Partial purification and characterization of dolichol phosphate mannose synthase from Entamoeba histolytica
Julio C. Villagómez-Castro2,
Carlos Calvo-Méndez2,3,
Arturo Flores-Carreón2,3 and
Everardo López-Romero1,2,3
2Instituto de Investigación en Biología Experimental, Facultad de Química, Universidad de Guanajuato, Apartado Postal 187, Guanajuato, Gto. 36000, México, and 3Departamento de Genética y Biología Molecular, CINVESTAV del IPN, Apartado Postal 14740, México, D.F. 07000, México
Received on April 20, 2000; revised on June 9, 2000; accepted on July 5, 2000.
 |
Abstract
|
---|
Dolichol phosphate mannose synthase, an essential enzyme in glycoprotein biosynthesis, was partially purified from E.histolytica by hydrophobic interaction and affinity chromatography with octyl Sepharose CL-4B and Affi-Gel 501, respectively. Reducing agents, particularly dithiothreitol, positively influenced enzyme activity and stability, indicating a role of sulfhydryl groups on the transferase function. Activity did not depend on phospholipids; however, it was significantly stimulated by phosphatidylethanolamine and to a lower extent by other common phospholipids. Mixtures consisting of activating phospholipids did not exert an additive effect. In vitro phosphorylation with a cAMP-dependent protein kinase resulted in enzyme activation. This alteration was not associated with a change in the Km for the substrate but rather with a 2.6-fold increase in Vmax. Phosphorylation in the presence of [
-32P]ATP resulted in strong labeling of two polypeptides, one of which exhibited the molecular mass reported for the enzyme from other organisms. Whether phosphorylation functions in vivo as a mechanism of regulation of dolichol phosphate mannose synthesis in E.histolytica remains to be determined.
Key words: dolichol phosphate mannose synthase/Entamoeba histolytica/protein glycosylation
 |
Introduction
|
---|
Dolichol phosphate mannose synthase (DPMS) catalyzes the transfer of mannose from GDP-Man to dolichol monophosphate (Dol-P) forming dolichol phosphate mannose (Dol-P-Man), a mannosyl donor in protein N- and O-glycosylation and in the biosynthesis of glycosylphosphatidylinositol membrane anchors (Menon et al., 1990
; Herscovics and Orlean, 1993
). The enzyme has been studied in a number of organisms and partially purified in a few cases (Heifetz and Elbein, 1977
; Jensen and Schutzbach, 1985
; Kruszewska et al., 1996
). Most of our knowledge of DPMS comes from studies in Saccharomyces cerevisiae. The yeast structural gene for the enzyme, DPM1, encodes a 30 kDa endoplasmic reticulum polypeptide (Beck et al., 1990
) which is essential for viability (Orlean et al., 1988
). Based on differences of DPMS gene sequences from various organisms and on the fact that it is an essential protein in yeast, the enzyme has been proposed as a potential target for inhibitors of pathogenic organisms (Colussi et al., 1997
). Both the native (Haselbeck, 1989
) and recombinant (Schutzbach et al., 1993
) DPMS from S.cerevisiae have been purified and some of their properties investigated.
Work in this laboratory has dealt with the characterization of essential glycosyl transferases for the synthesis of glycoproteins in E.histolytica, such as DPMS (Villagómez-Castro et al., 1998
) and the enzymes that catalyze the first two reactions of N-linked glycosylation (Vargas-Rodríguez et al., 1998
). To get further insight into these enzymes, here we report the partial purification and biochemical characterization of amoeba DPMS.
