Protein-denaturing Agents Research Articles

Studies of the topoisomerase II-mediated cleavage and religation reactions by use of a suicidal double-stranded DNA substrate

The cleavage and religation reactions of eukaryotic topoisomerase II were studied by use of a 5'-recessed DNA substrate containing a strong recognition sequence for the enzyme. Cleavage of the DNA substrate was suicidal, that is the enzyme was unable to religate the cleaved DNA due to a release of DNA 5' to the cleavage position. With this substrate cleavage products accumulated with time in the absence of protein-denaturing agents, and the cleavage reaction was not reversible with salt. The suicide cleavage complexes contained a kinetically competent topoisomerase II enzyme as determined by the enzyme's ability to perform intermolecular ligation of the cleaved DNA to a free 3'-hydroxyl end on another DNA strand. The efficiency of the religation reaction depended on the ability of the religation substrate to base pair to the DNA in the cleaved enzyme-DNA complex. Higher levels of religation were obtained with dinucleotides than with long DNA substrates. Mononucleotides also were efficiently religated, indicating an ability of the enzyme to mediate religation without making contacts to a long stretch of nucleotides 5' to the cleavage position.

The hepatitis B virus-encoded transcriptional trans-activator hbx appears to be a novel protein serine/threonine kinase

To study the functional mechanism of the hepatitis B virus (HBV) X (hbx) gene product, we have expressed the hbx protein in E. coli and purified it by HPLC. The purified hbx protein was shown to be active in trans-activating transcription directed by the LTR sequence of HIV-1. The hbx protein was found to have an intrinsic serine/threonine protein kinase activity. The hbx protein was detected in hepatitis B virions, and tryptic phosphopeptide maps of the hbx protein phosphorylated in the virion and of the in vitro phosphorylated bacterially expressed hbx protein were similar. Inactivation of the hbx protein by heat, protein-denaturing agents, or an ATP affinity analog, p-fluorosulfonyl-benzoyl 5′-adenosine, resulted in loss of both protein kinase activity and trans-activation activity. These results suggest that the HBV-encoded trans-activator hbx is a novel protein kinase.

Unusual stage-specific embryonic antigen (TEC-4) defined by a monoclonal antibody to embryonal carcinoma cells defective in the expression of embryoglycan.

Most developmentally regulated epitopes identified on embryonal carcinoma cells and murine preimplantation embryos are associated with a glycoprotein-bound large glycan called embryoglycan. To prepare monoclonal antibodies recognizing other, less immunogenic stage-specific embryonic epitopes, we used embryoglycan-negative embryonal carcinoma cells P19XT.1.1 as immunogen. One monoclonal antibody prepared by this strategy was found to react specifically with mouse embryonal carcinoma and embryo-derived stem cell lines. The target epitope, TEC-4, was found to be expressed on eggs and two-cell embryos but was undetectable on later stages of mouse embryos and adult mouse tissues. NaDodSO4/PAGE of immunoaffinity-isolated antigen revealed that TEC-4 epitope is associated with glycoproteins of apparent Mr 120,000 and 240,000. The epitope was resistant to oxidation by sodium periodate and to digestion by endoglycosidase F but was sensitive to treatment with protein-denaturing agents and proteases, which suggested that the epitope is located in the protein moiety of the molecule. In the course of retinoic acid-induced differentiation of embryonal carcinoma cells the epitope disappeared before the onset of morphological differentiation. The combined data indicate that TEC-4 is an unusual stage-specific embryonic antigen that may be amenable to direct genetic analysis.

A protein covalently bound to ColE1 DNA.

ColE1 DNA was isolated from Escherichia coli as a relaxation complex of supercoiled DNA and proteins. Treatment of the complex with either protein-denaturing agents (SDS, phenol etc.) or proteolytic enzymes converted the supercoiled DNA to an open-circular form (relaxation). The relaxation complex was separately labelled in vivo with [3H]Leu or [14C]Leu, [35S]Met or (32P)phosphate and extensively purified. Complete hydrolysis of the relaxed complex with DNase I and P1 nuclease produced a 36-kDa protein which, we believe, is covalently bound to ColE1 DNA. On the other hand, the relaxed complex was treated with tosylphenylalanylchloromethane-treated-trypsin and the DNA-peptide(s) produced was (were) isolated and digested with the nucleases as above. The resulting nucleotidylpeptide(s) was (were) isolated by DEAE-Sephadex chromatography. The only 5'-dCMP was released from the nucleotidylpeptide(s) by snake venom phosphodiesterase treatment. O-Phosphoserine was found in acid hydrolysates of the DNA-peptide(s). We suggest that in the relaxation event the 36-kDa protein becomes covalently linked to ColE1 DNA via a phosphodiester bond between dC and the serine residue.

