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1.
A Stevens 《Biochemical and biophysical research communications》1978,81(2):656-661
Using poly(A) as a substrate, an exoribonuclease has been purified from the high-salt wash of ribosomes of . The product of the reaction of the exoribonuclease is 5′ AMP. Hydrolysis of [3H](pA)3[14C](pA)n shows that both labels are released at the same rate, suggesting that the enzyme acts in a processive manner. Removal of the terminal phosphate of poly(A) with alkaline phosphatase reduces the rate of hydrolysis by 80%. Treatment of the terminally dephosphorylated poly(A) with polynucleotide kinase restores the activity. Two 5′ capped mRNA's have been tested and they are hydrolyzed slowly, if at all, by the enzyme. In contrast, phage T4 mRNA, ribosomal RNA, and encephalomyocarditis viral RNA are hydrolyzed at greater than 50% of the rate of poly(A). 相似文献
2.
M Sugiura K Segawa K Yoshinaga Y Fujio N Ito 《Biochemical and biophysical research communications》1977,76(3):739-745
extracts contain UDP-galactose:NAD+ 2-hexosyl oxidoreductase (1). ADP-ribose also serves as a substrate resulting in formation of NADH and an oxidized ADP-ribose derivative. Treatment of the oxidized product with NaBH4 followed by hydrolysis at pH 2 and 100° releases xylose as well as ribose. We conclude that ADP-D--D--3-pentosulose (ADP-3-ketoribose) is the product derived from ADP-ribose. 相似文献
3.
O2 uptake in spinach thylakoids was composed of ferredoxin-dependent and -independent components. The ferredoxin-independent component was largely 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) insensitive (60%). Light-dependent O2 uptake was stimulated 7-fold by 70 μM ferredoxin and both uptake and evolution (with O2 as the only electron acceptor) responded almost linearly to ferredoxin up to 40 μM. NADP+ reduction, however, was saturated by less than 20 μM ferredoxin. The affinity of O2 uptake for for O2 was highly dependent on ferredoxin concentration, with of less than 20 μM at 2 μM ferredoxin but greater than 60 μM O2 with 25 μM ferredoxin. O2 uptake could be suppressed up to 80% with saturating NADP+ and it approximated a competitive inhibitor of O2 uptake with a Ki of 8–15 μM. Electron transport in these thylakoids supported high rates of photophosphorylation with NADP+ (600 μmol ATP/mg Chl per h) or O2 (280 μmol/mg Chl per h) as electron acceptors, with ratios of 1.15–1.55. Variation in ratios with ferredoxin concentration and effects of antimycin A indicate that cyclic electron flow may also be occurring in this thylakoid system. Results are discussed with regard to photoreduction of O2 as a potential source of ATP in vivo. 相似文献
4.
5.
We have determined the ability of UV254nm-irradiated murine lymphoma cells to adapt their NAD+ metabolism to the increased NAD+ consumption for the poly ADP-ribosylation of chromatin proteins. Two murine lymphoma sublines with differential UV-sensitivity
and poly(ADP-ribose) turnover were used as a model system. The first subline, designated LY-R is UV254nm-sensitive and tumorigenic in DBA/2 mice. The second subline, LY-S is UV254nm-resistant and nontumorigenic. Following treatment of these cells with 2 mM benzamide, an inhibitor of the NAD+-utilizing enzyme poly(ADP-ribose) polymerase, NAD+ levels slowly increased up to about 160% of control levels after 3 hours. When benzamide was added to these cultures 20 min
after UV254nm irradiation, a dramatic transient increase of NAD+ levels was observed within 4 min in LY-R cells and more moderately in LY-S cells. At later times after UV254nm irradiation, the NAD+ levels increased in both sublines reaching up to 200% of the concentrations prior to benzamide treatment. These results demonstrate
an adaptative response of NAD+ metabolism to UV254nm irradiation. In parallel, we observed a differential repartitioning of ADP-ribosyl residues between the NAD+ and poly(ADP-ribose) pools of LY-R and LY-S cells that correlates with the differential UV sensitivity of these cells. 相似文献
6.
