scholarly journals Identity and origin of the ATPase activity associated with neuronal microtubules. II. Identification of a 50,000-dalton polypeptide with ATPase activity similar to F-1 ATPase from mitochondria.

1983 ◽  
Vol 96 (5) ◽  
pp. 1306-1315 ◽  
Author(s):  
D B Murphy ◽  
K T Wallis ◽  
R R Hiebsch
Keyword(s):  
Atpase Activity ◽  
Specific Activity ◽  
Gel Filtration ◽  
Membrane Vesicles ◽  
Cross Reactivity ◽  
Mitochondrial Atpase ◽  
Atpase Inhibitors ◽  
Native Gel ◽  
High Molecular Weight Proteins

We determined that the ATPase activity contained in preparations of neuronal microtubules is associated with a 50,000-dalton polypeptide by four different methods: (a) photoaffinity labeling of the pelletable ATPase fraction with [gamma-32P]-8-azido-ATP; (b) analysis of two-dimensional gels (native gel X SDS slab gel) of an ATPase fraction solubilized by treatment with dichloromethane; (c) ATPase purification by glycerol gradient sedimentation and gel filtration chromatography of a solvent-released ATPase fraction, (d) demonstration of the binding of affinity-purified antibody to the 50-kdalton polypeptide to ATPase activity in vitro. Beginning with preparations of microtubules we have purified the ATPase activity greater than 700-fold and estimate that the purified enzyme has a specific activity of 20 mumol Pi x mg-1 x min-1 and comprises 80-90% of the total ATPase activity associated with neuronal microtubules. With affinity-purified antibody we also demonstrate cross-reactivity to the 50-kdalton subunits of mitochondrial F-1 ATPase and show that the antibody specifically labels mitochondria in PtK-2 cells. Biochemical comparisons of the enzymes reveal similar but not identical subunit composition and sensitivity to mitochondrial ATPase inhibitors. These studies indicate that the principal ATPase activity associated with microtubules is not contained in high molecular weight proteins such as dynein or MAPs and support the hypothesis that the 50-kdalton ATPase is a membrane protein and may be derived from mitochondria or membrane vesicles with F-1-like ATPase activity.

scholarly journals Mitochondrial adenosine triphosphatase of the fission yeast, Schizosaccharomyces pombe 972h-. Changes in activity and inhibitor-sensitivity in response to catabolite repression

1976 ◽  
Vol 160 (2) ◽  
pp. 335-342 ◽  
Author(s):  
D Lloyd ◽  
S W Edwards
Keyword(s):  
Atpase Activity ◽  
Schizosaccharomyces Pombe ◽  
Adenosine Triphosphatase ◽  
Specific Activity ◽  
Early Stage ◽  
Gel Filtration ◽  
Growth Cycle ◽  
Mitochondrial Atpase ◽  
Submitochondrial Particles ◽  
Fourfold Increase

1. The specific activity of mitochondrial ATPase (adenosine triphosphatase) in extracts of Schizosaccharomyces pombe decreased 2.5-fold as the glucose concentration in the growth medium decreased from 50mM to 15mM. 2. During the late exponential phase of growth, ATPase activity doubled. 3. Sensitivity to oligomycin and Dio-9 as measured by values for I50(mug of inhibitor/mg of protein giving 50% inhibition) at pH 6.8 increased sixfold and ninefold respectively during the initial decrease in ATPase activity, and this degree of sensitivity was maintained for the remainder of the growth cycle. 4. Increased sensitivity to NN′-dicyclohexylcarbodi-imide, triethyltin and venturicidin was also observed during the early stage of glucose de-repression. 5. Smaller increases in sensitivity to efrapeptin, aurovertin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diaz-le, quercetin and spegazzinine also occurred. 6. The ATPase of glycerol-grown cells was less sensitive to inhibitors than that of glucose-repressed cells; change in values for I50 were not so marked during the growth cycle of cells growing with glycerol. 7. When submitochondrial particles from glycerol-grown cells were tested by passage through Sephadex G-50, a fourfold increase in activity was accompanied by increased inhibitor resistance. 8. Gel filtration of submitochondrial particles from glucose-de-repressed cells gave similar results, whereas loss of ATPase occurred in submitochondrial particles from glucose-repressed cells. 9. It is proposed that alterations in sensitivity to inhibitors at different stages of glucose derepression may be partly controlled by a naturally occuring inhibitor of ATPase. 10. The inhibitors tested may be classififed into two groups on the basis of alterations of sensitivity of the ATPase during physiological modification: (a) oligomycin, Dio-9, NN′-dicyclohexylcarbodi-imide, venturicidin and triethyltin, and (b) efrapeptin, aurovertin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, quercetin and spegazzinine.

