Isolation of plasma membranes from neurons grown in primary culture

Plasma membranes from chick embryo neuronal primary cultures were isolated after subjecting 5-day-old cells, previously surface labeled with either lactoperoxidase-catalyzed radioiodination or galactose oxidase/NaB3H4, to a freeze-thaw cycle. The cellular material adhering to the culture substratum was washed, and the "wash" fractions were pooled and centrifuged at 37,000g. The resulting pellet was resuspended in 3 ml of buffer, layered on 33 ml of 33% sucrose, and centrifuged at 105,000g. Radioactivity was recovered at the top of the gradient. Sedimentation of these fractions and biochemical studies revealed that the pellet was 20- and 12-fold enriched in (Na+,K+)-adenosinetriphosphatase and 5'-nucleotidase, respectively. The preparation was devoid of inner mitochondrial (succinate dehydrogenase), outer mitochondrial (monoamine oxidase), endoplasmic reticulum (glucose-6-phosphatase), outer mitochondrial (monoamine oxidase), endoplasmic reticulum (glucose-6-phosphatase), and Golgi (UDP galactose:N-acetylglucosamine galactosyltransferase) enzymatic markers. Ultrastructural studies showed that the membrane preparation was homogeneous and lacked mitochondria endoplasmic reticulum and lysosomes. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed the presence of 11 protein components with molecular masses ranging from 120 to 300 kDa. This method for the isolation of plasma membranes probably depends on the capacity of the cellular material to adhere to the culture substratum and to entrap intracellular organelles during the freeze-thaw cycle. The membrane preparation seems suitable for studying the function of high-molecular-weight protein components of neuronal plasma membranes.     

Electrofusion between heterogeneous-sized mammalian cells in a pellet: potential applications in drug delivery and hybridoma formation.

High-efficiency electrofusion between cells of different sizes was achieved by application of fusing electric pulses to cells in centrifuged pellets. Larger target cells (Chinese hamster ovary or L1210 cells) were stacked among smaller human erythrocytes or erythrocyte ghosts by sequential centrifugation at 700 g to form five-tier pellets in a specially designed centrifugation-electrofusion chamber. The membranes of erythrocytes and ghost were labeled with fluorescent membrane dye (1,1' dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine (Dil)), and the contents of ghosts were loaded with water-soluble fluorescent dye (42-kDa fluorescein isothiocyanate dextran (FITC-dextran)), to monitor heterogeneous cell fusion. Fusion efficiency was assayed by the extent of either membrane dye mixing or contents (FITC-dextran) mixing with target cells. Four rectangular electric pulses at 300 V and 80 microseconds each were found to give the optimal fusion results of approximately 80% heterogeneous fusion by the content-mixing assay and approximately 95% by the membrane-dye-mixing assay. Cell viability remained greater than 80% after electrofusion. Because of the electric breakdown of cell membranes at the beginning of the pulse, the pellet resistance and hence the partial voltage across the pellet reduced rapidly during the remaining pulse time. This voltage redistribution favored the survival of fused cells. The limited colloidal-osmotic swelling of cells in pellets enhanced cell-cell contact and increased the pellet resistance after each pulse. As a result, the partial voltage across the pellet was restored when the next pulse was applied. This redistribution of pulse voltage in the pellet system permitted the breakdown of cell membranes at a lower applied voltage threshold than that required for electrofusion of cells in suspension or in dielectrophoretic cell chains. The cell viability and soluble dye retention within cells (FITC-dextran) remained at the same high levels for 3 h when the cells were incubated in respective culture media with serum at 37 degrees C. Viability and dye retention decreased significantly within 30 min when cells were incubated in phosphate-buffered saline without serum. The pellet technique was applied to form hybridomas by fusion of larger SP2/0 murine myelomas with smaller naive mouse lymphocytes. An optimum of 173 +/- 70 hypoxanthine aminopterin thymidine (HAT)-selected clones of the hybridomas was obtained from 40,000 SP2/0 cells and 1.5 x 10(6) lymphocytes used in each trial. This high-efficiency fusion technique may be adapted to mediate drug and gene transfer to target cells ex vivo as well as to form hybrid cells with limited cell sources.     

