Isolation of cell membranes from Saccharomyces cerevisiae for evaluation of the protein composition

We describe a simple method for the isolation of membrane fractions from Saccharomyces cerevisiae yeasts, containing the complex of plasma membranes and cell walls. The method is based on cell disruption on an INBI flow disintegrator. This device spares subcellular structures, which simplifies the isolation of cell membranes. The membrane fraction obtained by this method was suitable for studies of protein composition of these structures by means of two-dimensional electrophoresis.     

Isolation and lipid composition of nuclear membranes from macronuclei of Tetrahymena pyriformis

A procedure was developed for isolation of macronuclei and nuclear membranes from the ciliated protozoan Tetrahymena pyriformis E, and the lipid composition of the isolated nuclear membranes was determined.This method involves cell lysis with octanol, separation of the nuclear membrane with 0.2 M phosphate–1M NaCl and purification on a discontinuous sucrose gradient. By phase-contrast and electron microscopic examinaton, our preparations were pure and preserved the typical nuclear membrane morphology: inner and outer nuclear membranes, and nuclear pore complexes. As for lipid distribution, the three major phospholipids in the membranes were phosphatidylcholine (31.0%), phosphatidylethanolamine (26.1%) and 2-aminoethylphosphonolipids (23.3%) and the molar ratio of a sterol-like lipid, tetrahymanol to phospholipid phosphorus was 0.036. These results were compared to other membrane fractions of Tetrahymena.     

Assembly,stability and integrity of basement membranes in vivo

Basement membranes are layered structures of the extracellular matrix which separate cells of various kinds from the surrounding stroma. One of the frequently recurring questions about basement membranes is how these structures are formed in vivo. Up to a few years ago, it was thought that basement membranes were formed spontaneously by a process of self-assembly of their components. However, it has now become clear that cell membrane receptors for basement membrane components are essential factors for the formation and stability of basement membranes in vivo. The present review highlights the modern concepts of basement membrane formation.     

Biochemical Approaches to Problems of Cellular Patterning*†

SYNOPSIS. The expression of intracellular patterning is perhaps nowhere more impressive than in the arrangements of structural elements associated with the cell surface in protozoa. The view is proposed that biochemical studies of protozoan plasma membranes and associated surface structures represent important contributions of potential significance for the understanding of the perpetuation, and expression of positional information at the intracellular level. Some recent work dealing with the isolation, identification, and metabolism of pellicular proteins in Tetrahymena is presented and discussed. Some integral membrane proteins have been identified by iodination and polyacrylamide gel electrophoresis. Labeling studies suggest heterogeneous turnover rates within the group of presently identified membrane proteins. High molecular weight proteins with some similarity to spectrin have been isolated from Tetrahymena epiplasm. It is suggested that the ciliate epiplasm is one example of membrane-associated, actomyosin-like systems found in a variety of cell types. The epiplasm may play a role in the positioning of surface-associated structures and in the control of cell shape.     

ZONAL CENTRIFUGATION OF NEURONAL PERIKARYA AND ISOLATION OF NEURONAL MEMBRANES RICH IN ACETYLCHOLINESTERASE

Abstract— A method is presented that allows the isolation of neuronal perikarya of high purity and in good yield from rat brain. To this procedure is coupled a second isolation step which affords neuronal membranes in preparative quantities. The arrangement of the two isolation steps in tandem ensures that the final preparation has a high degree of purity with respect to both its neuronal origin and its membrane content. The membranes so obtained do not constitute plasma membranes but rather represent intracellular structures that are predominantly derived from endoplasmic reticulum and contain mitochondrial elements as well. During the process of membrane purification AChE is enriched four-fold while cholinesterase is concurrently eliminated. The present method supplements the procedure reported by M organ , W olfe , M andel and G ombos (1971) for the isolation of synaptosomal plasma membranes. It may also open access to a useful source of brain AChE.     

Purification of Plasma Membrane from Acanthamoeba castellanii1

A simple method for isolation of plasma membrane from Acanthamoeba using self-generating gradients of Percoll is described. To obtain a membrane marker, intact amoebae were radioiodinated and the distribution of the radiolabel was followed through the plasma membrane isolation procedure. The purity of isolated plasma membrane was assessed by enrichment of radiolabel, by electron microscopy, and by enzymatic assays for contaminating membranes. As judged from enrichment of radiolabel, a 37-fold purification of plasma membrane was obtained. We estimate that 80% of the total protein was from plasma membrane and 10% from membrane-associated actin.     

