ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Vol. 198, No. 1, November, pp. 117-123, 1979
Solubilization and Properties of Polyprenyl Phosphate: GDP-D-Mannose Mannosyl Transferase’ PENNY L. CARLO AND CLARENCE Department
of Biochemistry,
University
of Wyoming,
L. VILLEMEZ Laramie,
Wyoming
82071
Received June 25, 1979 A soluble enzyme which catalyzes the formation of dolichyl p-D-mannosyl phosphate has been prepared from encysting cultures of Acanthamoeba castellanii. The enzyme is relatively specific for GDP-n-mannose in that GDP-D-glucose and various uridine nucleotides do not serve as substrates. Uridine diphosphate D-glucose is not an inhibitor at lOO-foldmolar excess concentration, but GDP-D-glucose, GDP, and GMP do inhibit the reaction at relatively high concentrations. The apparent K, for GDP-n-mannose is approximately 0.25 PM and that for dolichyl phosphate is approximately 3.3 PM. The enzyme has a pH optimum of 7.0, a temperature optimum of 27X, and requires a divalent cation. Magnesium, cobalt, and manganese salts will serve as cofactors but maximum activity is produced by Mn2+. No loss of activity is evident after storage for 2 weeks at -7O”C, but half the activity was lost within 3 days at O”C, and a third of the activity was lost within 2 weeks at -20°C.
The glycosylation of proteins in a variety of tissues involves lipid-linked oligosaccharides as substrates (see Refs. (1, 2) for reviews). The oligosaccharide pyrophosphate polyprenols are apparently formed by transferring mannosyl and glucosyl residues, from polyprenyl D-mannosyl phosphate and polyprenyl D-glucosyl phosphate among other substrates, to polyprenyl di-N-acetyl chitobiose pyrophosphate. Subsequent to glycosylation of the protein, the action of membrane-bound glycosidases removes the glucosyl and some of the peripheral mannosy1residues, leaving the core oligosaccharide attached N-glycosidically to the peptide through an N-acetyl D-glucosamine residue (3-4). Very little is known about the physical properties, specificity, and possible mechanisms of control, for individual enzymes in this biosynthetic pathway. One of the main reasons for this lack of knowledge has been the difficulty in separating individual enzymes from the membranes in which they are located. Until recently, there existed only one paper which reported the solubilization of two of these enzymes: polyprenyl phosphate:GDP-mannose transmannosylase I This work was supported by National Science Foundation Grant PCM 75-07629. 117
and polyprenyl phosphate:UDP-N-acetyl glucosamine transglycosylase (5). Those enzymes, in their reported state, appear to be unlikely candidates for purification and further experimentation because of low activity, instability, requisite exposure to detergent during preparation, and availability of source tissue. Recently, we reported the solubilization and basic properties of a polyprenyl phosphate:UDP-Glc transglucosylase which appears to be suitable for further study (6). This report provides a means of solubilization and outlines the basic properties of a second enzyme in this pathway which is quite active and relatively stable: a polyprenyl phosphate:GDP-mannose transmannosylase. MATERIALS
AND METHODS
Materials. Dolichyl phosphate, Grade III, was purchased from Sigma Chemical Company. Glass powder, 25 pm, was purchased from Heat Systems Ultrasonics, DEAE-cellulose from Bio-Rad, Silica gel G thin layer plates from Applied Science Laboratories. GDP-[Wmannose (12.6 Ci/mmol) and Biofluor were purchased from New England Nuclear. 1,6-Anhydro D-glucose (2,3,4-triacetate) was the gift of Dr. Paul Seib, Kansas State University. All other chemicals were reagent grade and purchased from commercial sources. Culture of organisms. Slants of Acanthamoeba
0003.9861/79/130117-07$02.00/O Copyright 6 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
118
CARLO AND VILLEMEZ
FIG. 1. Thin layer chromatography of 3H-labeled mannolipid. The butanol-soluble products from a larger scale version of the usual assay (see Materials and Methods) were chromatographed on silica gel G with a developing solvent of 65254 HCC13:CH30H:H20. Following development, 0.5~cm strips of the silica gel were scraped into a scintillation vial and counted. 0 Authentic [W]dolichyl /3-n-glucosyl phosphate, right y-axis; 0 3H-labeled mannolipid, left y-axis. castellanii (Neff), ATCC No. 30010, were originally obtained from the American Type Culture Collection. The organisms were grown in liquid culture at 30°C as described previously (7), except that the growth media contained 3% @glucose (instead of 1.5%). Encystment was induced, in the manner described previously (7), which consisted of removing the cells from the growth media and placing them in salts media for 2224 h. When harvested, the cells consisted of approximately 52% mature cysts, 31% immature cysts, and 17% trophozoites as determined by microscopic examination. Preparation of enzyme. The cells were washed once with homogenizing buffer (0.05 M Hepes,2 0.5 M sucrose, 0.005 M dithiothreitol, at pH 7.4), suspended in the same buffer to a concentration of 4.5 to 6.5 x 10’ cells/ml (total volume around 20 ml), 5 ml of glass beads was added, and sonicated for a total of 10 min at 80 w output with the 0.75in. probe of Braunsonic 1510sonic oscillator. The sonication was carried out in 30-s pulses, and temperature was monitored. An initial homogenate temperature of -2°C was produced with a CaCl,-ice slurry bath, and the homogenate temperature after 30-s sonication in the cooling bath was usually 5°C. The homogenate was allowed to cool to -2” before additional sonication. The supernatant solution from 2 Abbreviations used: Hepes, N-P-hydroxyethylpiperazine N ‘-2-ethanesulfonic acid.
