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 collection of vacuolar protein sorting mutants (vps mutants) in Saccharomyces cerevisiae comprises of 41 complementation groups. The vacuoles in these mutant strains were examined using immunofluorescence microscopy. Most of the vps mutants were found to possess vacuolar morphologies that differed significantly from wild-type vacuoles. Furthermore, mutants representing independent vps complementation groups were found to share aberrant morphological features. Six distinct classes of vacuolar morphology were observed. Mutants from eight vps complementation groups were defective both for vacuolar segregation from mother cells into developing buds and for acidification of the vacuole. Another group of mutants, represented by 13 complementation groups, accumulated a novel organelle distinct from the vacuole that contained a late-Golgi protein, active vacuolar H(+)-ATPase complex, and soluble vacuolar hydrolases. We suggest that this organelle may represent an exaggerated endosome-like compartment. None of the vps mutants appeared to mislocalize significant amounts of the vacuolar membrane protein alkaline phosphatase. Quantitative immunoprecipitations of the soluble vacuolar hydrolase carboxypeptidase Y (CPY) were performed to determine the extent of the sorting defect in each vps mutant. A good correlation between morphological phenotype and the extent of the CPY sorting defect was observed.
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PMID:Morphological classification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar compartment in class E vps mutants. 2279 87

Previous purification and characterization of the yeast vacuolar proton-translocating ATPase (H(+)-ATPase) have indicated that it is a multisubunit complex consisting of both integral and peripheral membrane subunits (Uchida, E., Ohsumi, Y., and Anraku, Y. (1985) J. Biol. Chem. 260, 1090-1095; Kane, P. M., Yamashiro, C. T., and Stevens, T. H. (1989) J. Biol. Chem. 264, 19236-19244). We have obtained monoclonal antibodies recognizing the 42- and 100-kDa polypeptides that were co-purified with vacuolar ATPase activity. Using these antibodies we provide further evidence that the 42-kDa polypeptide, a peripheral membrane protein, and the 100-kDa polypeptide, an integral membrane protein, are genuine subunits of the yeast vacuolar H(+)-ATPase. The synthesis, assembly, and targeting of three of the peripheral subunits (the 69-, 60-, and 42-kDa subunits) and two of the integral membrane subunits (the 100- and 17-kDa subunits) were examined in mutant yeast cells containing chromosomal deletions in the TFP1, VAT2, or VMA3 genes, which encode the 69-, 60-, and 17-kDa subunits, respectively. The steady-state levels of the various subunits in whole cell lysates and purified vacuolar membranes were assessed by Western blotting, and the intracellular localization of the 60- and 100-kDa subunits was also examined by immunofluorescence microscopy. The results suggest that the assembly and/or the vacuolar targeting of the peripheral subunits of the yeast vacuolar H(+)-ATPase depend on the presence of all three of the 69-, 60-, and 17-kDa subunits. The 100-kDa subunit can be transported to the vacuole independently of the peripheral membrane subunits as long as the 17-kDa subunit is present; but in the absence of the 17-kDa subunit, the 100-kDa subunit appears to be both unstable and incompetent for transport to the vacuole.
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PMID:Assembly and targeting of peripheral and integral membrane subunits of the yeast vacuolar H(+)-ATPase. 153 Sep 31

The preprotein translocase of Escherichia coli is a multisubunit enzyme with two domains, the peripheral membrane protein SecA and the membrane-embedded SecY/E protein. SecY/E has been isolated as a complex of three polypeptides, SecY, SecE, and band 1. We now present four lines of evidence that the active species of SecY/E is composed of a tightly associated complex of these three subunits: 1) antibodies to SecY efficiently precipitate SecY/E activity as well as all three polypeptides; 2) the proportions of SecY, SecE, and band 1 in the immunoprecipitates are the same as in the starting fraction; 3) the immunoprecipitable complex is not disrupted by treatment with either high salt or urea but is disrupted by brief incubation at 20 degrees C, and the kinetics of dissociation of both band 1 and SecE from SecY at 20 degrees C parallel the loss of translocation ATPase activity; 4) upon immunoprecipitation of similar units of activity of translocase from detergent solutions from either wild-type membranes or a SecY and SecE overproducer strain, the SecE and band 1 subunits are recovered in the same proportions. These data establish that the subunits of SecY/E are firmly associated and that it is the associated complex which is active for translocation.
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PMID:SecY, SecE, and band 1 form the membrane-embedded domain of Escherichia coli preprotein translocase. 153 82

