Purification and chemical characterization of the major glycoprotein of avian myeloblastosis virus.

The major glycoprotein of avian myeloblastosis virus (AMV) has been purified to an apparent state of homogeneity by gel filtration on a Sepharose 4B column in the presence of 6 m guanidine hydrochloride followed by dialysis against distilled water and then extraction with chloroform-methanol. The AMV glycoprotein remains soluble in the aqueous phase whereas contaminating proteins precipitate, either upon dialysis against distilled water or after treatment with chloroform-methanol.Carbohydrate, represented by glucosamine, mannose, galactose, fucose, and sialic acid, constitutes 40% of the weight of AMV glycoprotein. Glucosamine is the major carbohydrate component whereas fucose and sialic acid are present in relatively low amount. Amino acid analysis indicates a relatively high content of aspartic and glutamic acid, serine, threonine, and glycine. Based on SDS-polyacrylamide gel electrophoresis, a molecular weight value of 77,500 ± 500 was determined for AMV glycoprotein.     

Electrophoretic behavior of protein dodecyl sulfate complexes in the presence of various amounts of sodium dodecyl sulfate.

This report describes the relationship between the amount of sodium dodecyl sulfate present in a sample solution and the electrophoretic mobility of the protein-dodecyl sulfate complexes. In order to determine the extent of any conformational changes in the proteins and to establish a correlation between any of these structural changes and the electrophoretic behavior, visible absorption spectra and circular dichroism spectra were obtained for heme proteins in the presence of the same amounts of surfactants as used in electrophoresis.From the results obtained, it is apparent that the amount of sodium dodecyl sulfate present in the sample solution must be taken into consideration when performing a separation. Optimum experimental conditions are chosen for attaining enhanced separation and a maximized linear range of molecular weights of proteins that can be accurately determined.     

Amphiphilic properties of the avian myeloblastosis virus major glycoprotein, gp 80

The major glycoprotein (gp 80) from avian myeloblastosis virus (AMV) displays significant lipophilic properties, as shown by its strong interactions with acetylated uncharged decylamino agarose in hydrophobic chromatography. In effect, release from binding was achieved only by the added presence of a polarity reducing agent (ethylene glycol) and the strong anionic detergent sodium dodecyl sulfate. The hydrophobic behavior of the glycoprotein, coupled to the high content of hydrophilic carbohydrates, indicates its amphiphilic character. Confirmation of the amphiphilic nature of the AMV gp 80 was obtained by charge shift electrophoresis and crossed hydrophobic interaction immunoelectrophoresis. In both instances, the electrophoretic behavior of the glycoprotein was dependent on the presence of detergents. The AMV gp 80 displays the properties of integral membrane proteins.     

Physical and chemical properties of the major envelope glycoprotein gp85 from avian myeloblastosis virus

The major envelope glycoprotein gp85 of avian myeloblastosis virus, observed by electron microscopy as nearly spherical knobs projecting from the virus surface, was purified to homogeneity by gel filtration in 6 M guanidinium chloride followed by ion-exchange chromatography. The purified glycoprotein has a molecular weight of 80 000 from sedimentation equilibrium analysis. Glycoprotein gp85 contains approx. 45% carbohydrate including 25% N-acetylglucosamine, while the remaining weight consists of a polypeptide chain of approx. 45 000 daltons. Based on the oligosaccharide chain molecular weight data of Lai and Duesberg (Lai, M.M.C. and Duesberg, P.H. (1972) Virology 50, 359-372), the carbohydrate is calculated to be distributed between seven to nine oligosaccharide side chains. No self-association of gp85 was observed up to 2.0 mg/ml in dilute salt solution. The hydrodynamic properties of gp85 in dilute salt solution indicate a highly elongated molecule with an axial ratio of 7. One structural model which reconciles the hydrodynamic properties of gp85 with the nearly spherical architecture observed by electron microscopy requires the organization of the polypeptide chain and approx. 50% of the carbohydrate into a globular form. The remaining covalently linked oligosaccharides would by necessity extend outwardly from the globular structure as randomly oriented chains.     