The nucleocapsid of the lipid-containing double-stranded RNA bacteriophage phi 6 contains a protein skeleton consisting of a single polypeptide species.

The nucleocapsid of the enveloped double-stranded RNA bacteriophage phi 6 was isolated by extraction with the nonionic detergent Triton X-114 and subjected to disruption analysis with chelating and protein-denaturing agents. The subnucleocapsid particles were separated in rate-zonal sucrose gradients, and their ultrastructure and protein composition were analyzed. The role of divalent cations in the nucleocapsid structure was studied by using a precipitation assay of the isolated nucleocapsid proteins. The phi 6 nucleocapsid had a cagelike skeleton consisting of a single polypeptide species (P1). Two other proteins (P2 and P4) were associated with the P1 cage. These three early proteins were previously known to be involved in the RNA synthesis machinery of the virus. The stability of the nucleocapsid surface lattice consisting of protein P8 was dependent on Ca2+ ions.

Changes in antibody avidity after virus infections: detection by an immunosorbent assay in which a mild protein-denaturing agent is employed.

In titrating serum immunoglobulin G antibody to viruses by enzyme-linked immunosorbent assay, we used two rows of wells for serial twofold dilutions of the serum; in one row, a low concentration of a protein denaturant, 0.5 or 1.0 M guanidine hydrochloride, was added to the diluent so that the binding of low-avidity antibodies to viral antigens on the solid phase was inhibited. We then compared the antibody titration curves obtained in the two rows. We found that the addition of the reagent resulted in a parallel leftward shift of the curves and that the extent of the shift was greater in early than in late sera from all of the three infections studied (Japanese encephalitis virus, rotavirus, and rubella virus infections). This procedure may be useful for estimation of the avidity of antibody in serum and, with further evaluation, may prove to be applicable to single-serum diagnosis of virus infections.

A cell surface integral membrane glycoprotein of 85,000 mol wt (gp85) associated with triton X-100-insoluble cell skeleton.

The Triton X-100-insoluble skeleton of baby hamster kidney BHK cells consists of the nucleus, intermediate-size filaments, and actin fibers. By transmission electron microscopy, membrane fragments were found to be associated with these insoluble structures. When radioiodinated or [3H]glucosamine-labeled cells were extracted with 0.5% Triton, most plasma membrane glycoproteins were solubilized except for a glycoprotein with a molecular weight of 85,000 (gp85) that remained associated with the insoluble skeletons. Immunoprecipitation with a specific antiserum indicated that the gp85 is not a proteolytic degradation product of fibronectin, an extracellular matrix glycoprotein insoluble in detergent. A monoclonal antibody of BHK cells specific for gp85 was produced. Immunofluorescence analysis with this monoclonal antibody indicated that gp85 is not associated with the extracellular matrix, but is confined to the cell membrane. Both in fixed and unfixed intact cells, fluorescence was concentrated in dots preferentially aligned in streaks on the cell surface. Gp85 was found to behave as an integral membrane protein interacting with the hydrophobic core of the lipid bilayer since it was extracted from membrane preparations by ionic detergents such as SDS, but not by 0.1 N NaOH (pH 12) in the absence of detergents, a condition known to release peripheral molecules. Association of gp85 with the cell skeleton was unaffected by increasing the Triton concentration up to 5%, but it was affected when actin filaments were dissociated or when a protein-denaturing agent (6 M urea) was used in the presence of Triton, suggesting that protein-protein interactions are involved in the association of gp85 with the cell skeleton. We conclude that gp85 is an integral plasma membrane glycoprotein that might have a role in cell surface-cytoskeleton interaction.

