In:ernarional Journalfor Prrnted in Great Brrloin
Porasirology
Vd 21. No. 6, pp. 703-706.
1991
0
N-ACETYLGLUCOSAMINYLTRANSFERASE Z. KYOSSEV,*
B. BERGMANN,~
FROM
Oa&7519/91 $3.00 + 0.00 Pergamon Press plc 199 I Austroh Society for Parasitology
ASCARIS
SUUA4
E. OSSIKOVSKI* and R. D. WALTER-~-$
*Bulgarian Academy of Sciences, Central Laboratory of Helminthology, TDepartment of Biochemistry, Bernhard Nocht Institute for Tropical Medicine, D-2000 Hamburg 36, F. R. G.
1113Sofia, Bulgaria Bernhard-Nocht-StraDe
74.
(Received 29 November 1990; accepted 29 March 1991) OSSIKOVSKI E. and WALTER R. D. 1991. N-AcetylAscaris suum. International Journal for Parasirology 21: 703-706. The occurrence of N-acetylglucosaminyltransferase, the initial step in the synthesis of the carbohydrate moiety of N-linked glycoproteins, is demonstrated in the microsomal fraction of the nematode Ascaris suum. Phosphatidylglycerol stimulated enzyme activity three- to six-fold without affecting the K, values of either substrates, uridinediphospho-N-acetylglucosamine or dolichylphosphate. The K, values were determined to be about 12 PM and 100 pg ml-‘, respectively. The enzyme activity was strongly inhibited by tunicamycin acting as a competitive inhibitor with respect to the substrate uridinediphospho-N-glucosamine.
Abstract-Kuossav glucosaminyltransferase
INDEX KEY tunicamycin
BERGMANN B.,
Z.,
from
WORDS:
Ascaris
suum;
N-acetylglucosaminyltransferase;
INTRODUCTION
synthesis;
factor in glycosylation, have been detected in filarial worms (Comley & Jaffe, 198 1; Comley, Jaffe & Chrin, 1982; Walter, Ossikovski & Albiez, 1985). Here, we have investigated the N-acetylglucosaminyltransferase from Ascaris suum which is a convenient model for nematode parasites in general.
N-LINKED
glycoproteins, which function as structural proteins in membranes, specific receptors at the cell surface and secreted proteins, are known to play an essential role in host-parasite interaction. Particular oligosaccharides, added to certain proteins during glycosylation, are required for normal parasite function and survival in the host. An example of this is demonstrated by the effect of tunicamycin and other inhibitors of glycosylation on the ability of pathogenic protozoa to attach to and enter a host cell (Udeinya & Van Dyke, 1981; Villalta & Kierszenbaum, 1985; Nolan & Farrell, 1985; Zingales, Katzin, Arruda & Colli, 1985). Another example is illustrated by tunicamycin-resistant Leishmania mexicana amazonensis which display an increased virulence (Kink & Chang,1987). This is due to overproduction of Nacetylglucosaminyltransferase (a result of gene amplification), which is the enzyme that catalyses the first step in the synthesis of the carbohydrate moiety of Nglycoproteins. The increased virulence is therefore attributed to increased production of a N-linked glycoprotein, with the carbohydrate processing being the limiting factor. Little is known about the glycosylated proteins in filarial worms which play a role in the parasite-host relationship or about the synthesis of these proteins. Dolichols, as well as dolichol kinase, a regulatory enzyme providing dolichylphosphate as a rate-limiting
$ To whom all correspondence
N-glycoprotein
MATERIALS AND METHODS Chemicals. Uridinediphospho-N-acetyl-o-(U-“’C) glucosamine (11 GBq mmol-‘) was purchased from AmershamBuchler, Braunschweig, F.R.G. Dolichylmonophosphate, uridinediphospho-N-acetylglucosamine and phosphatidylglycerol were from Sigma, F.R.G. Deisenhofen, Tunicamycin was obtained from Boehringer, Mannheim, F.R.G. All other chemicals and solvents used were from Merck, Darmstadt, F.R.G., or from origins of highest purity commercially available. Enzyme preparation. Female Ascaris suum were collected at a local slaughterhouse and maintained at room temperature in RPM1 1640 medium until dissection. The following procedures were performed at WC. Ovary and uterus were excised for preparation of a microsomal fraction of Nacetylglucosaminyltransferase. The