Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.27.1 (RNase)
 16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. The isolated nuclei of the slime mould Physarum polycephalum contain an enzyme that will incorporated [adenine-3H] NAD+ into an acid-insoluble product, which is shown to be poly(ADP-ribose). 2. This incorporation has an optimum pH of 8.2 and a temperature optimum below 10degreesC. 3. Optimum stimulation is given by 15 mM-Mg2+. 4. 2-Mercaptoethanol or dithiothreitol also stimulates the incorporation, the latter at an optimum concentration of about 1 mM. 5. Under optimum conditions the Km value for the reaction is 0.28 mM at 15degreesC. Nicotinamide inhibits the incorporation with a Ki of 5.7 muM. 6. Exogenous DNA stimulates the incorporation by about 100%. 7. Preincubation of the nuclei with deoxyribonuclease, but not with ribonuclease, almost completely inactivates the incorporation of NAD+. 8. The enzyme is unstable at both 0degrees and 15degreesC in the absence of dithiothreitol. The presence of dithiothreitol at a concentration of 1 mM stabilizes the enzyme at both these temperatures. 9. The activity of this enzyme per nucleus was shown in three separate experiments to fall by about one-half in early S phase and then to rise to its pre-mitotic value after about 3 h, that is in late S phase. 10. The possible physiological function of this enzyme system is discussed.
 ...
PMID:Poly(adenosine diphosphate ribose) polymerase in Physarum polycephalum. 23 97

In Drosophila, multiple isoforms of alpha-glycerol-3-phosphate dehydrogenase (sn-glycerol-3-phosphate: NAD+ 2-oxidoreductase, EC 1.1.1.8) are produced in a tissue- and stage-specific manner. To understand the underlying molecular basis of these isoforms, we have sequenced a 5.8-kilobase region of the Drosophila genome that contains the entire Gpdh locus. Primer-extension and RNase protection assays show that the gene consists of eight exons and has a single transcription-start point. RNase protection mapping and comparison of the genomic sequence from three different cDNA clones reveal that three protein isoforms of glycerol-3-phosphate dehydrogenase are produced by alternative processing of 3' exons. Two of the isoforms differ from the third by the addition of either three or ten amino acids to their C-terminal ends. Transcripts corresponding to two of the isoforms are expressed during both larval and adult stages, while the third isoform is produced only in adults.
 ...
PMID:Structural characterization of the alpha-glycerol-3-phosphate dehydrogenase-encoding gene of Drosophila melanogaster. 250 Jun 60

We report the DNA sequence and in vivo transcription start of pdxB, which encodes a protein required for de novo biosynthesis of pyridoxine (vitamin B6). The DNA sequence confirms results from previous minicell experiments showing that pdxB encodes a 41-kilodalton polypeptide. RNase T2 mapping of in vivo transcripts and corroborating experiments with promoter expression vector pKK232-8 demonstrated that the pdxB promoter shares its -10 region with an overlapping, divergent promoter. Thus, pdxB must be the first gene in the complex pdxB-hisT operon. The steady-state transcription level from these divergent promoters, which probably occlude each other, is approximately equal in bacteria growing in rich medium at 37 degrees C. The divergent transcript could encode a polypeptide whose amino-terminal domain is rich in proline and glutamine residues. Similarity searches of protein data bases revealed a significant number of amino acid matches between the pdxB gene product and D-3-phosphoglycerate dehydrogenase, which is encoded by serA and catalyzes the first step in the phosphorylated pathway of serine biosynthesis. FASTA and alignment score analyses indicated that PdxB and SerA are indeed homologs and share a common ancestor. The amino acid alignment between PdxB and SerA implies that PdxB is a 2-hydroxyacid dehydrogenase and suggests possible NAD+, substrate binding, and active sites of both enzymes. Furthermore, the fact that 4-hydroxythreonine, a probable intermediate in pyridoxine biosynthesis, is structurally related to serine strongly suggests that the pdxB gene product is erythronate-4-phosphate dehydrogenase. The homology between PdxB and SerA provides considerable support for Jensen's model of enzyme recruitment as the basis for the evolution of different biosynthetic pathways.
 ...
PMID:Divergent transcription of pdxB and homology between the pdxB and serA gene products in Escherichia coli K-12. 268 Nov 52

