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:3.1.21.1 (DNase)
7,655 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human erythrocyte cytoskeletal ATPase was extracted with 0.2 mM ATP (pH 8.0) from Triton X-100 treated ghosts. The ATPase fraction contained mainly spectrin, actin, and band 4.1. When the ATPase fraction was applied to a Sepharose 4B column, 90% of the ATPase activity was recovered in a spectrin, actin, and band 4.1 complex fraction and none was detected in the spectrin fraction. A specific activity of the complex ATPase was 60-120 nmol/(mg protein X h). No ATPase activity was detected in the presence of EDTA. The presence of magnesium in the incubation medium was essential for the ATPase activity. The activity was activated by KCl and was almost completely inhibited by 10(-5) M free calcium in the presence of 0.2 mM MgCl2. The Ki for Ca2+ was 7 X 10(-7) M. Phalloidin and DNase 1, which affect actin, inhibited this K,Mg-ATPase activity by 95%, but cytochalasin B did not inhibit it. N-Ethylmaleimide activated the ATPase 1.6-fold. The order of affinity for nucleotides was ATP greater than ITP greater than CTP, ADP, AMP-PNP, GTP. A specific ATPase activity of purified actin was 50 nmol/(mg X h). These results suggest that the cytoskeletal ATPase is actin ATPase and the actin ATPase is activated by spectrin and band 4.1.
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PMID:Characterization of human erythrocyte cytoskeletal ATPase. 294 69

Epithelial and stromal cells were isolated from endometrium of Day 1 pseudopregnant rabbits by enzymatic digestion with trypsin or trypsin:collagenase:deoxyribonuclease. Dispersed cells were grown in RPMI 1640 supplemented with 10% whole or steroid-depleted fetal bovine serum (FBS). Epithelial and stromal cells reached confluency after 6 to 7 days in culture and showed specific characteristics. Cells could be differentiated according to morphology, growth patterns, electrophoretic patterns, and response to estrogen or progesterone. Hormonal stimulation of adenylate cyclase activity was measured in broken cell preparations by catalytic transformation of alpha-32P-adenosine triphosphate into 32P-adenosine 3'-5' cyclic monophosphate (cAMP). Adenylate cyclase activity was present in fresh endometrial tissue and in dispersed cells after 7 days in culture. The enzyme activity was significantly higher in stromal than in epithelial cells at all stimulation levels: basal (9.2 +/- 1.0 vs. 2.3 +/- 0.6, p less than 0.001) and guanosine triphosphate (GTP, 300 microM) (25.4 +/- 2.9 vs. 7.0 +/- 1.6, p less than 0.001). Net response to prostaglandin E2 (PGE2, 10 microM) was three times higher (p less than 0.001) in stromal (17 +/- 2) than in epithelial (5.0 +/- 1) cells. These results suggest that PGE2 can stimulate adenylate cyclase in rabbit endometrium and that the enzyme is preferentially localized in the stroma. Our results are in agreement with the hypothesis that cAMP formed in endometrium in response to PGE2 might be involved in the decidual reaction.
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PMID:Cell-specific localization of prostaglandin E2-sensitive adenylate cyclase in rabbit endometrium. 347 35

In the preceding paper [Riedel & Fasold (1987) Biochem. J. 241, 203-212] we have described a procedure for the preparation of nuclear-envelope vesicles (NE vesicles) from rat liver nuclei. These vesicles, which are largely free of components of the nuclear interior, were employed in an assay system in vitro to study protein translocation across the NE. We found that nuclear proteins such as histones, high-mobility-group proteins and acidic chromosomal proteins are specifically taken up and accumulated in the NE vesicles, whereas there is little or no affinity for non-nuclear proteins like immunoglobulin, myoglobin and cytochrome c. The kinetics of histone uptake into the NE vesicles are similar to those obtained for whole rat liver nuclei, and comparative studies with non-vesicular NEs prepared by deoxyribonuclease I-treatment (DNAase-NEs) indicate that the NE of the vesicles affects the uptake kinetics and increases the capacity for nuclear proteins. The uptake of histones into NE vesicles, but not the binding to DNAase-NEs, can be stimulated by GTP and GDP. Furthermore, we found that even very large molecules can be entrapped in the vesicles during their preparation. These results indicate that the NE vesicles might provide a useful system in vitro with which to investigate the structures and mechanisms involved in protein translocation across the NE.
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PMID:Nuclear-envelope vesicles as a model system to study nucleocytoplasmic transport. Specific uptake of nuclear proteins. 356 9

