Study of bromthymol blue-prealbumin in the blood serum of rats under hyperbaric oxygenation]

Three-fold increase of BTB-prealbumin (Rm 1.0) in rat serum following fierse convulsions under hyperbaric oxygenation (6 ati, 30-35 min) has been proved by disc electrophoresis. Glial S100-protein and 7-fold increase in the all-organ component of brain BTB-prealbumin were found by immunochemistry to appear in the serum of experimental rats. The consequences of disorders in the blood-brain barrier for non-specific, all-organ proteins and potentialities of protein output from the brain into the blood similarly to neurophysins under hyperbaric oxygenation are discussed.     

Capillary zone electrophoresis of collagen type I CNBr peptides in acid buffers

Collagen type-I CNBr peptides were separated under acidic conditions by capillary electrophoresis. Separation conditions were: 100 mM phosphate buffer pH 2.5, 50 cm × 50 μm capillary (placed in a cartridge), 8 kV, running time 30–45 min, detection by UV at 200 nm. The peptides were separated strictly by their molecular mass and the overall pattern was well comparable to RP-HPLC separations of these analytes. It is proposed that the separation mechanism may involve hydrophobic sorptions to the capillary wall.     

Substrate-containing gel electrophoresis: sensitive detection of amylolytic, nucleolytic, and proteolytic enzymes

Electrophoresis of hydrolytic enzymes under nondenaturing conditions on acrylamide gels containing the appropriate high-molecular-weight substrates entrapped on the gel has been explored as a general method for sensitive enzyme resolution and detection. Under electrophoresis conditions of optimal enzyme activity, the enzymes may bind tightly to the fixed substrate and can only migrate in the electrophoretic field as the substrate is hydrolyzed. When the gels after electrophoresis in this “binding mode” are stained with substrate-detecting reagents, clear tracks of enzyme migration are observed, and the length of each track is a function of the amount of enzyme present in that track. Multiple forms of a given enzyme activity have not been and are not likely to be observed under these conditions. Under electrophoresis conditions of minimal (or suboptimal) enzyme activity, the enzymes do not bind to the fixed substrate and their mobility in the electrophoretic field does not appear to be significantly affected by the presence of substrate. After electrophoresis in this “nonbinding mode” the gels are incubated under conditions of optimal enzyme activity to allow substrate hydrolysis to take place before they are stained with substrate-detecting reagents, and active enzymes are detected as clear bands. Multiple forms of a given activity which were resolved during electrophoresis in the nonbinding mode are reflected by the presence of individual bands. The substrate-containing gel electrophoresis technique does not appear to be amenable to precise quantification of enzymes. By comparing the length of the clear tracks or the degree of staining of the activity bands for a range of enzyme concentrations, however, it is possible to establish the smallest amount of enzyme that can unequivocally be detected under a given set of conditions; from such studies we estimate that the sensitivity of detection with the substrate-containing gel electrophoresis technique can be orders of magnitude better than that obtained with other methods. The levels of detection observed in the work presented here were about 50 pg for α-amylase run on starch-containing gels, 1 pg to 1 ng for nucleases run on DNA- or RNA-containing gels, and 100 pg to 10 ng for 11 different pure and crude protease preparations run on gels containing heat-denatured bovine serum albumin.     

改良逆转录环介导等温扩增检测技术快速实时检测H9亚型禽流感病毒

利用改良逆转录环介导等温扩增(RT-LAMP)技术建立一种快速、灵敏的检测方法用于H9亚型禽流感病毒检测。根据H9亚型禽流感病毒血凝素(HA)基因保守区序列中的8个区段设计6条特异性引物,在恒温条件下进行核酸扩增反应,并以琼脂糖凝胶电泳和目视检查绿色荧光两种方法对扩增结果进行判定。结果表明,RT-LAMP的最小检测限为100 fg,灵敏度比PT-PCR高100倍,且与H5亚型、H7亚型禽流感病毒,新城疫病毒(NDV)无交叉反应。目视检查绿色荧光与常规琼脂糖凝胶电泳的判定结果一致。整个扩增检测过程在35 min内即可完成。利用临床样本对RT-LAMP法进行验证,结果与RT-PCR一致。由实时浊度分析得到的标准曲线,计算出临床样本中的病毒质粒拷贝数均在2×107-2×104之间。因此,本研究建立的RT-LAMP方法快速、灵敏、特异性强,是H9亚型禽流感病毒的一种高效检测方法。     

Purification and characterization of beta-N-acetylhexosaminidase I2 from human liver.