Distinct platelet-activating factor binding sites in synaptic endings and in intracellular membranes of rat cerebral cortex

The binding of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC or PAF, platelet-activating factor) to synaptic plasma membranes, microsomal membranes, and other rat cerebral cortex subcellular fractions was studied. Using several PAF-binding antagonists, three distinct sites were identified. Two of them were in intracellular membranes (microsomes) and one in synaptic plasma membranes. Microsomal membranes were prepared after obtaining a 43,500 x g pellet from the postmitochondrial supernatant and subsequent centrifugation at 105,000 x g of the resulting supernatant. Most plasma membrane markers were retained in the 43,500 x g pellet (Sun, G.Y., Huang, H.-M., Kelleher, J.A., Stubbs, E.B., Sun, A. Y. (1988) Neurochem. Int. 12, 69-77). Microsomes were purified by density-gradient centrifugation and marker enzymes showed relatively very low contamination by plasma membrane markers. Myelin and mitochondria were devoid of specific PAF binding. A site displaying the highest PAF-binding affinity reported to date in all cells and membranes (KD = 22.5 +/- 1.7 pM and Bmax 8.75 = fmol/mg protein), was found in the microsomal fraction. There was a second binding site in microsomal fractions (KD = 25.0 +/- 0.8 nM and Bmax = 0.96 pmol/mg protein. Ca2+ decreases PAF affinity for the microsomal binding sites. The third binding site displays relatively low specific PAF binding and is present in synaptosomal plasma membranes. Moreover, displacement curves by a wide variety of PAF antagonists indicated different affinities for each of the binding sites described here. These results indicate that PAF-binding sites are heterogeneous in rat cerebral cortex, and they imply that the microsomal membrane sites may be involved, at least in part, in intracellular events such as gene expression.     

Detergent properties of water-soluble choline phosphatides. Selective solubilization of acyl-CoA:lysolecithin acyltransferase from thymocyte plasma membranes.

Several analogs of lysolecithin were found to solubilize human erythrocyte ghosts comparably or even better than other detergents. Derivatives with aliphatic chains of 12 to 14 carbons were most effective. The phosphorylcholine detergents apparently possess low protein-denaturing properties, since they, for the first time, allowed the solubilization of enzymatically active acyl-CoA:lysolecithin acyltransferase from thymocyte plasma membranes. The solubilized enzyme was not sedimented at 177,000 x g for 60 min and penetrated into Sepharose 6B gels. Low detergent concentration resulted in a selective extraction of the acyltransferase (about 70%) as compared to alkaline phosphatase, nucleotide pyrophosphatase, gamma-glutamyltransferase or Mg2+-ATPase (30 to 40%). The selectivity was reflected in sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of soluble and sedimentable membrane fractions; three bands of approximately 53, 84, and 94 x 10(3) daltons were enriched in the supernatants, whereas one band of about 68 x 10(3) daltons was concentrated in the pellet. The preferential extraction of acyltransferase may be related to particularly high affinity of lysolecithin analogs for this enzyme, which at higher concentrations was competitively inhibited by these detergents. The inhibitor constants ranged from 1400 micron for the C10 analog (ET-10-H) to 80 micron for the compound with 16 carbons (ET-16-H) per aliphatic chain.     

Isolation of plasma membrane vesicles, derived from transverse tubules, by selective homogenization of subcellular fractions of frog skeletal muscle in isotonic media.

