Carrier-free Electrophoresis Research Articles

Electrophoresis of particles and cells.

At ICES '99 particular emphasis was laid on electrophoresis of cells and particles dedicating a complete session to this topic. Challenged by this organizational strategy of Prof. Hashimoto 16 highly interesting abstracts had been submitted, of which 8 were presented orally. All of them showed that analytical as well as preparative electrophoresis of cells and particles have already reached a very high level of performance. Some possibilities of improving resolution were still proposed regarding analytical (3 abstracts) as well as preparative (2 abstracts) carrier free electrophoresis devices. All the other presentations, however, clearly demonstrated which tremendous impact on research and even on commercial production electrophoresis of cells and particles currently has. Outstanding results in chemical, biochemical and medical research were explained which would not have been obtained without carrier free electrophoresis technologies.

Separation by carrier-free electrophoresis of subpopulations of bovine alveolar macrophages

Carrier-free electrophoresis was used to separate subpopulations of bovine alveolar macrophages according to their electrical surface charges. The electrophoretic mobility of the macrophages changed significantly as a function of the time of cultivation in vitro. After 20 h, three subgroups were identified which differed in their specific contents of lysosomal hydrolases, α-naphthyl acetate esterase, and alkaline phosphodiesterase. By comparing the findings with results from the literature it was concluded that the subgroups correspond to different stages of maturation and that the fraction moving fastest may be the most mature fraction. The results were not influenced by phagocytosis and the presence or absence of Ca 2+.

Cellular aspects of retention and transport of inhaled soluble and insoluble actinide compounds in the rat lung

The binding of 241Am-hydroxide polymers (as models for readily soluble actinide compounds) to the cell components of rat lung was investigated using differential centrifugation, density gradient centrifugation, gel chromatography, carrier-free electrophoresis and electron microscopic autoradiography (with 241Pu). Irregularly shaped and spherical mixed (U, Pu)O 2 particles (as models for insoluble actinide compounds) were administered to rats by inhalation and intratracheal installation and the lung and organ retention was determined. Electron microscopic studies were performed with rat lung and with rat and bovine alveolar macrophages exposed to the actinide compounds in vitro. In the case of the mixed (U, Pu)O 2 particles the lung retention was independent of the particle shape and route of administration. Whereas 241Am administered as a hydroxide polymer was transferred rapidly from lung to skeleton and liver, only a few percent of the initial alveolar deposit was found in these organs after mixed oxide inhalation. It is concluded that all types of particles are stored primarily within phagolysosomes of alveolar macrophages. In the case of readily soluble compounds, the actinides are solubilized within these lysosomes and become bound to cytosolic ferritin in the alveolar macrophages. They are then released from the macrophages and probably cross the alveolar membranes as transferrin or as low-molecular-weight forms. Insoluble compounds remain within the lysosomes of alveolar macrophages, but there are indications of chemical damage to the lysosomal membranes, causing the particles to lie free in the cytoplasm.

Effects of Native and Phospholipid Treated Quartz on Bovine and Rat Alveolar Macrophages

Bovine and rat alveolar macrophages were obtained by post-mortem bronchoalveolar lavage. The standard test quartz DQ12 or another quartz type (Sicron F600) were used either in native form in serum-free medium or after treatment with Dipalmitoyl-lecithin (DPL). The dose-effect relationships were analyzed. The results for bovine and rat alveolar macrophages were similar. DPL treatment completely abrogated the cytotoxic effects of quartz, which were monitored by testing the viability of adherent cells and enzyme released into the culture medium. Results obtained by carrier-free electrophoresis indicate that bovine alveolar macrophages are composed of several subpopulations. After incubation with native, but not with DPL-treated quartz particles the electrophoretic pattern was markedly changed.

Intrahepatocellular site of the catabolism of heme and globin moiety of hemoglobin-haptoglobin after intravenous administration to rats.

