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:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Although most of the rat-liver AP (apurinic/apyrimidinic) endonuclease is in chromatin, some activity is found in microsomes. A quantitative assay of the microsomal AP endonuclease is described. The enzyme is a peripheral membrane protein that is located on the outside surface of microsomes. All the binding sites on the microsomes appear to have the same affinity for the AP endonuclease, suggesting the presence of receptors for the enzyme. The AP endonuclease is displaced from its membrane attachment by submicromolar concentrations of the karyophilic signal of SV-40 T antigen. The AP endonuclease receptors are likely to be on the cytosolic side of the endoplasmic reticulum. It is suggested that binding of the protein to these receptors might be the first step of the transport mechanism that enables the AP endonuclease to penetrate into the nucleus. The same mechanism utilizing the same receptors might be used by other karyophilic proteins, including SV-40 T antigen.
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PMID:The rat-liver microsomal AP endonuclease. The endoplasmic reticulum is presented as a net thrown into the cytosol to capture newly synthesized karyophilic proteins. 247 70

Outbred New Zealand white rabbits exhibit two phenotypes, 21H and 21L, corresponding to rates greater than or less than 1 nmol/min/mg, respectively, for liver microsomal progesterone 21-hydroxylase activity. In contrast, the inbred strain III/J exhibits only the 21L phenotype. Two 21H male New Zealand white rabbits were mated with several female III/J rabbits to produce a total 46 progeny. Both the 21H and 21L phenotypes were evident among male and female offspring in roughly equal numbers. Backcrosses between 21L progeny and III/J rabbits exhibit only the 21L phenotype, whereas 21H offspring yield both 21H and 21L progeny when backcrossed to the 21L inbred strain III/J. These results are consistent with autosomal dominant inheritance of the 21H phenotype. Analysis of Southern blots of genomic DNA digested with the restriction endonuclease KpnI reveals 20-, 13-, and 9-kb fragments that hybridize with a probe derived from the 3'-untranslated region of the 21-hydroxylase cDNA. The 13-kb band is not observed for strain III/J or 21L progeny of strain III/J crossed with 21H rabbits, but it is detected for both 21H fathers and 21H progeny indicating that the genetically determined difference of 21-hydroxylase expression is inherited cis to the gene for P450IIC5, the hepatic progesterone 21-hydroxylase. Electrophoretic analysis of P450IIC5 synthesized in vitro from mRNA isolated from 21L and 21H rabbits reveals that little or no P450IIC5 is synthesized from 21L mRNAs. A second immunoreactive, electrophoretically distinct protein is synthesized from both 21L and 21H mRNAs to a similar extent but in lesser amounts than P450IIC5. The second protein could represent either an allozymic form of the enzyme or the product of a distinct locus. Thus, it is likely that distinct structural genes for P450IIC5 contribute to the differences in P450-mediated metabolism in 21L as compared to 21H rabbits.
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PMID:Genetic contributions to the variation among rabbits of liver microsomal deoxycorticosterone synthesis. 257 May 49

Acetoxyoxirane, the epoxide of vinyl acetate and a potential reactive intermediate, was synthesized and characterized by 13C-nuclear magnetic resonance (13C-NMR) and mass spectroscopy. The compound induced lesions (endonuclease-sensitive and alkali-labile sites) in supercoiled PM2 DNA in vitro and was directly mutagenic toward Salmonella typhimurium TA100. The mutagenicity of the epoxide in phosphate buffer (pH 7.4, 37 degrees C) decreased, with an initial half-life of 2.8 minutes, and mutagenicity was completely abolished by addition of S-9 mix. Acetoxyoxirane did not induce unscheduled DNA synthesis on incubation with Syrian hamster embryo fibroblasts (SHE cells). These findings may possibly be explained by an effective inactivation of acetoxyoxirane by esterases when these are present in the biological system. This view is consistent with the lack of acetoxyoxirane detected in rat liver microsomal incubations of vinyl acetate.
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PMID:Synthesis and genotoxicity of acetoxyoxirane, the epoxide of vinyl acetate. 307 35

