Mild Lysis Research Articles

A simple and rapid procedure for the isolation of intact bovine rod outer segments (ROS)

A method is described which allows the rapid isolation and purification of intact outer segments (ROS) from cattle eyes. It requires very fresh retinal material and can be completed within less than 2 h of the death of the animals. Cattle eyes are dissected in the usual manner, the retinae are isolated and the ROS are separated from the rest of the retina by gentle vortexing and filtration through a nylon mesh. The resulting crude ROS suspension is purified on a discontinuous sucrose density gradient. Two fractions are otbtained, the major one consisting of mostly intact ROS, the minor one of RIS-ROS, i.e. of ROS which are still connected to part of their inner segment. The ROS are washed once and can be stored on ice for several days without loosing their intact plasma membrane. They can be transformed to leaky ROS by a quick freeze/thawing cycle or, if one wants unobstructed access to the interdiskal space, they can be subjected to a mild lysis treatment. The resulting ROS material is characterised using light microscopy, electron microscopy, light scattering, gel electrophoresis and absorption spectroscopy. It contains unusually low levels of 48k-protein and very high levels of G-protein. The latter cannot be washed out in the presence of GTP-γ-S, even in the case of leaky ROS.

Localization, biosynthesis, processing and isolation of a major 126 kDa antigen of the parasitophorous vacuole of Plasmodium falciparum

Monoclonal antibodies prepared against a 50 kDa antigen found in Plasmodium falciparum culture supernatants identify a 126 kDa polypeptide which can be localized by immunofluorescence and immunoelectronmicroscopy at the periphery of the schizonts. This polypeptide is released from the infected erythrocytes by mild saponin lysis and is probably a component of the parasitophorous vacuole. Pulse chase kinetic analysis demonstrated its disappearance from the parasitized red blood cell from 6 to 10 h after being synthesized and the concomitant appearance of the 50 kDa molecule in the culture supernatant. Purification of metabolically labeled, schizont infected cells demonstrated that spontaneous release of merozoites is needed for the processing of the 126 to the 50 kDa whereas reinvasion is not. Polyclonal antibodies were raised in rabbit against affinity purified 126 kDa protein. These antibodies, together with another 126 kDa specific monoclonal antibody have enabled us to characterize two other cleavage products of the 126 kDa antigen in culture supernatants, namely 47 and 18 kDa polypeptides. We believe that the processing of the 126 kDa protein into low molecular weight fragments reflects a proteolytic event which may participate in merozoite release.

Ｔｉｓｓｕｅ‐ｐｌａｓｍｉｎｏｇｅｎ Ａｃｔｉｖａｔｏｒによる血栓治療 Ｇｌｙｏｘｙｌ ａｇａｒｏｓｅ ｇｅｌ電気泳動による検討

We studied on detection of plasma fibrinogen derivatives of tissue-Plasminogen Activator (t-PA) mediated thrombosis with Glyoxyl agarose gel electrophoresis. Plasma proteins were immobilized into aldehyde-rich Glyoxyl phoresis. Plasma proteins were immobilized into aldehyde-rich Glyoxyl agarose with NaCNBH3 after electrophoresis, and immobilized fibrinogen derivatives were blotted with fluorescent anti-fibrinogen. Fibrinogen derivatives [Fibrin monomer, polymer (HMW); Fibrinogen (φ); Fragment X, Y (φ′); Fragment D, E (LMW)] were detected by the mobility on the gel and their concentrations were calculated by the fluorescent intensity of them. Standard plasma had no fluorescent blotting peak by this method. The change of fibrinogen levels of t-PA-mediated thrombosis (total 13 patients) were classified into 3 groups; Slightly lysis (7/13), Mild lysis (3/13), Systemic lysis (3/13) and the electrophorogram of thrombosic patient plasma was resemble to that of malignant hypertention rat, but only a few normal human had the HMW peak (1/7). It was concluded that Glyoxyl agarose gel electrophoretic method was very useful and sensitive for the detection of fibrinogen derivatives with small sample. This method could be used for the clinical samples, because immobilized proteins into gel were stable and possible to store for a few weeks.

