Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Oligomycin-sensitive particulate ATPase (MB ATPase) from L. donovani promastigotes was solubilized by chloroform treatment. Polyacrylamide gel electrophoresis revealed several protein bands, with the major one possessing ATPase activity. The solubilized enzyme had Mg2+-ATPase and Ca2+-ATPase but no K+-dependent alkaline phosphatase activity. The Mg2+-ATPase activity was stimulated by monovalent cations and was not sensitive to oligomycin. Hence it is referred to as F1 ATPase. It had optimum activity at pH 7.6 and 30 degrees C. The Arrhenius plot for MB ATPase was biphasic with activation energies (Ea) of 16.2 and 3.4 kcal mol-1, while F1 ATPase exhibited a linear plot with Ea = 10.1 kcal mol-1. Lineweaver-Burk plots were biphasic with Km values of 0.17 and 1.25 mM for MB ATPase and 0.18 and 1.33 mM for F1 ATPase. The enzyme could be preserved at -15 degrees C in Tris-SO2-(4)-EDTA-ATP-glycerol (t1/2 = 20 days).
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PMID:Solubilization and kinetic characterization of mitochondrial adenosine triphosphatase from Leishmania donovani promastigotes. 297 May 89

Posterior pituitary lobes from young pigs were fractionated by differential and sucrose-density-gradient centrifugation. The distributions of oxytocin and [8-lysine]-vasopressin were measured by bioassay and the distributions of neurophysin-I and -II by radioimmunoassays specific for each of these two proteins. Most of the hormone and neurophysin applied to the density gradient was localized in particles with the density expected of neurosecretory granules. However, the neurosecretory granules were separated into two bands (D and E). A close statistical correlation between the distributions of [8-lysine]-vasopressin and neurophysin-I, and of oxytocin and neurophysin-II on the gradients, suggested that in vivo porcine neurophysin-I binds [8-lysine]-vasopressin within one population of granules and porcine neurophysin-II binds oxytocin within another type of granule. However, there was no significant separation of oxytocin and vasopressin in fractions D and E. The molar ratios of hormones and neurophysins indicated that there was insufficient of either neurophysin to bind the [8-lysine]-vasopressin in the granule fractions or in the whole gland. Polyacrylamide-gel electrophoresis showed that only bands corresponding in mobility to porcine neurophysins-I, -II and -III were present in large amounts in the whole gland and in the granule fractions. The component with the mobility of neurophysin-III was, however, relatively enriched in whole young glands and granule fractions compared with adult gland extracts. It is suggested that the vasopressin that cannot be assigned to neurophysin-I may occur in (a) vesicles containing vasopressin but no neurophysin, (b) vesicles containing vasopressin and a protein that cannot be quantified by the radioimmunoassays used, such as porcine neurophysin-III, or (c) normal vasopressin-neurophysin granules which have accumulated extra vasopressin. Band E of the gradient was rich in adenosine triphosphatase activity, whereas band D possessed very little of this enzyme.
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PMID:Subcellular organization of neurophysins, oxytocin, (8-lysine)-vasopressin and adenosine triphosphatase in porcine posterior pituitary lobes. 426 6

1. Homogenates of neural lobes of bovine pituitary glands were fractionated by differential and density-gradient ultracentrifugation and the distribution of adenosine triphosphatase (ATPase) activity was studied. It was shown that all the activity was membrane-bound. 2. On the basis of ionic requirements the ATPase activity was grouped into three categories: (a) Mg(2+)-dependent, (b) Ca(2+)-dependent and (c) Mg(2+)+Na(+)+K(+)-dependent (ouabain-sensitive) ATPases. The activity in the absence of bivalent cations was negligible. The ratio between the activities of the three ATPases varied between the different subcellular fractions. 3. Preincubation of the subcellular fractions with deoxycholate increased the activity of the Mg(2+)+Na(+)+K(+)-dependent enzyme, whereas the Mg(2+)- and Ca(2+)-activated ATPases were either unaffected or slightly inhibited. Triton X-100 solubilized the Mg(2+)- and Ca(2+)-ATPases; however, the activity of the Mg(2+)+Na(+)+K(+)-ATPase was abolished by the concentration of Triton X-100 used. 4. All the subfractions displayed unspecific nucleotide triphosphatase activity towards GTP, ITP and UTP. These substrates inhibited the hydrolysis of ATP by all three ATPases. ADP also inhibited the ATPases. 5. Polyacrylamide-gel electrophoresis of extracts containing the Mg(2+)- and Ca(2+)-dependent ATPase activity solubilized by Triton X-100 revealed the presence of two enzymes; one activated by either Mg(2+) or Ca(2+) and the other activated only by Ca(2+). 6. In sucrose density gradients the distribution of vasopressin was different from that of all three types of ATPases. It is therefore suggested that the neurosecretory granules do not possess ATPase activity.
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PMID:Adenosine triphosphatase activity in the neural lobe of the bovine pituitary gland. 428 6

