Analysis of protein transport through the golgi in a reconstituted cell-free system

The processes which transport membrane proteins between compartments of the Golgi apparatus have been reconstituted in vitro using isolated Golgi fractions. This cell-free system allows a detailed analysis of protein transport not possible in intact cells. Transport of the membrane glycoprotein (G protein) of vesicular stomatitis virus (VSV) is measured from a “donor” to an “acceptor” Golgi fraction. The donor Golgi fraction is prepared from VSV-infected Chinese hamster ovary (CHO) mutant cells deficient in the glycosylation enzyme N-acetylglucosamine transferase I. “Acceptor” is prepared from uninfected wild-type CHO cells. Transport is measured by the addition of N-acetylglucosamine to G protein, which can occur only upon movement of G protein from donor to acceptor. Transport requires physiological pH and osmolarity, is dependent on nucleotide triphosphates, and is mediated by proteins both from cytosol and on the Golgi membranes. Protein movement is inhibited by the non-hydrolyzable GTP analogue, GTPγS. The process of transport proceeds through the budding, pinching off, targeting, and fusion of transport vesicles. In this system these vesicles are initially coated with a non-clathrin coat and are targeted with this coat intact. Several of the proteins which mediate transport have been characterized, and isolated to homogeneity. The successful development of this assay has led to the formulation of cell free assays for protein transport between other compartments. Comparison of these systems indicates that some common mechanisms of vesicular movement are used in transport between a variety of membrane compartments.     

Is there a direct relationship between oral astringency and human salivary protein binding?

For decades, it is believed that astringency is due to the polyphenol-induced complexation of proline-rich salivary proteins in the oral cavity. In order to compare for the first time the human sensory threshold concentrations and the salivary protein binding activity of a series of astringent stimuli, human saliva protein was incubated for 5 min at 37 °C in the presence of astringent food-derived compounds and, after micro-centrifugation, the amount of the target molecules in the supernatant was quantitatively determined by HPLC-UV/Vis. Significant protein binding was observed for (−)-epigallocatechin-3-gallate, (−)-gallocatechin-3-gallate, (+)-gallocatechin, and (−)-catechin-3-gallate, all of which containing at least one galloyl moiety in the molecule and exhibiting rather high sensory thresholds of more than 200 μmol/L. In comparison, (+)-catechin and procyanidin B2, both lacking in any galloyl function, showed only comparatively low binding activity and, most interestingly, quercetin-3-O-α-l-rhamnopyranosyl-(1 → 6)-β-d-glucopyranoside and 3-carboxymethyl-indole-1-N-β-d-glucopyranoside did not show any protein binding at all, although the later N- and O-glycosides exhibited extraordinarily low sensory threshold concentrations of less than 0.001 and 0.0003 μmol/L, respectively. The data give some first evidence that the quantity of the non-bound, “free” astringent stimulus in the saliva liquid might be more closely related to the sensory perception of astringency than the amount complexed or precipitated by proteins. It is therefore questionable as to whether oral perception of astringency is related to the complexation and/or precipitation of salivary proteins.     

Investigation of the use of ram horn hydrolysate for fungal protein production

Production of the fungus Aspergillus niger NRRL 330 was studied in submerged fermentation with ram horn hydrolysate (RHH) as substrate. The characteristics of RHH have been reported previously. The RHH was enriched by addition of glucose and KH₂PO₄. The effects of kinetic parameters on the biomass yield of the fungus were investigated. The optimal conditions for growth of A niger on RHH were initial pH 6.5, temperature 30 °C, fermentation time 96 h and agitation speed 150 rpm. Under these optimal conditions the initial carbohydrate content of RHH was reduced from 1.52 to 0.2% and the biomass yield was 8.9 g l^?1. The biomass contained about 48.1% protein, 5.2% fat and 9.4% ash (on a dry weight basis). The amino acid content of the biomass was compared with Food and Agricultural Organisation (FAO) and animal feed standards. The protein produced contained all the essential amino acids for animal feed, but the amounts of these amino acids were somewhat lower than those of FAO and soybean reference protein. However, the amino acid composition of the biomass was better than that of animal feed. The results with RHH were also compared with previously reported data on fungal mycelium grown on waste liquor substrate. In conclusion, it was found that RHH could be used as a substrate in the production of fungal protein for use as animal feed. Copyright © 2003 Society of Chemical Industry     