A new technique for isolating fragmented plasma membranes from skeletal muscle has been developed that is based on gentle mechanical disruption of selected homogenate fractions. (Na+ + K+)-stimulated, Mg2+-dependent ATPase was used as an enzymatic marker for the plasma membrane, Ca2+-stimulated, Mg2+-dependent ATPase as a marker for sarcoplasmic reticulum, and succinate dehydrogenase for mitochondria. Cell segments in an amber low-speed (800 x g) pellet of a frog muscle homogenate were disrupted by repeated gentle shearing with a Polytron homogenizer. Sarcoplasmic reticulum was released into the low-speed supernatant, whereas most of the plasma membrane marker remained in a white, fluffy layer of the sediment, which contained sarcolemma and myofibrils. Additional gentle shearing of the white low-speed sediment extracted plasma membranes in a form that required centrifugation at 100,000 x g for pelleting. This pellet, the fragmented plasma membrane fraction, had a relatively high specific activity of (Na+ + K+)-stimulated ATPase compared with the other fractions, but it had essentially no Ca2+-stimulated ATPase activity and only a small percentage of the succinate dehydrogenase activity of the homogenate. Experimental evidence suggests that the fragmented plasma membrane fraction is derived from delicate transverse tubules rather than from the thicker, basement membrane-coated sarcolemmal sheath of muscle cells. Electron microscopy showed small vesicles lined bu a single thin membrane. Hydroxyproline, a characteristic constituent of collagen and basememt membrane, could not be detected in this fraction.     

LUMENAL PLASMA MEMBRANE OF THE URINARY BLADDER : I. Three-Dimensional Reconstruction from Freeze-Etch Images

To determine the three-dimensional structure of the lumenal membrane of transitional epithelium, a study was made of sectioned, negatively stained, and freeze-etched specimens from intact epithelium and membrane fractions from rabbit urinary bladder. Particulate membrane components are confined to plaque regions within which the unit membrane is asymmetric, having a thicker outer leaflet. Transversely fractured freeze-etched plaques display a thick (~80 A), particulate lumenal leaflet and a thin (~40 A) cytoplasmic one. Four different faces of the two leaflets can be distinguished: two complementary, split, inner membrane faces exposed by freeze-cleaving the bilayer and two external (lumenal and cytoplasmic) membrane surfaces revealed by deep-etching. On the split, inner face of the lumenal leaflet appear polygonal plaques of hexagonally arranged particles. These fit into holes observed on the complementary, split, innerface of the cytoplasmic leaflet. The particles, which have a center-to-center spacing of ~160 A, also seem to protrude from the external surface of the lumenal leaflet, where their subunits (~50 A in diameter) are revealed by freeze-etching and negative staining. The plaques are separated from each other by smooth-surfaced regions, which cleave like simple lipid bilayers. Since the array of plaque particles covers only ~73% of the membrane surface area, whereas 27% is taken up by particle-free interplaque regions, the presence of particles cannot in itself entirely account for the permeability barrier of the lumenal membrane. Although no particles are observed protruding from the cytoplasmic surface of the membrane, cytoplasmic filaments are attached to it by short, cross-bridge-like filaments that seem to contact the particles within the membrane. These long cytoplasmic filaments cross-link adjacent plaques. Therefore, we suggest that at least one function of the particles is to serve as anchoring sites for cytoplasmic filaments, which limit the expansion of the lumenal membrane during distention of the bladder, thereby preventing it from rupturing. The particle-free interplaque regions probably function as hinge areas between the stiff plaques, allowing the membrane to fold up when the bladder is contracted.     

