Monomer of sodium and potassium ion activated adenosinetriphosphatase displays complete enzymatic function

The distribution of sodium and potassium ion activated adenosinetriphosphatase [(Na+ + K+)-ATPase] among the various oligomeric forms present in a given solution is assessed unambiguously by cross-linking with glutaraldehyde. Purified enzyme dissolved in a solution of a nonionic detergent, octaethylene glycol dodecyl ether, remains dispersed and unaggregated after removal of the bulk of the detergent. Increases in the aggregation of the enzyme, which have been previously observed upon the addition of substrates to such a solution, are found to be due to changes in ionic strength rather than a consequence of the initiation of turnover. Furthermore, conditions are described that produce solutions containing stable, enzymatically active mixtures of the smaller oligomers of the asymmetric unit, alpha beta. Cross-linking by glutaraldehyde while the enzyme is turning over demonstrates that at least one of these oligomers is responsible for the observed enzymatic activity. A determination of which oligomers are present in each fraction from a glycerol gradient demonstrates that the profiles of the enzymatic activity and the concentration of monomer coincide. In addition, the monomer can form the sodium-dependent, phosphorylated intermediate of the mechanism for the enzyme. Finally, a preparation of (Na+ + K+)-ATPase, dissolved in solutions of the same nonionic detergent, can be prepared in which the predominant species (greater than 85%) is the monomer. The enzyme in this solution exhibits high specific activity, and its apparent Michaelis constants for the cationic substrates are very similar to those of the purified, membrane-bound enzyme. It is concluded from these results that a monomer of the alpha beta asymmetric unit is fully capable of catalyzing (Na+ + K+)-ATPase activity, and hence active transport, in the native enzyme. A reassessment of proposed molecular mechanisms for active transport is made in light of these discoveries.     

A Rapid and Simple PCR-based Method for Analysis of Transgenic Fish using a Restricted Amount of Fin Tissue

The protocol described in this paper offers a simple and rapid method for PCR analysis of transgenes using a restricted amount of fin tissue from small-sized transgenic fish. A simple preparation of fin lysate using a buffer containing a low concentration of an ionic detergent, SDS (0.01%), followed by neutralization with a second buffer containing higher concentrations of non-ionic detergents NP40 (2%) and Tween 20 (2%) consistently provides a reliable quantity of high-quality DNA template for PCR amplification of transgenes. Based on this protocol, transgenic fish can be clearly distinguished from non-transgenic fish using PCR in a rapid and reproducible manner. Tedious DNA purifications are avoided while fidelity of amplification and efficient identification of transgenic fish are maintained.     

Isolation and Partial Characterization of Heat-denatured Products of Soybean 11S Globulin and Their Analysis by Electrophoresis

Heat denaturation of soybean 11S globulin was examined at 70° and 100°C in phosphate buffer (pH 7.6), at 0.01 and 0.5 ionic strength. Gel electrophoresis (Davis system) indicated that heat-denatured soybean 11S globulin contained two major components (buffer-soluble form). But they were not identified at 70°C-0.5 ionic strength. Gel filtration followed by SDS-gel electrophoresis showed that the major components were composed of a monomer and at least three of kinds of oligomers containing only an acidic subunit. Gel filtration of the precipitate formed at 100°C at 0.5 ionic strength gave two peaks. SDS-gel electrophoresis indicated that the first peak contained aggregates of highly polymerized subunits, and the second peak contained a monomer of basic subunit and seven kinds of oligomers with various proportions of basic subunits to an acidic subunit.     

Organic solvent extracted EmrE solubilized in dodecyl maltoside is monomeric and binds drug ligand

Ethidium multidrug resistance protein (EmrE) is a member of the small multidrug resistance family of proteins and is responsible for resistance to a diverse group of lipophilic cations. To examine the multimeric state(s), size-exclusion HPLC and sedimentation velocity experiments were performed with EmrE solubilized in N-dodecyl-beta-d-maltopyranoside (DM) detergent. EmrE was purified from Escherichia coli membranes using organic extraction with a 3:1 chloroform:methanol solvent followed by LH-20 chromatography and the recovered pure protein was re-solubilized in a buffer containing 2% DM. The purified protein was analyzed by SEC-HPLC to estimate the monodispersity and to determine the amount of bound detergent. The results show that EmrE is homogeneous in DM with a Stokes radius of 3.6nm compatible with that of a monomer. Sedimentation velocity experiments indicated that the EmrE preparation was monodisperse and supports the fact that the organic extracted protein solubilized in DM is monomeric. This monomeric form of the protein analyzed here is also shown to bind substrate in the micromolar range.     

