Protein--non-ionic detergent interaction. Interaction of bovine serum albumin with alkyl glucosides studied by equilibrium dialysis and infrared spectroscopy.

The binding isotherms of bovine serum albumin with octylglucoside and decyl glucoside were determined at 7 degrees C and 25 degrees C at pH 7.4 and ionic strength 0.1 M. The average number of detergent molecules bound was found to increase with increasing hydrocarbon chain length. Competitive binding indicates that alkylglycosides combine with the same sites as alkyl sulphates. Native bovine serum albumin has about 12 and 10 sites for non-ionic ligands at 7 degrees C and about 15 and 13 sites at 25 degrees C for octyl and decyl glucosides respectively. The values for standard free energy changes--delta G0, were calculated from the intrinsic association constants. Fourier-transformed infrared spectroscopy was used to study the effects of alkyl glucosides on the conformation of albumin. The results obtained indicate that there are no significant changes in protein structure.     

Interaction of the mouse and bovine myelin basic proteins and two cleavage fragments with anionic detergents

The binding of deoxycholate and dodecyl sulfate to the mouse and bovine myelin basic proteins and two peptide fragments, obtained by cleavage of the bovine basic protein at its single tryptophan residue, was examined. Complete equilibrium binding isotherms for both detergents were obtained by examining their binding to each of the polypeptides immobilized on agarose. The bulk of the binding of dodecyl sulfate was found to be highly cooperative, and at saturation all four polypeptides bound far more detergent than globular, water-soluble proteins. The sum of the dodecyl sulfate bound by each of the two bovine basic protein cleavage fragments was almost twice that bound by the intact protein at saturation, suggesting that cleavage of the bovine basic protein exposes sites for additional binding of dodecyl sulfate. At pH values below pH 8.0, an additional cooperative transition was observed below the critical micelle concentration of sodium dodecyl sulfate in the binding isotherms of all four polypeptides. The midpoint of this transition corresponded to an apparent pK of approximately 5.5; however, the destruction of 90% of the histidine residues in the bovine basic protein had no effect on this transition. At pH 9.2 and moderate ionic strength (I = 0.1), the bulk of the binding of deoxycholate to the mouse and bovine basic proteins occurred at and above the critical micelle concentration of the detergent; and saturation values of deoxycholate binding to these two proteins were considerably higher than that reported for globular, water-soluble proteins. In marked contrast to the results with dodecyl sulfate, neither cleavage fragment was observed to bind deoxycholate. The results suggest that the higher ordered structure of the bovine basic protein may play an important role in the binding of anionic amphiphiles to the protein.     

Interaction of lysozyme with dyes. II. Binding of bromophenol blue

The binding of lysozyme with bromophenol blue (BPB) at various dye concentrations and pH was carried out at 25 degrees C by equilibrium dialysis, ultraviolet (UV) difference and circular dichroism (CD) spectral techniques. Binding isotherms at pH 5.0 show non-cooperative binding at low dye concentrations, which change over to cooperative binding at higher concentrations indicating biphasic nature. However, binding isotherms at pH 7.0 and 9.0 show cooperative binding only, at all concentrations of the dye. The number of available binding sites decreases with the increase of pH. Gibbs free energy change, calculated on the basis of Wyman's binding potential concept, decreases with the increase of pH. Binding isotherms at pH 5.0 obtained at a lower temperature of 8 degrees C, also indicate the biphasic nature similar to those observed at 25 degrees C, but with a slight decreased strength of binding. The UV difference spectra of the complex do not show any distinct peaks in the 285 to 297 nm region eliminating any possible interaction of BPB with tryptophan and tyrosine residues of the lysozyme molecule. The CD spectra of lysozyme-BPB complex show a decrease in ellipticities with reference to native lysozyme in the near UV and far UV regions. This indicates that the lysozyme-BPB complex has a lower helical content probably due to the conformational changes induced into the native enzyme. The appearance of new positive peaks at 315 nm in the near UV region and at 592 nm in the visible region of the CD spectra may be due to the induced asymmetry into the BPB molecule as a result of its binding to a cationic residue (probably a lysine residue) of lysozyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ferritin. Binding of beryllium and other divalent metal ions