LOCALIZATION OF ENZYMES WITHIN MICROBODIES

Microbodies from rat liver and a variety of plant tissues were osmotically shocked and subsequently centrifuged at 40,000 g for 30 min to yield supernatant and pellet fractions. From rat liver microbodies, all of the uricase activity but little glycolate oxidase or catalase activity were recovered in the pellet, which probably contained the crystalline cores as many other reports had shown. All the measured enzymes in spinach leaf microbodies were solubilized. With microbodies from potato tuber, further sucrose gradient centrifugation of the pellet yielded a fraction at density 1.28 g/cm³ which, presumably representing the crystalline cores, contained 7% of the total catalase activity but no uricase or glycolate oxidase activity. Using microbodies from castor bean endosperm (glyoxysomes), 50–60% of the malate dehydrogenase, fatty acyl CoA dehydrogenase, and crotonase and 90% of the malate synthetase and citrate synthetase were recovered in the pellet, which also contained 96% of the radioactivity when lecithin in the glyoxysomal membrane had been labeled by previous treatment of the tissue with [¹⁴C]choline. When the labeled pellet was centrifuged to equilibrium on a sucrose gradient, all the radioactivity, protein, and enzyme activities were recovered together at peak density 1.21–1.22 g/cm³, whereas the original glyoxysomes appeared at density 1.24 g/cm³. Electron microscopy showed that the fraction at 1.21–1.22 g/cm³ was comprised of intact glyoxysomal membranes. All of the membrane-bound enzymes were stripped off with 0.15 M KCl, leaving the "ghosts" still intact as revealed by electron microscopy and sucrose gradient centrifugation. It is concluded that the crystalline cores of plant microbodies contain no uricase and are not particularly enriched with catalase. Some of the enzymes in glyoxysomes are associated with the membranes and this probably has functional significance.     

Isolation of plasma and nuclear membranes of thymocytes. I. Enzymatic composition and ultrastructure

The purpose of this work was to isolate thymocyte plasma membranes at high yield and purity to study specific surface molecules in their structural context. A procedure was developed in which 92-95% of the cells were disrupted by homogenization in a dense viscous medium, while nuclei remained intact. Differential centrifugation of the homogenate was avoided; instead, only a brief (2 h) centrifugation at equilibrium-density of membrane components was used. Five fractions were obtained, three by flotation. Membrane-bound enzymatic activities indicated a 60-80% yield of plasma membranes in the three floated membrane fractions, which comprised 1.6% of the homogenate protein. Enrichment factors for three ectoenzymes, alkaline phosphatase, gamma-glutamyltransferase, and ouabain-sensitive adenosine triphosphatase were respectively, 70-74, and 40-50 in the two lightest fractions. Nuclear membranes were then isolated from the remaining whole nuclei and were found to be enriched in esterase and NADH-cytochrome c reductase. Plasma membranes and light nuclear membranes appeared as pure unit-membrane vesicles in thin sections and freeze-etching electron microscopy. Some aggregation of intramembranous particles occurred in plasma membrane vesicles.     

DISTRIBUTION OF POLYSOMES IN MOUSE BRAIN TISSUE

Abstract— Polysomes were isolated from several different fractions of mouse brain tissue. After homogenization, the extract was centrifuged to yield a post-mitochondrial supernatant fraction and a pellet fraction. Sucrose gradient analysis of the material in the post-mitochondrial supernatant fraction indicated that 80 per cent of the ribosomes were present in polysomes and that little, if any, of the pellet fraction was present. Sucrose gradient analysis of the solution obtained after washing the pellet showed that very little polysomal material was present. The remaining pellet fraction was resuspended in a detergent mixture of deoxycholate-Tween 40. Sucrose gradient analysis of the resulting detergent-soluble solution indicated that large amounts of ribosomal material, in which 60–70 per cent of the ribosomes were associated in polysomes, were present.
In brain tissue from young animals, 20 per cent of the polysomes were found in the post-mitochondrial supernatant fraction whereas 80 per cent of the polysomes were released from the pellet fraction by detergent treatment. In contrast, in brain tissue from adult animals, 40 per cent of the polysomes were found in the post-mitochondrial supernatant fraction, whereas 60 per cent of the polysomes were released from the pellet fraction by detergent treatment.     

