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)

During the preparation of a suspension of dog kidney proximal tubules by collagenase treatment, an uptake of FITC-albumin was demonstrated. This process is attributed to the activation of receptor-mediated endocytosis leading to the appearance of FITC-albumin into intracellular vesicular structures. The isolation of brush border membrane vesicles (BBMV) from the dog kidney proximal tubules in suspension by the magnesium precipitation technique leads to the copurification of a large population of endosomes. These endosomes were separated from BBM vesicles by a technique involving wheat-germ agglutinin. The enrichment in BBM markers and in bafilomycin-sensitive ATPase activity was comparable in endosomes and BBM vesicles. However, the acridine orange acidification assay showed a V-type ATPase-dependent acidification in endosomes but not in BBMV, demonstrating a different orientation of the proton pumps in these structures. SDS-PAGE analysis also showed significant differences in protein pattern of vesicles and endosomes. The most notable difference was the presence of 42-44 kDa and 20-24 kDa proteins in BBMV and their complete absence in endosomes. Western blot analysis identified these proteins as actin and RhoA, among other small proteins, respectively. Western blot experiments also demonstrated a different distribution of beta-COP, beta-adaptin, and RhoGDI in vesicles and endosomes. The morphological aspect (electron microscopy) and sedimentation of endosomes in a 50% Percoll gradient identified these structures as "heavy endosomes" (buoyant density D = 1.036 g/ml). Flow cytometry analysis of heavy endosomes purified from tubules isolated in presence of FITC-albumin showed the presence of FITC-albumin in up to 92% of these intracellular organelles. Western blot analysis using anti-FITC and anti-collagenase antibodies allowed quantification of the FITC-albumin and collagenase A in the purified endosomes. Our results indicate that heavy endosomes are formed during the preparation of the proximal tubules following activation of receptor-mediated endocytosis, probably by soluble proteins. The suspension of tubules thus offers a experimental tool to study the protein reabsorption and traffic of endosomal vesicles in the proximal tubules.
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PMID:Isolation of heavy endosomes from dog proximal tubules in suspension. 869 8

Five major polypeptides of 70, 50, 47, 19 and 17 kDa and four minor polypeptides (100, 65, 45 and 39 kDa) become phosphorylated when clathrin-coated vesicles (CCV) from zucchini hypocotyls are incubated in [gamma 32P]Mg-ATP. After dissociation with 0.5 M Tris/HCl the CCV coat polypeptides were subjected to gel filtration in order to separate clathrin triskelions from beta-adaptin-containing fractions. Only the latter bore kinase activities, with phosphorylated polypeptides of 39 kDa in addition to the 50, 19-kDa and 17-kDa polypeptides just mentioned. Heparin, an inhibitor of casein kinase II, permitted the phosphorylation of only the 19-kDa and 17-kDa polypeptides. Staurosporine, an inhibitor of protein kinase c-like activities, prevented the phosporylation of the 70-kDa polypeptide. When recombined with the triskelions the beta-adaptin fractions achieved the phosphorylation of the 45-kDa and 70-kDa polypeptides. Because of its heat stability and calcium-binding properties we interpret the 45-kDa polypeptide as being a clathrin light chain. Antibodies raised against the 70-kDa group of heat-shock proteins (Hsp70) recognize a 70-kDa polypeptide in the beta-adaptin-containing fractions. Because this polypeptide only phosphorylates in the presence of triskelions we consider it to be the uncoating ATPase, which is known to aggregate upon dissociation of the CCV coat. Our results therefore indicate that zucchini CCV contain a number of phosphorylable polypeptides equivalent to the beta, mu and sigma adaptins of bovine brain. Just as in bovine brain CCV a casein-kinase-II-like activity is associated with the zucchini CCV 50/47-kDa polypeptides, further pointing to their identity as plant mu2/mu1 adaptin equivalents.
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PMID:Localization and properties of kinases in clathrin-coated vesicles from zucchini hypocotyls. 885 56

