Soluble Portion Research Articles

Preparative isolation of oligomeric procyanidins from Hawthorn leaves and flowers by HPLC

Hawthorn leaves and flowers (Crataegi folium cum flore) consist of the dried flower bearing branches of Crataegus monogyna Jacq. Emend. Lindm., C. laevigata (Poiret) and, more rarely, C. pentagyna Waldst. et Kit. Ex Willd., C. nigra Waldst. et Kit. and C. azarolus L. The oligomeric procyanidins (OPC) from Hawthorn leaves and flowers are considered to be in part responsible for the cardiotonic clinical activity of the herbal material. Effective methods for rapid isolation of these heterogenous oligomeric clusters with defined molecular weight as reference compounds are not available. Therefore the methanol soluble portion of the water fraction (MSF) of an acetone/water (7 + 3) extract of Hawthorn leaves and flowers was fractionated by a combination of MPLC on RP-18 material and preparative HPLC using a diol stationary phase. Effective isolation of procyanidins with a distinct degree of polymerization (DP) from dimers DP2 up to tridecamers DP13 was achieved. Exact mass measurements with negative ESI-TOF/MS were employed to confirm the respective structures of the isolated procyanidins. This method is highly suitable for fast and effective isolation of OPC reference material with defined degree of polymerization.

ChemInform Abstract: Structure of Prudomestin.

3,5,7-Trihydroxy-8-methoxy-2-(4-methoxyphenyl)-4H1-benzopyran-4-one, C17H1407, M r = 330.3, triclinic, P1, a = 6.579 (5), b = 8.441 (7), c = 13.893 (14) A, a = 101.92 (9), fl = 103.38 (6), y = 97.44 (6) °, V = 721 A. 3, Z = 2, Dx = 1.52 g cm -3, Mo Ka radiation, A = 0.71069 A, /z = 1.2 cm-1, F(000) = 344, T = 200 K, R = 0.076 for 1534 unique observed [1/o-(/)_>2.0] reflections. The definitive structure of prudomestin is established by this investigation. The molecule is essentially planar with the methyl groups projecting above and below the plane. The major intermolecular force within a layer of molecules in the solid state results from hydrogen bonding. Introduction. Prunus domestica Linn. is the tree of the well-known fruit plum and grows ideally in the western temperate Himalayas and is cultivated for its fruit in India. Its fruits are used as a mild laxative and demulcent and, in combination with other drugs, they are used in leucorrhoea, irregular menstruation and debility following miscarriage (Chopra, Nayar & Chopra, 1956). The prudomestin used in this study 0108-2701/92/061090-03506.00 was extracted from the wood of Prunus domestica Linn. The objective of this investigation was to provide an unambiguous identification of the structure of prudomestin. Experimental. The prudomestin was isolated from the hot-water soluble portion of the benzene solubles of an alcoholic extract from the heartwood of Prunus domestica Linn. collected from Baramulla, Kashmir, India. The compound was recrystallized from chloroform-petrol and separated as yellow needles. Data were collected with a Siemens P3R3 fourcircle diffractometer in co-28 mode. The crystal was held at 200 K with an Oxford Cryosystems Cryostream Cooler (version 2.4); this temperature was chosen because the crystal diffracted rather poorly under ambient conditions. Maximum 20 was 50 ° with scan range _+ 0.7 ° (28) around the K a l Kot2 angles, scan speed 3-29 ° mini , depending on the intensity of the 2 s prescan; backgrounds were measured at each end of the scan for 0.25 of the scan time. hkl ranges were 0/7, 9/10, 15/15 (unique reflections only). Three standard reflections were © 1992 International Union of Crystallography V I R I N D E R S. P A R M A R et al. 1091 Table 1. Atomic coordinates (x 104) and equivalent isotropic thermal parameters (/~2 x 103) Ucq is defined as one third of the trace of the orthogonalized U U tensor.

