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Query: EC:3.6.3.1 (Mg2+-ATPase)
 1,484 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mg2+-ATPase activity was identified in the cytosol of human erythrocytes. A partial purification of this activity was achieved by an initial DEAE-Sephadex column chromatography, followed by gel filtration on Sephadex G-100 and then a second DEAE-Sephadex chromatography procedure. The enzyme appeared in the void volume of the Sephadex G-100 column and was retained on an Amicon XM100A ultrafiltration membrane. The molecular weight of the enzyme was estimated to be 113 000 from SD gels. The above purification protocol yielded an enzyme with an optimal pH between 7.6 and 8.2. The enzyme activity increased linearly between 30 and 44 degrees C. It was stable for several months at -20 degrees C. Magnesium was essential for activity, but the rate attainable with Mn2+ was at least as great as that due to Mg2+. No other divalent cation was able to substitute for Mg2+ or Mn2+. Neither low nor high Ca2+ concentrations significantly affected the enzymatic activity. Substrate specificity studies showed that ATP was the preferred substrate followed by CTP (46% of the rate produced by ATP). Hydrolysis of GTP, UTP, ITP and ADP was less than 10% of the rate seen with ATP. No phosphatase, pyrophosphatase, phosphodiesterase, hexokinase, phosphofructokinase or adenylate cyclase activity could be detected in this enzyme preparation. Calmodulin, which stimulates the (Ca2+ + Mg2+)-ATPase of the human erythrocyte membrane, failed to enhance the Mg2+-ATPase activity. Of considerable interest, the activity of this Mg2+-ATPase was enhanced approximately 5-fold by low concentrations of mercuric ion, p-hydroxymercuribenzoate and DTNB, but was much less sensitive to iodoacetamide.
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PMID:Partial purification and characterization of a novel Mg2+-dependent ATPase present in the cytosol from human erythrocytes. 615 Jul 30

Membrane-bound enzyme activities and cardiac glycoside binding were determined in red blood cell membrane preparations from patients with myotonic dystrophy and in age matched controls. Na+-K+-activated ATPase activity was significantly increased in myotonic patients. [3H]Ouabain binding to erythrocyte membranes was also significantly increased in myotonic dystrophy patients. The Mg2+-ATPase (ouabain-insensitive) was, however, unchanged. The K+-stimulated paranitrophenyl phosphatase (KPNPPase) activity was markedly enhanced in myotonic patients as compared to controls. The kinetic analysis showed a marked change in Vmax of Na+-K+ ATPase with respect to the activation by Na+, K+ and ATP. However, the Km values were the same in control as well as in myotonic groups. The increased erythrocyte membrane Na+-K+-ATPase activity, KPNPPase and [3H]ouabain binding in myotonic patients supports the hypothesis that generalized membrane abnormality may be involved in pathogenesis of the human myotonic dystrophy.
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PMID:Erythrocyte membrane abnormalities in human myotonic dystrophy. 624 57

A procedure is described for the preparation of a membrane fraction enriched in basal-lateral plasma membranes from gastric mucosa. Gastric glands isolated from rabbit were employed as starting material, greatly reducing contamination from non-glandular cell types. The distribution of cellular components during the fractionation procedure was monitored with specific marker enzymes. (Na+ + K+)-ATPase, ouabain-sensitive K+-stimulated p-nitrophenyl-phosphatase and histamine-stimulated adenylate cyclase were used as markers for basal-lateral membranes. These three markers were similarly distributed during both differential and equilibrium density gradient centrifugation. The enriched membrane fraction contained more than 30% of the total initial activities of the three basal-lateral membrane markers which were purified better than 11-fold with respect to protein. (Na+ + K+)-ATPase activity was resolved from the activities of acid phosphatase, pepsin, Mg2+-ATPase, cytochrome c oxidase, NADPH-cytochrome c reductase, glucose-6-phosphatase, (K+ + H+)-ATPase, DNA and RNA.
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PMID:An enriched preparation of basal-lateral plasma membranes from gastric glandular cells. 626 84

N-Ethylmaleimide was employed as a surface label for sarcolemmal proteins after demonstrating that it does not penetrate to the intracellular space at concentrations below 1.10(-4) M. The sarcolemmal markers, ouabain-sensitive (Na+ +K+)-ATPase and Na+/Ca2+-exchange activities, were inhibited in N-ethylmaleimide perfused hearts. Intracellular activities such as creatine phosphokinase, glutamate-oxaloacetate transaminase and the internal phosphatase site of the Na+ pump (K+-p-nitrophosphatase) were not affected. Almost 20% of the (Ca2+ +Mg2+)-ATPase and Ca2+ pump were inhibited indicating the localization of a portion of this activity in the sarcolemma. Sarcolemma purified by a recent method (Morcos, N.C. and Drummond, G.I. (1980) Biochim. Biophys. Acta 598, 27-39) from N-ethylmaleimide-perfused hearts showed loss of approx. 85% of its (Ca2+ +Mg2+-ATPase and Ca2+ pump compared to control hearts. (Ca2+ +Mg2+)-ATPase and Ca2+ pump activities showed two classes of sensitivity to vanadate ion inhibition. The high vanadate affinity class (K1/2 for inhibition approx. 1.5 microM) may be localized in the sarcolemma and represented approx. 20% of the total inhibitable activity in agreement with estimates from N-ethylmaleimide studies. Sucrose density fractionation indicated that only a small portion of Mg2+-ATPase and Ca2+-ATPase may be associated with the sarcolemma. The major portion of these activities seems to be associated with high density particles.
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PMID:Localization of (Ca2+ + Mg2+)-ATPase, Ca2+ pump and other ATPase activities in cardiac sarcolemma. 628 90

