Regulation of the biosynthesis of N-acetylglucosaminylpyrophosphoryldolichol, feedback and product inhibition

The assembly of the core oligosaccharide region of asparagine-linked glycoproteins proceeds by means of the dolichol pathway. The first step of this pathway, the reaction of dolichol phosphate with UDP-GlcNAc to form N-acetylglucosaminylpyrophosphoryldolichol (GlcNAc-P-P-dolichol), is under investigation as a possible site of metabolic regulation. This report describes feedback inhibition of this reaction by the second intermediate of the pathway, N-acetylglucosaminyl-N-acetylglucosaminylpyrophosphoryldolichol (GlcNAc-GlcNAc-P-P-dolichol), and product inhibition by GlcNAc-P-P-dolichol itself. These influences were revealed when the reactions were carried out in the presence of showdomycin, a nucleoside antibiotic, present at concentrations that block the de novo formation of GlcNAc-GlcNAc-P-P-dolichol but not that of GlcNAc-P-P-dolichol. The apparent K(i) values for GlcNAc-P-P-dolichol and GlcNAc-GlcNAc-P-P-dolichol under basal conditions were 4.4 and 2.8 microM, respectively. Inhibition was also observed under conditions where mannosyl-P-dolichol (Man-P-dol) stimulated the biosynthesis of GlcNAc-P-P-dolichol; the apparent K(i) values for GlcNAc-P-P-dolichol and GlcNAc-GlcNAc-P-P-dolichol were 2.2 and 11 microM, respectively. Kinetic analysis of the types of inhibition indicated competitive inhibition by GlcNAc-P-P-dolichol toward the substrate UDP-GlcNAc and non-competitive inhibition toward dolichol phosphate. Inhibition by GlcNAc-GlcNAc-P-P-dolichol was uncompetitive toward UDP-GlcNAc and competitive toward dolichol phosphate. A model is presented for the kinetic mechanism of the synthesis of GlcNAc-P-P-dolichol. GlcNAc-P-P-dolichol also exerts a stimulatory effect on the biosynthesis of Man-P-dol, i.e. a reciprocal relationship to that previously observed between these two intermediates of the dolichol pathway. This network of inhibitory and stimulatory influences may be aspects of metabolic control of the pathway and thus of glycoprotein biosynthesis in general.     

Studies on the activation by dolichol-P-mannose of the biosynthesis of GlcNAc-P-P-dolichol and the topography of the GlcNAc-transferases concerned with the synthesis of GlcNAc-P-P-dolichol and (GlcNAc)2-P-P-dolichol: a review.

A review is presented of research carried out in this laboratory on two aspects of the dolichol pathway that may have regulatory influences on these events. (i) The validity of the phenomenon of the activation of the biosynthesis of GlcNAc-P-P-dolichol and (GlcNAc)2-P-P-dolichol by dolichol-P-mannose is supported by experiments carried out on the Thy-1-negative mouse lymphoma cell. While this cell cannot synthesize the activating compound, this capacity was retained and revealed upon the addition of exogenous dolichol-P-mannose. (ii) The topographical orientation of the GlcNAc-transferases that catalyze the biosynthesis of GlcNAc-P-P-dolichol and (GlcNAc)2-P-P-dolichol was investigated in microsomes from the liver of the embryonic chick using dolichol phosphate liposomes as an exogenous substrate. The formation of GlcNAc-P-P-dolichol and (GlcNAc)2-P-P-dolichol was inhibited by trypsinization under conditions where the native orientation of the microsome was maintained, as indicated by the latency of mannose-6-phosphatase. Both GlcNAc-lipids were detected on free liposomes after incubation with intact microsomes, and in the same proportions as found on the microsome. From these and other studies, evidence was obtained indicating the cytoplasmic orientation of the GlcNAc-transferases that catalyze the synthesis of the first two intermediates of the dolichol pathway.     