beta-N-Acetylhexosaminidase I2 was purified from human liver by a combination of concanavalin A chromatography, DEAE-cellulose chromatography, gel filtration and affinity chromatography on 2-acetamido-N-(6-aminohexanoyl)-2-deoxy-beta-D-glucopyranosylamine coupled to CNBr-activated Sepharose 4B. Its specific activity was 130 mumol/min per mg of protein compared with values of 150 and 320 mumol/min/mg of protein for beta-N-acetylhexosaminidases A and B purified from the same tissue. Km values for I2, A and B were 1.0 mM, 0.8 mM and 0.74 mM respectively. On gradient gel electrophoresis under non-denaturing conditions, hexosaminidase I2 behaved similarly to A and appeared to have an Mr between 100 000 and 110 000. beta-N-Acetylhexosaminidase I2 was resolved into two major polypeptides, of Mr 56 000 and 29 000, on SDS/polyacrylamide-gel electrophoresis under denaturing conditions. Immunoblotting with anti-(hexosaminidase alpha-subunit) serum confirmed that the 56 000-Mr component was the alpha-subunit and anti-(hexosaminidase B) serum reacted with the 29 000 Mr component. beta-N-Acetylhexosaminidase I2 more closely resembles form A than B, but the features of its structure that allow it to be separated from A on the basis of net charge have not yet been found.     

Effect of Triton X-100 on properties of acetylcholinesterase from human erythrocytes

The effect of Triton X-100 on catalytic properties of acetylcholinesterase from human erythrocytes under acetylcholine hydrolysis, on sensitivity of acetylcholinesterase to specific phosphoorganic inhibitors and eserine, and on the mobility and isoenyme spectrum under analytical electrophoresis in polyacrylamide gel is investigated. Triton X-100, independently on its concentration within 0.05-1.0%, slightly changes V and [S]opt values and increases Km value in 2-3 times. The inhibitory effect of Triton X-100 is mainly competitive, 0.5% Triton X-100 decreases bimolecular constant (kII) of the interaction of acetylcholinesterase with phosphoorganic inhibitor and eserine in 2.5-4 times. In the presence of phosphoorganic inhibitor, kII sharply decreased when 0.02% Triton X-100 was added, and then it did not change under the increase of Triton X-100 concentration up to 1.0%. On the basis of these data, an analytical method of estimating Triton X-100 content in protein solution is proposed. The introduction of 0.1% Triton X-100 into polyacrylamide gel results in considerable quantitative redistribution of acetylcholinesterase isoenzyme fractions and in the change of the mobility of one fraction under electrophoresis.     

Differential labeling of rat hepatic Golgi and serum very low density lipoprotein apoprotein B variants

The synthesis of apoB-100 and apoB-48 by rat liver was investigated by studying the apoB complement of very low density lipoproteins (VLDL) from hepatic perfusates and Golgi fractions. The relative amounts of apoB-100 and apoB-48 in perfusate and Golgi VLDL as determined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis were similar to those in serum VLDL. To investigate the relative rates of synthesis of the VLDL B proteins, rats were injected intraportally with tritiated amino acid, and hepatic Golgi and serum VLDL were isolated from 7.5 to 120 min later. In hepatic Golgi VLDL, apoB-100 and apoE were maximally labeled at 15 min after the tritiated amino acid pulse. In contrast, VLDL apoB-48 attained maximum radioactivity at 30 min after isotope injection. In serum VLDL, apoB-100 and apoE were maximally labeled at 30 min post-isotope injection, while activity in apoB-48 peaked at 60 min. The data suggest that the synthesis of the B proteins and incorporation into rat liver nascent VLDL are independently regulated. The differential labeling patterns of the VLDL B proteins may be explained by an intracellular pool of apoB-48 that is larger than that of apoB-100. An alternative explanation of the results is that apoB-100 is a precursor to apoB-48.     

The 57-Kilodalton Phosphoprotein of Spiroplasma melliferum Is Autophosphorylated

The partially purified 57-kDa protein of Spiroplasma melliferum was autophosphorylated when incubated with ATP in the presence of ZnCl₂. Autophosphorylation was also apparent by showing the in situ phosphorylation of the 57-kDa protein band separated by polyacrylamide gel electrophoresis under nondenaturing conditions. The autophosphorylation was affected neither by the pH of the reaction mixture nor by the presence of NaF. The steady state level of the phosphorylated 57-kDa protein remained constant for up to 15 min, suggesting the absence of a phosphoprotein phosphatase activity in the preparation. As the initial phosphorylation rate did not decrease upon a 100-fold dilution of the 57-kDa protein under constant substrate concentration, it is suggested that the autophosphorylation is an intramolecular process. Received: 7 June 1996 / Accepted: 6 July 1996     

The presence of two forms of the phosphocarrier protein HPr of the phosphoenolpyruvate:sugar phosphotransferase system in streptococci.