Cell ghosts have been prepared from mature chicken erythrocytes using 0.05% saponin. Such preparations are capable of incorporating label from [3H]UTP and provide a system, where the nucleus is permeable to nucleotides and macromolecules, for studying the low-level RNA synthesis characteristic of these cells. RNase A (50 micrograms/ml) eliminated all radioactivity binding to DE-81 filters, indicating that the product was RNA; and DNase (10 micrograms/ml) and actinomycin D (10 micrograms/ml) each inhibited UMP incorporation by 70%, suggesting that the synthesis was DNA-dependent. Polymerization was inhibited 90% by 0.1 microgram/ml alpha-amanitin, and maximum synthesis occurred in the presence of high salt (0.175 M KCl) and Mn2+ (0.5 mM). Polyacrylamide gel electrophoresis indicated that the newly synthesized RNA was heterogeneous in size, having a distribution from 5 to 60 S with a significant fraction migrating as 8-12 S. Approximately 15% of the total RNA was bound by an oligo(dT)-cellulose column, suggesting that some RNA processing was occurring, although attempts to detect the incorporation of label from [alpha-32P]GTP into a 5'-cap structure were unsuccessful. In comparison to RNA synthesis in reticulocyte nuclei, both the rate and extent of transcription in erythrocyte nuclei were much reduced. Moreover, about 25-30% of the reticulocyte nascent RNA was released from the nuclei during a 60-min incubation, while no release was observed for the erythrocyte nuclei. Hybridization of radiolabeled RNA to excess chicken DNA indicated that the majority (80%) of the in vitro transcripts were complementary to unique sequence DNA (C0t1/2 = 4.5 X 10(3)). When RNA synthesized by either erythrocyte or reticulocyte nuclei was hybridized to cDNA complementary to reticulocyte polysomal mRNA, about 8% of the reticulocyte nuclear RNA but less than 1% of the erythrocyte nuclear RNA were resistant to RNase A digestion. Taken together, these data suggest that nuclei prepared by saponin lysis of chicken erythrocytes synthesize messenger-like RNA via endogenous polymerase II activity. A fraction of this RNA is polyadenylated but contains few, if any, globin sequences or other transcripts found on reticulocyte polysomes.
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PMID:Synthesis of messenger-like RNA in avian erythrocyte nuclei. 390 27

A cytosolic factor that stimulates transcription in isolated nuclei was purified approximately 4000-fold to near homogeneity from rat liver. The molecular weight of the factor was determined as 47 000 by SDS-polyacrylamide gel electrophoresis. The factor had no detectable deoxyribonuclease and protease activity but showed ribonuclease inhibitor activity. The factor could stimulate transcription in isolated nuclei by 50% at about 3.0 ng and the maximal stimulation was about 100%. When [gamma-S]ATP and [gamma-S]GTP were included in the reaction, the factor stimulated the synthesis of RNA which was able to bind to a mercury-Sepharose column and about 80% of the bound RNA was sensitive to a low concentration of alpha-amanitin. When heparin was added before initiation to preincubation mixture containing RNA polymerases II and DNA, a small but definite incorporation of [14C]UTP was observed. The factor alone had no stimulatory effect on the heparin-resistant incorporation of [14C]UTP but, in the presence of two rat liver nuclear fractions, phosphocellulose 0.5 and 1 M KCl step fractions, could stimulate the incorporation above the level with the combination of the two nuclear fractions. Antibody raised against the factor inhibited accurate transcription from the adenovirus 2 major late promoter in a nuclear lysate from Ehrlich ascites tumor cells, and the inhibition was neutralized by the factor.
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PMID:Purification of a cytosolic factor from rat liver that stimulates transcription in isolated nuclei and its action on purified RNA polymerase II-DNA system. 407 43