reproductive tissues were homogenized with an all-glass homogenizer in 3 vols of buffer A containing 50 mM-Tris-HCI, pH 7.6, 5% (v/v) glycerol and 0.1 mM-phenylmethanesulphonyl fluoride. The homogenate was centrifuged at 10,000 g for 15 min and the resulting supernatant fraction recentrifuged at 100,000 g for 60 min. The microsomal pellet was resuspended in buffer A and stored at - 20°C until used for enzyme assays. Enzyme assyv. Enzyme activity was determined according to Kean (1980). The standard assay for N-acetylglucosaminyltransferase contained in a final volume of 0. I ml: 10 pg dolichylphosphate dispersed in 0.5% (v/v) Triton X-100, 100 mM-Tris-HCl, pH 7.2. and 12 PM IO mM-MgCl,,
should be addressed. 703
Z. KYOSSEV. B. BERGMANN. E. OSSIKOVSKI and R. D. WALTER
704
(SOnCi)-uridinediphospho-N-acetyl (‘“C) glucosamine. The reaction was initiated by addition of enzyme preparation, incubated at 37°C for 30 min and terminated by adding 1 ml of 6% (w/v) trichloroacetic acid and 0.6% (w/v) phosphotungstic acid. The precipitate was washed five times with this solution and the radioactivity in the residual pellet determined after extraction into 1 ml of chloroform/ methanol (2: 1, v/v). Incubations were performed in
duplicates, in controls the enzyme or dolichylphosphate were omitted or the incubation time restricted to zero. Enzyme
i
c d ci
3ooa
2ooo % g_,
activity is expressed in units (link of 1 pmol N-acetyl (‘4C)glucosamine to dolichylphosphate per min). Protein content was determined according to Bradford (1976) with bovine serum albumin as standard.
Initial experiments demonstrated that the Nacetylglucosaminyltransferase of Ascaris suum is associated with the microsomal fraction which is consistent with its localization in other organisms (Sharma, Lehle & Tanner, 1982; Panayotov & Zhivkov, 1984; Kean, 1985; Kaushal & Elbein, 1985; Mayer & Chen, 1985). The 100,000 g precipitate exhibited a specific activity of 1.6 f 0.4 pmol (mean f s. D., n = 4) min-’ mg-’ protein. This value is within the range of specific activities reported for the enzymes from microsomal fractions of the retina of the embryonic chick (Kean, 1985) and of the pupal instar of the stable fly (Mayer & Chen, 1985). The activity of the N-acetylglucosaminyltransferase was found to depend absolutely on the presence of divalent cations such as Mg” and Mr?+; optimal concentrations were found to be 10 and 1 mM, respectively. A similar dependence on divalent cations has been shown for the pig aorta enzyme (Kaushal & Elbein, 1985). In addition, the optimal pH range of 7.228.0 in TrissHC I
f
~~_---_.
1T
RESULTS AND DISCUSSION
10
20
50
i_-_i
100
Phosphatidygiycerol ( pg ml-‘) FIG. 1. Effect of phosphatidylglycerol on the activit! f the Nacetylglucosaminyltransferase. The standard assay conditions were employed except for the addition of phosphatidylglycerol as indicated.
for the Ascaris enzyme corresponds to pH optima reported for the enzymes of the pig aorta and of the pupal instar of the fly. Under standard assay conditions, the enzyme activity of the microsomal fractions of Ascaris was found to be linear for at least 30 min at 37°C. As shown in Fig. 1 the activity of the Ascaris enzyme was stimulated by phosphatidylglycerol with maximal stimulation at about 10 pg ml-‘. However, stimulation varied within preparations increasing the V,,,,, for both uridinediphosphoN-acetylglucosamine and dolichylphosphate by about three- to six-fold without affecting the K,,, values (Figs.
(b)
(a)
ib/ 3
-i
.c
Tc
e
ii=
E ‘ij E
2
I
/’
A
J
// I
I
0.1
0.2
l/UDP Glc NAc (FM)
31 l/Lkdiiylphosphate
( pg ml-‘)
FIG. 2. Stimulation of N-acetylglucosaminyltransferase by phosphatidylglycerol. A Lineweaver-Burk plot of the enzyme activity at a range of concentrations of (a) uridinediphospho-N-acetylglucosamine (UDPGlcNAc) (0) and (b) dolichylphosphate (0) and in the presence of 10 pg ml-’ phosphatidylglycerol (a). The enzyme was assayed as described in the Materials and Methods.