Normal and sickle cell erythrocyte membranes were examined for significant differences in their ATPase activities, thiobarbituric acid reactive products formed (measured relative to malondialdehyde), membrane protein polymerization, and number of protein-free sulfhydryl groups when treated with 0.5 mmol/L t-butylhydroperoxide (tBHP) for 30 minutes. Isolated sickle cell membranes treated with tBHP produced significantly greater inhibition in both their basal and calmodulin-stimulated Ca2+ + Mg(2+)-ATPase activities (75% inhibition in both cases) compared with that of control membranes. In addition, there was significantly more malondialdehyde formed from sickle cell membranes compared with control membranes. Oxidation caused greater protein polymerization in sickle cell membranes compared with normal membranes as demonstrated by the formation of high molecular weight polymers separated on sodium dodecyl sulfate polyacrylamide gels. The number of free sulfhydryl groups present in spectrin and actin decreased more in sickle cell membranes as measured by 3H-N-ethyl maleimide autoradiography and gel scanning. To prevent enzyme inhibition, erythrocyte membranes were treated with tBHP in the presence of 1 mmol/L ascorbate, a potential antioxidant, and 1 mmol/L desferal, an iron chelator. Both ascorbate and desferal added alone with tBHP were effective in preventing inhibition of the basal and calmodulin-stimulated Ca2+ + Mg(2+)-ATPase activities in normal membranes, but in sickle cell membranes only the addition of ascorbate and desferal together offered significant protection. The enhanced oxidation observed with sickle cell membranes can be mimicked in normal white membranes by adding hemoglobin, hemin, or ferrous chloride in the presence of tBHP. In contrast to hemoglobin, ferrous chloride has the ability to enhance membrane oxidation in the presence of ascorbate with or without tBHP. Furthermore, the addition of desferal to these membranes greatly decreased the iron-ascorbate-tBHP oxidation of erythrocyte membranes as determined by the sustained ATPase activities and the reduced formation of malondialdehyde. Maximal protection was provided by 1 mmol/L desferal in the presence of 1 mmol/L ascorbate, although some protection was observed even at 10 mumol/L, the lowest concentration tested. These results are discussed in light of the pro- and anti-oxidant effects of ascorbate in the absence and presence of iron and tBHP.
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PMID:Increased susceptibility of the sickle cell membrane Ca2+ + Mg(2+)-ATPase to t-butylhydroperoxide: protective effects of ascorbate and desferal. 153 18

The Kdp system of Escherichia coli, a transport ATPase with high affinity for potassium, is expressed when turgor pressure is low. Expression requires KdpD, a 99-kDa membrane protein, and KdpE, a 25-kDa soluble cytoplasmic protein. The sequences of KdpD and KdpE show they are members of the sensor-effector class of regulatory proteins: the C-terminal half of KdpD is homologous to sensors such as EnvZ and PhoR, and KdpE is homologous to effectors such as OmpR and PhoB. The predicted structure of KdpD suggests that it is anchored to the membrane by four membrane-spanning segments near its middle, with both C- and N-terminal portions in the cytoplasm. Subcellular fractionation confirms the expected location of the protein in the inner membrane. The N-terminal region has no homology to known proteins and is the site of mutations that make Kdp expression partially constitutive; this portion may serve to sense turgor pressure. Since several other sensor-effectors have been shown to mediate control through phosphorylation, this mechanism is proposed to control expression of Kdp.
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PMID:KdpD and KdpE, proteins that control expression of the kdpABC operon, are members of the two-component sensor-effector class of regulators. 153 88

