Membrane-associated Components Research Articles

Cytochemical properties of osteoblast cell membrane domains.

The interactions of osteoblasts with one another and with the extracellular milieu are of vital importance for cell function. These interactions are mediated by cell membrane-associated components. In the present work, we studied the distribution of several mediators known to be associated with the cell surface, using ultrastructural cytochemistry, to characterize the three cell membrane domains (osteoid, lateral, and vascular) of osteoblasts. Osteoblasts in neonatal rat calvariae were studied for cell surface distribution of alkaline phosphatase (APase), calcium-activated adenosine triphosphatase (Ca2+-ATPase), calcium, soybean agglutinin (SBA)-reactive sites, and peanut agglutinin (PNA)-reactive sites. APase was absent in the osteoid domain but was evenly distributed in the other domains. Ca2+-ATPase was found to be concentrated mainly in the lateral domains. In contrast, calcium was present in all cell membrane domains. Using lectins conjugated to horseradish peroxidase, we demonstrated that SBA binding sites were evenly distributed along the osteoblast cell membrane, whereas PNA binding sites were absent or minimally present in the osteoid and lateral domains but were evenly distributed in the vascular domain. These results suggest that the various functions of osteoblasts may be facilitated by specialized cell membrane domains which are cytochemically distinct. Previous reports have failed to demonstrate the cytochemical differences between the three domains of the osteoblast cell membrane.

Activation of the Superoxide Forming NADPH Oxidase in a Cell-free System by Sodium Dodecyl Sulfate: Absolute Lipid Dependence of the Solubilized Enzyme

The superoxide (O2-)-forming NADPH oxidase of resting macrophages can be activated in a cell-free system by certain anionic amphiphiles, such as sodium dodecyl sulfate (SDS). O2- production requires the cooperation of membrane-associated and cytosolic components. The membrane component can be solubilized by octyl glucoside yielding a highly active oxidase preparation. High performance gel filtration of the solubilized oxidase on Superose 12 in the presence of 40 mM octyl glucoside leads to the total loss of enzymatic activity. This can be restored in previously inactive eluate fractions by with N-ethylmaleimide or heat (60 degrees C)-inactivated total solubilized membrane. Oxidase activity, that becomes evident upon reconstitution, is eluted from Superose 12 with molecules in the Mr range of 300,000-71,000. The material with reconstitutive capacity is completely dissociated from the oxidase, eluting with molecules in the Mr range of 71,000-11,000. The Superose 12 elution profile of the material responsible for reconstitution coincides with that of membrane-derived phospholipid. Also, the reconstitutive capacity of total solubilized membrane and that of the Mr 71,000-11,000 region of the Superose eluate are recovered in a chloroform extract prepared by the method of Bligh and Dyer. It is concluded that loss of oxidase activity by gel filtration at a high octyl glucoside concentration is the consequence of delipidation. NADPH oxidase activity, revealed by reconstitution of Superose 12 fractions with exogenous phospholipid, correlates closely with the elution profile of cytochrome b559. Reconstitution of activity of delipidated oxidase can also be achieved with natural non-macrophage phospholipids and with synthetic phospholipids. Reconstitution of NADPH oxidase activity by lipids is governed by the following rules: (a) phospholipids are effective; lysophospholipids and neutral lipids are not; (b) phospholipids with polar heads represented by choline, ethanolamine, and serine, as well as cardiolipin, are effective; phosphatidylinositol is much less active; (c) phospholipids with unsaturated fatty acid residues are capable of reconstitution while saturated acyl residues do not confer activity; this specificity appears not to be related to the transition temperature of the phospholipids.

Genetic reconstitution of the high-affinity L-arabinose transport system.

Expression plasmids containing various portions of araFGH operon sequences were assayed for their ability to facilitate the high-affinity L-arabinose transport process in a strain lacking the chromosomal copy of this operon. Accumulation studies demonstrated that the specific induction of all three operon coding sequences was necessary to restore high-affinity L-arabinose transport. Kinetic analysis of this genetically reconstituted transport system indicated that it functions with essentially wild-type parameters. Therefore, L-arabinose-binding protein-mediated transport appears to require only two inducible membrane-associated components (araG and araH) in addition to the binding protein (araF).