Localization of glycoproteins within erythrocyte membranes of sheep. A freeze-etching and biochemical study

Freeze-fractured membranes of ghost red cells obtained from sheep blood contain randomly distributed particles which are 80–100 Å in diameter. After treatment of the ghosts with 0.1 M phosphate buffer, pH 7.0, the particles form clusters. Sonication of the ghost membranes with clustered particles leads to the formation of a few vesicles which are formed from membrane areas which were either largely particle free or contained clusters of particles. These two kinds of vesicles were separated by centrifugation on a sucrose density gradient. Glycoprotein analysis of the vesicles showed that vesicles without particles contain less glycoprotein than vesicles with particles. In agreement with ref. 1 (Tillack, T. W., Scott R. E. and Marchesi, V. T. (1972) J. Exp. Med. 135, 1209–1220), these results suggest that some of the particles exposed in freeze-etched membranes consist of glycoprotein.     

Isolation of Outer Membranes with an Ordered Array of Surface Subunits from Acinetobacter

A method has been developed for the isolation of outer membranes from Acinetobacter sp. strain MJT/F5/199A. Washed cells were broken in a French press and, after deoxyribonuclease and ribonuclease treatment, removal of intact cells, and four washes in 20 mosmol phosphate buffer, pH 7.4, with centrifugation at 25,000 x g for 10 min, preparations of cell wall fragments from which almost all pieces of plasma membrane had been removed resulted. Treatment of the cell walls with lysozyme and further washing, in the presence of 20 mM MgCl(2), yielded preparations of outer membranes. Electron microscopy of freeze-etched preparations shows that a regular pattern of subunits is present on the outer surfaces of intact cells. After negative staining, these subunits are visible on isolated walls and outer membranes; they can be removed by brief treatment with papain. In section, the cell wall structure is that typical of gram-negative bacteria, but the subunits are not detectable on the surface of the outer membrane. The outer membrane retains the appearance of a "unit membrane" in the cell wall, isolated outer membrane, and papain-treated outer membrane fractions. Both cell walls and outer membranes contain a high percentage of protein (76 and 84%, respectively) and not more than 5% carbohydrate, of which glucose and galactose are constitutents. The outer membranes of this Acinetobacter thus differ in structure and composition from those of bacteria in the Enterobacteriaceae.     

The structure of membrane crystals of the light-harvesting chlorophyll a/b protein complex

Membrane crystals of the light-harvesting chlorophyll a/b protein complex from pea chloroplasts were investigated using electron microscopy and image analysis. The membrane crystals formed upon precipitation of the detergent-solubilized complex with mono- and divalent cations in the presence of small amounts of Triton X-100. The crystalline fraction contained two polypeptides of 25,000 and 27,000 mol wt. Freeze-dried and freeze-etched specimens showed a periodic honeycomb structure on the surface of membrane crystals. Double replicas of freeze-fractured sheets showed a hexagonal lattice of particles on both fracture faces. Image analysis of negatively stained membrane crystals suggested that they had threefold rather than sixfold symmetry in projection. A projection map at 20-A resolution revealed two triangular structural units of opposite handedness per crystallographic unit cell. The structural units appeared to be inserted bidirectionally into the membrane, alternating in orientation perpendicular to the membrane plane.     

Structure of plasma membrane in radicles from cotton seeds

Summary Cortical cells in the apical region of excised radicles from cotton (Gossypium hirsutum L. var. M-8) seeds of differing moisture content (6, 12, or 16%) were examined in thin sections of vapor-fixed tissue and in freeze-etched replicas of unfixed tissue with transmission electron microscopy (TEM). The structure of the plasma membrane, as revealed in chemically-fixed tissue, had the tripartite feature characteristic of unit membranes. Regions of the plasma membrane appearing as discontinuities or breaks were shown by stereo tilting to represent an optical artifact caused by image superposition of curved regions of the plasma membrane contained within the thickness of a thin section. In freeze-etched preparations two complementary-type images of the plasma membrane were consistently observed. The asymmetric distribution of membrane-associated particles (MAPS) in the P and E fracture faces suggests that the structure of plasma membranes in cells of dry seeds resembles that of plasma membranes in germinating seeds. The physiological significance of these findings is that leaching of ions and small molecules during seed imbibition does not appear to involve passage through disorganized membranes in the radicle.     

