Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The plasma membrane of the kidney brush border is composed of two compositionally distinct microdomains, microvilli and clathrin-coated pits. To study their assembly we have immunolocalized brush border marker proteins in the developing proximal tubule epithelium of the neonatal rat and compared their time and site of appearance with those of basolateral markers, Na-K-ATPase and fodrin. The proteins studied were dipeptidyl peptidase IV (DPPIV) (microvilli), actin and villin (microvillar cytoskeletal proteins), glycoprotein 330 (gp330) (coated pits), and clathrin (coated pit cytoskeleton). Although apical microvilli and coated pits were first seen in the stage III nephron, many brush border markers including DPPIV, actin, and clathrin appeared earlier in the development and initially were not polarized. Only during stage III did they become concentrated at the apical membrane. Villin first appeared in the stage III proximal tubule where it was located diffusely in the cytoplasm and in lysosomes as well as along the apical membrane. It did not completely colocalize with actin until stage IV. Gp330 first appeared during stage III and from the beginning was restricted to the apical clathrin-coated membrane domains and endosomes. The results demonstrate that 1) the expression of renal brush border proteins during development is asynchronous, and 2) unlike the basolateral plasmalemmal domain, which is established early in nephrogenesis, brush border assembly occurs later, approximately coinciding with the onset of glomerular filtration.
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PMID:Assembly of distinctive coated pit and microvillar microdomains in the renal brush border. 173 97

A comprehensive survey of 11 peptidases, all of which are markers for renal microvillar membranes, has been made in membrane fractions prepared from pig choroid plexus. Two fractionation schemes were explored, both depending on a MgCl2-precipitation step, the preferred one having advantages in speed and yield of the activities. The specific activities of the peptidases in the choroid-plexus membranes were, with the exception of carboxypeptidase M, lower than in renal microvillar membranes: those of aminopeptidase N, peptidyl dipeptidase A ('angiotensin-converting enzyme') and gamma-glutamyltransferase were 3-5-fold lower, those of aminopeptidase A and endopeptidase-24.11 were 12-15 fold lower, and those of dipeptidyl peptidase IV and aminopeptidase W were 50-70-fold lower. Carboxypeptidase M had a similar activity in both membranes. Alkaline phosphatase and (Na+ + K+)-activated ATPase were more active in the choroid-plexus membranes. No activity for microsomal dipeptidase, aminopeptidase P and carboxypeptidase P could be detected. Six of the peptidases and (Na+ + K+)-activated ATPase were also studied by immunoperoxidase histochemistry at light- and electron-microscopic levels. Endopeptidase-24.11 and (Na+ + K+)-activated ATPase were uniquely located on the brush border, and the other two peptidases appeared to be much more abundant on the endothelial lining of microvessels. Dipeptidyl peptidase IV and aminopeptidase W were also detected in microvasculature. Pial membranes associated with the brain and spinal cord also stained positively for endopeptidase-24.11, aminopeptidase N and peptidyl dipeptidase A. The immunohistochemical studies indicated the subcellular fractionation did not discriminate between membranes derived from epithelial cells (i.e. microvilli) and those from endothelial cells. The possible significance of these studies in relation to neuropeptide metabolism and the control of cerebrospinal fluid production is discussed.
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PMID:Membrane peptidases in the pig choroid plexus and on other cell surfaces in contact with the cerebrospinal fluid. 265 79

Particles found in bovine seminal vesicle secretion were enriched by centrifugation. They varied in size and morphology and contained Mg2+,Ca2+-activated ATPase, aminopeptidase A, alanyl aminopeptidase, gamma-glutamyl transpeptidase and dipeptidyl peptidase IV activities. Hyperactivation of sperm motility and the acrosome reaction were induced by these particles in epididymal spermatozoa suspended in a modified Ringer medium. The hyperactivation, analysed with a microscopic slide test, started within minutes of exposure to membrane particles and continued for 3-4 h, during which time spermatozoa underwent the acrosome reaction. Acrosome staining, phase-contrast microscopy and transmission electron microscopy revealed that the acrosome reaction started within 60 min at 37 degrees C and affected up to 80% of spermatozoa in 4 h. These membrane particles differed from those reported previously in other species in enzyme composition, function and organ of origin.
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PMID:Effect of secretory particles in bovine seminal vesicle secretion on sperm motility and acrosome reaction. 357 75