N- Acetylglucosaminyltransferase
(see Kyossev 62 Ossikovski, in press). Results presented here clearly demonstrate that the N-acetylglucosaminyltransferase plays a regulatory role in the synthesis of N-linked glycoproteins of A. suum as it is reported from other organisms.
-i
/
S
Acknowledgement-This investigation received support from the Alexander von Humboldt-Stiftung (E.O.) and the Deutsche Forschungsgemeinschaft (Z.K.).
4
i
e
705
from A. suum
& V
REFERENCES
2
I
I
0.1
0.2
i/UDP Glc NAc (PM)
FIG. 3. Inhibition of N-acetylglucosaminyltransferase by tunicamycin. A Lineweaver-Burk plot of the enzyme activity at a range of concentrations of uridinediphospho-Nacetylglucosamine (0) and in the presence of 10 ng ml-’ (A)
and 50 ng ml-’ (0) tunicamycin. The enzyme activity was assayed as described in the Materials and Methods.
2a,b). Stimulation of enzyme activity by phosphatidylglycerol without changes in the K, values has also been reported for the pig aorta enzyme (Kaushal & Elbein, 1985), whereas in the case of the retina enzyme of embryonic chick the stimulation involved increased and decreased K,,, values for uridinediphospho-N-acetylglucosamine and dolichylphosphate, respectively (Kean, 1985). The apparent K, values for the Ascaris enzyme were determined in the absence and presence of phosphatidylglycerol to be12.1 * 4pM(mGm f s.o.,n=7)and98 f 59pg ml-’ (mean & s D, n=5) for uridinediphospho-Nacetylglucosamine and dolichylphosphate, respectively. The Km value for uridinediphospho-N-acetylglucosamine as shown here is similar to that reported for the yeast enzyme (Sharma et al., 1982), whereas those determined for the enzymes from pig, chick and fly were l&100-fold lower (Kaushal & Elbein, 1985; Kean, 1985; Mayer & Chen, 1985). The antibiotic tunicamycin, structurally related to N-acetylglucosamine and reported to block glycoprotein synthesis by inhibiting the N-acetylglucosaminyltransferase reaction (Elbein, 1984), was also found to be a potent inhibitor of the enzyme from A. suum. The type of inhibition was competitive with respect to uridinediphospho-N-acetylglucosamine (Fig. 3) and the inhibition constant for this tunicamycin preparation was calculated to be 9.2 & 1.6 ng ml-’ (mean f s. D., n=4). Synthesis of dolichylphosphate-N-acetylglucosamine and also its conversion to dolichylphosphate-NJ”-diacetylchitobiose have recently been demonstrated for the related nematode Ascaridiu galli
BRADFORDM. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72: 248-254. COMLEY J. C. W. & JAFFE J. J. 1981. Isoprenoid biosynthesis in adult Brugia pahangi and Dirofilaria immitis. Journal of Parasitolog~Ll~ 609-616. COMLEYJ. C. W.. JAFFE J. J. & CHRIN L. R. 1982. Glvcosvl transferase activity in homogenates of adult DiroJilaria immitis. Molecular and Biochemical Parasitology 5: 19-21. ELBEIN A. D. 1984. Inhibitors of the biosynthesis and processing of N-linked oligosaccharides. CRC Critical Reviews in Biochemistry 16: 2149. KAUSHAL G. P. & ELBEIN A. D. 1985. Purification and properties of UDP-GlcNAc: dolichyl-phosphate GlcNAcl-phosphate transferase. Activation and inhibition of the enzyme. Journal of Biological Chemistry 260: 16,303S 16,309. KEAN E. L. 1980. Stimulation by GDP-mannose of the biosynthesis of N-acetylglucosaminylpyrophosphoryl polyprenols by the retina. Journal of Biological Chemistry 255: 1921-1927. KEAN E. L. 1985. Stimulation by dolichol phosphatemannose and phospholipids of the biosynthesis of Nacetylglucosaminylpyrophosphoryl dolichol. Journal of Biological Chemistry 260: 12,561-12,570. KINK J. A. & CHANG K. P. 