Soluble Oligomers of the Pore-forming Toxin Cytolysin A from Escherichia coli Are Off-pathway Products of Pore Assembly

The α-pore-forming toxin Cytolysin A (ClyA) is responsible for the hemolytic activity of various Escherichia coli and Salmonella enterica strains. Soluble ClyA monomers spontaneously assemble into annular dodecameric pore complexes upon contact with membranes or detergent. At ClyA monomer concentrations above ∼100 nm, the rate-limiting step in detergent- or membrane- induced pore assembly is the unimolecular reaction from the monomer to the assembly-competent protomer, which then oligomerizes rapidly to active pore complexes. In the absence of detergent, ClyA slowly forms soluble oligomers. Here we show that soluble ClyA oligomers cannot form dodecameric pore complexes after the addition of detergent and are hemolytically inactive. In addition, we demonstrate that the natural cysteine pair Cys-87/Cys-285 of ClyA forms a disulfide bond under oxidizing conditions and that both the oxidized and reduced ClyA monomers assemble to active pores via the same pathway in the presence of detergent, in which an unstructured, monomeric intermediate is transiently populated. The results show that the oxidized ClyA monomer assembles to pore complexes about one order of magnitude faster than the reduced monomer because the unstructured intermediate of oxidized ClyA is less stable and dissolves more rapidly than the reduced intermediate. Moreover, we show that oxidized ClyA forms soluble, inactive oligomers in the absence of detergent much faster than the reduced monomer, providing an explanation for several contradictory reports in which oxidized ClyA had been described as inactive.     

Activity staining of nucleolytic enzymes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis: Use of aqueous isopropanol to remove detergent from gels

The sensitivity with which RNase and DNase activity can be detected after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) varies widely, depending upon the particular SDS preparation used for electrophoresis. (See also [10.], Anal. Biochem. 100, 357–363.) Sensitivity of detection is greatly increased by using buffered 25% isopropanol, rather than buffer alone, to wash detergent from gels after electrophoresis. Thus it is routinely possible to detect bovine pancreatic RNase A at the picogram level. Use of isopropanol improved activity staining of RNases with each of the 10 SDS preparations examined, including one containing 32% tetradecyl sulfate and 4% hexadecyl sulfate, and reduced the variability from preparation to preparation observed when buffer alone was used to remove SDS. Other water-organic cosolvent binary mixtures can be used but none shows advantages over aqueous isopropanol when sensitivity of detection as well as availability and cost of organic solvent are considered.     

Physicochemical characterization of the 68 000-dalton protein of bovine neurofilaments

The 68 000-dalton protein from bovine neurofilaments was purified by a combination of chromatography on DEAE-cellulose and on hydroxylapatite in buffers containing 8 M urea. Although the separation of this protein from the other proteins of the neurofilament appeared to be hampered by a mixed association of the several components, a nearly homogeneous product was obtained for study. Sedimentation equilibrium experiments in buffers containing 8 M urea showed the molecule to be a monomer with a molecular weight of 70 600 +/- 2000. Circular dichroic spectra taken under the same conditions gave no evidence of residual alpha-helix. Molecular sieve chromatography in 8 M urea on controlled-pore glass showed that the molecule eluted at an unexpectedly small volume. The small elution volume did not depend significantly on protein concentration and is unlikely to be the result of intermolecular association. Rather, the monomer probably has a conformation more rigid or extended than a classical random coil. When dialyzed into 0.01 M tris(hydroxymethyl)aminomethane/1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid/0.1 mM dithioerythritol, pH 8.5, the protein does not assemble into filaments. Sedimentation velocity reveals that under these conditions it consists mainly of a 4.8S molecular species, containing few large particles; sedimentation equilibrium shows that it is composed of oligomers, the smallest present in significant concentration having a molecular weight approximately that of a trimer. Circular dichroism measurements lead to the interpretation that the molecule has refolded in this buffer into a structure that has approximately 55% alpha-helix. Assembly into filamentous particles resembling neurofilaments occurs when the protein is dialyzed against 0.1 M 2-(N-morpholino)ethane-sulfonic acid/0.1% beta-mercaptoethanol/1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid/0.17 M NaCl, pH 6.5. We suggest that the oligomeric species present in 0.01 M tris(hydroxymethyl)aminomethane may frequently be present in solubilized preparations of intermediate filaments and may represent an intermediate in the assembly process.     