1. Plasma membranes were isolated from crude nuclear sediments from mouse and rat liver by a rate-dependent centrifugation through a sucrose density gradient contained in the ;A' type zonal rotor. 2. The membranes were further purified by isopycnic centrifugation, and characterized enzymically, chemically and morphologically. 3. When the plasma-membrane fraction of sucrose density 1.17g/cm(3) was dispersed in a tight-fitting homogenizer, two subfractions of densities 1.12 and 1.18 were obtained by isopycnic centrifugation. 4. The light subfraction contained 5'-nucleotidase, nucleoside diphosphatase, leucine naphthylamidase and Mg(2+)-stimulated adenosine triphosphatase activities at higher specific activities than unfractionated membranes. The heavy subfraction was deficient in the above enzymes but contained higher Na(+)+K(+)-stimulated adenosine triphosphatase activity. 5. The light subfraction contained twice as much phospholipid and cholesterol, and three times as much N-acetylneuraminic acid relative to unit protein weight as the heavy subfraction. Polyacrylamide-gel electrophoresis indicated differences in protein composition. 6. Electron microscopy showed the light subfraction to be vesicular. The heavy subfraction contained membrane strips with junctional complexes in addition to vesicles.
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PMID:Fractionation of liver plasma membranes prepared by zonal centrifugation. 431 49

The Ca2+-stimulated, Mg2+-dependent ATPase from rat liver plasma membranes was solubilized using the detergent polyoxyethylene 9 lauryl ether and purified by column chromatography using Polybuffer Exchanger 94, concanavalin A-Sepharose 4B, and Sephadex G-200. The molecular weight of the enzyme, estimated by gel filtration in the presence of the detergent on a Sephadex G-200 column, was 200,000 +/- 15,000. The enzyme was purified at least 300-fold from rat liver plasma membranes and had a specific activity of 19.7 mumol/mg/min. Polyacrylamide gel electrophoresis under nondenaturing conditions of the purified enzyme indicated that the enzymatic activity correlated with the major protein band. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, one major band in the molecular weight range of 70,000 +/- 5,000 was seen. The isoelectric point of the purified enzyme was 6.9 +/- 0.2 as determined by analytical isoelectric focusing. The enzyme was activated by Ca2+ with an apparent half-saturation constant of 87 +/- 2 nM for Ca2+. Calmodulin and trifluoperazine at the concentration of 1 microgram/ml and 100 microM, respectively, had no effect on the enzymatic activity.
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PMID:Purification of (Ca2+-Mg2+)-ATPase from rat liver plasma membranes. 614 50

The removal of tightly bound nucleotides from mitochondrial F1-ATPase was found to affect the inhibition by ADP and chemical reactivity toward 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-C1) and sulfhydryl reagents. Preincubation of nucleotide-depleted F1 with 40 microM ADP in the presence of ethylenediaminetetraacetic acid (EDTA) resulted in a 51% inhibition of the steady-state level of ATPase activity whereas only a 25% inhibition was observed for native F1. Both partially inhibited states of the enzyme could be reversed by the subsequent addition of ATP. Measurement of [14C]ADP binding to nucleotide-depleted F1 in the presence of EDTA reveals three equivalent ADP binding sites with a Kd of 0.45 microM, and a fourth site of lower affinity. The sulfhydryl reagents 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide (NEM) were found to inhibit the ATPase activity of nucleotide-depleted F1 but not native F1 or nucleotide-depleted F1 in the presence of ADP or ATP. Polyacrylamide gel electrophoresis of nucleotide-depleted F1 labeled with [14C]NEM gave a 2-fold increase in incorporation into the (alpha + beta) subunits and a 7-fold increase in label in the gamma subunit after 90 min compared to when ADP was present during the reaction. ADP binding to the noncatalytic sites enhanced the rate of inhibition of nucleotide-depleted F1 by NBD-C1 about 2-fold while retarding the subsequent intramolecular transfer from an essential phenol group to an amino group about 2.8-fold. The results suggest a conformational change in F1 caused by changes in nucleotide--protein interaction at the noncatalytic sites.
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PMID:Changes in chemical properties of mitochondrial adenosinetriphosphatase upon removal of tightly bound nucleotides. 622 55

In the presence of Mg2+ or Ca2+ the membranes of the anaerobic glycolytic bacterium Lactobacillus casei hydrolyze 0.1-0.2 mumole ATP/min/mg of protein with a pH optimum 6.4. This activity is inhibited by N,N'-dicyclohexylcarbodiimide and is insensitive to oligomycin, ouabain, vanadate and hydroxylamine. A soluble ATPase was isolated and purified from L. casei membranes. The specific activity of this ATPase is 3.0-4.0 mumole ATP/min/mg of protein. The enzyme homogeneity was established by analytical polyacrylamide gel disc electrophoresis and by analytical centrifugation (S20, omega = 12 +/- 0,5). The molecular weight of the enzyme is 270 000. Polyacrylamide gel electrophoresis of ATPase denaturated by 1% SDS and 8 M urea in the presence of SDS revealed one type of subunits with Mr = 43 000. These subunits could not be separated by isoelectrofocusing in polyacrylamide gel in the presence of 8 M urea and migrated as a single peptide with pI at 4.2. The experimental results suggest that the soluble ATPase from L. casei consists of six identical subunits with Mr of 43 000.
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PMID:[Characteristics of subunit composition of H+-ATPase from the anaerobic bacterium Lactobacillus casei]. 622 21