Phosphodiesterase I [EC 3.1.4.1] was purified from normal human urine in a highly purified state free from phosphodiesterase II, RNase, DNase I, DNase II, and phosphatase by column chromatographies of DEAE-Toyopearl, butyl-Toyopearl, Affi-Gel blue, and Sephadex G-150. The molecular weight of the enzyme was 1.9 x 10(5) and the pH optimum around 9.0 with p-nitrophenyl deoxythymidine 5'-phosphate as the substrate. The enzyme hydrolyzed the 3'-5' linkage of various dinucleoside monophosphates at approximately the same rate and the phosphodiester bonds of cyclic 3',5'-mononucleotides to produce mononucleoside 5'-phosphate. The enzyme also hydrolyzed ADP to 5'-AMP and Pi, ATP to 5'-AMP and PPi, and NAD+ to 5'-AMP and NMN. The enzyme activity was abolished by removal of metal ions with EDTA, and the metal-free enzyme was reactivated on the addition of Zn2+. The enzyme activity was also abolished by some reducing agents and the inhibition was reversed by Zn2+. The metal-free enzyme was less stable than the native enzyme, and Zn2+ and Co2+ restored the stability of the metal-free enzyme to the level of the native enzyme. The enzyme degraded oligonucleotides and high molecular nucleotides stepwise from the 3'-termini to give 5'-mononucleotides. The enzyme hydrolyzed single-stranded DNA more preferentially than double-stranded DNA. The enzyme also nicked superhelical covalently closed circular phi X174 DNA to yield first open circular DNA and then linear DNA.
 ...
PMID:Phosphodiesterase I in human urine: purification and characterization of the enzyme. 282 85

Native disulphide-bonded prolactin (band III) was distinguished from reduced prolactin (band II) and intermediate unstable disulphide-linked conformations by: (a) faster mobility of the former in sodium dodecyl sulphate/polyacrylamide gel electrophoresis, and (b) high-pressure liquid chromatography analyses of tryptic-digested peptides derived from prolactin in various conformations during its refolding pathway from reduced, unfolded to native conformation. The electrophoretic separation has been used to examine the state of disulphide bonding in newly synthesised prolactin translated from bovine pituitary mRNA in a rabbit reticulocyte translation system supplemented with nuclease-treated dog pancreatic microsomal membranes. The formation of correct disulphide pairing in prolactin (band III), synthesised in the in vitro translation system in the presence of pancreatic microsomes, required the presence of a thiol oxidant such as oxidised glutathione during the translation. The action of thiol oxidants on the in vitro biosynthesised and microsomally processed prolactin were both dose-dependent and catalytic; non-thiol oxidants such as NAD+ and NADP+ were ineffective. Examination of the time course of addition of oxidised glutathione to translating lysates showed that efficient and correct disulphide pairing in newly biosynthesised prolactin occurred when the oxidant was present co-translationally, but much lower yields of correctly disulphide-bonded prolactin were obtained when the oxidant was added after translation and processing were complete. The presence of protein-disulphide isomerase in dog pancreatic microsomes, employed in the in vitro translation system to process preprolactin, was demonstrated by (a) two-dimensional polyacrylamide gel electrophoresis of the membrane proteins, and (b) enzymic activity to accelerate reactivation of scrambled ribonuclease. Protein-disulphide isomerase activity was latent in intact microsomal vesicles, full activity being expressed upon sonication. A procedure has been devised to prepare pancreatic microsomal vesicles depleted of protein-disulphide isomerase which are active in processing and segregating in vitro biosynthesised prolactin. These membranes in the presence of low concentrations of oxidised glutathione are less active but in the presence of saturating levels of oxidised glutathione are fully competent in forming correct disulphide bridges in newly synthesised prolactin.
 ...
PMID:Studies on the formation of intrachain disulphide bonds in newly biosynthesised bovine prolactin. Role of protein-disulphide isomerase. 406 47

