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)

Characterization and quantification of the Hxt2 (hexose transport) protein of Saccharomyces cerevisiae indicate that it is one of a set of differentially expressed high-affinity glucose transporters. The protein product of the HXT2 gene was specifically detected by antibodies raised against a synthetic peptide encompassing the 13 carboxyl-terminal amino acids predicted by the HXT2 gene sequence. Hxt2 migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a broad band or closely spaced doublet with an average M(r) of 47,000. Hxt2 cofractionated with the plasma membrane ATPase, Pma1, indicating that it is a plasma membrane protein. Hxt2 was not solubilized by high pH or urea but was solublized by detergents, which is characteristic of an integral membrane protein. Expression of the Hxt2 protein was measured under two different conditions that produce expression of high-affinity glucose transport: a medium shift from a high (2.0%) to a low (0.05%) glucose concentration (referred to below as high and low glucose) and growth from high to low glucose. Hxt2 as measured by immunoblotting increased 20-fold upon a shift from high-glucose to low-glucose medium, and the high-affinity glucose transport expressed had a strong HXT2-dependent component. Surprisingly, Hxt2 was not detectable when S. cerevisiae growing in high glucose approached glucose exhaustion, and the high-affinity glucose transport expressed under these conditions did not have an HXT2-dependent component. The role of Hxt2 in growth during aerobic batch culture in low-glucose medium was examined. An hxt2 null mutant grew and consumed glucose significantly more slowly than the wild type, and this phenotype correlated directly with appearance of the Hxt2 protein.
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PMID:Physiological characterization of putative high-affinity glucose transport protein Hxt2 of Saccharomyces cerevisiae by use of anti-synthetic peptide antibodies. 824 39

Recent reports have shown the presence of a ouabain-like inhibitor of Na+/K(+)-ATPase in humans. We have purified a bovine hypothalamic Na+/K(+)-ATPase inhibitory factor (HIF) by using affinity chromatography combined with HPLC. This inhibitor has a molecular weight of 584 as determined by ion-spray mass spectrometry, making it isobaric with ouabain. Glycosidase treatment or acid hydrolysis of HIF released only L-rhamnose, the hexose isomer found in ouabain, as detected by chiral GC/MS. Additionally, enzymatically generated desrhamnosyl HIF was found to have a molecular weight of 438, as does ouabagenin, the aglycone of ouabain. HIF and its aglycone were indistinguishable from ouabain and ouabagenin, respectively, by reversed-phase HPLC retention times. However, derivatization with naphthoylimidazole followed by HPLC revealed different retention times for naphthoylation products of HIF and ouabain. Subsequent CD spectroscopy on isolated naphthoylation products of HIF and ouabain confirmed that they were different. This study provides chromatographic and spectroscopic evidence that ouabain and HIF are isomeric cardenolides. The structural difference is presumed to account for the significant differences in biological properties observed for HIF and ouabain.
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PMID:Physicochemical characterization of a ouabain isomer isolated from bovine hypothalamus. 839 62

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

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

Most or all mammalian cells contain vanadium at a concentration of 0.1-1.0 microM. The bulk of the vanadium in cells is probably in the reduced vanadyl (IV) form. Although this element is essential and should be present in the diet in minute quantities, no known physiological role for vanadium has been found thus far. In the years 1975-1980 the vanadate ion was shown to act as an efficient inhibitor of Na+,K(+)-ATPase and of other related phosphohydrolyzes as well. In 1980 it was observed that vanadate vanadyl, when added to intact rat adipocytes, mimics the biological actions of insulin in stimulating hexose uptake and glucose oxidation. This initiated a long, currently active, field of research among basic scientists and diabetologists. Several of the aspects studied are reviewed here.
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PMID:Insulin-like actions of vanadate are mediated in an insulin-receptor-independent manner via non-receptor protein tyrosine kinases and protein phosphotyrosine phosphatases. 892 46

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

We analyzed the vacuolar acidification in response to elevated hydrostatic pressure in Saccharomyces cerevisiae. The vacuolar pH, defined using 6-carboxyfluorescein, was directly measured in a hyperbaric chamber with a transparent window under high hydrostatic pressure. The vacuole of strain X2180 became acidified at the onset of pressurization to an extent dependent on the magnitude of pressure applied. A pressure of 40-60 MPa transiently reduced the vacuolar pH by about 0.33 within 4 min. The transient acidification was observed in the presence of D-glucose, D-fructose, or D-mannose as a carbon source, but not 3-o-methyl-D-glucose, ethanol, or glycerol, suggesting that the generation of CO2 was involved in the process. A vma3 mutant defective in vacuolar acidification showed no reduction of vacuolar pH when hydrostatic pressure was applied. This result indicates that the transient vacuolar acidification induced by elevated hydrostatic pressure is mediated through the function of the vacuolar H+-ATPase.
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PMID:Vacuolar acidification in Saccharomyces cerevisiae induced by elevated hydrostatic pressure is transient and is mediated by vacuolar H+-ATPase. 968 Mar 7