The intracellular site of incorporation and degradation of heme and globin moiety of hemoglobin-haptoglobin in rat liver cells was investigated in vivo. Hemoglobin-haptoglobin, administered intravenously to rats, is cleared from the circulation and incorporated exclusively into liver parenchymal cells through the receptor specific for the molecule (Kino, K., Tsunoo, H., Higa, Y., Takami, M., Hamaguchi, H., and Nakajima, H. (1980) J. Biol. Chem. 255, 9616-9620). Intracellular distribution of radioactivity was determined after intravenous administration of [3H-Heme,14C-Globin]hemoglobin-haptoglobin to rats. The doubly labeled hemoglobin-haptoglobin was incorporated first in organelles of lower anodic mobility in carrier-free electrophoresis and of low density (density range, 1.05-1.07 g/ml) in Percoll density gradient centrifugation recovered in Golgi subfractions of the liver cells in a substantially intact form. In the subsequent stages, these organelles progressively acquired a higher anodic mobility as well as higher density, presumably through fusion with other organelles. In the resulting organelles of higher anodic mobility in electrophoresis and high density (density range, 1.07-1.15 g/ml) in Percoll, the hemoglobin-haptoglobin first dissociated symmetrically into two 82,000-dalton subunits having intact heme, and then the organelles containing only 3H radioactivity but no 14C radioactivity were separated by electrophoresis. Most of the 3H radioactive materials in these organelles are identified as intact [3H]heme. These investigations suggest that the heme moiety of hemoglobin-haptoglobin in the organelles is detached from globin-haptoglobin and binds to another carrier protein prior to conversion of heme to bilirubin.

Studies on the lysosomal binding of 141Ce, 239Np, 239Pu and 241Am in rat and syrian hamster liver using carrier-free electrophoresis

The binding of 141Ce, 239Np, 139Pu and 241Am in the liver of rats and Syrian hamsters, following injection in essentially monomeric form, was analyzed by carrier-free electrophoresis at 4–9 days and several months after radionuclide injection. In contrast to density gradient methods lysosomes can be clearly separated from other cell organelles by carrier-free electrophoresis. These, and previous results from this and other laboratories confirm that lysosomes are the main initial binding site for these four radionuclides in the livers of rats and Syrian or Chinese hamsters. Light microscopic autoradiography showed that at all the time intervals studied 241Am was more or less uniformly distributed in the liver of all three species. Thus, the changes in the electrophoretic pattern of the nuclides, observed at later time periods in hamsters, cannot be explained by gross redistribution phenomena such as accumulation in macrophages.

Association of 239 Pu with Lysosomes in Rat, Syrian Hamster, and Chinese Hamster Liver as Studied by Carrier-Free Electrophoresis and Electron Microscopic Autoradiography with 241 Pu

The binding of injected monomeric plutonium in the liver of rats, Syrian hamsters, and Chinese hamsters (species which show profound differences in their ability to eliminate 239Pu from the liver) was investigated by carrier-free electrophoresis using 239Pu and electron microscopic autoradiography with 241Pu. These studies are part of a program designed to obtain a better understanding of the mechanisms of the clearance of transuranium elements from liver of different mammals and man. Between 4 and 9 days after nuclide injection, a clear correlation between the majority of the 239Pu and lysosomal enzymes was observed when the mitochondrial-lysosomal (ML) fraction of the livers was analyzed by carrier-free electrophoresis. In the two hamster species, a second 239Pu peak exists from the beginning and increases with time to comprise 50% of the total radioactivity at later times. During electron microscopic examination 4 days after 241Pu injection, beta tracks were frequently observed over globular structures resembling dense bodies in Chinese hamster liver. They were also observed frequently over chromatin-rich portions of the cell nuclei. These results, together with those from previous density gradient studies, show that lysosomes are the primary deposition site for 239Pu in the liver cytoplasm of these three rodent species. The hypothesis of a morphologic transformation of these lysosomes with time in hamster liver and of rapid bulk exocytosis of the lysosomes in rats are still possible explanations for the extreme differences in the elimination among the three species.

Recognition of a positive MLR within 4 hours using a carrier free electrophoresis system.

Indicator cells--tanned, surface stabilized sheep erythrocytes--were incubated for 1 h in supernatants of 3 h MLCs. Their electrophoretic mobility was measured by an analytical, carrier free electrophoresis system. The change in their mobility compared with an appropriate control was calculated in per cent and correlated with the conventional measured MLR-cpm. The correlation of the two quantities is statistically highly significant (p less than 0.01). Furthermore, the difference of the electrophoretic mobility values of the group of HLA-D-identical and the groups of HLA-D-haploidentical or -different donors is significant beyond the 1% level (p less than 0.0005). Our method enables, therefore recognition of a positive or negative MLC after only 4 h. Typing for HLA-D-determinants seems to be possible. This could be of great importance for histocompatibility testing and organ transplantation.