Hepatic endocytosis is characterized by a division of labor between the different cell types with respect to endocytosis, which is mediated by receptors expressed on their cell surface. We have investigated the expression of GTPases of the rab family in rat liver parenchymal and endothelial cells. Small GTPases of the rab protein family control distinct steps of intracellular transport both in the secretory and the endocytic pathway. As controls have been employed the normal rat kidney (NRK) cell line and brain tissue, neuronal cells are known to express high levels of components of the endocytic machinery (clathrin, adaptins, dynamin, uncoating adenosine triphosphatase, etc.). Endothelial cells were found to express four to seven times more rab4, rab5, and rab7 than parenchymal cells. A similar relationship was found between the endocytic rates in the two cell types; the rate of internalization from the plasma membrane of mannose receptors in rat liver endothelial cells was 2.3 pools/min, whereas the corresponding value for internalization of the galactose receptor in parenchymal liver cell was 0.27 pools/min (comparable with the rate of transferrin internalization in NRK cells). Both immunofluorescence and subcellular fractionation experiments showed that rab5 and rab7 were associated with compartments along the endocytic pathway. Brain tissue showed a similar high expression of endocytic components (rab4, rab5, and rab7) as liver endothelial cells, whereas lower values were found in NRK cells. We also analyzed the following proteins involved in endocytosis: clathrin, alpha-adaptin, beta-adaptin, and rabaptin-5. These proteins showed the same pattern of expression as the rab proteins. In conclusion, the results obtained with liver cells corroborate the data obtained in transfected cells and support the notion that rab proteins may be involved in controlling the endocytic rate in liver cells.
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PMID:The expression of endosomal rab proteins correlates with endocytic rate in rat liver cells. 914 39

gamma-Adaptin and clathrin heavy chain were identified on tubulovesicles of gastric oxyntic cells with the anti-gamma-adaptin monoclonal antibody (MAb) 100/3 and an anti-clathrin heavy chain MAb (MAb 23), respectively. In Western blots, crude gastric microsomes from rabbit and rat and density gradient-purified, H-K-ATPase-rich microsomes from these same species were immunoreactive for gamma-adaptin and clathrin. In immunofluorescent labeling of isolated rabbit gastric glands, anti-gamma-adaptin and anti-clathrin heavy chain immunoreactivity appeared to be concentrated in oxyntic cells. In primary cultures of rabbit oxyntic cells, the immunocytochemical distribution of gamma-adaptin immunoreactivity was similar to that of the tubulovesicular membrane marker in oxyntic cells, the H-K-ATPase. Further biochemical characterization of the tubulovesicular gamma-adaptin-containing complex suggested that it has a subunit composition that is typical of that for a clathrin adaptor: in addition to the gamma-adaptin subunit, it contains a beta-adaptin subunit and other subunits of apparent molecular masses of 50 kDa and 19 kDa. From solubilized gastric microsomes from rabbit, gamma-adaptin could be copurified with the major cargo protein of tubulovesicles, the H-K-ATPase. Thus this tubulovesicular coat may bind directly to the H-K-ATPase and may thereby mediate the regulated trafficking of the H-K-ATPase at the apical membrane of the oxyntic cell during the gastric acid secretory cycle. Given the similarities of the regulated trafficking of the H-K-ATPase with recycling of cargo through the apical recycling endosome of many epithelial cells, we propose that tubulovesicular clathrin and adaptors may regulate some part of an apical recycling pathway in other epithelial cells.
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PMID:Identification of clathrin and clathrin adaptors on tubulovesicles of gastric acid secretory (oxyntic) cells. 957 99

Clathrin and the gamma-adaptin subunit of the AP-1 clathrin adaptor have been previously identified on H-K-ATPase-rich tubulovesicles from gastric acid secretory (oxyntic) cells [C. T. Okamoto, S. M. Karam, Y. Y. Jeng, J. G. Forte, and J. Goldenring. Am. J. Physiol. 274 (Cell Physiol. 43): C1017-C1029]. We further characterized this AP-1 adaptor from rabbit and hog tubulovesicles biochemically and immunologically. Clathrin coat proteins were stripped from purified tubulovesicular membranes and fractionated by hydroxyapatite chromatography. The AP-1 adaptor appears to elute at 200 mM sodium phosphate, based on the presence of proteins in this fraction that are immunoreactive with antibodies against three of the four subunits of this heterotetrameric complex: the gamma-, mu1-, and sigma1-adaptin subunits. Although the putative beta-adaptin subunit in this fraction is not immunoreactive with the anti-beta-adaptin monoclonal antibody (MAb), this beta-adaptin is immunoreactive with polyclonal antibodies (PAbs) directed against the peptide sequence Gly625-Asp-Leu-Leu-Gly-Asp-Leu-Leu-Asn-Leu-Asp-Leu-Gly-Pro-Pro- Val640 , a region conserved between beta1- and beta2-adaptins that is thought to be involved in the binding of clathrin heavy chain. Immunoprecipitation of the AP-1 adaptor complex from this fraction with anti-gamma-adaptin MAb 100/3 resulted in the coimmunoprecipitation of the beta-adaptin that did not react with the anti-beta-adaptin MAb but did react with the anti-beta-adaptin PAbs. In contrast, immunoprecipitation of the AP-1 adaptor complex from crude clathrin-coated vesicles from brain resulted in the coimmunoprecipitation of a beta-adaptin that was recognized by both the anti-beta-adaptin MAb and PAbs. These results suggest that the tubulovesicular AP-1 adaptor complex may be distinct from that found in the trans-Golgi network and may contain an immunologically distinct beta-adaptin. This immunologically distinct beta-adaptin may be diagnostic of apical tubulovesicular endosomes of epithelial cells.
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PMID:An immunologically distinct beta-adaptin on tubulovesicles of gastric oxyntic cells. 981 81