 |
Results
|
---|
Purification of DPMS
Following elution with 1% Igepal CA-630, DPMS emerged from the Octyl-Sepharose CL-4B column as a sharp peak of activity well beyond the main protein peak which appeared in the KCl-wash (Figure 1). Most active fractions were pooled and after reduction with DTT the sample was subjected to affinity chromatography in Affi-Gel 501. Following elution with a continuous gradient of DTT, a peak of DPMS activity was resolved between 12 and 14 mM whereas most protein emerged in the washing eluate (Figure 2). Most active fractions were pooled and after removal of DTT they were used for enzyme characterization. Results of purification are summarized in Table I. Purification and recovery of DPMS activity after the final step were 18.7-fold and 37%, respectively. For these estimations, the Brij 35 pellet and not the whole homogenate was considered as the starting material since in the latter mannose is transferred to products other than Dol-P-Man. In terms of protein recovery, enzyme purification should be over 3-fold higher considering that total protein decreased from 121 mg in the whole homogenate (not shown) to 35.8 mg in the Brij 35 pellet. Polyacrylamide gels (10%) stained with silver nitrate revealed the presence in the affinity-purified samples of 10 polypeptides in the range of 26.4134.5 kDa. These included a protein of 31.5 kDa, a molecular mass closely comparable to that of DPMS from other organisms (not shown; see below). It is worth noting that the difficulty to obtain large amounts of trophozoites and also instability of DPMS precluded purification of the active polypeptide to homogeneity.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 1. Separation of DPMS by hydrophobic interaction. Detergent-free enzyme was applied on a column (1.7 x 4.9 cm) of Octyl Sepharose CL-4B equilibrated with buffer D. After washing with the same buffer, the sample was eluted with a discontinuous 05% gradient of Igepal CA-630 in buffer C (broken line). Elution of protein (open symbols) and DPMS activity (solid symbols) were monitored as described in the text.
|
|

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 2. Separation of DPMS by affinity chromatography. DTT-free enzyme was applied on a column (1 x 9 cm) of Affi-Gel 501 equilibrated with buffer E. After washing with the same buffer, the sample was eluted with a continuous gradient of 020 mM DTT in buffer E as indicated. Elution of protein (open symbols) and DPMS activity (solid symbols) were monitored as described in the text.
|
|
Effect of reducing agents on DPMS activity and stability
The presence of a number of thiol-dependent enzymes in Entamoeba species is well documented (Que and Reed, 1997
). Therefore, we considered it important to determine whether the amoeba DPMS was affected by common reducing agents. Early experiments revealed that activity of fresh preparations of the solubilized enzyme increased by only 1320% in the presence of either ß-mercaptoethanol, cysteine, or DTT at optimal concentrations of 1.25 mM, 2.5 mM, and 5 mM, respectively (not shown). However, enzyme stability at 4°C markedly increased when 5 mM DTT was added shortly after DPMS solubilization, as judged from the preservation of 78% of activity after 3 days of storage as compared with only 13% in the control (Figure 3). More important, however, was the observation that addition of DTT during assay of activity of samples that had been stored for 8 days at 4°C either in the absence or in the presence of this reductant restored enzyme activity to levels that represented about 7484% of the initial value. Both ß-mercaptoethanol and cysteine were less efficient in stabilizing and/or restoring the activity of samples that had been kept in the presence of the corresponding reductants (not shown). When tested on samples purified by affinity chromatography, DTT increased enzyme activity about 5-fold at 24 mM. Higher concentrations up to 8 mM did not result in further stimulation (not shown).

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 3. Effect of DTT on DPMS activity and stability. Samples of detergent- and DTT-free solubilized DPMS were incubated at 4°C in the presence of either buffer (open and solid circles) or 5 mM DTT (open and solid triangles). After the times indicated, activity was assayed either without further addition (open symbols) or after addition (solid symbols) of 5 mM DTT.
|
|
Reconstitution of DPMS activity by phospholipids
The ability of a number of phospholipids with different head groups to stimulate DPMS activity was determined. When tested individually, PC, PI, and PE increased enzyme activity by 1.3-, 1.8-, and 2.3-fold, respectively, at the optimum concentrations indicated in Figure 4. PG was inhibitory at all concentrations tested. When added in combination, mixtures were prepared as to have 100 µg of total phospholipid per assay, maintaining equal proportions of each component. Stimulation by the PE-PI mix (1.5-fold) was comparable to that obtained by PE alone (1.6-fold). Other mixtures were inhibitory in the range of 42% for PE-PC-PG-PI to 69% for PE-PG (Table II).