Phospholipid and fatty acid composition of tetrodotoxin receptor-rich membrane fragments from Electrophorus electricus

To study the interaction of voltage-sensitive Na +-channels with membrane lipids, the phospholipid and fatty acid composition of highly purified membrane fragments from the remarkably differentiated plasma membrane of Electrophorus electricus has been analyzed. After density gradient fractionation and carrier free electrophoresis, fractions with up to 30 pmol tetrodotoxin binding/mg protein can be obtained, which may correspond to a 50% pure preparation of the extrasynaptic part of the excitable face. Phospholipid classes and cholesterol are separated by one-dimensional thin-layer chromatography in acidic and alkaline solvent systems. The following mean molar contents are found: 40% phosphatidylcholine, 23% phosphatidylserine, 30% phosphatidylethanolamine and 7% sphingomyelin. In a series of 11 animals, significant deviations from these mean values have been observed. The fatty acid composition of the phospholipids has been determined by gas chromatography. Phosphatidylcholine contains more than 50% 16:0, and about 20% unsaturated fatty acids in the C-18 group. Compared to other plasma membrane fractions, this phospholipid is the least differentiated. By contrast, phosphatidylethanolamine and phosphatidylserine show many characteristics in different membrane fractions, especially in their unsaturated components representing more than 50%. 22:6, as the major constituent in these fractions, accounts for a quarter to a third of all fatty acids in these fractions. 18:0 is the main saturated component in these two phospholipids with abundances of typically a quarter or less of all fatty acids. Knowledge of the lipid composition of these excitable membranes may help to conserve binding and structural properties when analyzing lipid-sensitive Na +-channels in vitro. It is also useful as a guideline for systematic reconstitution studies.

Isoenzymes of Cysteine Synthase in the Cyanobacterium Synechococcus 6301

Summary Cysteine synthase (O-acetyl-L-serine sulfhydrolase; EC 4.2.99.9) was isolated from the cyanobacterium Synechococcus 6301. Isoenzymes could be separated by DEAE-cellulose chromatography and by electrophoretic separation on disc gels and by carrier-free electrophoresis. Both enzymes had a molecular weight of 56,000, a pH optimum of 7.5, and a temperature optimum of 50 °C. K m -values for both isoenzymes were found to be around 0.8 mM for sulfide and 0.7 mM for O-acetyl-L-serine. Both isoenzymes catalyzed an isotopic exchange reaction between cysteine and sulfide with pH optima around 9. A model for the cysteine synthase reaction is presented.

Recognition of a positive MLC reaction after four hours.

EMT indicator cells were incubated for 1 h in the supernatant of a 3 h MLC. Their electrophoretic mobility was then measured by an analytical, carrier-free electrophoresis system. The alteration of their mobility against a medium-standard was calculated in per cent and correlated with the conventional measured MLR-cpm. According to our results the correlation of the two quantities is statistically highly significant. This method could enable recognition of a positive or negative MLC after only 4 h. This fact could be of great importance for histocompatibility testing and transplantation.

The protein-chromophore bond in B phycoerythrin from Porphyridium cruentum. Radiosulfur labeling experiments.

Red algae of the species Porphyridium cruentum were grown in a minimum sulfate medium containing 35SO42-. 35S-labeled phycoerythrin was extracted. B Phycoerythrin, b phycoerythrin and R phycocyanin could be separated from other proteins by using a carrier-free electrophoresis on columns. The final ratio A545/A280 of B phycoerythrin thus obtained was greater than or equal to 5. 35S-labeled B phycoerythrin was digested proteolytically with trypsin and pepsin. The resulting 35S-containing bilipeptides were separated by isoelectric focusing. Zones of enhanced chromophore concentration always showed an enhanced radioactivity. Peptide fractions with a low molar ratio sulfur/chromophore (1.1-1.8) were purified to remove sucrose and the carrier ampholyte. A modified, optimized Edman degradation followed. A butylacetate-soluble, red Edman product was obtained that contained most of the chromophore and the bulk of the radioactivity. This product was purified by two-dimensional thin-layer chromatography. The main spot of the chromatogram was subjected to acidic hydrolysis. The major part of the radioactivity in the hydrolysate cochromatographed with cysteine. That proves cysteine to be the binding amino acid in all cases investigated.

Homogeneous cell populations from rabbit kidney cortex. Proximal, distal tubule, and renin-active cell isolated by free-flow electrophoresis.

