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 genetically obese Zucker rat (fa/fa) is an animal model with severe insulin resistance of the skeletal muscle. We investigated whether a defect of insulin-dependent glucose transporter (GLUT 4) translocation might contribute to the pathogenesis of the insulin-resistant state. fa/fa rats, lean controls (Fa/Fa) as well as normal Wistar rats were injected intraperitoneally with insulin and were killed after 2 or 20 min, respectively. Subcellular fractions were prepared from hind-limb skeletal muscle and were characterized by determination of marker-enzyme activities and immunoblotting applying antibodies against alpha 1 Na+/K+ ATPase. The relative amounts of GLUT 1 and GLUT 4 were determined in the fractions by immunoblotting with the respective antibodies. Insulin induced an approximately two-fold increase of GLUT 4 in a plasma membrane and transverse tubule enriched fraction and a decrease in the low density enriched membrane fraction in all three groups of rats. There was a high individual variation in GLUT 4 translocation efficiency within the groups. However, no statistically significant difference was noted between the groups. No effect of insulin was detectable on the distribution of GLUT 1 or alpha 1 Na+K+ ATPase. The data suggest that skeletal muscle insulin resistance of obese Zucker rats is not associated with a lack of GLUT 4 translocation.
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PMID:Insulin-induced translocation of GLUT 4 in skeletal muscle of insulin-resistant Zucker rats. 815 Feb 26

We have quantitated and studied the topology of isoforms of the Na+/K(+)-ATPase and of the glucose transporter in rat adipocyte plasma membranes. Adipocytes were incubated with or without insulin for 15 min. Sheets of native plasma membrane, with the cytoplasmic face exposed, were prepared by adsorption to EM grids. Grids were incubated in parallel with monoclonal antibodies against the Na+/K(+)-ATPase isoforms alpha 1 and alpha 2, and the glucose transporter isoforms GLUT1 and GLUT4, followed by immunogold labeling, negative staining and quantitation by counting of the gold particles in electron micrographs. In addition, the distribution of glucose transporters and Na+/K(+)-ATPase isoforms in subcellular membrane fractions prepared by an established fractionation procedure was monitored by Western blotting. We found that the Na+/K(+)-ATPases and the glucose transporters were confined to the planar part of the plasma membrane, without association to caveolar invaginations. The vast majority of the Na+/K(+)-ATPase molecules in the adipocyte plasma membrane were of the alpha 2 isoform; GLUT4 was the dominating glucose transporter isoform. The total number of Na+/K(+)-ATPase molecules labeled in the plasma membrane was 3.5 x 10(5) per cell, independent of insulin stimulation. Concomitantly, insulin increased GLUT4 labeling sevenfold to a value of 3.5 x 10(5) per cell.
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PMID:Quantitation of Na+/K(+)-ATPase and glucose transporter isoforms in rat adipocyte plasma membrane by immunogold labeling. 827 Dec 73

We have investigated the mechanism by which amiloride and 5-(N-ethyl-N-isopropyl)amiloride (EIPA) inhibit glucose-stimulated medium acidification in the fission yeast Schizosaccharomyces pombe. The addition of glucose to an unbuffered suspension of cells results in the extrusion of acid. This process was inhibited by diethylstilbestrol (DES), an inhibitor of the H(+)-ATPase (IC50 71 microM), and also by amiloride (IC50 824 microM) and EIPA (IC50 203 microM). The presence of 100 mM NaCl reduced the degree of inhibition observed for amiloride and EIPA, but had no effect on inhibition by DES. N-Methylglucosamine partially protected the cells against the effect of amiloride, but choline chloride did not, suggesting that sodium may be important in the action of amiloride. To establish the site of action of amiloride and EIPA, ATP hydrolysis assays were performed on isolated plasma membranes. H(+)-ATPase activity was inhibited by orthovanadate, but not by amiloride or EIPA. However, both amiloride and EIPA were found to inhibit the incorporation of radioactivity from labelled glucose in S. pombe, with IC50 values of 879 and 272 microM for amiloride and EIPA respectively. Again, 100 mM NaCl was found to reduce the effectiveness of inhibition. Amiloride had no effect on the uptake of 2-deoxyglucose under the same conditions, indicating that amiloride does not inhibit the glucose transporter. We propose that amiloride and EIPA disrupt glucose-induced acidification by inhibiting glucose metabolism.
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PMID:Amiloride and 5-(N-ethyl-N-isopropyl) amiloride inhibit medium acidification and glucose metabolism by the fission yeast Schizosaccharomyces pombe. 843 59

