Structural studies of cytochrome reductase. Improved membrane crystals of the enzyme complex and crystallization of a subcomplex

Membrane crystals of mitochondrial ubiquinol: cytochrome c reductase of improved size and long-range order and of the cytochrome bc1 subcomplex have been obtained by a dialysis method. The enzyme--Triton X-100 complex was mixed with Triton phospholipid micelles and the Triton slowly removed by dialysis for 48 hours at pH 5.5 at room temperature or above. The effect of varying the pH and temperature on the shape, size and order of the crystals is described.     

Effect of pH on molecular weight and size of fulvic acids in drainage water from peaty grassland in NW Netherlands

Summary The effect of pH on the molecular weight and size of fulvic acids (FA) in a peaty ditch water was studied by dialysis, Sephadex G-25 gel filtration and ultrafiltration. The results of these techniques of fractionating according to particle size indicated that the molecular weight and size of FA decrease with decreasing pH. The ratio of the light absorbances at 250 and 365 nm (E2/E3) of ditch water increased with decreasing pH corresponding with the results of dialysis, gel filtration and ultrafiltration of FA.Our results do not correspond with the ideas of the macromolecular structure of soil FA as developed by Schnitzer¹⁶.     

Cloning, expression, and refolding of a secretory protein ESAT-6 of Mycobacterium tuberculosis

A DNA encoding the 6-kDa early secretory antigenic target (ESAT-6) of Mycobacterium tuberculosis was inserted into a bacterial expression vector of pQE30 resulting in a 6x His-esat-6 fusion gene construction. This plasmid was transformed into Escherichia coli strain M15 and effectively expressed. The expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in cell lysate. The inclusion bodies were solubilized with 8M urea or 6M guanidine-hydrochloride at pH 7.4, and the recombinant protein was purified by Ni-NTA column. The purified fusion protein was refolded by dialysis with a gradient of decreasing concentration of urea or guanidine hydrochloride or by the size exclusion protein refolding system. The yield of refolded protein obtained from urea dialysis was 20 times higher than that from guanidine-hydrochloride. Sixty-six percent of recombinant ESAT-6 was successfully refolded as monomer protein by urea gradient dialysis, while 69% of recombinant ESAT-6 was successfully refolded as monomer protein by using Sephadex G-200 size exclusion column. These results indicate that urea is more suitable than guanidine-hydrochloride in extracting and refolding the protein. Between the urea gradient dialysis and the size exclusion protein refolding system, the yield of the monomer protein was almost the same, but the size exclusion protein refolding system needs less time and reagents.     

Metabolism of hybridoma cells and antibody secretion at high cell densities in dialysis tubing.

The experimental setup, consisting of a bundle of dialysis tubing 2.5 mm in diameter [10-15 kD cutoff, mean pore size 25 A, 20 microns (dry) and 40 microns (wet) wall thickness] inserted into a 1-l glass bioreactor supplied with oxygen and pH electrodes, a porous gas distributor, a sampling tube, and a holder for the eight pieces of dialysis tubing, was developed to investigate the properties and the microenvironment of hybridoma cells enclosed in the tubing during their batch cultivation. The concentrations of low-molecular-weight medium components were the same inside and outside the tubing, and it was possible to control the microenvironment of the cells in the tubing easily. The cell damage caused by mechanical stress was less in the dialysis tubing than in stirred spinner flasks. The influence of the initial cell density in the range from 4 X 10(5) to 1 X 10(8) cells ml-1 and the cultivation time were evaluated according to the total and viable cell concentrations and the cell/cell fragment size distributions. Furthermore, the cell membrane properties, glucose consumption rate, lactate, ammonia and lipid storage material, and the monoclonal antibody production rates as well as intracellular enzyme activities in the culture medium were measured and compared to those in reference cultures in spinner flasks with the same inoculum at low initial cell densities. In dialysis tubing in a concentration range of 5 X 10(6) to 10(8) cells ml-1, the total and viable concentrations of cells remained the same during cultivation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties of fructose 1,6-diphosphate aldolases from spores and vegetative cells of Bacillus cereus

