Isolation and characterization of plasma membranes from the adrenal medulla.

Abstract– The isolation of a plasma membrane fraction from the bovine adrenal medulla and its characterization are described. The plasma membranes are enriched 13-fold in AChE, a plasma membrane marker, and represent 0.7% of the homogenate membrane protein. The yield of these membranes is typically 10-12% by the criterion of the percentage of total membrane bound AChE in the homogenate. The membranes were characterized as to their polypeptide, phospholipid and cholesterol content and compared with chromaffin vesicle, mitochondrial and microsomal membranes by these parameters. Two enzymatic components of the plasma membranes, ATPase and adenylate cyclase, were also studied. Calcium ATPase activity is 2.5-fold higher than magnesium ATPase activity, appears to be the result of a single enzyme, and is a genuine component of the plasma membranes. The magnesium stimulated activity appears to have at least two enzymatic components, one of which may be identical to the calcium ATPase. Adenylate cyclase is a plasma membrane component, but may not be uniquely localized there, as it is rather unstable throughout the fractionation procedure. It is stimulated by GTP (0.7-fold at 10^?5M), GPP(NH)P (4.8-fold at 10^?5M) and sodium fluoride (4.6-fold at 10^?2M). It is refractory to stimulation by all other compounds tested.     

A Rapid and Reproducible Hcmogenization Procedure for the Isolation of Plasma Membranes from Rat Liver

A simplified modification of the Neville procedure for the isolation of plasma membranes from rat liver is described in which cells are broken by low-shear homogenizetion with a Polytron homogenizer. Plasma membranes are recovered from the homogenates by differential and discontinuous sucrose gradient centrifugetions. The procedure provides plasma membrane fractions enriched 25-fold for AMPase, a marker enzyme for the plasma membrane of rat liver, with a combined contamination from endoplasmic reticulum, Golgi apparatus and mitochondria of less than 10% The procedure is uncomplicated, reproducible, and yields enzymatically active plasma membrane fractions of high purity.     

Purification of plasma membrane from Acanthamoeba castellanii

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 plasma membranes from cultured glioma cells and application to evaluation of membrane sphingomyelin turnover

A rapid and reliable method for the isolation of plasma membranes and microsomes of high purity and yield from cultured glioma cells is described. The procedure involves disruption by N2 cavitation, preliminary separation by centrifugation in Tricine buffer, and final separation on a gradient formed from 40% Percoll at pH 9.3. Enzyme and chemical markers indicated greater than 60% yield with six- to eightfold enrichment for plasma membranes and greater than 25% yield with three- to fourfold enrichment for a microsomal fraction consisting mainly of endoplasmic reticulum. The final fractions were obtained with high reproducibility in less than 1 h from the time of cell harvesting. Application of this procedure to human fibroblasts in culture is assessed. The isolation procedure was applied to investigations of synthesis and turnover of sphingomyelin and phosphatidylcholine in plasma membranes of glioma cells following incubation for 4-24 h with [methyl-3H]choline. These studies indicated that radioactivity from phosphatidylcholine synthesized in microsomes from exogenous choline may serve as a precursor of the head-group of sphingomyelin accumulating in the plasma membrane.     

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.     

STUDIES ON ACETYLCHOLINESTERASE OF RAT BRAIN SYNAPTOSOMAL PLASMA MEMBRANES

Abstract— A fluorimetric assay has been used to examine some kinetic properties of AChE from synaptosomal plasma membranes prepared from rat brain. The AChE bound to the plasma membranes was compared to that solubilized with Triton X-100 and found to be essentially the same with respect to Michaelis constant and inhibitor constants for several AChE inhibitors. The two forms of the enzyme had slightly different pH optima. The kinetic studies revealed no evidence that synaptosomal plasma membrane AChE has allosteric properties. The solubilized enzyme was further purified by affinity chromatography.     

PRiMA: the membrane anchor of acetylcholinesterase in the brain.