The protein, HPr, a necessary component of the phosphoenolpyruvate phosphotransferase system (PTS) in bacteria, was purified from Streptococcus salivarius by column chromatography. The purified preparation gave only one band when analyzed by sodium dodecylsulfate gel electrophoresis or by isoelectric focusing in polyacrylamide gel (pI = 4.85). However, electrophoresis in Tris-containing buffers under non-denaturing conditions revealed 2 bands that could be phosphorylated by PEP in the presence of enzyme I of the PTS or by ATP with the HPr kinase. Homogeneous preparations of these 2 forms could be obtained by preparative electrophoresis. Each preparation exhibited only 1 band when analyzed by electrophoresis under non-denaturing conditions, indicating that the doublet observed before preparative electrophoresis was not an electrophoretic artefact. The electrophoretic mobility of each protein was not modified following heat-treatment at 100 degrees C for 20 min or storage at -40 degrees C for several months. Both HPr proteins catalyzed in vitro the PEP-dependent phosphorylation of glucose, but at a rate slightly lower than that observed with a preparation of HPr containing both forms of the protein. Both forms were also able to transfer the phosphate group from PEP to the other specific PTS proteins known in S salivarius. Rabbit polyclonal antibodies directed against each form reacted with both proteins. The presence of the 2 forms of HPr was detected in fresh cellular extracts of S salivarius; however, their intracellular ratio varied according to growth conditions. A doublet was also found in many other streptococcal species tested (S mutans, S sobrinus, S sanguis, S thermophilus, S bovis, S rattus) and also in L lactis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrophoretically monodisperse cytochrome c oxidases

A discontinuous gradient polyacrylamide gel electrophoresis under nondenaturing conditions has been used to demonstrate monodispersity of procaryotic and eucaryotic cytochrome c oxidase preparations. Alkaline treated bovine enzyme which contains nine subunits as analysed by subsequent discontinuous SDS-polyacrylamide gel electrophoresis is a monodisperse dimer in 0.1% Triton X-100 and a monomer in 0.1% dodecyl maltoside. The Mr-values corrected for bound detergent are 286,000 in Triton X-100 and 152,000 in dodecyl maltoside respectively. The two-subunit bacterial cytochrome c oxidase of Paracoccus denitrificans is proved to be a monomer with a corrected Mr of 76,000 in both nonionic detergents Triton X-100 and dodecyl maltoside.     

Differentiating the effects of microwave and heat on tissue proteins and their crosslinking by formaldehyde

Summary Alkaline phosphatase activity in mouse liver blocks, cooled by an ice-bath, decreased by 50% in 5 min of microwave irradiation (280 W). This loss of protein tertiary structure has been mirrored by ultrastructural changes in the same tissue. Microwave irradiation did not produce clevage or polymerization of lysozyme or haemoglobin. Protein formaldehyde reaction mixtures produced protein polymers between 0° and 40°C which could be separated by SDS-polyacrylamide gel electrophoresis. Microwave irradiation of lysozyme or haemoglobin plus formaldehyde on icebath up to 30 min produced a similar electrophoretic pattern. When lysozyme or haemoglobin plus formaldehyde was heated to 60°C for 30 min, the protein polymers migrated faster on electrophoresis, suggesting a smaller hydrodynamic volume than expected due to intramolecular crosslink formation, not opened up under the conditions of electrophoresis.     

Purification and properties of the membrane-bound hydrogenase from N2-fixing Alcaligenes latus