Photodegradation of some ethylene–norbornene random copolymers

We investigated the photodegradation of specific ethylene-norbornene random copolymers (ENRC) – a group of substances which has been attracting much attention in recent years. ENRCs having various contents of norbornene were studied. After irradiation, each sample was separated into chloroform-insoluble and soluble portions. The chloroform-insoluble portion was weighed and analyzed by FTIR. The chloroform-soluble portion was analyzed by size elimination chromatography (SEC), FTIR and 1H NMR. The yield of the chloroform-insoluble portion increased with increased irradiation time. Formyl, formate, acyl, hydroxy groups and a carbon–carbon double bond were formed by photo-irradiation. Apparently, hydrogen atoms bound to the tertiary and secondary carbon atom in the parent ENRCs are abstracted. From these results, it is suggested that auto-oxidation results from photo-irradiation of the ENRCs. In ENRCs with similar stereoregularity, the degree of photodegradation increases with increasing norbornene content.

ChemInform Abstract: Astertarone B, a Hydroxy-Triterpenoid Ketone from the Roots of Aster tataricus L.

The roots of Aster (A.) tataricus L. (Compositae) are used as the Chinese crude drug Asteris Radix (Japanese name: shion) and are combined in various traditional Chinese prescriptions for use as cough medicines, expectorants and diuretics 1) . Constituents of the root extracts have been investigated, and the presence of a triterpenoid ketone, shionone (shion-21-en-3-one), 2—4) seven triterpenoid glycosides, aster saponins A—G, 5—7) and three monoterpenoid glycosides, shionosides A—C, 7,8) have been reported. Our recent study on the methanol extract of A. tataricus roots has led to the isolation and structural elucidation of a new triterpenoid ketone, astertarone A (D: A-friedoeuph-21-en-3-one), along with epishionol (shion-21-en-3b -ol). 9) In this paper, we report the isolation and characterization of yet another new triterpenoid ketone, designated astertarone B (1), in addition to spinasterone [(22E)-5a-stigmasta-7,22-dien-3-one], from the extract. Column chromatography of the hexane soluble portion of the methanol extract of A. tataricus roots on silica gel afforded a triterpenoid ketone fraction. Crystallization of the ketone fraction from acetone‐methanol, followed by preparative HPLC of the filtrate portion, eventually yielded compound 1 and spinasterone. Identification of spinasterone was performed by spectral comparison with an authentic compound. Compound 1, with a molecular formula of C30H50O2 determined from its high-resolution mass spectrum (HR-MS) (M 1 , m/z 442.3836), gave IR absorptions at 3473 (hydroxyl group), 1712 (ketone), and 833 cm 21 (trisubstituted double bond). Compound 1 displayed two olefinic methyl singlets at d 1.61 and 1.69, with an olefinic methine signal at d 5.10 (br t, J57.0 Hz), which correspond to one isopropylidene group, and six tertiary methyl singlets at d 0.82, 0.89, 0.90,

Membrane Localization of β-Amyloid 1–42 in Lysosomes: A POSSIBLE MECHANISM FOR LYSOSOME LABILIZATION

beta-Amyloid peptide (Abeta42) is the core protein of amyloid plaque in Alzheimer disease. The intracellular accumulation of Abeta42 in the endosomal/lysosomal system has been under investigation for many years, but the direct link between Abeta42 accumulation and dysfunction of the endosomal/lysosomal system is still largely unknown. Here, we found that both in vitro and in vivo, a major portion of Abeta42 was tightly inserted into and a small portion peripherally associated with the lysosomal membrane, whereas its soluble portion was minimal. We also found that the Abeta42 molecules inserted into the membrane tended to form multiple oligomeric aggregates, whereas Abeta40 peptides formed only dimers. Neutralizing lysosomal pH in differentiated PC12 cells decreased the lysosomal membrane insertion of Abeta42 and moderated Abeta42-induced lysosomal labilization and cytotoxicity. Our findings, thus, suggest that the membrane-inserted portion of Abeta42 accumulated in lysosomes may destabilize the lysosomal membrane and induce neurotoxicity.