The substrate specificity of diadenosine 5',5"'-P1,P4-tetraphosphate pyrophosphohydrolase from Physarum polycephalum for dinucleoside polyphosphates has been determined by high-performance liquid chromatography (HP-LC). Elution of a strong anion-exchange resin with a pH and ionic strength gradient of ammonium phosphate separates a series of monoadenosine and diadenosine polyphosphates. Most of the corresponding guanine nucleotides are also resolved on this HPLC system. One mole each of Ap4A and Gp4G is symmetrically hydrolyzed to 2 mol of ADP and GDP, respectively. Ap3A, Ap5A, Ap6A, and Ap4 are hydrolyzed, and in each case ADP is one of the products. Gp3G, Gp5G, Gp6G, and Gp4 are also substrates, and in each case GDP is one of the products. AMP, ADP, ATP, Ap2A, ADPR, GMP, GDP, GTP, NAD+, and NADP+ are not substrates. No hydrolysis of the cap dinucleotides m7Gp3Am and m7Gp3Cm was detected by HPLC. Diadenosine tetraphosphate pyrophosphohydrolase preparations were also assayed for adenylate kinase, nucleotide diphosphate kinase, NAD(P)+ pyrophosphohydrolase, phosphodiesterase, cyclic nucleotide phosphodiesterase, phosphatase, and ribonuclease activities. These enzymic activities were not detectable in diadenosine tetraphosphate pyrophosphohydrolase. The symmetrical hydrolysis of Ap4A and Gp4G is an unique catalytic property that distinguishes diadenosine tetraphosphate pyrophosphohydrolase from P. polycephalum from diadenosine tetraphosphate phosphohydrolases from other organisms.
 ...
PMID:Diadenosine 5',5"'-P1,P4-tetraphosphate pyrophosphohydrolase from Physarum polycephalum. Substrate specificity. 629 57

Poly(ADP-ribose) synthetic activity in isolated nucleoli from rapidly growing mouse ascites tumor cells and ADP-ribosylation of the nucleolar proteins in vitro were studied. The specific activity of the synthesis in the nucleoli was significantly higher than that in the chromatin. The optimum magnesium and NAD+ concentrations, and the effect of RNase treatment on the reaction in the nucleoli were also distinctly different from those in the chromatin. Hydrolysis of the reaction product of the nucleoli with snake venom phosphodiesterase and with calf thymus poly(ADP-ribose) glycohydrolase yielded 5'-AMP and 2'-(5"-phosphoribosyl))5'-AMP, and ADP-ribose, respectively. The average chain length of the polymer formed in the nucleoli was found to be about 4 as a whole, but the distribution was heterogenous, from 1.2 to over 12. Analysis of ADP-ribosylated proteins in the nucleoli by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that several non-histone proteins with molecular weights of over 100,000 were highly ADP-ribosylated compared with other proteins including histones. This pattern was also different from that of the chromatin. These experimental results demonstrate that the nucleoli are independent from the chromatin as regards poly(ADP-ribose) synthesis in vitro.
 ...
PMID:Poly(ADP-ribose) synthesis in nucleoli and ADP-ribosylation of nucleolar proteins in mouse ascites tumor cells in vitro. 728 63

Isolated nuclei incubated with [14C]protein hydrolysate are shown to incorporate labelled amino acids into the acid-insoluble fraction. Purified chromatin and the complex of DNA with firmly bound proteins possess similar ability. The optimum pH of the reaction is 6.5-7.0, 2 mM MgCl2 stimulates incorporation, the temperature optimum is 37-40 degrees C. Chloramphenicol depresses incorporation by 70%, puromycin by 40%, cycloheximide does not affect the chromatin activity. Incorporation does not depend on the presence of ATP or GTP, and is substantially inhibited by deoxyribonuclease but not by ribonuclease treatment of chromatin or of the nuclei. Specific activity of firmly bound chromatin non-histone proteins is higher than that of labile bound ones; histones are not labelled. After pronase treatment of proteins radioactivity changes to an acid-soluble state. The molecular weight of isolated labelled polypeptides is about 6000 as shown by gel filtration and the analysis of NH2-terminal amino acids. Labelled polypeptides firmly bound to DNA consist of 7-10 amino acids. Specific activity of proteins firmly bound to DNA increases linearly with the time of incubation of chromatin with [14C]protein hydrolysate, the activity curve of labile bound non-histone proteins has a distinct sygmoid character. The polypeptide-synthesizing activity of rat liver chromatin increases between 9 h and 21 h after partial hepatectomy. Irradiation with 800 rads 30 min before the operation prevents activation of amino acid incorporation. From nine amino acids studied alanine, methionine, lysine, tyrosine and arginine are not incorporated in the system described. Glutamic acid is polymerized most effectively. Glutamine, asparagine and glycine are incorporated 7-8 times less. The data are given indicating that the incorporation is not random when an amino acid mixture is present. Preincubation of chromatin with NAD+ but not with its analogues increases the polypeptide-synthesizing activity of chromatin. The activation is prevented by thymidine and nicotinamide. Storage (18 h at 2-4 degrees C) brings about a complete loss of the polypeptide-synthesizing activity of chromatin. The ability of 'old' chromatin to incorporate amino acids can be restored by preincubating it with NAD+. Storage of chromatin in the presence of 5 mM adenosine 3',5'-monophosphate (cAMP) does not result in decrease of the polypeptide-synthesizing activity. It is assumed that poly-(ADP-ribose) is the energy source for amino acid activation in the system described.
 ...
PMID:Polypeptide-synthesizing activity of eukaryotic chromatin. Properties, dependence on poly(ADP-ribose) and connection with the cell cycle. 737 37