Fructose 1,6-diphosphate aldolase from cells of Bacillus cereus appears to be typical Class II aldolase as judged by its functional and physical properties. Spore and vegetative cell aldolase had similar enzymatic, immunochemical, and heat resistance properties in the absence of calcium, but they differed in their thermal stabilities in the presence of calcium, their Stokes' radii, their mobility in acrylamide gel electrophoresis, and their molecular weights. The pH optimum for both enzymes was 8.5, and their K(m) with respect to substrate was 2 x 10(-3)m. Highly purified spore and vegetative cell aldolases were both heat labile with half-lives of 4 min at 53 C and pH 6.4. In the presence of 3 x 10(-2)m solution of calcium ions, the stability of the spore protein increased 12-fold but the vegetative form became more heat labile. The enhanced stability of the spore aldolase was not diminished by dialysis or gel filtration but was lost after chromatography on diethylaminoethyl cellulose at pH 7.4. Aldolase from vegetative cells exists in an equilibrium mixture of two molecular weights, 115,000 and 79,000 in the approximate ratio of 1:4, respectively. The molecular weight of spore aldolase is 44,000. Spore aldolase was more mobile during electrophoresis than its vegetative cell counterpart because of its smaller size.     

Which electrodic solutions for immobilized pH gradients?

Carrier ampholytes covering a pH range corresponding to, or narrower than, the span of the immobilized pH gradient (IPG) are a most suitable electrodic solution for IPGs. They are able to collect, and completely remove from the gel, much higher amounts of non-buffering ions than are solutions of acidic and basic amino acids. This makes it possible to directly run IPGs just after their polymerization, without the need of a washing step to remove catalysts and unreacted Immobiline monomers. The same applies most advantageously when the gel formulation includes urea and/or detergents. Ions contributed by the sample solution are also prevented from casting high-conductivity ridges around the electrodes, without any need either of a dialysis step or of an increased slab size with pH plateaus. The migration of the sample proteins toward their equilibrium position is faster in the presence of carrier ampholytes. The effective concentrations of the latter are in the range 0.3-1%.     

Structural studies on acid unfolding and refolding of recombinant human interferon gamma

Interferon gamma is distinguished from other types of interferons in its instability upon acid treatment, as demonstrated by a loss of antiviral activity. Acid unfolding and refolding experiments were performed with recombinant DNA derived human interferon gamma. When the protein was subjected to unfolding and refolding, the refolded protein showed two peaks (peaks I and II) in gel filtration which have been shown to differ in size, structure, and antiviral activity. When the smaller, peak II, form was unfolded by dialysis against 0.01 M HCl containing 0.1 M NaCl (pH 2) and refolded by dialysis against various solvents at neutral pH, it re-formed as peak II but also generated peak I, and the ratio of the two forms was dependent on protein concentration and solvent conditions. Higher protein concentrations and higher ionic strength led to a greater ratio of peak I to peak II. Phosphate buffers caused precipitation of peak I. Since peak II is 4-8 times more active than peak I in the antiviral bioassay, generation of peak I by acid treatment of peak II should lead to a decrease in antiviral activity.     

Solvent exchange of buried water and hydrogen exchange of peptide NH groups hydrogen bonded to buried waters in bovine pancreatic trypsin inhibitor

Solvent exchange of 18O-labeled buried water in bovine pancreatic trypsin inhibitor (BPTI), trypsin, and trypsin-BPTI complex is measured by high-precision isotope ratio mass spectrometry. Buried water is labeled by equilibration of the protein in 18O-enriched water. Protein samples are then rapidly dialyzed against water of normal isotope composition by gel filtration and stored. The exchangeable 18O label eluting with the protein in 10-300 s is determined by an H2O-CO2 equilibration technique. Exchange of buried waters with solvent water is complete before 10-15 s in BPTI, trypsin, and BPTI-trypsin, as well as in lysozyme and carboxypeptidase measured as controls. When in-exchange dialysis and storage are carried out at pH greater than or equal to 2.5, trypsin-BPTI and trypsin, but not free BPTI, have the equivalent of one 18O atom that exchanges slowly (after 300 s and before several days). This oxygen is probably covalently bound to a specific site in trypsin. When in-exchange dialysis and storage are carried out at pH 1.1, the equivalent of three to seven 18O atoms per molecule is associated with the trypsin-BPTI complex, apparently due to nonspecific covalent 18O labeling of carboxyl groups at low pH. In addition to 18O exchange of buried waters, the hydrogen isotope exchange of buried NH groups H bonded to buried waters was also measured. Their base-catalyzed exchange rate constants are on the order of NH groups that in the crystal are exposed to solvent (static accessibility greater than 0) and hydrogen-bonded main chain O, and their pH min is similar to that for model compounds. The pH dependence of their exchange rate constants suggests that direct exchange with water may significantly contribute to their observed exchange rate.     