a l-h centrifugation at 223,800g was collected and used as enzyme. Enzyme assay. The reaction mixture consisted of 10 ~1 dolichyl phosphate (211 FM in 5% Triton X-100), 5 gl MnCl, (0.1 M), 28 ~10.2 M Hepes pH 6.6,5 ~1GDP[3H]mannose (approximately 130,000 cpm, 9.3 pmol), and 50 ~1 enzyme solution for a total volume of 0.1 ml and a reaction pH of 7.0. The mixture was reacted for 15 min at 3O”C, and the reaction terminated by the addition of 0.6 ml butanol (water saturated) and mixing. Analysis consisted of adding 0.5 ml H,O (butanol saturated), mixing, separating the phases by centrifugation, collecting the butanol phase, and washing the butanol phase in the same manner with 1 ml H,O (butanol saturated). The butanol phase was placed in a scintillation vial with 10 ml scintillation counting solution, and radioactivity determined. Acid and base hydrolysis. Mild acid hydrolysis was accomplishedby adding the radioactive sample (in 0.1 ml butanol) to 0.15 ml n-propanol followed by an addition of 0.25 ml 0.04 M trifluoroacetic acid (aqueous), and heating in a sealed tube at 92°C for 20 min. Mild base hydrolysis was accomplished with two different sets of conditions: reacting with 3 M NH,OH in 80% ethanol at room temperature for 20 min (8), or reacting with 0.1 M KOH in 1:l toluene-methanol at 0” for 2 h (9). More stringent conditions for base hydrolysis consisted of reacting the sample with 0.1 M NaOH for 20 min at 65°C (10). Analytical methods. Protein was estimated by the method of Bradford (11) using P-glucosidase as a standard. Radioactivity was determined using Biofluor as a liquid scintillation counting mixture, and a NuclearChicago Unilux II-A liquid scintillation counting system. The counting efficiency was approximately 49%, as determined using [3H]toluene as an internal standard. Results reported are the averages of duplicate samples. The range of the duplicate samples did not exceed 5% of the mean value. RESULTS
Structure
of Product
The solubilized enzyme, when incubated with GDP-[3H]mannose, alone or with Triton X-100 added, catalyzes the formation of insignificant quantities of 3H-labeled mannolipid. But, when dolichyl phosphate is added to the reaction substantial amounts of 3H-labeled mannolipid are produced. The 3H-labeled mannolipid consists of a single radioactive peak upon thin layer chromatography on silica gel with two solvent systems (6525~4 HCCl,-CH,OH-H,O; and 25:15:4:2 HCQ-CH,OH-acetic acid-H20). The mobility of the radioactive mannolipid is identi-
SOLUBLE GDP-MANNOSE MANNOSYL TRANSFERASE
/ 24
“A, 0
n 36 Fraction
4s (3ml
60
PROPERTIES
119
r^__ 72
84
each)
FIG. 2. DEAE-cellulose chromatography of 3H-labeled mannolipid. The combined butanol extracts from six standard reactions (see Materials and Methods) were applied to a DEAE-cellulose (acetate form) column (1.5 x 9.0 cm), eluted first with 99% CH,OH, and then with a 0 to 50 mM gradient of ammonium acetate in 99% CH,OH. The 99% CH,OH eluate was collected in bulk; radioactivity determination of an aliquot indicated that less than 1% of the applied radioactivity had eluted. The salt gradient eluate was collected in 3-ml fractions and 0.3-ml aliquots were used for radioactivity determination. The radioactive peak was collected, evaporated to dryness, dissolved in 1:l toluene: CH,OH, subjected to mild alkaline hydrolysis (see Materials and Methods), and the chromatography, described above, was repeated. 0 Original chromatography; 0 chromatography of alkaline-treated material.
cal to that of authentic dolichyl p-D-glucosyl phosphate (Fig. 1). Upon column chromatography with DEAE-cellulose, the 3H-labeled mannolipid binds quantitatively to the column, no detectible radioactivity is recovered upon elution with 99% CH,OH, and the radioactivity is eluted as a single component with a O-50 mM ammonium acetate gradient at a salt concentration of around 30 mM (Fig. 2). The single radioactive peak represented an essentially quantitative recovery of the radioactivity applied to the column. Mild alkaline treatment (0.1 M KOH, O”, 2 h) did not alter the chromatographic properties of the 3H-labeled mannolipid (Fig. 2). Mild acid hydrolysis, 0.02 M trifluoroacetic acid at 92°C for 20 min, resulted in 93% hydrolysis. The water-soluble radioactivity resulting from mild acid hydrolysis migrated as a single peak with a mobility identical to that of D-mannose upon paper chromatography (Whatman No. 1; 10~42ethyl acetatepyridine-H,O).
