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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)

When a plasma membrane preparation isolated from rat liver was incubated with [gamma-32P]ATP and Mg2+, protein-bound 32P increased rapidly, followed by a gradual decrease. The time course suggested the existence of membrane-bound kinase(s) and phosphatase(s) phosphorylating and dephosphorylating endogenous proteins. The extent of phosphorylation was not affected by inclusion of cyclic AMP in the reaction mixture. The extent of the maximum phosphorylation was dependent on membrane concentration, owing to rapid hydrolysis of ATP by the membrane-bound ATPase activity. Thus, phosphorylation proceeded further on repeated addition of ATP. Both phosphorylation and dephosphorylation were stimulated by Mg2+, an effective rate of phosphorylation being obtained at 15 mM. Pi up to 20 mM stimulated phosphorylation with little effect on the rate of dephosphorylation. At higher phosphate concentrations, the maximum 32P-incorporation decreased again, and at 100 mM, dephosphorylation was prevented significantly. Autoradiography after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea revealed six main phosphorylated bands, two of which (Band 3 and 5) were partly extractable with 1 M NaCl. In the presence of 100 mM Pi, very strong phosphorylation of Band 5 (about 23,000 daltons) was noted, and a new strongly labeled band (Band P, about 20,000 daltons) was observed. It was concluded that the phosphoproteins in the membrane may be turned over at different rates and high concentrations of Pi may affect the turnover rate of some phosphoproteins, probably through interference with the phosphatase.
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PMID:Protein phosphorylation and dephosphorylation in liver plasma membrane. Effect of inorganic phosphate. 627 66

1. The efflux of Na in dialysed axons of the squid has been used to monitor the sidedness of the interactions of the Na pump with Na(+) ions, K(+) ions and ATP. The axons were under conditions such that most of the Na efflux went through the Na pump by means of a complete cycle of ATP hydrolysis.2. With 310 mm-K(i) (+), 70 mm-Na(i) (+) and 10 mm-K(+) artificial sea water (ASW) more than 97% of the Na efflux was abolished by removal of ATP. The efflux of Na was stimulated by ATP with a K((1/2)) of about 200 mum. This is similar to the K((1/2)) of 150 mum found for the ATP dependence of a ouabain-sensitive Na,K-ATPase activity in membrane fragments isolated from squid optical nerves.3. A 100-fold reduction in the ATP concentration (from 3-5 mm to 30-50 mum) increased the apparent affinity of the Na pump for K(o) (+) about 8-fold. In addition, the maximal rate of K(o) (+)-stimulated Na efflux was reduced by a similar factor. Analogous results were seen in axons dialysed with 310 mm-K(i) (+) or without K(i) (+).4. The relative effectiveness of external monovalent cations as activators of the Na efflux was a function of the ATP concentration inside the axon. With 3-5 mm-ATP the order of effectiveness was K(+) > NH(4) (+) > Rb(+). With 30-50 mum-ATP the sequence was NH(4) (+) >> K(+) >> Rb(+). These results were not affected by the removal of K(i) (+).5. When the ATP concentration was 3 mm and the Na(i) (+) concentration 70 mm, the removal of K(i) (+) produced a slight and reversible increase in the total efflux of Na (15%) and no change in the ATP-dependent Na efflux. When the ATP concentration was reduced to 30-50 mum, or the Na(i) (+) concentration lowered to 5-10 mm, the removal of K(i) (+) reversibly increased the total and the ATP-dependent efflux of Na. The largest increase in Na efflux was seen when both ATP and Na(i) (+) were simultaneously reduced. The ATP-dependent extra Na efflux resulting from the exclusion of K(i) (+) was abolished by 10(-4)m-ouabain in the sea waters.6. The increase in the ATP-dependent Na efflux observed in axons dialysed with 0 K(i) (+) + 10 mm-K(+) ASW was not seen in axons perfused with 310 mm-K(i) (+) + 450 mm-K(+) ASW. However, both experimental conditions gave rise to a similar (and small) ATP-independent and ouabain-insensitive efflux of Na. This indicates that the effects on the Na pump of removing K(i) (+) are not due to the simultaneous membrane depolarization. In addition, it suggests that K(i) (+) has an inhibitory effect on the Na pump, and that that effect is antagonized by Na(i) (+) and ATP.7. The present results are consistent with the idea that the same conformation of the Na pump (and Na,K-ATPase) can be reached by interaction with external K(+) after phosphorylation and with internal K(+) before rephosphorylation. This enzyme conformation produces an enzyme-K complex from which K(+) ions are not easily released unless high concentrations of ATP are present. This also stresses a non-phosphorylating regulatory role of ATP.
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PMID:The effects of ATP on the interactions between monovalent cations and the sodium pump in dialysed squid axons. 627 35

