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 DNA replicase activity of the complex between bovine thymus DNA polymerase alpha and RNA primase was markedly decreased after the purification by ssDNA-cellulose column chromatography. In an attempt to restore the activity by supplementing some fractions eliminated from the purified enzyme, we found that a fraction eluted from the column by increasing salt concentration and 30% ammonium sulfate precipitates of the phosphocellulose-step enzyme possessed a high ability to restore the replicase activity. Thus, the factors were purified to near homogeneity from the two sources and the properties were examined. Both factors were heat-labile and trypsin-sensitive, possessed a native molecular mass of approximately 150-200 kDa as judged by Sephacryl S-200 column chromatography, and were composed of two polypeptides of 146 kDa and 47 kDa on SDS/polyacrylamide gel electrophoresis, indicating that they were an identical protein. The factor, which did not show any DNA polymerase or primase activities by itself, stimulated approximately 20-fold the replicase activity of purified DNA-polymerase-alpha-primase at a very low concentration (10 ng/50 microliter). The factor did not affect the deoxyribonucleotide polymerizing activity of the enzyme complex at all, but specifically stimulated the primase activity only. Thus, we designated the factor as primase-stimulating factor. Although varying the template concentration did not significantly affect the mode of stimulation, increasing the concentration of substrate for primer synthesis (ATP) markedly decreased the extent of stimulation. Thus, the stimulating factor seems to decrease the substrate concentration required for the primase reaction as well as increasing threefold the maximum activity attained by varying the substrate concentration. So far, no ATPase activity has been detected in the factor.
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PMID:Purification and properties of a specific primase-stimulating factor of bovine thymus. 283 71

Six weeks after induction of diabetes, the rate of ouabain-sensitive 86Rb+ accumulation, a parameter which reflects Na+ + K+-ATPase pumping activity, was significantly reduced in endoneurial preparations of sciatic nerve from untreated diabetic rats compared with that in control rats (Trial, 1, 0.19 +/- 0.09 versus 0.48 +/- 0.13 pmol/min per mg wet weight of tissue, p less than 0.001; Trial 2, 0.27 +/- 0.16 versus 0.47 +/- 0.18, p less than 0.01). This decrease in ouabain-sensitive 86Rb+ uptake was not observed in nerves from diabetic rats maintained on sorbinil (an aldose reductase inhibitor) or myo-inositol diets. Protein kinase C activity was demonstrated in the soluble fraction of a sciatic nerve homogenate by assaying for lipid-activated, Ca+-dependent phosphorylation of calf thymus histone. No significant difference in the time course of kinase C activity was observed between cytosol fractions of nerve homogenates from control and diabetic rats (control, 6.22 +/- 0.97 pmol 32P incorporated/mg cytosol protein in 50 min; diabetic, 5.32 +/- 0.71). Three low molecular weight neural proteins (each with Mr less than 29,000) were identified as substrates for protein kinase C.
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PMID:Reduced Na+ + K+-ATPase activity in peripheral nerve of streptozotocin-diabetic rats: a role for protein kinase C? 284 Mar 14

