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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)

Na, K-ATPase has been analysed by electron microscopy to obtain information about the structure of the enzyme and its organization within the membrane. Following negative staining the membrane-bound enzyme was observed as surface particles which on the basis of their size and frequency and the enzymatic and chemical composition of the membranes are interpreted as protomers (alpha beta-units). Freeze-fracture electron microscopy revealed the enzyme as intramembrane particles. Quantitative electron microscope studies suggested that the intramembrane particles are oligomers of the protein unit that forms the surface particles. Following reconstitution of the enzyme into phospholipid vesicles it was demonstrated that similar intramembrane particles represent a protein unit which transports sodium and potassium. Vanadate and magnesium induced the formation of two-dimensional crystals in the membrane fragments of the purified Na, K-ATPase. Further information regarding the shape and dimensions of the protomer was obtained through analysis of electron micrographs of negatively stained crystals with optical diffraction and image reconstruction methods.
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PMID:Ultrastructure of the Na, K-ion pump. 631 Aug 27

Rabbit renal (Na+ + K+)-ATPase (EC 3.6.1.3) was purified and incorporated into phosphatidylcholine liposomes. Freeze-fracture analysis of the reconstituted system reveals intramembrane particles formed by (Na+ + K+)-ATPase molecules which are randomly distributed on concave and convex fracture faces. The reconstituted (Na+ + K+)-ATPase performs active Na+,K+-transport. The distribution of particles as well as the rate of active transport are directly proportional to the (Na+ + K+)-ATPase protein concentration used for reconstitution, while the total amount of sodium and potassium ions exchanged by ATP per volume vesicle suspension reaches maximum when each vesicle contains on the average more than two particles. (Na+ + K+)-ATPase pretreated with ouabain or vanadate yields the same particle density and vesicle size as control enzyme. However, detergent-denatured enzyme loses its ability to form intramembrane particles or to increase the vesicle size indicating that the lipids surrounding the protein part of the molecule are essential for the reconstitution process. The vesicle diameter increases as a function of the number of particles per vesicle. Histograms of the size distribution become wider with increasing intramembrane particle density and tend to show more than one maximum.
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PMID:Characterization of (Na+ + K+)-ATPase liposomes. I. Effect of enzyme concentration and modification on liposome size, intramembrane particle formation and Na+,K+-transport. 632 84

Negative staining of purified spinach dicyclohexylcarbodiimide (DCCD) sensitive ATPase revealed a population of 110 A subunits attached by stalks to short string-like aggregates. The interpretation of these data is that 110 A CF1 are attached by stalks to an aggregate of CF0. The CF1-CF0 complex was incorporated into phospholipid vesicles; freeze-fracture analysis of this preparation revealed a homogeneous population of particles spanning the lipid bilayer; those averaged 96 A in diameter. The DCCD binding proteolipid (apparent molecular weight 7500), an integral component of CF0, was isolated from membranes by butanol extraction and was incorporated into phospholipid vesicles. Freeze-fracture analysis of the DCCD-binding proteolipid/vesicle preparation revealed a population of particles averaging 83 A in diameter suggesting that the DCCD-binding proteolipid self-associates in lipid to form a stable complex. This complex may be required for proton transport across chloroplast membranes in vivo. The size difference between CF0 and DCCD-proteolipid freeze-fracture particles may be related to differences in polypeptide composition of the two complexes.
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PMID:Structural analysis of the isolated chloroplast coupling factor and the N,N'-dicyclohexylcarbodiimide binding proteolipid. 645 5

A density gradient centrifugation method for the isolation of the surface membrane complex from Paramecium tetraurelia cells is presented. The resulting "pellicles" consist predominantly of the somatic cell membrane and the underlying alveolar membranes. Marker enzyme activities for other cell components are low and SDS-polyacrylamid-gel electrophoreses indicate the presence of only minor amounts of ciliary and secretory proteins. Pellicles were prepared from different strains: (a) Exocytosis-capable strains with the normal set of exocytotic organelles ("trichocysts") docked to the cell membrane (strains 7S, K 401, and 9-18 degrees C), (b) exocytosis-uncapable strains (although with normal trichocyst attachment: nd 9-27 degrees C, nd 6, nd 7) and (c) strain from tam 38 with empty docking sites and rare, defective, free trichocysts. A Ca2+-stimulated ATPase was present in the pellicles from all strains with Km (CA2+) values between 0.19 to 0.88 mM Ca2+ and Vmax between 286 to 787 nMoles Pi/mg protein/min. Km and Vmax was identical for all strains of group (a). Vmax was significantly lower for all strains of group (b) and still lower for group (c). Similar group differences were found for Km (except for strain nd 6). Freeze-fracture analysis shows that the disruption of the membrane-to-membrane attachments during fractionation is paralleled by the disarrangment of the regular arrays ("rings", "rosettes") of membrane-integrated particles.
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PMID:Isolation of surface membranes from normal and exocytotic mutant strains of Paramecium tetraurelia. Ultrastructural and biochemical characterization. 645 17

