Microdetermination of selenium in protein fractions isolated by analytical methods

A method has been developed for the determination of selenium associated with proteins in chromatographic fractions, in polyacrylamide gels, and on nitrocellulose membranes after transfer. This method involves the complete digestion of samples in the purest nitric and perchloric acids and takes advantage of the specificity afforded by the 99% pure 2,3-diaminonaphthalene. The use of these and other reagents of highest purity produces very low blank values and allows a detection limit as low as 0.76 picomoles (60 picograms) of selenium. Quantitative recoveries of selenium in glutathione peroxidase and low coefficients of variation were obtained.     

Listeria antigens and their isolated protein fractions

Antigens have been obtained from standard Listeria strains 1 and 4a by alcohol precipitation, sonication and multiple freezing and thawing. From these antigens protein fractions, immunologically identical as shown by the precipitation test and immunoelectrophoresis and similar in their chemical composition, have been isolated. The immunogenic properties of the fractions are determined by the polysaccharide-protein complex represented by amino acids (proline, histidine, lysine) and monosaccharides (ribose, glucose, arabinose), common for the fractions of three antigens. In experimental studies on mice and rabbits the protein fraction has been shown to produce a protective effect. After receiving this fraction in three injections and the subsequent challenge with the suspension of Listeria culture in a dose of 500 million cells, the animals have been found to develop no Listeria infection. After autoclaving the protein fraction looses its immunogenic properties, but retains the specificity of chemical groups and produces an allergenic effect as shown in experiments on guinea pigs and rabbits.     

Immunomodulating effect of protein fractions isolated from bifidobacteria

The screening of the immunomodulating activity (IMA) of different protein fractions isolated from bifidobacteria was carried out and the capacity of these fractions for changing the proliferative activity of immunocompetent cells was evaluated. Soluble proteins were extracted from lyophilized and sonicated bacterial mass of B. bifidum strain 1 in Na2HPO4 (pH 8) in a water bath at 65 degrees C for 30 minutes. After the formation and removal of nucleic acid sediment the resulting supernatant fluid was dialyzed, its adsorption spectra were analyzed and the fluid was fractionated in a specially proposed device for preparative electrophoresis. Protein fractions were tested for IMA on spleen cells of CBA mice in the reaction of lymphocyte blast-transformation by the level of the inclusion 3H thymidine. The analysis of IMA of protein fractions revealed that their high-molecular components produced a pronounced dose-dependent effect on the proliferative activity of spleen cells. The fractions containing low-molecular components were either inactive (fraction 4) or active only in the maximum dose (fraction 5).     

Immunological properties of isolated protein fractions from Artemia ribosomes

Acid-soluble ribosomal proteins from cysts of Artemia salina were separated by high-resolution polyacrylamide gel electrophoresis at pH 4.3. Three distinct protein bands, occurring in different parts of the electrophoretic pattern, were used for immunization in rabbits, and the γ-globulin fractions of the antisera were prepared. These preparations produced precipitation lines in agarose gel with protein extracted from whole 80S ribosomes and from 60S and 40S ribosomal subunits. With γ-globulin preparations from non-immune or anti-ovalbumin sera no reactions were obtained.     

Laser Raman spectroscopic studies of ocular lens and its isolated protein fractions.

The water-soluble proteins of the bovine lens were separated on a column of Sephadex G-200 into five fractions designated as alpha-, beta1-, beta2-, and gamma-crystallin. Laser Raman scattering studies on these isolated proteins (both in the lyophilized state and in solution) and insoluble albuminoid reveal that they contain predominantly antiparallel pleated sheet structure in the main chains and that sulfhydryl groups are highly localized in gamma-crystallin. This light-scattering technique was also applied to probe the homogeneity of protein structure in the intact lens. The analysis of the scattered light selectively collected from various parts of the lens indicated that these proteins also exist in an antiparallel beta structure throughout the entire lens. However, the central (nucleus) and outer (cortex) portions have somewhat different amino acid composition. Based on the relative intensities of the lines at 624 (phenylalanine) and 644 cm-1 (tyrosine), it is concluded that the nuclear part has the highest concentration of gamma-crystallin and that the content of alpha-crystallin increases significantly from the nucleus to the cortex. By examining the Raman spectra in the 2582 cm-1 and the amide I and III regions, we have demonstrated that the sulfhydryl groups and the beta conformation of the lens proteins are unaffected in the conversion of transparent to totally opaque lens by heat denaturation at 100 degrees. This means that the opacification of a lens does not necessarily involve the oxidation of sulfhydrul groups or conformation changes.     