Bone marrow-derived leukocytes of murine epidermis can express two phenotypes: typical Langerhans cells, which are Ia+ and Thy-1-, and a recently discovered second population that is Thy-1+ and Ia-. To verify that these phenotypes are expressed by two different cell types, and to help understand their lineage and function, we have studied morphology and reactivity with a large panel of antibodies. Dual antibody immunofluorescence combined with electron microscopy showed that Thy-1+ and Ia+ cells were each distributed in a regular fashion and formed adjacent dendritic systems in or close to the basal layer. Double-labeling studies with anti-Ia and a second monoclonal antibody revealed that all Langerhans cells expressed F4/80 (macrophage), Mac-1 (C3bi receptor), and 2.4G2 (Fc receptor), as well as the thymus leukemia (TL) and heat-stable (M1.69/16) antigens. A large fraction expressed S100 and all exhibited membrane ATPase and nonspecific esterase. In contrast, Thy-1+ cells lacked all these features of Langerhans cells, except that a minority were strongly reactive with 2.4G2. Thy-1+ cells also lacked differentiation antigens of most other types of leukocytes, except they were rich in asialo GM1. By electron microscopy, Thy-1+ cells had cytoplasmic granules that were similar in structure and in their aryl sulfatase content to those previously described in natural killer cells. The granules were enlarged in beige mice, suggesting a lysosomal origin, and were present in mast cell-deficient W/Wv mice, indicating no relation to mast cells. We conclude that Thy-1+ epidermal cells are thoroughly distinct from Langerhans cells. On the basis of morphology and phenotype, they may represent a type of tissue natural killer cell. Thy-1+ natural killer cells are now being identified in several nonlymphoid sites, such as gut epithelium and the livers of mice given adjuvants. If Thy-1+ epidermal cells prove to be natural killer cells, it is noteworthy that they represent a resident population regularly distributed in the basal layer of all mouse strains. The notion that Thy-1+ epidermal cells are immature natural killer cells is intriguing in light of recent evidence that Ia+ Langerhans cells are also immature with respect to accessory cell function. The epidermis may not have the functional capacities of a lymphoid organ, but it could contribute immature cells important for both natural and acquired resistance.
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PMID:The Thy-1-bearing cell of murine epidermis. A distinctive leukocyte perhaps related to natural killer cells. 286 Dec 45

We have established a novel procedure to purify calf thymus DNA polymerase delta from cytoplasmic extracts. The enzyme has typical properties of DNA polymerase delta including a 3' - greater than 5' exonuclease activity and efficiently replicates natural occurring genomes such as primed single-stranded M13 DNA and single-stranded porcine circovirus DNA, this last one thanks to an associated or contaminating primase activity. A processivity of at least a thousand bases was evident and this in the apparent absence of proliferating cell nuclear antigen. The enzyme was purified through a procedure that allows the simultaneous isolation of DNA polymerase delta, DNA polymerase alpha-primase and a DNA dependent ATPase. All these enzymes coeluted from a phosphocellulose column. After chromatography on hydroxylapatite DNA polymerase delta separated from the coeluting DNA polymerase alpha and DNA dependent ATPase. Separation of the latter two was achieved on heparin-Sepharose. DNA polymerase delta was further purified by heparin-Sepharose and fast protein liquid chromatography. Purified DNA polymerase delta was resistant to the DNA polymerase alpha inhibitors BuPdGTP and BuAdATP and did not react with DNA polymerase alpha monoclonal and polyclonal antibodies. Based on this isolation protocol we can start to test biochemically the hypothesis whether DNA polymerase delta and DNA polymerase alpha might act coordinately at the replication fork as leading and lagging strand replicases, respectively.
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PMID:Calf thymus DNA polymerase delta: purification, biochemical and functional properties of the enzyme after its separation from DNA polymerase alpha, a DNA dependent ATPase and proliferating cell nuclear antigen. 289 82

Vertebrate nonmuscle myosins contain two phosphorylatable light chains. The maximum rate, Vmax, of the actin-activated adenosinetriphosphatase (ATPase) of unphosphorylated calf thymus myosin was found to be about 100 nmol/(min X mg), the same as that of thymus myosin with two phosphorylated light chains. However, the Kapp (actin concentration required to achieve 1/2 Vmax) of the unphosphorylated myosin was 15-20-fold greater than that of the phosphorylated myosin. When actin complexed with either skeletal muscle tropomyosin or calf thymus tropomyosin was used, the values for Vmax were about the same as those obtained with F-actin. In the presence of skeletal muscle tropomyosin, the Kapp of the unphosphorylated myosin was only 2-3-fold greater than that of the phosphorylated myosin, and in the presence of thymus tropomyosin, there was about a 5-fold difference in their Kapp values. Thus, light chain phosphorylation regulates the actin-activated ATPase of thymus myosin not by increasing Vmax but rather by decreasing the Kapp of this myosin for actin. These rather small differences in Kapp suggest that other proteins may be involved in the regulation of the actin-activated ATPase of thymus myosin. Regulated actin (actin plus skeletal muscle troponin-tropomyosin) was used to examine possible effects of thin-filament regulatory proteins. In the presence of calcium, phosphorylation caused only a slight increase in Vmax and a 2-fold decrease in Kapp of the regulated actin-activated ATPase of thymus myosin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Phosphorylation of thymus myosin increases its apparent affinity for actin but not its maximum adenosinetriphosphatase rate. 293 21

