Stimulation as well as inhibition by antibiotics of the formation of GlcNAc-lipids of the dolichol pathway

The antiobiotics, diumycin, amphomycin, bacitracin, and showdomycin have been shown previously to block the synthesis of GlcNAc-P-P-dolichol and GlcNAc-GlcNAc-P-P-dolichol. In view of inconsistencies in the literature concerning the sites of inhibition, we have reinvestigated the influence of these drugs on the formation of these early intermediates of the dolichol pathway. Unexpectedly, when the individual products of the reactions were examined, instead of inhibition, showdomycin and bacitracin were found to stimulate the formation of GlcNAc-P-P-dolichol, and diumycin stimulated at low concentrations. Three derivatives of showdomycin were examined with similar results, showing stimulations of GlcNAc-P-P-dolichol formation of up to two-fold over controls. Amphomycin specifically inhibited GlcNAc-P-P-dolichol formation, an effect that was reversed by a high concentration of dolichyl phosphate. In contrast, with the exception of amphomycin, each compound directly inhibited the formation of GlcNAc-GlcNAc-P-P-dolichol. Using chemically synthesized GlcNAc-P-P-dolichol as substrate, the kinetics of inhibition of GlcNAc-GlcNAc-P-P-dolichol formation by showdomycin, bacitracin and diumycin was examined. The apparent Ki values calculated from these studies indicated that showdomycin was the most active inhibitor. These findings provide a new understanding of the action of these compounds on the GlcNAc-transferases of the dolichol pathway. © 1998 Rapid Science Ltd     

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     

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.     

Glucuronosyl-N-acetylglucosaminyl pyrophosphoryldolichol. Formation in SV40-transformed human lung fibroblasts and biosynthesis in rat lung microsomal preparations.

Incubation of SV40-transformed human lung fibroblasts with [3H]glucosamine for 1 h. followed by chloroform:methanol extraction and thin layer chromatographic analysis, revealed the presence of a major radioactive lipid that was isolated and characterized as GIcUA-(1 leads to 4)-GlcNAc-P-P-dolichol. An identical lipid was formed in smaller quantities under similar incubation conditions in several fibroblastic lines, HeLa cells, and in mouse L cells. Rat lung microsomal preparations catalyze the synthesis of the disaccharide lipid in the following sequence of reactions: UDP-[3H]GlcNAc + dolichol-P leads to [3H]GlcNAc-P-P-dolichol (1) [3H]GlcNAc-P-P-dolichol + UDP-[14C]GlcUA leads to [14C]GlcUA-[3H]GlcNAc-P-P-dolichol (2) The double-labeled lipid was identical to the lipid isolated from SV40-transformed fibroblasts with regard to its behavior on thin layer and silicic acid chromatography. Further, the double-labeled disaccharide released from the lipid by mild acid hydrolysis was identical to GlcUA-(1 leads to 4)-GlcNAc in its chromatographic and electrophoretic behavior and in its composition. The occurrence of a polyprenol derivative of GlcUA-(1 leads to 4)-GlcNAc suggests a possible role for this lipid in the biosynthesis of the repeating disaccharide units of proteoglycans, such as heparin.     

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.     

Polyisoprenyl phosphate specificity of UDP-GIcNAc:undecaprenyl phosphate N-acetylglucosaminyl 1-P transferase from E.coli

N-Acetyl-D-glucosaminylpyrophosphorylundecaprenol (GlcNAc-P-P-Und),an intermediate in the biosynthesis of the enterobacterial commonantigen in E.coli and some O-antigen chains in gram-negativebacteria, is formed by the transfer of GlcNAc 1-P from UDP-GlcNActo Und-P, analogous to the reaction forming GlcNAc-P-P-dolichol(GlcNAc-P-P-Dol) in mammalian cells. Since the micro-somal enzymefrom animal cells exhibits a strong preference for Dol-P, whichcontains a saturated     

