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

The epithelial layer lining the proximal convoluted tubule of mammalian kidney contains a brush border of numerous microvilli. These microvilli appear in structure to be very similar to the microvilli on epithelial cells of the small intestine. Microvilli found in both the small intestine and the proximal convoluted tubules in kidney have a core bundle of actin filaments bundled by the accessory proteins villin and fimbrin. Along the length of intestinal microvilli, lateral links can be observed to connect the core bundle of actin filaments to the membrane. These cross-bridges are comprised of a 110-kDa calmodulin complex which belongs to a class of single-headed myosin molecules, collectively referred to as myosin-1. We now report that an analogous calmodulin-binding polypeptide of 105 kDa has been identified in rat kidney cortex. The 105-kDa polypeptide is preferentially found in purified kidney brush borders, can be extracted with ATP, and co-elutes with calmodulin on gel filtration and anion exchange chromatography. Fractions containing the 105-kDa polypeptide exhibit a modest ATPase activity in buffer containing CaCl2. The partially purified 105-kDa polypeptide will bind iodinated calmodulin and will sediment with F-actin in buffer containing ethylene glycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or Ca2+. The addition of ATP partially reverses this association with F-actin. These results indicate that myosin-1, in addition to its presence in intestinal brush borders, is present in the brush border of kidney. We also provide preliminary evidence to indicate that the 105-kDa polypeptide is not restricted to tissues possessing a brush border.
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PMID:Identification of the microvillar 110-kDa calmodulin complex (myosin-1) in kidney. 183 82

Colchicine- and vinblastine-induced depolymerization of microtubules (MTs) in the intestinal epithelium of rats and mice resulted in significant delivery of three apical membrane proteins (alkaline phosphatase, sucrase-isomaltase, and aminopeptidase N) to the basolateral membrane domain. In addition, typical brush borders (BBs) occurred at the basolateral cell surface, consisting of numerous microvilli that contained the four major components of the cytoskeleton of apical microvilli (actin, villin, fimbrin, and the 110-kD protein). Formation of basolateral microvilli required polymerization of actin and proceeded at glycocalyx-studded plaques that resembled the dense plaques located at the tips of apical microvilli. BBs from the basolateral membrane became internalized into BB-containing vacuoles which served as recipient organelles for newly synthesized apical membrane proteins. The BB vacuoles fused with each other and finally were inserted into the apical BB. Polarized distribution of Na+,K+-ATPase, a basolateral membrane protein, was not affected by drug-induced depolymerization of MTs. These observations indicate that Golgi-derived carrier vesicles (CVs) containing apical membrane proteins are vectorially guided to the apical cell surface by a retrograde transport along MTs. MTs are uniformly oriented towards a narrow space underneath the apical terminal web (termed subterminal space) that contains MT-organizing properties and controls polarized alignment of MTs. In contrast to apical CVs, targeting of basolateral CVs appears to be independent of MTs but demands a barrier at the apical membrane domain that prevents basolateral CVs from apical fusion (transport barrier hypothesis).
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PMID:Role of microtubules in polarized delivery of apical membrane proteins to the brush border of the intestinal epithelium. 256 63

We have used two actin-binding proteins of the intestinal brush border, TW 260/240 and villin, to examine the effects of filament cross-linking and filament length on myosin-actin interactions. TW 260/240 is a nonerythroid spectrin that is a potent cross-linker of actin filaments. In the presence of this cross-linker we observed a concentration-dependent enhancement of skeletal muscle actomyosin ATPase activity (150-560% of control; maximum enhancement at a 1:70-80 TW 260/240:actin molar ratio). TW 260/240 did not cause a similar enhancement of either acto-heavy meromyosin (HMM) ATPase or acto-myosin subfragment-one (S1) ATPase. Villin, a Ca2+-dependent filament capping and severing protein of the intestinal microvillus, was used to generate populations of actin filaments of various lengths from less than 20 nm to 2.0 microns; (villin:actin ratios of 1:2 to 1:4,000). The effect of filament length on actomyosin ATPase was biphasic. At villin:actin molar ratios of 1:2-25 actin-activated myosin ATPase activity was inhibited to 20-80% of control values, with maximum inhibition observed at the highest villin:actin ratio. The ATPase activities of acto-HMM and acto-S1 were also inhibited at these short filament lengths. At intermediate filament lengths generated at villin:actin ratios of 1:40-400 (average lengths 0.26-1.1 micron) an enhancement of actomyosin ATPase was observed (130-260% of controls), with a maximum enhancement at average filament lengths of 0.5 micron. The levels of actomyosin ATPase fell off to control values at low concentrations of villin where filament length distributions were almost those of controls. Unlike intact myosin, the actin-activated ATPase of neither HMM nor S1 showed an enhancement at these intermediate actin filament lengths.
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PMID:Effects of actin filament cross-linking and filament length on actin-myosin interaction. 293 51