(1) A (K+ + H+)-ATPase containing membrane fraction, isolated from pig gastric mucosa, has been further purified by means of zonal electrophoresis, leading to a 20% increase in specific activity and an increase in ratio of (K+ + H+)-ATPase to basal Mg2+-ATPase activity from 9 to 20. (2) The target size of (Na+ + K+)-ATPase, determined by radiation inactivation analysis, is 332 kDa, in excellent agreement with the earlier value of 327 kDa obtained from the subunit composition and subunit molecular weights. This shows that the Kepner-Macey factor of 6.4 X 10(11) is valid for membrane-bound ATPases. (3) The target size of (K+ + H+)-ATPase is 444 kDa, which, in connection with a subunit molecular weight of 110000, suggests a tetrameric assembly of the native enzyme. The ouabain-insensitive K+-stimulated p-nitrophenylphosphatase activity has a target size of 295 kDa. (4) In the presence of added Mg2+ the target sizes of the (K+ + H+)-ATPase and its phosphatase activity are decreased by about 15%, while that for the (Na+ + K+)-ATPase is not significantly changed. This observation is discussed in terms of a Mg2+-induced tightening of the subunits composing the (K+ + H+)-ATPase molecule.
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PMID:Studies on (K+ + H+)-ATPase. VI. Determination on the molecular size by radiation inactivation analysis. 630 21

Phosphorylation of chicken gizzard myosin light chain in myofibril and its effect on myofibrillar ATPase activity were investigated in the contracted state of myofibrils. When myofibrils were incubated for two hours at 30 degreeds C with ATP, magnesium and calcium, the myosin light chain was phosphorylated by endogenous light-chain kinase. Standing overnight, the phosphorylated light chain was dephosphorylated by endogenous light-chain phosphatase. Control myofibril had much higher ATPase activity than phosphorylated and phosphorylated-dephosphorylated myofibrils. It was very interesting that the phosphorylated and phosphorylated-dephosphorylated myofibrils were quite similar in ATPase activity. However, phosphorylated myofibril differed from phosphorylated-dephosphorylated myofibril in Ca2+ dependency of Mg2+-ATPase activity. The phosphorylated-dephosphorylated myofibril was not affected by the presence or absence of Ca2+. In contrast, phosphorylated myofibril apparently showed a negative Ca2+-sensitivity. On the other hand, the results indicating that the superprecipitation gel formed by phosphorylated-dephosphorylated myosin could not be dissolved in 0.6 M NaCl, suggest that the phosphorylation-dephosphorylation process of the actomyosin system in gizzard myofibril results in stronger actin-myosin interaction.
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PMID:Properties of phosphorylated myofibrils from gizzard smooth muscle. 644 50

In pursuit of the characterization of the recently discovered flippase mode of operation of the anion transporter (band 3, AE1) of the human erythrocyte membrane, the transbilayer translocation (flip) of a fluorescently labeled, membrane-intercalated long-chain alkyl phosphate, 10-(alpha-napthyl)-1-decyl-phosphate (NDP) was investigated. In contrast to the alkyl sulfonates and esters of phosphatidic acid studied as yet, NDP moves exclusively via band 3. NDP is, however, dephosphorylated at the inner membrane surface by a cytoplasmic phosphatase likely to interact specifically with endofacial membrane structures of the erythrocyte. This phosphatase shares characteristic inhibitor sensitivities with protein tyrosine phosphatases present in the erythrocyte interior. Vanadate as an inhibitor of NDP dephosphorylation provided a means to study the kinetic properties and patterns of inhibition (by inhibitors of anion exchange) and stimulation (by proteolysis of band 3 and aliphatic alcohols) of the flip of NDP. NDP is also an inhibitor of the exchange of hydrophilic anions via band 3, while hydrophilic anions interfere with the flip of NDP. The results are compared with the characteristics of the flip, via Band 3, of other amphiphilic anions and of the exchange of hydrophilic anions. Attempts are presented to understand the low flip rate of long-chain amphiphilic anions on the basis of their molecular properties and the thermodynamics of the "transition state" of the flip process.
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PMID:Band 3-mediated flip-flop and phosphatase-catalyzed cleavage of a long-chain alkyl phosphate anion in the human erythrocyte membrane. 974 99