Mild alkaline hydrolysis (3 M NH,OH in 80% ethanol, 30 min, room temperature) resulted in less than 10% hydrolysis. Under more stringent conditions of alkaline hydrolysis (0.1 M NaOH, 65”C, 20 min), approximately 90% of the radioactive mannolipid was hydrolyzed. Paper chromatography of the water-soluble radioactivity resulting from alkaline hydrolysis indicated approximately 10% migrated with Dmannose, and approximately 90% migrated as D-mannosy1 phosphate (Fig. 3). Properties
of the Enzyme
There are indications that the soluble enzyme catalyzing the formation of dolichyl /3-D-[3H]mannosyl phosphate is, in Go, membrane associated. The 223,SOOgparticulate fraction obtained from the usual enzyme preparation contains enzymes which catalyze the formation of 3H-labeled mannolipid. One of the products produced by particulate enzyme catalysis has identical mobility upon
CARLO
AND VILLEMEZ
FIG. 3. Products of alkaline hydrolysis. Approximately 8600 cpm 3H-labeled mannolipid was dissolved in 0.45 ml 1-propanol to which was added 0.05 ml 1 M NaOH. The reaction was allowed to proceed for 20 min at 65”C, then terminated by the addition of 0.5 ml 0.1 M CH,COOH (10). Following the reaction, approximately 90% of the radioactivity was water soluble and this fraction was chromatographed on Whatman No. 1 paper with 7:3 ethanol:1 M ammonium acetate (pH 7.5). Four-centimeter strips were placed in a scintillation vial and counted. Another standard (not shown), l,&mhydro u-glucose, moved 1 cm faster than the u-mannose standard.
The pH optimum of the solubilized enzyme is approximately pH 7.0 (Fig. 5), the temperature optimum is approximately 27°C (Fig. S), and the solubilized enzyme is relatively stable (Table II). Under standard reaction conditions, the production of dolichyl P-D-[3H]mannosyl phosphate is linear for approximately 15 min and no additional product is produced after 30 min (at which point the yield is usually 35-50%). No breakdown of product is evident after 4 h of reaction. Individual enzyme preparations occasionally produce much greater yields, approaching 90%. The reaction rate is linearly related to protein concentration up to 0.4 mg protein/ml, and continues to increase, although in a nonlinear fashion, up to 1.4 mg protein/ml. If the solubilized enzyme is dialyzed overnight, the reaction rate is linearly related to protein concentration up to at least 1.4 mg protein/ml; the lack of inhibition, at higher concentrations, of the enzyme solution that has been dialyzed indicated the presence of low molecular weight inhibitors in the regular enzyme preparation. Two of the inhibitors are probably GDP and GMP since both inhibit the production of dolichyl P-DTABLE
I
CATION SPECIFICITY”
thin layer chromatography and identical susceptibility to acid and base hydrolysis as does the product of soluble enzyme catalysis. However, there are also other 3H-labeled mannolipids (probably oligosaccharide lipids) produced when the particulate enzyme is used. Sonication of the particulate enzyme solubilizes additional enzyme activity (about 50% of original activity), the characteristics of which are identical to the soluble enzyme used in these studies. That the enzyme requires a divalent cation is suggested by the fact that in the presence of 2 mM EDTA only 65 cpm of organic soluble radioactivity results and in an identical reaction containing 2 InM EDTA and 5 mM MgCl,, 7065 cpm of dolichyl p-D-[3H]mannosyl phosphate results. Of the cations tested only Co2+, Mn*+, and Mg2+ support significant levels of enzyme activity (Table I), of which Mn2+ is the best cofactor (Fig. 4).
Enzyme activity Addition
(cpm)
None Hi&l, cusoa KC1 FeCl, NaCl CaCl, ZnSO, FeS04 I%$& CoCl, MnCl,
400 69 77 339 342 346 375 412 720 1938 9231 13226
a The enzyme was prepared and assayed as described under Materials and Methods except that MnCl, was deleted from the reaction mixture and the enzyme solution was dialyzed overnight. The indicated substances were added to the reaction mixture at a final concentration of 1 mM.
SOLUBLE GDP-MANNOSE MANNOSYL TRANSFERASE
30000
PROPERTIES
121
/
/
FIG. 4. Divalent metal concentration-enzyme activity relationship. The enzyme was prepared and assayed as indicated under Materials and Methods with the indicated concentrations of either MgCl, (A), CoCl, (O), or MnClz (0) present in the reactions. The enzyme was dialyzed before use.
[3H]mannosyl phosphate. At concentrations of 0.01, 0.1, and 1.0 MM, GDP inhibits the reaction rate 81, 96, and 98%, respectively. At the same concentrations, GMP inhibits the reaction rate 19, 44, and 84%, respectively. Neither GDP nor GMP will reverse the reaction under the conditions we have used. GMP is probably a competitive inhibitor, by analogy to UMP in a similar system forming dolichyl p-D-ghXosy1 phosphate (6). The K, for GDP-Dmannose is around 0.25 PM (three separate determinations with three different enzyme preparations
FIG. 6. Temperature optimum. With the exception that MgCI, was employed (instead of MnCl,), the assays were done as described under Materials and Methods at the indicated temperature.
resulted in values of 0.21, 0.23, and 0.28 PM), whereas the K, for dolichyl phosphate is around 3.3 PM (Fig. 7). The enzyme is relatively specific for GDP-D-mannose since GDP-D-glucose, UDP-D-xylose, and UDPD-glucuronic acid do not serve as substrates for the production of radioactive glycolipid. There is an enzyme in the preparation catalyzing the production of dolichyl /3-D-glucosyl phosphate from UDP-D-glucose and dolichyl TABLE II ENZYME STABILITY" Enzyme activity (% of original)
L 5
6
7
8
9
PH
FIG. 5. pH optimum. The enzyme was assayed in the manner described under Materials and Methods at the DH indicated.
Storage time (days)
4°C
-20°C
-70°C
2 3 5 7 14
71 47 30 22 12
97 91 82 71 64
nd 99 99 96 106
” The enzyme was prepared and assayed as described under Materials and Methods when fresh and after storage, for the indicated time at the indicated temperatures. When stored at 3o”C, the enzyme retains 74, 69, 57, and 23% of the original activity after 10, 20, 30, and 102 min, respectively.
