Rapid purification of protein kinase C from rat brain. A novel method employing protamine-agarose affinity column chromatography

We describe a rapid purification of protein kinase C from rat brain cytosol employing a specific substrate, protamine-coupled to agarose. Sequential chromatography on DEAE-Sephacel, phenyl-Sepharose CL-4B, and protamine-agarose columns resulted in a 1,500-fold purification of protein kinase C. SDS-PAGE analysis of the purified enzyme resolved a doublet protein of 77-80 kDa. This doublet was recognized by a polyclonal antiserum against protein kinase C. Proteolytic digestion of each protein band generated similar peptide fragments. The underlying principle of the protamine sulfate purification method was also clarified. Protamine can serve as a Ca2+/phospholipid-independent substrate. We demonstrate phosphorylation of protamine on the column; phosphorylated protamine did not bind the enzyme with the same affinity and this covalent modification was most probably responsible for releasing the bound enzyme from the column after addition of Mg2+ and ATP. The C kinase inhibitor, H7, inhibits protamine phosphorylation in a dose-dependent fashion but does not prevent binding of the enzyme to a protamine-agarose column. We therefore conclude that protamine interacts with the active center of the enzyme enabling it to be phosphorylated, upon which it loses its binding affinity for C kinase.     

rfaP mutants of Salmonella typhimurium

Salmonella typhimurium rfaP mutants were isolated and characterised with respect to their sensitivity towards hydrophobic antibiotics and detergents, and their lipopolysaccharides were chemically analysed. The rfaP mutants were selected after diethylsulfate mutagenesis or as spontaneous mutants. The mutation in two independent mutants SH7770 (line LT2) and SH8551 (line TML) was mapped by cotransduction with cysE to the rfa locus. The mutants were sensitive to hydrophobic antibiotics (clindamycin, erythromycin and novobiocin) and detergents (benzalkoniumchloride and sodium dodecyl sulfate). Analysis of their lipopolysaccharides by chemical methods and by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed that their saccharide portion was, to a large extent, of chemotype Rc with small proportions of material containing a more complete core oligosaccharide and O-specific chains. Only 2.5 mol phosphate/mol lipopolysaccharide was found whereas the phosphate content of the lipopolysaccharide of a galE mutant strain was 4.8 mol. Thus the rfaP mutant lipopolysaccharides lacked more than two phosphate residues. Assessment of the location of phosphate groups in rfaP lipopolysaccharides revealed the presence of at least 2 mol phosphate in lipid A, indicating that the core oligosaccharide was almost devoid of phosphate. The chemical, physiological and genetic data obtained for these mutants are in full agreement with those reported earlier for rfaP mutants of Salmonella minnesota.     

A complex of GRB2-dynamin binds to tyrosine-phosphorylated insulin receptor substrate-1 after insulin treatment.

Insulin drives the formation of a complex between tyrosine-phosphorylated IRS-1 and SH2-containing proteins. The SH2-containing protein Grb2 also possesses adjacent SH3 domains, which bind the Ras guanine nucleotide exchange factor Sos. In this report, we examined the involvement of another SH3 binding protein, dynamin, in insulin signal transduction. SH3 domains of Grb2 as GST fusion proteins bound dynamin from lysates of CHO cells expressing wild-type insulin receptor (IR) (CHO-IR cells) in a cell-free system (in vitro). Immunoprecipitation studies using specific antibodies against Grb2 revealed that Grb2 was co-immunoprecipitated with dynamin from unstimulated CHO-IR cells. After insulin treatment of CHO-IR cells, anti-dynamin antibodies co-immunoprecipitated the IR beta-subunit and IRS-1, as tyrosine-phosphorylated proteins and PI 3-kinase activity. However, purified rat brain dynamin did not bind directly to either the IR, IRS-1 or the p85 subunit of PI 3-kinase in vitro. Together, these results suggest that in CHO-IR cells, insulin stimulates the binding of dynamin to tyrosine-phosphorylated IRS-1 via Grb2 and that IRS-1 also associates with PI 3-kinase in response to insulin. This complex formation was reconstituted in vitro using recombinant baculovirus-expressed IRS-1, GST-Grb2 fusion proteins and dynamin peptides containing proline-rich sequences. Furthermore, dynamin GTPase activity was found to be stimulated when an IRS-1-derived phosphopeptide, containing the Grb2 binding site, was added to the dynamin-Grb2 complex in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preliminary characterization of two thymus-dependent xenoantigens from mouse lymphocytes.