Electron microscopic and biochemical techniques were used to study the cellular localization of the ATP-dependent, IP3-sensitive, Ca2+ store in the glucose- and phosphatidylinositol(PI) agonist-sensitive hamster insulinoma cell line HIT-T15. Scanning electron microscopy revealed conspicuous shape changes of the microvilli following stimulation of these cells with bombesin or thapsigargin. These changes closely resemble those previously shown to accompany stimulation of hexose transport in adipocytes with insulin [J. Cell. Physiol. 142 (1990) 1-14]. Using a hydrodynamic shearing technique for the isolation of microvilli, two cell surface-derived vesicle fractions were prepared containing 80% of the total cellular Ca(2+)-storing activity. In contrast, subcellular fractionation using normal homogenization with a glass/teflon homogenizer yielded the well-known distribution of the Ca(2+)-storing activity which is then predominantly recovered within the microsomal fraction. The surface-derived vesicle fraction was clearly distinguished from the microsomal fraction by its high content of Na+/K(+)-ATPase and an immunoreactive fragment of the GluT-1 glucose transporter isoform which both are not detectable in the microsomal fraction isolated from homogenates from sheared cells. The Ca2+ uptake properties of the cell surface-derived vesicle fractions including the vanadate, A23187, and thapsigargin sensitivity were found to be identical with those described for the microsomal Ca2+ stores of various cell types. Inositol 1,4,5-trisphosphate (IP3) at 1 microM induced a maximal release of 35-40% of the stored Ca2+ from these vesicles.
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PMID:The IP3-sensitive calcium store of HIT cells is located in a surface-derived vesicle fraction. 846 84

In this study we have used wortmannin, a highly specific inhibitor of phosphatidylinositol (PI) 3-kinase, to assess the role of this enzyme on GLUT1 glucose carrier distribution and glucose transport activity in myoblasts from two skeletal-muscle cell lines, L6E9 and Sol8. As detected in L6E9 cells, myoblasts exhibited basal and insulin-stimulated PI 3-kinase activities. Incubation of intact myoblasts with wortmannin resulted in a marked inhibition of both basal and insulin-stimulated PI 3-kinase activities. L6E9 and Sol8 myoblasts showed basal and insulin-stimulated glucose transport activities, both of them inhibited by wortmannin in a dose-dependent manner (IC50 approximately 10-20 nM). Concomitantly, immunofluorescence analysis revealed that 1 h treatment with wortmannin led to a dramatic intracellular accumulation of GLUT1 carriers (the main glucose transporter expressed in L6E9 and Sol8 myoblasts) in both cell systems. The effect of wortmannin on GLUT1 cellular redistribution was independent of the presence of insulin. The cellular distribution of two structural plasma-membrane components such as beta 1-integrin or the alpha 1 subunit of the Na(+)-K(+)-ATPase were unaffected by wortmannin in both the absence and the presence of insulin. As a whole, our results indicate that PI 3-kinase is necessary to basal and insulin-stimulated glucose transport in L6E9 and Sol8 myoblasts. Moreover, immunofluorescence assays suggest that in both cellular models there is a constitutive GLUT 1 trafficking pathway (independent of insulin) that involves PI 3-kinase and which, when blocked, locks GLUT1 in a perinuclear compartment.
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PMID:Disruption of GLUT1 glucose carrier trafficking in L6E9 and Sol8 myoblasts by the phosphatidylinositol 3-kinase inhibitor wortmannin. 852 58