Rat liver homogenates in 0.1 M Tris, pH 7.5, were heated to 80 degrees C, cooled immediately, and centrifuged at 24,000 X g, and 7Be2+ was added to the supernatant. Twenty-five per cent of the radioactivity was bound to a single protein. It was purified to homogeneity and identified to be ferritin as judged by different criteria. These were sucrose density gradient centrifugation, electrophoresis in polyacrylamide gel of the native or sodium dodecyl sulfate-treated protein, reactivity to antibodies, isoelectric focusing, and total amino acid composition. Comparative study of the ability of ferritin or apoferritin to bind Cd2+, Zn2+, Cu2+, and Be2+ was conducted by using a gel equilibrium technique, Centifree micropartition technique, and microcentrifuge desalting technique. Ferritin could be saturated with Cd2+ or Zn2+ or Cu2+ but not with Be2+ even after 800 g atoms of Be2+ were bound. None of the bound Be2+ was dialyzable at 4 degrees C in 0.05 Tris acetate buffer, pH 8.5, but at pH 6.5 over 80% of the bound metal ion was dialyzed after 72 h. By contrast, apoferritin bound similar amounts of all four metal ions, some of which were dialyzable. By spectrophotometric titrations at pH 6.5 of Be2+ with sulfosalicylic acid (SSA), BeKDSSA was calculated to be 5.0 X 10(-6) M and by competition of sulfosalicyclic acid and ferritin for Be2+ the BeKDferritin was calculated to be 6.8 X 10(-6) M.     

Reduced lysozyme in solution and its interaction with non-ionic surfactants.

Reduced lysozyme at pH 2.5 bound poly(oxyethylene) alkylethers in two steps and the maximum bound amount Qmax of the surfactant reached as large as 0.5-0.7 mole per mole amino acid residue in the cooperative binding step. Binding isotherms were well superimposed when surfactant concentrations were normalized by respective values of the critical micelle concentration, cmc. In terms of the onset concentrations of the cooperative binding C*, hydrophobicity of reduced lysozyme was quantitatively defined as RT In (cmc/C*) which amounted to 670 J per mole surfactant and was unique to the protein irrespective of the kind of surfactant. Qmax could be used as another measure of the hydrophobicity of the protein. The binding isotherms were evaluated by two methods: equilibrium dialysis and surface tension. Their results were consistent with each other and rather complementary. Reduced lysozymes were molecularly dispersed at pH below 2.5 in 0.01 M NaCl but aggregation took place as pH increased. The aggregates could not be dissociated on dilution nor by the addition of nonionic surfactants but by lowering pH. The irreversible nature of the aggregation was reasonably interpreted with a model based on the 'entangled' arrangement of the beta-sheets, which could account for the irreversible aggregation of unfolded proteins in general.     

Hydroxylaminolysis of penicillin binding componenets is enzymatically catalyzed.

The hydroxylaminolysis of the penicilloyl moiety from [14C]penicillin G binding component (PBC) complexes of the Bacillus subtilis D-alanine carboxypeptidase and of the mixture of PBC's of Staphylococcus aureus was inhibited by denaturation of the complexes by heat (55 degrees), detergent (1% sodium dodecyl sulfate), or trichloroacetic acid. The kinetics of inhibition by denaturation were comparable to those of the inhibition of [14C]penicillin G binding to the PBC's and of carboxypeptidase activity of the B. subtilis enzyme under identical denaturing conditions. These data establish that the hydroxylaminolysis is an enzymatically catalyzed process suggesting that penicillin G is bound to an enzymatically active site. Treatment of the denatured [14C]penicillin G-carboxypeptidase complex with sodium borohydride or at pH 12 resulted in the release of the penicilloyl moiety. These results are consistent with a carboxylic ester bond for the penicilloyl-PBC instead of a thiolester linkage as was initially presumed.     