The nitrogen-fixing, aerobic hydrogen-oxidizing bacterium Alcaligenes latus forms hydrogenase when growing lithoautotrophically with hydrogen as electron donor and carbon dioxide as sole carbon source or when growing heterotrophically with N2 as sole nitrogen source. The hydrogenase is membrane-bound and relatively oxygen-sensitive. The enzymes formed under both conditions are identical on the basis of the following criteria: molecular mass, mobility in polyacrylamide gel electrophoresis, Km value for hydrogen (methylene blue reduction), stability properties, localization, and cross-reactivity to antibodies raised against the 'autotrophic' hydrogenase. The hydrogenase was solubilized by Triton X-100 and deoxycholate treatment and purified by ammonium sulfate precipitation and chromatography on Phenyl-Sepharose C1-4B, DEAE-Sephacel and Matrix Gel Red A under hydrogen to homogeneity to a specific activity of 113 mumol H2 oxidized/min per mg protein (methylene blue reduction). SDS gel electrophoresis revealed two nonidentical subunits of molecular weights of 67 000 and 34 000, corresponding to a total molecular weight of 101 000. The pure enzyme was able to reduce FAD, FMN, riboflavin, flavodoxin isolated from Megasphaera elsdenii, menadione and horse heart cytochrome c as well as various artificial electron acceptors. The reversibility of the hydrogenase function was demonstrated by H2 evolution from reduced methyl viologen.     

Electron spin resonance study of the role of nitrosyl-heme in the activation of guanylate cyclase by nitrosoguanidine and related agonists.

One major component of lens plasma membrane is a glycoprotein that SDS-polyacrylamide gel electrophoresis shows to possess an apparent molecular weight of 26,000. When this protein is solubilized in low ionic strength buffers containing SDS, and heated to 100° for 1 to 3 min prior to electrophoresis, conversion into high molecular weight aggregate results. The heat lability of this protein is greatly enhanced if it solubilized and heated in buffers containing 0.1 M NaCl. At this ionic strength, incubation for 3 h at 38° results in conversion of 20% of the protein into high melecular weight aggregates. Most other membrane proteins isolated from lens membrane are insensitive to heat treatment. It is concluded that temperature and ionic strength must be recorded and controlled carefully when using SDS-polyacrylamide gel electrophoresis to study this membrane protein.     

Phosphorylated proteins from anuran intestinal microvilli membranes--I. Relations with alkaline phosphatase

The degree of phosphorylation of intestinal microvilli membrane proteins in an adult amphibian, Rana esculenta, was investigated under various experimental conditions. The microvilli protein phosphorylation rate rapidly increases during the first 4 min of incubation in a medium containing [gamma-32P]ATP. This increase is slower afterwards. Cyclic nucleotides (cyclic AMP, cyclic GMP) and sorbitol do not modify the microvilli protein phosphorylation rate. On the contrary, this phosphorylation rate significantly decreases in the presence of L-lysine, when its concentration in the incubation medium is greater than 25 mM. The time course of phosphorylation confirms the inhibitory effects of L-lysine (100 mM). The microvilli membrane proteins were distinguished by polyacrylamide gel electrophoresis. In heated samples, electrophoresis followed by an radioautograph systematically reveals the existence of a very phosphorylated protein with a mol. wt of 86 kDa. The phosphorylation of this protein is partially inhibited by L-lysine (100 mM). The very phosphorylated protein could be the monomer of alkaline phosphatase. The dimer (170 kDa) is visualized on electrophoretograms by its catalytic activity. In mammals, several authors have established a correlation between phosphorylation of the microvilli membrane proteins and the intensity of intestinal calcium absorption. Such a control is presently being investigated in adult Rana esculenta.     

Substrate Preference of γ-Glutamyltransferase from Caps of Fruiting Bodies of Lentinus edodes

Three kinds of proteins (BA-1, BA-2 and BA-3) allergenic to the IgE antibody of allergenic individuals were isolated from buckwheat seeds. These three proteins were essentially homogeneous as judged by both polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis. The amino acid composition of BA-1 and BA-2 was very similar, and the molecular weight of each allergenic protein was between 8000–9000 by SDS-polyacrylamide gel electrophoresis. One of them was a trypsin inhibitor, and their immunoreactivity was quite stable to heating at 100°C for 60 min.     

Properties of Purified Staphylococcal β-Hemolysin

Purification of beta-hemolysin was achieved by ammonium sulfate precipitation, Sephadex G-100 gel filtration, carboxymethyl cellulose column chromatography, and density gradient electrophoresis. Active fractions eluted from carboxymethyl cellulose contained at least one nonhemolytic protein, and omission of this step was not detrimental to the purification process. Density gradient electrophoresis yielded approximately 1.6 mg of highly active purified beta-hemolysin per liter of culture supernatant liquid. Purified beta-hemolysin gave a single line on gel double diffusion and immunoelectrophoresis. A single symmetrical peak formed in the analytical ultracentrifuge, and the sedimentation coefficient was calculated to be 1.7S. The purified beta-hemolysin was stable at 4 C and could be lyophilized. Magnesium cations were required for full expression of beta-hemolytic activity. beta-Hemolysin was lethal for rabbits when injected intravenously in amounts between 40 and 160 mug. Crude beta-hemolysin was more stable than purified beta-hemolysin when heated at 60 C for 30 min. Purified beta-hemolysin lost almost all of its activity on subsequent heating at 100 C for 10 min.     