1987. Tunicamycin-resistant Leishmania mexicana amazonensis: expression of virulence associated with an increased activity of N-acetylglucosaminyltransferase and amplification of its presumptive gene. Proceedings of the National Academy of Sciences of the United States of America 84: 1253-1257. MAYER R. T. & CHEN A. C. 1985. N-Acetylglucosaminyl transferases from the pupal instar of the stable fly, Stomoxys calcitrans. Archives of Insect Biochemistry and Physiology 2: 161-179. NOLAN T. J. & FARRELLJ. P. 1985. Inhibition of in viva and in vitro infectivity of Leishmania donovani by tunicamycin. Molecular and Biochemical Parasitology 16: 127-135. PANAYOTOVB. N. & ZHIVKOV V. I. 1984. Biosynthesis of dolichyl pyrophosphate N-acetylglucosaminyl intennediates in liver microsomes from hibernating ground squirrels (Citellus citellus). International Journal of Biochemistry 16: 239-242. SHARMAC. B., LEHLEL. & TANNER W. 1982. Solubilization and characterization of the initial enzymes of the dolichol pathway from yeast. European Journal of Biochemistry 126: 319-325. UDEINYAI. J. &VAN DYKE K. 1981. Concurrent inhibition by tunicamycin of glycosylation and parasitemia in malaria parasites (Plasmodium falciparum) cultured in human erythrocytes. Pharmacology 23: 165-170. VILLALTA F. & KIERSZENBAUM F. 1985. The effect of
706
Z. KYOSSEV, B. BERGMANN, E. OSSIKOVSKI and R. D. WALTER
swainsonine on the association of Trypanosoma cruti with host cells. Molecular and Biochemical Parasitology 16: I-10. WALTER R. D., OSSIKOVSKIE. & ALBIEZ E. J. 1985. Dolichol kinase in Ascaris sum and Onchocerca volvulus. Molecular and Biochemical Parasitology 14: 2 1 l-2 17.
ZINCALESB., KATZIN A.M., ARRUDA M. V. & COLLI W. 1985. Correlation of tunicamycin-sensitive surface glycoproteins from Trypanosoma cruzi with parasite interiorization into mammalian cells. Molecular and Biochemical Parasitolog,t 16: 21-34.
Porasirology
Vd 21. No. 6, pp. 703-706.
1991
0
N-ACETYLGLUCOSAMINYLTRANSFERASE Z. KYOSSEV,*
B. BERGMANN,~
FROM
Oa&7519/91 $3.00 + 0.00 Pergamon Press plc 199 I Austroh Society for Parasitology
ASCARIS
SUUA4
E. OSSIKOVSKI* and R. D. WALTER-~-$
*Bulgarian Academy of Sciences, Central Laboratory of Helminthology, TDepartment of Biochemistry, Bernhard Nocht Institute for Tropical Medicine, D-2000 Hamburg 36, F. R. G.
1113Sofia, Bulgaria Bernhard-Nocht-StraDe
74.
(Received 29 November 1990; accepted 29 March 1991) OSSIKOVSKI E. and WALTER R. D. 1991. N-AcetylAscaris suum. International Journal for Parasirology 21: 703-706. The occurrence of N-acetylglucosaminyltransferase, the initial step in the synthesis of the carbohydrate moiety of N-linked glycoproteins, is demonstrated in the microsomal fraction of the nematode Ascaris suum. Phosphatidylglycerol stimulated enzyme activity three- to six-fold without affecting the K, values of either substrates, uridinediphospho-N-acetylglucosamine or dolichylphosphate. The K, values were determined to be about 12 PM and 100 pg ml-‘, respectively. The enzyme activity was strongly inhibited by tunicamycin acting as a competitive inhibitor with respect to the substrate uridinediphospho-N-glucosamine.
Abstract-Kuossav glucosaminyltransferase
INDEX KEY tunicamycin
BERGMANN B.,
Z.,
from
WORDS:
Ascaris
suum;
N-acetylglucosaminyltransferase;
INTRODUCTION
synthesis;
factor in glycosylation, have been detected in filarial worms (Comley & Jaffe, 198 1; Comley, Jaffe & Chrin, 1982; Walter, Ossikovski & Albiez, 1985). Here, we have investigated the N-acetylglucosaminyltransferase from Ascaris suum which is a convenient model for nematode parasites in general.