Site of stimulation by mannosyl-P-dolichol of GlcNAc-lipid formation by microsomes of embryonic chick retina

Mannosyl-P-dolichol (man-P-dol) has been shown to stimulate the early reactions of the dolichol pathway, specifically, the biosynthesis of GlcNAc-P-P-dol and GlcNAc-GlcNAc-P-P-dol, and thus may play a regulatory role in glycoprotein biosynthesis. The site of action of man-P-dol has previously been suggested to be the GlcNAc-transferase concerned with the formation of the monoglucosaminyl derivative. Since the concentration of the chitobiosyl compound also increases as a result of the presence of man-P-dol, the immediate site of the activation was reexamined. The effect of man-P-dol on the formation of GlcNAc-GlcNAc-P-P-dol using GlcNAc-P-P-dol synthesizedin situ or added exogenously as the substrate was investigated. In addition, the distribution of radioactivity in the glucosaminyl constituents of the products under the stimulatory conditions was determined. The results of these studies supported the conclusion that the stimulation of GlcNAc-lipid synthesis by man-P-dol is due to the enhanced synthesis of GlcNAc-P-P-dol. It is not a result of the activation of the GlcNAc-transferase catalyzing the attachment of the second GlcNAc residue for the biosynthesis of the chitobiosyl derivative.Abbreviations GlcNAc-P-P-dol N-acetylglucosaminylpyrophosphoryldolichol - GlcNAc-GlcNAc-P-P-dol N-acetylglucosaminyl-N-acetylglucosaminylpyrophosphoryldolichol; - chito N-N-diacetylchitobiose - man-P-dol mannosylphosphoryldolichol - TX-100 triton X-100 - Tes 2-{[tris-(hydroxymethyl)-methyl]-amino}-ethanesulfonic acid     

Inhibition of lipid-linked saccharide synthesis by bacitracin.

The antibiotic bacitracin was found to inhibit the incorporation of mannose and GlcNAc from their respective sugar nucleotides into lipid-linked saccharides. The inhibition of both systems was apparent in the aorta particulate enzyme system but it was much more pronounced with the solubilized enzyme system. In both cases, GlcNAc incorporation into Dol-P-P-GlcNAc was more sensitive than mannose incorporation into Dol-P-Man, with 50% inhibition being seen at about 0.1–0.2 mm antibiotic. Bacitracin inhibition of mannose incorporation appeared to be overcome at high concentrations of dolichyl phosphate but, in these cases, an unexplained stimulation was observed. However, GlcNAc inhibition could not be overcome by high concentrations of dolichol phosphate, metal ion, or both together. Thus, the mechanism of inhibition by bacitracin is not clear. Bacitracin also inhibited the transfer of mannose from GDP-mannose to lipid-linked oligosaccharides and to glycoprotein in the particulate enzyme, as well as the transfer of radioactivity from Dol-P-Man or from lipid-linked oligosaccharides to glycoprotein. Thus, bacitracin apparently blocks each of the steps in the lipid-linked pathway. In yeast spheroplasts, bacitracin inhibited the incorporation of [¹⁴C]mannose into Dol-P-Man, into lipid-linked oligosaccharides, and into glycoprotein. However, in this case, the antibiotic also blocked the incorporation of leucine into protein. Bacitracin also inhibited the cell-free synthesis of mannosyl-phosphoryl-decaprenol in Mycobacterium smegmatis with 50% inhibition being observed at a concentration of about 0.5 mm.     