scholarly journals Identity and Origin of the ATPase activity associated with neuronal microtubules. I. The ATPase activity is associated with membrane vesicles.

1983 ◽  
Vol 96 (5) ◽  
pp. 1298-1305 ◽  
Author(s):  
D B Murphy ◽  
R R Hiebsch ◽  
K T Wallis
Keyword(s):  
Molecular Weight ◽  
Atpase Activity ◽  
High Molecular Weight ◽  
Brain Tissue ◽  
Membrane Vesicles ◽  
Microtubule Associated Proteins ◽  
In Vitro Assembly ◽  
Microtubule Protein ◽  
Associated Proteins

Microtubule protein purified from brain tissue by cycles of in vitro assembly-disassembly contains ATPase activity that has been postulated to be associated with microtubule-associated proteins (MAPs) and therefore significant for studies of microtubule-dependent motility. In this paper we demonstrate that greater than 90% of the ATPase activity is particulate in nature and may be derived from contaminating membrane vesicles. We also show that the MAPs (MAP-1, MAP-2, and tau factors) and other high molecular weight polypeptides do not contain significant amounts of ATPase activity. These findings do not support the concept of "brain dynein" or of MAPs with ATPase activity.

Setaria cervidual specific phosphatase: characterization and its effect on eosinophil degranulation

Parasitology ◽  
2009 ◽  
Vol 136 (8) ◽  
pp. 895-904 ◽  
Author(s):  
S. RATHAUR ◽  
R. RAI ◽  
E. SRIKANTH ◽  
S. SRIVASTAVA
Keyword(s):  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Gel Filtration ◽  
Specific Protein ◽  
Enzyme Concentration ◽  
Cross Reactivity ◽  
Filarial Parasite ◽  
Significant Activity ◽  
Protein Tyrosine

SUMMARYSetaria cervi, a bovine filarial parasite contains significant acid phosphatase (AcP) activity in its various life stages. Two forms of AcP were separated from somatic extract of adult female parasite using cation exchange, gel filtration and concavalin affinity chromatography. One form having a molecular mass of 79 kDa was characterized as dual specific protein tyrosine phosphatase (ScDSP) based on substrate specificity and inhibition studies. With various substrates tested, it showed significant activity in the order of phospho-L-tyrosine>pNPP>ADP>phospho-L-serine. Inhibition by orthovanadate, fluoride, molybdate, and zinc ions further confirms protein tyrosine phosphatase nature of the enzyme. Km and Vmax determined with various substrates were found to be 16·66 mM, 25·0 μM/ml/min with pNPP; 20·0 mM, 40·0 μM/ml/min with phospho-L-tyrosine and 27·0 mM, 25·0 μM/ml/min with phospho-L-serine. KIwith pNPP and sodium orthovanadate (IC5033·0 μM) was calculated to be 50·0 mM. Inhibition with pHMB, silver nitrate, DEPC and EDAC suggested the presence of cysteine, histidine and carboxylate residues at its active site. Cross-reactivity withW. bancrofti-infected sera was demonstrated by Western blotting. ScDSP showed elevated levels of IgE in chronic filarial sera using ELISA. Underin vitroconditions, ScDSP resulted in increased effector function of human eosinophils when stimulated by IgG, which showed a further decrease with increasing enzyme concentration. Results presented here suggest thatS. cerviDSP should be further studied to determine its role in pathogenesis and the persistence of filarial parasite.

