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Query: UNIPROT:O95477 (membrane-bound)
 29,236 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An improved aqueous two-phase polymer method has been developed for the isolation of sperm plasma membranes by manipulating various parameters that influence markedly the purity as well as yield of the membrane. The method consists of hypotonic shock of intact spermatozoa with 1.25 mM EDTA to dissociate the plasma membrane and dispersion of these cells to a two-phase polymer system consisting of 5.5% 252-Kd dextran and 4.2% 20-Kd polyethylene glycol prior to centrifugation at 9700 X g for 30 min when the two polymer phases are separated; the membrane fraction sediments at the interphase. The resulting membrane fraction was purified further by repeating the two-phase fractionation step. The yield of the membranes was approx. 35-40%, based on the recovery of the membrane-bound marker enzymes alkaline phosphatase and 5'-nucleotidase. The isolated membranes showed a high degree of purity as evidenced by phase contrast and electron microscopic studies and analyses of marker enzymes characteristic of cellular organelles. The yield and purity of the membranes have been found to be markedly dependent on the conditions of the hypotonic shock, obtained as a function of, EDTA concentration and on the molecular sizes of the dextran and polyethylene glycol that constitute the two-phase polymer system, as well as on the centrifugal force used for the sedimentation of the membrane.
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PMID:Factors influencing the yield and purity of goat sperm plasma membranes isolated by means of an aqueous two-phase polymer system. 244 37

A panel of 14 monoclonal antibodies (McAb) against hematopoietic cell surface antigens was applied on mononuclear blood or bone marrow cells from 40 cases of acute leukemia in order to compare immunoenzymatic staining (IE) (alkaline phosphatase) of fixed cells with immunofluorescence staining (IF) of unfixed suspended cells. According to the immunological results, 25 cases were phenotyped as ALL and 15 cases as AML. Cases with blast crisis secondary to chronic myelogenous leukemia (CML-BC) were not represented in this series. In all ALL cases the two methods gave an identical antigenic distribution. In 20 our of 21 cases of non-T-cell ALL, a B-cell progenitor origin was demonstrated by a positive staining reaction with the anti-CD19 McAb AB1 or HD37, and in 10 cases additionally with the anti-CD20 McAb B1 or 1F5. In contrast to the results obtained with IF, IE revealed a poor preservation of the AB1 epitope on CD19, whereas the HD37 epitope was equally well demonstrated by both methods. In 15 cases of AML the distribution of positive versus negative cells with IE or IF was identical for all McAb except J5 (anti-CALLA) (CD10) and B1 (CD20). Thus, 10/15 AML cases expressed CALLA with IE compared to 2/15 with IF. The corresponding figures for B1 were 5/15 and 0/15, respectively. Accordingly, normal myeloid precursor cells were CALLA-positive with IE but negative with IF. The discrepancy probably reflects the fact that, whereas both intracytoplasmatic and membrane-bound antigens are exposed in IE, only the latter are in IF. If the alteration of antigenic accessibility after fixation is considered, IE can safely be used for immunophenotyping of acute leukemia.
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PMID:Immunological typing of acute leukemias: immunoenzymatic staining of fixed cells compared with immunofluorescence staining of unfixed cells in suspension. 245 10

The clinical course and hematologic changes of three dogs with lymphocytosis of cells morphologically resembling large granular lymphocytes are presented. Hemograms from all dogs showed leukocytosis with marked lymphocytosis. Lymphocytes were characterized by abundant basophilic cytoplasm containing distinct granules which varied in size and number. Electron microscopically the granules were membrane-bound with an electron-dense core. Lymphocytes from one dog were positive for alkaline phosphatase activity, and lymphocytes from another dog were positive for alpha naphthyl butyrate esterase activity. Lymphocytes from one dog were positive for surface receptors for the crystalline fraction portion of gamma immunoglobulins.
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PMID:Lymphocytosis of large granular lymphocytes in three dogs. 246 45