To analyze mechanisms of liver repopulation, we transplanted normal hepatocytes into syngeneic rats deficient in dipeptidyl peptidase IV activity. When isolated hepatocytes were injected into splenic pulp, cells promptly migrated into hepatic sinusoids. To examine whether transplanted hepatocytes entered liver plates and integrated with host hepatocytes, we analyzed sharing of hepatocyte-specific gap junctions and bile canaliculi. Colocalization studies showed gap junctions uniting adjacent transplanted and host hepatocytes in liver plates. Visualization of bile canalicular domains in transplanted and host hepatocytes with dipeptidyl peptidase IV and ATPase activities, respectively, demonstrated hybrid bile canaliculi, which excreted a fluorescent conjugated bile acid analogue. These results indicate that transplanted hepatocytes swiftly overcome mechanical barriers in hepatic sinusoids to enter liver plates and join host cells. Integration into liver parenchyma should physiologically regulate the function or disposition of transplanted hepatocytes and benefit applications such as gene therapy.
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PMID:Integration of transplanted hepatocytes into host liver plates demonstrated with dipeptidyl peptidase IV-deficient rats. 759 42

The effects of gentamicin treatment on functions of the plasma membrane-bound proteins in situ were investigated in primary culture of rabbit proximal tubular cells (PTC), a recognized model of renal epithelial cells. Activities of apical and basolateral enzymes, activities of phosphate, glucose and alanine sodium-coupled transport systems and leakage of the cytosolic enzyme lactate dehydrogenase (LDH) were determined in PTC grown in glucose-free culture medium as confluent monolayers and incubated with the aminoglycoside. Gentamicin altered in a concentration- and time-dependent manner the activity of dipeptidyl peptidase IV (DPP IV), neutral aminopeptidase (NAP), Na+K(+)-ATPase and the Vmax of sodium-dependent glucose and phosphate uptake, whereas gamma-glutamyl-transpeptidase (GGT) and sodium-dependent alanine uptake were unaffected. Identical concentration of gentamicin was required to induce LDH leakage and cell functions impairment. In contrast, under short time exposure, a condition where the enzyme activities were untouched, mercuric chloride inhibited to a similar extent the activity of the three sodium-coupled transport systems. These data suggest that whereas alterations in membrane fluidity might mediate the effects of gentamicin on membrane functions, the inhibition of transports by mercuric chloride rather reflects an effect on sodium permeability of the apical membrane. They also suggest that study of Na(+)-coupled transports in proximal tubular cells grown in primary culture is a simple and sensitive in vitro model to assess drug-induced nephrotoxicity.
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PMID:Primary culture of rabbit proximal tubules as a cellular model to study nephrotoxicity of xenobiotics. 810 56

Clonal cell lines have been established from primary fetal rat intestinal epithelial cells by stable transfection with plasmids containing either the simian virus 40 (SV40) large T-antigen gene under the control of the heavy metal inducible metallothionein promoter (pMTWt) or the thermolabile SV40 T-antigen gene under the control of the SV40 early promoter (pZipSVtsa58). pMTWt-transfected cells produced sufficient T-antigen to allow them to proliferate both when the metallothionein promoter was induced and uninduced. No differences were observed in the pattern of intestinal epithelial markers expressed when the cells were cultured in the presence or absence of inducing agent (zinc). In contrast, fetal rat intestinal epithelial cells transfected with pZipSVtsa58 were immortalized conditionally; cells proliferated at 32 degrees C but ceased to proliferate between 48 and 72 h of culture at 39 degrees C. Four of these cell lines were characterized in detail; they showed microvilli and tight junctions as well as dome formation and expressed functional and biochemical markers of intestinal epithelial cells, including keratins 8, 19, and 21, aminopeptidase N, and dipeptidyl peptidase IV. One cell line, 2/4/A1, expressed in addition a low level of lactase and sucrase-isomaltase. The amount and/or activity of some of these markers changed during the switch from the proliferative to the nonproliferative state (switch from culture at 32 to 39 degrees C), resulting in a more differentiated phenotype and mimicking similar changes taking place during intestinal epithelial cell differentiation in vivo.
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PMID:Conditionally immortalized intestinal epithelial cells: novel approach for study of differentiated enterocytes. 839 82