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 4. Effect of phospholipids on DPMS activity. Enzyme activity was assayed in the presence of increasing concentrations of the indicated phospholipids. All other components of the mixture remained unchanged.
|
|
Effect of in vitro phosphorylation on DPMS activity
It has been described that mammalian DPMS is stimulated by phosphorylation with cAMP-PK (Banerjee et al., 1987
) and that the enzyme is associated with a 32 kDa phosphoprotein (Banerjee et al., 1999
). We considered it important to determine whether this was also the case for the amoeba DPMS. Results (Table III) indicated that after treatment with ATP and cAMP-PK, DPMS activity increased about 4.8-fold over the mock incubation. Addition of a specific inhibitor of protein kinase reduced DPMS activity to values close to the control thus confirming that enzyme activation depended on the kinase activity. Interestingly, incubation in the absence of the exogenous PK resulted in an almost 2-fold stimulation suggesting the presence of an endogenous kinase activity. As expected, cAMP-PK alone had no effect on enzyme activity.
The analysis of protein phosphorylation after incubation of DPMS preparations with [
-32P]ATP and cAMP-PK revealed the presence of two strongly labeled polypeptides showing molecular masses of 31.6 (D) and 26.9 (E) kDa. In addition to these, three other faintly phosphorylated proteins of 43.6 (A), 38.4 (B), and 35.4 (C) kDa were detected (Figure 5, lane 1). Addition of the kinase inhibitor substantially reduced labeling of proteins D and E whereas polypeptides A, B, and C were no longer observed (Figure 5, lane 2). In the absence of the kinase, phosphorylation of protein D was barely detectable whereas polypeptide E remained significantly labeled (Figure 5, lane 3).

View larger version (118K):
[in this window]
[in a new window]
|
Fig. 5. Phosphorylation pattern of affinity-purified enzyme preparations by cAMP-PK. The affinity-purified DPMS was incubated at 37°C with [ 32P]ATP and the other components of the phosphorylation mixture in the presence (lanes 1 and 2) or absence (lane 3) of cAMP-PK and in the presence of the cAMP-PK inhibitor (lane 2). After 1 h, proteins were precipitated with TCA and processed as described in the text to reveal the labeled polypeptides by autoradiography.
|
|
Finally, to investigate whether activation by phosphorylation altered the enzyme affinity for the substrate, Km for GDP-Man and Vmax were determined under phosphorylation conditions as well as in conditions where the kinase was omitted. Activity of both enzyme preparations followed parallel Michaelis-Menten kinetics but that of the phosphorylated enzyme was substantially higher (Figure 6A). Apparent Km values for GDP-Man were 5.0 and 6.5 µM for the unphosphorylated and phosphorylated DPMS, respectively. Corresponding Vmax values were 50 and 133 pmol mannose incorporated/min/mg protein (Figure 6B).

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 6. Effect of substrate concentration on activity of phosphorylated and unphosphorylated DPMS. The affinity-purified enzyme was incubated at 37°C with (solid symbols) or without (open symbols) cAMP-PK and the other components of the phosphorylation mixture as described in Materials and methods. After 30 min, GDP-[14C]Man was added at the indicated concentrations and incubation continued for another 30 min. After this time, the mannolipid was extracted and quantified. (A) Enzyme velocity versus substrate concentration; (B) Lineweaver-Burk plots of data from (A). Lines drawn resulted from a least-squares analysis of data. Here, activity is expressed as pmol of mannose incorporated into Dol-P-Man/min/mg protein.
