1. In a crude particulate fraction from yeast mannose is transferred from GDP-mannose to an endogenous "lipid" fraction, the monophosphates of dolichol-14 to dolichol-18. The same membrane fraction also catalyzes the mannosylation of glycoproteins. More than 80 of the total radioactivity in the glycoprotein fraction obtained from GDP-[14C]mannose is released by /?-elimination. The small-sized radioactive products of /?-elimination are mannose, mannobiose and mannotriose.2. Ageing of the particulate fraction leads to a drastic loss of mannosyl transfer activity from GDP-[14C]mannose to dolichol monophosphate. To the same extent the amount of [14C]mannose obtained after /?-elimination of the glycoproteins decreases. The amount of radioactive mannobiose and mannotriose is, however, much less affected.3. With decreasing GDP-mannose concentrations the amount of [14C]mannose obtained after /&elimination increases as compared to the amount of radioactive mannobiose and mannotriose. The Km-value for the incorporation of mannosyl groups directly linked to serine and/or threonine, therefore, is lower than the Km-value for the transfer of subsequent mannosyl residues.4. 6. The results are consistent with the assumption that dolichol monophosphate is involved in the mannosylation of a specific position in yeast glycoproteins, i.e. in the formation of mannosyl linkages to serine and/or threonine. The subsequent mannosylation proceeds directly from GDP-mannose in a reaction obligatorily requiring Mn2+.7. After /?-elimination of the methanol-insoluble material mannosylated in the presence of dolichol-monophosphate [14C]mannose the residual insoluble radioactivity is to a large extent transformed into dialyzable material by pronase. Mannose, therefore, is transferred from dolicholmonophosphate mannose also to glycoprotein positions not involving OH-groups of serine or threonine.Glycosylated lipids play a role in the biosynthesis of glycoproteins in animals [l -51, fungi [6,7] and plants [S]. The lipophilic component in all these cases is probably dolichol monophosphate [2,4,9,10].In Succhuvomyces cerevisiue the lipophilic mannosyl acceptor has been shown by mass spectroscopy to consist of the monophosphates of the whole family of dolichols with 14 to 18 isoprene units [lo]. From -~ his 60th birthday. mannosylated dolichol monophosphates the mannosyl group is transferred by yeast membranes to glycoproteins. Preliminary evidence has been presented [7] threonine, only the mannosyl residue directly linked to Dedicated to Professor Dr 0. Hoffmann-Ostenhof on Abbreviations. Dolichol-P, dolichol monophosphate; dolichol-P-Man, dolichol-monophosphate-mannose ; GDPthat within oligomannose chains linked to serine/ Man, guanosine-diphosphate-mannose.Eur. J. Biochem. 46 (1974)

No abstract

BAY 10-8888 is a cyclic β-amino acid that is related to cispentacin and that has antifungal activity. Candida albicans cells accumulated BAY 10-8888 intracellularly to a concentration about 200 that in the medium when grown in media with a variety of nitrogen sources. In complex growth medium, BAY 10-8888 transport activity was markedly reduced and was paralleled by a decrease in its antifungal activity. Uptake of BAY 10-8888 was mediated by an H+-coupled amino acid transporter with specificity for branched-chain amino acids (isoleucine, leucine, and valine) and showed a KT (Michaelis constant of the transport reaction) of 0.95 mM and a V max of 18.9 nmol × min−1 × 107cells−1. Similar to the transport of natural amino acids in Saccharomyces cerevisiae, the transport of BAY 10-8888 into the cell was unidirectional. Efflux occurred by diffusion and was not carrier mediated. Inside the cell BAY 10-8888 inhibited specifically isoleucyl-tRNA synthetase, resulting in inhibition of protein synthesis and cell growth. Intracellular isoleucine reversed BAY 10-8888-induced growth inhibition. BAY 10-8888 was not incorporated into proteins. BAY 10-8888 inhibited isoleucyl-tRNA synthetase with the same concentration dependency as protein biosynthesis in intact cells assuming 200-fold accumulation.