Characterization and partial purification of two enzymes transferring N-acetylglucosamine to dolichyl monophosphate and ribonuclease A

Two N-acetylglucosamine (GlcNAc) transferases which catalyze the incorporation of GlcNAc into GlcNAc-P-P-dolichol (dolichol enzyme) and into bovine pancreatic ribonuclease A (RNAseA enzyme) were solubilized from the rat liver microsomes in a non-ionic detergent, Triton X-100. Both enzyme activities were adsorbed on activated CH-Sepharose 4B, and could be eluted with a linear KCl gradient. Two enzyme activities were separated by this column with the dolichol enzyme eluting before the RNAseA enzyme. A 49-fold and 136-fold purification was achieved for the dolichol and the RNAseA enzyme, respectively. The addition of exogeneous dolichyl phosphate resulted in a 3-5-fold stimulation of the purified dolichol enzyme, but did not affect the purified RNAseA enzyme. The addition of RNAseA stimulated only the RNAseA enzyme. Whereas, tunicamycin could inhibit only the dolichol enzyme. The purified dolichol enzyme had a Km of 14 X 10(-6) M for UDP-GlcNAc and the reaction was saturated with about 0.25 M dolichyl phosphate. The purified RNAseA enzyme had a Km of 4.55 X 10(-6) M for UDP-GlcNAc and was saturated with about 0.36 mM RNAseA. The pH optima and the metal ion requirement for the two enzymes were different. These results suggest that because of the different properties of these two enzymes they may have distinct functions regarding the core glycosylation of N-linked glycoproteins. It is well established that the dolichol enzyme catalyzes the formation of the first dolichol-linked intermediate GlcNAc-P-P-dolichol, whereas according to the present finding, the RNAseA enzyme may catalyze the transfer of GlcNAc directly from UDP-GlcNAc into acceptor protein.     

Studies on bacterial cell wall inhibitors: II. Inhibition of peptidoglycan synthesis in vivo and in vitro by amphomycin

Amphomycin has been reported by the present authors to be a selective inhibitor of cell wall peptidoglycan synthesis in Bacillus cereus T (ōmura, S., Tanaka, H., Shinohara, M., ōiwa, R. and Hata, T. (1975) Chemotherapy 5, 365–369). Investigations were carried out to clarify the target of amphomycin.Amphomycin (10 μg/ml) lysed growing cells of B. cereus T, and inhibited peptidoglycan synthesis, accompanied by accumulation of uridine diphosphate-N-acetylmuramyl (UDP-MurNAc) peptides. The nucleotide precursors that accumulated in cells of Staphylococcus aureus FDA 209P in the presence of amphomycin were identified as UDP-MurNAc-L-Ala-D-Glu-L-Lys-D-Ala-D-Ala, UDP-MurNAc-L-Ala and UDP-MurNAc. In the experiments using a particulate enzyme system of Bacillus megaterium KM, amphomycin inhibited the polymerization of UDP-MurNAc-L-Ala-D-Glu-meso-diaminopimelic acid-D-Ala-D-Ala (UDP-MurNAc-pentapeptide) and UDP-N-acetylglucosamine, and also inhibited the formation of lipid intermediates, but did not inhibit the cross-linking, the last step of peptidoglycan synthesis. Unlike bacitracin, amphomycin did not lyse protoplasts of B. megaterium KM.We conclude that the site of action of amphomycin is the formation of MurNAc-(pentapeptide)-P-P-lipid from MurNAc-pentapeptide and undecaprenol (lipid) phosphate.     

Inhibition of lipid-linked mannose and mannoprotein synthesis in yeast by diumycin in vitro

Diumycin, a phosphoglycolipid antibiotic, inhibits different mannosyl transfer reactions in yeast. Using membrane preparations, the drug effectively inhibited the formation of dolichyl phosphate mannose (DolP-Man); 50% inhibition was observed at approximately 10 microgram/ml. To a lesser extent also mannosyl transfer from DolP-Man to protein decreased in presence of diumycin. Both mannosyl transfer to protein-serine/threonine acceptor sites as well as into positions within the asparagine-linked polymannose part of the yeast mannoprotein are inhibited to about 60% under conditions where DolP-Man formation is blocked. DolP-Man synthesis as well as mannosyl transfer from DolP-Man to protein are also inhibited by diumycin using solubilized enzymes and exogenous acceptor substrates. Glycosyltransfer reactions from GDP-mannose either to protein-serine/threonine-linked mannose (formation of short manno-oligosaccharides) or to dolichyl-diphosphate-linked chitobiose (formation of lipid-linked trisaccharide) are not inhibited by diumycin under conditions where DolP-Man synthesis is blocked by the antibiotic. The inhibitory action of diumycin on DolP-Man formation does not seem to be competitive with respect to dolichyl phosphate, since it cannot be overcome by higher concentrations of dolichyl phosphate.     