The relationship between von willebrand factor antigen and fibronectin in human plasma, endothelial cells and fibroblasts in culture

The distribution of von Willebrand factor antigen (vWFAg) and fibronectin (Fn) in endothelial cells and in fibroblasts in culture was examined using immunohistological methods. Extracellular matrices were studied after removal of cells by mild hypotonic lysis. Co-distribution of these antigens was also examined after culture of fibroblasts in the presence of endothelial cell conditioned medium or of exogenous vWFAg. The presence of intracellular vWFAg and its association with extracellular Fn in confluent endothelial cells was confirmed. Furthermore, both antigens were detected in matrices from fibroblasts grown in the presence of human plasma and endothelial cell medium. vWFAg was not found, however, in the absence of endothelial cell conditioned medium or in experiments using human serum in place of plasma. Cross-linking of vWFAg was examined using quantitative and qualitative electrophoretic methods. Levels of vWFAg in serum from patients with severe Haemophilia A were approximately 50% of those found in the corresponding plasma. Furthermore, vWFAg was reduced to similar levels in serum from normal blood to which heparin was added. The lowest levels of vWFAg were found in the presence of sufficient heparin to totally inhibit the formation of fibrin. In contrast, levels of Fn remained unchanged in these circumstances. The results did not support the view that vWFAg was cross-linked to Fn in plasma by thrombin-activated factor XIII. In addition, immunoelectrophoresis of cultured endothelial cell products did not demonstrate cross-linking of vWFAg to Fn. The data are consistent with the concept that deposition of vWFAg on the subendothelium is dependent on viable endothelial cells or on another product of these cells.

Role for topoisomerases in the release of DNA into the detergent-soluble fraction of eukaryotic cells.

Detergent-soluble DNA is the fraction (2-4%) of DNA that is released into the supernate upon mild detergent lysis. It is nonmitochondrial in origin. It labels efficiently with deoxy[3H]ribonucleosides and the labeling is prevented by inhibitors of polymerase alpha and ribonucleotide reductase. In previous publications we have characterized detergent-soluble DNA from splenocytes of immunologically activated mice. In this publication we show that incorporation of [3H]thymidine into detergent-soluble DNA is prevented by pretreatment with novobiocin, 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), and teniposide (VM26), three inhibitors of type II topoisomerases. Camptothecin, an inhibitor of type I topoisomerases, also reduces incorporation of [3H]thymidine but only to 50% of control levels. In addition to affecting incorporation of [3H]thymidine, preincubation with the topoisomerase II inhibitors m-AMSA and VM26 alters the amount of DNA recovered in the detergent-soluble fraction. At low concentrations of m-AMSA the amount of detergent-soluble DNA increases somewhat, whereas at higher drug concentrations a marked decrease is observed. Treatment with VM26 results in diminished amounts of DNA being released into the detergent-soluble fraction as well. However, maximal inhibition of detergent-soluble DNA release by VM26 requires the presence of camptothecin. Therefore, we suggest that topoisomerases play an important role in making a small part of lymphocyte chromatin detergent labile. Furthermore, these results are consistent with recent studies demonstrating a role for topoisomerases in yeast replication. Thus, the newly synthesized portion of detergent-soluble DNA may arise as DNA replication intermediates not yet stabilized into mature chromatin.

Isolation ofMycoplasma gallisepticum membranes by a mild alkaline-induced lysis of nonenergized cells

A procedure is described for the preparation of purified membranes from late-exponential or stationary phase cells ofMycoplasma gallisepticum. NonenergizedM. gallisepticum cells were lysed in an isosmotic NaCl-Tris buffer at pH 8.5, conditions that seem to interfere with cell volume regulation. Electron microscopy, chemical, density gradient, and enzymatic analyses of the membrane preparation showed the membranes to be free of cytoplasmic contaminants and partially sealed.

Regulation of membrane peptides by the Pseudomonas plasmid alk regulon.