REFOLD: an analytical database of protein refolding methods

The expression and harvesting of proteins from insoluble inclusion bodies by solubilization and refolding is a technique commonly used in the production of recombinant proteins. To bring clarity to the large and widespread quantity of published protein refolding data, we have recently established the REFOLD database (http://refold.med.monash.edu.au), which is a freely available, open repository for protocols describing the refolding and purification of recombinant proteins. Refolding methods are currently published in many different formats and resources--REFOLD provides a standardized system for the structured reporting and presentation of these data. Furthermore, data in REFOLD are readily accessible using a simple search function, and the database also enables analyses which identify and highlight particular trends between suitable refolding and purification conditions and specific protein properties. This information may in turn serve to facilitate the rational design and development of new refolding protocols for novel proteins. There are approximately 200 proteins currently listed in REFOLD, and it is anticipated that with the continued contribution of data by researchers this number will grow significantly, thus strengthening the emerging trends and patterns and making this database a valuable tool for the scientific community.     

Alterations in the protein pattern of subcellular fractions isolated from Paramecium cells suppressed in phagocytosis.

SDS-PAGE and quantitative densitometric analysis revealed alterations in the protein pattern of subcellular fractions (100,000 x g) isolated from Paramecium aurelia (299s axenic) cells suppressed in phagocytosis as compared with the control. Two different agents were used to block phagocytosis: the beta-adrenergic antagonist-1-propranolol (200 microM) and inhibitor of calmodulin-dependent processes--trifluoperazine (20 microM). More than 40 polypeptides were identified in the cytosolic (soluble) fractions S1 and S2. A considerable decrease in band intensity was found for three polypeptides: by 60% for 87 kDa band, 52% for 75 kDa and 37% for 42 kDa in comparison to the control, when S2 fractions from propranolol-treated cells of equal load were quantified. TFP treatment evoked only a small decrease in the intensity of the same bands: 9%, 10% and 6%, respectively. The 42 kDa band was identified by Western blot analysis and chemiluminiscent detection to be actin. This result suggests that actin may be a primary target of pharmacological agents used in this study to inhibit Paramecium phagocytic activity.     

Microdetermination of cadmium and EDTA in protein solutions

Two simple high-precision methods have been developed for the microassay of cadmium and of the di-sodium salt of EDTA (ethylenediamine-tetraacetic acid). Chief interest centered on analyzing solutions containing from 0 to 10 γ of Cd or, alternatively, from 0 to 27 γ of EDTA in the presence of 0–48 mg of renal protein, the corresponding sensitivities being indicated by standard errors of estimate, ± 0.13 γ Cd and ± 0.76 γ EDTA, respectively.Cadmium was assayed as the dithizonate, the light absorption of this complex at 610 mμ being a linear function of Cd over the range specified.Assays of EDTA were based on the principle that this chelating agent increases light absorption in Cd solutions at 610 mμ by blocking the formation of the cadmium-dithizonate complex. Light absorption at this wavelength was found to be linearly related to EDTA.Digestion with hydrochloric acid and removal of excess HCl, in vacuo, was employed for deproteinization, instead of procedures more commonly used. This resulted in a considerably simplified extraction procedure as well as in high accuracy for cadmium and EDTA estimates as determined by very extensive statistical analysis of the basic experimental data reported in a separately available appendix. Incomplete factorial arrangements and their analysis of variance, along with appropriate regression techniques, yielded calibration charts suitable for routine application.     

Fraction I protein concentration in plants by analytical ultracentrifuge.

The fraction I protein content of plants was determined by centrifuging extracts in the analytical ultracentrifuge until the protein boundary was well separated. The refractive index increase across the boundary was determined with the interference optical system and converted to fraction I protein concentration with the factor 0.245 mg/ml/fringe.     

Control by TSH of protein turnover in thyroid subcellular fractions.

The action of TSH on protein turnover in various subcellular fractions has been investigated in dog thyroid slices incubated in vitro. The results suggest a general inhibition by TSH of protein catabolism. Using double labeline (3/ and 14C) of the proteins, an increase of the disappearance of some labeled material from the microsomal fraction in the presence of TSH has been observed. The protein nature of this material has been established by testing its susceptibility to hydrolysis by trypsin. The fact that the microsomal pellet had to be treated by triton X 100 before hydrolysis by trypsin could occur, suggests that the material is probably enclosed in, or protected by membrane vesicles. Its high molecular weight and its ability to be immunoprecipitated by an antithyroglobulin serum suggest that the microsomal protein, the disappearance of which is stimulated by TSH, is thyroglobulin or one of its subunits. It is suggested that our results reflect the acceleration by TSH of the vectorial transfer of thyroglobulin through the membranes of the endoplasmic reticulum to the colloid space.     