Isolation of detergent-resistant membranes (DRMs) from Escherichia coli

Lipid rafts or membrane microdomains have been proposed to compartmentalize cellular processes by spatially organizing diverse molecules/proteins in eukaryotic cells. Such membrane microdomains were recently reported to also exist in a few bacterial species. In this work, we report the development of a procedure for membrane microdomain isolation from Escherichia coli plasma membranes as well as a method to purify the latter. The method here reported could easily be adapted to other gram-negative bacteria, wherein the isolation of this kind of sub-membrane preparation imposes special difficulties. The analysis of isolated membrane microdomains might provide important information on the nature and function of these bacterial structures and permit their comparison with the ones of eukaryotic cells.     

Preparation of Secretory Vesicle-Free Plasma Membranes by Isopycnic Sucrose Gradient Fractionation of Neutrophils Purified by the Gelatin Method

Isolated human neutrophils serve as a model for the in vitro study of host defensive processes as well as the cell biology and biochemistry of primary human cells. We demonstrate that the requirements of the gelatinbased procedure for neutrophil isolation from whole blood induces the complete loss of secretory vesicles from in vitro isolated populations, whereas isolation by a dextran-based methodology results in the preservation of this organelle. Following density fractionation of cellular cavitates, examination of commonly employed plasma membrane marker activities yielded subcellular localization patterns that were indistinguishable between dextran- or gelatin-isolated populations, indicating both populations to be otherwise comparable in terms of the relative complexity and large-scale organization of plasma membranes. Given that the cell surface upregulation of secretory vesicles is implicated as an initial requirement of neutrophil activation as well as an intrinsic feature of neutrophil priming, we show that dextran and gelatin-isolated neutrophils may be considered to occupy functionally nonactivated and primed cellular states, respectively. These differences in phenotype can be exploited in specific ways. We suggest that the gelatin method has technical advantages with regard to the study of neutrophil plasma membranes. In particular, results from this study indicate the gelatin method to be a reliable and effective preparatory technique appropriate for tandem use with density fractionation procedures to achieve rapid isolation of plasma membranes that are uncontaminated by secretory organelles.     

Preparation of apical plasma membranes from cells grown on coverslips. Electron microscopic investigations of the protoplasmic surface

We introduce here a simple method which permits an efficient isolation of apical plasma membranes from tissue culture cells and the electron microscopic examination of their protoplasmic surfaces by use of the platinum/carbon replica technique. Different procedures were tested with regard to the efficiency of isolation and preservation of ultrastructure. Best results were obtained by prestabilization of cell surfaces with low concentrations of tannic acid prior to isolation. To demonstrate the possible applications and versatility of the method, studies were done on virus-infected cells in combination with immunocytochemical labeling. With this model system, we show that it is possible to correlate the structures seen on the cytoplasmic surface of the plasma membrane with the distribution of virus antigens at the cell surface labeled with immunogold markers prior to preparation.     

Isolation of sea urchin embryo cell surface membranes on polycationic beads

Summary Blastula cell surface membranes of the sea urchin, Strongylocentrotus purpuratus, were isolated on polycationic beads by a method modified from Jacobson and Branton (1977) and Jacobson (1980). This study represents the first application of this procedure to an embryonic system. Embryo cells were attached to polylysine-coated polyacrylamide beads and lysed, leaving the embryo cell surface membranes still attached to the beads, and cytoplasmic particles were washed free of the exposed inner surfaces of the membranes. Cell surface membrane sheets were desorbed from the beads and collected by centrifugation. Approximately 8% and 5% of the cell surface membranes of dissociated embryo cells were recovered on the beads and in the membrane pellet, respectively. Specific activities of [³H]concanavalin A-binding and of the cell surface marker enzymes, alkaline phosphatase and Na⁺/K⁺ ATPase, were 16-, 19-, and 32-fold higher, respectively, in the cell surface membrane fraction than in the embryo cell homogenate. Membranes were relatively free of cytoplasmic contaminants as judged from electron micrographs and enzyme analysis. Activities in the membrane fraction of the cytoplasmic marker enzymes, cytochrome c oxidase, catalase, acid phosphatase, NADP- and NADPH-cytochrome c reductase, and acetylcholinesterase, were substantially less than homogenate levels. The entire procedure can be completed in 4 h. Since this cell surface membrane isolation technique relies only on the tendency of a negatively charged cell to adhere to a positively charged surface, it is less likely than most other methods to exhibit species and developmental stage specificity and should prove useful in the study of the developmental role of embryonic stage-specific membrane components.     