Solubilization of Membrane-Associated Cross-Protection Factor(s) of Pasteurella multocida

Pasteurella multocida harvested from the blood of turkeys dying of experimental fowl cholera were purified by centrifugation and lysed. The soluble and membrane-associated components of the bacteria were separated by centrifugation. Nonionic (octylglucoside) and zwitterionic (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate; CHAPS) detergents were tested for their abilities to solubilize the cross-protection factor(s) (CPF) from the membrane-associated component. Protection studies in turkeys showed that optimum solubilization was by 1.0% octylglucoside and 0.5% CHAPS. Antibodies from turkeys made against solubilized membrane-associated CPF passively cross-protected poults against challenge. Ion exchange chromatography of detergent-solubilized CPF resulted in elution of two protein-containing peaks, each of which conferred active immune protection.

Nucleotide sequence of the osmoregulatory proU operon of Escherichia coli.

The sequence of 4,362 nucleotides encompassing the proU operon of Escherichia coli was determined. Three open reading frames were identified whose orientation, order, location, and sizes were in close accord with genetic evidence for three cistrons (proV, proW, and proX) in this operon. Similarities in primary structure were observed between (i) the deduced sequence of ProV with membrane-associated components of other binding-protein-dependent transport systems, in the nucleotide-binding region of each of the latter proteins, and (ii) that of ProW with integral membrane components of the transport systems above. The DNA sequence data also conclusively established that ProX represents the periplasmic glycine betaine-binding protein. Two copies of repetitive extragenic palindromic sequences were identified beyond the 3' end of the proX gene. The primer extension technique was used to identify the 5' ends of proU mRNA species that are present in cells grown at high osmolarity; the results suggest that at least some of the osmotically induced proU transcripts have a long leader region, extending as much as 250 base pairs upstream of the proV gene. Evidence was also obtained for the existence of a sequence-directed bend in DNA in the upstream regulatory region of the proU operon.

Endothelial Albumin Binding Proteins Are Membrane-associated Components Exposed on the Cell Surface

The heterobifunctional, photoactivatable, thiol-cleavable cross-linker sulfosuccinimidyl 2-(p-azido-salicylamido)ethyl-1,3'-dithiopropionate (SASD) was radioiodinated and used to determine whether endothelial albumin binding proteins (ABP) recently identified (Ghinea, N., Fixman, A., Alexandru, D., Popov, D., Hasu, M., Ghitescu, L., Eskenasy, M., Simionescu, M., and Simionescu, N. (1988) J. Cell Biol. 107, 231-239) are plasma membrane-associated components exposed on the cell surface. Microvascular endothelial cells (MEC) freshly isolated from rat epididymal fat were incubated with 125I-2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (ASD)-albumin conjugate which upon photolysis by UV light was cross-linked to the receptor proteins. By cleaving the disulfide linkages of the cross-linker with 5% beta-mercaptoethanol and the ligand-receptor interactions with 0.1% sodium dodecyl sulfate, the radioiodinated ASD moiety remained attached to the receptor peptides which were further detected by 5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. In parallel, samples were examined by ligand blotting with albumin-gold complex. The results showed that in these experimental conditions ABP are represented by two major peptides of 31 and 18 kDa and two minor bands of 73 and 56 kDa. Densitometric scanning showed that the two major bands constitute more than 70% of the total ABP. The four peptides were not apparent if the samples were not UV-irradiated. The binding of the radioiodinated ligand to ABPs was reduced by approximately 82% in the presence of excess competitive unlabeled albumin. When MEC were incubated with unlabeled SASD and exposed to UV light, the autoradiographic banding pattern obtained was similar to that of either radioiodinated receptor proteins or MEC not treated with SASD. This indicated that the four albumin binding peptides are distinct proteins of the endothelial cell plasmalemma.

Marmoset red blood cell receptor for membrane-associated complement components is not related to human CR1: Partial characterization of the C3-binding proteins responsible for the spontaneous rosette formation between marmoset red blood cells and human leukocytes