Farzin Farzaneh Colin K. Pearson 《Biochemical and biophysical research communications》1978,84(3):537-543
We have studied the synthesis of poly(ADP-ribose) by nuclei isolated from embryos at different stages of development. Determination of the total chain length of poly(ADP-ribose) molecules by hydroxylapatite column chromatography generally gave higher values than when the radioactive portions of these molecules, synthesized , were measured by poly(ethyleneimine)-cellulose thin layer chromatography, after snake venom phosphodiesterase digestion. The results show that most of the poly(ADP-ribose) synthesized is a covalent elongation of molecules previously initiated . 相似文献
7.
Chun-Yen Lai Qi-Chang Xia Poonam T. Salotra 《Biochemical and biophysical research communications》1983,116(1):341-348
Renatured, S-carboxymethylated subunit A1 of cholera toxin possess the ADP-ribose transferase activity (Lai, et.al., Biochem. Biophys. Res. Commun. 1981, , 1021). In the absence of acceptor self ADP-ribosylation of A1 subunit was observed. Stoicheometric incorporation of ADP-ribose moiety was achieved in 20 min at room temperature in a 0.1 – 0.2M PO4(Na) buffer, pH 6.6. On incubation of the complex with polyarginine, 75% of the enzyme-bound ADP-ribose moiety was transferred to the acceptor in 25 min. The ADP-ribosylated A1 was stable at low pH, and on cleavage with BrCN, the ADP-ribose moiety was found associated with peptide Cn I, the COOH-terminal fragment of A1 subunit. On further fragmentation with cathepsin D, a dodecapeptide containing ADP-ribose moiety was isolated whose structure was determined as: Asp-Glu-Glu-Leu-His-Arg-Gly-Tyr-Arg1-Asp-Arg-Tyr. The Arg1 in the peptide was indicated to be the site of ADP-ribosylation. 相似文献
8.
J Germershausen D Goodman E W Somberg 《Biochemical and biophysical research communications》1978,82(3):871-878
RNA (guanine-7) methyltransferase, partially purified from mycelia, catalyzed the transfer of the methyl group from S-adenosylmethionine to the 5′ terminus of both poly A(+) RNA and reovirus unmethylated mRNA. RNase T2 digestion of the methylated poly A(+) RNA from yielded the “cap” structures m 7G(5′)pppAp and m 7G(5′)pppGp in a ratio of 2:1 respectively. RNase T2 digestion of the methylated reovirus mRNA yielded m 7G(5′)pppGp exclusively. The absence of mRNA 2′-0-methyltransferase activity in the enzyme preparation is consistent with the absence of 2′-0-methylation in mRNA [Seidel, B. L. and Somberg, E. W. (1978) Arch. Biochem. Biophys. , 108–112]. This is the first isolation of an eucaryotic, cellular RNA (guanine-7) methyltransferase that has been shown to methylate homologous substrate. 相似文献
9.
Poly(adenosine diphosphate ribose) metabolism and regulation of myocardial cell growth by oxygen
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Control of the rate of cardiac cell division by oxygen occurs most probably by altering the redox state of a control substance, e.g. NAD(+)right harpoon over left harpoonNADH. NAD(+) (and not NADH) forms poly(ADP-ribose), an inhibitor of DNA synthesis, in a reaction catalysed by poly(ADP-ribose) polymerase. Lower partial pressure of oxygen, which increases the rate of division, would shift NAD(+)-->NADH, decrease poly(ADP-ribose) synthesis, and increase DNA synthesis. Chick-embryo heart cells grown in culture in 20% O(2) (in which they divide more slowly than in 5% O(2)) did exhibit greater poly(ADP-ribose) polymerase activity (+83%, P<0.001) than when grown in 5% O(2). Reaction product was identified as poly(ADP-ribose) by its insensitivity to deoxyribonuclease, ribonuclease, NAD glycohydrolase, Pronase, trypsin and micrococcal nuclease, and by its complete digestion with snake-venom phosphodiesterase to phosphoribosyl-AMP and AMP. Isolation of these digestion products by Dowex 1 (formate form) column chromatography and paper chromatography allowed calculation of average poly(ADP-ribose) chain length, which was 15-26% greater in 20% than in 5% O(2). Thus in 20% O(2) the increase in poly(ADP-ribose) formation results from chain elongation. Formation of new chains also occurs, probably to an even greater degree than chain elongation. Additionally, poly(ADP-ribose) polymerase has very different K(m) and V(max.) values and pH optima in 20% and 5% O(2). These data suggest that poly(ADP-ribose) metabolism participates in the regulation of heart-cell division by O(2), probably by several different mechanisms. 相似文献
10.