During replication of polyoma DNA in isolated nuclei, RNA was found attached to the 5' ends of growing progeny strands. This RNA starts with either ATP or GTP and can be labeled at its 5' end with (32)P from beta-labeled nucleotides. Digestion of progeny strands with pancreatic DNase released (32)P-labeled RNA that, on gel electrophoresis, gave a distinct peak in the position expected for a decanucleotide. We believe that this short RNA is involved in the initiation of the discontinuous synthesis of DNA and propose the name "initiator RNA" for it. The covalent linkage of initiator RNA to 5' ends of growing DNA chains was substantiated by the finding that (32)P was transferred to ribonucleotides by alkaline hydrolysis of purified initiator RNA obtained by DNase digestion of polyoma progeny strands synthesized from [alpha-(32)P]dTTP. While initiator RNA was quite homogeneous in size, it had no unique base sequence since digestion with pancreatic RNase of initiator RNA labeled at its 5' end with (32)P released a variety of different [(32)P]oligonucleotides. The switch from RNA to DNA synthesis during strand elongation may thus depend on the size of initiator RNA rather than on a specific base sequence.
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PMID:Initiator RNA in discontinuous polyoma DNA synthesis. 437 33

Rat liver mitochondria isolated in sucrose-N-tris(hydroxymethyl)methyl-2-aminoethane-sulphonic acid (TES) incorporated [(3)H]UTP into RNA for 1h. Incorporation was inhibited 50% by 1mug of actinomycin D/ml, 1mug of acriflavine/ml and 0.5mug of ethidium bromide/ml but was insensitive to rifampicin, rifamycin SV, streptovarcin and deoxyribonuclease. After the first 10min of incubation, the synthesis was insensitive to ribonuclease. RNA synthesis by mitochondria isolated in sucrose-EDTA was insensitive to actinomycin D and sensitive to ribonuclease during the first 10min of the incubation but thereafter the sensitivities were the same as for mitochondria isolated in sucrose-TES. In a hypo-osmotic medium the relative extent of incorporation of the four ribonucleoside triphosphates into RNA was CTP>UTP=ATP>>GTP. In an iso-osmotic medium the incorporation of CTP and GTP decreased. All four nucleotides were incorporated into RNA in a DNA-dependent process, as indicated by the inhibition by actinomycin D. In addition, CTP and ATP were incorporated into the CCA end of mitochondrial tRNA. ATP was also incorporated into an unidentified acid-insoluble compound, which hydrolysed in alkali to a product that was not ATP, ADP or 5'- or 2(3')-AMP. Atractyloside inhibited the incorporation of ATP into RNA with 50% inhibition at 2-3nmol/mg of protein. The [(3)H]UTP-labelled RNA had peaks of 16S and 13S characteristic of mitochondrial rRNA. In addition a peak at 20-21S was observed as well as heterogeneous RNA sedimenting throughout the gradient. The synthesis of all these species was inhibited by actinomycin D, indicating that rat liver mitochondrial DNA codes for mitochondrial rRNA as well as other as yet unidentified species.
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PMID:Synthesis of ribonucleic acid by isolated rat liver mitochondria. 440 94