122
CARLO AND VILLEMEZ
FIG. ‘7. Kinetics of mannosyl transferase reaction. The enzyme was prepared and assayed as indicated under Materials and Methods except that the indicated concentrations of GDP-D-13H]mannose and dolichyl phosphate were used in incubations of 6-min duration and dialyzed enzyme was used. The dimension of S is micromolar, and of V is picomolesJ6mini50 d of enzyme. Rates using dolichyl phosphate (0) as variable substrate are the values on the left y-axis and the upper values of the x-axis. Rates obtained from varying GDPmannose concentrations (0) are the values on the right y-axis and the lower values of the x-axis. Lines were obtained by linear regression.
phosphate (6). However, in addition to the difference in properties, it is evident that the catalytic activity utilizing UDP-Bglucose and that utilizing GDP-Dmannose represent different enzymes since the addition of UDP-D-glucose at 0.01 mM (loo-fold concentration excess) has no effect on the formation of dolichyl p-D-[3H]mannosyl phosphate from GDP-D-[3H]mannose. Although GDPD-ghCOSe is not a substrate for the solubilized enzyme, it is an inhibitor since at 0.01 mM (lOO-fold concentration excess) the rate of production of dolichyl p-~-[~Hlmannosyl phosphate is reduced approximately 50%. DISCUSSION The evidence presented here indicates that only one product results from the soluble enzyme-catalyzed reaction between dolichyl phosphate and GDP-D-mannose and suggests that a single enzyme is the catalyst. (a) Upon thin layer chromatography (Fig. 1) or DEAE-cellulose chromatography (Fig. 2) only one radioactive peak is observed.
(b) The 3H-labeled mannolipid reacts in a uniform manner with acid and base, producing [3H]mannose and [3H]mannosyl phosphate, respectively. (c) There are no evident anomalies in the enzymic properties, i.e., temperature optimum, pH optimum, kinetic behavior, and inactivation with storage. The evidence presented here also indicates that the product of catalysis is dolichyl p-D-mannosyl phosphate. (a) Solvent partition before and following acid hydrolysis indicate that the 3H-labeled product is a glycosyl derivative of a hydrophobic moiety. (b) The only sugar produced upon mild acid hydrolysis is D-mannose. (c) That the lipid moiety is dolichyl phosphate is indicated by: The enzyme catalyzes no reaction with GDP-D-mannose unless dolichyl phosphate is added; the reaction rate as a function of dolichyl phosphate concentration indicates that this compound participates in the reaction; the product binds to DEAEcellulose in low ionic strength solution and is desorbed upon increasing ionic strength indicating the presence of a negative charge, i.e., a phosphate group. The chromatographic mobility with thin layers of silica gel and columns of DEAE-cellulose is that observed previously for dolichyl D-glycosyl phosphates. (d) The acid lability indicates that D-mannose is attached to dolichyl phosphate through the phosphate group and the anomeric carbon of the D-mannose as does the formation of mannosyl phosphate in relatively high yield during alkaline hydrolysis. (e) The resistance to mild alkaline hydrolysis, and also the chromatographic mobility, is linked to dolichol indicate that D-IIXinnOSe by a phosphodiester rather than a pyrophosphate bridge. (f) The production of mannosyl phosphate upon hydrolysis with 0.1 M NaOH at 65°C indicates that the glycosidic linkage is p. Stable, soluble enzymes are a valuable research property that can be used as synthetic tools in the preparation of scarce substrates and for the investigation of enzymic properties as they relate to metabolism and its control. Previous attempts in this laboratory to solubilize enzymes that catalyze glycolipid intermediate reactions, using this organism and others and various detergents
SOLUBLE
GDP-MANNOSE
MANNOSYL
as solubilizing agents, have only been partially successful. However using controlled sonication, we have recently solubilized a polyprenyl phosphate:UDP-Glc glucosyl transferase (61, and what appear to be polyprenyl phosphate:UDP-iV-acetyl glucosamine N-acetyl glucosaminyl transferase and a dolichol kinase (Villemez and Carlo, unpublished). It is possible that controlled sonication is the method of choice in solubilizing this type of enzyme. REFERENCES 1. WAECHTER, C. J. (1976)Annu. Rev. Biochem. 45, 95-112. 2. PARODI, A. J., AND LELOIR, L. F. (1979) Biochim. Biophys. Acta 559, l-37.
TRANSFERASE
PROPERTIES
123
3. CHEN, W. W., AND LENNARZ, W. J. (1978)J. Biol. Chem. 253, 5774-5779. 4. CHEN, W. W., AND LENNARZ, W. J. (1978)J. Biol. Chem. 253, 5780-5785. 5. HEIFETZ, A., AND ELBEIN, A. D. (1977) J. Biol. Chem. 252, 3057-3063. 6. VILLEMEZ, C. L., ANDCARLO, P. L. (1979)J. Biol. Chem,. 254, 4814-4819. 7. NEFF, R. J., RAY, S. A., BENTON, W. F., AND WILBORN, M. (1964) in Methods in Cell Physiology (Prescott, D. M., ed.), Vol. 1, pp. 55-83, Academic Press, New York. 8. HOPP, A. E., ROMERO, P. A., DALEO, G. R., AND LEZICA, R. P. (1978) Eur. J. Biochem. 84,561571. 9. LESTER, R. L., ANDSTEINER, M. R. (1968)J. Biol. Chem. 243, 4889-4893. 10. HERSCOVICS, A., WARREN, C. D., JEANLOZ, R. W. (1974) FEBS Lett. 45, 312-317. 11. BRADFORD, M. M. (1976)Anal. Biochem. 72,248.