Interactions of free and immobilized myelin basic protein with anionic detergents

The interaction of free and immobilized myelin basic protein (MBP) with sodium deoxycholate (DOC) and sodium dodecyl sulfate (NaDodSO4) was studied under a variety of conditions. Free MBP formed insoluble complexes with both detergents. Analysis of the insoluble complexes revealed that the molar ratio of detergent/MBP in the precipitate increased in a systematic fashion with increasing detergent concentration until the complex became soluble. At pH 4.8, equilibrium dialysis studies indicated that approximately 15 mol of NaDodSO4 could bind to the protein without precipitation occurring. Regardless of the surfactant, however, minimum protein solubility occurred when the net charge on the protein-detergent complex was between +18 and -9. Complete equilibrium binding isotherms of both detergents to the protein were obtained by using MBP immobilized on agarose. The bulk of the binding of both detergents was highly cooperative and occurred at or above the critical micelle concentration. At I = 0.1, saturation levels of 2.09 +/- 0.15 g of NaDodSO4/g of protein and 1.03 /+- 0.40 g of DOC/g of protein were obtained. Below pH 7.0 the NaDodSO4 binding isotherms revealed an additional cooperative transition corresponding to the binding of 15-20 mol of NaDodSO4/mol of protein. Affinity chromatography studies indicated that, in the presence of NaDodSO4 (but not in its absence), [125I]MBP interacted with agarose-immobilized histone, lysozyme, and MBP but did not interact with ovalbumin-agarose. These data support a model in which the detergent cross-links and causes precipitation of MBP-anionic detergent complexes. Cross-linking may occur through hydrophobic interaction between detergents electrostatically bound to different MBP molecules.     

Thermodynamics of binding interactions between bovine beta-lactoglobulin A and the antihypertensive peptide beta-Lg f142-148

The binding capacity of bovine beta-lactoglobulin variant A (beta-Lg A) for six peptides derived from beta-Lg was evaluated using an ultrafiltration method under the following conditions: pH 6.8, 40 degrees C, and a beta-Lg A/peptide molar ratio of 1:5. Only peptides beta-Lg f102-105, f142-148, and f69-83 bound in significant amounts to beta-Lg A corresponding to 1.5, 1.1, and 0.7 mol of peptide per mole of beta-Lg A, respectively. The interaction between beta-Lg A and the antihypertensive peptide beta-Lg f142-148 was investigated further by isothermal titration calorimetry. The binding isotherms at pH 6.8 and 25 degrees C confirmed that beta-Lg f142-148 bound to beta-Lg A and that the interaction followed a sequential three-site binding model with constants of association of 2 x 10(3), 1 x 10(3), and 0.4 x 10(3) M(-1) for the first, second, and third binding sites, respectively. The enthalpy of binding was exothermic for the first and second binding sites and endothermic for the third binding site. Binding of the peptide to all three sites was spontaneous as shown by the negative free energy values. These results show for the first time that beta-Lg A can bind bioactive peptides. This potential could be exploited to transport bioactive peptides and protect them in the gastrointestinal tract following their oral administration as nutraceuticals.     

Parameters of interaction of sodium dodecyl sulfate with human hemoglobin

It was shown that sodium dodecyl sulfate at concentrations not exceeding the critical micelle concentrations (0-1.9 mM) induced the conversion of oxy- and methemoglobin but not deoxyhemoglobin to hemichrome. The concentration dependences of hemichrome formation were represented as Hill plots, and the parameters of detergent binding were estimated. OxyHb in 20 mM potassium-phosphate buffer, pH 6.8, has two groups of binding sites: the first group is characterized by the Hill constant n1 = 2 and the concentration of half saturation [SDS]50 = 0.8 mM, and the second group is characterized by the Hill constant n2 = 8 and [SDS]50 = 0.9 mM. In the case of metHb one group of binding sites with the Hill constant n = 2 and half saturation concentration [SDS]50 = 0.2 mM was observed. An increase in environmental pH to 7.9 decreased the affinity of Hb for SDS. It is suggested that primary binding sites for SDS in oxyHb coincide with the anion-binding center of the Hb molecule. The interaction of the detergent with these binding sites induced a structural transition of the hemoprotein molecule. As a result of this transition, secondary binding sites were exposed. In a model system (hemin--imidazole in ethanol solution), the enthalpy of the transition of hemin from a high-spin to a low-spin state was estimated to be 47 +/- 7 kJ/mol.     