Prostaglandin E2 at concentrations of 5.6 mM and 56 mM, in presence of EDTA 2.2 mM, did not affect the patterns of respiration and coupling, registered by means of polarographic procedure, in isolated rat liver mitochondria. Activation of phosphorylating and uncoupled respiration of mitochondria was noted in incubation mixtures containing EDTA 2.2 microM, Ca2+ 0.2 microM, prostaglandin E2 5.6 microM. In absence of EDTA prostaglandin E2, at concentrations of 5.6 microM and 56 microM, caused uncoupling of the oxidative phosphorylation and in presence of Ca2+--it caused uncoupling and inhibition of mitochondrial respiration, decrease in the rate of phosphorylation and in the ATPase reaction was well as in the efficiency of mitochondrial proton pump, connected with Ca2+ transport. At concentrations 2.8 microM, 5.6 microM, 14 microM protaglandin E2 increased the proton conductivity of the mitochondrial membrane in presence of K+ and valinomycine. The data obtained suggest that the effect of prostaglandin E2 on the energy functions of mitochondria depends on presence of Ca2+ in the incubation mixture.
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PMID:[Effect of prostaglandin E2 on energy metabolism in isolated rat liver mitochondria]. 628 14

Antibodies have been produced, in three rabbits, to Na/K-ATPase purified from guinea pig renal outer medulla. Each rabbit produced antibodies to both the alpha (catalytic) and the beta (glycoprotein) subunits of Na/K-ATPase. The titers of the anti-alpha and anti-beta antibodies varied with time and between rabbits. None of the antisera inhibited Na/K-ATPase activity under various preincubation conditions. A method is presented for separating small amounts of anti-alpha subunit from anti-beta subunit antibodies. There was no cross-reactivity of antibodies to one subunit with the other subunit. The alpha subunit of the Na/K-ATPase was cleaved into a 41,000-dalton peptide (that contains the ATP phosphorylating site) and a 58,000-dalton hydrophobic peptide as described by Castro and Farley (Castro, J., Farley, R.A., 1979, J. Biol. Chem. 254: 2221-2228). Anti-alpha antibodies from all of the rabbits reacted with both proteolytic fragments. The anti-guinea pig Na/K-ATPase antisera (pooled) cross-reacted with the alpha subunit of Na/K-ATPase from human, cow, dog, rabbit, rat, mouse, turtle, and toad; and with the beta subunit from human, rat, and mouse. The loci of cross-reactivity were investigated using partially purified canine kidney Na/K-ATPase cleaved with trypsin as described above. The anti-sera from rabbits 1 and 2 cross-reacted with the 41,000-dalton peptide from the dog but very little with the 58,000-dalton peptide. No cross-reactivity was observed with antiserum from rabbit 3 to either fragment. Guinea pig kidney RNA was translated in a rabbit reticulocyte lysate system followed by immunoprecipitation with the antisera. The molecular weight of the cell-free synthesized alpha chain was 96,000 daltons. Its identity was established with purified anti-alpha antibodies and by immunocompetition with purified Na/K-ATPase and Ca-ATPase. Translation of the beta subunit was not detected in this system.
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PMID:Characteristics of antibodies to guinea pig (Na+ + K+)-adenosine triphosphatase and their use in cell-free synthesis studies. 628 56

The various protein components of a reversible phosphorylating system regulating smooth muscle actomyosin Mg-ATPase activity have been purified. The enzyme catalyzing phosphorylation of smooth muscle myosin, myosin-kinase, requires Ca2+ and the Ca2+-binding protein calmodulin for activity and binds calmodulin in a ratio of 1 mol calmodulin to 1 mol of myosin kinase. Myosin kinase can be phosphorylated by the catalytic subunit of cyclic AMP (cAMP)-dependent protein kinase, and phosphorylation of myosin kinase that does not have calmodulin bound results in a marked decrease in the affinity of this enzyme for Ca2+-calmodulin. This effect is reversed when myosin kinase is dephosphorylated by a phosphatase purified from smooth muscle. When the various components of the smooth muscle myosin phosphorylating-dephosphorylating system are reconstituted, a positive correlation is found between the state of myosin phosphorylation and the actin-activated Mg-ATPase activity of myosin. Unphosphorylated and dephosphorylated myosin cannot be activated by actin, but the phosphorylated and rephosphorylated myosin can be activated by actin. The same relationship between phosphorylation and enzymatic activity was found for a chymotryptic peptide of myosin, smooth muscle heavy meromyosin. The findings reported here suggest one mechanism by which Ca2+ and calmodulin may act to regulate smooth muscle contraction and how cAMP may modulate smooth muscle contractile activity.
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PMID:Regulation of smooth muscle contractile proteins by calmodulin and cyclic AMP. 629 Feb 74