As a tetramer, acetylcholinesterase (AChE) is anchored to the basal lamina of the neuromuscular junction and to the membrane of neuronal synapses. We have previously shown that collagen Q (ColQ) anchors AChE at the neuromuscular junction. We have now cloned the gene PRiMA (proline-rich membrane anchor) encoding the AChE anchor in mammalian brain. We show that PRiMA is able to organize AChE into tetramers and to anchor them at the surface of transfected cells. Furthermore, we demonstrate that AChE is actually anchored in neural cell membranes through its interaction with PRiMA. Finally, we propose that only PRiMA anchors AChE in mammalian brain and muscle cell membranes.     

THE EFFECTS OF TETRAPHENYLBORON ON THE ISOLATION OF NEURONAL AND GLIAL CELLS FROM GUINEA-PIG BRAIN

Abstract— The addition of 20 mM-tetraphenylboron to the tissue-softening medium of a neuronal and glial cell isolation procedure results in a 2-fold and 5-fold increase in the yield of neuronal and glial-enriched material respectively. The purity of the isolated fractions appears unaltered. While tetraphenylboron strongly inhibits protein synthesis, carbonic anhydrase activity and structural integrity appear unaffected by tetraphenylboron. The potential use of this compound in cell isolations is discussed.     

A rapid method of isolation of platelet membranes for radioligand and enzymatic assays

A simple and rapid procedure for isolation of the total platelet membrane fraction by chromatography on Sepharose CL 6B has been developed. This method allows a rapid (25-30 min) one-step separation of the membrane fraction on 26 x 150 mm columns with a 20-21 mg recovery. The high degree of purity of membrane preparations was confirmed by a radioligand assay using [3H]adenosine and L-[3H]glutamic acid. The purity of membrane preparations is comparable with that of membrane fractions obtained by standard ultracentrifugation methods. The homogeneity of the membrane fraction was demonstrated by using marker enzymes of plasma, microsomal and mitochondrial membranes. This finding is very important in that it allows the isolation of fractions differing in their protein content with no effect on the method reproducibility. The high utility of the membrane preparations in receptor studies was demonstrated for high affinity binding sites for [3H]adenosine and L-[3H]glutamic acid.     

Isolation of synaptic plasma membrane from brain by combined flotation-sedimentation density gradient centrifugation

A method is described for the subcellular fractionation of brain to obtain a preparation highly enriched in synaptic plasma membranes. The enriched fraction is recovered from the interface of a two-step sucrose density gradient on which a hypotonically lysed crude mitochondrial fraction from brain has been separated by simultaneous sedimentation and flotation centrifugation. Enzyme marker activities associated with the neuronal plasma membrane are enriched in the synaptic plasma membrane-containing fraction while less than 10% of enzyme markers associated with the major probable contaminants, myelin and mitochondria, are found in the same fraction. Morphological examination of the enriched fraction suggests that about 80% of the profiles are recognisably synaptic in origin. Compared to previously described methods for obtaining synaptic plasma-enriched fractions of equivalent purity, the procedure reported here is simpler, shorter, and of greater capacity.     

Plasma membranes of human neutrophils: a one-step isolation procedure by cell disruption in paraffin oil

Plasma membranes of high purity and good yield have been prepared from human polymorphonuclear neutrophils by a one-step procedure involving disruption of cells suspended in paraffin oil and forced by pressure through an annular slit. This results in a band floating above the oil which is composed of large sheets of plasma membranes. Enrichment values for the plasma membrane marker alkaline phosphatase and 125I-labeled protein after surface labeling performed at the whole cell level were 23-fold and 22-fold, respectively. Contamination of the plasma membrane by other organelles was negligible and approximately 2 mg of membrane protein was obtained from 10(9) neutrophils. The procedure is very fast and the use of paraffin oil avoids lengthy high-speed centrifugation. The technique also allows isolation of granules devoid of plasma membrane and can probably be applied to other cell types.     