Chloride ion binding to human plasma albumin from chlorine-35 quadrupole relaxation.

The 35Cl nuclear magnetic quadrupole relaxation enhancement on binding of chloride ions to human plasma albumin (HPA) has been studied under conditions of variable temperature, pH, ionic strength, protein, and sodium dodecyl sulfate concentration. A small number (less than 10) of chloride ions, most of which are bound to the primary detergent binding sites, contribute a major portion of the relaxation enhancement (greater than 80% at neutral pH). A comparison of the pH dependence of the relaxation rate with the hydrogen ion titration curve, which was determined and analyzed, identified ten lysyl and arginyl residues as being involved in the chloride ion binding. These data, in conjuction with NaDodSO4 titrations at different pH values and the amino acid sequence of HPA, suggests that the high-affinity chloride-binding sites are doubly cationic at neutral pH. An irreversible dimerization at acidic pH and 5 x 10(-5) m HPA was detected. The data also indicate the presence of internal modes of motion in the expanded forms of the HPA molecule, probably an independent reorientation of domains. The rate of exchange of chloride ions was shown to be much higher than the corresponding intrinsic relaxation rate in the temperature range 2--26 degrees C and pH values ranging from 4.0 to 10.5. No indications of protein-protein interaction could be found up to the physiological concentration of ca. 6 x 10(-4)m HPA at either neutral or alkaline pH. The mechanistic basis for HPA's exceptional capacity for binding of inorganic anions was discussed.     

Thermodynamic studies on the interaction between sodium n-dodecyl sulphate and histone H2B

The thermodynamic parameters for the interaction of the anionic detergent sodium n-dodecyl sulphate (SDS) with H2B at pH 3.2, 6.4 and 10 have been measured at 27 degrees C and 37 degrees C by equilibrium dialysis to determine the Gibbs energies of detergent binding. The data have been used to obtain the enthalpy of interaction from the temperature dependence of the equilibrium constants from the Van't Hoff relation. The enthalpy of interaction between H2B and SDS is endothermic at pH 3.2, 6.4 and 10. The shapes of the enthalpy curves at pH 3.2 and 10 show some small exothermic contribution which probably indicates folding of H2B. The interactions of H2B-SDS are dominated by the increase in entropy on detergent binding. The larger negative free energy, enthalpy and entropy changes at pH 6.4 are consistent with greater denaturation relative to pH 3.2 and 10.     