The glucose transporter of Trypanosoma brucei procyclic forms was characterized and compared with its bloodstream form counterpart. Measuring the glucose consumption enzymatically, we determined a saturable uptake process of relatively high affinity (Km = 80 microM, Vmax = 4 nmol min-1 10(-8) cells), which showed substrate inhibition at glucose concentrations above 1.5 mM (Ki = 21 mM). Control experiments measuring deoxy-D-[3H]Glc uptake under zero-trans conditions indicated that substrate inhibition occurred on the level of glycolysis. Temperature-dependent kinetics revealed a temperature quotient of Q10 = 2.33 and an activation energy of Ea = 64 kJ mol-1. As shown by trans-stimulation experiments, glucose uptake was stereospecific for the D isomer, whereas L-glucose was not recognized. Inhibitor studies using either the uncoupler carbonylcyanide-4-(trifluoromethoxy)phenylhydrazone (5 microM), the H+/ATPase inhibitor N,N'-dicyclohexylcarbodiimide (20 microM), the ionophor monensin (1 microM), or the Na+/K+-ATPase inhibitor ouabain (1 mM) showed insignificant effects on transport efficiency. The procyclic glucose transporter was subsequently enriched in a plasma-membrane fraction and functionally reconstituted into proteoliposomes. Using Na+-free conditions in the absence of a proton gradient, the specific activity of D-[14C]glucose transport was determined as 2.9 nmol min-1 (mg protein)-1 at 0.2 mM glucose. From these cumulative results, we conclude that glucose uptake by the procyclic insect form of the parasite occurs by facilitated diffusion, similar to the hexose-transport system expressed in bloodstream forms. However, the markedly higher substrate affinity indicates a differential expression of different transporter isoforms throughout the lifecycle.
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PMID:Glucose uptake occurs by facilitated diffusion in procyclic forms of Trypanosoma brucei. 861 69

As demonstrated previously, liver acini draining the blood from intraportally transplanted pancreatic islets in streptozotocin-diabetic rats are altered in various respects. The hepatocytes in these acini store glycogen and/or fat, and they show an increase in proliferation as well as in apoptotic activity. Thus, they are phenotypically similar to carcinogen-induced preneoplastic liver foci (glycogen-storing foci and sometimes also mixed cell foci). By means of catalytic enzyme histochemistry or immunohistochemistry, we investigated the activity of key enzymes of alternative pathways of carbohydrate metabolism and some additional marker enzymes (well known from studies on preneoplastic hepatic foci) in the altered liver acini surrounding the islet isografts. In addition, the expression of glucose transporter proteins 1 and 2 (GLUT-1 and GLUT-2) were investigated immunohistochemically. The activities of hexokinase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase, and glucose-6-phosphate dehydrogenase were increased, whereas the activities of glycogen phosphorylase, adenylate cyclase, glucose-6-phosphatase, and membrane-bound adenosine triphosphatase were decreased in the altered liver acini. The expression of GLUT-2 was also decreased. GLUT-1 and glutathione S-transferase placental form were not expressed, and the activities of glycogen synthase and gamma-glutamyl-transferase remained unchanged. All changes of the enzyme activities were in line with the well known effects of insulin and resembled alterations characteristic of preneoplastic liver foci observed in different models of hepatocarcinogenesis. It remains to be clarified in long-term experiments whether or not these foci represent preneoplastic lesions and may proceed to neoplasia.
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PMID:Altered liver acini induced in diabetic rats by portal vein islet isografts resemble preneoplastic hepatic foci in their enzymic pattern. 864 65