Uncoating of clathrin-coated vesicles requires the J-domain protein auxilin for targeting hsc70 to the clathrin coats and for stimulating the hsc70 ATPase activity. This results in the release of hsc70-complexed clathrin triskelia and concomitant dissociation of the coat. To understand the complex role of auxilin in uncoating and clathrin assembly in more detail, we analyzed the molecular organization of its clathrin-binding domain (amino acids 547-813). CD spectroscopy of auxilin fragments revealed that the clathrin-binding domain is almost completely disordered in solution. By systematic mapping using synthetic peptides and by site-directed mutagenesis, we identified short peptide sequences involved in clathrin heavy chain and AP-2 binding and evaluated their significance for the function of auxilin. Some of the binding determinants, including those containing sequences 674DPF and 636WDW, showed dual specificity for both clathrin and AP-2. In contrast, the two DLL motifs within the clathrin-binding domain were exclusively involved in clathrin binding. Surprisingly, they interacted not only with the N-terminal domain of the heavy chain, but also with the distal domain. Moreover, both DLL peptides proved to be essential for clathrin assembly and uncoating. In addition, we found that the motif 726NWQ is required for efficient clathrin assembly activity. Auxilin shares a number of protein-protein interaction motifs with other endocytic proteins, including AP180. We demonstrate that AP180 and auxilin compete for binding to the alpha-ear domain of AP-2. Like AP180, auxilin also directly interacts with the ear domain of beta-adaptin. On the basis of our data, we propose a refined model for the uncoating mechanism of clathrin-coated vesicles.
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PMID:Molecular and functional characterization of clathrin- and AP-2-binding determinants within a disordered domain of auxilin. 1273 33

We recently reported that megalin is subjected to regulated intramembrane proteolysis (RIP) and includes 1) protein kinase C (PKC)-regulated, metalloprotease-mediated ectodomain shedding producing a membrane-bound megalin COOH-terminal fragment (MCTF) and 2) gamma-secretase-mediated cleavage of the MCTF producing a soluble megalin intracellular domain (MICD). Based on studies of RIP of other receptors, the MICD is predicted to target to the nucleus and regulate gene expression. To determine whether RIP of megalin regulates proximal tubule gene expression, we stably expressed the transfected MCTF (tMCTF) or transfected MICD (tMICD) in opossum kidney proximal tubule (OKP) cells and examined the resulting phenotype. Immunoblotting and immunocytochemical analysis of tMCTF cells showed the tMCTF was expressed and constitutively processed by gamma-secretase. Analysis of specific protein expression in tMCTF- and tMICD-transfected cells using Western blot showed endogenous megalin and Na(+)/H(+) exchanger 3 (NHE3) protein expression to be dramatically lower than that of control cells. Expression of other proteins including myosin VI, beta-adaptin, and the Na-K-ATPase appeared unchanged. Analysis of specific mRNA expression using quantitative real-time PCR showed megalin and NHE3 mRNA levels were significantly lower in tMCTF- and tMICD-transfected cells compared with controls. Inhibition of gamma-secretase activity in tMCTF cells resulted in an 8- to 10-fold recovery of megalin mRNA within 4 h. These data show that the COOH-terminal domain of megalin regulates expression of specific proteins in OKP cells and provides the first evidence that RIP of megalin may be part of a signaling pathway linking protein absorption and gene expression in proximal tubule.
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PMID:The COOH terminus of megalin regulates gene expression in opossum kidney proximal tubule cells. 1849 14

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