Romanowsky dyes and the Romanowsky-Giemsa effect. 4. Binding of azure B to DNA

We investigated the binding of azure B to DNA (calf thymus) over a wide range of concentrations of the dye (CF) and the nucleic acid (CN) using absorption spectroscopy [CF and CN represent the total concentrations of the ye (F) and the mononucleotide units (N) of the DNA, respectively]. The binding isotherms of the dye to DNA in aqueous solutions were determined. In addition, we analysed the composition of insoluble DNA/azure B precipitates that are formed in presence of an excess of azure B. These precipitates are of particular interest, because Giemsa staining is usually performed using high dye concentrations. Azure B easily forms dimers in aqueous solutions. When determining the binding isotherms, the equilibrium between free monomers and dimers must be taken into account. Therefore, we determined the dimerisation constant (Kd) of azure B from the concentration dependency of its absorption spectra in water at the standard temperature T = 298 K (25 degrees C), Kd = 6.5 X 10(3) M-1 (experimental conditions: tris buffer, pH 7.2; concentration of Na ions, CNa = 0.002 M). As the CNa value increases, the dimerisation constant rises rapidly. When the azure B concentration is very low and there is an excess of DNA, ordinary Scatchard and Langmuir isotherms are observed. Monomer dye cations are bound to DNA, these cations being in equilibrium with free monomers in the solution. In order to obtain the Scatchard binding constant (Ks) and the binding parameter (n) spectroscopically, it is necessary to determine the extinction coefficient (epsilon Fb) of the monomer bound (b) dye molecules (F) at one analytical wave number (upsilon a). The three constants can be determined simultaneously using an iterative technique that combines Scatchard isotherms and the Benesi-Hildebrand extrapolation, CN----infinity. We obtained Ks = 1.8 X 10(5) M-1 and n = 0.18 (25 degrees C; tris buffer, pH 7.2; CNa = 0.002 M). At very low dye (CF) and competitor (CNa) concentrations, only 18% of the anionic binding sites of the DNA are capable of binding the dye cations. With increasing CNa values the concentration of bound azure B cations decreases rapidly. The Na cations displace the bound dye cations and act as a competitor. The Ks value also greatly depends on the competitor concentration (CNa).(ABSTRACT TRUNCATED AT 400 WORDS)     

delta-Aminolevulinic acid synthetase from rat liver mitochondria. Purification and properties.

delta-Aminolevulinic acid synthetase has been purified from liver mitochondria of young, uninduced rats. After nonionic detergent solubilization of mitochondrial inner membrane-matrix fractions, the enzyme was purified to a specific activity of approximately 2,000 nmol of delta-aminolevulinic acid formed/h/mg of protein at 30 degrees C, by means of ammonium sulfate precipitation, diethylaminoethyl cellulose chromatography, Sephacryl chromatography, and preparative gel electrophoresis. The purified enzyme preparation thus obtained was apparently homogeneous as judged by its migration as a single band with a molecular weight of 58,000 +/- 6,000 upon electrophoresis in sodium dodecyl sulfate polyacrylamide gels. The native enzyme probably exists as a dimer with a molecular weight of approximately 120,000. A pH optimum of 7.5 and an isoelectric point of 4.5 were also determined. Both monovalent cations and hemin strongly inhibited the activity of the purified enzyme.     

Determination of partial unfolding enthalpy for lysozyme upon interaction with dodecyltrimethylammonium bromide using an extended solvation model

The interactions of dodecyltrimethylammonium bromides (DTABs) with hen egg lysozyme have been investigated at pH = 7.0 and 27 degrees C in phosphate buffer by isothermal titration calorimetry. DTAB interacts endothermically and activate lysozyme. The endothermicity of the lysozyme-DTAB interaction is in marked contrast to the exothermic interactions between sodium dodecyl sulphate (SDS) and lysozyme which have been attributed to specific binding between the anionic sulphate head groups and cationic amino acid residues. The enthalpies of interaction between the cationic surfactant (DTAB) and lysozyme are dominated by the endothermic unfolding of the native structure followed by an exothermic solvation of the lysozyme-DTAB complex by the addition of extra DTAB. A new direct calorimetric method to follow protein denaturation, and the effect of surfactants on the stability of proteins was introduced. The extended solvation model was used to reproduce the enthalpies of lysozyme-DTAB interaction over the whole range of DTAB concentrations. The solvation parameters recovered from the new equation, attributed to the structural change of lysozyme and its biological activity. At low concentrations of DTAB, the binding is mainly electrostatic, with some simultaneous interaction of the hydrophobic tail with nearby hydrophobic patches on the lysozyme. These initial interactions presumably cause some protein unfolding and expose additional hydrophobic sites. The DTAB-induced denaturation enthalpy of lysozyme is 86.46 +/- 0.02 kJ mol(-1).     

A thrombin receptor in resident rat peritoneal macrophages.