Limited proteolysis was used to identify regions on the heavy chains of calf thymus myosin which may be involved in ATP and actin binding. Assignments of the various proteolytic fragments to different parts of the myosin heavy chain were based on solubility, gel filtration, electron microscopy, and binding of 32P-labeled regulatory light chains. Chymotrypsin rapidly cleaved within the head of thymus myosin to give a 70,000-dalton N-terminal fragment and a 140,000-dalton C-terminal fragment. These two fragments did not dissociate under nondenaturing conditions. Cleavage within the myosin tail to give heavy meromyosin occurred more slowly. Cleavage at the site 70,000 daltons from the N-terminus of the heavy chain caused about a 30-fold decrease in the actin concentration required to achieve half-maximal stimulation of the magnesium-adenosinetriphosphatase (Mg-ATPase) activity of unphosphorylated thymus myosin. The actin-activated ATPase activity of this digested myosin was only slightly affected by light chain phosphorylation. Actin inhibited the cleavage at this site by chymotrypsin. In the presence of ATP, chymotrypsin rapidly cleaved the thymus myosin heavy chain at an additional site about 4000 daltons from the N-terminus. Cleavage at this site caused a 2-fold increase in the ethylenediaminetetraacetic acid-ATPase activity and 3-fold decreases in the Ca2+- and Mg-ATPase activities of thymus myosin. Thus, cleavage at the N-terminus of thymus myosin was affected by ATP, and this cleavage altered ATPase activity. Papain cleaved the thymus myosin heavy chain about 94,000 daltons from the N-terminus to give subfragment 1.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effects of proteolysis on the adenosinetriphosphatase activities of thymus myosin. 295 19

The effects of light chain phosphorylation on the actin-activated ATPase activity and filament assembly of calf thymus cytoplasmic myosin were examined under a variety of conditions. When unphosphorylated and phosphorylated thymus myosins were monomeric, their MgATPase activities were not activated or only very slightly activated by actin, but when they were filamentous, their MgATPase activities were stimulated by actin. The phosphorylated myosin remained filamentous at lower Mg2+ concentrations and higher KC1 concentrations than did the unphosphorylated myosin, and the myosin concentration required for filament assembly was lower for phosphorylated myosin than for unphosphorylated myosin. By varying the myosin concentration, it was possible to have under the same assay conditions mostly monomeric myosin or mostly filamentous myosin; under these conditions, the actin-activated ATPase activities of the filamentous myosins were much greater than those of the monomeric myosins. The addition of phosphorylated myosin to unphosphorylated myosin promoted the assembly of unphosphorylated myosin into filaments. These results suggest that phosphorylation may regulate the actomyosin-based motile activities in vertebrate nonmuscle cells by regulating myosin filament assembly.
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PMID:Filament assembly and regulation of the actin-activated ATPase activity of thymus myosin. 297 5