Binding interactions between the membrane-associated vitamin K-dependent carboxylase and its prothrombin and factor X substrates have been investigated in liver microsomes. Both substrates are firmly attached to microsomal membrane fragments which also harbor the carboxylase. In vitro 14CO2 gamma-carboxylation of these substrates, triggered by reduced vitamin K1H2, resulted in release of 14C-labeled prothrombin precursors from the membrane fragments, but no release of 14C-labeled factor X precursors could be demonstrated, which suggested a difference in early processing of these substrates by the carboxylase. Warfarin treatment of rats resulted in a 3-fold increase in the membrane concentration of factor X antigens and a 20-fold increase in 14C gamma-carboxylation of the membrane pool of factor X carboxylase substrates. There was a dose-response relationship between the amount of drug administered to the rats and 14C labeling of the membrane pool of factor X carboxylase substrates. On the other hand, the membrane concentration of prothrombin antigens did not increase in response to the drug, and 14CO2 gamma-carboxylation of the membrane pool of prothrombin carboxylase substrates was the same in warfarin and saline-treated rats. The results demonstrate significant differences in the interaction between the carboxylase and its prothrombin and factor X substrates. It appears that the different interactions result from binding of the prothrombin and the factor X precursors to separate microsomal membrane proteins that are involved in the gamma-carboxylation reaction. Warfarin appears to induce the factor X precursor-specific but not the prothrombin precursor-specific binding proteins, which suggests a new mechanism for the action of warfarin. These binding proteins may be under different genetic control. Treatment of the prothrombin and the factor X carboxylase substrates with endonuclease H showed that the rat prothrombin and the human factor X carboxylase substrates are high mannose glycoproteins. The human prothrombin and the rat factor X carboxylase substrates did not, on the other hand, change their migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels after endonuclease H treatment. The data demonstrate differences in the glycoprotein nature of the rat and the human carboxylase substrates.
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PMID:Early processing of prothrombin and factor X by the vitamin K-dependent carboxylase. 329 Feb 18

The identity of the primary in vitro translation products of fetal sheep nuchal ligament elastin mRNA was confirmed as two distinct polypeptides of 63 Kdal and 65 Kdal in both rabbit reticulocyte and wheat germ extract cell-free translation systems. Both polypeptides were co-translationally processed by a microsomal membrane signal peptidase, with the removal of 20-25 amino acid residues. A single (3,5 kb) RNA species encodes both tropoelastin polypeptides. Restriction endonuclease mapping of sheep genomic DNA by hydridization with two radiolabelled genomic DNA fragments containing sequences coding for sheep tropoelastin (pSE1-1,3 and pSE1-0.7,) indicated the presence of a single elastin gene. The elastin gene copy number was further quantitated by comparison of hybridisation of pSE1-1.3 and pSE1-0.7 to slot-blots and Southern transfers of sheep genomic DNA and to standard curves constructed with each clone. These results clearly demonstrate that each of these sequences is represented only once per haploid genome, suggesting that the two tropoelastin polypeptides are products of a single elastin gene.
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PMID:The gene coding for tropoelastin is represented as a single copy sequence in the haploid sheep genome. 360 4

1. After dimethylnitrosamine (DMNA) administration to mice, the content of poly(A)-containing RNA decreases rapidly in the postmicrosomal fraction of the liver. We report here that the loss of free mRNA is not a result of increased nucleolytic activity. On the contrary, a decreased activity of microsomal endonuclease, assayed by its effect on polyribosomal mRNA, was demonstrated already 15 min after the administration of DMNA at 37.5 mg/kg body wt. The loss of activity was more pronounced in the rough than in the smooth membranes. Total detergent-released microsomal nucleases, as assayed by use of labelled poly(U) as substrate, showed a less rapid decline. No corresponding increase in enzyme activities was observed in the postmicrosomal fraction. 2. The dimethylnitrosamine effect on the microsomal endonuclease was not accounted for by altered lysosomal contamination of the microsomal fraction. 3. No early effect of dimethylnitrosamine administration was found on the cytoplasmic ribonuclease inhibitor.
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PMID:Non-involvement of nucleolytic activities in the early effect of dimethylnitrosamine on the content of free mRNA in mouse liver. 627 31

We have determined the molecular genetic basis of congenital adrenal hyperplasia due to 21-hydroxylase (21-OHase) deficiency. This common disorder of cortisol biosynthesis is HLA-linked. The haplotype HLA-(A3);Bw47;DR7 is strongly associated with 21-OHase deficiency and always carries a null allele at the locus encoding the C4A (Rodgers) form of the fourth component (C4) of complement. It seemed likely that this haplotype carries a deletion encompassing the genes encoding both C4A and 21-OHase. We hypothesized that the HLA-linked defect involved a structural gene for the adrenal microsomal cytochrome P-450 specific for steroid 21-hydroxylation. Using a plasmid with a 520-base-pair bovine adrenal cDNA insert encoding the middle third of the cytochrome P-450 polypeptide, we compared hybridization patterns in DNA from normal and 21-OHase-deficient individuals. Normal human DNA yielded two fragments that hybridized with the probe after digestion with either restriction endonuclease EcoRI [12- and 14-kilobase (kb) fragments] or Taq I (3.7 and 3.2 kb). One of these bands (the first mentioned in each digest) was absent in DNA from a cell line derived from a patient homozygous for HLA-Bw47. DNA from six unrelated patients homozygous for 21-OHase deficiency who were heterozygous for HLA-Bw47 yielded diminished relative intensity of the 3.7-kb Taq I band in five patients, consistent with a heterozygous deletion, and complete disappearance of the 3.7-kb band in one. This deletion segregated with HLA-Bw47 in a large pedigree carrying 21-OHase deficiency and HLA-Bw47. Thus, 21-OHase deficiency sometimes results from the deletion of a specific cytochrome P-450 gene and sometimes, presumably, from smaller mutations. This gene is probably located very near the C4A gene.
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PMID:HLA-linked congenital adrenal hyperplasia results from a defective gene encoding a cytochrome P-450 specific for steroid 21-hydroxylation. 633 10