Anomalous behavior of human leukocyte interferon subtypes on polyacrylamide gel electrophoresis in the presence of dodecyl sulfate

35S-labeled human leukocyte interferon (IFN) subtypes produced in a cell-free system derived from Escherichia coli were analyzed by polyacrylamide gel electrophoresis in the presence of SDS (SDS-PAGE). Some IFN subtypes anomalously showed lower electrophoretic mobilities than those expected from their formula molecular masses. The results with hybrid IFNs and esterification suggest that this anomaly of IFN subtypes on SDS-PAGE is due to the introduction of one or two negative charges in the middle of the molecule.     

Subunit structure of the major human erythrocytes glycoprotein: depolymerization by heating ghosts with sodium dodecyl sulfate

Heating human erythrocyte ghosts with sodium dodecyl sulfate (SDS) at 100° was found to cause depolymerization of the major membrane glycoprotein. The molecular weight of the heat-induced product was found to be about half that of the precursor and to have an identical surface charge density in SDS. These findings were obtained by analysis of Ferguson plots derived from SDS- polyacrylamide gel electrophoresis in which the retardation coefficients and free mobilities of the two glycoprotein forms were compared. Based on these findings, we propose that previous conflicts regarding the molecular weight of this glycoprotein can be resolved by reference to the isolation and/or solubilization conditions.     

Growth of Enterobacter cloacae in the presence of 25% sodium dodecyl sulfate.

The growth of Enterobacter cloacae in 25% sodium dodecyl sulfate is described. The bacteria appeared to tolerate sodium dodecyl sulfate rather than metabolize it. The process was energy dependent, and cell lysis occurred during stationary phase. Extreme detergent resistance may be characteristic of the genus Enterobacter.     

Anomalous behavior of bovine alpha s1- and beta-caseins on gel electrophoresis in sodium dodecyl sulfate buffers

Electrophoresis in the presence of sodium dodecyl sulfate (SDS) provides a relatively simple means of determining molecular weights of proteins. This technique relies on the validity of a correlation between some function of Mr and the mobility of the protein through the gel matrix. However, bovine caseins (especially alpha s1-casein) have lower mobilities than expected on the basis of their known Mr. The binding of SDS to both alpha s1-casein (Mr 23,600) and beta-casein (Mr 24,000) reached a maximum at the slightly low value of 1.3 g SDS/g protein. Gel-filtration chromatography showed, however, that the alpha s1-casein:SDS complex was larger than the beta-casein:SDS complex at pH 6.8 or 7.0, but that they were similar in size at pH 2.9 or 3.0. Circular dichroism spectra indicated that the low helical structure content of both alpha s1- and beta-casein increased with the addition of SDS and/or decreasing the pH to 1.5. 13C NMR results showed that SDS bound to alpha s1- and beta-casein in the same way as it did to bovine serum albumin. Either esterification or dephosphorylation followed by amidation of alpha s1-casein increased its mobility in SDS-gel electrophoresis, but neither modification affected beta-casein mobility. These and other results indicate that the low electrophoretic velocity of alpha s1-casein in SDS-gel electrophoresis results from its unexpectedly large hydrodynamic size. This is caused by localized high negative charges on certain segments of alpha s1-casein, which would induce a considerable amount of inter- and intrasegmental electrostatic repulsion, leading to an expanded or extended structure for portions of the alpha s1-casein molecule in the presence of SDS. It is clear that the conformation, and hence the equivalent radius, of an SDS:protein complex is determined by the sequence of amino acids in the protein and that, a priori, it cannot be anticipated that the electrophoretic mobility of such a complex will bear more than a casual relationship to the Mr of the protein.     

NMR structure of human apolipoprotein C-II in the presence of sodium dodecyl sulfate

The structure and protein-detergent interactions of apolipoprotein C-II (apoC-II) in the presence of SDS micelles have been investigated using circular dichroism and heteronuclear NMR techniques applied to (15)N-labeled protein. Micellar SDS, a commonly used mimetic of the lipoprotein surface, inhibits the aggregation of apoC-II and induces a stable structure containing approximately 60% alpha-helix as determined by circular dichroism. NMR reveals the first 12 residues of apoC-II to be structurally heterogeneous and largely disordered, with the rest of the protein forming a predominantly helical structure. Three regions of helical conformation, residues 16-36, 50-56, and 63-77, are well-defined by NMR-derived constraints, with the intervening regions showing more loosely defined helical conformation. The structure of apoC-II is compared to that determined for other apolipoproteins in a similar environment. Our results shed light on the lipid interactions of apoC-II and its mechanism of lipoprotein lipase activation.     