Inhibitors of branched‐chain amino acid biosynthesis—by inhibiting acetolactate synthase (ALS)—represent the most active group of herbicidal compounds to date (Shaner, D.L., Recent Adv. Phytochem. 23 (1989) 227–61). A microbial screening technique has been developed to investigate known and possible new ALS‐inhibitors. Escherichia coli mutant FD 1062, which expresses only valine‐resistant ALS II isoenzyme as the solely branched‐chain amino acid synthesizing isoenzyme, has been used extensively to optimize known and to screen for new chemical classes of ALS‐inhibitors, respectively. Herbicidal compounds like sulfonylureas, triazolopyrimidines, pyrimidylsalicylates, carbamoylpyrazolines, sulfonylimino‐azinyl‐heteroazoles, sulfonylamide azines, and substituted sulfonyldiamides, respectively, are active on minimal medium with Ki‐values which resemble the rank order of biological activity of these compounds in the greenhouse. Interestingly, herbicidal imidazolinones are not at all inhibitory on E.coli strain FD 1062 in vivo although, of course, they exert high activity on isolated bacterial ALS. Similarly, N‐protected valylanilides, pyrimidyl mandelic acids, benzenesulfonyl carboxamides, and uhiquinone‐O are inactive in the bacterial assay but have been shown by other methods to act as ALS inhibitors. Additionally, reversal experiments can he performed to exclude, e.g. artificial inhibitory effects of test compounds. Moreover, a thin‐layer biogram application technique opens the opportunity to test mixtures of chemicals. From green plant cell cultures (Catharanthus roseus) ALS has been isolated and characterized in terms of inhibition by sulfonylureas, imidazolonones, triazolopyrmidines, salicylated, and carbamoylpyrazolines, imidazolinones, triaiolopyritnidines, salicjdates, and carbarnoylpj˜razolines, respectively. All five types show biphasic slow tight binding kinetics with Stedy state I50values of 0.5 nm (sulforneturon), 1.9 nm (triazolopjirimidine), 8.3 nm (salicylate), 23 nm (imazapyr), and 135 nm (carbamoylpyrazoline),respectiuely . Isolated ALS from Saccharomyces cerevisiae is equallv well blocked by herbicidal ALS inhibitors although with different I50values ( triazoiopyrimidine, 21 nm, sulforneturon, 70 nm, salicylate, 21 μm, imaiapyr, 38μm, and carbamoylpyrazoline, 148 μm ). Surprisingly, biphasic kinetics could not be observed with the yeast enzjtme although slow binding hehauiour was clearly established.

The first mannosyl unit of manno-oligosaccharides of fungal mannoproteins is transferred in a dolichyl-phosphate-dependent reaction sequence to serineithreonine residues of the protein. The two membrane-bound enzymes catalyzing this transfer in the yeast Saccharomyces cerevisiae have been solubilized by detergents. The enzyme transferring mannose from guanosine diphosphate mannose to dolichyl phosphate has been purified 18-fold when based on membrane protein and 140-fold when based on total cell protein. The enzyme transferring mannose from dolichyl phosphate mannose to protein has been purified 48-fold and 380-fold, respectively. A HC1-treated cellwall mannoprotein from yeast served as acceptor protein for the second enzyme.The solubilized enzyme catalyzing the formation of dolichyl diphosphate mannose has a K,,, for guanosine diphosphate mannose of 7 x M and is saturated with about 0.15 mM yeast dolichyl phosphate. The metal requirement, pH-optima, and the detergent concentration necessary for optimal activity have been determined for both solubilized enzymes. (2) Subsequent mannosyl residues of manno-oligosaccharides up to four mannose residues long are transferred to the serine/threonine-linked mannose directly from GDP-Man as donor [4,5]. Of the various enzymes involved in glycosyl transfer reactions with dolichyl phosphates as glycosyl acceptors or donors, only few have been solubilized so far [6-101. They are all part of the dolichol pathway leading to the formation of N-glycosidic linkages.Abbreviations. GDP-Man, guanosine diphosphate mannose, Dol, dolichyl; Man, mannose; GlcNAc, N-acetylglucosamine; UDP-GlcNAc, uridine diphosphate N-acetylglucosamine.