scholarly journals 131 PEROXIDATIVE CHALLENGE OF PLASMA MEMBRANE VESICLES FROM BELL PEPPER FRUIT LEADS TO DECREASED H+ATPase ACTIVITY

HortScience ◽  
1994 ◽  
Vol 29 (5) ◽  
pp. 447c-447
Author(s):  
Darlene M. Cowart ◽  
Robert L. Shewfelt
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Fruits And Vegetables ◽  
Chilling Injury ◽  
Membrane Vesicles ◽  
Thiobarbituric Acid ◽  
Plasma Membrane Vesicles ◽  
Water Control ◽  
Solubilized Enzyme

-Lipid peroxidation has been proposed as an important factor in chilling injury of susceptible fruits and vegetables. The effect of in vitro peroxidative challenge on H+ATPase activity in intact plasma membrane vesicles and solubilized enzyme was determined by incubation with (1) deionized water (control), (2) Fe3+-ascorbate, and (3) lipoxygenase (LOX) + phospholipase A2(PLA2) for 0, 30, and 60 min. Enzyme activity increased throughout the incubation period with no accumulation of thiobarbituric acid-reactive substances (TBA-RS) in the control, but vesicles challenged by the peroxidative systems showed significant increases in TBA-RS and decreases in membrane-bound H+ATPase activity. Greater losses in H+ATPase activity were observed in solubilized enzyme than in intact vesicles. The results indicate that loss of H+ATPase activity due to chemical modification of the protein rather than changes in membrane fluidity and suggest that modification is away from the active site.

scholarly journals A V-ATPase drives active, electrogenic and Na+-independent Cl- absorption across the gills of Eriocheir sinensis

1995 ◽  
Vol 198 (3) ◽  
pp. 767-774 ◽  
Author(s):  
H Onken ◽  
M Putzenlechner
Keyword(s):  
Atpase Activity ◽  
Specific Activity ◽  
Short Circuit ◽  
Specific Inhibitor ◽  
Membrane Vesicles ◽  
Short Circuit Current ◽  
Eriocheir Sinensis ◽  
Similar Distribution ◽  
Fluorescence Studies ◽  
F1fo Atpase

Using biochemical and electrophysiological techniques, we have examined the proposal that an H+-ATPase is involved in Cl- uptake across the gills of the Chinese crab Eriocheir sinensis. Bafilomycin A1 (1 µmol l-1), a specific inhibitor of V-ATPases, was used to investigate the importance of this H+-translocating enzyme in Cl- transport across the gill. In homogenates of ion-transporting posterior gills, we found the activity of a bafilomycin-sensitive V-ATPase to be markedly higher than in the anterior gills, which are not involved in ion transport. A similar distribution was found for the Na+/K+- and the mitochondrial F1Fo-ATPase. After differential and density centrifugation, the specific activity of the V-ATPase was enriched by a factor of 5. Neither Na+/K+- and F1Fo-ATPase activities nor acid phosphatase activity copurified with the bafilomycin-sensitive ATPase activity, indicating that at least the major portion of V-ATPase activity is not of basolateral, mitochondrial or lysosomal origin. In fluorescence studies, using Acridine Orange or Oxonol V as dyes, membrane vesicles displayed ATP-dependent proton transport and membrane potential generation, which were markedly reduced in the presence of bafilomycin. In addition to these biochemical studies, we mounted split lamellae of posterior gills in an Ussing-type chamber and measured the negative short-circuit current (Isc), which was shown to reflect active, electrogenic, Na+-independent and ouabain-insensitive Cl- absorption. After the addition of 1 µmol l-1 bafilomycin to the external bath, this Isc was reduced to about 50­60 % of its original value. Concomitantly, the conductance of the preparation decreased by about 13 %. From these results, we conclude that an apical V-ATPase drives electrogenic Cl- uptake across the posterior gills of the Chinese crab.