Poly(A) polymerase activity is induced during vaccinia virus infection of HeLa cells. The enzyme is maximally induced at 3.5 h postinfection. Partial purification frees the preparation of RNase activity and RNA polymerase activity. ATP is the substrate for poly(A) synthesis. A small amount of poly(A) is produced from added adenosine diphosphate due to the production of ATP by an adenylate kinase present in the preparation. The incorporation of ATP into poly(A) is dependent on divalent cations (Mg(2+) or Mn(2+)) and is not inhibited by UTP, CTP, or GTP. Poly(U) stimulates ATP incorporation; poly(A) and poly(C) have little effect on ATP incorporation, and poly(dT) is extremely inhibitory. RNA prepared from HeLa cells and from the partially purified poly(A) polymerase (the enzyme preparation contains endogenous RNA [Brakel and Kates]) stimulates ATP incorporation by poly(A) polymerase which was subjected to DEAE-cellulose chromatography. RNase's, pancreatic and T(1), inhibit the production of poly(A). DNase has little effect. Poly(U) is able to stimulate poly(A) production in the presence of T(1) RNase.
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PMID:Poly(A) polymerase from vaccinia virus-infected cells. I. Partial purification and characterization. 441 6

1. The 105000g supernatant fraction of rat liver catalyses the incorporation of ribonucleotides from ribonucleoside triphosphates into polyribonucleotide material. The reaction requires Mg(2+) ions and is enhanced by the addition of an ATP-generating system and RNA, ATP, UTP and CTP but not GTP are utilized in this reaction. In the case of UTP, the product is predominantly a homopolymer containing 2-3 uridine residues, and there is evidence that these may be added to the 3'-hydroxyl ends of RNA or oligoribonucleotide primers. 2. The microsome fraction of rat liver incorporates ribonucleotides from ATP, GTP, CTP and UTP into polyribonucleotide material. This reaction requires Mg(2+) ions and is enhanced slightly by the addition of an ATP-generating system, and by RNA but not DNA. Supplementation of the reaction mixture with the three complementary ribonucleoside 5'-triphosphates greatly increases the utilization of a single labelled ribonucleoside 5'-triphosphate. The optimum pH is in the range 7.0-8.5, and the reaction is strongly inhibited by inorganic pyrophosphate and to a much smaller degree by inorganic orthophosphate. It is not inhibited by actinomycin D or by deoxyribonuclease. In experiments with [(32)P]UTP in the absence of ATP, GTP and CTP, 80-90% of (32)P was recovered in UMP-2' or -3' after alkaline hydrolysis of the reaction product. When the reaction mixture was supplemented with ATP, GTP and CTP, however, about 40% of the (32)P was recovered in nucleotides other than UMP-2' or -3'. Although the reactions seem to lead predominantly to the synthesis of homopolymers, the possibility of some formation of some heteropolymer is not completely excluded.
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PMID:Chain extension of ribonucleic acid by enzymes from rat liver cytoplasm. 568 1

We employed the photoaffinity probe 8-azido-adenosine 5'-triphosphate (aATP) to identify the nuclear envelope (NE) nucleosidetriphosphatase activity (NTPase) implicated in control of RNA transport. The photoprobe was hydrolyzed at rates comparable to those for ATP, with a Michaelis constant of 0.225 mM. Photolabeling was dependent upon UV irradiation (300-nm max) and was not affected by quercetin. Unlabeled ATP or GTP competed with [32P]aATP in photolabeling experiments, and UTP was a less effective competitor, paralleling the substrate specificity of the NTPase. Incubation of NE with aATP led to a UV, time, and concentration dependent irreversible inactivation of NTPase. The inactivation could be blocked by ATP or GTP. Polyacrylamide gel electrophoresis and autoradiography of photolabeled NE showed selective, UV-dependent labeling of a 46-kDa protein with both [gamma-32P]aATP and [alpha-32P]aATP. This band was not labeled with [gamma-32P]ATP. Since the NE NTPase implicated in RNA transport is modulated by RNA, we examined the effects of RNA on the labeling process. Removal of RNA from the NE preparations (by RNase/DNase digestion) reduced NTPase by 30-40% and eliminated photolabeling of the 46-kDa band. Addition of yeast RNA to such preparations increased NTPase activity to control levels and selectively reinstated photolabeling of the 46-kDa band. These results suggest that the 46-kDa protein represents the major NTPase implicated in RNA transport.
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PMID:Photoaffinity labeling of the major nucleosidetriphosphatase of rat liver nuclear envelope. 608 95


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