Preliminary characterization of two mouse thymus-dependent (T) lymphocyte xenoantigens, T25 and T200, which are selectively labelled by lactoperoxidase-catalysed iodination of T-cells, is described. Both molecules are membrane-bound glycoproteins. Fractionation of membrane vesicles prepared from BW5147 lymphoma cells by sedimentation through sucrose density gradients show that antigens T25 and T200 are in fractions enriched with plasma membrane. Moreover antigen T200 is partially degraded when viable cells are treated briefly with low concentrations of trypsin. Both molecules are efficiently solubilized in buffers containing sodium deoxycholate or Nonidet P-40, as measured by failure to sediment at 100000g for 60min. However, gel filtration on Sepharose 6B showed the presence of aggregated material in Nonidet P-40 extracts which was not found in deoxycholate-solubilized membranes. After solubilization in detergent, antigens T25 and T200 bind to, and may be specifically eluted from, columns of pea lectin--Sepharose or concanavalin A--Sepharose. Both molecules are heterogeneous when examined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. As judged by its binding to columns of pea lectin, at least part of the heterogeneity of mouse thymocyte antigen T25 resides in its carbohydrate moiety.     

Lipopolysaccharides in liver injury: molecular mechanisms of Kupffer cell activation

Endogenous gut-derived bacterial lipopolysaccharides have been implicated as important cofactors in the pathogenesis of liver injury. However, the molecular mechanisms by which lipopolysaccharides exert their effect are not entirely clear. Recent studies have pointed to proinflammatory cytokines such as tumor necrosis factor-alpha as mediators of hepatocyte injury. Within the liver, Kupffer cells are major sources of proinflammatory cytokines that are produced in response to lipopolysaccharides. This review will focus on three important molecular components of the pathway by which lipopolysaccharides activate Kupffer cells: CD14, Toll-like receptor 4, and lipopolysaccharide binding protein. Within the liver, lipopolysaccharides bind to lipopolysaccharide binding protein, which then facilitates its transfer to membrane CD14 on the surface of Kupffer cells. Signaling of lipopolysaccharide through CD14 is mediated by the downstream receptor Toll-like receptor 4 and results in activation of Kupffer cells. The role played by these molecules in liver injury will be examined.     

Age-dependent changes in rat liver microsomal and mitochondrial NADPH-dependent lipid peroxidation.

Purified outer membrane proteins O-8 and O-9 were able to bind to the peptidoglycan sacculi in sodium dodecyl sulfate solution. Binding was stimulated by lipopolysaccharide, that of protein O-9 being stimulated more remarkably. Proteins which had been heated in sodium dodecyl sulfate solution did not bind to the peptidoglycan sacculi even in the presence of lipopolysaccharide, while heated lipopolysaccharide stimulated the binding of non-heated proteins. The removal by pronase of the lipoprotein covalently bound to the peptidoglycan sacculi did not change the protein binding ability of the sacculi.     

Characterization of rapidly labelled rat liver ribonucleic acid showing high affinity for columns of methylated albumin on kieselguhr

Most of the rapidly labelled RNA from rat liver submitted to column chromatography on methylated albumin on kieselguhr remains tightly bound to the column and can only be recovered by elution with m-ammonia. The tightly bound RNA is composed mainly of DNA-like RNA. The binding capacity is dependent not only on base composition but also on molecular size: the heavier RNA molecules show a greater affinity to the column than do the lower-molecular-weight components. Rapidly labelled mouse liver and Saccharomyces cerevisiae RNA show similar behaviour to rat liver RNA on columns of methylated albumin on kieselguhr.     