scholarly journals Potassium uptake and release by human blood platelets

Blood ◽  
1976 ◽  
Vol 48 (2) ◽  
pp. 185-197
Author(s):  
JS Wiley ◽  
J Kuchibhotla ◽  
CC Shaller ◽  
RW Colman
Keyword(s):  
Time Course ◽  
Specific Activity ◽  
Blood Platelets ◽  
Gel Filtration ◽  
Lactic Dehydrogenase ◽  
Human Platelets ◽  
First Order Kinetics ◽  
A Minor ◽  
Human Blood Platelets

Thrombin is known to reduce the K+ content of human platelets, but the subcellular origin of the lost K+ is not known. The effect of aggregating agents on K+ release was studied in platelets labeled in plasma by preincubation with 42KCI. Platelets were separated from plasma by gel filtration through Sepharose 2B equilibrated with K+ - free Tyrode's buffer. Platelet K+ was 116nEq/10(8) platelets, of which 23% was found to be extracellular immediately after gel filtration. K+ influx was 65 nEq/10(8) platelets/hr at pH 7.5 and was more rapid at pH 7.9. About 70% of cell K+ exchanged with plasma in 4 hr with first- order kinetics, while a minor fraction of about 30% exchanged with a slower time course. This slowly exchanging fraction of platelet K+ was thought to arise from heterogeneity in the platelet population. Epinephrine and ADP aggregated gel-filtered platelets and released serotonin, but with loss of only 5%-10% of cell K+ and no beta- glucuronidase. In contrast, thrombin released up to 30% of platelet K+, whether aggregation occurred or was prevented by not stirring the cells. The specific activity of K+ released by all aggregating agents was identical to the specific activity of total platelet K+. Thrombin (0.01–0.2 NIH U/ml) released serotonin and also beta-glucuronidase (an enzyme of the alpha-granule), and there was a linear relation between release of K+ and this enzyme (r = 0.88). No lysis of platelets occurred, since lactic dehydrogenase was not detected. Pretreatment of platelets with aspirin in vitro inhibited thrombin-induced release of serotonin but had no effect on the loss of K+ or beta-glucuronidase. In contrast, the ingestion of aspirin by mouth inhibited the release of serotonin, beta-glucuronidase, and K+ by thrombin. The data suggested that the K+ loss induced by thrombin was primarily derived from release of alpha-granules and that these organelles contained about 20% of the total platelet K+ in a freely exchangeable and nonsequestered state.

Isoform-independent heart rate-related variation in cardiac myofibrillar Ca(2+)-activated Mg(2+)-ATPase activity

1996 ◽  
Vol 270 (5) ◽  
pp. C1271-C1276 ◽  
Author(s):  
W. Rouslin ◽  
C. W. Broge
Keyword(s):  
Heart Rate ◽  
Beef Cattle ◽  
Atpase Activity ◽  
Specific Activity ◽  
Myosin Atpase ◽  
Curvilinear Relationship ◽  
Mitochondrial Atpase ◽  
Myofibrillar Atpase ◽  
Cardiac Myosin ◽  
Myofibrillar Atpase Activity