A strategy for purifying glutathione S-transferase in the presence of sodium dodecyl sulfate

Glutathione S-transferase (GST) is widely used to prepare and purify GSTtagged fusion proteins. Although GST improves protein solubility, detergents must often be used to achieve protein solubilization from bacterial lysates. However, purification of GST by affinity chromatography cannot be achieved in the presence of even low concentrations of the detergent sodium dodecyl sulfate (SDS). Here we show that 2-methyl-2,4-pentanediol (MPD) can prevent SDS from interfering with purification of GST, thus enabling purification of proteins that require SDS to improve their solubility.     

Dissociation and unfolding of GCN4 leucine zipper in the presence of sodium dodecyl sulfate.

The dissociation and unfolding behavior of the GCN4 leucine zipper has been studied using SDS titration. Circular dichroism (CD) spectra showed that the alpha-helix content of the leucine zipper (20 microM) decreased during the sodium dodecyl sulfate (SDS) titration. However, the alpha-helix content of the leucine zipper still remained significant in the presence of 1 mM SDS, with little change detected when the SDS concentration further increased to 2 mM. The dimer dissociation of the leucine zipper is also a co-operative process during SDS titration; with no dimer remaining when SDS concentration reached 1 mM, as shown by electrophoresis and the the theta(222)/theta(208) ratio. Our results indicate that SDS efficiently induces leucine zipper dimer dissociation with the monomers still partially folded. The experimental results provide important evidence for the previous model that partial helix formation precedes dimerization in coiled coil folding.     

Characterization of the adenosine triphosphatase of avian myeloblastosis virus.

The ATPase of avian myeloblastosis virus (AMV) is not a recognizable cellular enzyme. It hydrolyzes ATP, GTP, ITP, UTP, and dCTP at equal rates, is inhibited by high concentrations of dithiothreitol, and is partially inhibited by 1 × 10^?5mp-chloromercuribenzoic acid (PCMB) and p-chloromercuribenzene sulfonate acid (PCMBS). The inhibition by the mercurials is reversed by increasing the concentration of PCMB or PCMBS to 1 × 10^?3m. The enzyme requires phospholipid for activity. Incubation with phospholipase C inhibits activity and subsequent addition of lecithin-containing saturated fatty acids partially restores activity, whereas lecithin-containing unsaturated fatty acids further inhibit activity.     

Redox-dependent subunit dissociation of Azotobacter vinelandii hydrogenase in the presence of sodium dodecyl sulfate

Hydrogenases catalyze the reversible activation of dihydrogen. We have previously demonstrated that the purified hydrogenase from the nitrogen-fixing microorganism Azotobacter vinelandii is an alpha beta dimer (98,000 Da) with subunits of 67,000 (alpha) and 31,000 (beta) daltons and that this enzyme contains iron and nickel. The enzyme can be purified anaerobically in the presence of dithionite in a fully active state that is irreversibly inactivated by exposure to O2. Analysis of this hydrogenase by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) following boiling in SDS yields two protein staining bands corresponding to the alpha and beta subunits. However, when this enzyme was treated with SDS (25-65 degrees C) for up to 30 min under anaerobic/reductive conditions and then analyzed by anaerobic SDS-PAGE, a protein staining band corresponding to an apparent molecular mass of 58,000 Da was observed that stained for hydrogenase activity. Analysis of the 58,000-Da activity staining band by a Western immunoblot or a second aerobic SDS-polyacrylamide gel revealed that this protein actually consisted of both the alpha and beta subunits. Thus, the activity staining band (apparent 58,000 Da) represents the 98,000-Da dimer migrating abnormally on SDS-PAGE. Treatment of the anaerobically purified hydrogenase with SDS under aerobic conditions or under anaerobic conditions with electron acceptors prior to electrophoresis resulted in no activity staining band and the separated alpha and beta subunits. A. vinelandii hydrogenase was also purified under aerobic conditions in an inactive O2 stable form that can be activated by removal of oxygen followed by addition of reductant. This enzyme (as isolated), the activated form, and the reoxidized form were analyzed for their stability toward denaturation by SDS. We conclude that the dissociation of the A. vinelandii hydrogenase subunits in SDS is controlled by the redox state of the enzyme suggesting an important role of one or more redox sites in controlling the structure of this enzyme.