Isolation of separate apical,lateral and basal plasma membrane from cells of an insect epithelium. A procedure based on tissue organization and ultrastructure

The tissue used in this study was the midgut of the tobacco hornworm larva, Manduca sexta. The midgut epithelium is a single layer of cells resting on a thin basal lamina and underlying discontinuous muscle layer. The epithelial cells are of two main types, goblet and columnar cells, joined together by the septate junctions characteristic of insect epithelia. From this tissue we were able to isolate four distinct plasma membrane fractions; the lateral membranes, the columnar cell apical membrane, the goblet cell apical membrane and a preparation of basal membranes from both cell types. The lateral membranes were isolated by density gradient centrifugation following gentle homogenization of the midgut hypotonic medium, which caused the cells to rupture at their apical and basal surfaces, releasing long segments of lateral membranes still joined by their septate junctions. For isolation of apical and basal membranes the tissue was disrupted by ultrasound, based on the light microscopic observation that carefully controlled ultrasound can be used to disrupt each cell in layers starting at the apical surface. The top layer contained the columnar cell apical membrane, which consists of microvilli forming a brush border covering the lumenal surface of the epithelium. The second layer contained the goblet cell apical membrane, which is invaginated to form a cavity occupying the apical half of the cell, and the third layer contained the basal membranes. As each layer was stripped off the epithelium it was collected and the plasma membrane purified by differential or density gradient centrifugation. For all four membrane fractions, the isolation procedure was designed to preserve the original structure of the membrane as far as possible. This allowed electron microscopy to be used to follow each step in the isolation procedure, and to identify the constituents of each subcellular preparation. Although developed specifically for M. sexta midgut, these techniques could readily be modified for use on other epithelia.     

Tetrahymena pyriformis as a Model System for Membrane Studies*†

SYNOPSIS. Tetrahymena pyriformis is an exceptionally useful subject for studying metabolic interrelationships among intracellular membranes. Its advantages include the striking differences in lipid composition among the cell's various functionally distinct membrane systems, indicating a pronounced lipid specificity at the membrane sites. The magnitude of these differences permits analysis of the mechanisms underlying the specificity. Even more valuable is the unique physical isolation of ciliate surface membranes from the cytoplasm of the cell. In contrast to the almost immediate equilibration of newly made lipids with preexisting lipids found in most cells, Tetrahymena surface membranes have a lipid turnover slow enough to be conveniently analyzed. Finally, the well-studied responses of Tetrahymena to such physiological stresses as heat and starvation may be used to evaluate the effects of environmental factors on membrane formation.     

Outer membrane proteins of thebacteroides fragilis group

The Triton-insoluble outer membrane proteins of members of the obligately anaerobicBacteroides fragilis group were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The application of previously published methods for the preparation of outer membranes from these anaerobes led to relatively low recoveries of outer membrane material. Additionally, the outer membrane recovered was not qualitatively or quantitatively representative of the total outer membrane. A method previously devised for the isolation of the outer membrane proteins fromEscherichia coli was used. By this method, the members of theB. fragilis group were found to possess a striking capacity to alter their cell-surface proteins in response to cultivation in different media.     

A rapid method for the isolation of L-cell surface membranes using an aqueous two-phase polymer system

Summary A dextran-polythylene glycol aqueous two-phase system has been used to separate cell surface membranes from other cellular organelles. The surface membranes have been identified on the basis of morphology, content of Na⁺, K⁺-ATPase, and presence of surface antigen as detected by a⁵¹Cr release method. Contamination of the surface membrane preparations by smooth endoplasmic reticulum, mitochondria, and nuclei has been found to be minimal. An average of 6.5% of the total protein was found in the membrane fraction. Less than two hours is required to isolate the membrane fraction after preparation of a Dounce homogenate. Fractionation by aqueous two-phase polymer systems appears to be a rapid and effective method for the isolation of surface membranes.     

Separation of intact and damaged hepatocytes in sucrose following non-enzymatic liver perfusion

This study deals with isolation of rat hepatocytes by a non-enzymatic method and the separation of intact and damaged cells in sucrose medium. Low speed centrifugation in isotonic sucrose medium of a hepatocyte suspension obtained by mechanical desaggregation of liver pre-perfused with EDTA solution results in the formation of a cell pellet which contains two different layers. A darker layer contains hepatocytes with intact plasma membranes. Their respiratory activity and xenobiotic metabolism are close to those of the cells isolated by collagenase perfusion. The study of distribution of lipophilic cation tetraphenylphosphonium (TPP⁺) indicates a predominantly mitochondrial localization of TPP⁺ in the intact cells following non-enzymatic and collagenase isolation. Hepatocytes in the upper layer have damaged plasma membranes. As a result they lose the potential to accumulate TPP⁺, and have low rates of endogenous respiration and biotransformation activity. Addition of exogenous NADPH restores the capability to metabolize xenobiotics. Washing and incubation of these hepaticytes in an intracellular type medium results in restoration of uncoupler-stimulated oxygen consumption and generation of membrane potential in the presence of a succinate substrate. These properties are close to those of hepatocytes permeabilized by digitonin treatment. Thus, the procedure allows the simultaneous isolation of both intact and permeabilized hepatocytes with functionally active intracellular structures without the use of relatively expensive chemicals such as collagenase and Percoll.Abbreviations 4-OHBP 4-hydroxybiphenyl - BP biphenyl - BSA bovine serum albumin - DNP 2,4-dinitrophenol - EDTA ethylendiamintetraacetate - NADPH nicotinamide adenine dinucleotide phosphate reduced - p-NA p-nitroanisole - p-NPh p-nitrophenol - TPP⁺ tetraphenylphosphonium     