Action of heparin on protein fractions of isolated nuclei and on their DNA content.

Isolated nuclei of rat hepatocytes were incubated with 0.05% sodium heparinate for 2 to 10 min. Alterations in the nuclei were controlled biochemically, determining the amounts of DNA and histones, and by cytophotometric methods determining the amounts of total and nonhistone proteins and DNA. Under the selected experimental conditions 95% of histones are bound already after 5-min incubation with heparin; nonhistone proteins of cell nuclei remain unchanged. The blockade of histones is followed by DNA diffusion into the incubation medium. Experiments with nuclear staining with alcian blue proved the specificity of heparin binding with histones and showed that heparin-histone complex remains in the nuclei, and its histones lose their extractability with 0.25 n HCl.     

Localization of proteoglycan core protein in subcellular fractions isolated from rat chondrosarcoma chondrocytes

Chondrocytes from the Swarm rat chondrosarcoma were pulse-labeled with [3H]serine for 30 min and chased, in the presence of cycloheximide, for times up to 300 min. The movement of newly synthesized core protein precursor of the proteoglycan through elements of the endoplasmic reticulum and Golgi complex was examined. Rough and smooth microsome fractions were obtained by centrifuging postmitochondrial supernatants from cell homogenates on discontinuous sucrose gradients. The core protein precursor was identified in subcellular fractions by (a) immunoprecipitation with an antiserum directed against the hyaluronate binding region of the core protein and the link protein and (b) its size on polyacrylamide gels. Labeled core protein precursor decreased from the microsomes with a t1/2 of 60 +/- 8 min, nearly the same as for the appearance of label in completed proteoglycan monomer (t1/2 = 58 +/- 13 min), consistent with a precursor-product relationship. After correcting for incomplete recovery of the core protein precursor in the microsomal fractions and for cross-contamination of the smooth microsomes by elements of rough endoplasmic reticulum, the redistribution of core protein precursor and completed proteoglycan in the intracellular compartments and of labeled extracellular proteoglycan were fit to a three-compartment model. A t1/2 of 98 +/- 7 min for the loss of core protein precursor from the rough microsomes and a t1/2 = 10 +/- 4 min for the completed proteoglycan in the intracellular compartment (Golgi and secretory vesicles) was obtained. The data indicate that at least 70% of the intracellular transit time for the core protein precursor is spent in the rough endoplasmic reticulum. The addition of glycosaminoglycan chains followed by secretion from the cell occurs relatively rapidly, occupying less than 30% of the total intracellular dwell time.     

Application of enzymehistochemical methods to isolated subcellular fractions and to sucrose-ficoll density gradients

Summary To compare histochemical and biochemical determinations of enzyme activities, enzymehistochemical procedures are applied to sections of pellets of subcellular fractions. These investigations are of value to determine the subcellular localization of histochemically demonstrable enzyme activities and to test the homogeneity of an isolated fraction. In homogenating duckling liver a great part of the endothelial cells is not destructed and consequently is found in the nuclear fraction. Kupffer cell lysosomes land in the heavy mitochondrial fraction, whereas hepatocyte lysosomes are chiefly found in the light mitochondrial fraction. -Glucuronidase activity shows a preferentially microsomal localization. Application of enzymehistochemical staining reactions to discontinuous gradients and comparison with biochemical data provides additional information about the validity of an enzymehistochemical reaction. In rat liver the tetrazolium reductases show a distinctly dual localization: activity in the mitochondrial band and in microsomal bands. As to their localization in different bands of the gradients non-specific esterases demonstrate a clear pH-dependency.     

Investigation of selenium distribution in subcellular fractions of human liver by neutron activation analysis

Selenium is an important and essential trace element to living systems. In the article, two methods of instrumental neutron activation analysis and hydride generation-atomic fluorescence spectrometry were applied to determine Se in biological samples and the accuracy was evaluated by several reference materials. The subcellular distribution of selenium in human liver samples, which were obtained from normal subjects who had an accidental death, was investigated by differential centrifugation combined with INAA. Selenium was mainly enriched in mitochondria, nuclei, and cytosol. Almost half of the total Se content existed in nuclei as a result of the large amount in liver and the high Se concentration. Generally, the highest Se concentration in the mitochondrial fractions of each liver sample suggested that Se had important functions in this liver component.