Membrane isolation on polylysine-coated beads. Plasma membrane from HeLa cells

HeLa cell plasma membranes have been purified after binding cells to polylysine-coated polyacrylamide beads. Cell attachment to beads and membrane recovery were maximal in a sucrose-acetate buffer, pH 5.0, at 25 degrees C. Measurements of ouabain-sensitive NaK-adenosine triphosphatase, membrane-bound 125I-wheat germ agglutinin, and chemical analyses showed that membranes on beads were of comparable or greater purity than membranes isolated by conventional methods. Because the isolation procedure is rapid (approximately 2.5 h), and produces membranes whose protoplasmic surfaces are fully exposed, it should be a useful supplement to standard isolation techniques.     

Coupling polylysine to glass beads for plasma membrane isolation

Solid glass beads for use in isolating cell membranes were coated with a stable, covalently attached layer of polylysine. The optimal conditions for coating the bead surface were established and the beads were tested by measuring the attachment of human erythrocyte plasma membranes. When compared to other beads, such as those with absorbed polylysine or protamine, none retained red-cell membranes as well as glass beads with covalently linked polylysine.     

Transbilayer mapping of membrane proteins using membranes isolated on polylysine-coated polyacrylamide beads

Erythrocyte and HeLa cell plasma membranes were isolated on polylysinecoated polyacrylamide beads and the transbilayer disposition of their proteins was investigated.When membranes of intact erythrocytes were isolated on beads and then labelled by lactoperoxidase-catalysed iodination, their labelling pattern was similar to that of inside-out vesicles in solution.When the membranes of intact HeLa cells were isolated on beads and then labelled by galactose oxidase-[³H]borohydride treatment, no glycoprotein or glycolipid sugars were accessible. On the other hand, when the HeLa cell membranes were isolated on beads and then labelled by the lactoperoxidase-catalysed iodination, all of the major membrane proteins were iodinated. These experiments confirmed for HeLa cell membranes what had previously been shown for erythrocyte membranes: when the membranes of intact cells are isolated on beads, the accessibility of their surfaces to enzymatic probes is the same as would be expected of inside-out vesicles in suspension. Double-label experiments, in which the HeLa cell membranes were labelled first on the intact HeLa cells and again after isolation on beads, identified several     

Plasma membrane isolation on DEAE-Sephadex beads

A much-simplified method for the purification of plasma membranes of cultured cells is presented, based upon the attachment of viable cells to nitrocellulose-treated DEAE-Sephadex beads, and their subsequent shearing by hypotonic lysis, agitation on a vortex mixer and sonication. The method is suggested by an older procedure involving attachment to poly-(L-lysine)-coated glass or polyacrylamide beads; the preparation involved in the present method, however, is considerably easier, more rapid and less expensive. Recovery of L-cell plasma membrane marker enzyme activities is approx. 25%, while contamination by internal membrane markers is much less than 1%.     

Retention of lipid asymmetry in membranes on polylysine-coated polyacrylamide beads

Phosphatidylcholine-specific exchange protein from calf liver was used to study the asymmetry and transmembrane movement of phosphatidylcholine in rat erythrocyte membranes isolated on polylysine-coated beads. While confirming previously published results for sealed ghosts, we found that for membranes attached to beads, where the cytoplasmic surface is exposed, about 36% of the total phosphatidylcholine is readily available for exchange, while the remaining 64% is exchangeable at a much slower rate. This indicates that the relative transbilayer asymmetry of phosphatidylcholine is largely maintained when red cell membranes are isolated on beads. On the other hand, transmembrane movement of phosphatidylcholine is decreased in membranes attached to cationized beads: the half-time for equilibration of phosphatidylcholine between the two monolayers of the membrane is 8 h for membranes on beads, compared to 1.5 h for sealed ghosts. Our results indicate that polylysine-derivatized beads are a useful tool for studying asymmetric properties of biological membranes.     

Schistosoma mansoni: surface membrane isolation by polycationic beads

The Schistosoma mansoni surface membrane complex was isolated by binding polycationic beads to the worm surface in a sucrose- or sorbitol-acetate buffer, pH 5.0, at 4 C. The ratio of incorporation [3H]cholesterol/[14C]arachidonic acid was measured as well as the specific activities of the alkaline phosphatase (EC 3.1.3.1), Type I phosphodiesterase (EC 3.1.4.1), and Ca2+-adenosine triphosphatase (EC 3.6.1.3). The results indicated that membranes isolated on beads were of comparable or greater purity than membranes isolated by sucrose gradient centrifugation. The isolation procedure was rapid (30 min) and produced membrane fractions whose cytoplasmic surfaces were probably exposed.     

Improved method for isolation of plasma membrane on cationic beads. Membranes from Dictyostelium discoideum

The plasma membrane from Dictyostelium discoideum was routinely purified 35-fold by an improved technique using beads coated with positively charged polymers. Cells were attached to the beads and bare regions between the cells were neutralized with a polyanion. The neutralization decreased contamination of the bare regions by intracellular proteins released when cells were disrupted to leave behind beads coated by plasma membrane. The neutralization increased the purification as measured by membrane-bound ¹²⁵I-labeled concanavalin A. Contamination by markers for various intracellular components was markedly decreased. Various bare-site neutralization reagents were evaluated and gave different results depending upon their charge density and molecular weight. The pH of the neutralization was critical. The optimum pH for cell attachment to beads, 5.0, had little effect as regards bare-site neutralization. A new procedure is given that optimizes the essential features for the plasma membrane isolation on beads.     