Activity of Extracts and Procesterol from Calotropis gigantea against Entamoeba histolytica

Extracts from the root bark of Calotropis gigantea were subjected to bioactivity-guided fractionation using growth inhibitory effects against Entamoeba histolytica. The n-hexane soluble portion of the chloroform extract showed in vitro antiamoebic activity against the HK-9 strain of Entamoeba histolytica. Chromatographic separation of the chloroform extract afforded the known compound, procesterol, which showed activity against E. histolytica.

Potent α-glucosidase inhibitors from the roots of Panax japonicus C. A. Meyer var. major

Bioassay-guided fractionation of the CHCl 3 soluble portion of the roots of Panax japonicus C. A. Meyer var. major afforded an active fraction with inhibitory activity against baker’s yeast α-glucosidase with an IC 50 value 1.02 mg/mL. Furthermore, the active fraction isolated contained three previously unreported polyacetylenes, designated panaxjapynes A–C, together with 11 other compounds, including four polyacetylenes, five phenolic compounds, a sesquiterpenoid, and a sterol glucoside. The structures of the compounds were elucidated by spectroscopic and chemical methods. Compared with the control acarbose (IC 50 677.97 μM), six compounds were shown to be more potent α-glucosidase inhibitors with IC 50 values in the range 22.21–217.68 μM.

The Heterohexameric Complex Structure, a Component in the Non-classical Pathway for Fibroblast Growth Factor 1 (FGF1) Secretion

Fibroblast growth factors (FGFs) are key regulators of cell proliferation, tumor-induced angiogenesis, and migration. FGFs are essential for early embryonic development, organ formation, and angiogenesis. FGF1 also plays an important role in inflammation, wound healing, and restenosis. The biological effects of FGF1 are mediated through the activation of the four transmembrane phosphotyrosine kinase fibroblast growth factor receptors in the presence of heparin sulfate proteoglycans and, therefore, require the release of the protein into the extracellular space. FGF1 is exported through a non-classical release pathway involving the formation of a specific multiprotein complex. The protein constituents of this complex include FGF1, S100A13, and the p40 form of synaptotagmin 1 (Syt1). Because FGF1 plays an important role in tumor formation, it is clear that preventing the formation of the multiprotein complex would be an effective strategy to inhibit a wide range of cancers. To understand the molecular events in the FGF1 release pathway, we studied the FGF1-S100A13 tetrameric and FGF1-S100A13-C2A hexameric complex structures, which are both complexes possibly formed during the non-classical pathway of FGF1 release.

Activation and Stiffness of the Inhibited States of F1-ATPase Probed by Single-molecule Manipulation

F(1)-ATPase (F(1)), a soluble portion of F(o)F(1)-ATP synthase (F(o)F(1)), is an ATP-driven motor in which gammaepsilon subunits rotate in the alpha(3)beta(3) cylinder. Activity of F(1) and F(o)F(1) from Bacillus PS3 is attenuated by the epsilon subunit in an inhibitory extended form. In this study we observed ATP-dependent transition of epsilon in single F(1) molecules from extended form to hairpin form by fluorescence resonance energy transfer. The results justify the previous bulk experiments and ensure that fraction of F(1) with hairpin epsilon directly determines the fraction of active F(1) at any ATP concentration. Next, mechanical activation and stiffness of epsilon-inhibited F(1) were examined by the forced rotation of magnetic beads attached to gamma. Compared with ADP inhibition, which is another manner of inhibition, rotation by a larger angle was required for the activation from epsilon inhibition when the beads were forced to rotate to ATP hydrolysis direction, and more torque was required to reach the same rotation angle when beads were forced to rotate to ATP synthesis direction. The results imply that if F(o)F(1) is resting in the epsilon-inhibited state, F(o) motor must transmit to gamma a torque larger than expected from thermodynamic equilibrium to initiate ATP synthesis.