Purification and characterization of amphiphilic trehalase from rabbit small intestine

Rabbit intestinal trehalase (alpha,alpha-trehalose glucohydrolase, EC 3.2.1.28) was solubilized with Triton X-100 and purified in the presence of EDTA. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis in the presence of Triton X-100 or SDS. It showed amphiphilic properties on gel filtration. polyacrylamide gel electrophoresis, charge-shift electrophoresis and phenyl-Sepharose chromatography. Its molecular weight was estimated to be about 330 000 by gel filtration under nondenaturing conditions and in the presence of Triton X-100, the value being in satisfactory agreement with the sum of the weight of one Triton X-100 micelle and twice the molecular weight (105 000) of purified hydrophilic trehalase which had been deprived of the anchor segment. The two purified trehalases gave almost the same molecular weights (about 75 000) on SDS-polyacrylamide gel electrophoresis. These results suggest that intestinal trehalase consists of two subunits with a molecular weight of 75 000 and that its anchor segment is small (less than 5000). Triton X-100 extracts freshly prepared from intestinal microvilli essentially showed one form of trehalase, which behaved on phenyl-Sepharose and Con A-Sepharose chromatography in the same manner as purified amphiphilic trehalase.     

Some physicochemical characteristics of an immune lymphocyte product which inhibits the multiplication of toxoplasma within mouse macrophages.

In response to antigenic stimulation, the splenic lymphocytes from Toxoplasma-infected mice produce a factor which is called by the authors the Toxoplasma growth inhibitory factor (Toxo-GIF) and which inhibits the multiplication of Toxoplasma within nonimmune macrophages in vitro. In this study, partial characterization of murine Toxo-GIF was examined using Sephadex G-100 gel-filtration, isoelectric focusing, zonal electrophoresis and heat and enzymatic treatment. Peak activity of Toxo-GIF was found in a Sephadex G-100 fraction with a similar molecular size to that of the ovalbumin. The molecular weight of Toxo-GIF was calculated to be between 38,000 and 55,000. Toxo-GIF was stable to heating at 56 C for 30 min but lost its activity at 80 for 30 min or by exposure to pH values of 5 and 2. Exposure of Toxo-GIF to water-insoluble chymotrypsin or neuraminidase markedly decreased its ability to induce enhanced microbicidal activity of cultured macrophages, suggesting that Toxo-GIF was a glycoprotein. Furthermore, Toxo-GIF migrated in a region cathodal to mouse albumin on agar zone electrophoresis. Isoelectric focusing of active Sephadex fractions showed a well-defined peak of Toxo-GIF activity with an isoelectric point of pH 4.9 to 5.9.     

Identification of different quaternary structures of beef heart cytochrome-c oxidase by two-dimensional polyacrylamide gel electrophoresis

A two-dimensional gel electrophoresis is described to identify different quaternary structures of the heart cytochrome-c oxidase. Bovine enzyme was purified and separated by discontinuous gradient polyacrylamide gel electrophoresis under nondenaturing conditions in the 1st dimension into several discrete complexes and thereupon shown to be heterodisperse in Triton X-100 and dodecyl maltoside. A discontinuous SDS-polyacrylamide gel electrophoresis in the 2nd dimension was used to determine the subunit composition of the isolated complexes. One of these represents the intact enzyme with 12 different polypeptides while the others have an incomplete subunit composition.     

Labeling of platelet surface proteins with 125I by the iodogen method

A procedure for the 125I-iodination of platelet suspensions is described. The procedure utilizes Iodogen, a solid-phase oxidizing agent similar to chloramine-T. Platelets were labeled under a variety of conditions, including in the presence of 0.1% albumin, and showed between 7 and 28% incorporation of 125I. Best labeling results were obtained at low platelet concentrations (3-5 x 10(8) platelets/ml), short reaction times (15 min), and with 2-ml glass vials coated with 100 micrograms of Iodogen. Analysis of the labeled platelet proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography revealed that the same major protein bands were labeled by this procedure as were labeled by the lactoperoxidase procedure. At low platelet concentrations, the Iodogen procedure gives twice the amount of iodine incorporation.