Immobilization of hydrogenase on glass beads

Hydrogenase from $\text{Clostridium pasteurianum}$ was immobilized on glass beads by four different methods. The sensitivity of the native and bound enzyme to oxygen was examined. Hydrogenase bound to succinyl glass proved to be the most stable to oxygen. All bound enzymes were active with ferredoxin as a substrate and evolved hydrogen in a chloroplast-ferredoxin-hydrogenase system driven by light.     

Immobilization of uricase on protamine bound to glass beads and its application to determination of uric acid

Uricase was found to be stabilized by protamine from salmon testis. Protamine was then bound to controlled-pore glass beads aminohexyl CPG 500 using glutaraldehyde. Microbial uricase was readily immobilized on the protamine bound to glass beads. The immobilized uricase proved to be stable even at 70 degrees C, whereas free uricase was inactivated at 45 degrees C and showed activity over a broader pH range than free uricase. Automated analysis of uric acid was facilitated using the immobilized uricase. The standard curve for uric acid was linear in the range of 2 to 10 micrograms/sample and passed through the origin. This automated procedure was also applicable to the determination of uric acid in human serum. Protamine bound to glass beads is expected to be useful for the simple immobilization and stabilization of enzymes.     

One-step isolation of plasma membrane proteins using magnetic beads with immobilized concanavalin A

We have developed a simple method for isolating and purifying plasma membrane proteins from various cell types. This one-step affinity-chromatography method uses the property of the lectin concanavalin A (ConA) and the technique of magnetic bead separation to obtain highly purified plasma membrane proteins from crude membrane preparations or cell lines. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. When these ConA magnetic beads were used to enrich plasma membranes from a crude membrane preparation, this procedure resulted in 3.7-fold enrichment of plasma membrane marker 5′-nucleotidase activity with 70% recovery of the activity in the crude membrane fraction of rat liver. In agreement with the results of 5′-nucleotidase activity, immunoblotting with antibodies specific for a rat liver plasma membrane protein, CEACAM1, indicated that CEACAM1 was enriched about threefold relative to that of the original membranes. In similar experiments, this method produced 13-fold enrichment of 5′-nucleotidase activity with 45% recovery of the activity from a total cell lysate of PC-3 cells and 7.1-fold enrichment of 5′-nucleotidase activity with 33% recovery of the activity from a total cell lysate of HeLa cells. These results suggest that this one-step purification method can be used to isolate total plasma membrane proteins from tissue or cells for the identification of membrane biomarkers.     

Continuous culture of mixed oral flora on hydroxyapatite-coated glass beads.

Methods for initiating and perpetuating a culture of mixed oral flora on hydroxyapatite (HT)-coated glass beads are described. Preliminary characterization of the resultant flora showed that species common in human dental plaque were present. The composition of the flora could be manipulated by altering cultural conditions. Scanning electron micrographs demonstrated that the microbes grew in densely packed microcolonies on the surface of the HT bead. A procedure for continuous culture of colonized HT beads in glass columns is described. This technique should make it possible to utilize experimental protocols employing continuous or interrupted flow of materials.     

Continuous culture of mixed oral flora on hydroxyapatite-coated glass beads.

Methods for initiating and perpetuating a culture of mixed oral flora on hydroxyapatite (HT)-coated glass beads are described. Preliminary characterization of the resultant flora showed that species common in human dental plaque were present. The composition of the flora could be manipulated by altering cultural conditions. Scanning electron micrographs demonstrated that the microbes grew in densely packed microcolonies on the surface of the HT bead. A procedure for continuous culture of colonized HT beads in glass columns is described. This technique should make it possible to utilize experimental protocols employing continuous or interrupted flow of materials.     

Recovery of streptokinase by immunoprecipitation with anti-SK polyclonal antibodies bound to porous glass beads (SIKUGR)

Polyclonal antibodies against streptokinase (SK) were bound covalently to porous glass beads (SIKUG^R) and used for direct recovery of SK from fermentation broth. This process does not present problems for the recovery of the inmunoconjugates and more than 90% of the precipitated protein being SK.SIGUK^R is trademark of Schott Glaswerke, Mainz, FRG.     

Immobilization of human thrombomodulin on glass beads and its anticoagulant activity.

Human thrombomodulin (TM) was for the first time immobilized on glass beads by the reaction between the carboxyl group of TM and the amino group of glass beads using water-soluble carbodiimide. Immobilized human TM exhibited both anticoagulant activity and inhibition of platelet aggregation of human blood.     

Platelet retention by albuminated glass and polystyrene beads.

Ex vivo platelet retention by albuminated glass and polystyrene beads has been evaluated as a function of flow rate, bead surface area, blood exposure time and albumin treatment. The stability of the albumin coatings as well as scanning electron microscopy of the various surfaces before and after blood exposure has also been included. Results indicate that platelet retention is sensitive to changes in the above parameters and that albumin pretreatment of different substrates can decrease platelet retention. This decrease is substrate dependent in that platelet retention is different for the albuminated glass and polystyrene substrates. Chemical analysis of the substrate materials by X-ray photoelectron spectroscopy (XPS) as well as bulk chemical analysis is also reported.