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)

The behavior of Ca2+-ATPase from sarcoplasmic reticulum in detergent solution was compared with that of Ca2+-ATPase which had been cleaved in half by limited trypsin digestion. Attempts to dissociate the fragments (I and II) with an excess of detergent micelles demonstrated that fragments I and II are structurally dependent upon each other, and that they must be denatured in order to be dissociated. Partial dissociation of the fragmented ATPase was found to occur in the bile salt detergents, deoxycholate and cholate, and optical data showed that there was an accompanying change in conformation. No dissociation of the fragmented ATPase was observed in nonionic detergents. The fragmented ATPase retained the same specific activity and stability as the intact ATPase under a variety of conditions when solubilized in Tween 80 or dodecyl octaoxyethylene glycol monoether. The data demonstrate that the noncovalent interactions that maintain the native conformation of the ATPase are not affected by either trypsin cleavage or solubilization in nonionic detergent solution.
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PMID:Behavior of fragmented calcium (II) adenosine triphosphatase from sarcoplasmic reticulum in detergent solution. 15 20

Isolated sarcoplasmic reticulum vesicles from rabbit white muscle were separated into a light (15--20% of total microsomes) and a heavy (80--85%) fraction by density gradient centifugation. The ultrastructure, chemical composition, enzymic activities and localization of membrane components in the vesicles of both fractions were investigated. From the following results it was concluded that both fractions are derived from the membranes of the sarcoplasmic reticulum system of the muscle: (i) The protein pattern of both fractions is essentially the same, except for different ratios of acidic, Ca2+-binding proteins. (ii) The 105000 dalton protein of the light fraction cross-reacts immunologically with the Ca2+-dependent ATPase of the heavy fraction. (iii) Ca2+-dependent ATPase, although of different specific activity, is found in both fractions. After rendering the vesicles leaky, specific activities in both fractions reach the same value. The light fraction was found to consist of "inside-out" vesicles by the following criteria: (i) No Ca2+ accumulation can be measured and the Ca2+-dependent ATPase activity is low and variable. (ii) The rate of trypsin digestion is lower and, compared to the heavy microsomes, a different ratio of degradation products is obtained. (iii) The sarcoplasmic reticulum membrane has a highly asymmetrical lipid distribution. This distribution of aminophospholipids is opposite to that in vesicles of heavy fraction. The light sarcoplasmic reticulum fraction has a higher phospholipid to protein ratio than the heavy one. This is consistent with the possibility that the two fractions derive from different parts of the sarcoplasmic reticulum system.
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PMID:Studies on the heterogeneity of sarcoplasmic reticulum vesicles. 15 48

A method for primary culture of ovine myometrial cells is described. After dissection, myometrium of ewe uteri was digested in trypsin and collagenase. The cells were preplated for 1 h at 37 degrees C. The non-attached cells were grown in appropriate medium supplemented with 2% fetal calf serum. They had a doubling time of 3 days, reached confluency at 10 days and did not exhibit contact inhibition. Cultures were maintained up to 22 days. Characterization of the cells was achieved by electron microscopy, analysis of myosin in cell extracts and assessment of hormone sensitivity. The cells were found to contain myofilaments, characteristic of smooth muscle. A high content of myosin (6--13%) was demonstrated on SDS-polyacrylamide gel electrophoresis: this was confirmed by ATPase activity assay. Cells responded to estradiol stimulation by increased protein synthesis, and bound [3H]estradiol in a specific and saturable way. These results suggest that myometrial cells grown in primary culture should provide a useful model for studying the hormonal control of contractile protein synthesis.
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PMID:Myometrial cells in primary culture: characterization and hormonal profile. 15 21

Rat liver lysosomes were lysed and subfractionated by differential centrifugation through 0.2M-NaCl to yield a membranous pellet. This membrane fraction contains less than 20% of the lysosomal protein, adenosine triphosphatase activity of about 1.2mumol/min per mg of protein, 120nmol of thiol groups/mg of protein and at least 16 protein and glycoprotein bands on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The gel patterns of membranes isolated from lysosomes after treatment with (1) [125I]iodidehydrogen peroxide-lactoperoxidase, (2) toluene 2,4-di-isocyanate-activated bovine serum albumin, (3) trypsin and (4) subtilisin indicate that most of the membrane proteins are exposed to the cytoplasm. These exposed proteins are candidates for intracellular receptors which recognize either substances that are to be degraded or vesicles containing those substances.
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PMID:Properties of the membrane proteins of rat liver lysosomes. The majority of lysosomal membrane proteins are exposed to the cytoplasm. 15 36