Vol. 198, No. 1, November, pp. 117-123, 1979
Solubilization and Properties of Polyprenyl Phosphate: GDP-D-Mannose Mannosyl Transferase’ PENNY L. CARLO AND CLARENCE Department
of Biochemistry,
University
of Wyoming,
L. VILLEMEZ Laramie,
Wyoming
82071
Received June 25, 1979 A soluble enzyme which catalyzes the formation of dolichyl p-D-mannosyl phosphate has been prepared from encysting cultures of Acanthamoeba castellanii. The enzyme is relatively specific for GDP-n-mannose in that GDP-D-glucose and various uridine nucleotides do not serve as substrates. Uridine diphosphate D-glucose is not an inhibitor at lOO-foldmolar excess concentration, but GDP-D-glucose, GDP, and GMP do inhibit the reaction at relatively high concentrations. The apparent K, for GDP-n-mannose is approximately 0.25 PM and that for dolichyl phosphate is approximately 3.3 PM. The enzyme has a pH optimum of 7.0, a temperature optimum of 27X, and requires a divalent cation. Magnesium, cobalt, and manganese salts will serve as cofactors but maximum activity is produced by Mn2+. No loss of activity is evident after storage for 2 weeks at -7O”C, but half the activity was lost within 3 days at O”C, and a third of the activity was lost within 2 weeks at -20°C.
The glycosylation of proteins in a variety of tissues involves lipid-linked oligosaccharides as substrates (see Refs. (1, 2) for reviews). The oligosaccharide pyrophosphate polyprenols are apparently formed by transferring mannosyl and glucosyl residues, from polyprenyl D-mannosyl phosphate and polyprenyl D-glucosyl phosphate among other substrates, to polyprenyl di-N-acetyl chitobiose pyrophosphate. Subsequent to glycosylation of the protein, the action of membrane-bound glycosidases removes the glucosyl and some of the peripheral mannosy1residues, leaving the core oligosaccharide attached N-glycosidically to the peptide through an N-acetyl D-glucosamine residue (3-4). Very little is known about the physical properties, specificity, and possible mechanisms of control, for individual enzymes in this biosynthetic pathway. One of the main reasons for this lack of knowledge has been the difficulty in separating individual enzymes from the membranes in which they are located. Until recently, there existed only one paper which reported the solubilization of two of these enzymes: polyprenyl phosphate:GDP-mannose transmannosylase I This work was supported by National Science Foundation Grant PCM 75-07629. 117
and polyprenyl phosphate:UDP-N-acetyl glucosamine transglycosylase (5). Those enzymes, in their reported state, appear to be unlikely candidates for purification and further experimentation because of low activity, instability, requisite exposure to detergent during preparation, and availability of source tissue. Recently, we reported the solubilization and basic properties of a polyprenyl phosphate:UDP-Glc transglucosylase which appears to be suitable for further study (6). This report provides a means of solubilization and outlines the basic properties of a second enzyme in this pathway which is quite active and relatively stable: a polyprenyl phosphate:GDP-mannose transmannosylase. MATERIALS
AND METHODS
Materials. Dolichyl phosphate, Grade III, was purchased from Sigma Chemical Company. Glass powder, 25 pm, was purchased from Heat Systems Ultrasonics, DEAE-cellulose from Bio-Rad, Silica gel G thin layer plates from Applied Science Laboratories. GDP-[Wmannose (12.6 Ci/mmol) and Biofluor were purchased from New England Nuclear. 1,6-Anhydro D-glucose (2,3,4-triacetate) was the gift of Dr. Paul Seib, Kansas State University. All other chemicals were reagent grade and purchased from commercial sources. Culture of organisms. Slants of Acanthamoeba
0003.9861/79/130117-07$02.00/O Copyright 6 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
118
CARLO AND VILLEMEZ
FIG. 1. Thin layer chromatography of 3H-labeled mannolipid. The butanol-soluble products from a larger scale version of the usual assay (see Materials and Methods) were chromatographed on silica gel G with a developing solvent of 65254 HCC13:CH30H:H20. Following development, 0.5~cm strips of the silica gel were scraped into a scintillation vial and counted. 0 Authentic [W]dolichyl /3-n-glucosyl phosphate, right y-axis; 0 3H-labeled mannolipid, left y-axis. castellanii (Neff), ATCC No. 30010, were originally obtained from the American Type Culture Collection. The organisms were grown in liquid culture at 30°C as described previously (7), except that the growth media contained 3% @glucose (instead of 1.5%). Encystment was induced, in the manner described previously (7), which consisted of removing the cells from the growth media and placing them in salts media for 2224 h. When harvested, the cells consisted of approximately 52% mature cysts, 31% immature cysts, and 17% trophozoites as determined by microscopic examination. Preparation of enzyme. The cells were washed once with homogenizing buffer (0.05 M Hepes,2 0.5 M sucrose, 0.005 M dithiothreitol, at pH 7.4), suspended in the same buffer to a concentration of 4.5 to 6.5 x 10’ cells/ml (total volume around 20 ml), 5 ml of glass beads was added, and sonicated for a total of 10 min at 80 w output with the 0.75in. probe of Braunsonic 1510sonic oscillator. The sonication was carried out in 30-s pulses, and temperature was monitored. An initial homogenate temperature of -2°C was produced with a CaCl,-ice slurry bath, and the homogenate temperature after 30-s sonication in the cooling bath was usually 5°C. The homogenate was allowed to cool to -2” before additional sonication. The supernatant solution from 2 Abbreviations used: Hepes, N-P-hydroxyethylpiperazine N ‘-2-ethanesulfonic acid.
