Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.3.5.1 (succinate dehydrogenase)
 8,177 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The plasmalemma and hyaline ectoplasm together constitute the sensory and motor organ of macrophages. The purpose of this study was to isolate this cell fraction in order to analyze it biochemically and functionally. Brief sonification of warmed rabbit lung macrophages caused release of heterodisperse hyaline blebs and filopodia, which were easily collected by differential centrifugation. Viewed in the electron microscope, these structures consisted of membrane-bounded sacs principally containing actin filaments. Some contained secondary lysosomes. They were enriched threefold over whole cell homogenates in specific adenylate cyclase activity and in trichloroacetic-acid-precipitable (125)I when derived from cells labeled with 125(I) by means of a lactoperoxidase-catalyzed reaction. These markers were found to have identical isopycnic densitites when macrophage homogenates were subjected to sedimentation in a focusing sucrose density gradient system, and these markers had densities distinct from those of other cytoplasmic organelles. These markers were therefore assumed to be associated with macrophage plasma membranes. The specific beta- glucuronidase activity of the bleb fraction was similar to that of homogenates, but the blebs had considerably lower specific succinic dehydrogenase activity and RNA content, and DNA was undetectable. Electrophoresis of blebs solubilized in sodium dodecyl sulfate on polyacrylamide gels revealed polypeptides co-migrating with macrophage actin-binding protein, myosin, and actin; blebs also had EDTA-activated adenosine triphosphatase activity characteristic of myosin. The concentrations of actin-binding protein and myosin were higher in blebs than in cells or cytoplasmic extracts, whereas actin concentrations were similar (relative to extracts) or only slightly greater (than in cells). Blebs and intact cells had high lactate dehydrogenase activities in the presence but not the absence of Triton X-100. Blebs and cells oxidased 1-[(14)C]glucose, and the rate of glucose oxidation was increased substantially in the presence of latex beads. We conclude that intact sacs of plasmalemma encasing contractile proteins and cytoplasmic enzymes can be isolated from macrophages. They are enriched in myosin and actin-binding protein, indicating that the contractile apparatus is regulated in the cell periphery. These structures have the capacity to respond to environmental signals. We suggest the name "podosomes" for them because of their resemblance to macrophage pseudopodia. We propose that podosome formation results from rapid dissolution of the cortical gel when the membrane is in an actively extended configuration.
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PMID:Peripheral hyaline blebs (podosomes) of macrophages. 92 88

Two-dimensional gel electrophoresis (2-D) was used to resolve the plasma membrane proteins from cultured human retinal pigment epithelial cells. The cells were metabolically labeled either with [35S]methionine to reveal proteins in general or with [3H]glucosamine or [3H]fucose which are more specific for glycoprotein visualization. The cell surface proteins were also iodinated, using the lactoperoxidase--glucose oxidase technique. These labeled membranes were separate into plasma membrane-enriched fractions by subjecting the water-shocked postnuclear supernatant to a discontinuous sucrose-density gradient. The five resulting membrane fractions were assayed for protein, RNA (microsomes), galactosyltransferase (Golgi membranes), 5'-nucleotidase (plasma membranes), and succinate dehydrogenase (mitochondrial membranes) and were examined by electron microscopy. The plasma membranes were enriched with minimal contamination at the 0.6-0.85 M (F2) and 0.85-1.0 M (F3) sucrose interfaces based on these biochemical and morphological criteria. Examination of 2-D autoradiographic profiles of F2 and F3 showed that approximately 180 proteins or protein subunits had incorporated [35S]methionine. Certain proteins were also labeled by [3H]glucosamine and [3H]fucose, and surface-labeled by iodination. This was especially true of 17 different high-molecular-weight (43-139 X 10(3) MW) very acidic glycoproteins which formed a constellation of spots. These glycoproteins, as well as others, were also seen in the whole-cell acidic glucosamine-labeled 2-D profiles, where about 150 proteins were detected. A total of 39 proteins were catalogued, of which 34 were detectable in the plasma membrane-enriched fractions. The results show that the use of subcellular fractionation, specific precursors, and labeling techniques aids in the detection and characterization of minor proteins in 2-D gels.
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PMID:Isolation and characterization of plasma membrane proteins of cultured human retinal pigment epithelium. 243 67