Isolation of plasma membranes and Golgi apparatus from a single chicken liver homogenate

Chicken liver plasma membranes, minimally contaminated with Golgi apparatus-derived vesicles, were prepared from a low-speed (400 g) pellet by means of flotation in isotonic Percoll solution, followed by a hypotonic wash and flotation in a discontinuous sucrose gradient. Based on the analysis of suitable marker enzymes, alkaline phosphatase and alkaline phosphodiesterase, two plasma membrane fractions were isolated with enrichments, depending on the equilibrium density and marker of 28-97 and with a total yield of 4-5%. Golgi apparatus fractions were prepared by flotation of microsomes, obtained from the same homogenate as the low-speed pellet, in a discontinuous sucrose gradient. The trans-Golgi marker galactosyltransferase was 27-fold enriched in a fraction of intermediate density (d=1.077-1.116 g/ml). Approximately 12% of galactosyltransferase was recovered in the membranes equilibrating d=1.031-1.148 g/ml. Contamination with plasma membrane fragments was low in the light (d=1.031-1.077 g/ml) and intermediate density Golgi vesicles. The isolation of purified plasma membranes and Golgi vesicles from one liver homogenate will enable future studies on receptor cycling between these cell organelles.     

Localization and Solubilization of the Respiratory Nitrate Reductase of Bacillus stearothermophilus

Membranes were isolated from Bacillus stearothermophilus 2184D by lysozyme digestion of the cell wall and subsequent differential centrifugation. Observations with the electron microscope indicate that such membranes are relatively intact and have a typical membrane appearance. Nitrate will preferentially oxidize the cytochrome b of such membranes. Approximately 80% of the total respiratory nitrate reductase activity of whole cells can be localized in the washed membrane fraction and the process of membrane isolation results in a sixfold purification of this enzyme. Of several detergents tested, sodium dodecyl sulfate, Triton 114, and Triton X-100 are most effective in converting reduced methyl viologen-nitrate reductase to a form which will not pellet at 130,000 x g. Density gradient analysis reveals that such detergent-mediated solubilization converts virtually all membrane protein to a form of lighter density.     

Isolation and Partial Characterization of Plasmalemma from Quiescent Schwann Cells in Denervated Cat Sciatic Nerve

Fractions enriched in plasma membranes have been obtained from peripheral nerves enriched 89% in quiescent Schwann cells. Fractions were prepared from the intrafascicular tissue of desheathed distal stumps of cat sciatic nerve 8-10 weeks after transection and suture in the upper thigh. Tissue enriched in Schwann cells was minced, homogenized, and centrifuged to remove nuclei and undispersed tissue. Centrifugation of the resulting supernatant produced a pellet that was osmotically shocked, layered over a discontinuous sucrose gradient, and recentrifuged. Fractions enriched in plasma membrane (PM) markers were pooled, osmotically shocked for 16 h, layered over a second discontinuous sucrose density gradient, and recentrifuged. Membrane fractions (0.6 M:0.85 M and 0.85 M:1.0 M interfaces) contained a homogeneous population of unilamellar vesicles free of myelin. The 0.85 M fraction was enriched in 5'-nucleotidase, 2',3'-cyclic nucleotide 3'-phosphohydrolase. and specific [3H]ouabain binding, 4.8-, 3.0-, and 5.7-fold over the crude homogenate, respectively. These fractions also demonstrated low enzyme activities for succinate dehydrogenase, lactate dehydrogenase, and glucose-6-phosphatase (9, 13, and 15% of control values, respectively). Protein yield of the PM fraction (0.85 M) was approximately 0.6 mg/g of denervated nerve. This preparation should be suitable to characterize the surface properties of Schwann cells free of neuronal regulation.     