Cells from all the human B-lymphoblastoid cell lines tested and most human monocytes form rosettes with marmoset red blood cells (MaRBC). Because previous reports suggested the involvement of complement components in this phenomenon, the mechanism of rosette formation and the eventual similarities between the MaRBC receptor and the CR1 receptor present on human erythrocytes have been analyzed herein. The binding of MaRBC to human leukocytes strongly differs from the immune adherence phenomenon: rabbit anti-human CR1 did not react with MaRBC and the MaRBC receptor-binding activity is Ca 2+-dependent. Rosette formation required intact energy metabolism and cytoskeleton integrity of leukocytes. Our attempts to purify the receptor from MaRBC membranes revealed the absence of CR1. Nevertheless, C3-binding proteins were isolated by selective desorption by Sepharose iC3 column chromatography. A three-band pattern was observed under reduced conditions with 74,000, 70,000, and 53,000 molecular weights. It was not possible to further separate these components. This protein complex inhibited the rosette phenomenon between MaRBC and both Raji and U-937 cells, exhibited a very poor cofactor activity, and had no decay-accelerating activity toward the classical C3 convertase. This material did not cross-react with antibodies directed to human proteins. These results showed that erythrocytes from new world monkeys do not express a receptor analogous to the human CR1, but expressed C3-binding protein with low cofactor activity that could recognize membrane-associated complement components.

A new marker for identifying quail cells in embryonic avian chimeras: a quail-specific antiserum.

The characterization of cell behavior in quail chick chimeras has greatly increased our knowledge of the ontogeny of embryonic cell populations and the role of cell-cell interactions in development. We sought to extend the value of avian chimeras by producing a marker that would recognize cell surface components and that could be used instead of the traditional nuclear marker to identify quail cells within chimeras. We describe here a quail-specific antiserum produced by injecting chickens with a membrane fraction of 6-10-day quail embryos. By use of peroxidase coupling of a second antibody, serum reactivity was tested in tissue sections of normal quail and chick embryos and of somitic mesoderm and neural tube chimeras. The primary time period examined was 6-10 days of development. At these stages, the antiserum recognizes only quail cells and stains both plasma membrane-associated and cytoplasmic cell components. The latter characteristics allow the identification of quail axons in chimeras and facilitate visualization of quail cells at low magnification. We show that antiserum staining can also be used to identify quail cells in culture and can be combined with orthograde HRP labeling of neurons.

Activation of the respiratory burst oxidase in a fully soluble system from human neutrophils.

The O2(-)-forming respiratory burst oxidase is present in a dormant state in a fully soluble system containing both cytosol and a deoxycholate extract of membranes from resting human neutrophils. Sodium dodecyl sulfate at low concentrations converts this soluble dormant oxidase into its catalytically active form. The Vmax for the activated oxidase was 2.1 mumol of O2-/min/mg of membrane protein. Michaelis constants for NADPH and NADH (38 microM and 1.7 mM, respectively) were similar to those measured previously in other systems. Oxidase activity was not detected after sodium dodecyl sulfate treatment of systems containing solubilized neutrophil membranes obtained from patients with X-linked chronic granulomatous disease. These results suggest that the deoxycholate extract contains both the resting oxidase and those membrane-associated components needed for its activation, all in functioning states.

Visualization of proteoglycans and membrane-associated components in rat incisor predentine and dentine using ruthenium hexammine trichloride

Ruthenium hexammine trichloride (RHT) used as a probe to visualize anion groups in the predentine and dentine of rat incisors, showed a complex distribution pattern including: (i) intercellular proteoglycans (Pg), detected in the predentine as granules 10–15 nm in diameter and as filaments. Non-aggregating Pg was observed in the spaces between collagen fibres as an amorphous group substance. The dentine included smaller RHT-positive granules, observed after thin-section demineralization; (ii) pericellular aggregates, 30–50 nm in diameter, which were absent at the onset of dentinogenesis but appeared when the mantle dentine started to mineralize; (iii) discontinuous staining of the cell coat along the plasma-membrane of the odontoblast process and of the membrane itself. All these RHT-positive components might be Pg and/or sialoglyconjugates and glyco- or phospholipids present on the plasma membrane.

Effect of trifluoperazine on the action of insulin in rat adipocytes.

Trifluoperazine (TFP), a potent inhibitor of calmodulin action, at a concentration of 12 microM decreased the stimulating effects of insulin on 1) fat cell pyruvate dehydrogenase (PDH) activation, 2) generation/action of PDH activator by adipocyte plasma membranes, and 3) insulin-induced loss of insulin receptors, without altering spermine-induced activation of fat cell PDH or preventing insulin stimulation of glucose oxidation. In addition to these effects on insulin action, TFP abolished several biological actions of the insulin-generated PDH stimulator from liver particulate fractions. These actions include fat cell PDH activation and decrease in receptors. These data indicate that TFP inhibits both membrane-associated and intracellular components of insulin action. The results suggest involvement of calcium-binding protein (calmodulin) and/or phospholipid dependent-calcium activated protein kinase C in some of the actions of insulin in fat cells. The insulin effect on glucose oxidation appears to be less dependent on these mediators.