A comparison of the self-association behavior of the plant cyclotides kalata B1 and kalata B2 via analytical ultracentrifugation

The recently discovered cyclotides kalata B1 and kalata B2 are miniproteins containing a head-to-tail cyclized backbone and a cystine knot motif, in which disulfide bonds and the connecting backbone segments form a ring that is penetrated by the third disulfide bond. This arrangement renders the cyclotides extremely stable against thermal and enzymatic decay, making them a possible template onto which functionalities can be grafted. We have compared the hydrodynamic properties of two prototypic cyclotides, kalata B1 and kalata B2, using analytical ultracentrifugation techniques. Direct evidence for oligomerization of kalata B2 was shown by sedimentation velocity experiments in which a method for determining size distribution of polydisperse molecules in solution was employed. The shape of the oligomers appears to be spherical. Both sedimentation velocity and equilibrium experiments indicate that in phosphate buffer kalata B1 exists mainly as a monomer, even at millimolar concentrations. In contrast, at 1.6 mm, kalata B2 exists as an equilibrium mixture of monomer (30%), tetramer (42%), octamer (25%), and possibly a small proportion of higher oligomers. The results from the sedimentation equilibrium experiments show that this self-association is concentration dependent and reversible. We link our findings to the three-dimensional structures of both cyclotides, and propose two putative interaction interfaces on opposite sides of the kalata B2 molecule, one involving a hydrophobic interaction with the Phe6, and the second involving a charge-charge interaction with the Asp25 residue. An understanding of the factors affecting solution aggregation is of vital importance for future pharmaceutical application of these molecules.     

Efficient extraction of proteins from woody plant samples for two-dimensional electrophoresis

Protein extraction from plant samples is usually challenging due to the low protein content and high level of contaminants. Therefore, the 2-DE pattern resolution is strongly influenced by the procedure of sample preparation. Efficient solubilization of proteins strictly depends on the chaotrope and detergent in the extraction buffer. Despite the large number of detergents that have been developed for the use in protein extraction and IEF, there is no single compound able to efficiently extract proteins from any source. Hence, optimization has to be performed for each type of sample. We tested several chaotrope/detergent combinations to achieve optimal solubilization and separation of proteins from Norway spruce [Picea abies (L.) H. Karst.] needles and European beech (Fagus sylvatica L.) leaves and roots. The same chaotrope mixture (7 M urea, 2 M thiourea) was found to be suitable for the extraction and separation of proteins from all samples. Nonetheless, the efficiency of the surfactants tested varied between samples so that optimal extraction and separation was achieved with different detergents or combination of detergents for each sample. The 2-DE separation of spruce needle proteins was optimal in a mixture of two zwitterionic detergents (2% CHAPS and 2% decyl dimethylammonio propanesulfonate). Beech proteins were best separated in buffers containing sugar-based detergents (2% n-octyl beta-D-glucopiranoside in the case of leaf samples and 2% dodecyl maltoside for the root samples). IEF was performed in buffers with the same composition as the extraction buffer except for the root proteins that were better focused in a buffer containing 2% CHAPS.     

Human proton coupled folic acid transporter is a monodisperse oligomer in the lauryl maltose neopentyl glycol solubilized state

The human proton coupled folic acid transporter PCFT is the major import route for dietary folates. Mutations in the gene encoding PCFT cause hereditary folic acid malabsorption, which manifests itself by compromised folate absorption from the intestine and also in impaired folate transport into the central nervous system. Since its recent discovery, PCFT has been the subject of numerous biochemical studies aiming at understanding its structure and mechanism. One major focus has been its oligomeric state, with some reports supporting oligomers and others a monomer. Here, we report the overexpression and purification of recombinant PCFT. Following detergent screening, n-Dodecyl β-D-maltoside (DDM) and lauryl maltose neopentyl glycol (LMNG) were chosen for further work as they exhibited the most optimal solubilization. We found that purified detergent solubilized PCFT was able to bind folic acid, thus indicating a functionally active protein. Size exclusion chromatography showed that PCFT in DDM was polydisperse; the LMNG preparation was clearly monodisperse but with shorter retention time than the major DDM peak. To assess the oligomeric state negative stain electron microscopy was performed which showed a particle with the size of a PCFT dimer.     

Equilibrium between monomers and oligomers of soluble Ca2+-ATPase during the functional cycle

Molecular sieve HPLC shows that soluble sarcoplasmic reticulum Ca2+-ATPase at low concentrations of the non-ionic detergent octaethylene glycol monododecyl ether exists as monomers in equilibrium with dimers and higher oligomers. Binding of vanadate or ATP as well as phosphoenzyme turnover shifts the equilibrium towards the monomer. This suggests that the Ca2+-pump cycle can occur without transient self-association of Ca2+-ATPase peptides.     

Purification and characterization of human erythrocyte glucose transporter in decylmaltoside detergent solution.