A mutated membrane protein of vesicular stomatitis virus has an abnormal distribution within the infected cell and causes defective budding.

Two temperature-sensitive (ts) mutants of the M protein of vesicular stomatitis virus (tsG31 and tsG33) are defective in viral assembly, but the exact nature of this defect is not known. When infected cells are switched from nonpermissive (40 degrees C) to permissive (32 degrees C) temperatures in the presence of cycloheximide, tsG33 virus release increased by 100-fold, whereas tsG31 release increased only by 10-fold. Thus, the tsG33 defect is more reversible than that of tsG31. Therefore, we investigated how the altered synthesis and cellular distribution of tsG33 M protein correlates with the viral assembly defect. At 32 degrees C tsG33 M protein is stained diffusely in the cell cytoplasm and later at the budding sites. In contrast, at 40 degrees C the mutant M protein formed unusual aggregates mostly located in the perinuclear regions of virus-infected cells and partially colocalized with G protein in this region. In temperature shift-down experiments, M can be disaggregated and used to some extent for nucleocapsid coiling and budding, which correlates with the virus titer increase. M aggregates also formed after shift-up from 32 to 40 degrees C, indicating a complete dependence of M aggregation on the temperature. Biochemical analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting revealed that at 40 degrees C M protein is detected exclusively in pellet fractions (nuclear and cytoskeleton components), whereas at 32 degrees C M protein is mainly in the cytoplasmic soluble fractions. Furthermore, when the temperature is raised from 32 to 40 degrees C, the distribution of M protein tends to shift from the soluble to the pellet and cytoskeletal fractions. Electron micrographs of immunoperoxidase-labeled M protein showed that at 40 degrees C M aggregates are often associated with the outer nuclear membranes as well as with vesicular structures. No nucleocapsid coiling was observed in these cells, whereas coiling and budding were seen at 32 degrees C in cells where M protein was partly associated with the plasma membrane. We suggest that the tsG33 M protein mutation may produce a reversible conformational alteration which causes M protein to aggregate at 40 degrees C, therefore inhibiting the proper association of M protein with nucleocapsids and budding membranes.     

Activation of guinea pig polymorphonuclear leukocytes with soluble stimulators leads to nonrandom distribution of NADPH oxidase in the plasma membrane

Guinea pig polymorphonuclear leukocytes (PMN) were briefly activated with soluble stimulators such as sodium myristate (SM) or phorbol myristate acetate (PMA) and then disrupted by the nitrogen cavitation method to study the subcellular distribution of NADPH oxidase, which is responsible for O2 - generation. Fc-receptor and 5'-nucleotidase activities were measured as plasma membrane markers. 1) The homogenate was first fractionated by differential centrifugation. The O2- -generating activity of PMN activated either by SM or PMA was recovered in a 2 X 10(4) g pellet which contained a large amount of granules and about 50% of the plasma membrane markers, but not in a 1 X 10(5) g pellet which consisted of plasma membranes and few granules. 2) Further separation of the 2 X 10(4) g pellet from PMA-activated PMN was attempted by an iso-osmotic Percoll density gradient centrifugation. The O2- -generating activity was recovered in light fractions in which plasma membrane markers were found, but neither in specific nor in azurophil granules. The 1 X 10(5) g pellet showed a similar distribution of the plasma membrane markers to that of the 2 X 10(4) g pellet, except that the peak of the O2- -generating activity was much smaller on an identical density gradient. The results showed that NADPH oxidase is located in the plasma membranes precipitated by centrifugation at 2 X 10(4) X g but not in the ones precipitated at 1 X 10(5) X g. The results suggest that the plasma membrane of activated PMN has a mosaic distribution of NADPH oxidase.     