Intramembranous localization of alkaline phosphatase (orthophosphoric monoester phosphohydrolase, alkaline optimum, EC 3, 1, 3, 1; AlPase) was observed biochemically in Bacillus megaterium KM grown in 1% polypeptone medium containing 0.5% NaCl at 37 degrees C under aerobic conditions and harvested at the latter logarithmic phase. AlPases from B. megaterium have been separated into soluble and membrane-bound forms by the centrifugation after cell disruption by sonication. The membrane-bound enzyme was further fractionated to two forms by phase separation using a non-ionic detergent, Triton X-114; one was successfully solubilized into the aqueous phase and the other remained in the Triton phase. Both AlPases of sonication- and Triton-solubilized forms were partially purified by gel filtration and anion-exchange column chromatographies. Their molecular weights were different (52,000 for soluble and 66,000 for Triton-solubilized forms) and the Vmax of the sonication-solubilized enzyme (227 nmol/min/mg protein) was 11-fold higher than that of the Triton-solubilized one although similar Km values (1.7 and 2.3 mM) were observed. Optimum pH of these enzymes tended to shift to a neutral range during the purification steps. These results suggest the multiplicity of AlPase anchoring to the membranes; 1) sonication-solubilized form which may be buried within the membrane lipids by its hydrophobic peptide and solubilized by the cell disruption, 2) detergent-solubilized form which may be bound loosely to the membrane by its hydrophobic domain and solubilized due to the amphiphilicity of enzyme protein, and 3) insolubilized form which may be bound fast to the membrane by its strong hydrophobicity and also have the function of enzymatic ability.
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PMID:[Biochemical studies on intramembranous localization of alkaline phosphatase in Bacillus megaterium KM]. 251 45

In order to investigate the membrane activities underlying development of neural cells, a histochemical localization of Ca2+-ATPase, Mg2+-ATPase and alkaline phosphatase (AlPase) activities in the rat cerebellar cortex during postnatal development was carried out. In the developing cerebellar cortex, ATPase activity was mainly associated with the plasma membranes of Purkinje and granular cells. This activity appeared in the immature Purkinje cells at birth and was proportionally increased throughout postnatal development. It was observed that the ATPase activity of migratory granular cells during a critical period from 3 and 15 postnatal days was increased in a funicular pattern in the developing cerebellar cortex. Conversely, peak AlPase activity in the developing cerebellar cortex was localized in the proliferative external granular cells until 7 postnatal days. Apparently, these phosphatase activities were not present in Bergmann glial fibers during the course of granular cell migration. The present findings were taken to indicate that neuronal cells in the cerebellar cortex have acquired a membrane-bound ATPase which can participate in Ca2+ transport or ATP metabolism during the course of early postnatal development.
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PMID:Histochemical localization of Ca2+, Mg2+-ATPase of the rat cerebellar cortex during postnatal development. 252 32

The SecY protein is a membrane-bound factor required for bacterial protein export and embedded in the cytoplasmic membrane by its 10 transmembrane segments. We previously proposed a topology model for this protein by adapting the Manoil-Beckwith TnphoA approach, a genetic method to assign local disposition of a membrane protein from the enzymatic activity of the alkaline phosphatase (PhoA) mature sequence attached to the various regions. SecY-PhoA hybrid proteins with the PhoA domain exported to the periplasmic side of the membrane have been obtained at the five putative periplasmic domains of the SecY sequence. We now extended this method to apply it to follow export of the newly synthesized PhoA domain. Trypsin treatment of detergent-solubilized cell extracts digested the internalized (unfolded) PhoA domain but not those exported and correctly folded. One of the hybrid proteins was cleaved in vivo after export to the periplasm, providing a convenient indication for the export. Results of these analyses indicate that export of the PhoA domain attached to different periplasmic regions of SecY occurs rapidly and requires the normal functioning of the secY gene supplied in trans. Thus, this membrane protein with multiple transmembrane segments contains multiple export signals which can promote rapid and secY-dependent export of the PhoA mature sequence attached to the carboxyl-terminal sides.
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PMID:Export of Escherichia coli alkaline phosphatase attached to an integral membrane protein, SecY. 253 43