a l-h centrifugation at 223,800g was collected and used as enzyme. Enzyme assay. The reaction mixture consisted of 10 ~1 dolichyl phosphate (211 FM in 5% Triton X-100), 5 gl MnCl, (0.1 M), 28 ~10.2 M Hepes pH 6.6,5 ~1GDP[3H]mannose (approximately 130,000 cpm, 9.3 pmol), and 50 ~1 enzyme solution for a total volume of 0.1 ml and a reaction pH of 7.0. The mixture was reacted for 15 min at 3O”C, and the reaction terminated by the addition of 0.6 ml butanol (water saturated) and mixing. Analysis consisted of adding 0.5 ml H,O (butanol saturated), mixing, separating the phases by centrifugation, collecting the butanol phase, and washing the butanol phase in the same manner with 1 ml H,O (butanol saturated). The butanol phase was placed in a scintillation vial with 10 ml scintillation counting solution, and radioactivity determined. Acid and base hydrolysis. Mild acid hydrolysis was accomplishedby adding the radioactive sample (in 0.1 ml butanol) to 0.15 ml n-propanol followed by an addition of 0.25 ml 0.04 M trifluoroacetic acid (aqueous), and heating in a sealed tube at 92°C for 20 min. Mild base hydrolysis was accomplished with two different sets of conditions: reacting with 3 M NH,OH in 80% ethanol at room temperature for 20 min (8), or reacting with 0.1 M KOH in 1:l toluene-methanol at 0” for 2 h (9). More stringent conditions for base hydrolysis consisted of reacting the sample with 0.1 M NaOH for 20 min at 65°C (10). Analytical methods. Protein was estimated by the method of Bradford (11) using P-glucosidase as a standard. Radioactivity was determined using Biofluor as a liquid scintillation counting mixture, and a NuclearChicago Unilux II-A liquid scintillation counting system. The counting efficiency was approximately 49%, as determined using [3H]toluene as an internal standard. Results reported are the averages of duplicate samples. The range of the duplicate samples did not exceed 5% of the mean value. RESULTS
Structure
of Product
The solubilized enzyme, when incubated with GDP-[3H]mannose, alone or with Triton X-100 added, catalyzes the formation of insignificant quantities of 3H-labeled mannolipid. But, when dolichyl phosphate is added to the reaction substantial amounts of 3H-labeled mannolipid are produced. The 3H-labeled mannolipid consists of a single radioactive peak upon thin layer chromatography on silica gel with two solvent systems (6525~4 HCCl,-CH,OH-H,O; and 25:15:4:2 HCQ-CH,OH-acetic acid-H20). The mobility of the radioactive mannolipid is identi-
SOLUBLE GDP-MANNOSE MANNOSYL TRANSFERASE
/ 24
“A, 0
n 36 Fraction
4s (3ml
60
PROPERTIES
119
r^__ 72
84
each)
FIG. 2. DEAE-cellulose chromatography of 3H-labeled mannolipid. The combined butanol extracts from six standard reactions (see Materials and Methods) were applied to a DEAE-cellulose (acetate form) column (1.5 x 9.0 cm), eluted first with 99% CH,OH, and then with a 0 to 50 mM gradient of ammonium acetate in 99% CH,OH. The 99% CH,OH eluate was collected in bulk; radioactivity determination of an aliquot indicated that less than 1% of the applied radioactivity had eluted. The salt gradient eluate was collected in 3-ml fractions and 0.3-ml aliquots were used for radioactivity determination. The radioactive peak was collected, evaporated to dryness, dissolved in 1:l toluene: CH,OH, subjected to mild alkaline hydrolysis (see Materials and Methods), and the chromatography, described above, was repeated. 0 Original chromatography; 0 chromatography of alkaline-treated material.
cal to that of authentic dolichyl p-D-glucosyl phosphate (Fig. 1). Upon column chromatography with DEAE-cellulose, the 3H-labeled mannolipid binds quantitatively to the column, no detectible radioactivity is recovered upon elution with 99% CH,OH, and the radioactivity is eluted as a single component with a O-50 mM ammonium acetate gradient at a salt concentration of around 30 mM (Fig. 2). The single radioactive peak represented an essentially quantitative recovery of the radioactivity applied to the column. Mild alkaline treatment (0.1 M KOH, O”, 2 h) did not alter the chromatographic properties of the 3H-labeled mannolipid (Fig. 2). Mild acid hydrolysis, 0.02 M trifluoroacetic acid at 92°C for 20 min, resulted in 93% hydrolysis. The water-soluble radioactivity resulting from mild acid hydrolysis migrated as a single peak with a mobility identical to that of D-mannose upon paper chromatography (Whatman No. 1; 10~42ethyl acetatepyridine-H,O).