In the present study we have used immunogold labeling of ultrathin sections of the intact chicken and human intestinal epithelium to obtain further insight into the molecular structure of the brush-border cytoskeleton. Actin, villin, and fimbrin were found within the entire microvillus filament bundle, from the tip to the basal end of the rootlets, but were virtually absent from the space between the rootlets. This suggests that the bulk of actin in the brush border is kept in a polymerized and cross-linked state and that horizontally deployed actin filaments are virtually absent. About 70% of the label specific for the 110-kD protein that links the microvillus core bundle to the lipid bilayer was found overlying the microvilli. The remaining label was associated with rootlets and the interrootlet space, where some label was regularly observed in association with vesicles. Since the terminal web did not contain any significant amounts of tubulin and microtubules, the present findings would support a recently proposed hypothesis that the 110-kD protein (which displays properties of an actin-activated, myosin-like ATPase) might also be involved in the transport of vesicles through the terminal web. Label specific for myosin and alpha-actinin was confined to the interrootlet space and was absent from the rootlets. About 10-15% of the myosin label and 70-80% of the alpha-actinin label was observed within the circumferential band of actin filaments at the zonula adherens, where myosin and alpha-actinin displayed a clustered, interrupted pattern that resembles the spacing of these proteins observed in other contractile systems. This circular filament ring did not contain villin, fimbrin, or the 110-kD protein. Finally, actin-specific label was observed in close association with the cytoplasmic aspect of the zonula occludens, suggesting that tight junctions are structurally connected to the microfilament system.
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PMID:Organization of the actin filament cytoskeleton in the intestinal brush border: a quantitative and qualitative immunoelectron microscope study. 341 73

A highly reproducible method is described for simultaneous preparation of the rabbit intestinal BBMV and BLMV. When BLMV were purified on continuous sorbitol gradient the final enrichment was over ten-fold for both Na+, K(+)-ATPase and K(+)-activated phosphatase. BBMV were separated using repeated precipitation with magnesium and EGTA. Marker enzymes of the brush border membrane also showed over ten-fold enrichment. There was no contamination with other cellular organelles. The characteristics of the preparations were checked by SDS gel electrophoresis, which showed distinct protein patterns for both membranes; monoclonal antibodies for villin reacted with the BBMV but not with BLMV, showing the absence of contamination of the BLMV fraction. The quality of the membranes, assessed by D-glucose transport, showed good functional properties for both BBMV and BLMV. Orientation of the vesicles was checked by permeabilizing with SDS buffered with BSA and showed that BLMV are oriented inside out and BBMV right side out. The model simulates normal transepithelial intestinal transport at the level of membrane vesicles and could be applied in transport studies in physiology and pharmacology.
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PMID:Simultaneous preparation of rabbit intestinal brush border and basolateral membrane vesicles. 785 46

Membrane vesicles were isolated from the basolateral domains of pig and normal human colonocytes. The activity of the ouabain-sensitive K(+)-activated phosphatase, the basolateral membrane marker, was enriched 13-fold in these membrane vesicles over the original homogenate. The membranes displayed cross-reactions with antibodies to the (Na+/K+)ATPase and the RLA class I major histocompatibility antigen, both known indicators of the basolateral membrane. There was negligible contamination by other organelles and the luminal membrane, as revealed by marker-enzyme analysis and Western blotting, using an antibody to villin. The vesicles transported D-glucose in a cytochalasin B-inhibitable Na(+)-independent manner, with a Km of 28.1 +/- 0.8 mM and Vmax. of 3.1 +/- 0.4 nmol/s per mg of protein. The transport was inhibited by 2-deoxy-D-glucose and 3-O-methyl-D-glucose, but not by L-glucose or methyl-alpha-D-glucose. Probing the colonocyte basolateral membranes with an antibody against the C-terminus of the human liver GLUT 2 produced a cross-reaction at 52 kDa. These properties indicate the presence of a GLUT 2 isoform on the basolateral membranes of human and pig colonocytes.
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PMID:Preparation and characterization of basolateral membrane vesicles from pig and human colonocytes: the mechanism of glucose transport. 839 17