Cell-surface Processing of the Metalloprotease Pro-ADAMTS9 Is Influenced by the Chaperone GRP94/gp96

A disintegrin-like and metalloprotease domain with thrombospondin type 1 motifs 9 (ADAMTS9) is a highly conserved metalloprotease that has been identified as a tumor suppressor gene and is required for normal mouse development. The secreted ADAMTS9 zymogen undergoes proteolytic excision of its N-terminal propeptide by the proprotein convertase furin. However, in contrast to other metalloproteases, propeptide excision occurs at the cell surface and leads to decreased activity of the zymogen. Here, we investigated the potential cellular mechanisms regulating ADAMTS9 biosynthesis and cell-surface processing by analysis of molecular complexes formed by a construct containing the propeptide and catalytic domain of pro-ADAMTS9 (Pro-Cat) in HEK293F cells. Cross-linking of cellular proteins bound to Pro-Cat followed by mass spectrometric analysis identified UDP-glucose:glycoprotein glucosyltransferase I, heat shock protein gp96 (GRP94), BiP (GRP78), and ERdj3 (Hsp40 homolog) as associated proteins. gp96 and BiP were present at the cell surface in an immunoprecipitable complex with pro-ADAMTS9 and furin. Treatment with geldanamycin, an inhibitor of the HSP90alpha family (including gp96), led to decreased furin processing of pro-ADAMTS9 and accumulation of the unprocessed pro-ADAMTS9 at the cell surface. gp96 siRNA down-regulated the levels of cell-surface pro-ADAMTS9 and furin, whereas the levels of cell-surface pro-ADAMTS9, but not of cell-surface furin, were decreased upon treatment with BiP siRNA. These data identify for the first time the cellular chaperones associated with secretion of an ADAMTS protease and suggest a role for gp96 in modulating pro-ADAMTS9 processing.

Crystal Structures Of Yeast Mitochondrial ATPase with Uncoupling Mutations

Yeast mitochondrial ATP synthase is a transmembrane protein responsible for synthesis of more than 90% of ATP under aerobic conditions. The water soluble portion of ATP synthase, F1, is composed of five subunits with stoichiometry α3β3γδe and has a combined molecular weight of 360 kDa. The three active sites of ATPase are formed at the interfaces between alternating α- and β-subunits. ATPase is capable of efficient ATP hydrolysis, accompanied by rotation of central stalk subunits, γδe, within the α3β3 core and is capable of ATP synthesis if central subunits are forced to rotate in the opposite direction. A number of mutations in ATP synthase have been identified that result in the uncoupling of catalytic function and proton flow across the mitochondrial membrane. These uncoupling mutations cluster at the interface between γ-subunit and α3β3 catalytic core of ATPase. In this work, four X-ray crystal structures of ATPase with single amino acid substitutions αN67I, αF405S, βV279F, and γI270T were solved at resolutions ranging from 3.2 A to 2.74 A. This study will present a structural comparison of the mutant structures with the wild type structures to understand the mechanism of coupling. However, the crystal structures likely represent the ground state of catalytic reaction cycle while the mutations may result in notable distortions of enzyme structure during other stages of catalytic cycle. Additionally, the uncoupling of the ATPase may be caused by changes in the energy of interaction between the portions that are rotating in the molecular machine thereby altering transition to the higher energy states. Structural based hypotheses are presented to explain the role of these residues in the coupling of the enzyme. Supported by NIH R01GM0662223

Proteomic Analysis of Soluble Proteins Important in Pediatric Acute Lymphoblastic Leukemia.