Isolated myocytes were prepared from adult canine hearts using a combined technique of myocardial perfusion followed by incubation with collagenase. More than 60% of the cells routinely excluded trypan blue dye. Disruption of the myocytes was accomplished using high pressure nitrogen cavitation. After differential and sucrose gradient centrifugation, the peak sarcolemmal fraction averaged 100-fold enrichment in ouabain-inhibited K+-stimulated p-nitrophenyl phosphatase and 82-fold in ouabain-inhibited (Na+,K+)-ATPase. These sarcolemmal membranes are enriched in phospholipid phosphorus (1.98 mumol/mg of protein) and more than 4-fold in sphingomyelin and cholesterol. Polyacrylamide gels revealed three major protein peaks at 50,000, 91,000, and 140,000 apparent molecular weights. This work demonstrates the feasibility of preparing highly pure cardiac sarcolemma from isolated adult myocytes. The problem of cellular cross-contamination due to heterogeneity of cell types in whole myocardial tissue has been circumvented. The level of enrichment exceeds all reported preparations of cardiac sarcolemma from whole myocardium and cultured myocytes. This preparation should prove to be useful as an in vitro model for studies of physiological, pharmacological, and pathological perturbations of sarcolemmal structure and function.
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PMID:Preparation and properties of highly enriched cardiac sarcolemma from isolated adult myocytes. 624 86

The cell surface complex (Detering et al., 1977, J. Cell Biol. 75, 899-914) of the sea urchin egg consists of two subcellular organelles: the plasma membrane, containing associated peripheral proteins and the vitelline layer, and the cortical vesicles. We have now developed a method of isolating the plasma membrane from this complex and have undertaken its biochemical characterization. Enzymatic assays of the cell surface complex revealed the presence of a plasma membrane marker enzyme, ouabain-sensitive Na+/K+ ATPase, as well as two cortical granule markers, proteoesterase and ovoperoxidase. After separation from the cortical vesicles and purification on a sucrose gradient, the purified plasma membranes are recovered as large sheets devoid of cortical vesicles. The purified plasma membranes are highly enriched in the Na+/K+ ATPase but contain only very low levels of the proteoesterase and ovoperoxidase. Ultrastructurally, the purified plasma membrane is characterized as large sheets containing a "fluffy" proteinaceous layer on the external surface, which probably represent peripheral proteins, including remnants of the vitelline layer. Extraction of these membranes with Kl removes these peripheral proteins and causes the membrane sheets to vesiculate. Polyacrylamide gel electrophoresis of the cell surface complex, plasma membranes, and Kl-extracted membranes indicates that the plasma membrane contains five to six major proteins species, as well as a large number of minor species, that are not extractable with Kl. The vitelline layer and other peripheral membrane components account for a large proportion of the membrane-associated protein and are represented by at least six to seven polypeptide components. The phospholipid composition of the Kl-extracted membranes is unique, being very rich in phosphatidylethanolamine and phosphatidylinositol. Cholesterol was found to be a major component of the plasma membrane. Before Kl extraction, the purified plasma membranes retain the same species-specific sperm binding property that is found in the intact egg. This observation indicates that the sperm receptor mechanisms remain functional in the isolated, cortical vesicle-free membrane preparation.
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PMID:Isolation and partial characterization of the plasma membrane of the sea urchin egg. 625 14

Polyacrylamide gel electrophoresis was used to investigate the relation of the soluble thiamine triphosphatase activity of various rat tissues to other phosphatases. This technique separated the thiamine triphosphatase of rat brain, heart, kidney, liver, lung, muscle and spleen from alkaline phosphatase (EC 3.1.3.1), acid phosphatase (EC 3.1.3.2) and other nonspecific phosphatase activities. In contrast, the hydrolytic activity for thiamine triphosphate in rat intestine moved identically with alkaline phosphatase in gel electrophoresis. Thiamine triphosphatase from rat liver and brain was also separated from alkaline phosphatase and acid phosphatase by gel chromatography on Sephadex G-100. This gave an apparent molecular weight of about 30,000 and a Stokes radius of 2.5 nanometers for brain and liver thiamine triphosphatase. The intestinal thiamine triphosphatase activity of the rat was eluted from the Sephadex G-100 column as two separate peaks (with apparent molecular weights of over 200,000 and 123,000) which exactly corresponded to the peaks of alkaline phosphatase. The isoelectric point (pI) of the brain thiamine triphosphatase was 4.6 (4 degrees C). The partially purified thiamine triphosphatase from brain and liver was highly specific for thiamine triphosphate. The results suggest that, apart from the intestine, the rat tissues studied contain a specific enzyme, thiamine triphosphatase (EC 3.6.1.28). The specific enzyme is responsible for most of the thiamine triphosphatase activity in these tissues. Rat intestine contains a high thiamine triphosphatase activity but all of it appears to be due to alkaline phosphatase.
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PMID:The relation of the soluble thiamine triphosphatase activity of various rat tissues to nonspecific phosphatases. 627 68


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