ATP-depletion in renal cultured cells has been used as a model for studying various cytoskeletal and functional alterations induced by renal ischemia. This communication explores the reversibility of these effects utilizing a novel method [1] that depleted ATP (ATP-D) to 2% of control within 30 minutes and caused complete recovery (REC) of ATP in one hour. Under confocal microscopy, ATP-D (30 min) caused thinning of F-actin from the microvilli, cortical region, and basal stress fibers, with the concurrent appearance of intracellular F-actin patches. These changes were more pronounced after 60 minutes of ATP-D. One hour of REC following 30 minutes of ATP-D produced complete recovery of F-actin in each region of the cell. However, after 60 minutes of ATP-D, a heterogeneous F-actin recovery pattern was observed: almost complete recovery of the apical ring and microvilli, thinned cortical actin with occasional breaks along the basolateral membrane, and a dramatic reduction in basal stress fiber density. The time course of cortical actin and actin ring disruption and recovery coincided with a drop recovery in the transepithelial resistance and the cytoskeletal dissociation and reassociation of the Na,K-ATPase. Additionally, the microvilli retracted into the cells during ATP-D, a process that was reversed during REC. Triton extraction and confocal microscopy demonstrated that villin remained closely associated with microvillar actin during both ATP-D and REC. These distinctive regional differences in the responses of F-actin to ATP depletion and repletion in cultured renal epithelial cells may help to clarify some of the differential tubular responses to ischemia and reperfusion in the kidney.
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PMID:Actin and villin compartmentation during ATP depletion and recovery in renal cultured cells. 858 43

Acute hypertension provokes a rapid decrease in proximal tubule sodium reabsorption with a decrease in basolateral membrane sodium-potassium-ATPase activity and an increase in the density of membranes containing apical membrane sodium/hydrogen exchangers (NHE3) [Y. Zhang, A. K. Mircheff, C. B. Hensley, C. E. Magyar, D. G. Warnock, R. Chambrey, K.-P. Yip, D. J. Marsh, N.-H. Holstein-Rathlou, and A. A. McDonough. Am. J. Physiol. 270 (Renal Fluid Electrolyte Physiol. 39): F1004-F1014, 1996]. To determine the reversibility and specificity of these responses, rats were subjected to 1) elevation of blood pressure (BP) of 50 mmHg for 5 min, 2) restoration of normotension after the first protocol, or 3) sham operation. Systolic hypertension increased urine output and endogenous lithium clearance three- to fivefold within 5 min, but these returned to basal levels only 15 min after BP was restored. Renal cortex lysate was fractionated on sorbitol gradients. Basolateral membrane sodium-potassium-ATPase activity (but not subunit immunoreactivity) decreased one-third to one-half after BP was elevated and recovered after BP was normalized. After BP was elevated, 55% of the apical NHE3 immunoreactivity, smaller fractions of sodium-phosphate cotransporter immunoreactivity, and apical alkaline phosphatase and dipeptidyl-peptidase redistributed to membranes of higher density enriched in markers of the intermicrovillar cleft (megalin) and endosomes (Rab 4 and Rab 5), whereas density distributions of the apical cytoskeleton protein villin were unaltered. After 20 min of normalized BP, all the NHE3 and smaller fractions of the other apical membrane proteins returned to their original distributions. These findings suggest that the dynamic regulation of proximal tubule sodium transport by acute changes in BP may be mediated by rapid reversible regulation of sodium pump activity and relocation of apical sodium transporters.
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PMID:Reversible effects of acute hypertension on proximal tubule sodium transporters. 957 7

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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