N-LINKED
glycoproteins, which function as structural proteins in membranes, specific receptors at the cell surface and secreted proteins, are known to play an essential role in host-parasite interaction. Particular oligosaccharides, added to certain proteins during glycosylation, are required for normal parasite function and survival in the host. An example of this is demonstrated by the effect of tunicamycin and other inhibitors of glycosylation on the ability of pathogenic protozoa to attach to and enter a host cell (Udeinya & Van Dyke, 1981; Villalta & Kierszenbaum, 1985; Nolan & Farrell, 1985; Zingales, Katzin, Arruda & Colli, 1985). Another example is illustrated by tunicamycin-resistant Leishmania mexicana amazonensis which display an increased virulence (Kink & Chang,1987). This is due to overproduction of Nacetylglucosaminyltransferase (a result of gene amplification), which is the enzyme that catalyses the first step in the synthesis of the carbohydrate moiety of Nglycoproteins. The increased virulence is therefore attributed to increased production of a N-linked glycoprotein, with the carbohydrate processing being the limiting factor. Little is known about the glycosylated proteins in filarial worms which play a role in the parasite-host relationship or about the synthesis of these proteins. Dolichols, as well as dolichol kinase, a regulatory enzyme providing dolichylphosphate as a rate-limiting
$ To whom all correspondence
N-glycoprotein
MATERIALS AND METHODS Chemicals. Uridinediphospho-N-acetyl-o-(U-“’C) glucosamine (11 GBq mmol-‘) was purchased from AmershamBuchler, Braunschweig, F.R.G. Dolichylmonophosphate, uridinediphospho-N-acetylglucosamine and phosphatidylglycerol were from Sigma, F.R.G. Deisenhofen, Tunicamycin was obtained from Boehringer, Mannheim, F.R.G. All other chemicals and solvents used were from Merck, Darmstadt, F.R.G., or from origins of highest purity commercially available. Enzyme preparation. Female Ascaris suum were collected at a local slaughterhouse and maintained at room temperature in RPM1 1640 medium until dissection. The following procedures were performed at WC. Ovary and uterus were excised for preparation of a microsomal fraction of Nacetylglucosaminyltransferase. The reproductive tissues were homogenized with an all-glass homogenizer in 3 vols of buffer A containing 50 mM-Tris-HCI, pH 7.6, 5% (v/v) glycerol and 0.1 mM-phenylmethanesulphonyl fluoride. The homogenate was centrifuged at 10,000 g for 15 min and the resulting supernatant fraction recentrifuged at 100,000 g for 60 min. The microsomal pellet was resuspended in buffer A and stored at - 20°C until used for enzyme assays. Enzyme assyv. Enzyme activity was determined according to Kean (1980). The standard assay for N-acetylglucosaminyltransferase contained in a final volume of 0. I ml: 10 pg dolichylphosphate dispersed in 0.5% (v/v) Triton X-100, 100 mM-Tris-HCl, pH 7.2. and 12 PM IO mM-MgCl,,
should be addressed. 703
Z. KYOSSEV. B. BERGMANN. E. OSSIKOVSKI and R. D. WALTER
704
(SOnCi)-uridinediphospho-N-acetyl (‘“C) glucosamine. The reaction was initiated by addition of enzyme preparation, incubated at 37°C for 30 min and terminated by adding 1 ml of 6% (w/v) trichloroacetic acid and 0.6% (w/v) phosphotungstic acid. The precipitate was washed five times with this solution and the radioactivity in the residual pellet determined after extraction into 1 ml of chloroform/ methanol (2: 1, v/v). Incubations were performed in
duplicates, in controls the enzyme or dolichylphosphate were omitted or the incubation time restricted to zero. Enzyme
i
c d ci
3ooa
2ooo % g_,
activity is expressed in units (link of 1 pmol N-acetyl (‘4C)glucosamine to dolichylphosphate per min). Protein content was determined according to Bradford (1976) with bovine serum albumin as standard.
