephosphorylation of Dol-P and/or the polyprenyl pyrophosphate intermediates.

Another potential function for the brain enzyme could be to dephosphorylate the seven Dol-P molecules formed during the mannosylation and glucosylation reactions involving Man-P-Dol and Glc-P-Dol on the luminal leaflet of the ER (2, 47) to facilitate the transbilayer movement of dolichol. Although there is evidence that the transbilayer movement of Dol-P in phospholipid bilayers is very slow (48), the free long chain polyisoprenol may diffuse freely between the luminal leaflet and the cytoplasmic monolayer of the ER/Golgi following dephosphorylation. In this context, Wolf et al. (49) speculated that a recycling mechanism in which dolichyl diphosphate, discharged during the primary N-glycosylation event when the glucosylated precursor oligosaccharide is transferred from the carrier lipid to appropriate nascent polypeptides, may exit the ER via bulk flow. Dolichyl diphosphate would then be converted to dolichol in the Golgi apparatus by the sequential action of dolichyl diphosphate and Dol-Pases. Both of these activities have been found in light microsomal/Golgi fractions in brain (19, 49). Free dolichol formed by this scheme could more readily diffuse to the cytoplasmic leaflet and be retrieved by the ER, where it could be rephosphorylated by dolichol kinase (26, 50, 51) and utilized for another round of Glc3Man9GlcNAc2-P-P-Dol biosynthesis.

An interesting result emerging from the examination of the specificity of the purified enzyme was that although it is not a nonspecific phosphomonoesterase, the pig brain phosphatase catalyzes the enzymatic dephosphorylation of Dol-P and PA with very similar catalytic efficiencies. In related studies, Dol-Pase activity in rat liver plasma membranes was found to be inhibited by PA and lyso-PA (35). This may have been a nonspecific inhibitory effect, since Dol-P dephosphorylation was also inhibited by phosphatidylcholine and phosphatidylethanolamine. Phosphatidate competitively inhibited Dol-P hydrolysis catalyzed by soy bean microsomes (37), but based on differences in thermolability and sensitivity to Mn2+ and NEM, DeRosa and Lucas (16) concluded that Dol-Pase activity in oviduct microsomes was distinct from PAase activity.

These observations and the results of this study raise interesting questions about the relationship between the purified enzyme described here and the various enzymes that dephosphorylate PA reported previously. Multiple forms of PA phosphatase activities have been found in yeast and mammalian tissues, and it has been proposed that they are involved in the de novo synthesis of triacylglycerols and membrane phospholipids (52) as well as signal transduction (53). The Dol-Pase described here is readily distinguished from PAP1, the cytosolic/ER form, by its subcellular location and its requirement for Mg2+ (54). The properties of yeast PA phosphatase (55, 56) are similar to the cytosolic and microsomal PA phosphatase activities in liver but differ from the purified pig brain Dol-Pase with respect to its Mg2+ requirement and insensitivity to fluoride ions. A neutral, Mg2+-independent Dol-Pase with the same properties as the pig brain enzyme described here has also been detected in microsomes from S. cerevisiae.2

The pig brain Dol-Pase shares some characteristics with the Mg2+-independent PA phosphatase (54). PAP2 was purified from porcine thymus by Kanoh and co-workers (57) and originally reported to be 83 kDa, but the size was later revised to be 35 kDa after the cDNA was cloned (58). The thymus PAP2 has a native molecular size (218 kDa) similar to that of Dol-Pase when estimated by gel filtration2 but is insensitive to 10 mM NEM and only partially inhibited by 20 mM NaF. In contrast to PAP2, the pig brain Dol-Pase is inhibited 80% by 10 mM NEM and over 90% by 20 mM NaF (Figs. 6 and 8). Dol-Pase purified from brain also is clearly distinguished from PAP2 on the basis of their differential sensitivities to phenylglyoxal, diethylpyrocarbonate, and Mn2+ ions. Dol-Pase is inhibited 80% by 2 mM MnCl2, 100% by 25 mM phenylglyoxal, and 70% by 5 mM DEPC (Figs. 6 and 8), whereas PAP2 is considerably less sensitive to these compounds (57, 59).

