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)

In a previous study [K. Lange and U. Brandt (1993) FEBS Lett. 320, 183-188], we showed that the bulk of the ATP-dependent IP3-sensitive Ca2+ store of the hamster insulinoma cell line, HIT-T15, resides in cell surface-derived vesicles most likely of microvillar origin. The origin and orientation of these vesicles suggested that Ca2+ storage is not due to a membrane-located Ca2+ pumping ATPase but rather to ATP-dependent Ca(2+)-binding within the vesicles. In this case, Ca2+, ATP and IP3 should have free access to the vesicle lumen. This hypothesis was tested. ATP-independent Ca2+ uptake occurred with biphasic kinetics. An initial rapid uptake, which was complete within 30 s, was followed by a slow linear uptake lasting about 10 min. The rapid component was shown by efflux experiments to have an equilibration half-time of about 4 s. This rapid Ca2+ efflux pathway was inhibited by externally applied La3+ (0.1 mM). A similar rapidly equilibrating La(3+)-sensitive Ca2+ pool was also present in vesicles which had been actively loaded with Ca2+ in the presence of ATP. The intravesicular distribution space of this labile Ca2+ pool was identical with that of the non-metabolizable hexose analogue 3-O-methyl-D-glucose, demonstrating that rapid Ca2+ uptake occurs into a true vesicular water space and is not due to binding. ATP and IP3 were also shown to enter the vesicles by an energy-independent pathway which is inhibited by the anion channel inhibitor, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; 0.5 mM). Both ATP-dependent Ca2+ uptake and IP3-induced Ca2+ release from preloaded vesicles were inhibited by DIDS. These findings clearly demonstrate that (1) the vesicle membrane is permeable to ATP and IP3 via anion channels, and (2) Ca2+ uptake into as well as IP3-induced Ca2+ release from the vesicles occur by passive diffusion through a cation channel which is not regulated by IP3. Consequently, the mechanisms for Ca2+ storage and IP3-induced Ca2+ release must be located in the vesicle lumen. Moreover, the microvillar diffusion-barrier concept, originally proposed for the regulation of hexose transport may also be valid for the receptor-operated regulation of cation and anion influx pathways.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Rapid uptake of calcium, ATP, and inositol 1,4,5-trisphosphate via cation and anion channels into surface-derived vesicles from HIT cells containing the inositol 1,4,5-trisphosphate-sensitive calcium store. 768 9

Glucokinase is distinguished from yeast hexokinase and low Km mammalian hexokinases by its low affinity for glucose and its cooperative behavior, even though glucose binding residues and catalytic residues are highly conserved in all of these forms of hexokinase. The roles of Ser-151 and Asn-166 as determinants of hexose affinity and cooperative behavior of human glucokinase have been evaluated by site-directed mutagenesis, expression and purification of the wild-type and mutant enzymes, and steady-state kinetic analysis. Mutation of Asn-166 to arginine increased apparent affinity for both glucose and ATP by a factor of 3. Mutation of Ser-151 to cysteine, alanine, or glycine lowered the Km for glucose by factors of 2-, 26-, and 40-fold, respectively, decreased Vmax, abolished cooperativity for glucose, and also decreased Km for mannose and fructose. The Ser-151 mutants had hexose Km values similar to those of yeast hexokinase, hexokinase I, and the recombinantly expressed COOH-terminal half of hexokinase I. However, the Ki values for the competitive inhibitors, N-acetylglucosamine and glucose-6-P, were unchanged, suggesting that Ser-151 is not important for inhibitor binding. Mutation of Ser-151 also increased the Km for ATP about 5-fold and abolished the enzyme's low ATPase activity, which indicates it is essential for ATP hydrolysis. The substrate-induced change in intrinsic fluorescence of S151A occurred at a much lower glucose concentration than that for wild-type enzyme. The results implicate a dual role for Ser-151 as a determinant of hexose affinity and catalysis, exclusive of the glucose-induced conformational change, and suggest that the low hexose affinity of glucokinase is dependent on interaction of Ser-151 with other regions of the protein.
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PMID:Human beta-cell glucokinase. Dual role of Ser-151 in catalysis and hexose affinity. 773 Mar 77

Understanding the molecular mechanisms involved in the regulation of glucose transport into human muscle is necessary to unravel possible defects in glucose uptake associated with insulin resistance in humans. Here we report a strategy to subfractionate human skeletal muscle biopsies (0.5 g) removed from vastus lateralis during a euglycemic insulinemic clamp procedure. A sucrose gradient separated total membranes into five fractions. Fraction 25 (25% sucrose) contained the plasma membrane markers alpha 1- and alpha 2-subunits of the Na(+)-K(+)-adenosinetriphosphatase and the GLUT-5 hexose transporter, recently immunolocalized to the cell surface of human skeletal muscle. The dihydropyridine receptor, a transverse tubule marker, was present exclusively in this fraction. The GLUT-4 glucose transporter was more concentrated in fraction 27.5 (27.5% sucrose) and largely diminished in plasma membrane markers. Open skeletal muscle biopsies were removed before and 30 min after clamping insulin to 550 pM. This increased GLUT-4 protein by 1.61-fold in fraction 25 and lowered it by 50% in fraction 27.5. Thus physiological concentrations of insulin induce translocation of glucose transporters from an internal membrane pool to surface membranes in human skeletal muscle.
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PMID:Insulin induces translocation of GLUT-4 glucose transporters in human skeletal muscle. 773 59

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

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


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