Several extracellular matrix (ECM) configurations involving type I collagen and Matrigel were examined for their ability to support differentiated function and polarity of cultured adult rat hepatocytes. Collagen sandwich- and Matrigel-based cultures yielded superior and comparable albumin secretion for at least 2 weeks. In collagen sandwich, hepatocytes were polygonal, and formed multicellular arrays. Collagen sandwich was also found to promote in vivo-like polarization of F-actin, cell adhesion molecules (E-cadherin), and lateral (Na+, K(+)-ATPase, glucose transporter) and apical (dipeptidyl peptidase, aminopeptidase) membrane polarity markers, but not the expression of the gap junction protein connexin 32 and the epidermal growth factor (EGF) receptor. In contrast, hepatocytes cultured in or on Matrigel were more rounded and formed aggregates. Matrigel-based cultures also elicited detectable levels of connexin and EGF receptor and an altered distribution of F-actin, E-cadherin, and apical and lateral membrane proteins. Composite sandwich configurations containing collagen I and Matrigel restored markers lacking in the collagen sandwich, and showed a variable morphology and membrane polarity. Hepatocyte polarity could thus be manipulated by the overall ECM composition. Furthermore, in composite sandwich cultures, these manipulations can be effected largely independent of changes in hepatocyte morphology and albumin secretion.
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PMID:Culture matrix configuration and composition in the maintenance of hepatocyte polarity and function. 874 35

Gentamicin nephrotoxicity may arise in part from alterations in the expression of genes critical for renal proximal tubule metabolism. We tested the hypothesis that gentamicin suppressed the gene expression of the Na+/Ca2+ exchanger (NaCaX), glucose transporter 1 (GLUT1) and alpha 1-subunit of Na(+)-K(+)-ATPase (alpha 1-NKA) in renal tubules. The products of these genes mediate Na(+)-dependent Ca2+ efflux, glucose efflux and influx, and ATP-dependent Na+ efflux across tubular basolateral membranes, respectively. After 10 days of gentamicin intoxication (40 mg/kg ip, twice daily), levels of mRNAs encoding NaCaX and the cognate protein declined. GLUT1 mRNA levels increased, although GLUT1 protein levels were also reduced. Moreover, whereas alpha 1-NKA mRNA levels remained unchanged, alpha 1-NKA protein levels were also reduced. We suggest that the higher GLUT1 mRNA level is part of the stress response to tubular injury. However, regardless of the mRNA level, the most consistent effect of gentamicin was reduction of specific protein levels. We propose that failure to translate high levels of mRNA into proportionally high levels of protein, as in the case of GLUT1, may attenuate the expression of stress response gene products, and thus diminish the possibility of recovery in gentamicin intoxication.
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PMID:Studies of renal injury. I. Gentamicin toxicity and expression of basolateral transporters. 877 84

This paper gives an overview of recent findings regarding erythrocyte transport. The main transport systems in erythrocyte are Na+, K(+)-ATPase, Ca(2+)-ATPase, anion transport by band 3 protein, water channel and glucose transporter. The kinetics of Na+, K(+)-ATPase had been investigated in detail in several studies and these findings were briefly summarized. The accumulated studies in Ca(2+)-ATPase suggest that the ATPase is also E1/E2 type enzyme. The band 3 protein is most densely distributed protein in the erythrocyte membrane. The anion exchange through band 3 protein is indispensable for CO2 transport from CO2 generating tissues to the lung. The development of molecular biology enabled to discover water channel. The existence of water channel explains the swift movement of water through erythrocyte membrane. The gene for water channel of erythrocyte is designated as AQP1 and is one of gene family, MIP, consisting of 20 genes. The glucose transporter was also identified in the erythrocyte membrane and was named as GLUT1. The transporters involved in Na+, K(+)-cotransport, Na(+)-Li+ countertransport, and Gardos effect remain to be identified.
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PMID:[Characteristic feature in transport of erythrocyte membrane]. 889 May 63


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