Core Extrusion of ironsulfur proteins with benzenethiol in aqueous urea/triton X-100 solutions: A study on the reactivity of different conformational states of succinate dehydrogenase

The aqueous DMF/Triton X-100/benzenethiol extrusion procedure by Kurtz [1] and investigated in detail in [2] has been modified by substituting urea for DMF as the protein-denaturing agent. This replacement allows for shorter reaction times, lower sensitivity to ionic strength [3] and for lower dilution factors. Preliminary experiments with spinach ferredoxin [4] demonstrated the identity of the extruded thiolate with respect to the synthetic analogue, and the feasibility of an easy and ready separation of the t001 Relationships between the Conformational State, E S1 m, and the Rate Constant of the Urea/Triton/Benzenethiol Extrusion Reaction for Succinate Dehydrogenase. E S1 m is determined by potentiometric titration of the EPR signal at g = 1.94 at liquid nitrogen temperature. Reaction rates for the extrusion reaction are determined by monitoring the increase in absorbance at 747 nm resulting from the addition of the protein to a suitable reaction medium, whose final composition is: 9 × 10 −6 M protein, 0.096 M benzenethiol, 4.73 M urea, 4.77 (% v/v) Triton X-100, 0.05 M Tris-HCI and enough effector or NaCl to give a final ionic strength of 0.0365 M. Effector E S1 m, mV Pseudo-first-order rate constant of the extrusion reaction, min −1 None −14 0.0769 Oxaloacetate +80 0.2631 Succinate +108 0.4444 products of the reaction by ion-exchange chromatography. The thiolate does not bind to a cellulosic anion exchanger, whereas the bound apoprotein as eluted with high-ionic strength buffer and can be chemically [5] or enzymatically [6] reconstituted to its native form. We used this technique in our investigation on the molecular basis of the modulation of the redox potentials of the electron-transferring groups in the complex ironsulfurflavoprotein succinate dehydrogenase. This protein exist in three different conformational states, or conformers, depending on the binding of different effectors to a site different from the catalytic one [7]. We thus distinguish between: the active; ligand-free enzyme; the active, succinate-stabilized enzyme, and the inactive complex between the protein and oxaloacetate. Different catalytic activities of the conformers depend on the different midpoint potential of the covalently bound flavin, which is high in the active conformers and low in the inactive one [8]. Also the redox midpoint potential of the tetrahedral FeS center S1 ( E S1 m) is affected by ligand binding. This parameter is related in Table I to the reaction rates measured for the extrusion of FeS structures from different conformers. Although a possible non-selectivity of the extrusion must be considered, rate constants for different conformers exactly parallel the figures obtained for E S1 m, increasing as this latter increases. The concentrations of both the denaturing agent (urea) and of the extruding one (micellar benzenethiol) being the same, the reaction rate only depends on the accessibility of the cluster to the hydrophobic extrudant. Thus binding of effectors which increase E S1 m also increases the hydrophobicity of the environment states of the cluster. This extends to different conformation states of the same protein previously inferred by comparing the redox properties of the same cluster in different proteins of known structure [9]. Since several recent reports present modifications of the thermodynamic properties of FeS centers in a number of proteins and relate these modifications to more or less subtle conformational changes, the approach we introduced may be of some interest.

Interactions of Pseudomonas aeruginosa lectins with Escherichia coli strains bearing blood group determinants.

Pseudomonas aeruginosa lectins interact with Escherichia coli strains O86B7 and O128B12, which possess B and H (O) blood group determinants, respectively. The interaction could be demonstrated by specific agglutination of the bacteria, by haemagglutination inhibition tests and by lectin-mediated peroxidase binding to the bacteria. The agglutination of E. coli O86B7 by the Pseudomonas galactose-binding lectin was inhibited by D-galactose and by the lipopolysaccharide extracted from E. coli O86B7. Similarly, the specific agglutination of E. coli O128B12 by the Pseudomonas mannose-binding lectin (which also binds L-fucose, L-galactose and D-fructose) was inhibited by D-mannose, L-fucose, L-galactose and D-fructose, as well as by athe lipopolysaccharide extracted from E. coli O128B12. The interaction between E. coli O128B12 and the Pseudomonas mannose-binding lectin was also demonstrated by lectin-mediated peroxidase binding to the bacterial surface. Peroxidase binding was also inhibited by the above-mentioned sugars and E. coli O128B12 lipopolysaccharide. Treatment of cells of the two E. coli strains with protein-denaturing agents did not reduce their agglutination by the Pseudomonas lectins. On the other hand, oxidation of the cell surface sugars by sodium metaperiodate or boiling the cells in the presence of 1% acetic acid for 1 h abolished their agglutination by the two lectins. It is, therefore, suggested that the Pseudomonas lectins interact with the B and H (O) blood group determinant sugars (D-galactose in E. coli O86B7 and L-fucose in E. coli O128B12) residing in the lipopolysaccharides of these E. coli strains.