Sheep T lymphocytes showed a cell surface magnesium-dependent adenosine triphosphatase (Mg2+-ATPase) reaction, which is reported to be characteristic of human B lymphocytes. In cryostat sections of lymph nodes, spleen and thymus, Mg2+-ATPase positive regions closely matched those labelled by sheep pan T monoclonal antibodies (Moab). An Mg2+-ATPase reaction was also found in fibroblastic recticulum cells of T cell regions in lymph nodes. Double labelling of cells from peripheral blood and peripheral lymph for Mg2+-ATPase and the pan T marker showed that 78% of the lymphocytes were positive for both of these markers. In cell suspensions enriched for B lymphocytes the percentage of cells positively labelled was decreased to 37%. Samples of each cell population which were labelled with a pan T Moab and analysed by flow microfluorometry revealed T cell levels which were not significantly different from those obtained by histochemical or immunohistochemical techniques. Less than 1% of lymphocytes positive for heavy and light chains of immunoglobulin (Ig) G were labelled with Mg2+-ATPase. Veiled cells in lymph and monocytes showed a cytoplasmic Mg2+-ATPase reaction.
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PMID:Mg2+-dependent adenosine triphosphatase: an enzyme marker for ovine T lymphocytes. 297 23

We report here the large scale purification of DNA topoisomerase II from calf thymus glands, using the unknotting of naturally knotted P4 phage DNA as an assay for enzymatic activity. Topoisomerase II was purified more than 1300-fold as compared to the whole cell homogenate, with 22% yield. Analysis of the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two bands of apparent molecular masses of 125 and 140 kDa. Tryptic maps of the two bands indicated that they derive from the same protein. Using these fragments, specific polyclonal antisera to topoisomerase II were raised in rabbits. Immunoblotting of whole cell lysates from various species indicated that topoisomerase II is well conserved among mammals and has a native subunit molecular mass of 180 kDa. Analytical sedimentation and gel filtration were used to determine a sedimentation coefficient of 9.8 S and a Stokes radius of 68 A. The calculated solution molecular mass of 277 kDa implies a dimer structure in solution. The purified topoisomerase II unknots P4 DNA in an ATP-dependent manner and is highly stimulated in its relaxation activity by ATP. A DNA-stimulated ATPase activity, as has been found with other type II topoisomerases, is associated with the purified enzyme. Approximate kinetic parameters for the ATPase reaction were determined to be: a Vmax of 0.06 nmol of ATP/(micrograms of protein) (min) and Km of 0.2 mM in the absence of DNA, and a Vmax of 0.2 nmol of ATP/(micrograms of protein) (min) and Km of 0.4 mM ATP in the presence of supercoiled plasmid DNA.
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PMID:Purification and characterization of a type II DNA topoisomerase from bovine calf thymus. 298 21

The present studies concern the effects of triiodothyronine (T3) on rat thymic and splenic NaK-ATPase activities. When hypothyroid rats were given a single injection of T3 (250 micrograms/100 g body weight), thymic NaK-ATPase activity increased by 18% at 24 h (p less than 0.005), and remained at this peak at 72 h. Injection of T3 (250 micrograms/100 g body weight, on alternate days X 3) significantly augmented NaK-ATPase activity from the thymus and spleen of hypothyroid rats by 19 and 49%, respectively. Two weeks of daily injections of T3 (1 microgram/100 g body weight) to hypothyroid rats significantly increased NaK-ATPase activity in the thymus and the spleen by 14 and 28%, respectively. Reverse T3 had no significant effect of thymic and splenic NaK-ATPase activities. Mg-ATPase and 5'-nucleotidase activities were not significantly affected by T3 in either tissue under different thyroid states. The lack of response of thymic and splenic NaK-ATPase to reverse T3, and Mg-ATPase and 5'-nucleotidase to T3, substantiates the specificity of the hormone action on NaK-ATPase. To investigate whether the T3-dependent increase in NaK-ATPase activity is a result of an increase in the number of enzyme units, the effect of T3 on the quantity of phosphorylated intermediate of NaK-ATPase was assessed in the partially purified membrane fraction. In the euthyroid state, NaK-ATPase activity and phosphorylated intermediate units were 33 and 43% higher, respectively than in the hypothyroid state, implying that the T3-dependent increase in NaK-ATPase activity is a result of an increase in the number of NaK-ATPase units.
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PMID:Thyroidal regulation of rat thymic and splenic (Na+ + K+)-adenosine triphosphatase. 300 78


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