Properties of Solubilized UDP-GlcNAc: Dolichyl Phosphate-GlcNAc-1-P-Transferase from Soybean Cultured Cells

The GlcNAc-1-P-transferase was solubilized from microsomal preparations of soybean cultured cells by treatment with 1% Triton X-100. The solubilized enzyme catalyzed the formation of dolichyl pyrophosphoryl-GlcNAc when incubated with UDP-GlcNAc and dolichyl phosphate. The GlcNAc-1-P-transferase activity was stimulated by the addition of phosphatidylglycerol and phosphatidylinositol, but was inhibited by phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. The K_m value for dolichyl-phosphate was 6.2 micromolar and that determined for UDP-GlcNAc was 0.42 micromolar. The pH optimum for the GlcNAc-1-P reaction was between 7.2 and 7.6; maximum activity occurred at about 10 millimolar Mg²⁺. The addition of unlabeled GDP-mannose or UDP-glucose considerably inhibited enzyme activity which could be restored to nearly the original value by addition of more dolichyl phosphate to the incubation mixture. On the other hand, the addition of unlabeled ADP-glucose and GDP-glucose enhanced the enzyme activity. This stimulation by these sugar nucleotides was found to be due to the protection of the substrate UDP-[³H]-GlcNAc from pyrophosphatase degradation. The GlcNAc-1-P-transferase reaction was very sensitive to tunicamycin and 50% inhibition required less than 1 microgram of antibiotic per milliliter. Amphomycin, showdomycin, and diumycin also inhibited this reaction but at higher concentrations.     

Mechanism of Action of Showdomycin

¹⁴C-Labelled showdomycin was rapidly taken up by Escherichia coli K-12 cells. The showdomycin uptake was highly temperature dependent, sensitive to azide and N-ethyl-maleimide, but was only partially inhibited by treatment with high concentration of iodoacetic acid.

The uptake of showdomycin was inhibited by a wide variety of nucleosides but not by purine and pyrimidine bases, nucleotides, ribose or ribose-5-phosphate. The inhibition of showdomycin uptake by adenosine was of a competitive type.

Since nucleosides inhibited the uptake of showdomycin but did not facilitate its efflux, they must play a role of inhibitors to the entry of the antibiotic into cells.

Removal of extracellular showdomycin by washing, or inhibition of its subsequent entry into cells by the addition of nucleosides or sulfhydryl compounds resulted in a rapid decrease in the intracellular level of the antibiotic during subsequent incubation.     

Mechanism of Action of Showdomycin

The effect of showdomycin on the syntheses of deoxyribonucleotides from various pyrimidine and purine derivatives was studied in cell-free systems from E. coli.

The formations of deoxycytidine phosphates, deoxyuridine phosphates, deoxyguanosine phosphates and deoxyadenosine phosphates from the corresponding ribonucleoside diphosphates were all inhibited by low concentrations of showdomycin. The formation of deoxythymidine phosphates from dUMP was also very susceptible to the antibiotic. These inhibitory actions of showdomycin could be reversed by a sulfhydryl compound (mercaptoethanol) but not by nucleosides, in contrast to a previous finding that the inhibitory action of this antibiotic on the cell growth was reversed by compounds belonging to both of these groups.