Mild alkaline hydrolysis (3 M NH,OH in 80% ethanol, 30 min, room temperature) resulted in less than 10% hydrolysis. Under more stringent conditions of alkaline hydrolysis (0.1 M NaOH, 65”C, 20 min), approximately 90% of the radioactive mannolipid was hydrolyzed. Paper chromatography of the water-soluble radioactivity resulting from alkaline hydrolysis indicated approximately 10% migrated with Dmannose, and approximately 90% migrated as D-mannosy1 phosphate (Fig. 3). Properties
of the Enzyme
There are indications that the soluble enzyme catalyzing the formation of dolichyl /3-D-[3H]mannosyl phosphate is, in Go, membrane associated. The 223,SOOgparticulate fraction obtained from the usual enzyme preparation contains enzymes which catalyze the formation of 3H-labeled mannolipid. One of the products produced by particulate enzyme catalysis has identical mobility upon
CARLO
AND VILLEMEZ
FIG. 3. Products of alkaline hydrolysis. Approximately 8600 cpm 3H-labeled mannolipid was dissolved in 0.45 ml 1-propanol to which was added 0.05 ml 1 M NaOH. The reaction was allowed to proceed for 20 min at 65”C, then terminated by the addition of 0.5 ml 0.1 M CH,COOH (10). Following the reaction, approximately 90% of the radioactivity was water soluble and this fraction was chromatographed on Whatman No. 1 paper with 7:3 ethanol:1 M ammonium acetate (pH 7.5). Four-centimeter strips were placed in a scintillation vial and counted. Another standard (not shown), l,&mhydro u-glucose, moved 1 cm faster than the u-mannose standard.
The pH optimum of the solubilized enzyme is approximately pH 7.0 (Fig. 5), the temperature optimum is approximately 27°C (Fig. S), and the solubilized enzyme is relatively stable (Table II). Under standard reaction conditions, the production of dolichyl P-D-[3H]mannosyl phosphate is linear for approximately 15 min and no additional product is produced after 30 min (at which point the yield is usually 35-50%). No breakdown of product is evident after 4 h of reaction. Individual enzyme preparations occasionally produce much greater yields, approaching 90%. The reaction rate is linearly related to protein concentration up to 0.4 mg protein/ml, and continues to increase, although in a nonlinear fashion, up to 1.4 mg protein/ml. If the solubilized enzyme is dialyzed overnight, the reaction rate is linearly related to protein concentration up to at least 1.4 mg protein/ml; the lack of inhibition, at higher concentrations, of the enzyme solution that has been dialyzed indicated the presence of low molecular weight inhibitors in the regular enzyme preparation. Two of the inhibitors are probably GDP and GMP since both inhibit the production of dolichyl P-DTABLE
I
CATION SPECIFICITY”
thin layer chromatography and identical susceptibility to acid and base hydrolysis as does the product of soluble enzyme catalysis. However, there are also other 3H-labeled mannolipids (probably oligosaccharide lipids) produced when the particulate enzyme is used. Sonication of the particulate enzyme solubilizes additional enzyme activity (about 50% of original activity), the characteristics of which are identical to the soluble enzyme used in these studies. That the enzyme requires a divalent cation is suggested by the fact that in the presence of 2 mM EDTA only 65 cpm of organic soluble radioactivity results and in an identical reaction containing 2 InM EDTA and 5 mM MgCl,, 7065 cpm of dolichyl p-D-[3H]mannosyl phosphate results. Of the cations tested only Co2+, Mn*+, and Mg2+ support significant levels of enzyme activity (Table I), of which Mn2+ is the best cofactor (Fig. 4).
Enzyme activity Addition
(cpm)
None Hi&l, cusoa KC1 FeCl, NaCl CaCl, ZnSO, FeS04 I%$& CoCl, MnCl,
400 69 77 339 342 346 375 412 720 1938 9231 13226
a The enzyme was prepared and assayed as described under Materials and Methods except that MnCl, was deleted from the reaction mixture and the enzyme solution was dialyzed overnight. The indicated substances were added to the reaction mixture at a final concentration of 1 mM.
SOLUBLE GDP-MANNOSE MANNOSYL TRANSFERASE
30000
PROPERTIES
121
/
/
FIG. 4. Divalent metal concentration-enzyme activity relationship. The enzyme was prepared and assayed as indicated under Materials and Methods with the indicated concentrations of either MgCl, (A), CoCl, (O), or MnClz (0) present in the reactions. The enzyme was dialyzed before use.
[3H]mannosyl phosphate. At concentrations of 0.01, 0.1, and 1.0 MM, GDP inhibits the reaction rate 81, 96, and 98%, respectively. At the same concentrations, GMP inhibits the reaction rate 19, 44, and 84%, respectively. Neither GDP nor GMP will reverse the reaction under the conditions we have used. GMP is probably a competitive inhibitor, by analogy to UMP in a similar system forming dolichyl p-D-ghXosy1 phosphate (6). The K, for GDP-Dmannose is around 0.25 PM (three separate determinations with three different enzyme preparations
FIG. 6. Temperature optimum. With the exception that MgCI, was employed (instead of MnCl,), the assays were done as described under Materials and Methods at the indicated temperature.
resulted in values of 0.21, 0.23, and 0.28 PM), whereas the K, for dolichyl phosphate is around 3.3 PM (Fig. 7). The enzyme is relatively specific for GDP-D-mannose since GDP-D-glucose, UDP-D-xylose, and UDPD-glucuronic acid do not serve as substrates for the production of radioactive glycolipid. There is an enzyme in the preparation catalyzing the production of dolichyl /3-D-glucosyl phosphate from UDP-D-glucose and dolichyl TABLE II ENZYME STABILITY" Enzyme activity (% of original)
L 5
6
7
8
9
PH
FIG. 5. pH optimum. The enzyme was assayed in the manner described under Materials and Methods at the DH indicated.
Storage time (days)
4°C
-20°C
-70°C
2 3 5 7 14
71 47 30 22 12
97 91 82 71 64
nd 99 99 96 106
” The enzyme was prepared and assayed as described under Materials and Methods when fresh and after storage, for the indicated time at the indicated temperatures. When stored at 3o”C, the enzyme retains 74, 69, 57, and 23% of the original activity after 10, 20, 30, and 102 min, respectively.