The feasibility of liver repopulation with hepatocytes has been shown, although clinical applications demand significant hepatic replacement. To show whether portal vascular bed in large animals could accomodate a greater cell number, we analyzed liver repopulation in syngeneic Fischer 344 rats deficient in dipeptidyl peptidase IV. This system allowed localization of transplanted normal hepatocytes in liver or various ectopic sites, as well as dual studies for analysis of gene expression. Interestingly, the product of a dipeptidyl peptidase IV substrate inactivated bile canalicular adenosine triphosphatase (ATPase) activity in normal but not in dipeptidyl peptidase IV-deficient rats, which allowed localization of dipeptidyl peptidase IV-deficient hepatocytes in normal rat liver for additional reversed transplantation systems. Further studies with genetically marked cells showed that because of the size difference between hepatocytes and portal vein radicles, intrasplenically transplanted cells were distributed in periportal areas (zone 1) in mice, whereas in larger animals (rats or rabbits) cells were also distributed downstream to midlobular (zone 2) or perivenous (zone 3) areas. Transplantation of an escalating number of hepatocytes showed that adult rats tolerated intrasplenic injection of a large cell number in single sessions (up to 1 X 10(8), approximately 10% to 15% of the host hepatocyte mass). Morphometric analysis of recipient livers showed survival of a significantly greater cell number with incorporation in host liver plates. At 4 weeks, transplantation of 2 x 10(7) hepatocytes into adult rats led to a survival of 1.4 +/- 1.0 x 10(6) transplanted cells/cm3 liver, whereas after transplantation of 5 x 10(7) cells or 7.5 x 10(7) cells, the number of surviving transplanted cells in the liver significantly increased to 4.1 +/- 1.4 x 10(6) transplanted cells/cm3 liver (mean, 2.9-fold; P<.003) and 5.5 +/- 1.3 x 10(6) transplanted cells/cm3 liver (mean, 3.9-fold; P<.003), respectively. When cells were injected in greater numbers, transplanted hepatocytes retained normal function and produced more serum albumin or hepatitis B surface antigen in deficient hosts. These data indicate the feasibility in larger animals of significant liver repopulation with hepatocyte transplantation. Use of dipeptidyl peptidase IV-deficient rats should help further analysis of mechanisms in liver repopulation.
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PMID:Studies of liver repopulation using the dipeptidyl peptidase IV-deficient rat and other rodent recipients: cell size and structure relationships regulate capacity for increased transplanted hepatocyte mass in the liver lobule. 861 28