In the present study, we compared the activities of the cardiac myofibrillar Ca(2+)-activated Mg(2+)-ATPase and the content of cardiac muscle mitochondrial ATPase inhibitor protein (IF1) of several mammalian species covering broad ranges of body mass and heart rate, i.e., from beef cattle to mouse. The cardiac myofibrillar ATPase from each species was assayed over a range of pCa values at pH 7.4. While the cardiac myofibrillar ATPase from all species examined showed essentially identical Ca2+ concentration dependencies with the ATPase in each species activating steeply between pCa 6.5 and 5.5, the maximal ATPase specific activity reached varied considerably from species to species, and this variation was largely independent of the predominant cardiac myosin ATPase isoform present. Thus, while adult beef cattle, pig, dog, and rabbit all contain predominantly the slow cardiac myosin ATPase isoform the cardiac myofibrillar ATPase specific activities of these four species varied over approximately a fourfold range. Moreover, there was a fairly smooth curvilinear relationship between maximum Ca(2+)-activated myofibrillar ATPase activity and median conscious heart rate for the slow cardiac myosin ATPase-possessing species examined. This smooth continuum also extended to include two species possessing the fast cardiac myosin ATPase isoform, rat and mouse. This relationship between myofibrillar ATPase activity and heart rate that appears to be applicable to a broad range of species suggests that the myofibrillar ATPase is specifically modeled or fine-tuned to the kinetic (heart rate) demand of each species and, within slow and fast heart rate ranges, is essentially independent of myosin ATPase isoform per se. Only hearts containing predominantly the slow myosin ATPase isoform contained functional levels of IF1. Finally, while it has been reported that the ratio of myosin Ca(2+)-ATPase to actomyosin Mg(2+)-ATPase activity is a good index of the percent of the fast myosin ATPase in rabbit myofibrillar preparations, we found that this relationship may be applicable to only some species.

scholarly journals Ca2+-Dependent Interaction of S100A1 with F1-ATPase Leads to an Increased ATP Content in Cardiomyocytes

2007 ◽  
Vol 27 (12) ◽  
pp. 4365-4373 ◽  
Author(s):  
Melanie Boerries ◽  
Patrick Most ◽  
Jonathan R. Gledhill ◽  
John E. Walker ◽  
Hugo A. Katus ◽  
...  
Keyword(s):  
Energy Metabolism ◽  
Atpase Activity ◽  
Cardiac Contractility ◽  
Gel Filtration ◽  
Dependent Manner ◽  
Atp Content ◽  
Atp Production ◽  
Rat Hearts

ABSTRACT S100A1, a Ca2+-sensing protein of the EF-hand family that is expressed predominantly in cardiac muscle, plays a pivotal role in cardiac contractility in vitro and in vivo. It has recently been demonstrated that by restoring Ca2+ homeostasis, S100A1 was able to rescue contractile dysfunction in failing rat hearts. Myocardial contractility is regulated not only by Ca2+ homeostasis but also by energy metabolism, in particular the production of ATP. Here, we report a novel interaction of S100A1 with mitochondrial F1-ATPase, which affects F1-ATPase activity and cellular ATP production. In particular, cardiomyocytes that overexpress S100A1 exhibited a higher ATP content than control cells, whereas knockdown of S100A1 expression decreased ATP levels. In pull-down experiments, we identified the α- and β-chain of F1-ATPase to interact with S100A1 in a Ca2+-dependent manner. The interaction was confirmed by colocalization studies of S100A1 and F1-ATPase and the analysis of the S100A1-F1-ATPase complex by gel filtration chromatography. The functional impact of this association is highlighted by an S100A1-mediated increase of F1-ATPase activity. Consistently, ATP synthase activity is reduced in cardiomyocytes from S100A1 knockout mice. Our data indicate that S100A1 might play a key role in cardiac energy metabolism.

Stimulation-associated redistribution of H+-K+-ATPase activity in isolated gastric glands

1987 ◽  
Vol 252 (4) ◽  
pp. G458-G465 ◽  
Author(s):  
T. Urushidani ◽  
J. G. Forte
Keyword(s):  
Atpase Activity ◽  
Membrane Vesicles ◽  
Apical Plasma Membrane ◽  
Plasma Membrane Vesicles ◽  
Gastric Glands ◽  
Transport Pathways ◽  
Isolated Gastric Glands ◽  
Kcl Transport