NAD is normally regarded as a redox molecule or as the substrate for ADP-ribosylation reactions. In this study, we describe the rapid metabolism of NAD by Percoll-gradient-purified lettuce chloroplasts and show that the adenine moiety can be incorporated into RNA in a dark-activated reaction that senses the redox state of the cytochrome b6f complex. Isolated chloroplasts rapidly metabolised radiolabelled NAD+ to 5'-AMP (within seconds) and adenosine during a 60-min incubation in vitro; the products were analysed by high-performance liquid chromatography. No radiolabelled ADP-ribose was detected. Radioactivity was incorporated into trichloroacetic-acid-insoluble material during this period, with approximately 2-4-fold more incorporation occurring in the dark. Most of this radiolabel was rendered acid-soluble by dilute alkaline digestion at 37 degrees C, yielding an approximately equal mixture of 2'-AMP and 3'-AMP, and by RNase digestion, identifying the acid-insoluble radioactive material as RNA. Protein-bound ADP-ribose would have yielded 5'-AMP and/or oligomeric/polymeric ADP-ribose after alkali digestion. The utilisation of NAD metabolites for RNA synthesis was restricted to the thylakoid compartment of the chloroplast. The use of a variety of electron-transport inhibitors such as 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, bromanil (tetrabromo-1,4-benzoquinone), electron donors (dithiothreitol), electron acceptors (ferricyanide) and an uncoupler showed that the incorporation of radiolabel from NAD into acid-insoluble material was favoured when the cytochrome b6f complex was in the oxidised state (as pertaining to incubations in the dark).
 ...
PMID:NAD turnover and utilisation of metabolites for RNA synthesis in a reaction sensing the redox state of the cytochrome b6f complex in isolated chloroplasts. 750 45

The enzyme 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-Isomerase (3 beta HSD) catalyzes the conversion of delta 5-3 beta-hydroxysteroids to delta 4-3-ketosteroids, an essential step in the biosynthesis of all biologically active steroid hormones. We previously reported the isolation of three distinct mouse cDNAs for 3 beta HSD (3 beta HSD I, II, and III) and tissue-specific expression of their mRNAs. 3 beta HSD I is expressed only in gonads and adrenal glands, and 3 beta HSD II and III are expressed in both liver and kidneys. In the current study, we present data which demonstrate that transiently expressed 3 beta HSD I and 3 beta HSD III proteins can catalyze the conversion of the delta 5-steroids, pregnenolone and dehydroepiandrosterone, to their respective delta 4-3-ketosteroids, progesterone and androstenedione. They also can dehydrogenate the 3 beta-hydroxy group of the 5 alpha-reduced steroid 5 alpha-androstanediol to yield dihydrotestosterone in the presence of the cofactor NAD+. The Km values of the expressed 3 beta HSD I (for each of these substrates) were all below 0.2 microM. Km values of 3 beta HSD III were greater for all substrates, with the greatest increase observed for pregnenolone, which was over 10-fold greater. Both forms of expressed protein can catalyze the reduction of dihydrotestosterone to 5 alpha-androstanediol in the presence of the cofactor NADH, but with considerably higher Km values (5.5 microM for form I and 6.8 microM for form III). The observed maximum velocity of form I was much higher for all substrates examined. RNase protection and immunoblot analysis of expressed 3 beta HSD I and III indicate that the difference in maximum velocity reflect differences in the steady state levels of mRNA and amounts of protein. In addition, the expressed 3 beta HSD III protein analyzed by Western blot has a lower mobility than the 3 beta HSD I protein, both similar in mol wt to the 3 beta HSD proteins detected in mouse liver and adrenal glands, respectively. These data demonstrate that an isoform of 3 beta HSD expressed in liver and kidney has the capacity to convert delta 5-3 beta-hydroxysteroids to delta 4-3-ketosteroids. The data suggest that a homologous human 3 beta HSD isoform could play an important role in cases of genetic deficiency of the gonadal and adrenal isoform.
 ...
PMID:Enzyme characteristics of two distinct forms of mouse 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase complementary deoxyribonucleic acids expressed in COS-1 cells. 847 48


1 2 Next >>