Abstract 1448Poster Board I-471In acute leukemia (AL), some clinical symptoms may be caused by soluble factors secreted by AL cells into the bone marrow (BM) microenvironment. On the other hand, AL cells are often dependent on the microenviroment of the host, with most AL cells dying during first days after transferred to in vitro. It is not known whether soluble factors are responsible for some host-AL interactions since most of the attention is paid to the biology of the leukemic cells themselves rather than the soluble portion of BM microenvironment.We aimed at identifying proteins in BM plasma of children with lymphoblastic AL (ALL), which may be responsible for ALL aggressiveness or for microenvironment-mediated survival of ALL cells. We used proteomic techniques to compare BM plasma at the diagnosis of ALL with non-malignant controls (BM plasma from patients with no signs of malignant disease with a BM check-up more than one year after BM transplantation or healthy donor blood plasma).BM plasma and blood samples were analyzed by protein microarray and/or by two-dimensional electrophoresis (2D PAGE). Protein microarray enabled a simultaneous detection of 79 proteins per sample (mainly cytokines and chemokines). The aim of 2D PAGE was to compare the complex patterns of between ALL and controls and to search for differences among unknown or unexpected molecules.We tested 79 soluble proteins in patients (n=15) and control samples (BM plasma, n=9 and blood plasma, n=5) by protein microarray. We detected 23 proteins with a significantly different concentration in patients (p<0.05). Of these, only 2 proteins TIMP-1 and LIF differed with a high level of significance after Bonferroni correction was applied (p<0.00064). The other differences need to be confirmed or excluded in a validation cohort. We observed no difference between control blood and control BM plasma. Both LIF and TIMP-1 are likely to be produced by non-leukemic cells given the low level of expression by leukemic cells in expression profiling. Causal relationship between LIF and TIMP-1 expression, as well as their importance for cell “stemness” which have been proven in other systems, need to be confirmed in leukemia.Before 2D PAGE, BM plasma was immuno-depleted from 12 abundant proteins by affinity chromatography. This improved considerably the separation of proteins on 2D PAGE and increased the number of detectable minor plasma proteins with possible biologic importance. We identified 393 protein spots per gel. Of these, 16 proteins spots were significantly different between the BM plasma from ALL and controls.The differently expressed spots were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF). The MALDI-TOF identified the concentration of haptoglobin to be increased in ALL, whereas hemoglobin alpha, hemoglobin beta, catalase, retinol binding protein, peroxiredoxin, ferritin, and carbonic anhydrase were identified as more abundant in controls. At the time of abstract submission, we confirmed elevated concentration of haptoglobin in patients by immuno-turbidometry (8 TEL-AML1, 12 hyperdiploid, 20 non-hyperdiploid or fusion gene ) and compared with 10 control samples. Ferritin concentration, on the other hand, was higher in control samples, as confirmed by Enzyme-linked imunoassay (ELISA).Presented data provide an insight into leukemic BM environment. The role of the observed differences in soluble factors in leukemia pathogenesis, diagnostic and therapeutic potentialis being investigated. Supported by : GAUK 7543/2007, IGA MZCR NR/9531-3 DisclosuresNo relevant conflicts of interest to declare.

Optimization of the method of crosslinked portion determination in irradiated polyamide-6

Given the absence of a conventional determination method for polyamide-6 (PA-6) gels, the optimal conditions for gel content determination were investigated in crosslinked bulk PA-6 by dissolving the sample in 90% formic acid. The sample was wrapped in glass fabric and treated using three selected modes. Based on statistical testing of the results, a two-step procedure consisting of slowly stirring the sample immersed in solvent at room temperature in an orbital shaker for 48 h followed by solvent exchange after 24 h was chosen for extraction of the uncrosslinked, soluble portion. A correction was applied for the fraction of the glass fabric lubricant extractable by the solvent when calculating the crosslinked portion. Gel content of ∼70 wt.% was determined. The relative standard deviation was less than 5%.

Structural determinants underlying constitutive dimerization of unoccupied human follitropin receptors

The human follitropin receptor (hFSHR) is a G protein-coupled receptor (GPCR) central to reproductive physiology that is composed of an extracellular domain (ECD) fused to a serpentine region. Using bioluminescence resonance energy transfer (BRET) in living cells, we show that hFSHR dimers form constitutively during their biosynthesis. Mutations in TM1 and TM4 had no effect on hFSHR dimerization, alone or when combined with mutation of Tyr110 in the ECD, a residue predicted to mediate dimerization of the soluble hormone-binding portion of the ECD complexed with FSH (Q. Fan and W. Hendrickson, Nature 433:269–277, 2005). Expressed individually, the serpentine region and a membrane-anchored form of the hFSHR ECD each exhibited homodimerization, suggesting that both domains contribute to dimerization of the full-length receptor. However, even in the context of only the membrane-anchored ECD, mutation of Tyr110 to alanine did not inhibit dimerization. The full-length hFSHR and the membrane-anchored ECD were then each engineered to introduce a consensus site for N-linked glycosylation at residue 110. Despite experimental validation of the presence of carbohydrate on residue 110, we failed to observe disruption of dimerization of either the full-length hFSHR or membrane-anchored ECD containing the inserted glycan wedge. Taken altogether, our data suggest that both the serpentine region and the ECD contribute to hFSHR dimerization and that the dimerization interface of the unoccupied hFSHR does not involve Tyr110 of the ECD.