N-Ethylmaleimide (NEM), a thiol reagent which has a structure related to the aglycone moiety of showdomycin, was also found to be a potent inhibitor of both the reduction of CDP and the methylation of dUMP as showdomycin. A mercurial thiol reagent, p-chloromercuribenzoic acid (PCMB), however, was found to be inactive against the methylation of dUMP although the salvage synthesis of dUMP was inhibited by low concentrations of this reagent.

The formations of deoxythymidine phosphates and of deoxyuridine phosphates from their respective pyrimidine bases and a deoxyribosyl donor were quite resistant to showdomycin.     

Stimulation by dolichol phosphate-mannose and phospholipids of the biosynthesis of N-acetylglucosaminylpyrophosphoryl dolichol

Dolichol phosphate-mannose (dol-P-mannose) has been shown previously to stimulate the reaction: dolichol phosphate + UDP-[3H]GlcNAc----[3H]GlcNAc-P-P-polyprenols (Kean, E. L. (1982) J. Biol. Chem. 257, 7952-7954). Further studies on this phenomenon are described, using microsomes from the retina of the embryonic chick as the major source of enzyme. Neither dolichol-P-glucose nor retinyl-P-mannose showed this stimulatory activity. Phosphatidylglycerol also stimulated this same process and was most active among a variety of phospholipids which were tested, in accord with previous reports. The presence of GDP-2-deoxy-2-fluoro-D-mannose or GTP had no effect on the reaction. The apparent activation constant for dolichol-P-mannose was 2.2 microM, and for phosphatidylglycerol, 401 microM. The major product (90% or greater) obtained under basal and stimulatory conditions was GlcNAc-P-P-dolichol and the site of the stimulatory effect was the glucosaminyltransferase catalyzing the formation of this compound. The effects of stimulation on the kinetic properties were similar for both activators: increases in the Vmax of the reactions of 7-10-fold; increases in apparent Km for UDP-GlcNAc of 5-7-fold; a 3-fold decrease in apparent Km for dolichol-phosphate. When present together, a mutual inhibition of stimulation was observed compared to the additive effect from dol-P-mannose or phosphatidylglycerol alone. Although a substrate for the reaction, dolichol phosphate repressed the stimulation by dolichol-P-mannose but not that by phosphatidylglycerol. Dol-P-glucose, while not an activator of the reaction, acted as a negative modifier of the stimulation by dol-P-mannose by acting as a competitive inhibitor of the stimulation. The stimulatory phenomenon was observed in microsomes prepared from a variety of tissues from the embryonic chick and from postnatal tissue after partial delipidation. The addition of pyrophosphatase inhibitors did not bring about stimulation of GlcNAc-lipid synthesis, but did enhance the effect. These studies extend the previous observations of the participation of dolichol-P-mannose and phosphatidylglycerol as allosteric activators of GlcNAc-lipid synthesis and indicate additional aspects of metabolic regulation of the dolichol pathway.     

Inhibitors of glycoprotein biosynthesis block fertilization in the hamster

The nature and control of changes in surface carbohydrates in capacitating hamster spermatozoa were analysed by using five inhibitors of glycoprotein biosynthesis in an in vitro fertilization system. Epididymal spermatozoa were treated with amphomycin, bacitracin, tunicamycin, 2-deoxyglucose, and 2-deoxy-2-fluoro-D-glucose either during the entire period of capacitation or briefly at the end of capacitation before exposing to Con A-coated agarose beads or hamster eggs with or without their zonae pellucidae. Untreated 4½-5-hr spermatozoa exhibited nearly 100% fertilization and became bound to Con A-agarose beads mainly along the length of their flagellae, resulting in the formation of clumps on the beads. In the presence of inhibitors of glycosylation, spermatozoa did not bind to Con A-agarose beads or zona-intact oocytes and they did not fuse with the zona-free oocytes. Sperm-zona binding was also inhibited by UDP-galactose and UDP-N-acetylglucosamine, but not by UDP-glucose. Sperm motility was not damaged by these inhibitors, and zona-intact and zona-free oocytes pretreated with these inhibitors underwent normal fertilization with untreated spermatozoa. These results further strengthen the view that glycoproteins on the sperm surface may be required during different stages of fertilization, including sperm-egg fusion.     