Ultrastructural localization of acetylcholinesterase in the synganglion of the tick,Dermacentor variabilis (Say)

Summary Light- and electron-microscopic enzyme cytochemistry was used to localize acetylcholinesterase (AChE) activity in the synganglion (brain) of the tick Dermacentor variabilis. High AChE activity was observed throughout the neuropil as well as adjacent to most neuronal perikarya. Intracellular activity was not observed by light microscopy. By electron microscopy, reaction product was localized at the plasma membrane of glia and neurons. Enzyme activity was not associated with the olfactory globuli neurons. In other types of neurons, small amounts of reaction product were observed in the Golgi apparatus and nuclear envelope. Large neurosecretory neurons contained activity that appeared to be associated with deep invaginations of the plasma membrane as well as intracellular membranes. AChE activity was also associated with processes of both neurons and glia. In most peripheral nerves AChE activity was associated with virtually all axons. Clearly then, AChE is associated with glia and non-cholinergic neurons as well as with presumed cholinergic neurons. The widespread localization and large amounts of AChE in the tick brain exceeds that reported for other invertebrates and vertebrates. As has been suggested for other animals, AChE in the tick brain may have functions in addition to its known role in cholinergic neurotransmission.     

ISOLATION OF POSTSYNAPTIC DENSITIES FROM RAT BRAIN

Most synapses in the central nervous system exhibit a prominent electron-opaque specialization of the postsynaptic plasma membrane called the postsynaptic density (PSD). We have developed a procedure for the isolation of PSDs which is based on their buoyant density and their insolubility in N-lauroyl sarcosinate. Treatment of synaptic membranes with this detergent solubilizes most plasma membranes and detaches PSDs from the plasma membrane so that they can be purified on a density gradient. Isolated PSDs appear structurally intact and exhibit those properties which characterize them in tissue. The isolated PSDs are of the size, shape, and electron opacity of those seen in tissue; they stain with both ethanolic phosphotungstic acid and bismuth iodide-uranyl lead and the fraction contains cyclic 3',5'-phosphodiesterase activity. Quantitative electron microscope analysis of the PSD fraction gives an estimated purity of better than 85%. Inasmuch as the PSD is associated primarily with dendritic excitatory synapses, our PSD fraction represents the distinctive plasma membrane specialization of this specific synaptic type in isolation.     

The Isolation and Characterization of Plasma Membranes From Calf Thymocytes

A simple method is described for the isolation and characterization of plasma membranes from calf thymocytes. The procedure involves extraction of thymocytes in a hypotonic medium containing borate and EDTA. Membrane ghosts, obtained by centrifugation of the cell lysate, are purified by passage through a column containing glass beads. The purity of plasma membranes was checked by chemical analysis, by assay of marker enzymes and also by electron microscopy. Polyacrylamide gel electrophoresis of the calf thymocyte plasma membrane produced a number of protein bands as well as a major band which stained for carbohydrate. The method is rapid and could be applied to isolate plasma membranes from nucleated cells of various types in large quantities.     

The isolation and characterization of plasma membranes from calf thymocytes.

A simple method is described for the isolation and characterization of plasma membranes from calf thymocytes. The procedure involves extraction of thymocytes in a hypotonic medium containing borate and EDTA. Membrane ghosts, obtained by centrifugation of the cell lysate, are purified by passage through a column containing glass beads. The purity of plasma membranes was checked by chemical analysis, by assay of marker enzymes and also by electron microscopy. Polyacrylamide gel electrophoresis of the calf thymocyte plasma membrane produced a number of protein bands as well as a major band which stained for carbohydrate. The method is rapid and could be applied to isolate plasma membranes from nucleated cells of various types in large quantities.     

A new method for the isolation of renal basement membranes.

A method is described for the isolation of basement membranes from rabbit renal cortex in which the detergent N-lauroyl sarcosine is used as the disruptive agent. The isolated membranes have been compared with membranes prepared using ultrasonication and they were comparable both in terms of purity and gross chemical composition. Glomerular and tubular basement membranes were isolated by first separating glomeruli from tubules by density gradient centrifugation followed by detergent treatment of the separated tissues. The detergent method has the advantage that the basement membranes retained their native structure to a large degree, whereas sonicated membranes were severely fragmented. Collagen fibres were a significant contaminant in both preparations and were revealed more clearly by negative staining than by examination of thin sections. Studies with the detergent-treated membrane revealed that a few proteins, which seemed to be membrane components, were extracted with 1 M NaCl and that these proteins were lost from the basement membranes during sonication used in the conventional isolation procedure.     