Waggoner et al. (60) have also reported a pI < 4 for an anionic form of PAP2, while a pI of 5.2 was determined for purified Dol-Pase from pig brain (Fig. 4). The anionic form of PAP2 may also be sialylated (60), but in contrast to the pig brain polyisoprenyl phosphate phosphatase described here, Waggoner et al. (61) found Dol-P to be a very poor substrate for PAP2. These authors reported that no hydrolysis was seen when purified PAP2 was incubated with Dol-P for 1-4 h. However, 5 mM NEM, which partially inhibits the Dol-Pase activity described in this paper (Fig. 8), was included in the incubation mixture. Dol-Pase also differs from the ecto-PAase reported by Perry et al. (62), which is insensitive to 10 mM NEM. Considering all of these enzymological differences, it seems unlikely that the pig brain Dol-Pase is identical to the previously characterized PAP2 enzymes. These results further emphasize the importance for workers interested in Dol-P or PA (as well as ceramide phosphate and sphingosine phosphate) metabolism to be aware that microsomal fractions from mammalian cells contain multiple phosphatases capable of dephosphorylating these lipophilic phosphomonoesters.

In conclusion, the first procedure for the effective purification of a neutral Dol-Pase from pig brain microsomes is described. The enzyme is an intrinsic membrane glycoprotein that is capable of dephosphorylating Dol-P and PA with similar catalytic efficiencies. Although the porcine phosphatase is not a nonspecific phosphomonoesterase, future molecular studies will be required to establish the exact role this enzyme plays in Dol-P metabolism and/or possibly the dephosphorylation of other lipid phosphomonoesters in vivo.

    ACKNOWLEDGEMENTS

We thank Dean Crick, Jeff Rush, and Tessie McNeely for helpful suggestions throughout the course of this work.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM36065 (to C. J. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger This work was completed in partial fulfillment of doctoral dissertation requirements for this author. Current address: Dept. of Pathology and Laboratory Medicine, 272 John Morgan Bldg., University of Pennsylvania School of Medicine, Philadelphia, PA 19104.

§ To whom correspondence should be addressed.

1 The abbreviations used are: Dol-P, dolichyl monophosphate; Dol-Pase, dolichyl monophosphate phosphatase; WGA, wheat germ agglutinin; PA, phosphatidic acid; ER, endoplasmic reticulum; PEG, polyethylene glycol; PAGE, polyacrylamide gel electrophoresis; DEPC, diethylpyrocarbonate; NEM, N-ethylmaleimide; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.