The binding properties of dynein arms to the A- and B-tubules of outer doublets of cilia from Tetrahymena pyriformis were examined, with the following results: 1. When 30s dynein purified from Tetrahymena cilia was added to doublets deficient in dynein arms, it bound to both A- and B-tubules almost equally and formed arms along the edges. The overall length of arms bound to the A-tubule was 22 +/- 3 nm, and that of arms bound to the B-tubule was 24 +/- 3 nm. Each arm bound to the A- and B-tubules was pointed toward the base at angles of 55 degrees +/- 7 degrees and 48 degrees +/- 7 degrees, respectively. In the presence of sufficient amounts of dynein, the arms along the A- and B-tubules were located at intervals of 22.8 +/- 1.5 nm and 22.5 +/- 1.7 nm, respectively. 2. On adding ATP, only the arms bound to the B-tubule were dissociated from the doublet decorated with arms on both sides. The dissociated arms rebound themselves to the B-tubule after hydrolysis of the ATP. When several doublets decorated with arms along both A- and B-tubules were arrayed side by side, the interdoublet spacing increased from 14 +/- 2 nm to 17 +/- 2 nm on addition of ATP. 3. The turbidity of a suspension of trypsin [EC 3.4.21.4]-treated axonemes decreased rapidly on addition of ATP, then recovered partially. Observations by dark-field microscopy and electron microscopy showed that the doublets which had slid out from the axonemes on ATP addition formed large aggregates after hydrolysis of the ATP. The dynein arms were also solubilized from the axonemes upon addition of ATP, and rebound themselves to the B-tubule after hydrolysis of the added ATP. 4. The double-reciprocal plot for the ATPase [EC 3.6.1.3] activity of the trypsin-treated axonemes against ATP concentration was composed of two straight lines, from which the Km values were estimated to be 1.0 and 12.7 micrometer. The dependence of the decrease in turbidity of the axonemal suspension on ATP concentration indicated that the binding of ATP to sites with an apparent dissociation constant of 1 micrometer induced dissociation of the arms from the B-tubule.
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PMID:Binding of 30s dynein with the B-tubule of the outer doublet of axonemes from Tetrahymena pyriformis and adenosine triphosphate-induced dissociation of the complex. 15

Dicyclohexylcarbodiimide (DCCD) inhibits the (Ca2+)ATPase, Ca2+ uptake by sarcoplasmic reticulum vesicles and Ca2+ binding to the (Ca2+)ATPase from sarcoplasmic reticulum. Ca2+ (at micron concentrations) specifically protects against DCCD inhibition. The inhibition can, therefore, be readily demonstrated only in the presence of Ca2+ chelating agents such as EGTA. In the presence of EGTA, the ionophore A-23187 increased the sensitivity to DCCD. The ionophore also increased the phosphorylation of the enzyme by inorganic phosphate in the presence of Mg2+. These results indicate that tightly bound Ca2+ is located in a hydrophobic region of the enzyme which is not accessible to EGTA. Complete inhibition of the (Ca2+)ATPase is accompanied by binding of 4--5 nmol of [14C]DCCD per mg of ATPase protein in the absence of Ca2+ compared with 2 nmol bound per mg in the presence of Ca2+ with no ATPase inhibition. Assuming a molecular weight of 100 000 for the ATPase monomer, about 1 nmol of DCCD inhibits 4 nmol of ATPase. This result suggests that the minimal functional unit of the enzyme is a tetramer. Following trypsin digestion of the [14C]DCCD-labeled ATPase most of the radioactivity appears in the 20 000-dalton fragment. We propose that DCCD reacts with the Ca2+-binding site of the ATPase.
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PMID:Inhibition of the (Ca2+)ATPase from sarcoplasmic reticulum by dicyclohexylcarbodiimide: evidence for location of the Ca2+ binding site in a hydrophobic region. 15 44

The effect of trypsin on gastric (H+ + K+)-ATPase and K+-phosphatase was studied. Loss of both enzymic activities was biphasic, consisting of a fast and slow phase. Several peptides were produced from the original 105,000-dalton region of the sodium dodecyl sulfate electrophoretic separation, but only two, 87,000 and 47,000 daltons, were labeled following incubation with [gamma-33P]ATP. After a 30-min hydrolysis, 35% of the original peptide remained unaltered and appeared to be a glycoprotein. ATP and ADP abolished the second phase of tryptic inactivation of both activities and only two peptides, of 78,000 and 30,000 daltons, were found on the acrylamide gel in addition to the original 105,000-dalton region, neither of which was labeled by [gamma-33P]ATP. The protection was specific for these nucleotides, AMP, beta, gamma-methylene ATP, TTP, and pNPP being ineffective. Na+ and K+ at high concentrations reduced the rate of loss of activity but no change in the peptides produced was found. The level of phosphoenzyme was increased 2-fold by trypsin treatment, whereas the quantity of K+-sensitive phosphoenzyme remained relatively constant. Thus, the 105,000-dalton region is heterogeneous, consisting of a catalytic subunit (the active site is on a 47,000-dalton fragment), a glycoprotein, and another 105,000-dalton peptide. The action of trypsin is initially to prevent interconversion of a K+-insensitive to a K+-sensitive form of the phosphoenzyme, thus inhibiting hydrolysis.
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PMID:The action of trypsin on the gastric (H+ + K+)-ATPase. 15 59