Initial experiments demonstrated that the Nacetylglucosaminyltransferase of Ascaris suum is associated with the microsomal fraction which is consistent with its localization in other organisms (Sharma, Lehle & Tanner, 1982; Panayotov & Zhivkov, 1984; Kean, 1985; Kaushal & Elbein, 1985; Mayer & Chen, 1985). The 100,000 g precipitate exhibited a specific activity of 1.6 f 0.4 pmol (mean f s. D., n = 4) min-’ mg-’ protein. This value is within the range of specific activities reported for the enzymes from microsomal fractions of the retina of the embryonic chick (Kean, 1985) and of the pupal instar of the stable fly (Mayer & Chen, 1985). The activity of the N-acetylglucosaminyltransferase was found to depend absolutely on the presence of divalent cations such as Mg” and Mr?+; optimal concentrations were found to be 10 and 1 mM, respectively. A similar dependence on divalent cations has been shown for the pig aorta enzyme (Kaushal & Elbein, 1985). In addition, the optimal pH range of 7.228.0 in TrissHC I
f
~~_---_.
1T
RESULTS AND DISCUSSION
10
20
50
i_-_i
100
Phosphatidygiycerol ( pg ml-‘) FIG. 1. Effect of phosphatidylglycerol on the activit! f the Nacetylglucosaminyltransferase. The standard assay conditions were employed except for the addition of phosphatidylglycerol as indicated.
for the Ascaris enzyme corresponds to pH optima reported for the enzymes of the pig aorta and of the pupal instar of the fly. Under standard assay conditions, the enzyme activity of the microsomal fractions of Ascaris was found to be linear for at least 30 min at 37°C. As shown in Fig. 1 the activity of the Ascaris enzyme was stimulated by phosphatidylglycerol with maximal stimulation at about 10 pg ml-‘. However, stimulation varied within preparations increasing the V,,,,, for both uridinediphosphoN-acetylglucosamine and dolichylphosphate by about three- to six-fold without affecting the K,,, values (Figs.
(b)
(a)
ib/ 3
-i
.c
Tc
e
ii=
E ‘ij E
2
I
/’
A
J
// I
I
0.1
0.2
l/UDP Glc NAc (FM)
31 l/Lkdiiylphosphate
( pg ml-‘)
FIG. 2. Stimulation of N-acetylglucosaminyltransferase by phosphatidylglycerol. A Lineweaver-Burk plot of the enzyme activity at a range of concentrations of (a) uridinediphospho-N-acetylglucosamine (UDPGlcNAc) (0) and (b) dolichylphosphate (0) and in the presence of 10 pg ml-’ phosphatidylglycerol (a). The enzyme was assayed as described in the Materials and Methods.
N- Acetylglucosaminyltransferase
(see Kyossev 62 Ossikovski, in press). Results presented here clearly demonstrate that the N-acetylglucosaminyltransferase plays a regulatory role in the synthesis of N-linked glycoproteins of A. suum as it is reported from other organisms.
-i
/
S
Acknowledgement-This investigation received support from the Alexander von Humboldt-Stiftung (E.O.) and the Deutsche Forschungsgemeinschaft (Z.K.).
4
i
e
705
from A. suum
& V
REFERENCES
2
I
I
0.1
0.2
i/UDP Glc NAc (PM)
FIG. 3. Inhibition of N-acetylglucosaminyltransferase by tunicamycin. A Lineweaver-Burk plot of the enzyme activity at a range of concentrations of uridinediphospho-Nacetylglucosamine (0) and in the presence of 10 ng ml-’ (A)
and 50 ng ml-’ (0) tunicamycin. The enzyme activity was assayed as described in the Materials and Methods.