An active Ca2+-stimulated, Mg2+-dependent adenosinetriphosphatase (Ca2+-ATPase) isolated from rabbit skeletal muscle sarcoplasmic reticulum membranes has been incorporated into dilauroyl-, dimyristoyl-, dipentadecanoyl-, dipalmitoyl-, and palmitoyloleoylphosphatidylcholine bilayers by using a newly developed lipid-substitution procedure that replaces greater than 99% of the endogenous lipid. Freeze--fracture electron microscopy showed membranous vesicles of homogeneous size with symmetrically disposed fracture-face particles. Diphenylhexatriene fluorescence anisotropy was used to define the recombinant membrane phase behavior and revealed more than one transition in the membranes. Enzymatic analysis indicated that saturated phospholipid acyl chains inhibited both overall ATPase activity and Ca2+-dependent phosphoenzyme formation below the main lipid phase transition temperature (Tm) of the lipid-replaced membranes. At temperatures above Tm, ATPase activity but not phosphoenzyme formation was critically dependent on acyl chain length and thus bilayer thickness. No ATPase activity was observed in dilauroylphosphatidylcholine bilayers. Use of the nonionic detergent dodecyloctaoxyethylene glycol monoether demonstrated that the absence of activity was not due to irreversible inactivation of the enzyme. Increased bilayer thickness resulted in increased levels of activity. An additional 2-fold rise in activity was observed when one of the saturated fatty acids in dipalmitoylphosphatidylcholine was replaced by oleic acid, whose acyl chain has a fully extended length comparable to that of palmitic acid. These results indicate that the Ca2+-ATPase requires for optimal function a "fluid" membrane with a minimal bilayer thickness and containing unsaturated phospholipid acyl chains.
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PMID:Effect of lipid membrane structure on the adenosine 5'-triphosphate hydrolyzing activity of the calcium-stimulated adenosinetriphosphatase of sarcoplasmic reticulum. 645 19

The correlation between structures of chemicals and their inducibility for megamitochondrial formation was investigated. Since the chemical structure universal to the inducers of megamitochondria previously reported (cuprizone and isonicotinic acid derivatives) is the carbazoyl group (-CONHNH2), semicarbazide (NH2NHCONH2) was tested first. Then, hydrazine (NH2NH2) was tested, replacing the carbazoyl group of semicarbazide by an amino group (-NH2). The present study demonstrates that (1) megamitochondria were induced in mouse and rat hepatocytes by feeding the animals with a diet containing semicarbazide or hydrazine, suggesting that the carbazoyl group was not essential for megamitochondrial formation; (2) hydrazine-induced megamitochondrial formation was a reversible process. Coupling efficiencies and activities of ATPase and cytochrome oxidase of megamitochondria induced by hydrazine were slightly decreased, while the activity of monoamine oxidase was moderately decreased. Freeze-fracture electron microscopy revealed particle-free regions in the outer membranes of megamitochondria fixed with glutaraldehyde at 22 degrees C; the regions disappeared at 25 degrees C, indicating that the temperature of the liquid crystalline to gel state lipid phase transition in the megamitochondrial outer membrane was elevated. It is speculated that chemical structure of inducer of megamitochondria could be simplified to NH2-G (G, substituting group).
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PMID:Induction of megamitochondria in the mouse and rat livers by hydrazine. 661 23