The fluorescence decay of the plasma membrane calmodulin-activated Ca2(+)-ATPase from the erythrocyte was measured for the first time. The availability of a novel procedure for on-line blank subtraction in frequency-domain lifetime data acquisition (G.G. Reinhart, B. Feddersen, D. Jameson and E. Gratton, Biophys. J. 57 (1990) 189a) permitted the elimination of background interference from detergent-solubilized purified plasma membrane ATPase samples. The fluorescence decay of the erythrocyte Ca2(+)-ATPase was measured in the absence of Ca2+, or in the presence of Ca2+ or Ca2+ plus calmodulin. In the three different experimental conditions the fluorescence decay was very heterogeneous and could be best described by Lorentzian distributions of lifetime values. In the absence of Ca2+ the decay was described by a broad lifetime distribution centered at 4.4 ns with a width of 3.2 ns, indicating heterogeneity of tryptophan microenvironments in the ATPase. Calcium ion binding promoted an 11% increase in the center and a 27% decrease in the width of the distribution. By contrast, addition of calmodulin in the presence of Ca2+ caused a 15% decrease in the center of the distribution, revealing structural difference between calmodulin-activated and Ca2(+)-activated states of the ATPase. These results indicate the usefulness of on-line blank subtraction in frequency-domain lifetime measurements to investigate conformational changes in detergent-solubilized membrane protein samples.
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PMID:Time-resolved fluorescence of erythrocyte plasma membrane Ca2(+)-ATPase in different functional states. 153 98

In obesity, impaired glucose tolerance (IGT), non-insulin-dependent diabetes mellitus (NIDDM), and gestational diabetes mellitus (GDM), defects in glucose transport system activity, contribute to insulin resistance in target tissues. In adipocytes from obese and NIDDM patients, we found that pretranslational suppression of the insulin-responsive GLUT4 glucose transporter isoform is a major cause of cellular insulin resistance; however, whether this process is operative in skeletal muscle is not clear. To address this issue, we performed percutaneous biopsies of the vastus lateralis in lean and obese control subjects and in obese patients with IGT and NIDDM and open biopsies of the rectus abdominis at cesarian section in lean and obese gravidas and gravidas with GDM. GLUT4 was measured in total postnuclear membrane fractions from both muscles by immunoblot analyses. The maximally insulin-stimulated rate of in vivo glucose disposal, assessed with euglycemic glucose clamps, decreased 26% in obesity and 74% in NIDDM, reflecting diminished glucose uptake by muscle. However, in vastus lateralis, relative amounts of GLUT4 per milligram membrane protein were similar (NS) among lean (1.0 +/- 0.2) and obese (1.5 +/- 0.3) subjects and patients with IGT (1.4 +/- 0.2) and NIDDM (1.2 +/- 0.2). GLUT4 content was also unchanged when levels were normalized per wet weight, per total protein, and per DNA as an index of cell number. Levels of GLUT4 mRNA were similarly not affected by obesity, IGT, or NIDDM whether normalized per RNA or for the amount of an unrelated constitutive mRNA species. Because muscle fibers (types I and II) exhibit different capacities for insulin-mediated glucose uptake, we tested whether a change in fiber composition could cause insulin resistance without altering overall levels of GLUT4. However, we found that quantities of fiber-specific isoenzymes (phopholamban and types I and II Ca(2+)-ATPase) were similar in all subject groups. In rectus abdominis, GLUT4 content was similar in the lean, obese, and GDM gravidas whether normalized per milligram membrane protein (relative levels were 1.0 +/- 0.2, 1.3 +/- 0.1, and 1.0 +/- 0.2, respectively) or per wet weight, total protein, and DNA. We conclude that in human disease states characterized by insulin resistance, i.e., obesity, IGT, NIDDM, and GDM, GLUT4 gene expression is normal in vastus lateralis or rectus abdominis. To the extent that these muscles are representative of total muscle mass, insulin resistance in skeletal muscle may involve impaired GLUT4 function or translocation and not transporter depletion as observed in adipose tissue.
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PMID:Gene expression of GLUT4 in skeletal muscle from insulin-resistant patients with obesity, IGT, GDM, and NIDDM. 153 55