Hull-less Barley Varieties: Storage Proteins and Amino Acid Distribution in Relation to Nutritional Quality

Barley (Hordeum vulgare L.) has had an important impact on human nutrition. Hull-less barley is a genetically improved type that has been widely used in recent years. Six Brazilian hull-less barley varieties (IAC-IBON 214-82; IAC 8612-421; IAC 8501-31; IAC 8501-12; IAPAR 39-ACUMAI; IAC 8501-22) were analyzed for storage protein constituents, amino acid contents, and similarity among the hull-less barley varieties. Albumins, globulins, prolamins I and II, and glutelins were extracted and separated by SDS-PAGE. The total protein amino acid contents of the flour were also determined for each variety by TLC and HPLC. Variations in intensity and appearance and disappearance of protein bands were observed among the varieties suggesting genetic variability. However, the amino acid profile did not indicate any major variations in the amino acid concentrations. The high lysine and threonine total concentrations detected in the seeds of the hull-less barley varieties encouraged an investigation into the regulation of amino acid metabolism and storage protein synthesis.     

Structure of soya glycinin and conglycinin gels

Gels of glycinin and conglycinin formed at various heating temperatures, in the absence and presence of 0.2M sodium chloride were characterised by transmission electron microscopy. In distilled water both proteins formed gels consisting of strands with a thickness of 10–15 nm. The strands of glycinin were very regular and cross sections of strands showed a hollow cylindrical structure. In the presence of sodium chloride, glycinin formed an aggregated gel structure at 85°C, but at 95°C an ordered strand structure was formed. Dissociation of the quaternary structure on heating and reassociation of subunits into regular strands were considered the most probable mechanisms for strand formation from glycinin. The aggregated structure at 85°C was interpreted as a transient state prior to dissociation. Conglycinin rich gels were less regular and more crosslinked than gels of glycinin. Also, the strands of conglycinin showed a complex mode of aggregation possibly in the form of double spirals. The addition of sodium chloride caused a denser and more aggregated structure at 75 and 85°C, but the effects were not as drastic as in the case of glycinin. Heating temperature had only minor effects on the gel structure in the range studied.     

Nephelometric study of salivary protein–tannin aggregates

Grape seed procyanidins were fractionated through different degrees of polymerisation, and human saliva was purified and separated into two fractions: one was mostly α‐amylase and the other was essentially proline‐rich proteins (PRPs). The interaction of these proteins with the procyanidin compounds was assayed using nephelometry, and the influence of several factors was investigated, such as degree of polymerisation, pH and concentrations of both protein and tannin. The same experiments were performed with bovine serum albumin (BSA). The amount of insoluble aggregates, resulting from the formation of polyphenol–protein aggregates, increased quickly up to a maximum value which thereafter remained practically unchanged. pH was set at 5.0 for all further assays, since it was the nearest value to that encountered in human saliva (pH 5.6–7.9), where proteins were stable and had a maximum ability to bind and precipitate procyanidin oligomers. These proteins were shown to have a strong affinity for procyanidin oligomers and were unable to resolubilise the polyphenol–protein aggregates when present in excess. PRPs required a much lower content to bind all the tannins (400 µg of procyanidin oligomers) than BSA and especially α‐amylase (48, 60 and 132 µg respectively). The procyanidin's ability to bind PRPs, BSA and α‐amylase increased with its average molecular weight. This ability increased regularly for PRPs up to 4500 Da, whereas the ability to bind the globular proteins decreased beyond 3400 Da. © 2001 Society of Chemical Industry     