122
CARLO AND VILLEMEZ
FIG. ‘7. Kinetics of mannosyl transferase reaction. The enzyme was prepared and assayed as indicated under Materials and Methods except that the indicated concentrations of GDP-D-13H]mannose and dolichyl phosphate were used in incubations of 6-min duration and dialyzed enzyme was used. The dimension of S is micromolar, and of V is picomolesJ6mini50 d of enzyme. Rates using dolichyl phosphate (0) as variable substrate are the values on the left y-axis and the upper values of the x-axis. Rates obtained from varying GDPmannose concentrations (0) are the values on the right y-axis and the lower values of the x-axis. Lines were obtained by linear regression.
phosphate (6). However, in addition to the difference in properties, it is evident that the catalytic activity utilizing UDP-Bglucose and that utilizing GDP-Dmannose represent different enzymes since the addition of UDP-D-glucose at 0.01 mM (loo-fold concentration excess) has no effect on the formation of dolichyl p-D-[3H]mannosyl phosphate from GDP-D-[3H]mannose. Although GDPD-ghCOSe is not a substrate for the solubilized enzyme, it is an inhibitor since at 0.01 mM (lOO-fold concentration excess) the rate of production of dolichyl p-~-[~Hlmannosyl phosphate is reduced approximately 50%. DISCUSSION The evidence presented here indicates that only one product results from the soluble enzyme-catalyzed reaction between dolichyl phosphate and GDP-D-mannose and suggests that a single enzyme is the catalyst. (a) Upon thin layer chromatography (Fig. 1) or DEAE-cellulose chromatography (Fig. 2) only one radioactive peak is observed.
(b) The 3H-labeled mannolipid reacts in a uniform manner with acid and base, producing [3H]mannose and [3H]mannosyl phosphate, respectively. (c) There are no evident anomalies in the enzymic properties, i.e., temperature optimum, pH optimum, kinetic behavior, and inactivation with storage. The evidence presented here also indicates that the product of catalysis is dolichyl p-D-mannosyl phosphate. (a) Solvent partition before and following acid hydrolysis indicate that the 3H-labeled product is a glycosyl derivative of a hydrophobic moiety. (b) The only sugar produced upon mild acid hydrolysis is D-mannose. (c) That the lipid moiety is dolichyl phosphate is indicated by: The enzyme catalyzes no reaction with GDP-D-mannose unless dolichyl phosphate is added; the reaction rate as a function of dolichyl phosphate concentration indicates that this compound participates in the reaction; the product binds to DEAEcellulose in low ionic strength solution and is desorbed upon increasing ionic strength indicating the presence of a negative charge, i.e., a phosphate group. The chromatographic mobility with thin layers of silica gel and columns of DEAE-cellulose is that observed previously for dolichyl D-glycosyl phosphates. (d) The acid lability indicates that D-mannose is attached to dolichyl phosphate through the phosphate group and the anomeric carbon of the D-mannose as does the formation of mannosyl phosphate in relatively high yield during alkaline hydrolysis. (e) The resistance to mild alkaline hydrolysis, and also the chromatographic mobility, is linked to dolichol indicate that D-IIXinnOSe by a phosphodiester rather than a pyrophosphate bridge. (f) The production of mannosyl phosphate upon hydrolysis with 0.1 M NaOH at 65°C indicates that the glycosidic linkage is p. Stable, soluble enzymes are a valuable research property that can be used as synthetic tools in the preparation of scarce substrates and for the investigation of enzymic properties as they relate to metabolism and its control. Previous attempts in this laboratory to solubilize enzymes that catalyze glycolipid intermediate reactions, using this organism and others and various detergents
SOLUBLE
GDP-MANNOSE
MANNOSYL
as solubilizing agents, have only been partially successful. However using controlled sonication, we have recently solubilized a polyprenyl phosphate:UDP-Glc glucosyl transferase (61, and what appear to be polyprenyl phosphate:UDP-iV-acetyl glucosamine N-acetyl glucosaminyl transferase and a dolichol kinase (Villemez and Carlo, unpublished). It is possible that controlled sonication is the method of choice in solubilizing this type of enzyme. REFERENCES 1. WAECHTER, C. J. (1976)Annu. Rev. Biochem. 45, 95-112. 2. PARODI, A. J., AND LELOIR, L. F. (1979) Biochim. Biophys. Acta 559, l-37.
TRANSFERASE
PROPERTIES
123
3. CHEN, W. W., AND LENNARZ, W. J. (1978)J. Biol. Chem. 253, 5774-5779. 4. CHEN, W. W., AND LENNARZ, W. J. (1978)J. Biol. Chem. 253, 5780-5785. 5. HEIFETZ, A., AND ELBEIN, A. D. (1977) J. Biol. Chem. 252, 3057-3063. 6. VILLEMEZ, C. L., ANDCARLO, P. L. (1979)J. Biol. Chem,. 254, 4814-4819. 7. NEFF, R. J., RAY, S. A., BENTON, W. F., AND WILBORN, M. (1964) in Methods in Cell Physiology (Prescott, D. M., ed.), Vol. 1, pp. 55-83, Academic Press, New York. 8. HOPP, A. E., ROMERO, P. A., DALEO, G. R., AND LEZICA, R. P. (1978) Eur. J. Biochem. 84,561571. 9. LESTER, R. L., ANDSTEINER, M. R. (1968)J. Biol. Chem. 243, 4889-4893. 10. HERSCOVICS, A., WARREN, C. D., JEANLOZ, R. W. (1974) FEBS Lett. 45, 312-317. 11. BRADFORD, M. M. (1976)Anal. Biochem. 72,248.