Resident rat peritoneal macrophages possess 6 x 10(2) high-affinity binding sites per cell for bovine thrombin with a Kd of 11 pM, and 7.5 x 10(4) low-affinity sites with a Kd of 5.8 nM. These binding sites are highly specific for thrombin. Half-maximal binding of 125I-labeled bovine thrombin is achieved after 1 min at 37 degrees C, and after 12 min at 4 degrees C. The reversibly bound fraction of the ligand dissociates according to a biexponential time course with the rate constants 0.27 and 0.06 min-1 at 4 degrees C. Part of the tracer remains cell-associated even after prolonged incubation, but all cell-associated radio-activity migrates as intact thrombin upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The bound thrombin is minimally endocytosed as judged by the resistance to pH 3 treatment, and the receptor does not mediate a quantitatively important degradation of the ligand. The binding is not dependent on the catalytic site of thrombin, since irreversibly inactivated thrombin also binds to the receptor. 125I-labeled thrombin covalently cross-linked to its receptor migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a Mr 160,000, corresponding to an approximate receptor size of Mr 120,000.     

Protein chromatography on adsorbents with hydrophobic and ionic groups. Chromatography of dodecyl sulphate-solubilized proteins of the human erythrocyte membrane on N-(3-carboxypropionyl)aminodecyl-sepharose.

Human erythrocyte 'ghosts' were solubilized in 0.5% (w/v) sodium dodecyl sulphate at pH 4.0(I = 0.012 mol/I). At a loading of 1-2 mg of protein/ml of column volume, all of membrane proteins were adsorbed to a column of CPAD [N-(3-carboxypropionyl)-aminodecyl]-Sepharose at pH 4.0 (I = 0-012 mol/1) and room temperature (22 degrees C). Many proteins were subsequently desorbed by raising the pH or by including sodium dodecyl sulphate continuously in the eluting buffer. Experiments with a series of adsorbents homologous with CPAD-Sepharose, in which the length of the hydrocarbon chain was varied, provided strong evidence of hydrophobic interactions, in addition to ionic interactions, in the binding of these proteins to CPAD-Sepharose. Elution with increasing-pH gradients at different concentrations of sodium dodecyl sulphate showed that glycophorin (the major sialoglycoprotein) was eluted in the void volume, at recoveries close to 100%, when the detergent concentration was greater than or equal to 0.3% (w/v). Protein E, the major protein, was desorbed late in the pH gradient even at a high (0.5%, w/v) concentration of the detergent, and was always incompletely desorbed, the maximum recovery recorded being 40%. Spectrin (the high-molecular-weight polypeptide pair) did not behave in a well-defined manner, and was found widely distributed among the effluent fractions under all the conditions that were tested.     

Urokinase binds to a plasminogen activator inhibitor type-2-like molecule in placental microvillous membranes

Placental microvillous membranes exhibited saturable binding of urokinase-type plasminogen activator with plateau achieved by 30 min at 4 degrees C and 10 min at 37 degrees C. The binding was essentially irreversible. The capacity was about 8 pmol urokinase per mg membrane protein. Half-maximal displacement of 125I-labelled urokinase was achieved with about 1.0 nM unlabelled urokinase when using 75 micrograms membrane protein/ml. 125I-labelled urokinase did not bind when treated with diisopropylfluorophosphate to block the catalytic activity. Single-chain urokinase (prourokinase), devoid of catalytic activity, did not bind. Catalytically active tissue-type plasminogen activator did compete with 125I-labelled urokinase for binding although less efficiently than urokinase. Binding activity remained in the 100,000 x g pellet after treatment of the membranes with 3 M KCl, alkaline stripping at pH 12 or extraction by the detergent Triton X-100. The binding was essentially blocked by antibodies against plasminogen activator inhibitor-type-2 (PAI-2). Sodium dodecyl sulfate polyacrylamide gel electrophoresis of solubilized membranes with bound 125I-labelled urokinase showed that the urokinase-PAI-2 complexes largely migrated in fractions corresponding to a very large Mr although no clearly defined peaks were observed. It is suggested that PAI-2 occurs in a form anchored to syncytiotrophoblast microvilli, possibly to the cytoskeleton.     