A protein with lectin activity in penetration glands of Diplostomum pseudospathaceum cercariae

Homogenates of Diplostomum pseudospathaceum cercariae agglutinated mouse erythrocytes. The haemagglutination could be inhibited by certain glycoconjugates containing beta-1,3- and beta-1,4-glycan chains and also by some simple saccharides. The most potent inhibitors were heparin and some other glycosaminoglycans, bacterial lipopolysaccharides, laminarin (a beta-1,3-glucan) and lactulose. After electrophoresis of cercarial proteins, a dominant double band appeared in the 22-24 kDa region of gels. On blots, this protein bound labelled laminarin and it was also one of the few proteins recognised by mouse antibodies raised against cercarial haemagglutinins. In addition, mouse polyclonal antibodies against the beta-1,3-glucan-binding protein bound exclusively to the 22-24 kDa region on Western blots. Histochemistry revealed strong binding of labelled laminarin to cercarial penetration glands; this reaction was fully blocked by unlabelled laminarin. Other labelled glycoconjugates such as heparin, hyaluronic acid and a bacterial lipopolysaccharide also bound to the glands. Immunohistochemistry confirmed the localisation of the beta-1,3-glucan-binding protein in penetration glands. Reaction of the cercarial protein with immunoglobulins from non-immunised mice was observed on both nitrocellulose membranes and histological sections; this could be blocked by laminarin in incubation buffers. We consider the cercarial haemagglutinin to be a lectin which is identical with the 22-24 kDa beta-1,3-glucan-binding protein. According to the binding specificity and localisation we speculate on a role of this lectin in cercarial penetration into the host, probably as a tissue recognition or antibody rendering factor.     

Quantitation of metal cations bound to membranes and extracted lipopolysaccharide of Escherichia coli

Inductively coupled plasma emission spectroscopy was used to quantitate the metal cations bound to outer and cytoplasmic membranes and to extracted lipopolysaccharide from several Escherichia coli K12 strains. The outer membrane was found to be enriched in both calcium and magnesium relative to the cytoplasmic membrane. Both membranes contained significant levels of iron, aluminum, and zinc. The multivalent cation content of the lipopolysaccharide resembled that of the intact outer membrane. Lipopolysaccharide extracted from wild-type k12 strains contained higher levels of Mg than Ca regardless of the growth medium, but the medium used for growth did affect the relative amounts of bound Mg as well as the levels of the minor cations iron, aluminum, and zinc. In contrast, lipopolysaccharide isolated from a deep rough mutant strain, D21f2, contained more Ca than Mg. Electrodialysis of lipopolysaccharide from wild-type k12 strains removed 1 mol of Mg per mol of lipopolysaccharide but did not significantly affect the level of other bound metal ions. Dialysis of lipopolysaccharide against sodium (ethylenedinitrilo)tetraacetate removed most of the Mg and Ca, resulting in a sodium salt. The equimolar replacement of divalent cations with sodium in the sodium salt resulted in a net loss of counterion change. The sodium salt was dialyzed against either tris(hydroxymethyl)aminomethane hydrochloride, CaCl2, MgCl2, or TbCl3, and the resulting lipopolysaccharide salts were analyzed for their ionic composition. It was shown that tris(hydroxymethyl)aminomethane and Ca can replace some but not all of the Na bound to the sodium salt, but all of the other multivalent cations tested replaced Na, resulting in uniform lipopolysaccharide salts. Lipopolysaccharide isolated from the deep rough mutant strain D21f2 was also converted into a sodium salt. Relative to the wild-type lipopolysaccharide, Na was able to neutralize the anionic charge to a greater extent in the mutant lipopolysaccharide. Our results suggest that the loss of specific groups in the core region of the lipopolysaccharide from the mutant strain results in a more open structure that allows the binding of larger cations and of more monovalent cations.     