Congenital adrenal hyperplasia due to 21-hydroxylase (21-OH) deficiency is HLA-linked. The haplotype HLA-(A3);Bw47;DR7 is strongly associated with 21-OH deficiency and always carries a null allele at the complement C4A (Rodgers) locus. It seemed likely that this haplotype carries a deletion encompassing both the C4A and 21-OH loci. We hypothesized that the HLA-linked defect involved a structural gene for the adrenal microsomal cytochrome P-450 specific for steroid 21-hydroxylation. We isolated a plasmid with a 520 bp bovine adrenal cDNA insert encoding the middle third of the P-450 peptide. When human DNA was digested with Taq I restriction endonuclease and hybridized with the cDNA probe, DNA from 13 unrelated normal individuals yielded two hybridizing bands of equal intensity at 3.7 and 3.2 kb. The upper band was not present in DNA from a patient homozygous for Bw47. DNA from six unrelated patients heterozygous for Bw47 yielded, in five, diminished relative intensity of the upper band consistent with a heterozygous deletion, and complete disappearance of the upper band in one. Thus 21-OH deficiency sometimes results from the deletion of a gene and sometimes, presumably, from smaller mutations. This gene is probably located very near the C4A gene encoding the 4th component of complement.
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PMID:Molecular cloning of steroid 21-hydroxylase. 633 60

Using polymerase chain reaction (PCR), we have isolated a cDNA that encodes a rat liver carboxylesterase. This novel enzyme, designated hydrolase C, is structurally very similar to hydrolase B, a microsomal carboxylesterase expressed in rat liver and kidney. Hydrolase B and C are 96% identical in nucleotide sequence and 93% identical in deduced amino acid sequence. Both enzymes have an 18-amino-acid signal peptide at the N-terminus. The C-terminus of hydrolase B and C contains an HXEL consensus sequence for retaining proteins in the endoplasmic reticulum. As expected, when the cDNA encoding hydrolase C was expressed in a baculovirus/Sf21 cell system, the recombinant enzyme was localized in the endoplasmic reticulum. Hydrolase B and C both have putative N-linked glycosylation sites at Asn1 and Asn61. The active site of hydrolase B and C appears to be composed of a nucleophile, Ser203, a basic residue, His448, and an acidic residue, either Asp97 or Glu228. Based on cloning experiments, restriction endonuclease mapping and Northern blotting, hydrolase B is expressed in both rat liver and kidney, whereas hydrolase C is expressed predominantly, perhaps exclusively, in liver. When expressed in Escherichia coli, hydrolase C was catalytically inactive and unstable, but when expressed in the baculovirus/Sf21 cell system hydrolase C it was stable and catalytically active toward 1-naphthylacetate and esters of para-nitrophenol. Hydrolase C is the fourth member of the rat carboxylesterase family to be cloned and sequenced. In terms of nucleotide and deduced amino acid sequence, hydrolase C is highly similar to hydrolase B, but differs from hydrolase B in terms of its catalytic activity and tissue distribution. Recombinant hydrolase C has properties similar to those described for esterase RL2, which was purified from rat liver microsomes by Hosokawa et al. (Arch. Biochem. Biophys. 277, 219-227, 1990), although additional studies will be required to establish conclusively the identity of this enzyme. The high degree of sequence identity (96%) between hydrolase B and C, particularly in the 3' untranslated region, suggests that the genes encoding these two carboxylesterases evolved by duplication and divergence of a common ancestral gene.
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PMID:Cloning and expression of hydrolase C, a member of the rat carboxylesterase family. 787 88

Internucleosomal DNA fragmentation (DNA laddering) and formation of apoptotic bodies have long been considered characteristic features of apoptosis. However, recent work has shown that formation of high molecular weight DNA fragments precedes internucleosomal cleavage and may involve mechanisms that differ from those responsible for DNA laddering. Here, we show that glucocorticoid treatment of human thymocytes stimulated the formation of high molecular weight DNA fragments by Ca(2+)- and endonuclease-mediated mechanisms. Either the removal of Ca2+ from the medium or pretreatment of the cells with the intracellular Ca2+ chelator, BAPTA-AM, prevented the formation of large DNA fragments. Further, treatment of the thymocytes with the microsomal Ca(2+)-ATPase inhibitor, thapsigargin, which caused a sustained increase in intracellular Ca2+ concentration, was in itself sufficient to activate high molecular weight DNA fragmentation. Our results show that Ca(2+)-dependent mechanisms promote the multistep chromatin cleavage in human thymocyte apoptosis.
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PMID:Involvement of Ca2+ in the formation of high molecular weight DNA fragments in thymocyte apoptosis. 803 2


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