Acute arterial hypertension provokes a rapid decrease in proximal tubule (PT) Na+ reabsorption, increasing flow to the macula densa, the signal for tubuloglomerular feedback. We tested the hypothesis, in rats, that Na+ transport is decreased due to rapid redistribution of apical Na+/H+ exchangers and basolateral Na+ pumps to internal membranes. Arterial pressure was increased 50 mmHg by constricting various arteries. We also tested whether transporter internalization occurred when PT Na+ reabsorption was inhibited with the carbonic anhydrase inhibitor benzolamide. Five minutes after initiating either natriuretic stimuli, cortex was removed, and membranes were fractionated by density gradient centrifugation. Urine output and endogenous lithium clearance increased threefold in response to either stimuli. Acute hypertension provoked a redistribution of apical Na+/H+ exchanger NHE3, alkaline phosphatase, and dipeptidyl peptidase IV to higher density membranes enriched in the intracellular membrane markers. Basolateral membrane Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) activity decreased 50%, 25-30% of the alpha 1-and beta 1-subunits redistributed to higher density membranes, and the remainder is attributed to decreased activity of the transporters. Benzolamide did not alter Na+ transporter activity or distribution, implying that decreasing apical Na+ uptake does not initiate redistribution or inhibition of basolateral Na(+)-K(+)-ATPase. We conclude that PT natriuresis provoked by acute arterial pressure is mediated by both endocytic removal of apical Na+/H+ exchangers and basolateral Na+ pumps as well as decreased total Na+ pump activity.
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PMID:Rapid redistribution and inhibition of renal sodium transporters during acute pressure natriuresis. 876 20

The Triton-insoluble complex from porcine lung membranes has been separated into two distinct subfractions visible as discrete light-scattering bands following buoyant density-gradient centrifugation in sucrose. Both of these detergent-insoluble complexes were enriched in the glycosyl-phosphatidylinositol (GPI)-anchored ectoenzymes alkaline phosphatase, aminopeptidase P and 5'-nucleotidase, and both complexes excluded the polypeptide-anchored ectoenzymes angiotensin-converting enzyme, dipeptidyl peptidase IV and aminopeptidases A and N. The GPI-anchored proteins in both complexes were susceptible to release by phosphatidylinositol-specific phospholipase C. Both complexes were also enriched in cholesterol and glycosphingolipids, and in caveolin/VIP21, although only the higher-density fraction was enriched in the plasmalemmal caveolar marker proteins Ca(2+)-ATPase and the inositol 1,4,5-trisphosphate receptor. Among the annexin family of proteins, annexins I and IV were absent from the two detergent-insoluble complexes, annexin V was present in both, and annexins II and VI were only enriched in the higher-density fraction. When the mental chelator EGTA was present in the isolation buffers, annexins II and VI dissociated from the higher-density detergent-insoluble complex and only a single light-scattering band was observed on the sucrose gradient, at the same position as for the lower-density complex. In contrast, in the presence of excess calcium only a single detergent-insoluble complex was isolated from the sucrose gradients, at an intermediate density. Thus the detergent-insoluble membrane complex can be subfractionated on the basis of what appears to be calcium-dependent, annexin-mediated, vesicle aggregation into two distinct populations, only one of which is enriched in plasmalemmal caveolar marker proteins.
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PMID:Isolation and characterization of two distinct low-density, Triton-insoluble, complexes from porcine lung membranes. 892 Sep 95

Incorporation into plasmids of genes conferring resistance to aminoglycoside antibiotics such as hygromycin B is currently utilized for selection in experiments involving gene transfer in eukaryotic cells. Using a subclone of Caco-2 cells stably transfected with an episomal plasmid containing the hygromycin resistance gene, we observed that transformed cells subcultured in the presence of hygromycin B exhibit, compared with the same cells subcultured in antibiotic-free medium, a sixfold increase in the rates of glucose consumption and lactic acid production and dramatic changes, at mRNA and protein level, of the expressions of sucrase-isomaltase and hexose transporter GLUT-2, which are downregulated, contrasting with an upregulation of hexose transporter GLUT-1. This occurs without significant modifications of the differentiation status of the cells, as demonstrated by the normal expression of villin, ZO-1, dipeptidyl peptidase IV, or Na(+)-K(+)-ATPase. The plasmid copy number is, however, the same, whether or not the cells are cultured in the presence of hygromycin B. These results draw attention to the need to consider antibiotic-dependent alterations of metabolism and gene expression in transfection experiments.
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PMID:Selecting agent hygromycin B alters expression of glucose-regulated genes in transfected Caco-2 cells. 961 75


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