Quantitative Modeling of the High-Throughput Production and In Vivo Kinetics of (Drug-Encapsulating) Liposomes

In developing liposomes for in vivo use, it is important to design the liposomes to have optimal in vivo kinetics, and it is also necessary to identify optimal high-throughput production conditions for these liposomes. Previous work has not definitively established the general relationship between liposomes'' configuration and composition, and their in vivo kinetics. Also, no straightforward method exists to calculate optimal liposome high-throughput production conditions for specific liposome compositions. This work presents first-principles quantitative correlations describing liposomes'' in vivo drug leakage and vascular mass transfer kinetics. This work further presents a simple quantitative model relating specific liposome compositions to ideal high-throughput production parameters. The results have implications for the identification of promising liposome compositions via high-throughput screening methodologies, as well as the design and optimization of high-throughput reactors for liposome production.     

Spectral and chemical evidence for the formation of zinc-porphyrin in aged, initially metal-free, porphyrin-IX solutions

The appearance of new additional bands, at 580 and 630 nm, in the fluorescence spectra of porphyrins, upon the aging of their aqueous solutions, was investigated for four porphyrin systems: The deutero, the hemato, the hemato-derivative, and the proto. The conditions for quantitative conversion to the new spectra were sought. To that end the concentration levels investigated were adjusted to have each porphyrin system in a monomer-dominant state. The species fluorescing at 580 nm was denoted f580 and its rate of appearance was found to fit first-order kinetics, for each porphyrin, the range of the rate constants being 0.03-0.10 hr-1. Two key issues were probed: (a) The identity of f580, the effort directed towards zincporphyrin. (b) Its origins--a contaminant unmasked, or a species newly formed, during the aging. Testing f580 for zincporphyrin properties, spectral at neutral pH, acid/base de/remetalation, EDTA competition, and chromatography gave in each case positive results, allowing the identification of f580 as zincporphyrin. On the basis of the data obtained and the known sources for the contamination of aqueous solutions by zinc, the case is made for zinc porphyrin formation upon aging. The relevance of the phenomena to the therapeutic activities of porphyrins is discussed.     

Kinetic analysis of rhodamines efflux mediated by the multidrug resistance protein (MRP1)

Characterization of rhodamine 123 as functional assay for MDR has been primarily focused on P-glycoprotein-mediated MDR. Several studies have suggested that Rh123 is also a substrate for MRP1. However, no quantitative studies of the MRP1-mediated efflux of rhodamines have, up to now, been performed. Measurement of the kinetic characteristics of substrate transport is a powerful approach to enhancing our understanding of their function and mechanism. In the present study, we have used a continuous fluorescence assay with four rhodamine dyes (rhodamine 6G, tetramethylrosamine, tetramethylrhodamine ethyl ester, and tetramethylrhodamine methyl ester) to quantify drug transport by MRP1 in living GLC4/ADR cells. The formation of a substrate concentration gradient was observed. MRP1-mediated transport of rhodamine was glutathione-dependent. The kinetics parameter, k(a) = V(M)/k(m), was very similar for the four rhodamine analogs but approximately 10-fold less than the values of the same parameter determined previously for the MRP1-mediated efflux of anthracycline. The findings presented here are the first to show quantitative information about the kinetics parameters for MRP1-mediated efflux of rhodamine dyes.     

Ultrafast fluorescence study on the location and mechanism of non-photochemical quenching in diatoms

The diatom algae, responsible for at least a quarter of the global photosynthetic carbon assimilation in the oceans, are capable of switching on rapid and efficient photoprotection, which helps them cope with the large fluctuations of light intensity in the moving waters. The enhanced dissipation of excess excitation energy becomes visible as non-photochemical quenching (NPQ) of chlorophyll a fluorescence. Intact cells of the diatoms Cyclotella meneghiniana and Phaeodactylum tricornutum, which show different NPQ induction kinetics under high light illumination, were investigated by picosecond time-resolved fluorescence under dark and NPQ-inducing high light conditions. The fluorescence kinetics revealed that there are two independent sites responsible for NPQ. The first quenching site is located in an FCP antenna system that is functionally detached from both photosystems, while the second quenching site is located in the PSII-attached antenna. Notwithstanding their different npq induction and reversal kinetics, both diatoms showed identical NPQ via both mechanisms in the steady-state. Their fluorescence decays in the dark-adapted states were different, however. A detailed quenching model is proposed for NPQ in diatoms.     