|
|
 |
Discussion
|
---|
Efforts to overcome the problem of amoeba DPMS instability resulted in partial stabilization of the enzyme by thiol-protecting compounds. While these agents did not significantly alter the activity of freshly extracted DPMS, activity of samples that had been stored for up to 8 days at 4°C either in the absence or presence of DTT and appeared to be inactive, was restored by DTT to levels that represented over 70% of the initial value. Cysteine and ß-mercaptoethanol were less efficient in restoring activity of samples incubated in the presence of the corresponding reductants. DTT was thus a critical component of assay mixtures as it increased activity of affinity-purified DPMS up to 4- to 5-fold at optimum concentration. These results indicate that activity of amoeba DPMS depends on functional thiol groups. Whether these are at or close the catalytic domain is not presently known. This property allowed us to partially purify DPMS affinity chromatography with Affi-Gel 501, an agarose derivative that forms covalent mercaptide bonds with free sulfhydryls. A 19-fold enzyme enrichment over the starting material was achieved after the second step. In most experiments, values of specific activity of affinity-purified DPMS ranged from 1000 to 5000 cpm/min/mg protein, a variation most likely depending on the state of oxidation of the enzyme as well as on other properties that will be discussed later.
Solubilized or purified glycosyltransferases are differently affected by phospholipids bearing distinct head groups. Thus, it has been described that mammalian DPMS is active in the presence of PE and of phospholipid mixtures of PE and PC (Jensen and Schutzbach, 1985
). Similarly, the yeast enzyme was active when reconstituted with PE but showed little activity in PC bilayers (Schutzbach and Zimmerman, 1992
). The plant GlcNAc-1-P transferase (GPT), on the other hand, is stimulated by PG and PI and inhibited by PC, PE, and PS (Kaushal and Elbein, 1985
). Work from this laboratory has shown that activity of GPT and GlcNAc transferases from E.histolytica is not stimulated by PE, PC, and PI while it is inhibited by PG (Vargas-Rodríguez et al., 1998
). Here, DPMS was activated by common phospholipids in an increasing order of efficiency of PC<PI<PE whereas PG was inhibitory. Stimulation was not additive as the effect of the PE-PI mix was comparable to that exerted by PE alone. Activating phospholipids did not counteract the effect of PG since mixtures containing the latter remained inhibitory. Phospholipid effect on amoeba DPMS is thus reminiscent of that from yeast and mammalian cells. Removal of phospholipids during enzyme purification may thus be another important factor influencing stability and recovery of amoeba DPMS.
Banerjee et al. (1987)
first demonstrated that in vitro phosphorylation of mammalian microsomes enhanced DPMS activity by 4080% and that this change was associated with a 2-fold increase in Vmax with no significant alteration in the apparent Km for GDP-Man. Later, it was described that a fungal DPMS was also activated by cAMP-PK (Kruszewska et al., 1996
). In agreement with these findings, incubation of amoeba DPMS under phosphorylation conditions resulted in enzyme stimulation which was not related with an increased affinity for the substrate but to a 2.6-fold increase in the Vmax value. A difference with the mammalian enzyme was a larger variation in the magnitude of activation, ranging from 40 to 380% in several experiments. This may be due to variations in the extent of endogenous phosphorylation of DPMS in the different enzyme preparations.
Examination of the pattern of protein phosphorylation revealed the labeling of several polypeptides with molecular weights in the range of 26.9 to 43.6 kDa. One of these, polypeptide D, exhibited a molecular weight closely comparable to that estimated for yeast (Beck et al., 1990
), fungal (Kruszewska et al., 1996
) and mammalian (Banerjee et al., 1999
) DPMS. This labeled species was barely detectable when the kinase was omitted or otherwise blocked by a specific inhibitor. A protein with this molecular weight was also detected by denaturing electrophoresis of partially purified DPMS (not shown). The nature of the other 32P-labeled polypeptides has not been determined. However, polypeptide A may well correspond to the kinase itself, for which a molecular mass of 43 kDa has been estimated by SDS-PAGE (Sigma Bulletin). Likewise, polypeptide E is likely to correspond to an inhibitor-sensitive, endogenous protein kinase. This polypeptide would be expected to remain labeled under conditions where the exogenous kinase is omitted. The endogenous phosphorylating activity would explain the 2-fold activation of DPMS observed in the absence of the commercial kinase.