Activation of acid ribonuclease from bovine brain by aluminum.

An acid ribonuclease (optimum pH 6.0) has been purified from bovine brain in a five-step procedure. The preparation appeared homogeneous on SDS-polyacrylamide gel electrophoresis. The molecular size of the acid ribonuclease is 70 kDa and it is a dimeric protein with a subunit molecular size of 35 kDa. The acid RNase was activated by aluminum at low concentration. Preincubation of the acid RNase with 10 microM increased the specific activity of the enzyme 2.3-fold at acid pH, while the effect of aluminum was much weaker at alkaline pH under otherwise the same conditions. A stoichiometry of 1: 1 for the binding aluminum to brain acid RNase was estimated. None of the enzyme-bound aluminum was dissociated by dialysis against 50 mM HEPES, pH 7.0 at 4 degrees C for 24 h. Citrate, EDTA, NaF, and apotransferrin abolished the effects of aluminum on the enzyme. Ribonucleic acid also protected the enzyme against the activation caused by aluminum. These results suggest that accumulation of aluminum in brain may change the regulation of ribonucleic acid metabolism.     

胶束化色胺酮的制备及表征

目的通过制备胶束化色胺酮,增加色胺酮的溶解度,并进一步提高其生物利用度。方法:以酸敏感的腙键连接聚乙二醇和色胺酮,并通过透析法,将聚乙二醇化色胺酮进一步制备成胶束。用动态光散射法测定胶束的粒径分布用透射电镜观察胶束的形貌。通过芘荧光探针法测定胶束的临界胶束浓度。测定胶束在不同pH下的药物释放情况(pH5.5和7.4)。采用薄层色谱法和高效液相色谱法研究腙键的断裂行为。通过CCK-8法比较生理pH和酸性pH下,色胺酮和聚乙二醇化色胺酮胶束(PTMs)对MCF-7细胞的体外细胞毒性。结果:与色氨酸相比,PTMs的溶解度提高了1493倍。制备的胶束粒径为228.8 nm,PDI为0.1,形貌为球形。PTMs的临界胶束浓度为3.5×10^-7mol/L,较低的CMC值表明制备的胶束稳定性高,便于进一步使用。腙键可在酸性条件下发生断裂,且在pH 5.5下,12 h内95%的色胺酮从胶束中释放,而在生理pH下(pH 7.4),药物释放缓慢。在生理条件下胶束的细胞毒性低于色胺酮,说明胶束化色胺酮可降低药物毒性及胶束在生理条件下有一定的稳定性。而在pH 5.5时,色胺酮胶束与色胺酮的细胞毒性相近表明胶束可响应肿瘤细胞内的低pH值成功实现药物释放。结论:胶束化色胺酮不仅能有效改善色胺酮的溶解度,有利于进一步提高其生物利用度,而且是一种很有应用前景的肿瘤靶向前药。     

Preparation and Properties of Nanocolloidal Rhenium Sulfide Solution for Lymphoscintigraphic Methods of Micrometastase Examination

A method for the preparation of a nanocolloidal solution of rhenium sulfide is proposed. It includes the following stages: interaction of ammonium perrhenate and sodium thiosulfate in an aqueous solution of gelatin in an acidic medium with heating to 70–80°C; neutralization to pH 7.0; dialysis against saline; desalination and concentrating by ultrafiltration. The resulting colloidal solution contains 85% of particles with an optimal size of 80–100 nm and can be used as a diagnostic tool for lymphoscintigraphy of sentinel lymph nodes in patients with cancer and other diseases.     

