Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.3.5 (5'-nucleotidase)
 3,167 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Plasma membranes were prepared from the human lymphocyte cell line WIL23A by hypotonic swelling, Dounce homogenization, differential and equilibrium centrifugation. The resulting vesiculated membrane fragments were found to have densities of 1.10 and 1.17 g/ml, and were defined by lactoperoxidase mediated whole cell iodination, L-[3H] fucose incorporation, 5'-nucleotidase activity (EC 3.1.3.5) and electron micrographic visualization. Recovery of plasma membrane from whole cell homogenates was estimated to be approximately 30-35% as judged by the recovery of 125I-labeled cell surface protein. When plasma membranes were prepared from cells which had been incubated for 18 h in the presence of 0.5 muCi/ml [3H] thymidine such that greater than 10(9) acid insoluble counts could be demonstrated in the whole cell homogenates, no [3H] thymidine label and presumably, therefore, no DNA, could be shown to be coincident with either the 1.10 or 1.17 density. Similar experiments with [3H] uridine suggested that 90% of the plasma membranes did not contain RNA, while 10% remained questionable.
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PMID:Investigation of human lymphocyte plasma membrane associated nucleic acid. 126 57

The subcellular origin of membranes from rat skeletal muscle that contain insulin-responsive glucose transporters was investigated. Rat skeletal muscle membranes were prepared by isopycnic centrifugation in sucrose gradients. In vivo insulin treatment increased the content of GLUT-4 glucose transporters in the 25% sucrose fraction (enriched in the plasma membrane marker 5'-nucleotidase) and decreased it in the 35% sucrose fraction (devoid of plasma membrane markers). The possibility of endothelial cell membrane contamination in these fractions was investigated using a mouse monoclonal antibody, MRC OX-43, raised against a cell surface protein specific to rat vascular endothelium. MRC OX-43 did not react with any of the muscle membrane fractions, but did recognize a protein of around 100 kDa in extracts of human endothelial cells and rat aorta. An antibody to the dihydropyridine receptor of skeletal muscle, IIC12, was used to determine the presence of transverse tubules in these fractions. IIC12 reacted positively with a 180-kDa protein in purified rat transverse tubules. In contrast, this antibody did not cross-react with the 25% or 35% sucrose fractions. The 25% sucrose fraction was devoid of calsequestrin and ryanodine receptor, cisternal sarcoplasmic reticulum markers. However, small amounts of these proteins were detected in the 35% sucrose fraction. The results suggest that the 25% sucrose fraction represents plasma membranes, while the 35% sucrose fraction is an insulin-sensitive intracellular fraction that contains, but is not enriched in, sarcoplasmic reticulum cisternae. The results further show that insulin-induced recruitment of GLUT-4 transporters in skeletal muscles can be demonstrated independently of GLUT-4 recruitment in endothelial cells.
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PMID:Characterization of glucose transporter-enriched membranes from rat skeletal muscle: assessment of endothelial cell contamination and presence of sarcoplasmic reticulum and transverse tubules. 198 44

Intact rat fat cells exposed to 12.5 microM [gamma-32P]ATP incorporate label into specific proteins within minutes. By solubilizing the reaction mixture with SDS which by passes the subcellular fractionation steps, the labeled proteins can be identified in autoradiographs of SDS-PAGE gels. The most prominently labeled protein has an Mr of 42,000. Localization of this component to the cell surface can be made on the basis of inhibition of phosphorylation by addition of a protein derived from the rat brain with protein kinase inhibitory property, susceptibility of the phosphorylated protein to tryptic digestion, whereas the unphosphorylated protein is unaffected by digestion with trypsin (15 min), inhibition of phosphorylation of this protein after brief exposure to melittin, and the consistent observation that more label is associated with the 42,000 Mr band in homogenates and permeabilized cells than in comparable numbers of intact cells exposed to the same amount of label. A 42,000 Mr phosphoprotein is also present in mitochondria which is most likely the alpha subunit of pyruvate dehydrogenase. To rule out the possibility that the cell surface protein might be a mitochondrial contaminant from broken cells, 32Pi-labeled and [gamma-32P]ATP-labeled cells were solubilized with Triton and chromatographed on a rabbit anti-pyruvate dehydrogenase antibody-Sepharose 4B column. A single labeled peak was detected upon elution of the bound fraction only in the 32Pi-labeled sample, and not in the [gamma-32P]ATP-labeled sample. Subcellular fractionation studies of intact cells labeled with [gamma-32P]ATP showed differences in the recovery of phosphoproteins of 42,000 Mr depending on whether a continuous sucrose gradient (27.6-54.1%, g/ml) or a discontinuous sucrose gradient (16, 35 and 48%, g/ml) was used. Phosphoproteins of 42,000 Mr were located in the mitochondrial and membrane fractions collected by discontinuous sucrose gradient separation, whereas a phosphoprotein of 42,000 Mr was found primarily in the mitochondrial fraction after continuous sucrose gradient separation. By 5'-nucleotidase activity measurements, the latter approach appears to result in the isolation of a heavy fragment of the plasma membrane with the mitochondrial light fraction which is 42,000 in Mr and labeled. Finally, comparison of the autoradiographs of two-dimensional (2D) gels (isoelectric focusing followed by 10% SDS-PAGE) show different isoelectric points for 42,000 Mr components in [gamma-32P]ATP- and 32Pi-labeled cells.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Characterization of the major phosphoprotein and its kinase on the surface of the rat adipocyte. 377 93