Detoxification of lipopolysaccharide (LPS) by egg white lysozyme

Abstract Recent studies carried out by our group suggest that lysozyme binds to bacterial lipopolysaccharide with a high affinity to produce a complex, and inhibits various biological activities of lipopolysaccharide. Although the basic structure of lipopolysaccharide is independent of the species and strains of Gram-negative bacteria, many structural factors such as O-antigenic polysaccharide, lipid A, substituted groups, and associated molecules, affect the biological activities of lipopolysaccharide. In this study, we prepared lysozyme/lipopolysaccharide complexes using various structures of lipopolysaccharide and compared the activity and physiochemical properties. Native and dansylated lysozyme were found to bind to all tested lipopolysaccharides. The mitogenic activity and TNF production by all tested lipopolysaccharides were significantly reduced by complex formation in vitro. Administration of the complex prepared by various lipopolysaccharides produced significantly less quantities of TNF in the septic shock model. These results suggested that binding of lysozyme to lipopolysaccharide is important for the host both in pathophysiological responses to lipopolysaccharides and in the modification of lipopolysaccharide biological activity.     

Use of a zwitterionic detergent for the restoration of the antibody binding capacity of immunoblotted Francisella tularensis lipopolysaccharide.

A method for the partial restoration of the antibody binding capacity of Francisella tularensis lipopolysaccharide (LPS) following denaturation (dissociation) in boiling sodium dodecyl sulfate (SDS) is described. The method relies on the presence of a zwitterionic detergent in the matrix of an SDS-polyacrylamide gel and in the transfer buffer during an immunoblot. F. tularensis LPS, which had lost its earlier capacity to bind to a particular monoclonal antibody in the normal blot procedure, did bind following the addition of the zwitterionic detergent to the polyacrylamide gel and transfer buffer. A number of detergents were tested but most success in restoring antibody binding was achieved with Zwittergent 3-08. This simple modification to the immunoblot procedure proved helpful in identifying a monoclonal antibody specific to hot phenol-extracted F. tularensis LPS.     

A specific insulin receptor and tyrosine kinase activity in the membranes of Neurospora crassa

Cells of the wall-less ("slime") strain of Neurospora crassa possess specific high affinity insulin binding sites on their cell surface. 125I-labeled bound insulin was not displaced from these cells by insulin-like growth factor II (IGF-II), and was only weakly displaced by IGF-I and proinsulin. Cross-linking of 125I-labeled insulin with N. crassa cells using disuccinimidyl suberate resulted in the labeling of a single band of ca. 67 kDa m.w. on a polyacrylamide gel. Two proteins of ca. 66 and 59 kDa m.w. were purified from detergent solubilized plasma membrane preparations by passage over an insulin-agarose affinity matrix. Antibodies against an autophosphorylation site on the human and Drosophila insulin receptors (anti P2) immunoprecipitated a single phosphoprotein of ca. 50 kDa m.w. from detergent solubilized plasma membranes, which possessed protein tyrosine kinase activity when histone H2 was used as substrate.     

Effects of detergent on the binding of solubilized sodium channels to immobilized wheat germ agglutinin: structural implications

The binding of the solubilized voltage-dependent sodium channel from rat brain to immobilized wheat germ agglutinin (WGA) is detergent-dependent. When solubilized in sodium cholate, only 11% of total recovered channels bound to a WGA-Sepharose column. When solubilized in Triton X-100 or CHAPS, however, 80% and 60% could bind, respectively. The effect of cholate on sodium channel binding is relatively specific: the major rat brain glycoproteins which bind to immobilized WGA are roughly the same in either Triton or cholate, as analyzed by SDS gel electrophoresis. The structural implications for the channel are discussed.     