In summary, results presented here demonstrate that at least three enzyme properties may be critical for regulation of amoeba DPMS in vitro: (1) its dependence on reduced thiol groups, (2) the influence of phospholipids on catalytic activity, and (3) the enzyme activation by protein phosphorylation. The in vivo significance of these properties remains to be determined.
 |
Materials and methods
|
---|
Organism and culture conditions
E.histolytica, strain HM1:IMSS, was maintained and propagated as previously described (Villagómez-Castro et al., 1998
).
Buffers
The following buffers were used: 50 mM Tris/HCl buffer, pH 7.5 (buffer A) containing either 5% (buffer B) or 10% (buffer C) glycerol, buffer C plus 2 M KCl (buffer D), and buffer C plus 0.25% Igepal CA-630 (buffer E).
Preparation of enzyme fractions
Preparation of trophozoite membranes and extraction of DPMS with the nonionic detergent Igepal CA-630 were carried out according to previously described protocols (Villagómez-Castro et al., 1998
).
Assay of DPMS activity
Unless otherwise stated, reaction mixtures contained 7.5 mM MgCl2, 4 µg Dol-P, 4 mM DTT, 0.01% Igepal CA-630, 0.01 µCi (about 22,000 cpm) of GDP-[14C]Man (sp. act., 55 mCi/mmol), the enzyme fraction (3060 µg protein), and buffer A in a total volume of 300 µl. After 30 min of incubation at 37°C, the reaction was stopped and the mannolipid was extracted as described previously (Villagómez-Castro et al., 1998
). Unless otherwise indicated, activity was expressed as radioactivity (cpm) incorporated into Dol-P-Man in one min. Specific activity was referred to one mg of protein.
Purification of DPMS
(1) Hydrophobic interaction with Octyl Sepharose CL-4B. Detergent in the solubilized enzyme preparation was removed by passing the sample (1214 ml) through a column (2.5 x 24.5 cm) of Bio-Beads SM-2 adsorbent equilibrated with buffer B. After elution with the same buffer, the enzyme was recovered in the Vo eluate and slowly mixed with KCl at a final concentration of 2 M. The sample was subjected to chromatography in a column (1.7 x 4.9 cm) of Octyl Sepharose CL-4B equilibrated with buffer D and successively eluted with buffer D, and buffer C containing 0, 1, 2.5, and 5% of Igepal CA-630. DPMS activity and protein content were determined in 30 µl aliquots of each fraction. Most active fractions were pooled, slowly mixed with DTT at final concentration of 20 mM, and incubated for 12 h at 4°C in the dark to reduce DPMS. Excess DTT was removed by passing the sample through a column (2.5 x 30 cm) of Bio-Gel P-6 equilibrated and eluted with buffer E.
(2) Affinity chromatography in Affi-Gel 501. The DTT-free sample was subjected to chromatography in a column (1 x 9 cm) of Affi-Gel 501 equilibrated with buffer E. The sample was eluted with buffer E followed by a continuous gradient of 020 mM DTT in the same buffer. DPMS activity and protein content were determined in 100 µl aliquots of each fraction. Most active fractions were pooled and after removal of DTT by filtration in Bio-Gel P-6, they were used for the analysis of enzyme properties.