Biosynthesis of glycoproteins in Candida albicans: activity of mannosyl and glucosyl transferases

The enzymes dolichol phosphate glucose synthase and dolichol phosphate mannose synthase (DPMS), which catalyze essential steps in glycoprotein biosynthesis, were solubilized and partially characterized in Candida albicans. Sequential incubation of a mixed membrane fraction with increasing concentrations of Nonidet P-40 released a soluble fraction that transferred glucose from UDP-Glc to dolichol phosphate glucose and minor amounts of glucoproteins in the absence of exogenous dolichol phosphate. Studies with the soluble fraction revealed that some properties were different from those previously determined for the membrane-bound enzyme. Accordingly, the soluble enzyme exhibited a twofold higher affinity for UDP-Glc and a sixfold higher affinity over the competitive inhibitor UMP, and the transfer reaction was fourfold more sensitive to inhibition by amphomycin. On the other hand, a previously described protocol for the solubilization of mannosyl transferases that rendered a fraction exhibiting both DPMS and protein mannosyl transferase (PMT) activities operating in a functionally coupled reaction was modified by increasing the concentration of Nonidet P-40. This resulted in a solubilized preparation enriched with DPMS and nearly free of PMT activity which remained membrane bound. DPMS solubilized in this manner exhibited an absolute dependence on exogenous Dol-P. Uncoupling of these enzyme activities was a fundamental prerequisite for future individual analysis of these transferases.     

Biosynthesis of N-acetylglucosamine-P-P-dolichol, the committed step of asparagine-linked oligosaccharide assembly.

Asparagine-linked glycosylation is initiated by the synthesis of N-acetylglucosaminylpyrophosphoryl dolichol (GlcNAc-P-P-dolichol), which is extended by a series of glycosyltransferases to yield Glc3Man9GlcNAc2-P-P-dolichol (where Glc is glucose and Man is mannose). The oligosaccharide unit is then transferred en bloc to asparagine residues of nascent polypeptides in the lumen of the rough endoplasmic reticulum. The question here is whether GlcNAc-P-P-dolichol biosynthesis is a fixed process unaffected by cellular events, or a regulated reaction responsive to cellular requirements for glycoprotein biosynthesis. Several lines of evidence indicate that the latter is the case and that GlcNAc-P-P-dolichol biosynthesis may be subject to multiple forms of regulation. Recent information about the N-acetylglucosamine-1-P transferase (GPT) responsible for this reaction and the cloning of cDNA candidates for this enzyme have provided further insight into these mechanisms. This review will examine current hypotheses dealing with GPT and its role in the committed step of asparagine-linked glycosylation.     

Conformational change on calcium binding by the lipopeptide antibiotic amphomycin. A C.D. and monolayer study

The acidic linear lipopeptide amphomycin is a calcium dependent antibiotic which is thought to bind to carrier lipids such as dolichol monophosphate. The actual role of Ca++ is not definitely established and in this article we have examined the peptides interactions with a range of divalent cations. By CD we have shown that a conformational change is induced by Ca++, Sr++ and Ba++ but not by Mg++, Zn++, Cd++ or Gd+++. Monolayer studies show a decrease in molecular area and an increase in film stability when the subphase contains Ca++. The ensemble of results provides preliminary evidence for the formation of a beta hairpin structure on ion binding (Ka (Ca++) = 2.4 x 10(3)M-1) which could enhance amphomycin's bilayer solubility.     