Smooth muscle cell (SMC) migration is an early response to vascular injury and contributes to the development of intimal thickening. Upregulation of several components of the plasminogen activator (PA) system has been documented after vascular injury. Utilizing a Transwell filter assay system and human umbilical vein SMCs, we sought to define the role of four different PA system components on SMC migration and matrix invasion: (1) PAs, (2) plasmin, (3) PA receptors, and (4) PA clearance receptors (ie, low density lipoprotein receptor-related protein [LRP]). Addition of active two-chain urokinase-type PA (UPA) stimulated random migration (192 +/- 30% of control, 0.36 nmol/L, P < .001). The stimulation was inhibited by pretreatment with diisopropylfluorophosphate, PA inhibitor type 1 (PAI-1), or aprotinin, a plasmin inhibitor. Augmented migration was also observed with either low-molecular-weight UPA or the amino terminal fragment of UPA (ATF), with the effects being additive. Stimulation by ATF alone, however, was not inhibited by aprotinin. The stimulatory effect was not specific for UPA, in that tissue-type PA (TPA) also increased migration (169 +/- 9% of control, 10 nmol/L, P < .001); the augmentation was inhibited by pretreatment with DFP, PAI-1, or aprotinin and was additive to the UPA effect. Antibodies to the UPA receptor but not 5'-nucleotidase (another glycosylphosphatidylinositol-anchored cell surface protein) inhibited baseline and UPA-stimulated migration. Similarly, both UPA and TPA stimulated invasion of a collagen gel; this augmentation was inhibited by aprotinin, whereas antibodies to the UPA receptor reduced baseline invasion. Finally, we tested whether inhibition of LRP function, which mediates internalization of PA/inhibitor complexes, affected either process. Both antibodies to LRP and recombinant receptor associated protein, a known inhibitor of ligand binding to the LRP, significantly inhibited migration but did not affect collagen gel invasion. These data demonstrate the ability of several components of the PA system to modulate SMC migration and invasion in vitro via plasmin-dependent and -independent mechanisms.
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PMID:Contrasting effects of plasminogen activators, urokinase receptor, and LDL receptor-related protein on smooth muscle cell migration and invasion. 885 24

Surface adhesion plays an essential part in the survival of the commensal organism Streptococcus gordonii in the oral cavity as well as during opportunistic infections such as endocarditis. At least two types of cell surface protein involved in adhesion are found on the surface of Gram-positive bacteria: those anchored via an LPXTG motif by the enzyme sortase A (SrtA) and those associated with the cell surface by, as yet, unknown mechanisms. In srtA(-) mutants, LPXTG-containing proteins have been shown to be released rather than cross-linked to the cell wall. We have therefore used 2D gel electrophoresis of released proteins from an srtA(-) mutant as well as the wild-type strain, followed by peptide identification by MS, to identify a set of novel proteins predicted to be present on the surface of S. gordonii DL1. This includes two large LPXTG-linked proteins (SGO_0707 and SGO_1487), which both contain tandemly repeated sequences similar to those present in known fibrillar adhesins. A 5'-nucleotidase and a protein with a putative collagen-binding domain, both containing LPXTG motifs, were also identified. Anchorless proteins with known chaperone, stress response and elongation factor functions, apparently responsible for bacterial binding to keratinocytes and saliva-coated surfaces in the absence of the LPXTG-linked adhesins, were also associated with the cell surface. These data reveal a range of proteins to be present on the S. gordonii DL1 cell surface, the expression of which plays an important role in adhesion to epithelia and which represent likely candidates for novel virulence factors in S. gordonii.
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PMID:Identification of novel LPXTG-linked surface proteins from Streptococcus gordonii. 1938 83