A new principle in biospecific affinity chromatography used for purification of cobalamin-binding proteins

To avoid using protein-denaturing agents for desorption, when purifying cobalamin-binding protein by biospecific affinity chromatography, an affinity column has been prepared where cobalamin is attached through a temperature-labile linkage to insolubilized 3,3′-diaminodipropylamine. On desorption the protein is obtained in solution saturated with cobalamin. The method has been used for purification of intrinsic factor and transcobalamin I.

Studies on the gelation reaction of Limulus lysate (pre-gel). III. Influences of various substances on the gelation reaction of Limulus lysate (author's transl)

Effect of nucleic acid-related substances, amino acids, saccharides, protein-denaturing or -precipitating agents, SH reagents, reducing agents, biological products, urine, and saliva, on the gelation reaction of limulus lysate was examined. Most of them have no effect on the gelation reaction, but some inhibition was observed in benzyl alcohol, ATP of nucleic acid-related substances, cysteine, and mercaptoethanol among SH reagents. Protein-denaturing agents inhibited the gelation reaction at the concentration higher than a certain level. Saliva, urine, and biologicd products showed no inhibition of the gelation reaction. It was also found that the limulus test was applicable to the detection of endotoxin in products at various stages of manufacturing process.

Mechanism of the salicylate hydroxylase reaction: IV. Fluorometric analysis of the complex formation

1. 1.|Binary and ternary complexes between apoenzyme of salicylate hydroxylase (salicylate, NADH: oxygen oxidoreductase (1-hydroxylating, 1-decarboxylating)) and components such as FAD, substrate and NADH were fluorometrically studied to determine dissociation constant and stoichiometric ratio of each component. This was based on the changes in fluorescence intensity when the apoenzyme was mixed with these components. 2. 2.|A protein-denaturing agent or an alkali treatment was shown to depress the fluorescence emission of the apoenzyme with the red shift of the maximum. 3. 3.|The addition of FAD to the apoenzyme resulted in a marked decrease in both FAD and protein fluorescences. 4. 4.|Either substrate or NADH formed 1 :1 complex with the apoenzyme. This was indicated by both quenching of protein fluorescence and enhancement of substrate or NADH fluorescence. 5. 5.|The holoenzyme combined specifically with substrate to form a fluorescent ternary complex in which the ratio of apoenzyme, FAD and substrate was 1:1:1. 6. 6.|Upon formation of these complexes, the blue shift occurred in the fluorescence spectrum of protein, salicylate, NADH or FAD.

Cortical ultrastructure of Paramecium aurelia. Studies on isolated pellicles.

Two methods have been devised for the isolation of large quantities of purified pellicles (cortical layers) of Paramecium aurelia. Pellicles isolated by both procedures, when examined by electron microscopy, were found to contain ciliary basal bodies, two types of cortical membranes, ribbons of microtubules, kinetodesmal fibers, and elements of the infraciliary lattice system. By electron microscopy, the extent of preservation of the various cortical structures when pellicles are isolated by each method has been characterized. Pellicles isolated in both ways have been utilized to investigate cortical morphology of Paramecium. Both phase-contrast and electron microscopic observations have been made. Many new ultrastructural features were observed and are reported herein. An interesting result of this study is the discovery in stock CD that the structure of cortical territories (the territory is the functional unit of cortical morphogenesis and physiology) may vary within a single organism. Features which show variation include number of parasomal sacs, microtubular ribbons, and basal bodies (and therefore cilia) per territory, number of microtubules per ribbon, and length of kinetodesmal fibers. The possible significance of these variations, with respect to territory replication, is discussed. In addition, preliminary observations on the solubility of various cortical organelles in the presence of a number of protein-denaturing agents are reported.

Separation of the fertilization membrane in Urechis and sea urchin eggs as a phenomenon caused by breakage of hydrogen bonds : I. Action of mercaptide-forming, alkylating, oxidizing and protein-denaturing agents