Phosphorylation of DPMS
Phosphorylation was carried out in a buffer containing the purified enzyme (44 µg of protein), 7.5 mM MgCl2, 4 mM DTT, 10 µg Dol-P, 20 µg PE, 0.2 mM ATP, 0.01% Igepal CA-630, 12.5 units of cAMP-PK, and buffer A in a total volume of 200 µl. Control mixtures in which ATP and/or cAMP-PK were omitted or the cAMP-PK inhibitor was added were run in parallel. After 30 min of incubation at 37°C, 0.01 µCi of GDP-[14C]Man were added and the volume was adjusted to 300 µl with the same buffer. Mixtures were post-incubated for 30 min at 37°C and the mannolipid was extracted and quantified as described previously (Villagómez-Castro et al., 1998
).
To analyze the polypeptides phosphorylated by cAMP-PK, reaction mixtures essentially equal to those described above were prepared except that cold ATP was substituted by 5 µCi of [
-32P]ATP (sp. act., >6000 Ci/mmol). After 1 h of incubation at 37°C, proteins were precipitated with 10% TCA, washed twice with ethanol:acetone (1:1) and separated by denaturing electrophoresis in a 12.5% SDS-polyacrylamide gel according to standard protocols (Laemmli, 1970
). A gel run in parallel was stained with silver (Merril et al., 1984
) and radioactive polypeptides were revealed after exposure for 72 h at 70°C to 20 x 25 cm X-Omat LS Kodak films.
Determination of protein
Protein content was determined by the modified Lowrys method (Lowry et al., 1951
) using the Bio-Rad DC protein assay kit with bovine serum albumin as standard.
Chemicals
Guanosine diphosphate mannose [mannose-114C] and adenosine 5'-triphosphate [
-32P] were obtained from American Radiolabeled Chemicals (St. Louis, MO). GDP-Man, dolichol monophosphate, dithiothreitol, Igepal CA-630, the catalytic subunit of cAMP-dependent protein kinase, the protein kinase inhibitor, and all phospholipids were from Sigma Chemical Co. (St. Louis, MO). Affi-Gel 501, Bio-Gel P-6, and Bio-Beads SM-2 were purchased from Bio-Rad Laboratories (Hercules, CA). Octyl Sepharose CL-4B was obtained from Pharmacia Biotech (Uppsala, Sweden). All other products were from reliable commercial sources.
 |
Acknowledgments
|
---|
This work was partially supported by Grants Nos. 45810050041PN and 458100527818N from the Consejo Nacional de Ciencia y Tecnología (CONACyT), México. All authors are National Investigators, México.
 |
Abbreviations
|
---|
cAMP-PK, catalytic subunit of cAMP-dependent protein kinase; Dol-P, dolichol monophosphate; Dol-P-Man, dolichol phosphate mannose; DPMS, dolichol phosphate mannose synthase; DTT, dithiothreitol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol.
 |
Footnotes
|
---|
1 To whom correspondence should be addressed at: IIBE, Facultad de Química, Universidad de Guanajuato, Apartado Postal 187, Guanajuato, Gto. 36000, México 
 |
References
|
---|
Banerjee,D.K., Kousvelari,E.E. and Baum,B.J. (1987) cAMP-mediated protein phosphorylation of microsomal membranes increases mannosylphosphodolichol synthase activity. Proc. Natl. Acad. Sci. USA, 84, 63896393.[Abstract]
Banerjee,D.K., DaSilva,J.J. and Bigio,B. (1999) Mannosylphosphodolichol synthase activity is associated with a 32 kDa phosphoprotein. Biosci. Rep., 19, 169177.[ISI][Medline]
Beck,P.J., Orlean,P., Albright,C., Robbins,P.W., Getting,M.J. and Sambrook,J.F. (1990) The membrane topology and ER localization signal of S. cerevisiae dolichol-phosphate-mannose synthase. J. Cell Biol., 111, 378 [abstract].