The ars operon of the conjugative R-factor R773 encodes an oxyanion pump that catalyzes extrusion of arsenicals from cells of Escherichia coli. The oxyanion translocation ATPase is composed of two polypeptides, the catalytic ArsA protein and the intrinsic membrane protein, ArsB. The topology of regions of the ArsB protein in the inner membrane was determined using a variety of gene fusions. Random gene fusions with lacZ and phoA were generated using transposon mutagenesis. A series of gene fusions with blaM were constructed in vitro using a beta-lactamase fusion vector. To localize individual segments of the ArsB protein, a ternary fusion method was developed, where portions of the arsB gene were inserted in-frame between the coding regions for two heterologous proteins, in this case a portion of a newly identified arsD gene and the blaM sequence encoding the mature beta-lactamase. The location of a periplasmic loop was determined from V8 protease digestion of an ArsA-ArsB chimera. From analysis of data from 26 fusions, a topological model of the ArsB protein with 12 membrane-spanning regions is proposed.
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PMID:Membrane topology of the ArsB protein, the membrane subunit of an anion-translocating ATPase. 153 22

Acylphosphatase, purified from human erythrocytes, actively hydrolyzes the acylphosphorylated intermediate of human red blood cell membrane Ca(2+)-ATPase. This effect occurred with acylphosphatase amounts (up to 10 units/mg membrane protein) that fall within the physiological range. Furthermore, a very low Km value, 3.41 +/- 1.16 (S.E.) nM, suggests a high affinity in acylphosphatase for the phosphoenzyme intermediate, which is consistent with the small number of Ca(2+)-ATPase units in human erythrocyte membrane. Acylphosphatase addition to red cell membranes resulted in a significant increase in the rate of ATP hydrolysis. Maximal stimulation (about 2-fold over basal) was obtained at 2 units/mg membrane protein, with a concomitant decrease in apparent Km values for both Ca2+ and ATP. Conversely, similar amounts of acylphosphatase significantly decreased (by about 30%) the rate of Ca2+ transport into inside-out red cell membrane vesicles, albeit that reduced apparent Km values for Ca2+ and ATP were also observed in this case. A stoichiometry of 2.04 Ca2+/ATP hydrolyzed was calculated in the absence of acylphosphatase; in the presence of acylphosphatase optimal concentration, this ratio was reduced to 0.9. Acylphosphatase activity, rather than just protein, was essential for all the above effects. Taken together these findings suggest that, because of its hydrolytic activity on the phosphoenzyme intermediate, acylphosphatase reduces the efficiency of the erythrocyte membrane Ca2+ pump. A possible mechanism for this effect is that the phosphoenzyme is hydrolyzed before its transport work can be accomplished.
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PMID:Effects of acylphosphatase on the activity of erythrocyte membrane Ca2+ pump. 164 13

Basement membrane proteins such as the heparan sulfate proteoglycan (HSPG) are secreted in a polarized fashion from the basolateral membrane of epithelial cells. We have used the microtubule-disrupting drug colchicine to study the role of the microtubule network in directing constitutive secretion to the basolateral membrane of LLC-PK1 renal epithelial cells. Microtubule depolymerization induced by colchicine resulted in fragmentation and redistribution of fluorescently labeled trans-Golgi membranes. Increased immunofluorescent staining of HSPG was associated with these dispersed Golgi cisternae. The biosynthetic processing of HSPG was not significantly altered by the loss of microtubules or by the dispersal of the Golgi elements. The most striking effect of microtubule disruption was the loss of polarity of HSPG secretion. Immunoprecipitation studies showed that HSPG was secreted from both apical and basolateral surfaces of LLC-PK1 cells treated with colchicine, and a similar result was found for the delivery of laminin, another basement membrane protein. In contrast, there was no change in the distribution of an integral basolateral membrane protein, Na(+)-K(+)-ATPase, following colchicine treatment. Our results provide the first demonstration that microtubules are involved in the constitutive trafficking of basolateral secretory proteins. These data also suggest that there may be an inherent difference in the targeting or delivery of membrane and secretory proteins to the basolateral cell surface.
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PMID:Disruption of microtubules alters polarity of basement membrane proteoglycan secretion in epithelial cells. 165 Jan 39


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