Identification of livG, a membrane-associated component of the branched-chain amino acid transport in Escherichia coli.

Branched-chain amino acids are transported into Escherichia coli by two osmotic shock-sensitive systems (leucine-isoleucine-valine and leucine-specific transport systems). These high-affinity systems consist of separate periplasmic binding protein components and at least three common membrane-bound components. In this study, one of the membrane-bound components, livG, was identified. A toxic analog of leucine, azaleucine, was used to isolate a large number of azaleucine-resistant mutants which were defective in branched-chain amino acid transport. Genetic complementation studies established that two classes of transport mutants with similar phenotypes, livH and livG, were obtained which were defective in one of the membrane-associated transport components. Since the previously cloned plasmid, pOX1, genetically complemented both livH and livG mutants, we were able to verify the physical location of the livG gene on this plasmid. Recombinant plasmids which carried different portions of the pOX1 plasmid were constructed and subjected to complementation analysis. These results established that livG was located downstream from livH with about 1 kilobase of DNA in between. The expression of these plasmids was studied in minicells; these studies indicate that livG appears to be membrane bound and to have a molecular weight of 22,000. These results establish that livG is a membrane-associated component of the branched-chain amino acid transport system in E. coli.

Subspecies- and species-specific antigens of Leishmania mexicana characterized by monoclonal antibodies.

Monoclonal antibodies have been produced that are specific for the reference stocks of Leishmania mexicana species and subspecies L. mexicana mexicana(L11, M379), L. mexicana amazonensis (WR303, H6, LV72), and L. mexicana pifanoi (L20). The specificities of these antibodies were confirmed by analyses employing an indirect radioimmune binding assay and 107 stocks of New World Leishmania. The molecules associated with these species- and subspecies-specific determinants have been characterized by Western blot analysis and consist of mainly low m.w. (11,000 to 50,000) membrane-associated components.

Basement membrane diversity detected by monoclonal antibodies

Human fetal membranes or pepsin solubilized proteins thereof were used as immunogens in the production of monoclonal antibodies to basement membrane-associated components. Some of the antibodies obtained reacted with all basement membranes in indirect immunofluorescent-cent microscopy, others reacted with all epithelial but not with endothelial basement membranes, and yet other antibodies reacted only with certain epithelial basement membranes in these tests. The reactivities of the antibodies demonstrate that different basement membranes are (immuno)chemically different and contain unique components in addition to ubiquitous components such as type IV collagen and laminin

An autoradiographic study of the in-vivo incorporation of [ 3H]-palmitic acid into the dentine and enamel lipids of rat incisors, with a comparison of rapid-freezing freeze-substitution fixation and aldehyde fixation

Four and twenty-four hours after injection of the labelled precursor, there were twice as many silver grains scored with the cryotechniques as with conventional fixation. Absence of labelled-band formation in dentine, the diffusion of silver throughout the entire thickness of dentine as well as the layer of forming enamel. This suggests that lipids are membrane-associated components rather than true matrix-associated components.

Biosynthesis of hepatocyte endoplasmic reticulum proteins

Publisher Summary This chapter explores the application of rat hepatocyte that can serve as an excellent model for the study of the biosynthesis of endoplasmic reticulum (ER) membrane proteins. In the hepatocyte, as in all cell types, the biosynthetic machinery for the synthesis of secretory and membrane proteins includes both rough and smooth ER membrane-associated components, which affect a variety of co- and post-translational modifications of polypeptides synthesized in bound polysomes. Certain integral membrane proteins, such as cytochrome b 5 and NADH-cytochrome b 5 reductase, which are anchored to the membranes solely by a small carboxy terminal segment, are incorporated post-translationally into the ER membrane after their discharge from free polysomes into the cell sap. Other ER membrane proteins, however, only two of which have been demonstrated to be glycoproteins and to span the membrane, are synthesized in membrane-bound ribosomes and are inserted into the membrane during their synthesis. The fact that the concentrations of cytochrome b 5 and its reductase are much higher in the ER than in other membranes may reflect the affinity of these proteins for other components of the microsomal electron transport chains, such as those that are synthesized on bound polysomes.

Extensive homology between membrane-associated components of histidine and maltose transport systems of Salmonella typhimurium and Escherichia coli.