Seven hepatic phosphatases were histochemically investigated in male white rats (Rattus norvegicus) pretreated with chronic subtoxic doses of lead acetate. Lead has increased the activities of alkaline-, acid-, neutral-, adenosine mono- and glucose-6-phosphatase, but has markedly decreased the activity of membrane-bound Na+-K+, ATPase while the activity of mitochondrial Mg2+-ATPase was not altered. It has also produced heterogenous alterations in the distribution patterns, sites of the enzymatic activities and in the intensity of phosphatase activities among the same type of cells in the terminal afferent and efferent venules of the hepatic lobules. The obtained histochemical findings indicate that the alterations in the activities of hepatic phosphatases could be an adaptation to the metabolic, structural and functional changes in the organelles of hepatic cells due to lead intoxication.
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PMID:Histochemical demonstration of changes in the activity of hepatic phosphatases induced by experimental lead poisoning in male white rats (Rattus norvegicus). 1079 82

Francisella tularensis and related intracellular pathogens synthesize lipid A molecules that differ from their Escherichia coli counterparts. Although a functional orthologue of lpxK, the gene encoding the lipid A 4'-kinase, is present in Francisella, no 4'-phosphate moiety is attached to Francisella lipid A. We now demonstrate that a membrane-bound phosphatase present in Francisella novicida U112 selectively removes the 4'-phosphate residue from tetra- and pentaacylated lipid A molecules. A clone that expresses the F. novicida 4'-phosphatase was identified by assaying lysates of E. coli colonies, harboring members of an F. novicida genomic DNA library, for 4'-phosphatase activity. Sequencing of a 2.5-kb F. novicida DNA insert from an active clone located the structural gene for the 4'-phosphatase, designated lpxF. It encodes a protein of 222 amino acid residues with six predicted membrane-spanning segments. Rhizobium leguminosarum and Rhizobium etli contain functional lpxF orthologues, consistent with their lipid A structures. When F. novicida LpxF is expressed in an E. coli LpxM mutant, a strain that synthesizes pentaacylated lipid A, over 90% of the lipid A molecules are dephosphorylated at the 4'-position. Expression of LpxF in wild-type E. coli has no effect, because wild-type hexaacylated lipid A is not a substrate. However, newly synthesized lipid A is not dephosphorylated in LpxM mutants by LpxF when the MsbA flippase is inactivated, indicating that LpxF faces the outer surface of the inner membrane. The availability of the lpxF gene will facilitate re-engineering lipid A structures in diverse bacteria.
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PMID:Expression cloning and periplasmic orientation of the Francisella novicida lipid A 4'-phosphatase LpxF. 1646

During protein N-glycosylation, dolichyl pyrophosphate (Dol-P-P) is discharged in the lumenal monolayer of the endoplasmic reticulum (ER). Dol-P-P is then cleaved to Dol-P by Dol-P-P phosphatase (DPPase). Studies with the yeast mutant cwh8Delta, lacking DPPase activity, indicate that recycling of Dol-P produced by DPPase contributes significantly to the pool of Dol-P utilized for lipid intermediate biosynthesis on the cytoplasmic leaflet. Whether Dol-P formed in the lumen diffuses directly back to the cytoplasmic leaflet or is first dephosphorylated to dolichol has not been determined. Incubation of sealed ER vesicles from calf brain with acetyl-Asn-Tyr-Thr-NH(2), an N-glycosylatable peptide, to generate Dol-P-P in the lumenal monolayer produced corresponding increases in the rates of Man-P-Dol, Glc-P-Dol, and GlcNAc-P-P-Dol synthesis in the absence of CTP. No changes in dolichol kinase activity were observed. When streptolysin-O permeabilized CHO cells were incubated with an acceptor peptide, N-glycopeptide synthesis, requiring multiple cycles of the dolichol pathway, occurred in the absence of CTP. The results obtained with sealed microsomes and CHO cells indicate that Dol-P, formed from Dol-P-P, returns to the cytoplasmic leaflet where it can be reutilized for lipid intermediate biosynthesis, and dolichol kinase is not required for recycling. It is possible that the flip-flopping of the carrier lipid is mediated by a flippase, which would provide a mechanism for the recycling of Dol-P derived from Man-P-Dol-mediated reactions in N-, O-, and C-mannosylation of proteins, GPI anchor assembly, and the three Glc-P-Dol-mediated reactions in Glc(3)Man(9)GlcNAc(2)-P-P-Dol (DLO) biosynthesis.
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PMID:Recycling of dolichyl monophosphate to the cytoplasmic leaflet of the endoplasmic reticulum after the cleavage of dolichyl pyrophosphate on the lumenal monolayer. 1807 51


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