A new surfactant, 6-O-(N-heptylcarbamoyl)-methyl-alpha-D-glucopyranoside (HECAMEG, molar mass 335.38 g), was synthesized by a simple and low cost procedure from methyl-alpha-D-glucopyranoside. This surfactant is characterized by a high solubility in water (even at 0 degree C), ultraviolet light transparency in the region useful for protein detection, and a high critical micellar concentration (CMC = 19.5 mM), permitting fast elimination by dialysis. Furthermore, the surfactant is colorimetrically titratable by the anthrone technique and its weak interference in protein titration by the Lowry et al. procedure and the bicinchoninic method is easy to overcome. Two membrane proteins (NADH oxidase and succinate dehydrogenase) and a soluble enzyme (lactoperoxidase) retained full activity in the presence of HECAMEG below or above its CMC. The partial inhibition of beta-lactamase (soluble form) by HECAMEG above the CMC was probably only apparent and due to an interference of the surfactant with the substrate rather than a direct effect on the enzyme. HECAMEG was capable of extracting up to 75% of bacteriorhodopsin from the purple membrane of Halobacterium halobium in a nondenatured form as indicated by the spectral properties of the protein. It also solubilized spiralin from the Spiroplasma melliferum membrane with a great selectivity and efficiency, without detectable loss of antigenic properties. These data show that HECAMEG is a very mild surfactant, useful for membrane protein studies.
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PMID:Synthesis and characterization of 6-O-(N-heptylcarbamoyl)-methyl-alpha-D-glucopyranoside, a new surfactant for membrane studies. 275 88

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.
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PMID:Isolation of plasma membranes from neurons grown in primary culture. 282 51

A procedure for the isolation and separation of three different subfractions of plasma membrane from the cellular slime mould Dictyostelium discoideum is described. The cells were disrupted by freeze-thawing in liquid N(2) and plasma membranes were purified by equilibrium centrifugation in a sucrose gradient. The cell surface was labelled with radioactive iodide by using the lactoperoxidase iodination method. Alkaline phosphatase was identified as a plasma-membrane marker by its co-distribution with [(125)I]iodide. 5'-Nucleotidase, which has been widely described as a plasma-membrane marker enzyme in mammalian tissues, was not localized to any marked extent in D. discoideum plasma membrane. The isolated plasma membranes showed a 24-fold enrichment of alkaline phosphatase specific activity relative to the homogenate and a yield of 50% of the total plasma membranes. Determination of succinate dehydrogenase and NADPH-cytochrome c reductase activities indicated that the preparation contained 2% of the total mitochondria and 3% of the endoplasmic reticulum. When the plasma-membrane preparation was further disrupted in a tight-fitting homogenizer, three plasma-membrane subfractions of different densities were obtained by isopycnic centrifugation. The enrichment of alkaline phosphatase was greatest in the subfraction with the lowest density. This fraction was enriched 36-fold relative to the homogenate and contained 19% of the total alkaline phosphatase activity but only 0.08% of the succinate dehydrogenase activity and 0.34% of the NADPH-cytochrome c reductase activity. Electron microscopy of this fraction showed it to consist of smooth membrane vesicles with no recognizable contaminants.
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PMID:The isolation and subfractionation of plasma membrane from the cellular slime mould Dictyostelium discoideum. 415 70

1. The visible absorption spectrum of peroxidase II, isolated from the uterine tissue of oestradiol-treated rats, and some of its derivatives were recorded. The spectral properties of this enzyme are very similar to eosinophile peroxidase and lactoperoxidase, suggesting that these enzymes may have a similar form of haem as prosthetic group. 2. The uterine peroxidase is modified upon interaction with H2O2 and the difference spectrum of this modified enzyme is similar to that of complex II of lactoperoxidase. The modified enzyme was found to revert spontaneously to the native enzyme at rates which depended on the concentration of free enzyme and H2O2.
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PMID:Spectral properties of the oestrogen-induced rat uterus peroxidase II and some of its derivatives. 628 64

Lactoperoxidase-catalyzed radioiodination was used to study the arrangement of the component peptides of succinate-cytochrome c reductase with respect to the aqueous phases on each side of the mitochondrial inner membrane. Mitochondria depleted of their outer membrane and inside-out vesicles purified from submitochondrial particles by the lectin-affinity procedure (D'Souza, M. P., and Lindsay, J. G. (1981) Biochim. Biophys. Acta 640, 463-472) were iodinated using immobilized preparations of lactoperoxidase. The labeled membranes were solubilized in detergent and the succinate-cytochrome c reductase was purified by immunoprecipitation with specific IgG. Analysis of the radioiodine distribution after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and comparison with peptide stain patterns show that bands 2 (64 kilodaltons), 6 (30 kilodaltons), 9 (15 kilodaltons), and 11 (less than 10 kilodaltons) are labeled from the cytoplasmic surface of the membrane. Bands 1 (72 kilodaltons), 4 (48 kilodaltons), and 8 (20 kilodaltons) appear to be labeled on the matrix side of the membrane, while bands 3 (52 kilodaltons), 5 (35 kilodaltons), 7 (25 kilodaltons), and 10 (11 kilodaltons) are labeled from both sides of the membrane. Tentative identification of the labeled bands suggests that band 1 is the large subunit of succinate dehydrogenase. Bands 3 and 4 represent proteins which have been referred to as core proteins I and II. Bands 5 and 6 are the proteins associated with cytochromes b and c1, respectively; band 7 is the Rieske iron-sulfur protein.
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PMID:Labeling of succinate-cytochrome c reductase with 125I. Accessibility of the peptides to the aqueous phases on the cytosolic and matrix sides of the mitochondrial membrane. 628 97