Hypoglycaemic Potential Of Bridelia Micrantha (Hochst) Baill

The anti-diabetic activity of extracts and fractions from leaves of Bridelia micrantha was investigated in alloxan diabetic rats. Powdered leaves of B.micrantha were macerated with 80% MeOH for five days. The petroleum ether, CHCl3 and MeOH/H20 soluble portions were prepared from the crude aqueous MeOH extract and tested on the diabetic rats. Hyperglycaemia was induced by intraperitoneal injection of alloxan monohydrate (80mg/kg) and blood glucose monitored for seven days using a glucometer. The MeOH/H2O residue exhibited significant (p

Regulatory Mechanism of Matrix Metalloprotease-2 Enzymatic Activity by Factor Xa and Thrombin

Matrix metalloprotease (MMP)-2 plays a key role in many biological and pathological processes related to cell migration, invasion, and mitogenesis. MMP-2 is synthesized as a zymogen that is activated through either a conformational change or proteolysis of the propeptide. Several activating enzymes for pro-MMP-2 have been proposed, including metalloproteases and serine proteases. The mechanism of pro-MMP-2 activation by metalloproteases is well established, and the most studied activation mechanism involves cleavage of the propeptide by membrane type 1-MMP (MT1-MMP). In contrast, serine protease activation has not been thoroughly studied, although studies suggest that MT1-MMP may be involved in activation by thrombin and plasmin. Here, we demonstrate that factor Xa mediates MT1-MMP-independent processing of pro-MMP-2 in vascular smooth muscle cells and endothelial cells. Factor Xa and thrombin directly cleaved the propeptide on the carboxyl terminal sides of the Arg(98) and Arg(101) residues, whereas plasmin only cleaved the propeptide downstream of Arg(101). Moreover, processed MMP-2 showed enzymatic activity that was enhanced by intermolecular autoproteolytic processing at the Asn(109)-Tyr peptide bond. In addition to its role in activation, factor Xa rapidly degraded MMP-2, thereby restricting excessive MMP-2 activity. Thrombin also degraded MMP-2, but the degradation was reduced greatly under cell-associated conditions, resulting in an increase in processed MMP-2. Overall, factor Xa and thrombin regulate MMP-2 enzymatic activity through its activation and degradation. Thus, the net enzymatic activity results from a balance between MMP-2 activation and degradation.

Study of chain branching in natural rubber using size-exclusion chromatography coupled with a multi-angle light scattering detector (SEC-MALS)

The different rheological behaviour of natural rubber (NR) compared to industrial synthetic poly( cis-1,4-isoprene) (SR) has been attributed to the gel phase and long-chain branching. Previous studies on branching in NR were carried out using the fractionation technique by precipitation to obtain narrow molar mass distribution. In this study, chain branching of poly( cis-1,4-isoprene) in NR was characterised by size-exclusion chromatography coupled with an online multi-angle light scattering detector (SEC-MALS). The nanoaggregates adsorbed on the column packing interfered with branching characterisation for short and medium chains ( M w < 1000 kg/mol). Using a master curve of linear standard poly( cis-1,4-isoprenes), SEC-MALS revealed no or very little branching in the higher chains (1000 < M w < 10,000 kg/mol) of natural rubber contrary to previous studies. This study showed that the soluble portion of NR samples was composed of almost linear poly( cis-1,4-isoprene) and nanoaggregates with rather compact structures.