A single-cell suspension has been prepared from rabbit kidney cortex by using a Ca-binding medium and gentle mechanical forces. The suspension was subjected to carrier-free electrophoresis, and several cell fractions were obtained. Proximal and distal tubule cell populations could be identified by their morphology. Renin-containing cells were located by means of radioimmunoassay. The morphology of the cells and their vitality (uridine incorporation) are discussed.

Continuous-flow isoelectric focusing of mixed red blood cells of different species in an apparatus for carrier-free electrophoresis

Continuous-flow isoelectric focusing of mixed red blood cells of different species in an apparatus for carrier-free electrophoresis

Studies on deoxyribonucleoproteins. IX. Subfractions of deoxyribonucleoproteins from rat and mouse liver.

By carrier-free continuous electrophoresis, deoxyribonucleoprotein from rat and mouse liver could be separated into two subfractions. The more anodic fraction (DNP I), comprising 5 - 8 per cent of the total, contains fewer proteins (two types of histones only). [3H]Cyclophosphamide caused in vivo a 2.5 times higher alkylation of the DNA in in DNP I than of the DNA in DNP II. These and additional results led to the suggestion of a structural model with DNP I as a spacer in the deoxyribonucleoprotein fiber.

Studies on deoxyribonucleoproteins: VIII. Isolation and characterization of nonhistone proteins from euchromatic and heterochromatic deoxyribonucleoprotein of rat liver

Rat liver nuclei were fractionated into euchromatin and heterochromatin. Deoxyribonucleoproteins of these fractions were purified by continuous carrier-free electrophoresis and analyzed for their proteins, mainly the nonhistones. The ratio of nonhistones/DNA was found to be much higher in the purified deoxyribonucleoprotein of euchromatin than in the deoxyribonucleoprotein of heterochromatin. Comparative studies of the nonhistones derived from euchromatin and heterochromatin showed no clear indications for qualitative differences. By gel electrophoresis 13–14 bands were obtained, the molecular weight range was found to be in the range between 13 000 and 150 000. The main fractions of the nonhistones possessed isoelectric points from 4.5–6.5. The biological implications of these observations and the special methodical problems are discussed.

Nonspecific collagenolytic activity: Hydrolysis of native collagen by a mold protease

A proteolytic enzyme from culture fluids of Aspergillus oryzae was purified by a multistep procedure including carrier-free electrophoresis and DEAE-Sephadex chromatography. It was a homogenous protein, judging from results obtained by disc electrophoresis, immunoelectrophoresis and end-group determination. The enzyme hydrolysed native collagen at a slow rate. Electron microscopic investigations of the digest revealed triple-helical fragments remaining after degradation of the collagen molecule at its N-terminal end. The results thus contribute to a current discussion on collagenolysis by proteolytic enzymes other than specific collagenases.

Isolation of α-crystallin by means of continuous flow electrophoresis in a liquid film

Carrier-free continuous electrophoresis was applied for the separation of α-crystallin on a preparative scale. Every 24 hr 1g of the purified protein can be obtained. Purity was verified by means of polyacrylamide gel electrophoresis and sedimentation analysis. If necessary, leucine aminopeptidase activity may be removed from the α-crystallin preparation by gel chromatography on Sepharose 6-B.

A comparative study of hordein fractions

Further investigations of the fractionation of hordein by gel chromatography on Sephadex G-200 are described. The hordein fractions obtained by gel chromatography and those by continuous carrier-free electrophoresis were also characterized by means of continuous electrophoresis (or, respectively, gel chromatography), disc electrophoresis, amino acid analysis and molecular weight determination. The presence of six electrophoretically different fractions of hordein was firmly established. It was demonstrated that of the six hordein fractions only Fraction B has the property to yield reversibly aggregated molecules in urea medium. The remaining fractions are stable and behave as individual proteins. The correlation of the fractions obtained by the three methods employed is discussed. Further arguments for the necessity of a new classification of plant proteins based on their chemical and electrical properties rather than on their solubilities are presented.

Increased buffer density as a means of separating peroxidase isoenzymes by preparative continuous-flow carrier-free electrophoresis

The use of 5, 10, and 15% sucrose in the separating buffer in a preparative continuous-flow carrier-free electrophoresis system is described as it applies to the fractionation of peroxidase isoenzymes. The use of sucrose in the separating buffer greatly reduced diffusion of the isoenzymes during electrophoresis, which resulted in a better fractionation and a ceduction in the number of collecting tubes. Electrophoresis in 10% sucrose yielded fractions that contained the individual, separated, peroxidase isoenzymes.