Colussi,P.A., Taron,C.H., Mack,J.C. and Orlean,P. (1997). Human and Saccharomyces cerevisiae dolichol phosphate mannose synthases represent two classes of the enzyme, but both function in Schizosaccharomyces pombe. Proc. Natl. Acad. Sci. USA, 94, 78737878.[Abstract/Free Full Text]
Haselbeck,A. (1989) Purification of GDP mannose:dolichyl-phosphate O-ß-D-mannosyltransferase from Saccharomyces cerevisiae. Eur. J. Biochem., 181, 663668.[Abstract]
Heifetz,A. and Elbein,A.D. (1977) Solubilization and properties of mannose and N-acetylglucosamine transferases involved in formation of polyprenyl-sugar intermediates. J. Biol. Chem., 252, 30573063.[Abstract]
Herscovics,A. and Orlean,P. (1993) Glycoprotein biosynthesis in yeast. FASEB J., 7, 540550.[Abstract/Free Full Text]
Jensen,J.W. and Schutzbach,J.S. (1985) Activation of dolichyl-phospho-mannose synthase by phospholipids. Eur. J. Biochem., 153, 4148.[Abstract]
Kaushal,G.P. and Elbein,A.D. (1985) Purification and properties of UDP-GlcNAc-Dolichyl phosphate: GlcNAc-1-phosphate transferase: activation and inhibition of the enzyme. J. Biol. Chem., 260, 1630316309.[Abstract/Free Full Text]
Kruszewska,JS., Perlinska-Lenart,U. and Palamarczyk,G. (1996) Solubilization and one-step purification of mannosylphosphoryldolichol synthase from Trichoderma reseei. Acta Biochim. Pol., 43, 397401.[ISI][Medline]
Laemmli,U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680685.[ISI][Medline]
Lowry,O.H., Rosebrough,N.J., Farr,A.L. and Randall,R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265275.[Free Full Text]
Menon,A.K., Mayor,S. and Schwarz,R.T. (1990) Biosynthesis of glycosyl-phosphatidylinositol lipids in Trypanosome brucei: involvement of mannosyl-phosphoryldolichols as the mannose donor. EMBO J., 9, 42494258.[Abstract]
Merril,C.R., Goldman,D. and Van Keuren,M.L. (1984) Gel protein stains: silver stain. Methods Enzymol., 104, 441446.[ISI][Medline]
Orlean,P., Albright,C. and Robbins,P.W. (1988) Cloning and sequencing of the yeast gene for dolichol phosphate mannose synthase, an essential protein. J. Biol. Chem., 263, 1749917507.[Abstract/Free Full Text]
Que,X. and Reed,S.L. (1997) The role of extracellular cysteine proteinases in pathogenesis of Entamoeba histolytica invasion. Parasitol. Today, 13, 190194.[ISI]
Schutzbach,JS. and Zimmerman,J.W. (1992) Yeast dolichyl-phosphomannose synthase: reconstitution of enzyme activity with phospholipids. Biochem. Cell Biol.., 70, 460465.[ISI][Medline]
Schutzbach,J.S., Zimmerman,J.W. and Forsee,W.T. (1993) The purification and characterization of recombinant yeast dolichyl-phosphate-mannose synthase. Site-directed mutagenesis of the putative dolichol recognition sequence. J. Biol. Chem., 268, 2419024196.[Abstract/Free Full Text]
Vargas-Rodríguez,L., Villagómez-Castro,J.C., Flores-Carreón,A. and López-Romero,E. (1998) Identification and characterization of early reactions of asparagine-linked oligosaccharide assembly in Entamoeba histolytica. Int. J. Parasitol., 28, 13331340.[ISI][Medline]
Villagómez-Castro,J.C., Calvo-Méndez,C., Vargas-Rodríguez,L., Flores-Carreón,A. and López-Romero,E. (1998) Entamoeba histolytica: solubilization and biochemical characterization of dolichol phosphate mannose synthase, an essential enzyme in glycoprotein biosynthesis. Exp. Parasitol., 88, 111120.[ISI][Medline]