[4-14C]Cholesterol was incubated with an adrenocortical preparation in the presence of 16O2 and 18O2 devoid of significant 16O18O. Isolated (20R,22R)-20,22-dihydroxycholesterol was converted to a trimethylsilyl derivative and analyzed by gas chromatography - mass spectrometry to determine the isotope distribution of the oxygen atoms at C-20 and C-22. The ions of 289, 291, and 293 (comprising the C8 C-20 to C-27 side-chain and containing, respectively, 16O2, 16O18O, and 18O2) exhibited a binomial distribution indicating that the oxygen atoms of the vicinal glycol were drawn at random from the atomic pool of the oxygen molecules. If both side-chain hydroxyl groups had originated from the atoms of the same oxygen molecule, the ion of 291 would have been absent. 相似文献
11.
John I. Mc Cormick Sandra S. Susten James H. Freisheim 《Archives of biochemistry and biophysics》1981,212(1):311-318
L1210/R81 lymphoma cells are resistant to methotrexate (MTX) by virtue of a 35-fold elevation in dihydrofolate reductase and an inability to transport the folate antagonist drug effectively. In a phosphate-containing buffer there was little or no influx into the resistant cells at either 1 or 50 μm MTX. Replacement of this buffer with a 4-(2-hydroxyethyl)-1-piperazine-N′-2-ethanesulfonic acid-Mg2+ system resulted in an apparent influx of MTX into the resistant cells. Under these conditions, L1210/R81 cells achieved an apparent steady state at an extracellular MTX concentration of 50 μm. The apparent steady-state level of 5 nmol cells was well below the intracellular level of dihydrofolate reductase (45 nmol/109 cells). Efflux experiments at the apparent steady state indicated that 60% of the MTX was very rapidly removed from the cells by washing. Over the range of the experiment a further 20% of the MTX effluxed more slowly (). The apparent influx into the resistant cells at 5 μm MTX was inhibited 13% by sodium azide (100 μm) and initially stimulated, then inhibited, by p-chloromercuriphenylsulfonic acid (100 μm). 5-Methyltetrahydrofolate (100 μm) had little effect on the process while aminopterin (100 μm) was inhibitory (68%). Kt and V values of 2 × 10?5m and 0.31 nmol cells/min, respectively, were determined for the apparent influx in cells. Comparison of apparent MTX influx in the resistant cells with MTX transport in the sensitive cells indicates profound differences in the two processes. The evidence suggests that the apparent influx in the former cell line may consist of MTX binding to the cell membrane together with a small degree of MTX influx into the intracellular compartment. 相似文献
12.
M S Balakrishnan T R Sharp J J Villafranca 《Biochemical and biophysical research communications》1978,85(3):991-998
Oxygen-18 exchange out of [18O]Pi catalyzed by Mg2+-activated unadenylated glutamine synthetase from was followed by 31P-NMR in the presence of the other substrates, ADP and L-glutamine. The pattern of the in the species P18O4, P18O316O1, P18O216O2, P18O116O3, P16O4 during the exchange followed a binomial distribution consistent with indiscriminate removal of any of the four oxygens of Pi. The rate constant for exchange was 410±40 min?1 while the rate constant for net reaction (ATP formation) was 62±4 min?1. Thus exchange proceeds ~7 times faster than net reaction, a finding in accord with that of Stokes and Boyer ( (1976) , 5558) for the Mn2+-activated adenylylated glutamine synthetase. A model for the overall catalytic events first derived from rapid kinetic fluorescence experiments (Rhee and Chock, Proc. Natl. Acad. Sci. USA, (1976) , 476) was successfully used to fit the oxygen exchange data in this paper. 相似文献
13.