大鼠脑突触质膜糖皮质激素受体样抗原的免疫电镜研究

本文利用ABC金标记法(受体-抗受体中的单克隆抗体-生物素化马抗鼠IgG-金标链霉亲和素),首次在电镜下观察到大鼠脑突触质膜的外表面存在有糖皮质激素受体样抗原,为神经细胞膜上可能存在有糖皮质激素受体提供了形态学依据。     

Boar sperm cytoplasmic droplets: Their ultrastructure,their numbers in the epididymis and at ejaculation and their removal during isolation of sperm plasma membranes

Cytoplasmic droplets of the boar are progressively lost from the flagellum of boar spermatozoa during epididymal transit, at ejaculation and during the nitrogen cavitation technique for isolation of plasma membranes. Apparently very fragile, these structures are broken up in the fluids of the reproductive tract and in the buffer used during the nitrogen cavitation procedure. The maximal potential contamination of cytoplasmic droplet internal vesicular membranes in plasma membrane fractions was determined to be 2.2% of the entire membrane surface area collected. The highly sensitive silver-stained, two-dimensional (2-D) polyacrylamide (PAGE) gels of boar sperm plasma membranes did not reveal cytoplasmic droplet, internal membrane, marker polypeptides, further demonstrating the high purity of plasma membrane preparations. In addition, freeze-fracture demonstrates that the internal membranes of the cytoplasmic droplet show few intramembranous particles and these may contribute little protein to plasma membrane preparations. The presence of two forms of vesicular elements in boar sperm Cytoplasmic droplets (typical vesicles and collapsed vesicles) is described.     

A new method for the isolation of renal basement membranes

A method is described for the isolation of basement membranes from rabbit renal cortex in which the detergent $\text{N-}\text{lauroyl}$ sarcosine is used as the disruptive agent. The isolated membranes have been compared with membranes prepared using ultrasonication and they were comparable both in terms of purity and gross chemical composition. Glomerular and tubular basement membranes were isolated by first separating glomeruli from tubules by density gradient centrifugation followed by detergent treatment of the separated tissues.The detergent method has the advantage that the basement membranes retained their native structure to a large degree, whereas sonicated membranes were severely fragmented. Collagen fibres were a significant contaminant in both preparations and were revealed more clearly by negative staining than by examination of thin sections. Studies with the detergent-treated membrane revealed that a few proteins, which seemed to be membrane components, were extracted with 1 M NaCl and that these proteins were lost from the basement membranes during sonication used in the conventional isolation procedure.     

ISOLATION AND PROPERTIES OF THE PLASMA MEMBRANE OF KB CELLS

Plasma membranes from KB cells were isolated by the method of latex bead ingestion and were compared with those obtained by the ZnCl₂ method. Optimal conditions for bead uptake and the isolation procedure employing discontinuous sucrose gradient centrifugation are described. All steps of preparative procedure were monitored by electron microscopy and specific enzyme activities. The plasma membrane fraction obtained by both methods is characterized by the presence of the Na⁺ + K⁺-activated ATPase and 5'-nucleotidase, and contains NADPH-cytochrome c reductase and cytochrome b₅. The latter two enzymes are also present in lower concentrations in the microsomal fraction. Unlike microsomes which are devoid of the Na⁺ + K⁺-activated ATPase and which contain only traces of 5'-nucleotidase activity, the plasma membrane fraction contains only trace amounts of the rotenone-insensitive NADH-cytochrome c reductase but no cytochrome P-450, both of which are mainly microsomal components. Morphologically the plasma membrane fraction isolated by the latex bead method is composed of vesicles of 0.1–0.3 µm in diameter. On the basis of the biochemical and morphological criteria presented, it is concluded that the plasma membrane fraction isolated by the above methods are of high degree of purity.