1. The occlusion of rubidium ions by Na, K-ATPase has been investigated by suspending enzyme prepared from pig kidney outer medulla in media containing low concentrations of (86)Rb, forcing the suspensions rapidly through small columns of cation-exchange resin, and measuring the amounts of radioactivity emerging from the columns.2. When the suspension media contained 2 mM-ATP or ADP, or 15 mM-NaCl, the amounts of radioactivity emerging from the columns were greatly (and similarly) reduced, presumably because both nucleotides and sodium ions stabilized the enzyme in the E(1) form. (See p. 19 for definition of E(1) and E(2)). The extra radioactivity carried through the columns when nucleotides and sodium were absent was taken as a measure of the amount of rubidium occluded within the enzyme (in the E(2) form) when it emerged from the resin.3. By varying the flow rate, and therefore the time spent by the enzyme on the resin, and relating this to the amount of radioactivity emerging from the columns, we have been able to estimate the rate constant for the conformational change (E(2) --> E(1)) that allows the occluded rubidium ions to escape. At 20 degrees C, and in the absence of nucleotides, it is about 0.1 S(-1).4. The rate constant for rubidium release was the same in a sodium-containing as in a potassium-containing medium. The opposite effects of sodium and potassium ions on the poise of the equilibrium between the E(1) and the E(2) forms of the enzyme must, therefore, be due solely to opposite effects of these ions on the rate of conversion of E(1) to E(2).5. The rate constant for rubidium release was greatly increased by ATP and by ADP. Both nucleotides appeared to act at low-affinity sites and without phosphorylating the enzyme.6. Orthovanadate, in the presence of magnesium ions, stabilized the enzyme in the occluded-rubidium (E(2)Rb) form.7. Ouabain, in the presence of magnesium ions, prevented the occlusion of rubidium ions.8. We have measured the amount of rubidium occluded by the enzyme as a function of rubidium concentration, and estimate that at saturating rubidium concentrations about three rubidium ions can be occluded per phosphorylation site (or per ouabain-binding site).9. We have found that the occluded-rubidium form of the enzyme can also be formed by allowing rubidium ions to catalyse the hydrolysis of phosphoenzyme generated by the addition of ATP to enzyme suspended in a high-sodium medium.10. The properties of the occluded-rubidium form of the enzyme, and of the two routes that can lead to its formation, suggest that an analagous occluded-potassium form plays a central role in the transport of potassium ions through the sodium-potassium pump. This hypothesis is supported by a detailed consideration of the probable magnitudes of the rate constants of the individual reactions making up the two routes.
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PMID:Occlusion of rubidium ions by the sodium-potassium pump: its implications for the mechanism of potassium transport. 629 86

Transporting epithelia of insects are unlike most vertebrate epithelia in that they lack a serosa and possess septate junctions. The Na+/K+ pump is absent and an electrogenic K+ uniport pump is present in such insect epithelia as lepidopteran midgut, dipteran salivary glands, and many Malpighian tubules. The K+ pump is located in the apical plasma membrane and pumps K+ out of the cells. In midgut the transepithelial K+ transport is against a potential difference (PD) in excess of 120 mV and against a 10-fold K+ concentration difference in vivo. The pump uses a K+-modulated ATPase thought to be located in particles called K+ portasomes, which resemble the F1-F0 ATPase of phosphorylating membranes. Like F1-F0 particles the K+ portasomes are located on the cation input, electronegative, ATP-binding side of the membrane and appear to pump two cations for each MgATP2- hydrolyzed. We propose that in K+-transporting epithelia and in phosphorylating membranes running backwards the portasomes orient the binding of ATP with respect to a cation-gated channel in such a way that when MgATP2- is hydrolyzed P-i is separated from MgADP-; the 2 K+ or 2 H+ ions are no longer neutralized and are repelled from the channel to the opposite side of the membrane. We have isolated the K+ portasome-containing goblet cell apical membrane from larval Manduca sexta midgut and are attempting to isolate the K+ portasomes and K+-ATPase.
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PMID:Chemiosmotic potassium ion pump of insect epithelia. 629 16