The facilitative glucose transporter from human erythrocyte membrane, Glut1, was purified by a novel method. The nonionic detergent decylmaltoside was selected for solubilization on the basis of its efficiency to extract Glut1 from the erythrocyte membrane and its ability to maintain the protein in a monodisperse state. A positive, anion-exchange chromatography protocol produced a Glut1 preparation of 95% purity with little copurified lipid. This protein preparation exhibited cytochalasin B binding in detergent solution, as measured by tryptophan fluorescence quenching. The transporter existed as a monomer in decylmaltoside, with a Stokes radius of 50 A and a molecular mass of 147 kDa for the protein-detergent complex. We screened detergent, pH, additive, and lipid and have found conditions to maintain Glut1 monodispersity for 8 days at 25 degrees C or over 5 weeks at 4 degrees C. This Glut1 preparation represents the best available material for two- and three-dimensional crystallization trials of the human glucose transporter protein.     

Enhanced solubilization of membrane proteins by alkylamines and polyamines

Around 25% of proteins in living organisms are membrane proteins that perform many critical functions such as synthesis of biomolecules and signal transduction. Membrane proteins are extracted from the lipid bilayer and solubilized with a detergent for biochemical characterization; however, their solubilization is an empirical technique and sometimes insufficient quantities of proteins are solubilized in aqueous buffer to allow characterization. We found that addition of alkylamines and polyamines to solubilization buffer containing a detergent enhanced solubilization of membrane proteins from microsomes. The solubilization of polygalacturonic acid synthase localized at the plant Golgi membrane was enhanced by up to 9.9‐fold upon addition of spermidine to the solubilization buffer. These additives also enhanced the solubilization of other plant membrane proteins localized in other organelles such as the endoplasmic reticulum and plasma membrane as well as that of an animal Golgi‐localized membrane protein. Thus, addition of alkylamines and polyamines to solubilization buffer is a generally applicable method for effective solubilization of membrane proteins. The mechanism of the enhancement of solubilization is discussed.     

Post-synthetic and site-specific modification of endocyclic nitrogen atoms of purines in DNA and its potential for biological and structural studies

Site-specific modification of the N1-position of purine was explored at the nucleoside and oligomer levels. 2′-Deoxyinosine was converted into an N1-2,4-dinitrophenyl derivative 2 that was readily transformed to the desired N1-substituted 2′-deoxyinosine analogues. This approach was used to develop a post-synthetic method for the modification of the endocyclic N1-position of purine at the oligomer level. The phosphoramidite monomer of N1-(2,4-dinitrophenyl)-2′-deoxyinosine 9 was prepared from 2′-deoxyinosine in four steps and incorporated into oligomers using an automated DNA synthesizer. The modified base, N1-(2,4-dinitrophenyl)-hypoxanthine, in synthesized oligomers, upon treatment with respective agents, was converted into corresponding N1-substituted hypoxanthines, including N1-¹⁵N-hypoxanthine, N1-methylhypoxanthine and N1-(2-aminoethyl)-hypoxanthine. These modified oligomers can be easily separated and high purity oligomers obtained. Melting curve studies show the oligomer containing N1-methylhypoxanthine or N1-(2-aminoethyl)-hypoxanthine has a reduced thermostability with no particular pairing preference to either cytosine or thymine. The developed method could be adapted for the preparation of oligomers containing mutagenic N1-β-hydroxyalkyl-hypoxanthines and the availability of the rare base-modified oligomers should offer novel tools for biological and structural studies.     

Functional properties of acetylcholine receptor monomeric and dimeric forms in reconstituted membranes

The dimeric and monomeric forms of the acetylcholine receptor from $\text{Torpedo californica}$ electroplax have been purified in the presence of lipids and reconstituted. A spectroscopic method was applied to study the rapid kinetics of cation transport mediated by each of the reconstituted AcChR oligomers. Both the AcChR dimer and monomer responded to carbamylcholine by mediating cation transport on the time scale of a few milliseconds. The responses to carbamylcholine were blocked by histrionicotoxin and by desensitization, demonstrating that both forms manifest pharmacological properties observed $\text{in vivo}$. Analysis of the fast ion transport produced by various agonist concentrations yielded estimated rates of transport through a single receptor channel. These were comparable for the monomer and dimer and in agreement with those obtained for a preparation containing a mixture of both oligomers.     