Immunological characterization of a basement membrane-specific chondroitin sulfate proteoglycan

Reichert's membrane, an extraembryonic membrane present in developing rodents, has been proposed as an in vivo model for the study of basement membranes. We have used this membrane as a source for isolation of basement membrane proteoglycans. Reichert's membranes were extracted in a guanidine/3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate buffer followed by cesium chloride density-gradient ultracentrifugation under dissociative conditions. The proteoglycans were subsequently purified from the two most dense fractions (greater than 1.3 g/ml) by ion-exchange chromatography. Mice were immunized with the proteoglycan preparation and four mAbs recognizing the core protein of a high-density, buoyant chondroitin sulfate proteoglycan were raised. Confirmation of antibody specificity was carried out by the preparation of affinity columns made from each of the mAbs. Chondroitin sulfate proteoglycans (CSPGs) were purified from both supernatant and tissue fractions of Reichert's membranes incubated in short-term organ culture in the presence of radiolabel. The resultant affinity-purified proteoglycan samples were examined by gel filtration, SDS-PAGE, and immunoblotting. This proteoglycan is of high molecular weight (Mr = 5-6 x 10(5)), with a core protein of Mr = approximately 1.5-1.6 x 10(5) and composed exclusively of chondroitin sulfate chains with an average Mr = 1.6-1.8 x 10(4). In addition, a CSPG was purified from adult rat kidney, whose core protein was also Mr = 1.6 x 10(5). The proteoglycan and its core protein were also recognized by all four mAbs. Indirect immunofluorescence of rat tissue sections stained with these antibodies reveal a widespread distribution of this proteoglycan, localized specifically to Reichert's membrane and nearly all basement membranes of rat tissues. In addition to heparan sulfate proteoglycans, it therefore appears that at least one CSPG is a widespread basement membrane component.     

Protein phosphorylation associated with stimulation of rabbit gastric glands

Changes in protein phosphorylation associated with stimulation of acid secretion were investigated using isolated rabbit gastric glands labeled with 32P. The glands were stimulated by 100 microM histamine plus either 10 microM forskolin or 50 microM isobutylmethylxanthine, homogenized, and fractionated into a series of pellets: 40 X g, 5 min; 4000 X g, 10 min; 14,500 X g, 10 min; 48,200 X g, 90 min (microsomes); and supernatant. Stimulation induced a redistribution of H+/K+-transporting ATPase among the membrane fractions, i.e., a reduction in activity of the microsomal fraction, and a compensatory increase in the 4000 X g fraction. Further subfractionation of the 4000 X g pellet by Ficoll density gradient produced an 18% Ficoll layer, greatly enriched in the H+/K+-ATPase, and which is thought to be rich in apical membranes of parietal cells. SDS-polyacrylamide gel electrophoresis showed that the amount of 94 kDa peptide (the molecular size of the H+/K+-ATPase) was increased in the 18% Ficoll layer and decreased in the microsomal fraction by stimulation. Analysis of autoradiograms of the gels revealed that apparent changes in level of phosphorylation occurred in the 120, 94 and 80 kDa regions of the 18% Ficoll layer, and in the 94 kDa region of the microsomal fraction. The phosphorylation changes in the 94 kDa region may not reflect changes in specific activity of a single peptide but may be due to the heterogeneity of proteins in this region, which was demonstrated by selective heat treatment of the samples as well as two-dimensional electrophoresis. Phosphorylation of 120 kDa protein in the 18% Ficoll layer was clearly increased by stimulation, and this appeared to be associated with protein distribution changes as well as phosphorylation. The 80 kDa protein in the 18% Ficoll layer showed marked increased phosphorylation by stimulation, with little change in protein distribution. This 80 kDa protein was focused on two-dimensional gels as several sequential spots, with the most radioactive peptide focused toward the acidic side; thus, we propose isomeric forms of an 80 kDa protein with sequential phosphorylation sites. The phosphorylation changes observed in this study are considered to be important to the process of gastric acid secretion because they occurred in the putative apical membrane fractions in which biochemical and functional changes with stimulation have been demonstrated.