K+ transport by the epithelia of midgut, salivary glands, Malpighian tubules, sensory sensilla, possibly rectum, and other organs of certain insects appears to use a unique K+ ATPase. Ouabain inhibition of transport-related events has not been demonstrated in these epithelia. The K+ pump is unlike the Na-K;ump but resembles the H;ump of phosphorylating membranes in its transport orientation, efficient thermodynamics, speculated two K+ per one MgATP2- stoichiometry, electrogenicity, and structure. Older electrochemical, tracer flux, and conductance evidence suggested that the K+ pump was on the apical plasma membrane of transporting cells in these epithelia. New X-ray microanalytical studies (XMA), reveal that the K+ concentration in all cells is more than 100 mM. Together with new microelectrode data these XMA results confirm the apical K+ pump location, resolve the K+ transport sport route, and suggest that the goblet cell cavity facilitates the generation of a large apical PD which may be used in nutrient absorption and pH regulation. K+ portasomes, which resemble F1-Fo ATPase particles, stud these K+ transporting apical membranes and are though to be the unit of active K+ transport. We have suggested a K+ transport mechanism in which two cations (2K+) are abandoned in an isolated domain of the portasomes during ATP2-hydrolysis and are repelled to the opposite membrane side via a K+ channel. Small peptides hydrolysed from the delta-endotoxin of Bacillus thuringiensis inhibit the K+ transport and may be useful as K+ pump inhibitors, apical membrane probes and insecticides. Goblet cell apical membrane fragments (GCAM) as well as fragments from columnar cell apical membrane (CCAM), lateral membrane (LM) and basal membranes (BM) were isolated as clean fractions using ultrasound, aspiration, and both differential and density gradient centrifugation; purification was monitored by electron microscopy. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) reveals that GCAM, CCAM, LM and BM have very different protein compositions. Preliminary enzymology is consistent with the K+ ATPase being on the apical plasma membrane of the goblet cells of midgut and enveloping cells of sensilla.
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PMID:Potassium ion transport ATPase in insect epithelia. 631 92

Acanthamoeba myosin I heavy chain kinase activates the actin-activated Mg2+ -ATPase activity of the Acanthamoeba myosin I isoenzymes, myosins IA and IB, by phosphorylating a single site within the myosin heavy chain. In this paper, we report that myosin I heavy chain kinase also phosphorylates isolated turkey gizzard smooth muscle myosin light chains, gizzard smooth muscle heavy meromyosin, and intact gizzard smooth muscle myosin, all in the absence of Ca2+ and with specific activities close to those measured for purified Ca2+/calmodulin-dependent gizzard smooth muscle myosin light chain kinase. Myosin I heavy chain kinase incorporates a maximum of 2 mol of phosphate/mol of heavy meromyosin, both by itself and together with smooth muscle myosin light chain kinase (the light chain kinase alone incorporates 1.6 mol of phosphate/mol of heavy meromyosin). Both kinases phosphorylate intact smooth muscle myosin to a maximum of 2 mol of phosphate/mol of myosin. Myosin I heavy chain kinase fully activates the actin-activated Mg2+ -ATPase of both myosin and heavy meromyosin. Two-dimensional tryptic peptide maps of isolated light chains phosphorylated by myosin I kinase show the same phosphopeptide as for light chains phosphorylated by the light chain kinase. These results support the conclusion that myosin I heavy chain kinase phosphorylates gizzard smooth muscle myosin at the same site within the 20,000-Da light chain as does smooth muscle myosin light chain kinase. The results suggest that the amino acid sequence around the phosphorylation site within the heavy chain of Acanthamoeba myosin I isoenzymes may be similar to the primary sequence around the phosphorylation site within the smooth muscle myosin light chain.
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PMID:Phosphorylation and activation of smooth muscle myosin by Acanthamoeba myosin I heavy chain kinase. 632 1

Mixed anhydrids of AMP, ADP, ATP and IMP and mesitylene carboxylic acid (AMP-MC, ADP-MC, ATP-MC and IMP-MC) are efficient irreversible inhibitors of the Ca-ATPase activity of myosin and heavy meromyosin. The highest rate of inhibition is observed in the case of AMP-MC: at AMP-MC concentration of 1,5.10(-3) M the half inactivation time for heavy meromyosin varies in different protein preparations from 10 to 20 min. The rates of inhibition in the presence of ADP-MC and ATP-MC are roughly the same and are far lower than those for AMP-MC (half inactivation time is 1,5-2 hrs). However, in the latter case the inhibition is complete, the time of the analogs interaction with the protein being increased up to several hours. In the presence of IMP-MC the inhibition is also time-dependent but is never complete. A necessary condition for the manifestation of irreversible inhibition of the Ca-ATPase activity of TMM by phosphorylating analogs of the substrate is the presence of bivalent cations. No inhibition occurs in the presence of EDTA. An addition of ADP or ATP to the preincubation medium causes a sharp decrease of the inhibition rate (a protective effect), which suggests a specific interaction of the analogs with TMM at the substrate binding site.
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PMID:[Inhibition of the Ca-ATPase activity of heavy meromyosin by phosphorylating analogs of the substrate]. 644 61


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