The effect of cooking on proteins from acha (Digitaria exilis) and durum wheat

The effect of cooking on proteins from acha and durum wheat was assessed from an analysis of protein extractability, gel electrophoretic profiles, in-vitro protein digestibility (IVPD) and the amino acid compositions of wholemeal flour and residue proteins. Heating wholemeal flour samples at 100–140°C (t = 10–40 min) resulted in 0–30% and 45–55% decreases in acha and durum protein solubility, respectively. In general, high molecular weight (30–70 k Da) protein subunits were more susceptible to heat damage. For both cereals, sodium dodecyl sulphate (SDS; 10 g litre^?1) and/or dithiothrcitol (DTT; 10 mM ) increased protein solubility in unheated and heated samples. The IVPD index was 90–91% and was not significantly altered by cooking (100–120°C, t = 40 min). Cooking at extreme temperatures (140°C, t = 40 min) reduced the IVPD by 8% (P = 0.05). Osborne fractionation resulted in a durum or acha residue level of 7.8% or 55.2%. Treatment with solvent containing propanol, SDS and/or DTT at room temperature followed by SDS-polyacrylamide gel electrophoresis of non-solubilised proteins showed that the glutelin fraction of acha, with the exception of a 65 kDa subunit, was insoluble owing to strong inter-subunit hydrophobic and disulphide interactions. Wholemeal acha flour and residue protein showed a significantly greater level of hydrophobic and sulphur amino acids as well as glutamine which is associated with H-bonding. The possibility that cereal protein solubility is also dependent on protein-carbohydrate links is discussed.     

Preparation of an anion‐exchange adsorbent by the radiation‐induced grafting of vinylbenzyltrimethylammonium chloride onto cotton cellulose and its application for protein adsorption

Poly(vinylbenzyltrimethylammonium chloride)‐graft‐cotton cellulose, an anion‐exchange matrix, was synthesized by a mutual radiation‐induced grafting technique with a ⁶⁰Co γ‐radiation source. The grafted matrix was characterized by grafting yield estimation, elemental analysis, Fourier transform infrared spectroscopy, and scanning electron microscopy. The grafting yield decreased with the increase in the dose rate. However, the grafting yield and nitrogen content of grafted samples increased almost linearly with an increase in the total irradiation dose. To evaluate the performance of the grafted anion‐exchange matrix, the protein adsorption and elution behavior were investigated in a continuous column process under various experimental conditions, with bovine serum albumin used as a model protein. The binding and elution behavior of the anion‐exchange matrix depended on different experimental parameters, such as the grafting yield, ionic strength, pH of the medium, and amount of protein loaded. From a breakthrough curve, the equilibrium binding capacity and elution percentage of the grafted anion‐exchange matrix were estimated to be 40 mg/g and 94%, respectively. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 102: 5512–5521, 2006     

Solubility of amaranth seed proteins in sodium sulphate and sodium chloride: the main factor in quantitative extraction for analysis

Nitrogen was extracted more efficiently from amaranth seed with 0.04 M Na₂SO₄ (5% w/v) than with either 0.09 M or 0.17 M NaCl (5% or 10% w/v), despite both solutions having the same ionic strength (μ= 1). Solubility of saline soluble proteins (albumin ± globulin) was very poor in either water or 1M NaCl, but increased in 0.4M NaCl at alkaline pH between 7 and 10. Globulins were very soluble in 0.4M NaCl at a pH 9. Albumin was the main storage protein. Saline soluble proteins formed very weak gels.     

Fractionation and characterisation for antioxidant activity of hydrolysed whey protein

Whey protein isolate (WPI) was hydrolysed for 1 h using Alcalase, Protamex and Flavourzyme. Native WPI, hydrolysed WPI and two commercial WPI hydrolysates were subjected to fractionation by size exclusion chromatography. Antioxidant activity of WPI fractions was measured with a liposome‐oxidising system (50 µM FeCl₃/0.1 µM ascorbate, pH 7.0). Lipid oxidation was measured as thiobarbituric acid‐reactive substances (TBARS). Gel electrophoresis and amino acid analysis were run to identify the peptide composition. The influence of amino acid composition on antioxidant activity was evaluated using multivariate analysis methods (correlation analysis, principal component analysis, multiple linear regression and discriminant analysis). TBARS assays indicated the presence of antioxidant activity in all protein fractions, including non‐hydrolysed WPI. For native and hydrolysed WPI samples the first fraction (> 45 kDa) showed a higher TBARS inhibition effect (24–27%) when compared with lower‐molecular‐weight fractions and hydrolysate mixtures. In contrast, for commercial WPI hydrolysates a higher inhibitory effect was found in most of the lower‐molecular‐weight fractions (30–55%). The ability of WPI fractions to delay lipid oxidation was found to be related to the prevalence of histidine and hydrophobic amino acids. Copyright © 2004 Society of Chemical Industry