Electrophoretic mobility is a reporter of hairpin structure in single-stranded DNA oligomers

The electrophoretic mobilities of 24 single-stranded DNA oligomers, each containing 26 nucleotide residues, have been measured in polyacrylamide gels and in free solution. The mobilities observed at 20 degrees C differed by approximately 20% in polyacrylamide gels and by approximately 10% in free solution, even though the oligomers contained the same number of bases. Increasing the temperature or adding urea to the solution equalized the mobilities of the oligomers, suggesting that the variable mobilities observed at 20 degrees C are due to the formation of stable secondary structures, most likely hairpins. Thermal melting profiles were measured for eight oligomers in 40 mM Tris acetate buffer. The observed melting temperatures of most oligomers correlated roughly with the mobilities observed at 20 degrees C; however, one oligomer was much more stable than the others. The melting temperatures of four of the oligomers were close to the values predicted by DINAMelt [Markham, N. R., and Zuker, M. (2005) Nucleic Acids Res. 33, W577-W581]; melting temperatures of the other oligomers differed significantly from the predicted values. Thermal melting profiles were also measured for two oligomers as a function of the Tris acetate buffer concentration. The salt concentration dependence of the melting temperatures suggests that 0.15 Tris+ ion per phosphate is released upon denaturation. Because the apparent number of Tris+ ions released is greater than that observed by others for the release of Na+ ions from similar hairpins, the results suggest that DNA hairpins (and, presumably, duplexes) bind more Tris+ ions than Na+ ions in solution.     

Self-assembly of a high molecular weight basement membrane heparan sulfate proteoglycan into dimers and oligomers

A high molecular weight basement membrane heparan sulfate proteoglycan, isolated from murine Englebreth-Holm-Swarm tumor, is seen in platinum replicas as an elongated flexible core (Mr = 450,000) consisting of a series of tandem globular domains from which extend, at one end, two to three heparan sulfate chains (average Mr = 80,000 each). This macromolecule will self-assemble into dimers and lesser amounts of oligomers when incubated in neutral isotonic buffer. These molecular species can be separated by zonal velocity sedimentation and assembly is seen to be time- and concentration-dependent. In rotary-shadowed platinum replicas the binding region is found at or near the end of the core at the pole opposite the origin of the heparan sulfate chains. Dimers are double-length structures and oligomers are seen as stellate clusters: in both, the heparan sulfate chains appear peripherally oriented. While isolated cores self-assemble, isolated heparan sulfate chains do not bind intact proteoglycans. Furthermore, proteolytic removal of a non-heparan sulfate containing core moiety destroys the ability of the proteoglycan monomer to form larger species or bind intact proteoglycan, further supporting the binding topography determined morphologically. These negatively charged macromolecular complexes may be important contributors to basement membrane structure and function.     

Electron-microscopic and chemical studies of oligomers in horse ferritin

Horse ferritin was fractionated both by starch-gel electrophoresis and by gel filtration on Sephadex G-200. Monomer fractions contained up to 98% of monomer and oligomer fractions up to 76% of oligomers as determined by quantitative electron microscopy. Percentages obtained from electron micrographs correlated well with analytical starch-gel electrophoretograms and ultracentrifuge patterns. Amino acid analyses of monomer- and oligomer-enriched fractions showed no significant differences. Ferritin oligomers did not apparently dissociate on dilution for electron microscopy or on storage. Apoferritin dimers were stable in 0·01m-phosphate buffer at dilutions down to 0·19mg./ml. as shown by ultracentrifugation. Chemical studies indicated that the intermolecular bonds in oligomers are resistant to a variety of reagents and conditions, including those that would be expected to attack disulphide, peptide and ester linkages respectively. Partial disaggregation was achieved at high pH values and in 67% (v/v) acetic acid. Centre-to-centre intermolecular distances in dimers were found to be about 100å. Three main types of trimer configuration were found and a variety of tetramers and pentamers. These configurations are described and discussed.     

Chemical and hydrodynamic characterization of the human leucocyte receptor for complement subcomponent C1q.

A procedure for preparation of the receptor for complement subcomponent Clq from human tonsil lymphocytes and the monocytic cell line U937 was developed. The procedure is suitable for isolation of several hundred micrograms of the receptor, Clq-R, and has yielded sufficient material for chemical and hydrodynamic characterization. Clq-R from tonsil lymphocytes behaves identically with that from U937 cells. Clq-R has a monomer Mr of 56,000, and is an acidic glycoprotein containing about 17% carbohydrate. The polypeptide chain length is estimated to be 416-448 amino acid residues, with two or three sites for N-linked glycosylation. Detergent-solubilized Clq-R exists as an elongated dimer (f/fo = 1.8), and does not bind a significant weight of detergent. The radioiodinated isolated receptor binds specifically and saturably to solid-phase Clq, but not to collagen, IgG, bovine serum albumin or complement component C3.