The objective of this work is to establish a procedure to study the stimulation-dependent membrane redistribution and properties of H+-K+-ATPase in an in vitro model system, rabbit isolated gastric glands. Stimulated (10(-4) M histamine plus 10(-5) M forskolin) and resting (10(-4) M metiamide) glands were homogenized and fractionated into PO (40 g, 5 min), P1 (400 g, 10 min), P2 (14,500 g, 10 min), P3 (48,200 g, 90 min), and supernatant, S3. Significant changes occurred in the distribution of our marker for H+-K+-ATPase (K+-p-nitrophenyl phosphatase) activity: a reduction in activity of P3 and a compensatory increment in P1. P3 showed valinomycin (Val)-dependent vesicular H+ uptake, while H+ uptake in P1 was Val independent. Direct measurements of ATPase revealed that H+-K+-ATPase activity of P3 was Val dependent and decreased by stimulation; H+-K+-ATPase activity of P1 was Val independent and increased by stimulation. Further density gradient purification of P1 showed that membranes lighter than 17% Ficoll contained higher specific H+-K+-ATPase activity, and the observed increase in H+-K+-ATPase associated with stimulation was more pronounced. Also, the lighter fractions from stimulated P1 had much latent H+-K+-ATPase activity that was unmasked by n-octylglucoside. The properties of membrane fractions from isolated glands were consistent with results obtained in vivo: high H+-K+-ATPase activity of P3 from resting glands corresponds to cytoplasmic tubulovesicles lacking KCl transport pathways; high activity of P1 from stimulated glands corresponds to apical plasma membrane vesicles containing KCl transport in addition to the H+-K+-ATPase, and full competency for the generation of HCl.

scholarly journals Potassium uptake and release by human blood platelets

Blood ◽  
1976 ◽  
Vol 48 (2) ◽  
pp. 185-197 ◽  
Author(s):  
JS Wiley ◽  
J Kuchibhotla ◽  
CC Shaller ◽  
RW Colman
Keyword(s):  
Time Course ◽  
Specific Activity ◽  
Blood Platelets ◽  
Gel Filtration ◽  
Lactic Dehydrogenase ◽  
Human Platelets ◽  
First Order Kinetics ◽  
A Minor ◽  
Human Blood Platelets

Abstract Thrombin is known to reduce the K+ content of human platelets, but the subcellular origin of the lost K+ is not known. The effect of aggregating agents on K+ release was studied in platelets labeled in plasma by preincubation with 42KCI. Platelets were separated from plasma by gel filtration through Sepharose 2B equilibrated with K+ - free Tyrode's buffer. Platelet K+ was 116nEq/10(8) platelets, of which 23% was found to be extracellular immediately after gel filtration. K+ influx was 65 nEq/10(8) platelets/hr at pH 7.5 and was more rapid at pH 7.9. About 70% of cell K+ exchanged with plasma in 4 hr with first- order kinetics, while a minor fraction of about 30% exchanged with a slower time course. This slowly exchanging fraction of platelet K+ was thought to arise from heterogeneity in the platelet population. Epinephrine and ADP aggregated gel-filtered platelets and released serotonin, but with loss of only 5%-10% of cell K+ and no beta- glucuronidase. In contrast, thrombin released up to 30% of platelet K+, whether aggregation occurred or was prevented by not stirring the cells. The specific activity of K+ released by all aggregating agents was identical to the specific activity of total platelet K+. Thrombin (0.01–0.2 NIH U/ml) released serotonin and also beta-glucuronidase (an enzyme of the alpha-granule), and there was a linear relation between release of K+ and this enzyme (r = 0.88). No lysis of platelets occurred, since lactic dehydrogenase was not detected. Pretreatment of platelets with aspirin in vitro inhibited thrombin-induced release of serotonin but had no effect on the loss of K+ or beta-glucuronidase. In contrast, the ingestion of aspirin by mouth inhibited the release of serotonin, beta-glucuronidase, and K+ by thrombin. The data suggested that the K+ loss induced by thrombin was primarily derived from release of alpha-granules and that these organelles contained about 20% of the total platelet K+ in a freely exchangeable and nonsequestered state.

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