Pseudomonas putida strains carrying the plasmid alk genes will grow on n-alkanes. Induced alk+ strains contain membrane activities for alkane hydroxylation and dehydrogenation of aliphatic primary alcohols. P. putida cytoplasmic and outer membranes can be separated by sucrose gradient centrifugation after disruption of cells by either mild detergent lysis or passage through a French press. Both the membrane component of alkane hydroxylase and membrane alcohol dehydrogenase fractionated with the cytoplasmic membrane. Induction of the alk regulon resulted in the appearance of at least three new plasmid-determined cytoplasmic membrane peptides of about 59,000 (59K), 47,000 (47K), and 40,000 (40K) daltons as well as the disappearance of a pair of chromosomally encoded outer membrane peptides of about 43,000 daltons. The 40K peptide is the membrane component of alkane hydroxylase and the product of the plasmid alkB gene because the alkB1029 mutation altered the properties of alkane hydroxylase in whole cells, reduced its thermal stability in cell extracts, and led to increased electrophoretic mobility of the inducible 40K peptide. These results are consistent with a model for vectorial oxidation of n-alkanes in the cytoplasmic membrane of P. putida.

An improved method for the preparation of undegraded polysomes and active messenger RNA from immature chicken erythrocytes

A simple method for the large-scale isolation of undegraded polysomes and active mRNA from immature hen erythrocytes is described. The method is based on the removal of white cells from heparinized blood by adsorption on sulfoethyl- or sulfopropyl-Sephadex. Red cells thus purified can be stored frozen and lysed in the presence of nonionic detergents. The yield of polysomes is about twice that obtained by the usual mild lysis procedures designed to prevent lysis of lysosomes from contaminating white cells.

Studies on the deoxyribonucleic acid‐bearing portion of the cell envelope of Escherichia coli

AbstractThe envelope components of nuclear bodies which were obtained from Escherichia coli W7 by a mild lysis method were investigated. By using 2,6‐diaminopimelic acid (DAP) as precursor which is incorporated only into peptidoglycan in this strain it was found that the particles contained about 14% of the murein layer of the cell. The percentage of phosphatidylethanolamine was enriched at the cost of the other phospholipids in the nuclear bodies compared to whole cells. If lipids were labelled with 3H‐palmitic acid the cytoplasmic and the outer membrane could be found after isopycnic centrifugation; however, when the cells were incubated with chloramphenicol, only the outer membrane was seen. The peptidoglycan and the proteins could be assigned only to the outer membrane. The DNA is also bound to the outer membrane. From these results it was concluded that (1) in all lysis methods the cytoplasmic membrane is more easily dissolved than the outer layers of the envelope, and (2) that there is a firm binding between DNA and the outer membrane in vivo.

Studies on the deoxyribonucleic acid-bearing portion of the cell envelope ofEscherichia coli

The envelope components of nuclear bodies which were obtained from Escherichia coli W7 by a mild lysis method were investigated. By using 2,6-diaminopimelic acid (DAP) as precursor which is incorporated only into peptidoglycan in this strain it was found that the particles contained about 14% of the murein layer of the cell. The percentage of phosphatidylethanolamine was enriched at the cost of the other phospholipids in the nuclear bodies compared to whole cells. If lipids were labelled with 3H-palmitic acid the cytoplasmic and the outer membrane could be found after isopycnic centrifugation; however, when the cells were incubated with chloramphenicol, only the outer membrane was seen. The peptidoglycan and the proteins could be assigned only to the outer membrane. The DNA is also bound to the outer membrane. From these results it was concluded that (1) in all lysis methods the cytoplasmic membrane is more easily dissolved than the outer layers of the envelope, and (2) that there is a firm binding between DNA and the outer membrane in vivo.

Size and structure of mitochondrial DNA from Physarum polycephalum.

One band of DNA with a buoyant density of 1.688 g cm−3 is found when isolated mitochondria of the slime mold Physarum polycephalum are incubated with deoxyribonuclease prior to lysis. The DNA consisted mainly of linear molecules up to about 20 μm in length. As many as 10% of the molecules were, however, of open circular conformation with a circumference of 19.1±0.5 μm. Mild lysis conditions favoured the isolation of DNA/protein complexes with no visible free ends. The data suggest that the mitochondrial DNA (mtDNA) of Physarum is a circle and that circularity may be maintained by DNA/protein interaction.