Isolation and properties of chloroperoxidase isozymes

A new purification method for chloroperoxidase from Caldariomyces fumago is described. This method involves dialysis, alumina gel adsorption, DEAE-cellulose chromatography at pH 6, and then at pH 3.85 and crystallization. Two isozymes have been isolated and one has been crystallized. The MWs estimated by gel filtration are 46000 for chloroperoxidase A and 40000 for chloroperoxidase B. The guaiacol peroxidation catalysed by these isozymes is proportional to their chlorination activity.     

Fractionation of the proteinases present in the endosperm of germinating seed of Scots pine, Pinus sylvestris

When fresh extracts of endosperms separated from germinating seeds of Scots pine were dialysed at 5°C, proteinase activity on haemoglobin at pH 3.7 showed only a small initial increase, proteinase activities on casein at pH 5.4 and at pH 7.0 increased several-fold, and all the corresponding inhibitor activities disappeared (Salmia and Mikola 1980, Physiol. Plant. 48: 126–130). To find out what happens during dialysis, both fresh and dialysed extracts were fractionated by gel chromatography on Sephacryl S-200. – The fresh extracts had a major proteinase peak (mol. wt. 42,000) with high activity at pH 3.7 and moderate activities at pH 5.4 and 7.0 (pine proteinase I) and a smaller peak (mol. wt. 30,000) with high activity at pH 5.4 and 7.0 and smaller activity at pH 3.7 (pine proteinase II). In dialysed extracts the situation was reversed: the peak of proteinase I was very small while the peak of proteinase II was very high. Apparently, proteinase I is largely inactivated during dialysis while the activity of proteinase II increases, at least partly due to destruction of inhibitors. – The two enzymes were -SH proteinases, as they were completely inhibited by p -hydroxymercuribenzoate; both of them were also inhibited by the endogenous proteinase inhibitors of resting pine seeds. Besides these enzymes, the endosperm extracts contained pepsin-like acid proteinase activity, which is not affected by the endogenous inhibitors. This enzyme activity was largely inactivated during dialysis.     

The Interaction of New Pyridylporphyrins with Bovine Serum Albumin

The interaction of meso-tetra(4-N-hydroxyethylpyridyl)porphyrin, meso-tetra(3-N-hydroxyethylpyridyl)porphyrin, and their zinc complexes with bovine serum albumin (BSA) was studied by electronic spectroscopy, CD, and equilibrium dialysis at pH 7.2. The titration of the porphyrins with BSA was accompanied by a decrease in light absorption and a bathochromic shift of the Soret band, as well as by the appearance of an isobestic point. The porphyrin interaction with BSA also led to the induction of positive CD spectra in the visible region, which is explained by the porphyrin sorption on the protein globule. The equilibrium dialysis helped in determining the stoichiometry of binding and the binding constants of the porphyrins under study with BSA using Scatchard plots. This interaction is nonspecific and reversible.     

A protease-like permeability factor in the guinea pig skin. 2. In vitro activation of the latent form permeability factor by weakly acidic phosphate buffer.

Conditions for the in vitro activation of the latent form of a protease-like permeability factor in the pseudoglobulin fraction from guinea pig skin were examined. (1) The factor was activated by dialysis against 67 mM phosphate buffer at pH 5.8--6.4, not at pH 7.0--8.0. (2) High salt concentration (200 mM or greater phosphate buffer or 67 mM phosphate buffer containing 200 mM or greater KCl or NaCl) prevented the activation at pH 6.2. (3) High osmotic pressure (sucrose at 1 M) did not affect activation at pH 6.2. (4) Reconversion of the activated permeability factor into an inactive form was not observed under high salt conditions, under which the latent permeability factor was stable in its own form. (5) The molecular size of the latent permeability factor was estimated as approx. 80 000 by Sephadex G-100 gel filtration at high salt concentration.     

Interaction of new pyridylporphyrins with bovine serum albumin

The interaction of meso-tetra(4-N-hydroxyethylpyridyl)porphyrin, meso-tetra(3-N-hydroxyethylpyridyl)porphyrin, and their zinc complexes with bovine serum albumin (BSA) was studied by electronic spectroscopy, CD, and equilibrium dialysis at pH 7.2. The titration of the porphyrins with BSA was accompanied by a decrease in light absorption and a bathochromic shift of the Soret band, as well as by the appearance of an isobestic point. The porphyrin interaction with BSA also led to the induction of positive CD spectra in the visible region, which is explained by the porphyrin sorption on the protein globule. The equilibrium dialysis helped in determining the stoichiometry of binding and the binding constants of the porphyrins under study with BSA using Scatchard plots. This interaction is nonspecific and reversible. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 2; see also http://www.maik.ru.     