Uptake of ant-derived nitrogen in the myrmecophytic orchid Caularthron bilamellatum

Background and Aims Mutualistic ant-plant associations are common in a variety of plant families. Some myrmecophytic plants, such as the epiphytic orchid Caularthron bilamellatum, actively form hollow structures that provide nesting space for ants (myrmecodomatia), despite a substantial loss of water-storage tissue. This study aimed at assessing the ability of the orchid to take up nitrogen from ant-inhabited domatia as possible trade-off for the sacrifice of potential water storage capacity. Methods Nitrogen uptake capabilities and uptake kinetics of (15)N-labelled compounds (NH(4)(+), urea and l -glutamine) were studied in field-grown Caularthron bilamellatum plants in a tropical moist forest in Panama. Plants were either labelled directly, by injecting substrates into the hollow pseudobulbs or indirectly, by labelling of the associated ants in situ. Key Results Caularthron bilamellatum plants were able to take up all tested inorganic and organic nitrogen forms through the inner surface of the pseudobulbs. Uptake of NH(4)(+) and glutamine followed Michaelis-Menten kinetics, but urea uptake was not saturable up to 2 mm. (15)N-labelled compounds were rapidly translocated and incorporated into vegetative and reproductive structures. By labelling ants with (15)N in situ, we were able to prove that ants transfer N to the plants under field conditions. Conclusions Based on (15)N labelling experiments we were able to demonstrate, for the first time, that a myrmecophytic orchid is capable of actively acquiring different forms of nitrogen from its domatia and that nutrient flux from ants to plants does indeed occur under natural conditions. This suggests that beyond anti-herbivore protection host plants benefit from ants by taking up nitrogen derived from ant debris.     

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.     

Kinetics of the reduction of tetrathionate by intact bacterial cells

The kinetics of the reduction of tetrathionate by washed nongrowing intact cells ofCitrobacter were studied. The rate of the reduction in most cases is not constant. The course of reduction usually resembles the first order kinetics within a limited, varying initial time period. This finding makes it possible to evaluate quantitatively the curves obtained experimentally of the relationship between concentration of the product and time, applyingequations valid for this reaction order, to determine the rate constant of the first order reaction and thus to determine the initial rate of the reduction of tetrathionate as the measure of the tetrathionate-reductaso activity of intact cells. Deviations from the first order kinetics, observed during later phases of the reduction are caused by excessive acidification of the medium. The effect of various experimental conditions on the initial rate of reduction of tetrathionate by the cells was also studied. The results presented served as a basis of a general method for the quantitative evaluation of the tetrathionate-reductase activity of intact bacterial cells.     

Product Inhibition in Native-State Proteolysis

The proteolysis kinetics of intact proteins by nonspecific proteases provides valuable information on transient partial unfolding of proteins under native conditions. Native-state proteolysis is an approach to utilize the proteolysis kinetics to assess the energetics of partial unfolding in a quantitative manner. In native-state proteolysis, folded proteins are incubated with nonspecific proteases, and the rate of proteolysis is determined from the disappearance of the intact protein. We report here that proteolysis of intact proteins by nonspecific proteases, thermolysin and subtilisin deviates from first-order kinetics. First-order kinetics has been assumed for the analysis of native-state proteolysis. By analyzing the kinetics of proteolysis with varying concentrations of substrate proteins and also with cleavage products, we found that the deviation from first-order kinetics results from product inhibition. A kinetic model including competitive product inhibition agrees well with the proteolysis time course and allows us to determine the uninhibited rate constant for proteolysis as well as the apparent inhibition constant. Our finding suggests that the likelihood of product inhibition must be considered for quantitative assessment of proteolysis kinetics.     