RNA polymerase from Bacillus subtilis: isolation of core and holo enzyme by DNA-cellulose chromatography

A new procedure for the purification of B. subtilis RNA polymerase, based on mild lysis of cells, low speed centrifugation, gel filtration, DEAE-Sephadex chromatography and affinity chromatography on DNA-cellulose, yields three forms of enzyme referred here as enzyme A, B and C. As revealed by SDS gel electrophoresis, enzyme A has the subunit structure of core polymerase plus some small polypeptides. Its catalytic properties are similar to those of core polymerase. Enzyme B has the composition of core polymerase. Both enzymes A and B can be stimulated by the addition of beta factor. Enzyme C has the holo-enzyme composition. The pattern of sensitivity of the three forms of enzyme towards KCl are very different: enzymes A and B, even at low concentration of salt, are inhibited with all the DNA templates tested, whereas enzyme C shows a pattern of stimulation specific for each DNA tested. The transcripts of the three enzymes on phage SPP1 DNA template have been analyzed by hybridization to the separated strands. Only enzyme C selectively transcribed the H strands.

Identification and radiochemical purification of the recA protein of Escherichia coli K-12.

The product of the recA gene of E. coli has been identified by labeling proteins synthesized in UV-treated cells after infection with specialized transducing phages carrying the recA gene. Following infection of UV-treated cells by lambda precA, which carries the recA+ gene, a major protein with a molecular weight of 43,000 is detected on polyacrylamide gels containing sodium dodecyl sulfate. This protein is also made after infection of suppressing hosts by lambda precA99, which carries an amber recA- mutation, but is not synthesized after infection of nonsuppressing hosts by this transducing phage. A spontaneous recatrevertant of lambda preca99 induces synthesis of this protein after infection of a nonsuppressing host. The product of the recA gene is a soluble protein found in a complex with a molecular weight of approximately 150,000 after mild detergent lysis of cells.

Isolation of free minus strands from Qβ-infected Escherichia coli

Free minus strands (minus strands not involved in a firm duplex structure) are produced in Escherichia coli infected with the RNA phage Q β. These minus strands can be extracted from the cells under conditions of mild lysis and low salt concentrations, and can be purified by electrophoresis on polyacrylamide gels. The free minus strands are fully competent as template for the Q β-replicase in the absence of host factors, directing the synthesis of plus strands.

The effect of lysozyme on DNA—Membrane association in Escherichia coli

The chromosome of Escherichia coli sediments three different ways in sucrose density gradients, depending upon the concentration of lysozyme used to lyse the cells, as follows: 1. (1) Low-S complex. In the presence of 2–400 μg/ml of egg white lysozyme with Brij-58-Na deoxycholate or Triton X-100, the chromosome sediments at 500–1100 S (low-S complex). When Brij-58 alone is used, some of the DNA sediments as a low-S complex and some sediments rapidly with the membrane fraction. 2. (2) Secondary DNA—membrane complex. In the presence of 800 μg/ml of egg white lysozyme, all the DNA sediments with the membrane fraction at greater than 4000 S regardless of which detergent is used. This DNA—membrane complex is due to the high concentration of lysozyme, for when a pre-formed low-S complex is exposed to a high concentration of lysozyme, it sediments with the membrane fraction. This association can also be seen in the presence of poly( l-lysine), ribonuclease, Mg 2+ or spermidine, indicating that lysozyme mediates the DNA—membrane association non-specifically as a polycation. An artificial association between exogenous coalfish DNA and an E. coli membrane fraction is induced by 800 μg/ml of lysozyme. 3. (3) Primary DNA—membrane complex. A concentration of T4 lysozyme as low as 0.01 μg/ml, or 20 lysozyme molecules/chromosome, can lyse cells. This is in contrast to 2 · 10 6 lysozyme molecules/chromosome corresponding to 800 μg/ml of egg white lysozyme. Under these mild lysis conditions, the chromosome sediments with the membrane fraction, suggesting a primary DNA—membrane association.