Interleukin-1 (IL-1) is known to be a hypoglycemic cytokine, but its mechanism of action is still unknown. Since the blood glucose levels depend on the amount of glucose entering and leaving the circulation, this work was conducted to test the hypothesis that the hypoglycemia observed with IL-1beta might result, at least partially, from a reduced intestinal glucose absorption. Male Sprague Dawley rats were injected intraperitoneally (i.p.) with IL-1beta, and a jejunal segment was perfused with [14C] 3-O-methylglucose for 5, 15, 25 and 40 min. Our results showed that IL-1beta significantly inhibited the mucosal uptake of this hexose and reduced its intestinal retention. The time course and the dose response effect for this cytokine were also determined. Studies on the effect of IL-1beta on the activity of the intestinal Na+-K+ ATPase demonstrated a significant inhibition of the pump. The effect of IL-1beta on the hexose transport across the brush border membrane may thus be attributed to its inhibitory effect on the Na+-K+ ATPase.
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PMID:Interleukin-1 beta inhibits the intestinal transport of [14C] 3-O-methylglucose in the rat. 982 69

Metabolic abnormalities observed in retina and in cerebral cortex were compared in diabetic rats and experimentally galactosemic rats. Diabetes or experimental galactosemia of 2 months duration significantly increased oxidative stress in retina, as shown by elevation of retinal thiobarbituric acid reactive substances (TBARS) and subnormal activities of antioxidant defense enzymes, but had no such effect in the cerebral cortex. Activities of sodium potassium adenosine triphosphatase [(Na,K)-ATPase] and calcium ATPase became subnormal in retina as well as in cerebral cortex. In contrast, protein kinase C (PKC) activity was elevated in retina but not in cerebral cortex in the same hyperglycemic rats. Dietary supplementation with an antioxidant mixture (containing ascorbic acid, Trolox, alpha-tocopherol acetate, N-acetyl cysteine, beta-carotene, and selenium) prevented the diabetes-induced and galactosemia-induced elevation of retinal oxidative stress, the elevation of retinal PKC activity and the decrease of retinal ATPases. In cerebral cortex, administration of the antioxidant diet also prevented the diabetes-induced decreases in (Na,K)-ATPase and calcium ATPases, but had no effect on TBARS and activities of PKC and antioxidant-defense enzymes. The results indicate that retina and cerebral cortex differ distinctly in their response to elevation of tissue hexose, and that cerebral cortex is more resistant than retina to diabetes-induced oxidative stress. The greater resistance to oxidative stress in cerebral cortex, as compared to retina, is consistent with the resistance of cerebral cortex to microvascular disease in diabetes, and with a hypothesis that oxidative stress contributes to microvascular disease in diabetes. Dietary supplementation with these antioxidants offers a means to inhibit multiple hyperglycemia-induced retinal metabolic abnormalities.
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PMID:Abnormalities of retinal metabolism in diabetes or experimental galactosemia. VI. Comparison of retinal and cerebral cortex metabolism, and effects of antioxidant therapy. 989 29

Effects of hyperglycemia (both diabetes and experimental galactosemia) on cardiac metabolism have been determined. In addition, the effect of supplemental antioxidants on these hyperglycemia-induced abnormalities of cardiac metabolism has been investigated. Diabetes or experimental galactosemia of 2 months duration in rats significantly increased oxidative stress in myocardium, as demonstrated by elevation of thiobarbituric acid reactive substances (TBARS) and lipid fluorescent products in left ventricle. Activity of protein kinase C (PKC) was elevated in the myocardium, and the activities of (Na,K)-ATPase and calcium ATPases were subnormal. Administration of supplemental antioxidants containing a mixture of ascorbic acid, Trolox; alpha-tocopherol acetate, N-acetyl cysteine, beta-carotene, and selenium prevented both the diabetes-induced and galactosemia-induced elevation of oxidative stress and PKC activity, and inhibited the decreases of myocardial (Na,K)-ATPase and calcium ATPases. The results show that these metabolic abnormalities are not unique to diabetes per se, but are secondary to elevated blood hexose levels, and supplemental antioxidants inhibit these metabolic abnormalities. Our findings suggest that antioxidants inhibit abnormal metabolic processes that may contribute to the development of cardiac disease in diabetes, and offer a potential clinical means to inhibit cardiac abnormalities in diabetes.
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PMID:Diabetes-induced metabolic abnormalities in myocardium: effect of antioxidant therapy. 1062 18


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