A strong homology was found between the amino acid sequences, deduced from DNA nucleotide sequences, of cytoplasmic membrane-associated components of the high affinity histidine transport system of Salmonella typhimurium (coded by the hisP gene) and the maltose-maltodextrin transport system of Escherichia coli (coded by the malK gene). When the HisP protein sequence was aligned with that of the NH2-terminal two-thirds of the MalK protein, 32% of the positions were identical, and an additional 35% were occupied by functionally similar amino acid residues. These results suggest that some, and possibly many, "periplasmic-binding protein-dependent" transport systems have evolved from a common ancestral system.

New methods of purifying and identifying parasitic diagnostic antigens: A review of the systematic and quantitative approach with the schistosoma model

Several antigen preparations intended for use in the serodiagnosis of schistosomiasis were reviewed. The efficacies of these antigens in terms of specific activities and cross-reactivities were compared. The idea of quantitation of the actual usable amount of antigens in various preparations was introduced. A systematic approach to the purification and identification of relevant antigens in the Schistosoma system was discussed. Use of the quantitative single-tube kinetic-dependent enzyme-linked immunosorbent assay in conjunction with a systematic approach to antigen purification allowed for the identification of substantial activities in membrane-associated antigens. Solubilization of the particulate- and presumably membrane-associated components resulted in significant amounts of highly sero-reactive antigens. Total reliance on aqueous-soluble parasitic antigens is questioned.

Translocation of Thy-1 antigen and a fluorescent lipid probe during lymphoblastoid cell interaction with Mycoplasma hyorhinis.

The translocation of membrane-associated components during in vitro surface interactions between BW5147 murine T-lymphoblastoid cells and Mycoplasma hyorhinis was investigated. Thy-1 antigen (shown to be selectively associated with mycoplasmas following their detachment from infected BW5147 cells) was found by immunoferritin techniques to be localized at the surface of organisms colonizing lymphoblastoid cells. These results are consistent with a transfer of Thy-1 to membranes of mycoplasmas on the lymphoid cell surface. The exchange of membrane lipid-associated components was further investigated with the fluorescent lipid probe 1,6-diphenyl-1,3,5-hexatriene (DPH). Fluorescence polarization of DPH was much less pronounced in uninfected BW5147 cells than in mycoplasmas--a result indicating markedly different probe environments. The increase of fluorescence intensity and the characteristic polarization in mycoplasmal supernatant fractions obtained after incubation of organisms with DPH-labeled BW5147 cells indicated translocation of DPH from lymphoid cells to mycoplasmas. Incorporation of the probe into mycoplasmas using supernatants from DPH-labeled BW5147 cells (incubated alone) was not demonstrated; this observation is consistent with, but does not prove, a contact-dependent process of exchange. Direct monitoring of the exchange of surface antigens and lipophilic probes may be useful in studying the interaction of hydrophobic surface glycoproteins with membranes and the role of membrane components in mycoplasma-host cell interactions.

Fractionation and characterization of the phosphoenolpyruvate: fructose 1-phosphotransferase system from Pseudomonas aeruginosa.

The initial reactions involved in the catabolism of fructose in Pseudomonas aeruginosa include the participation of a phosphoenolpyruvate:fructose 1-phosphotransferase system (F-PTS). Fractionation of crude extracts of fructose-grown cells revealed that both membrane-associated and soluble components were essential for F-PTS activity. Further resolution of the soluble fraction by both size exclusion and ion-exchange chromatography revealed the presence of only one component, functionally analogous to enzyme I. Enzyme I exhibited a relative molecular weight of 72,000, catalyzed the pyruvate-stimulated hydrolysis of phosphoenolpyruvate to pyruvate, and mediated the phosphorylation of fructose when combined with a source of enzyme II (washed membranes). No evidence for the requirement of a phosphate carrier protein, such as HPr, could be demonstrated. Thus, the F-PTS requires a minimum of two components, a soluble enzyme I and a membrane-associated enzyme II complex, and both were shown to be inducible. Reconstituted F-PTS activity was specific for phosphoenolpyruvate as a phosphate donor (Km, approximately -0.6 mM) and fructose as the sugar substrate (Km, approximately 18 microM). Components of the Pseudomonas F-PTS did not restore activity to extracts of deletion mutants of Salmonella typhimurium deficient in individual proteins of the PTS or to fractionated membrane and soluble components of the F-PTS of Escherichia coli. Similarly, membrane and soluble components of E. coli and S. typhimurium would not cross-complement the F-PTS components from P. aeruginosa.