Osmotically reactive plasma membrane vesicles prepared from rabbit kidney tubules by mild hypotonic lysis

Plasma membrane vesicles were obtained by hypotonic lysis in an ice-cold medium containing EDTA and MgCl 2. The vesicles were isolated by differential centrifugation. Compared to a total kidney homogenate, a 10–12-fold enrichment of trehalase and alkaline phosphatase (marker enzymes for renal brush border), and a 6-fold enrichment of (Na +:K +)-stimulated ATPase, (a marker enzyme for the basal and lateral plasma membrane of the tubule cell), was achieved. Contamination by other cell organelles was very low. The plasma membrane vesicles enclosed small amounts of the cytoplasmic enzymes lactate dehydrogenase and malate dehydrogenase, which exhibited full activity only after their release into the medium by sonication. Electron micrographs of this preparation showed microvilli with drumstick-like expansions, but also spherical vesicles. By measuring the distribution of radio-actively labelled compounds of different molecular weight in a packed sediment of the plasma membranes under isotonic conditions, an intravesicular volume of 82% or 9 μl/mg of membrane protein was estimated. The intravesicular volume decreased when the osmolality of the medium was augmented by raffinose. The scattering of light by the vesicular suspension could be used to monitor rapid volume changes. By this method, the following sequence of flux rates was established: glycerol>erythritol> adonitol>mannitol. The fluxes of LiCl, NaCl, and KCl were almost identical, but faster than those of adonitol and mannitol. The data indicate, that a large fraction of the plasma membrane isolated in this preparation have formed vesicles, and also that they have retained, as far as investigated, the permeability characteristics of the plasma membrane.

Role of gene 46 in bacteriophage T4 deoxyribonucleic acid synthesis.

In an attempt to establish whether Escherichia coli B infected with N130 (an amber mutant defective in gene 46) is recombination-deficient, the postinfection fate of (14)C-labeled N130 parental deoxyribonucleic acid (DNA) was followed, its amount in complex with the host cell membrane being determined in sucrose gradients after mild lysis of the infected cells. The parental DNA was found to undergo gradual detachment from the membrane during infection. Pulse-chase experiments similarly showed that newly synthesized DNA is normally attached to the host cell membrane and is detached by endonucleolytic breakage at a late stage of infection. The conclusion is that only attached DNA molecules are replicated by membrane-bound replicase, whereas those detached by endonucleolytic breakage are not. It thus seems that the gene 46 product controls the activity of a nuclease whose main function is recombination of DNA nicked by endonuclease, thereby attaching it to the host cell membrane. The rate of T4 DNA synthesis is apparently governed by the efficiency of recombination. Supporting evidence was found in experiments with the double mutant N130 x N134 (genes 46, 33).

Characterization of two circular satellite species of deoxyribonucleic acid in Rhodopseudomonas spheroides

DNA, isolated from a strain of Rhodopseudomonas spheroides, showed two components when banded in CsCl, with buoyant densities of 1.719 and 1.724 g/ml. The lighter band comprised about 8% of the total. Using a mild lysis procedure, it was possible to extract DNA of which 15–20% was circular. Circular DNA, isolated by dye-buoyant density centrifugation, contained two components with buoyant densities 1.718 and 1.724 g/ml. Sedimentation of the circular DNA gave two peaks, with S 20,w values of 78.2 S and 52.7 S; sedimentation in alkali, and in neutral solution after denaturation, showed that the first peak was closed circular and the second peak open circular DNA. Band-buoyant density sedimentation of the circular DNA in alkaline CsCl initially showed two peaks; after a time, each peak split into two. The alkaline buoyant densities of the four peaks were consistent with open and closed circles with neutral buoyant densities 1.718 and 1.724 g/ml. It is concluded that Rps. spheroides contains two circular satellite DNA species, with the same molecular weight of 70–75 × 10 6 daltons, but with slightly different base compositions.