The interaction between Beta-lactoglobulin and sodium N-dodecyl sulphate.

1. The binding of sodium n-dodecyl sulphate to beta-lactoglobulin was studied in the pH range 3.5-7.0 by equilibrium dialysis, ultracentrifugation and microcalorimetry. 2. At low binding concentrations (less than 30 bound surfactants anions per protein molecule) the complexes formed aggregates in solution. 3. At higher binding concentrations aggregation does not occur at low ionic strength (0.01 mol/litre), but continues at high ionic strength (0.1 mol/litre). 4. At 25 degrees C the enthalpy of interaction of sodium n-dodecyl sulphate with beta-lactoglobulin can be interpreted as the sum of the enthalpies of formation of a complex with 2 bound surfactant anions, with an enthalpy change of -9.5 kJ-mol-1 of bound surfactant, and complexes containing at least 22 bound surfactant anions, with limiting enthalpies per bound surfactant anion of -12.4 kJ-mol-1 at pH 3.5 and -3.25 kJ-mol-1 at pH 5.5. 5. The binding of surfactant and the enthalpy of interaction at pH 3.5 ARE NOT SIGNIFICANTLY AFFECTED BY THE ADDITION Of 8 M-urea. 6. The data indicate that at low binding concentrations the interaction is of an ionic nature, and is accompanied by a conformational change in the protein.     

Purification and characterization of the heterologously expressed trehalose/maltose ABC transporter complex of the hyperthermophilic archaeon Thermococcus litoralis.

We report the purification of the maltose/trehalose transporter complex MalFGK of the hyperthermophilic archaeon Thermococcus litoralis. The complex was expressed in Escherichia coli, solubilized in dodecyl maltoside and purified with the aid of a histidine tag on one of the membrane proteins. One hundred grams of cells yielded 3 mg of pure complex. The final product showed ATPase activity at 70 degrees C and was soluble at low detergent concentration. ATPase activity was not due to dissociation of the MalK subunit from the integral membrane proteins MalF and MalG but could not be further stimulated by trehalose/maltose binding protein (TMBP), be it the native protein as isolated from T. litoralis or the soluble engineered protein. The purified native TMBP was identified as a glycoprotein.     

Isoelectric focussing of the catalytic subunit of (Na,K)-ATPase from pig kidney

The catalytic subunit of sodium and potassium ion transport adenosine triphosphatase was isolated by sodium dodecyl sulfate-polyacrylamide electrophoresis and was subjected to isoelectric focussing on 3.5% acrylamide in 2% Triton X-100, 9 M urea, and 2% Bio-Lyte 3/10 from Bio-Rad Laboratories. At 20 degrees C this resolved 2 equal and closely spaced bands centered at pH 5.5 about 0.04 pH unit apart. The distribution of the polypeptide between the 2 bands came to a temperature-dependent equilibrium during focussing. At 15 degrees C predominantly the acidic band and at 25 degrees C predominantly the alkaline band appeared. Perhaps association of the nonionic detergent with the polypeptide resulted in its partitioning into bands corresponding to different physical states. A change of phase in a polypeptide-detergent complex might have altered its charge. To test functional homogeneity of the subunit in the native enzyme, the active center for ATP binding was covalently labeled with fluorescein isothiocyanate, an acidic ligand. Isoelectric focussing of the derivatized subunit at 20 degrees C showed displacement of all of the alkaline band to the position of the acidic band, which was fluorescent. Isoelectric focussing at 25 degrees C showed displacement of almost half of the alkaline band to the position of the acidic band, and both bands were fluorescent. The results suggest that all of the subunit accepted the fluorescent label and that derivatization slightly raised the temperature at which the polypeptide equilibrated between the 2 states. A few experiments on the calcium-dependent ATPase of sarcoplasmic reticulum indicated that it responded similarly.