Solubilization of phencyclidine receptors from rat cerebral cortex in an active ligand binding state

A phencyclidine (PCP) receptor binding site has been solubilized in an active ligand-binding state from rat cerebral cortical membranes with sodium deoxycholate. Optimal receptor solubilization occurs at a detergent/protein ratio of 0.5 (w/w); for 5 mg protein/ml solubilized with 0.25% sodium deoxycholate, about 60% of the protein and 25% of the receptor is solubilized. Specific binding of either [3H]-N-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) or [3H]MK-801 is measurable by filtration through Sephadex G-50 columns or glass fiber filters; more than 60% of the binding activity is stable after 48 h at 4 degrees C. In the presence of detergent, [3H]TCP binding exhibits a Kd of 250 nM, a Bmax of 0.56 pmol/mg protein, and a pharmacological profile consistent with that of the membrane-bound PCP receptor, although most drugs bind with affinities 2 to 8 fold lower than in membranes. Upon reduction of detergent concentration, binding parameters approximate those for the membrane-bound receptor ([3H]TCP binding: Kd = 48 nM, Bmax = 1.13 pmol/mg protein).     

The expression of lipopolysaccharide by strains of Shigella dysenteriae, Shigella flexneri and Shigella boydii and their cross-reacting strains of Escherichia coli

Strains of Shigella dysenteriae, Shigella flexneri and Shigella boydii express lipopolysaccharides, that enable the serotyping of strains based on their antigenic structures. Certain strains of S. dysenteriae, S. flexneri and S. boydii are known to share epitopes with strains of Escherichia coli ; however, the lipopolysaccharide profiles of the cross-reacting organisms have not been compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) lipopolysaccharides profiling. In the present study, type strains of these bacteria were examined using SDS-PAGE/silver staining to compare their respective lipopolysaccharide profiles. Strains of S. dysenteriae, S. boydii and S. flexneri all expressed long-chain lipopolysaccharide, with distinct profile patterns. The majority of strains of Shigella spp., known to cross-react with strains of E. coli , had lipopolysaccharide profiles quite distinct from the respective strain of E. coli . It was concluded that while cross-reacting strains of Shigella spp. and E. coli may express shared lipopolysaccharide epitopes, their lipopolysaccharide structures are not identical.     

Affinity chromatography of the bovine cerebral cortex A1 adenosine receptor

An approximate 140-fold purification of the A1 adenosine receptor of bovine cerebral cortex has been obtained via affinity chromatography. The affinity column consists of Affi-Gel 10 coupled through an amide linkage to XAC, a high-affinity A1 adenosine receptor antagonist. As assessed by [3H]XAC binding, bovine brain membranes solubilized with the detergent CHAPS had a specific binding activity of 1.1 pmol/mg protein. Interaction of solubilized A1 adenosine receptors with the XAC-Affi-Gel was biospecific and 30% of the receptor activity was bound by the gel. Demonstration of [3H]XAC binding in the material eluted from the column with R-PIA required insertion of receptor into phospholipid vesicles. The specific activity of the affinity column purified receptor was 146 +/- 22 pmol/mg protein with typically 5-15% of the bound receptor recovered. The purified receptor displayed high-affinity antagonist binding and bound agonists with the potency order expected of the bovine brain A1 adenosine receptor: R-PIA greater than S-PIA greater than NECA. In purified preparations, the photoaffinity probe [125I]PAPAXAC-SANPAH specifically labelled a protein of molecular mass 38,000 which has previously been shown to be the A1 adenosine receptor binding subunit.     