Phosphate binding to chromatophores of Rhodospirillum rubrum.

Equilibrium dialysis has been used to determine the binding of phosphate to chromatophores of Rhodospirillum rubrum. Assuming a complete exchange of the added 32Pi with endogenous phosphate, the saturation with phosphate retained in any form by chromatophores was reached at about 20 nmoles Pi per mg of bacteriochlorophyll. The retention of phosphate had a pH optimum at pH 6.5 to 6.8. At pH 8.0 only chromatophores which have not been liberated from DNA and RNA show a considerable retention of phosphate. However, illumination of chromatophores prior to dialysis in the presence of ADP leads to a retention of phosphate at pH 8.0 which persists during dark dialysis in the absence of added magnesium.     

The effects of pH on amphetamine-induced dopamine release as measured by in vivo dialysis

The release of endogenous dopamine (DA) from the rat corpus striatum before and after the administration of d-amphetamine sulphate (AMPH) was monitored by in vivo dialysis to compare the effects of perfusion media at pH 6.0 and at pH 7.4. Basal release of DA did not differ significantly at pH 6.0 (61.25 +/- 5.34 pg/sample, n = 4) or pH 7.4 (58.02 +/- 14.17 pg/sample, n = 4). The basal value of homovanillic acid (HVA) was not significantly reduced at pH 7.4 as compared with pH 6.0; while there was a significant reduction in 3,4-dihydroxyphenylacetic acid (DOPAC) at pH 7.4 as compared to pH 6.0. Intraperitoneal injection of 2.5 mg/kg AMPH resulted in a 21 fold increase in the concentration of DA appearing in subsequent dialysis samples. This increase in DA release was not significantly affected by the pH. Equally the decrease in DOPAC and HVA content following AMPH were also not altered by the pH. These present results differ from experiments using push-pull cannulae and suggest that responses observed with the two techniques may not be equivalent.     

Effects of mixing during acid addition on fractionally precipitated protein

Isoelectric fractionation of the two major proteins of soy is characterized. Fractions are acid precipitated and cen-trifugally collected at pH 6.0 (glycinin) and pH 4.8 (beta-conglycinin). Two extremes in the speed of acid addition (rapid, with no mixing, and slow, via acid dialysis, with complete mixing) are compared to determine their effects on the properties of the precipitate. Total protein yield, fraction composition, and aggregate microstructure do not depend significantly on the method of acid addition. Particle size distribution and hindered settling behavior do differ and are explained using a model of aggregate strength. The rapid acid addition produces larger primary particles, because of higher supersaturation, and yields larger aggregates, because of higher interparticle potential and stronger aggregates. Further aggregation in low-shear hindered settling is faster for the slowly precipitated aggregate because few of these bonding sites could survive the high-shear precipitator, whereas more can contribute to aggregation during hindered settling.     

Characterization of polypeptide chains of epidermal prekeratin and reconstituted filaments

Prekeratin was isolated from bovine snout epidermis with 0.1 M citric acid/sodium citrate buffer, pH 2.6 (buffer A). Filaments, 6.0-9.0 nm wide, were produced by dialysis against low ionic strength buffer A or by dissociating prekeratin in 8 M urea solution followed by dialysis against 0.005 M Tris-HCl buffer, pH 8.0. The polypeptide composition of both prekeratin and filaments was studied by four different SDS-polyacrylamide gel electrophoresis methods. The best resolution was obtained by Laemmli's technique in which both prekeratin and filaments were separated into three major and seven distinct minor bands of polypeptides. The major ones comprise approx. 70% of total polypeptides and their estimated molecular weights are 68 000, 54 000, and 50 000. The molecular weight of minor ones is in decreasing order 65 000, 63 000, 61 000, 58 000, 47 000, 44 000 and 42 000. It is proposed that the major polypeptides form the backbone structure of epidermal filaments and the minor polypeptides play a role in its stabilization.