Showdomycin,a nucleotide-site-directed inhibitor of (Na+ + K+)-ATPase

Showdomycin [2-(β-d-ribofuranosyl)maleimide] is a nucleoside antibiotic containing a maleimide ring and which is structurally related to uridine. Showdomycin inhibited rat brain (Na⁺ + K⁺)-ATPase irreversibly by an apparently bimolecular reaction with a rate constant of about 11.01·mol^?1·min^?1. Micromolar concentrations of ATP protected against this inhibition but uridine triphosphate or uridine were much less effective. In the presence of K⁺, 100 μM ATP was unable to protect against inhibition by showdomycin. These observations show that showdomycin inhibits (Na⁺ + K⁺)-ATPase by reacting with a specific chemical group or groups at the nucleotide-binding site on this enzyme. Inhibition by showdomycin appears to be more selective for this site than that due to tetrathionate or N-ethylmaleimide. Since tetrathionate is a specific reactant for sulfhydryl groups it appears likely that the reactive groups are sulfhydryl groups. The data thus show that showdomycin is a relatively selective nucleotide-site-directed inhibitor of (Na⁺ + K⁺)-ATPase and inhibition is likely due to the reaction of showdomycin with sulfhydryl group(s) at the nucleotide-binding site on this enzyme.     

Study on the biosynthesis of dolichol in yeast: recognition of the prenyl chain length in polyprenol reduction

We synthesized three water-soluble biotin-tagged compounds with different prenyl chain lengths, biotinylated farnesal (BF), biotinylated C(55)-polyprenal (BP55), and biotinylated C(80)-polyprenal (BP80), and examined their effects on in vitro dolichol synthesis from farnesyl diphosphate. BF and BP55 did not affect the dolichol synthesis, whereas BP80 inhibited the reduction pathway from polyprenol to dolichol, accompanied by a decrease in the entire polyprenol and dolichol synthesis. Comparison of BP80 with eighteen detergents, including Triton X-100, CHAPS, octylglucoside, deoxycholate, and Tween 80, revealed the specific effect of BP80 on the reduction pathway. On SDS-polyacrylamide gel electrophoresis, BP80 was detected in an associated form with a 50 kDa protein. These results suggest that the reduction of polyprenol to dolichol in the dolichol biosynthetic pathway proceeds with the recognition of the polyprenol chain length by a 50 kDa protein.     

Regulation of dolichol-linked glycosylation

In the majority of congenital disorders of glycosylation, the assembly of the glycan precursor GlcNAc₂Man₉Glc₃ on the polyprenol carrier dolichyl-pyrophosphate is compromised. Because N-linked glycosylation is essential to life, most types of congenital disorders of glycosylation represent partial losses of enzymatic activity. Consequently, increased availability of substrates along the glycosylation pathway can be beneficial to increase product formation by the compromised enzymes. Recently, we showed that increased dolichol availability and improved N-linked glycosylation can be achieved by inhibition of squalene biosynthesis. This review summarizes the current knowledge on the biosynthesis of dolichol-linked glycans with respect to deficiencies in N-linked glycosylation. Additionally, perspectives on therapeutic treatments targeting dolichol and dolichol-linked glycan biosynthesis are examined.     

Bacitracin: An inhibitor of the insulin degrading activity of glutathione-insulin transhydrogenase

The antibiotic bacitracin, a known inhibitor of insulin degradation by both isolated cells and subcellular organelles, inhibited the ability of purified glutathione-insulin transhydrogenase to split insulin into its constituent A and B chains. This inhibition was demonstrated by measuring the formation of insulin degradative products that were both soluble in 5% trichloroacetic acid and chromatographed as the separate chains of insulin on Sephadex G-50. At concentrations of 90 and 300 μM, bacitracin inhibited 50 and 90%, respectively, of the degrading activity of the purified enzyme. Similarly, degradation by crude liver lysates was inhibited 50 and 90% by 70 and 250 μM bacitracin, respectively. Kinetic studies indicated that this inhibition was by a complex mechanism that decreased both the Vmax and affinity of the enzyme for insulin. These data raise the possibility that the inhibition of glutathione-insulin transhydrogenase by bacitracin could account for part or all of the effects of this antibiotic on inhibition of insulin degradation by target cells.