Streptococcus mutans, an important aetiological agent of dental caries, is known to transport glucose via the phosphoenolpyruvate (PEP) phosphotransferase system (PTS). An alternative non-PTS glucose transport system in S. mutans Ingbritt was suggested by the increased ATP-dependent phosphorylation of glucose and the presence of higher cellular concentrations of free glucose in cells grown in continuous culture under PTS-repressed conditions compared to those resulting in optimal PTS activity. A method was developed for the preparation of membrane vesicles in order to study this system in the absence of PTS activity. These vesicles had very low activity of the cytoplasmic enzymes, glucokinase, pyruvate kinase and lactate dehydrogenase. This, coupled with the lack of glycolytic activity and the inability to transport glucose, suggested that the vesicles would also be deficient in PTS activity because of the absence of the general soluble PTS proteins, Enzyme I and HPr, required for the transport of all PTS sugars. Freeze-fracture electron microscopy and membrane H(+)-ATPase analysis indicated that over 90% of the vesicles had a right-side-out orientation. Vesicles from cells grown in continuous culture under PTS-dominant and PTS-repressed conditions both exhibited glucose counterflow. This indicates the presence of a constitutive non-PTS carrier in the organism capable of transporting glucose and utilizing ATP for glucose phosphorylation. Analysis of growth yields of cells grown under PTS-repressed and PTS-optimal conditions suggests that ATP, or an equivalent high energy molecule, must be involved in the actual transport process. This analysis is consistent with an ATP-binding protein model such as the Msm transport system reported by R. R. B. Russell and coworkers (J Biol Chem 267, 4631-4637), but it does not exclude the possibility of a separate permease for glucose.
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PMID:Vesicles prepared from Streptococcus mutans demonstrate the presence of a second glucose transport system. 800 May 34

In order to characterize the protein composition of the outer membrane of Borrelia burgdorferi, we have isolated inner and outer membranes by using discontinuous sucrose density step gradients. Outer and inner membrane fractions isolated by this method contained less than 1 and 2%, respectively, of the total lactate dehydrogenase activity (soluble marker) in cell lysate. More importantly, the purified outer membranes contained less than 4% contamination by the C subunit of F1/F0 ATPase (inner membrane marker). Very little flagellin protein was present in the outer membrane sample. This indicated that the outer membranes were relatively free of contamination by cytoplasmic, inner membrane or flagellar components. The outer membrane fractions (rho = 1.19 g/cm3) contained 0.15 mg (dry weight) of protein per mg. Inner membrane samples (rho = 1.12 g/cm3) contained 0.60 mg (dry weight) of protein per mg. Freeze-fracture electron microscopy revealed that the outer membrane vesicles contained about 1,700 intramembranous particles per micron 2 while inner membrane densities for inner and outer membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and nonequilibrium pH gel electrophoresis-SDS-PAGE analyses of inner and outer membrane samples revealed several proteins unique to the inner membrane and 20 proteins that localized specifically to the outer membrane. This analysis clearly shows that the inner and outer membranes isolated by this technique are unique structures.
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PMID:Isolation and partial characterization of Borrelia burgdorferi inner and outer membranes by using isopycnic centrifugation. 800 66

Structure and function of the marginal cell in the stria vascularis were studied by freeze-fracture, cytochemistry and immunohistochemistry with special regard to the ion transport of potassium. Freeze-fracture showed that marginal cells were connected by tight junctions beneath the scala media K(+)-NPPase cytochemistry showed that Na+, K(+)-ATPase was abundant on the basolateral infoldings of the marginal cell. Immunohistochemistry of a rat Isk protein, which has a property of a potassium channel, revealed that the rat Isk protein was localized at the endolymphatic surface of the marginal cell. These findings supported the 'one-pump' theory (Offner et al. Hear Res 1987; 29: 117-24).
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PMID:Morphological aspects of transport of potassium ion in the marginal cell. 838 17

The sensitivies of double-barrelled K(+)-selective micro-electrodes (KSMs) employing the low-impedance membrane cocktail based on the neutral K(+)-selective ion carrier valinomycin (Fluka, Cocktail B 60398) to the following 3 different classes of inhibitors of K+ transport were measured: (1) general metabolic inhibitors (dinitrophenol, potassium cyanide, sodium azide, rotenone, dicyclohexylcarbodiimide, salicylhydroxamic acid); (2) P-type ATPase inhibitors (vanadate, ouabain, amiloride, SCH 28080); and (3) anion-dependent K+ transport inhibitors (bumetanide, 4-acetamide-4-isothiocyanostilbene-2,2-disulphonic acid). Of the 12 inhibitors tested, only dinitrophenol had any significant effect on the response of KSMs to K+ activity. Comparison of the calibrations in solutions with and without 0.1 mM dinitrophenol showed that this inhibitor behaved as a 'classical' interferent whereby its contribution to the K+ activity signal was statistically significant at K+ activities of 36.0 mM and less. However, at higher K+ activities (97.0 mM), dinitrophenol interference was not significant. It was possible to correct for the DNP interference and to obtain measurements of intracellular K+ activity in insect muscles.
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PMID:Sensitivity of valinomycin-based K(+)-selective micro-electrodes to inhibitors of K+ transport. 853 96


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