The enzyme that transfers N-acetylglucosamine 1-phosphate from UDP-N-acetylglucosamine to dolichyl phosphate forming N-acetylglucosaminylpyrophosphoryl-dolichol was solubilized from a microsomal fraction of pig aorta and was partially purified on DEAE-cellulose [Heifetz, A., & Elbein, A. D. (1977) J. Biol. Chem. 252, 3057-3065], This enzyme was used to study the mode of action of tunicamycin, an N-acetylglucosamine containing antibiotic produced by Streptomyces lysosuperificus. At low concentrations of enzyme, the reaction was inhibited 50% or greater by antibiotic concentrations of 0.05-0.1 μg/mL, whereas higher enzyme concentrations required progressively more tunicamycin for inhibition. The inhibition was noncompetitive with respect to the concentration of UDP-N-acetvlslucosamine or of dolichyl phosphate. Tunicamycin also inhibited the reverse reaction; that is, it prevented the formation of UDP-.N-acetylglucosamine from N-acetylglucosaminylpyro-phosphoryldolichol. The inhibition was increased by prein-cubating the enzyme with antibiotic for up to 5 min before the addition of substrates. The addition of phosphatidylcholine, at concentrations up to 20 mM, did not affect the inhibition regardless of whether it was added during the preincubation or at the same time as the substrates. Tunicamycin did, however, bind to heat-denatured microsomal particles of aorta as shown by the fact that preincubation of antibiotic with these particles prevented the inhibition of the N-acetylglucos-amine-1-phosphate transferase. © 1979, American Chemical Society. All rights reserved.