The subcellular localization of the microbicidal nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and associated b-cytochrome was investigated in human neutrophils. In unperturbed neutrophils 85% of b-cytochrome and the major part of membrane-bound components of the NADPH oxidase co-sedimented with markers for specific granules and gelatinase. Using cytochrome b559 as a marker for membrane-bound components of the NADPH oxidase in quantitative studies we observed that, of the remaining 15%, the vast majority co-sedimented with latent alkaline phosphatase, a marker for a newly identified mobilizable intracellular compartment. Only a small fraction co-localized with the plasma membranes. Azurophil granules contained a protease activity which rapidly inactivated the NADPH oxidase components present in other membranes. Stimulation of the neutrophils with formyl-methionyl-leucyl-phenyl-alanine and leukotriene B4 which caused minimal degranulation of specific granules, resulted in translocation of b-cytochrome to the plasma membrane, concomitant with incorporation of alkaline phosphatase into the plasma membrane.
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PMID:Dual granule localization of the dormant NADPH oxidase and cytochrome b559 in human neutrophils. 254 92

Studies were performed to examine the lateral organization of the NADPH oxidase system in the plasma membrane of human neutrophils. Analysis of the subcellular fractionation of human neutrophils by isopycnic sedimentation of cavitated cell lysates suggested that there may be more than one population of plasma membrane vesicles formed upon cell disruption. One population (30-32% sucrose) contained surface accessible wheat germ agglutinin binding sites, alkaline phosphatase activity, and cytochrome b. Another population (34-36% sucrose) contained membrane-bound flavin and, when the cells were prestimulated with phorbol myristate acetate (PMA), NADPH-dependent superoxide generating activity. Approximately 25% of the neutrophil cytochrome b cosedimented with the heavy population, confirming our previous hypothesis (Parkos et al. (1985) J. Biol. Chem. 260, 6541-6547) that only a fraction of the total cellular cytochrome b is involved in superoxide production. The heavy plasma membrane fraction was also enriched in membrane associated actin and fodrin as detected by Western blot analysis. After extraction of the plasma membrane vesicles with detergent cocktails, the majority of superoxide generating activity remained associated with the detergent insoluble pellet. Western blot analysis demonstrated that the pellets were also enriched in actin. Further analysis of these pellets using rate-zonal detergent-containing sucrose density gradients indicated that the superoxide generating complex had an approximate sedimentation coefficient of 80 S, suggesting that the neutrophil superoxide generating system may form a complex on the plasma membrane which is associated with or somehow organized by the membrane skeletal matrix. This organization may be of functional relevance not only to the actual production of superoxide, but also to the targeting of microbicidal oxidants.
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PMID:The lateral organization of components of the membrane skeleton and superoxide generation in the plasma membrane of stimulated human neutrophils. 255 84

The effect of sodium butyrate was examined on the growth and phenotypic expression of a cell line derived from the ascitic fluid of an untreated patient with ovarian carcinoma. The chemical inducer of differentiation, sodium butyrate, markedly enhances the activity of the membrane-bound glycoprotein enzymes, alkaline phosphatase and gamma-glutamyl transpeptidase. The alkaline phosphatase corresponds to placental Regan type. Sodium butyrate (1 mM) alone has only a small inhibitory effect on cell growth. However, it was shown to potentiate the anti-proliferative effect of Adriamycin and to render the cells sensitive to cis-platinum.
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PMID:Sodium butyrate enhances the activities of membranal enzymes and increases drug sensitivity in a cell line from ascitic fluid of an ovarian carcinoma patient. 257 49

Two forms of alkaline phosphatase, extracted from human liver and named API1 and API3, are of high molecular mass, but API3 is the larger molecule and is membrane-bound while API1 is smaller and soluble. Enzyme kinetics are identical. It is suggested that API1 is produced from API3 by an endoprotease. We demonstrated the action of an endoprotease in human liver homogenate converting API3 into API1. In the absence of this enzyme no conversion occurred. This enzyme is active at an acidic pH (less than 6.5) in the presence of Ca.. or Mg.. -ions. It is inhibited by traces of EDTA. It is insensitive to diisopropyl fluoro-phosphate, to leupeptin and to reducing or oxidizing chemicals. At alkaline pH (8.6) its activity is rapidly destroyed. The enzyme is stable in acidic buffer. We conclude that API1 is indeed formed from API3 in the living cell by enzymatic conversion.
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PMID:Endoprotease in human liver transforming multiple forms of alkaline phosphatase. 265 95


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