Different molecular arrangements of the tetrameric annexin 2 modulate the size and dynamics of membrane aggregation

Annexin 2, a member of the annexin family of Ca²⁺-dependent membrane binding proteins is found in monomeric and heterotetrameric forms and has been involved in different membrane related functions. The heterotetrameric annexin 2 is composed of a dimer of S100A10, a member of the S100 family of Ca²⁺ binding proteins and two annexin 2 molecules ((Anx2-S100A10)₂). Different molecular models including tetramers and octamers in which S100A10 is localized in the centre of the complex with the annexin 2 molecules positioned around S100A10 had been proposed. Herein, the organization of the (Anx2-S100A10)₂ complex in conditions in which membranes are able to bridge was studied. We performed Cryo-electron microscopy observations of the tetrameric annexin 2 on the membrane surface, and study the S100A10 accessibility to antibodies by flow “cytometry”. We also studied the kinetics and size evolution of vesicle aggregates by dynamic light scattering. The results show that the protein is able to organize in three different arrangements depending on the presence of Ca²⁺ and pH and that the aggregation is faster in the presence of Ca²⁺ compared with the aggregation in its absence. In one arrangement the S100A10 molecule is exposed to the solvent allowing its interaction with other proteins. The presented results will serve as a molecular basis to explain some of the functions of the tetrameric annexin 2.     

Probing nature's knots: the folding pathway of a knotted homodimeric protein

The homodimeric protein YibK from Haemophilus influenzae belongs to a recently discovered superfamily of knotted proteins that has brought about a new protein-folding conundrum. Members of the alpha/beta-knot clan form deep trefoil knots in their native backbone structure, a topological feature that is currently unexplained in the protein-folding field. To help solve the puzzle of how a polypeptide chain can efficiently knot itself, the folding kinetics of YibK have been studied extensively and the results are reported here. Folding was monitored using probes for changes in both secondary and tertiary structure, and the monomer-dimer equilibrium was perturbed with a variety of solution conditions to allow characterisation of otherwise inaccessible states. Multiphasic kinetics were observed in the unfolding and refolding reactions of YibK, and under conditions where the dimer is favoured, dissociation and association were rate-limiting, respectively. A folding model consistent with all kinetic data is proposed: YibK appears to fold via two parallel pathways, partitioned by proline isomerisation events, to two distinct monomeric intermediates. These form a common third intermediate that is able to fold to native dimer. Kinetic simulations suggest that all intermediates are on-pathway. These results provide the valuable groundwork required to further understand how Nature codes for knot formation.     

Subunits of the chaperonin CCT interact with F-actin and influence cell shape and cytoskeletal assembly

The integrity of the cytoskeleton is closely linked to the oligomeric chaperonin containing TCP-1 (CCT) via the folding requirements of actin and tubulin, but the role of CCT in cytoskeletal organization remains unclear. We address this issue by analyzing the effects of targeting CCT subunits via siRNA and assessing their location/assembly state in cultured mammalian cells. Reducing levels of individual CCT subunits implicates CCT? in influencing cell shape and reduced levels of this subunit limit the cells' ability to recover from microfilament depolymerization. Conversely, cells displayed enhanced microtubule regrowth when CCT subunit levels were altered by siRNA. Some CCT subunits co-localize with F-actin, whilst all are predominantly monomeric in extracts enriched for the cytoskeleton. This provides compelling evidence that some CCT subunits as monomers can influence cytoskeletal organization/polymerization. Therefore the activity of CCT may well extend beyond the folding of newly synthesized polypeptides, representing a novel function for CCT subunits distinct from their role in the CCT oligomer.