2a,b). Stimulation of enzyme activity by phosphatidylglycerol without changes in the K, values has also been reported for the pig aorta enzyme (Kaushal & Elbein, 1985), whereas in the case of the retina enzyme of embryonic chick the stimulation involved increased and decreased K,,, values for uridinediphospho-N-acetylglucosamine and dolichylphosphate, respectively (Kean, 1985). The apparent K, values for the Ascaris enzyme were determined in the absence and presence of phosphatidylglycerol to be12.1 * 4pM(mGm f s.o.,n=7)and98 f 59pg ml-’ (mean & s D, n=5) for uridinediphospho-Nacetylglucosamine and dolichylphosphate, respectively. The Km value for uridinediphospho-N-acetylglucosamine as shown here is similar to that reported for the yeast enzyme (Sharma et al., 1982), whereas those determined for the enzymes from pig, chick and fly were l&100-fold lower (Kaushal & Elbein, 1985; Kean, 1985; Mayer & Chen, 1985). The antibiotic tunicamycin, structurally related to N-acetylglucosamine and reported to block glycoprotein synthesis by inhibiting the N-acetylglucosaminyltransferase reaction (Elbein, 1984), was also found to be a potent inhibitor of the enzyme from A. suum. The type of inhibition was competitive with respect to uridinediphospho-N-acetylglucosamine (Fig. 3) and the inhibition constant for this tunicamycin preparation was calculated to be 9.2 & 1.6 ng ml-’ (mean f s. D., n=4). Synthesis of dolichylphosphate-N-acetylglucosamine and also its conversion to dolichylphosphate-NJ”-diacetylchitobiose have recently been demonstrated for the related nematode Ascaridiu galli
BRADFORDM. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72: 248-254. COMLEY J. C. W. & JAFFE J. J. 1981. Isoprenoid biosynthesis in adult Brugia pahangi and Dirofilaria immitis. Journal of Parasitolog~Ll~ 609-616. COMLEYJ. C. W.. JAFFE J. J. & CHRIN L. R. 1982. Glvcosvl transferase activity in homogenates of adult DiroJilaria immitis. Molecular and Biochemical Parasitology 5: 19-21. ELBEIN A. D. 1984. Inhibitors of the biosynthesis and processing of N-linked oligosaccharides. CRC Critical Reviews in Biochemistry 16: 2149. KAUSHAL G. P. & ELBEIN A. D. 1985. Purification and properties of UDP-GlcNAc: dolichyl-phosphate GlcNAcl-phosphate transferase. Activation and inhibition of the enzyme. Journal of Biological Chemistry 260: 16,303S 16,309. KEAN E. L. 1980. Stimulation by GDP-mannose of the biosynthesis of N-acetylglucosaminylpyrophosphoryl polyprenols by the retina. Journal of Biological Chemistry 255: 1921-1927. KEAN E. L. 1985. Stimulation by dolichol phosphatemannose and phospholipids of the biosynthesis of Nacetylglucosaminylpyrophosphoryl dolichol. Journal of Biological Chemistry 260: 12,561-12,570. KINK J. A. & CHANG K. P. 1987. Tunicamycin-resistant Leishmania mexicana amazonensis: expression of virulence associated with an increased activity of N-acetylglucosaminyltransferase and amplification of its presumptive gene. Proceedings of the National Academy of Sciences of the United States of America 84: 1253-1257. MAYER R. T. & CHEN A. C. 1985. N-Acetylglucosaminyl transferases from the pupal instar of the stable fly, Stomoxys calcitrans. Archives of Insect Biochemistry and Physiology 2: 161-179. NOLAN T. J. & FARRELLJ. P. 1985. Inhibition of in viva and in vitro infectivity of Leishmania donovani by tunicamycin. Molecular and Biochemical Parasitology 16: 127-135. PANAYOTOVB. N. & ZHIVKOV V. I. 1984. Biosynthesis of dolichyl pyrophosphate N-acetylglucosaminyl intennediates in liver microsomes from hibernating ground squirrels (Citellus citellus). International Journal of Biochemistry 16: 239-242. SHARMAC. B., LEHLEL. & TANNER W. 1982. Solubilization and characterization of the initial enzymes of the dolichol pathway from yeast. European Journal of Biochemistry 126: 319-325. UDEINYAI. J. &VAN DYKE K. 1981. Concurrent inhibition by tunicamycin of glycosylation and parasitemia in malaria parasites (Plasmodium falciparum) cultured in human erythrocytes. Pharmacology 23: 165-170. VILLALTA F. & KIERSZENBAUM F. 1985. The effect of
706
Z. KYOSSEV, B. BERGMANN, E. OSSIKOVSKI and R. D. WALTER
swainsonine on the association of Trypanosoma cruti with host cells. Molecular and Biochemical Parasitology 16: I-10. WALTER R. D., OSSIKOVSKIE. & ALBIEZ E. J. 1985. Dolichol kinase in Ascaris sum and Onchocerca volvulus. Molecular and Biochemical Parasitology 14: 2 1 l-2 17.
ZINCALESB., KATZIN A.M., ARRUDA M. V. & COLLI W. 1985. Correlation of tunicamycin-sensitive surface glycoproteins from Trypanosoma cruzi with parasite interiorization into mammalian cells. Molecular and Biochemical Parasitolog,t 16: 21-34.