2 D. W. Frank and C. J. Waechter, unpublished observations.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Kornfeld, R., and Kornfeld, S. (1985) Annu. Rev. Biochem. 54, 631-664[CrossRef][Medline] [Order article via Infotrieve]
  2. Waechter, C. J. (1989) in Neurobiology of Glycoconjugates (Margolis, R. U., and Margolis, R. K., eds), pp. 127-149, Plenum Press, New York
  3. Harford, J. B., Waechter, C. J., and Earl, F. L. (1977) Biochem. Biophys. Res. Commun. 76, 1036-1043[CrossRef][Medline] [Order article via Infotrieve]
  4. Lucas, J. J., and Levin, E. (1977) J. Biol. Chem. 252, 4330-4336[Free Full Text]
  5. Hubbard, S. C., and Robbins, P. W. (1980) J. Biol. Chem. 255, 11782-11793[Abstract/Free Full Text]
  6. Carson, D. D., Earles, B. J., and Lennarz, W. J. (1981) J. Biol. Chem. 256, 11552-11557[Abstract/Free Full Text]
  7. Lennarz, W. J. (1985) Trends Biochem. Sci. 10, 248-251[CrossRef]
  8. Spiro, M. J., and Spiro, R. G. (1986) J. Biol. Chem. 261, 14725-14732[Abstract/Free Full Text]
  9. Pan, Y. T., and Elbein, A. D. (1990) Biochemistry 29, 8077-8084[CrossRef][Medline] [Order article via Infotrieve]
  10. Crick, D. C., and Waechter, C. J. (1994) J. Neurochem. 62, 247-56[Medline] [Order article via Infotrieve]
  11. Crick, D. C., Scocca, J. R., Rush, J. S., Frank, D. W., Krag, S. S., and Waechter, C. J. (1994) J. Biol. Chem. 269, 10559-10565[Abstract/Free Full Text]
  12. Rip, J. W., Rupar, C. A., Ravi, K., and Carroll, K. K. (1985) Prog. Lipid Res. 24, 269-309[CrossRef][Medline] [Order article via Infotrieve]
  13. Rossignol, D. P., Scher, M., Waechter, C. J., and Lennarz, W. J. (1983) J. Biol. Chem. 258, 9122-9127[Abstract/Free Full Text]
  14. Bhat, N. R., Frank, D. W., Wolf, M. J., and Waechter, C. J. (1991) J. Neurochem. 56, 339-344[CrossRef][Medline] [Order article via Infotrieve]
  15. Scher, M. G., Sumbilla, C. M., and Waechter, C. J. (1985) J. Biol. Chem. 260, 13742-13746[Abstract/Free Full Text]
  16. DeRosa, P. A., and Lucas, J. J. (1984) Arch. Biochem. Biophys. 234, 537-545[CrossRef][Medline] [Order article via Infotrieve]
  17. Sagami, H., Kurisaki, A., and Ogura, K. (1993) J. Biol. Chem. 268, 10109-10113[Abstract/Free Full Text]
  18. Burton, W. A., Scher, M. G., and Waechter, C. J. (1981) Arch. Biochem. Biophys. 208, 409-417[CrossRef][Medline] [Order article via Infotrieve]
  19. Scher, M. G., DeVries, G. H., and Waechter, C. J. (1984) Arch. Biochem. Biophys. 231, 293-302[CrossRef][Medline] [Order article via Infotrieve]
  20. Frank, D., and Waechter, C. J. (1990) FASEB J. 4, 1930 (abstr.)
  21. Miller, R. L., Collawn, J. F., Jr., and Fish, W. W. (1982) J. Biol. Chem. 257, 7574-7580[Abstract/Free Full Text]
  22. Adair, W. L., Jr., and Keller, R. K. (1985) Methods Enzymol. 111, 201-215[Medline] [Order article via Infotrieve]
  23. Danilov, L. L., Druzhinina, T. N., Kalinchuk, N. A., Maltsev, S. D., and Shibaev, V. N. (1989) Chem. Phys. Lipids 51, 191-203
  24. Dittmer, J. C., and Lester, R. L. (1964) J. Lipid Res. 5, 126-127
  25. McSweeney, G. P. (1965) J. Chromatogr. 17, 183-185[CrossRef]
  26. Sumbilla, C., and Waechter, C. J. (1985) Methods Enzymol. 111, 471-482[Medline] [Order article via Infotrieve]
  27. Rush, J. S., and Waechter, C. J. (1992) Anal. Biochem. 206, 328-333[CrossRef][Medline] [Order article via Infotrieve]
  28. Laemmli, U. K. (1970) Nature 227, 680-685[CrossRef][Medline] [Order article via Infotrieve]
  29. Wray, W., Boulikas, T., Wray, V. P., and Hancock, R. (1981) Anal. Biochem. 118, 197-203[CrossRef][Medline] [Order article via Infotrieve]
  30. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985) Anal. Biochem. 150, 76-85[CrossRef][Medline] [Order article via Infotrieve]
  31. Kates, M. (1986) Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 3, Part 2, Elsevier Science Publishers B.V., Amsterdam
  32. Monsigny, M., Roche, A-C., Sene, C., Maget-Dana, R., and Delmotte, F. (1980) Eur. J. Biochem. 104, 147-153[Medline] [Order article via Infotrieve]
  33. Sumbilla, C., and Waechter, C. J. (1985) Arch. Biochem. Biophys. 238, 75-82[CrossRef][Medline] [Order article via Infotrieve]
  34. Sakakihara, Y., and Volpe, J. J. (1985) J. Biol. Chem. 260, 15413-15419[Abstract/Free Full Text]
  35. Rip, J. W., Rupar, C. A., Chaudhary, N., and Carroll, K. K. (1981) J. Biol. Chem. 256, 1929-1934[Free Full Text]
  36. Belocopitow, E., and Boscoboinik, D. (1982) Eur. J. Biochem. 125, 167-173[Medline] [Order article via Infotrieve]
  37. Ravi, K., Rip, J. W., and Carroll, K. K. (1983) Biochem. J. 213, 513-518[Medline] [Order article via Infotrieve]
  38. Miles, E. W. (1977) Methods Enzymol. 47, 431-442[Medline] [Order article via Infotrieve]
  39. Idoyaga-Vargas, V., Belocopitow, E., Mentaberry, A., and Carminatti, H. (1980) FEBS Lett. 112, 63-66[CrossRef][Medline] [Order article via Infotrieve]
  40. Wedgewood, J. F., and Strominger, J. L. (1980) J. Biol. Chem. 255, 1120-1123