1. 1. Janus green dyes were effective in elevating the blisters in the sea urchin eggs without jelly layer. 2. 2. Alkylating, oxidizing and mercaptide-forming agents, and protein denaturants were effective upon Urechis and sea urchin eggs when properly employed. 3. 3. Iodoacetic acid caused the separation of the fertilization membrane only after entering the eggs in a neutral form. Iodoacetamide was slightly effective. 4. 4. Iodosobenzoic acid was potent in causing the membrane elevation like iodoacetic acid. Ferricyanide was effective only upon the Urechis eggs. Iodine affected the Urechis eggs and caused the blister formation in sea urchin eggs. 5. 5. PCMB, merthiolate and mercurochrome were potent in causing the membrane separation in Urechis eggs at pH 8.2. However, in sea urchin eggs, PCMB and mercurochrome were effective in an alkaline medium only after entering as the neutral form. Methyl mercuric nitrate caused the blisters in sea urchin eggs, while it caused an ordinary separation of the fertilization membrane in Urechis eggs. Structural difference of the egg surface in both species was suggested. 6. 6. Protein denaturants, such as guanidine and phenol, were potent in causing the separation of the fertilization membrane. 7. 7. A possible connection of vitelline and plasma membranes by means of hydrogen bondings was proposed.

The dissolution of silica from diatom walls

Silica walls of diatoms, prepared by digesting cultured cells with nitric acid, dissolved more rapidly in media of higher pH or at higher temperatures. Most of the studies reported here were carried out in Tris buffer at pH 9·0–9·3 and at 19°C. The rate of dissolution under these conditions was slightly less than in sea water at pH 8·0, since NaCl accelerates the dissolution of silica. The silica from cleaned diatom walls dissolved in Tris buffer until, after 3–4 weeks, the solution had become saturated at 40–50 mg Si/l. (85–110 mg SiO 2/l.). The rate of solution decreased with time, probably due to the presence of silica in different states of hydration. Living cells suspended in buffer in darkness remained viable for many weeks during which time very little silicon dissolved from their walls. However, every treatment which killed the cells (heat, protein-denaturing agents, organic solvents, breakage) resulted in an increase in the rate of silica dissolution, although this rato was never as great as from acid-cleaned walls. Experiments with EDTA indicated that a cationic inorganic factor was retarding silica dissolution from killed cells. Protreatment of cleaned silica walls with salts of Al, Be, Fe, Ga, Gd, Sc, Ti, Y and Yb at pH values between 2·0 and 9·0, and with Mn at pH values between 4·0 and 11·0, had various effects on the subsequent rate of dissolution of the silica at pH 9·0. At certain pH values each of these elements can evidently-combine superficially with amorphous silica and thereby retard its rate of dissolution. Pretreatment of cleaned silica walls with salts of La, Mo, Cr, at pH values between 2·0 and 9·0, and with Ca and Mg at pH values between 4·0 and 11·0, had no effect on their subsequent dissolution at pH 9·0, Al, Be, Fe, Ga, Gd and Y were all capable of combining with silica walls at pH 8·0, which is the pH of sea water, and thereby retarding their subsequent dissolution. A single determination showed that the walls adsorbed Al equivalent to 1 per cent of their weight. In retarding dissolution of the silica, Al was as effective on killed, but intact, cells as on acid-eleaned walls. The silica of nine samples of untreated diatomaceous earth of fresh-water and marine origins, ranging in age from Upper Cretaceous through Pliocene, was essentially insoluble under these conditions. Digestion in strong nitric acid freed only a small fraction of the silica for dissolution. The factor which retarded the initial dissolution of silica from natural marine phytoplankton could be removed completely by pretreating the cells with a chelation mixture of EDTA and oxalate. DHEG, which is reported to complex certain cations such as Fe, but to have no effect on Ca, or Mg, was equally effective. It can be concluded that certain adsorbed inorganic cations decrease the rate of dissolution of diatom-aceous silica. In living diatom cells there may be an additional mechanism for stabilizing silica walls.

Chain length specificity for pectin-methylesterase inhibition by anionic detergents

The possibility that plant auxins affect the methyl esterification of cell wall pectic substances has been suggested by Bennet-Clark (1955) and others. Recently Ordin, Cleland and Bonner (1957) have presented evidence that indolacetic acid (IAA) affects the rate of incorporation of methionine methyl groups into cell wall pectic substances. According to these workers, the findings support the hypothesis that esterification of carboxyl groups of pectin is involved in the auxin mechanism of cell expansion. These reports have suggested to us that a study of higher plant pectic enzymes and of chemical substances affecting their activity might provide new tools and information useful in studying possible auxin-pectic enzyme relationships. McColloch and Kertesz (1947) reported that soaps and alkyl aryl sulfonate detergents powerfully inhibited tomato pectin-methylesterase (PM) activity in vitro . However, since detergents are protein-denaturing agents, it was stated that the observed inhibition might be due to this effect. This communication reports a study of the effects of chain lengths of fatty acid salts and alkyl sulfates on their inhibition of PM.