Experientia 41 (1985), Birkh~iuser Verlag, CH~-010 Basel/Switzerland 623 sence of exogenous substrate protein (such as histone) was noted. In contrast, about 36% inhibition in the absence of Mg ~+ and 47 % inhibition in the presence of Mg 2+ on cyclic AMP-dependent protein kinases by this acidic protein was ob- served when arginine-rich histone was added to reaction mix- ture as a substrate. Such inhibition may be partially due to the strong interaction between histone and this acidic protein which may deprive a part of histone from phosphorylation by these kinases. Cyclic AMP-dependent protein kinases have been found in many mammalian tissues ~5, including brain. Therefore, the results of this study suggest that this acidic pro- tein may also play an important regulatory role on this type of kinases in brains. It is of great interest that the small acid pro- tein stimulates both enzymes, phosphoprotein phosphatases and megamodulin-dependent protein kinase 1, which in general carry out opposite functions. Therefore, the time sequence for the activation of both enzyme in vivo may be different to avoid antagonism. This acidic protein is a heat-stable factor since it retained origi- nal stimulatory effect on the same enzymes after being boiled at 100~ for 30 rain. However, its stimulatory activity was de- stroyed after being treated with trypsin at 37~ for 2 h, indi- cating that it indeed is a protein. Moreover, altered UV spectra of this acidic protein in the presence of Mg 2+ or Mn 2+ were noted, suggesting that the concomitant conformational transi- tion ~6 of this protein may occur through its binding with the cation to convert into its active forms, acidic protein-cation complexes, which may thereby modulate enzymes. 1 Acknowledgments. This work was supported by a grant (RR- 08229) from the National Institutes of Health, USA. W.N. Kuo is a recipient of a Distinguished Faculty Scholar Award from United Negro College Fund, Inc., USA. 2 Lin, Y.M., Molec. cell. Biochem. 45 (1982) 101. 3 Lin, Y.M., Liu, Y.P., and Cheung, W.Y., J. biol. Chem. 249 (1974) 4943. 4 Kuo, W.N., Cytobiose 36 (1983) 175. 5 Shoji, M., Brackett, N.L., Tse, J., Shapira, M., and Kuo, J.F., J. biol. Chem. 253 (1978) 3427. 6 Kuo, W.N., Biochem. biophys. Res. Commun. 114 (1983) 403. 7 Itano, T., Itano, R., and Penniston, J.T., J. Biochem. 189 (1980) 455. 8 Kuo, W.N., Experientia 39 (1983) 60. 9 Jamieson, G.A., Jr, and Vanaman, T.C., Biochem. biophys. Res. Commun. 90 (1979) 1048. 10 Kuo, W.N., unpublished observation. 11 Maeno, H., and Greengard, P., J. biol. Chem. 247 (1972) 3269. 12 Appleman, M.M., Birnbaumer, L., and Terres, H.N, J. biol. Chem. 116 (1966) 39. 13 Weber, K., and Osborn, M., J. biol. Chem. 244 (1969) 4406. 14 Winter, A., Ed, K., and Anderson, U.B., LKB Application Note 250 (1977). 15 Pasternak, Th., Cyclic AMP. Eds Robinson, Butcher, and Suther- land (1971) 127. 0014-4754/85/050622-0251.50 + 0.20/0 9 B[rkMuser Verlag Basel, 1985 Effects of diflubenzuron and tunicamycin on N-acetylglucosaminyl transferases in prepupae of the stable fly (Sto- moxys calcitrans) R.T. Mayer and A.C. Chen Veterinary Toxicology and Entomology Research Laboratory, Agricultural Research Service, US Department of Agriculture, P.O. Drawer GE, College Station (Texas 77841, USA), 16 March 1984 Summary. Tunicamycin, an antibiotic, and diflubenzuron, an insect growth regulator, were tested to determine their effects on N-acetylglucosaminyl transferase from S. ealcitrans prepupae. Diflubenzuron had no effect, but tunicamycin inhibited the transfer of GlcNAc-I-P from UDP-GlcNAc to dolicholmonophosphate with an I50 of 1.5-4 ng/ml. Key words. Stable fly; Stomoxys ealcitrans; diflubenzuron; tunicamycin; N-acetylglucosaminyl transferase; prepupae. Chitin synthesis inhibitors (CSI) such as diflubenzuron have been under investigation for a number of years now. Reports concerning the molecular mode of action of CSI are often con- flicting and contradictory 1. Initially, CSI were thought to inhi- bit chitin synthesis in insects by acting directly on chitin syn- thase 25. There are now several reports that show that CSI do not operate on chitin synthase when studied in cell-free enzyme preparations a,6.7. However, chitin synthesis is inhibited by diflubenzuron (DFB) and other CSI when in vitro organ culture or in vivo assays are used2-5, s, 9, and a buildup of UDP-N-acetylglucosamine (UDP- GlcNAc) is observed in tissues or insects that have been treated with CSI. In addition, Meola and Mayer 9 and DeLoach et alJ ~ showed that DFB acted as a cytostatic agent when topically applied to pupae of the stable fly. Conse- quently, it was suggested that the inhibition of chitin synthesis and buildup of UDP-GIcNAc in DFB-treated tissues and the cytostatic action of DFB might be explained by an effect on cell membrane permeability 1'~~ Indeed, recent studies with Harding-Passey melanoma cells showed that DFB significantly inhibited the uptake of certain nucleosides into those cells, which indicated that a membrane effect was involved H. It has been suggested that DFB might inhibit the N-acetyl- glucosamine-l-P (GIcNAc-I-P) transferases which transfer GlcNAc-I-P from UDP-GlcNAc to form GlcNAc-pyrophos- phoryl-dolichol (Dol.PP-GlcNAc), and which are involved in membrane synthesis 1. Inhibition of this enzyme system could alter membrane permeability and cause a buildup of UDP-Glc- NAc. We have investigated the GlcNAc-1-P transferases in prepupae of stable fly, and report here the effects of DFB on these en- zymes. The present studies were done in conjunction with stu- dies on the antibiotic tunicamycin, which has been reported to inhibit chitin ~2 and glycoprotein I3"I4 synthesis in insects. Our results are the first demonstration that tunicamycin inhibits in- sect GlcNAc-I-P transferases to prevent the formation of Dol. PP-GlcNAc, as it does in other organisms. Materials and methods. Chemicals. UDP-[glucosamine-6- ~H]GlcNAc (24 Ci/mmole) and UDP-[glucosamine-1- 14C]GlcNAc (35 mCi/mmole) were purchased, respectively, from New England Nuclear, Boston, MA and ICN Pharma- ceuticals, Inc., Irvine, CA. Betafluor scintillation cocktail was purchased from National Diagnostics, Somerville, N.J. Doli- chol monophosphate (Dol'P), 80-90% pure based on phos- phorus content, was purchased from Sigma Chemical Co., St. Louis, MO. A purified sample of tunicamycin was obtained from Dr J.D. Douros, Developmental Therapeutics Program, Chemotherapy, NCI, Bethesda, MD. Diflubenzuron (Dimi-