An O2-evolving Photosystem II subchloroplast preparation was obtained from spinach chloroplasts, using low concentrations of digitonin and Triton X-100. The preparation showed an O2 evolution activity equivalent to 20% of the uncoupled rate of fresh broken chloroplasts, but had no significant Photosystem-I-dependent O2 uptake activity. The preparation showed a chlorophyll ratio of 1.9 and a ratio of . Absorption spectra at room temperature and fluorescence emission spectra of chlorophyll at 77 K suggested a significant decrease in Photosystem I antenna chlorophylls in the O2-evolving Photosystem II preparation. 相似文献
14.
Exposure of mouse melanoma cells in culture to MSH (melanocyte stimulating hormone) results in a marked increase in tyrosinase (O-diphenyl: O2 oxidoreductase) activity following a lag period of 6–9 hours. Within 20 minutes after exposure of cells to MSH, the intracellular levels of cyclic AMP rise to levels which are ten times those of controls but fall to concentrations twice control values by 60 minutes. Transient increases in both protein and RNA synthetic rates also occur following MSH administration correlating in time with the dramatic but rapidly decaying increase in cellular cyclic AMP. The increase in tyrosinase activity observed in response to either MSH, dibutyryl cAMP, or theophylline, is completely suppressed by the addition of either cycloheximide (0.28 μg/ml) or actinomycin D (0.05 μg/ml) as is the basal activity of the enzyme. Results from 14C/3H leucine studies suggest that MSH may cause increased synthesis of tyrosinase. 相似文献
15.
C I Hong A Nechaev C R West 《Biochemical and biophysical research communications》1979,88(4):1223-1229
Superior antitumor activity of 1-β-D-arabinofuranosylcytosine (ara-C) conjugates of prednisolone and prednisone against L1210 leukemic mice, based on ara-C content, has encouraged us to synthesize 5′-(cortisone-21-phosphoryl)-1-β-D-arabinofuranosylcytosine (I) and 5′-(cortisone-21-phosphoryl)-1-β-d-arabinofuranosylcytosine (II) by condensation of N4,2′,3′-triacetyl-1-β-d-arabinofuranosylcytosine 5′-monophosphate with cortisol and cortisone in the presence of N,N′-dicyclohexylcarbodiimide at room temperature followed by removing the acetyl groups in 2 N methanolic ammonia in 20% yield. The conjugates I and II inhibited the growth of L1210 by 50% (ED50) at 0.25 μM and 0.07 μM, respectively, while ara-C showed ED50 0.1 μM. However, the conjugates I and II exhibited 287% and 238% of at 50 mg/kg/day × 5 doses against L1210 leukemic mice, respectively, while ara-C at 25 mg and 50 mg/kg/day × 5 gave the respective 127% and 110% of . 相似文献
16.
The relative effectiveness of oxidizing (.OH, H2O2), ambivalent (O2?) and reducing free radicals (e? and CO2?) in causing damage to membranes and membrane-bound glyceraldehyde-3-phosphate dehydrogenase of resealed erythrocyte ghosts has been determined. The rates of damage to membranebound glyceraldehyde-3-phosphate dehydrogenase ((enz)) were measured and the rates of damage to membranes ((mb)) were assessed by measuring changes in permeability of the resealed ghosts to the relatively low molecular weight substrates of glyceraldehyde-3-phosphate dehydrogenase. Each radical was selectively isolated from the mixture produced during gamma-irradiation, using appropriate mixtures of scavengers such as catalase, superoxide dismutase and formate. .OH, O2? and H2 O2 were approximately equally effective in inactivating membrane-bound glyceraldehyde-3-phosphate dehydrogenase, while e? and CO2? were the least effective. (enz) values of O2? and H2O2 were 10-times and of .OH 15-times that of e?. (mb) values were quite similar for e? and H2O2 (about twice that of O2?), while that of .OH was 3-times that of O2?. Hence, with respect to (mb): , and with respect to (enz): . The difference between the effectiveness of the most damaging and the least damaging free radicals was more than 10-fold greater in damage to the enzyme than to the membranes. Comparison between H2O2 added as a chemical reagent and H2O2 formed by irradiation showed that membranes and membrane-bound glyceraldehyde-3-phosphate dehydrogenase were relatively inert to reagent H2O2 but markedly susceptible to the latter. 相似文献
17.