Trypsin treatment of solubilized coupling factor-latent ATPase from Mycobacterium phlei alters its subunit structure and functional properties. This coupling factor exhibits ATPase activity following trypsin treatment. Concurrently, both the ability of the enzyme to rebind to membranes depleted of coupling factor and its capacity for coupled phosphorylation are lost. The native alpha (64 000 dalton) subunit undergoes limited proteolytic digestion, and the delta (14 000 dalton) subunit is partially lost. During the course of tryptic proteolysis, the coupling factor molecule may exist in one of ten unique structural state (e.g. the native, ATPase-inactive molecule exists in the alpha alpha alpha state). Rigorous analysis of the experimental data by theoretical modeling provided information concerning the intermediate structural states leading to the fully ATPase-activated alpha" alpha" alpha" state under different conditions of trypsin treatment. The theoretical models of structure-function relationships that best-represented the experimental data predicted that the native coupling factor molecule contains three copies of the alpha (64 000 dalton) form of the alpha subunit, that the alpha" (58 000 dalton) alpha subunit species contributes maximally and the alpha' (61 000 dalton) form about half-maximally to ATPase activity, that membrane rebinding ability is proportional to the number of native alpha subunits in the enzyme, and that at least one native alpha subunit/molecule is required for full expression of coupled phosphorylation. These results indicate an essential role for the alpha subunit in the regulation of ATPase activity and in the ability of the solubilized coupling factor to rebind to depleted membranes.
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PMID:Tryptic proteolysis of coupling factor-latent ATPase from mycobacterium phlei. Theoretical modeling of structure-function relationships. 15 57

The activation of the coupling factor-latent ATPase enzyme by tryptic proteolysis may resemble the activation of many proenzymes by limited proteolysis. The beta (53 000 dalton) subunit of solubilized coupling factor-latent ATPase from Mycobacterium phlei was selectively lost in some trypsin-treated samples. Since a concomitant loss of ATPase activity was not observed, the beta subunit may not be essential for ATPase catalytic activity. Treatment of solubilized coupling factor with chymotrypsin rapidly produced an A'-type (61 000 dalton) species from the native alpha (64 000 dalton) subunits with partial activation of the APTase enzyme. Secondary chymotryptic cleavage yielded an A"-type (58 000 dalton) species and a less-active enzyme. Storage of fresh coupling factor samples at -20degreeC in the presence of 4 mM MgCl2 with several freeze-thaw cycles resulted in loss of ATPase activity without apparent change in alpha subunit structure. Storage at 4 degrees C in the presence or absence of MgCl2 both decreased ATPase activity and generated A'-type alpha subunit species. Since presence was suspected. The peptide bonds first cleaved by trypsin, chymotrypsin, and the unknown protease are all apparantly located within the same small segment of alpha subunit polypeptide chain.
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PMID:Limited proteolysis of coupling factor-latent ATPase from Mycobacterium phlei. Effects of different enzymes and modifying agents. 15 59

We described previously the existence of a soluble ATPase activity in rat liver mitochondria [1]. The purification and catalytic properties have been described [2]. In a continuation of these experiments, we have studied the immunologic and structural properties of one molecular form of this enzyme : ATPase I. We have prepared the antiserum anti-ATPase I and demonstrated the purity of our enzyme preparation by immunodiffusion and immunoelectrophoresis. An immunohistochemical method also confirmed the localization of ATPase I in the soluble fraction of mitochondria. The molecular weight of ATPase I was measured by G 100 Sephadex gel filtration and was found to be 18,400; electrophoresis on polyacrylamide gels gave a value of 18,600. The pHi of ATPase I was found to be 7,2. Amino acid analysis showed high amounts of aspartic acid, glutamic acid, serine and glycine. The molecular weight calculated from the total amino acid residues was found to be 17,000. Alanine is the NH2 terminal amino acid. The peptide maps obtained after degrading ATPase I with cyanogen bromide or trypsin are in accordance with the methionine, lysine and arginine residues we found in the ATPase I molecule. ATPase I does not appear to be a glycoprotein.
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PMID:Studies of soluble rat liver mitochondrial acid ATPases. II. Structural and immunological properties of ATPase 1. 15 69


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