Soluble Receptor for Advanced Glycation Endproducts (sRAGE) Induces Human Monocyte Survival and Differentiation (38.23)

Abstract The receptor for advanced glycation end-products (RAGE) is a transmembrane receptor of the immunoglobulin super family or is the soluble portion of extracellular domain of RAGE (sRAGE). sRAGE is a competitive, negative regulator of transmembrane RAGE. RAGE and its ligands are overexpressed in a wide variety of diseases. However, the biological function of RAGE and sRAGE on human mononuclear phagocytes is unclear. Using flow cytometry, we found that sRAGE directly bound to human monocytes to mediate their survival and differentiation into macrophages, events that were dependent on cellular adhesion. In both monocytes and MDM, sRAGE treatment activated Akt, Erk and NFk-B signaling, and decreased caspase-3 activity. Interestingly, while sRAGE bound the surface of suspended pre-monocytic leukemic THP1 cells, it failed to stimulate intracellular signaling or cellular differentiation. Furthermore, we found sRAGE induced monocytes chemotaxis in vitro and recruited monocytes to the lung in vivo. Our data suggest a novel role for sRAGE in mononuclear phagocyte survival, differentiation, and cell recruitment of human monocytes. These observations suggest a potential role for sRAGE to modulate organ-based inflammation.

Human serum transferrin: a tale of two lobes. Urea gel and steady state fluorescence analysis of recombinant transferrins as a function of pH, time, and the soluble portion of the transferrin receptor.

Iron release from human serum transferrin (hTF) has been studied extensively; however, the molecular details of the mechanism(s) remain incomplete. This is in part due to the complexity of this process, which is influenced by lobe-lobe interactions, the transferrin receptor (TFR), the salt effect, the presence of a chelator, and acidification within the endosome, resulting in iron release. The present work brings together many of the concepts and assertions derived from previous studies in a methodical, uniform, and visual manner. Examination of earlier work reveals some uncertainty due to sample and technical limitations. We have used a combination of steady-state fluorescence and urea gels to evaluate the effect of conformation, pH, time, and the soluble portion of the TFR (sTFR) on iron release from each lobe of hTF. The use of authentic recombinant monoferric and locked species removes any possibility of cross-contamination by acquisition of iron. Elimination of detergent by use of the sTFR provides a further technical advantage. We find that iron release from the N-lobe is very sensitive to the conformation of the C-lobe, but is insensitive to the presence of the sTFR or to changes in pH (between 5.6 and 6.4). Specifically, when the cleft of the C-lobe is locked, the urea gels indicate that only about half of the iron is completely removed from the cleft of the N-lobe. Iron release from the C-lobe is most affected by the presence of the sTFR and changes in pH, but is unaffected by the conformation of the N-lobe. A model for iron release from diferric hTF is provided to delineate our findings.

Expression and purification of recombinant M-Pol I from Saccharomyces cerevisiae with α-1,6 mannosylpolymerase activity

Mannan outer chain N-glycan structures are yeast/fungal-specific typically found on secreted and cell wall glycoproteins. Mannan outer chains consist of an α-1,6 polymannose backbone attached to a Man 8–10(GlcNAc) 2 core. The backbone contains branches of α-1,2 mannose residues, terminated with α-1,3 mannose and decorated with α-1,2 mannose phosphate. Mannan biosynthesis starts in the Golgi with the initial polymerization of the α-1,6 linked mannose backbone by the M-Pol I complex. Constructs encoding soluble portions of the M-Pol I subunits, Mnn9p and Van1p from Saccharomyces cerevisae, were expressed in Pichia pastoris. Both subunits had to be expressed in the same strain to obtain the recombinant proteins. Recombinant M-Pol I was made only by the KM71 strain transformed with two vectors: one encoding Mnn9p and the other encoding Van1p. Soluble secreted M-Pol I was purified by sequential chromatography on DEAE–Trisacryl, GDP–Hexanolamine–Sepharose and Superdex 200. Characterization of the purified complex indicates that recombinant M-Pol 1 is a Mnn9p—Van1p heterodimer. Purified M-Pol I was active with α-1,6 mannobiose as acceptor and GDP–mannose as donor. HPLC identified five products confirmed to be 3–7 mannose residues long. Digestion with linkage-specific α-mannosidases revealed that the linkage formed is exclusively α-1,6. No α-1,2 mannosyltransferase activity, reported previously for M-Pol I immunoprecipitates from cell extracts was detected. These results provide further information on the role of M-Pol I in mannan biosynthesis.