William Trager: An Appreciation

SYNOPSIS. Additional information on host interactions with trypanosomatid membranes was obtained from studies of a monomorphic strain of Trypanosoma brucei harvested at peak parasitemia from intact and lethally irradiated rats. Pellets of trypanosomes were fixed briefly in glutaraldehyde and processed for thin section electron microscopy or freeze-cleave replicas. Observations of sectioned material facilitated orientation and comparison of details seen in replicas. Fracture faces of cell body and flagellar membranes as well as 3-dimensional views of the nuclear membrane were studied. Cell body membranes of 80% of the organisms from intact rats contained random arrays of intramembranous particles (IMP). Aggregated clusters of particles appeared on the fracture faces of 20% of the trypanosomes. Some of these membranes had nonrandomly distributed particles aligned in distinct rows on the outer fracture face of both cell body and flagellum. Many inner face fractures of the cell body membranes had a particle arrangement similar to the longitudinal alignment of cytoskeletal microtubules. No aggregated particle distribution was seen in membranes of trypanosomes harvested from lethally irradiated rats. Replicas of trypanosome pellets also had plasmanemes as a series of attached, empty, coated membrane vesicles. These structures were found in close association with, as well as widely separated from the parasites. The shedding of these vesicles and the variation of particles in cell body membranes are discussed in light of antibody-induced architectural and antigenic changes in surface properties of trypanosomatids. The convex face of the inner membrane of the nucleus also is covered with randomly arrayed particles. More IMP were observed on the inner than on the outer nuclear membranes. Images of nuclear pores were also seen. The importance of these structures in drug and developmental studies of trypanosomes is discussed. On fracture faces of the flagellar membrane there were miniature maculae adherentes, unique to the inner fracture face and occurring only at regions of membrane apposition between cell body and flagellum. Each cluster of particles exposed by the freeze-cleave method corresponds to an electron-dense plaque seen in thin section images. However, because of a unique fracture pattern, these plaques were not revealed on the apposing body membranes, as illustrated in thin sectioned organisms.     

Pattern formation inDrosophila melanogaster: The effects of mutations on polarity in the developing leg

Summary The effects of the mutations eyeless dominant (ey ^D) and shibire (shi) on bristle pattern in the legs ofDrosophila melanogaster were examined. Both mutations cause gaps in the intersegmental membranes which separate leg segments and often alter the position of these membranes. It was observed that pattern disturbances including reversed bristle polarity and duplication of structures such as sex combs and transverse rows were associated with defects in the intersegmental membranes. The alterations in bristle polarity and most of the duplication of structures could be accounted for by a segmentally reiterated gradient in the legs which controls bristle polarity and which requires the integrity of the intersegmental membrane. A computer simulation of this gradient model was devised which accounted for the observed results. The possible role of cell death as a cause of the gaps in the intersegmental membrane and of some of the pattern disturbances was examined.     

Rapid screening and scale-up of ultracentrifugation-free,membrane-based procedures for purification of His-tagged membrane proteins

This study develops procedures to rapidly screen conditions for purification of membrane proteins (MPs) using 96-well plates containing nickel-functionalized membranes. In addition to their application in the pharmaceutical industry, MPs are important components of new sensors, synthetic membranes, and bioelectronic devices. However, purification of MPs is challenging due to their hydrophobic exterior, which requires stabilization in amphipathic detergent micelles. We examined the extent of extraction of the light-driven sodium transporter, Krokinobacter eikastus rhodopsin 2 (KR2) heterologously expressed in Escherichia coli using different salts and maltoside-based detergents. The extraction was followed by subsequent affinity purification in membranes functionalized with Ni²⁺-nitrilotriacetate complexes that bind the His-tagged KR2. We also employed a hydrophobic chelator to separate detergent micelles from the aqueous phase as an initial isolation step prior to affinity purification. Unlike conventional resin-based capture, which can take a full day or more, the membrane-based screening of purification conditions takes only a few hours, and its scale-up involves changing from a 96-well format to a larger membrane module. The novelty of the method lies in utilizing membrane-based ultracentrifugation-free purification of MPs from cell membrane fragments; the optimized purification conditions from the screening method can potentially be applied to large-scale/conventional resin-based purification of MPs.     

Isolation of the plasma membrane from sea urchin embryos.

A method for isolation of sea urchin embryos plasma membranes is described. Purification of the obtained fraction was assayed by several enzymatic markers and electron microscopy. The isolated plasma membranes appear to be pure from contamination of other cell membranes (endoplasmic reticulum and mitochondria), and they can therefore be used for analytical studies on the composition and structure of plasma membrane.