Solubilization and enrichment of boar sperm membrane proteins

Boar sperm membranes are rather resistent to the solubilizing effect of some detergents. Deoxycholate, an ionic detergent, was efficient in solubilizing sperm proteins but some nonionic detergents like Triton X-100 displayed relatively poor capacity in rendering membrane proteins soluble. This may be due to sperm proteins being attached to submembraneous structures through bonds involving divalent cations, since mixtures of Triton X-100 and ethylenediamine tetraacetic acid (EDTA) were almost as efficient as deoxycholate in solubilizing membrane proteins. Since intact spermatozoa were directly treated with detergents the solubilized proteins comprised a mixture of intracellular and membrane components. To enrich for membrane proteins, affinity chromatography on columns containing different lectins was carried out. SDS polyacryiamide gel electrophoresis of sperm glycoproteins desorbed from the various lectin columns demonstrated that each lectin bound a unique set of components although most glycoproteins were recovered from two or more columns. Columns containing Lens culinaris hemagglutinin yielded more sperm glycoproteins than any of the other lectin columns examined. The predominant amount of the sperm proteins recovered from the Lens culinaris lectin column was membrane derived, as the majority of the proteins were integrated into liposomes. It is concluded that sperm membrane proteins are efficiently solubilized by detergent in the presence of a chelator and that most of the membrane glycoproteins can easily be enriched by affinity chromatography on a lectin column. Proteins obtained in this way should serve as excellent starting material for the isolation of individual sperm membrane proteins.     

A novel approach to the study of glycolipid receptors for viruses. Binding of Sendai virus to thin-layer chromatograms

A method for the binding of virus to a silica gel thin-layer chromatogram is presented. After development the chromatogram is overlayed with the 125I-labelled virus and the bound virus is autoradiographed. Alternatively, the unlabelled virus may be detected after exposure to monoclonal antibody and labelled anti-antibody. The Sendai virus strain used did not bind to brain gangliosides earlier proposed to be receptors, but bound to human erythrocyte gangliosides. This finding may be explained by the existence of Sendai virus variants with different receptor specificities.     

Photoreactive insulin analogues used to characterise the insulin receptor

Three photoreactive insulin analogues (“photoprobes”) have been prepared in which an aryl azide group was substituted at either the A1, B1 or B29 positions of the insulin molecule. When incubated with rat liver plasma membranes and irradiated all three photoprobes covalently labelled specific insulin binding sites within the membrane. SDS-polyacrylamide gel electrophoresis of plasma membranes covalently tagged with either of the three ¹²⁵I-photoprobes resolved one major specifically labelled polypeptide with an apparent molecular weight of 130,000. The labelled polypeptide migrated anomalously in SDS-polyacrylamide gels and a molecular weight of 90,000 for the polypeptide was determined from a ‘Ferguson’ plot using the combined results from gels of different acrylamide concentrations. Column chromatography of detergent solubilised photoprobe-labelled membranes indicated that the labelled polypeptide may be a subunit of a larger protein complex.     

High-performance hydrophobic interaction chromatography of proteins

A new, weakly hydrophobic, high-performance liquid chromatography column has been developed for the separation of native proteins based on their relative hydrophobicities. Starting with a covalently bound, hydrophilic polyamine matrix, packing materials were synthesized through acylation with anhydrides and acid chlorides of increasing chain length to obtain increasingly hydrophobic surfaces. Proteins in aqueous buffers were induced to bind hydrophobically to the columns by the use of high salt concentrations in the mobile phase. Elution was achieved by decreasing the ionic strength of the solvent in a linear gradient. A mixture of cytochrome c, conalbumin, and beta-glucosidase was used as a standard to test the resolving power of newly synthesized columns. On a 4-cm butyrate column, baseline resolution was achieved in 20 min with a gradient of 3.0 mu sodium sulfate in 0.1 M potassium phosphate buffer, pH 7.0, to water. The static loading capacity for each column was determined using a hemoglobin binding assay. Capacities normally ranged between 150 and 180 mg of hemoglobin per gram of support. Since proteins are not denatured in hydrophobic interaction chromatography, enzymes eluted from the column retained enzymatic activity. Samples of alpha-amylase and beta-glucosidase ranging in size from 10 to 200 micrograms were recovered from the butyrate column with greater than 92% enzymatic activity in all cases. In a single trial, the enzyme citrate synthase was recovered from the benzoate column with 92% retention of enzymatic activity.