Kiyoshi Nose Hiroshi Okamoto 《Biochemical and biophysical research communications》1983,111(2):383-389
A nick-translation reaction with DNA polymerase I (pol. I) was used to detect DNA breaks produced by chemical carcinogens. Normal human fibroblasts treated with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) in various doses were permeabilized with lysolecithin, and were nick translated in the presence of [3H]dCTP and pol. I. The radioactivity incorporated increased with MNNG concentration, and was directly proportional to the poly(ADP-ribose) synthetase activity. Other DNA-damaging agents such as bleomycin or 4-nitroquinoline 1-oxide also caused the nick translation rate to increase. When MNNG-treated cells were cultured in fresh medium containing no MNNG, the increase in the rate of nick translation in permeable cells became less and this decrease was abolished by addition of aphidicolin or cytosine arabinoside. The nick translation method described here may be a useful means for estimating intrinsic DNA breaks in cells treated with carcinogens. 相似文献
18.
Ramsay Richmond Barry Halliwell Jasbir Chauhan Andre Darbre 《Analytical biochemistry》1981,118(2):328-335
A mixture of xanthine or hypoxanthine and xanthine oxidase generates the superoxide radical, O2, and H2O2. In the presence of iron salts, O2 and H2O2 can interact to produce the hydroxyl radical, OH·. Superoxide-dependent formation of OH· can be measured by its ability to hydroxylate salicylate as followed by an improved colorimetric assay described in this paper. A more accurate analysis of OH· can be obtained using its ability to hydroxylate phenol, the hydroxylated products being separated and measured after derivatization using gas-liquid chromatography and electron-capture detection. The derivatization and separation techniques are described. 相似文献
19.
The presence of cytoplasmic poly(A) polymerase has been established in sea urchin eggs and four-cell embryos by subcellular fractionation and use of enucleate egg halves. ATP is the only ribonucleoside triphosphate incorporated. This incorporation is time dependent, contingent on input protein concentration, and immune to a variety of antimetabolites known to inhibit DNA-directed RNA synthesis. Both the unfertilized egg and the four-cell embryo cytoplasmic poly(A) polymerase activities display a preference for Mn2+. While oligo(A)4 is inactive as a primer, addition of and stimulates ATP incorporation. On a unit per milligram protein basis, the endogenous activity associated with cytoplasmic fractions obtained from nucleate and enucleate egg halves is 36 and 83% that obtained with the cytoplasmic fraction prepared from the unfertilized egg. In the presence of , both the nucleate and enucleate egg halves exhibit 81% of the activity associated with the unfertilized egg cytoplasmic fraction. The level of Mn2+ cytoplasmic poly(A) polymerase activity from the four-cell embryo is approximately 50% that of the unfertilized egg. This decrease does not appear to be due to either a postfertilization alteration in the subcellular localization of poly(A) polymerase or an increase in RNase activity. Supplementation with failed to restore the four-cell embryo cytoplasmic poly(A) polymerase potential to a level comparable to that of the unfertilized egg. Suppression of postfertilization protein synthesis by emetine, however, prevents this developmental decline in ATP incorporation thereby suggesting that postfertilization cytoplasmic poly(A) polymerase activity is subject to negative translational control. 相似文献
20.
P M Sinet A M Michelson A Bazin J Lejeune H Jerome 《Biochemical and biophysical research communications》1975,67(3):910-915
Glutathione peroxidase activity has been measured in erythrocytes from normal subjects and from trisomy 21 patients. The latter cases show about 50 % increase of this enzyme similar to the increase observed for superoxide dismutase (erythrocuprein) suggesting either localisation of the gene for glutathione peroxidase on chromosome 21 (as is the case for erythrocuprein) or regulation of this enzyme by intracellular levels of O2, H2O2 or superoxide dismutase. 相似文献