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1.
A chromatographic procedure for improved separation of deoxyribonucleosides and methylated deoxyribonucleosides is described. DNA was isolated from liver and small intestine of rats treated with [14C]dimethylnitrosamine ([14C]DMN) or ([3H]MNU), and the purified DNA was hydrolyzed enzymatically. The deoxyribonucleosides were chromatographed on an Aminex A-6 cation exchange column at 37°C with 0.4 M ammonium formate, pH 4.5, as eluant. In addition to showing the presence of the expected alkylated products, N7-methyldeoxyguanosine (determined as N7-methylguanine) and O6-methyldeoxyguanosine, several other minor methylated products were found in liver and intestinal DNA of rats treated with DMN or MNU. Two of these products are believed to be N3-methylthymidine and O4-methylthymidine. 相似文献
2.
Commercial [5-14C]mevalonate is shown to contain several radioactive impurities, which give artifactually high amounts of Hyamine bound, volatile acidic radioactivity when incubated with killed or living rat renal cortex slices, as compared with [5-14C]mevalonate purified either by liquid-liquid partition chromatography or through the enzymically generated by ion-exchange chromatography. The artifactual results were not diluted by incubation with increasing amounts of unlabelled mevalonate, whereas the and []cholesterol produced by rat renal cortex slices incubated with purified [5-14C]mevalonate were both diluted to the same extent by unlabelled mevalonate. It is concluded that is genuinely oxidized to , and that purification of substrate before its use is necessary. Production of and various []lipids from purified [5-14C]mevalonate, as a function of time and substrate concentration, by renal cortex and liver slices, is described. 相似文献
3.
[14C]Guanidine was observed in the urine after subcutaneous administration to rats of l-[guanidino-14C]arginine or l-[guanidino-14C]canavanine. []Hydroxyguanidine was additionally detected in the urine after injection of dl-[guanidino-14C]canavanine. These metabolites were characterized by high-voltage electrophoresis and paper chromatography, by enzymatic conversion of [14C]hydroxyguanidine to [14C]guanidine, and by repeated recrystallization of isolated urinary []guanidine as the picrate salt with no significant loss of specific activity. These experiments demonstrate that both l-arginine and l-canavanine can serve as precursors of guanidine in the rat. 相似文献
4.
The selenium-dependent formate dehydrogenase of Methanococcus vannielii was isolated from bacteria grown in the presence of []selenite. Purification under strictly anaerobic conditions resulted in the simultaneous enrichment of formate dehydrogenase activity, , and a brown chromophore that absorbs maximally at 380 nm. Acid hydrolysis of the enzyme after reduction with borohydride and alkylation with iodoacetamide, released a radioactive selenoamino acid derivative that was identified as []carboxymethyl-selenocysteine. This is the third selenoenzyme shown to contain selenocysteine. 相似文献
5.
B S Jacobson C G Kannangara P K Stumpf 《Biochemical and biophysical research communications》1973,51(2):487-493
A disrupted spinach chloroplast preparation readily synthesized from [2-14C]acetate under anaerobic conditions. It can be shown by degradation data that [14C]oleate is not a precursor of [14C]linolenate and that 7,10,13-hexadecatrienoic acid is the probable immediate precursor of the [14C]linolenate. 相似文献
6.
Transfer of N, N'-diacetylchitobiose from dolichyl diphosphate into a gray matter membrane glycoprotein 总被引:1,自引:0,他引:1
Gray matter and white matter membranes catalyze the transfer of label from UDP-N-acetyl-[] glucosamine into N-acetyl[]glucosaminyl-pyrophosphoryl-dolichol, N,N′-diacetyl []chitobiosyl-pyrophosphoryl-dolichol, and N-acetyl[]glucosamine-labeled glycoprotein. Gel filtration of the Pronase digests of gray matter N-acetyl[]glucosamine-labeled glycoprotein reveals two N-acetyl[]glucosamine-labeled glycopeptide fractions. One fraction (A) contains approximately eight glycose units. All of the radioactivity is at nonreducing termini and can be released by treatment with an exo-β-N-acetylglucosaminidase. A smaller N-acetyl[]glucosamine-labeled glycopeptide (B) is recovered in the elution volume expected for an asparaginyl disaccharide. Structural studies show that the labeled saccharide unit in glycopeptide B is N,N′-diacetyl[]chitobiose. The linkage between the -labeled disaccharide and the polypeptide has the properties of an N-glycosidic attachment to asparagine. Only the larger N-acetyl[]glucosamine-labeled glycopeptide (A) is found in Pronase digests of white matter membrane N-acetyl[]glucosamine-labeled glycoprotein after incubation with UDP-N-acetyl[]glucosamine. When gray matter membranes are incubated with UDP-N-acetyl[]glucosamine in the presence of tunicamycin or UMP, the labeling of glycolipid and the asparaginyl disaccharide is inhibited. UMP and tunicamycin have no effect on the transfer of N-acetyl[]glucosamine to external acceptor sites of the larger glycopeptide (A). The transfer of N,N′-diacetyl[]-chitobiose from carrier lipid to protein is observed when extensively washed membranes containing endogenous, prelabeled -labeled glycolipids are incubated in the presence or absence of unlabeled GDP-mannose. UMP treatment of the prelabeled membranes selectively discharged over 80% of the label from N-acetyl[]glucosaminyl-pyrophosphoryl-dolichol, but had no effect on the transfer of the -labeled disaccharide to protein. All of these results are concordant with transfer of N,N′-diacetylchitobiose from dolichyl diphosphate to gray matter glycoprotein. The major membrane glycoprotein labeled by the lipid-mediated []disaccharide transfer reaction has an apparent molecular weight of 24,000. Tunicamycin prevents the enzymatic labeling of the gray matter glycoprotein having an apparent molecular weight of 24,000. 相似文献
7.
Yukihiro Shoyama Yasuo Kishimoto 《Biochemical and biophysical research communications》1976,70(4):1035-1041
A doubly labeled 3-ketoceramide, [1-14C] lignoceroyl [1-3H2] 3-ketosphingosine ( ratio, 3.61) was injected into the left ventricle of rat heart. The ceramide isolated from the livers of the animals after 1 hr incubation contained an equal ratio of 3.60. This finding strongly supports the existence for direct conversion of 3-ketoceramide to ceramide in rat liver. 相似文献
8.
Catherine Ronin Simone Bouchilloux 《Biochimica et Biophysica Acta (BBA)/General Subjects》1978,539(4):470-480
In order to evaluate the possibility in a pig thyroid rough microsomal system of a transfer of pre-assembled sugar cores from sugar-lipids to protein, we have examined after incubation with GDP-[14C]Man the compounds bearing labeled saccharides and have determined some properties of their released saccharide moieties. The [14C]Man material specifically soluble in CHCl3/CH3OH/H2O, 10 : 10 : 3, behaved on DEAE-cellulose and when treated with hot alkali and alkaline phosphatase as a lipid pyrophosphate (sometimes accompanied by some ). Its saccharide moiety, released by mild acid, exhibited properties (molecular size, sensitivity to α-mannosidase, affinity for concanavalin A and charge modification introduced by a strong reductive alkaline treatment) pointing to a polymannosylated N,N′-diacetylchitobiose containing an average of nine monosaccharide units (from six to twelve). The [14C]mannosylated glycoproteins have represented all the polymeric label remaining after lipid extraction. From the susceptibility of their pronase glycopeptides to a differential reductive alkaline hydrolysis, it was concluded that their label belonged mainly to N-glycosidically linked units. Released saccharides exhibited the same properties as those from lipids, a result substantiating the possibility raised from previous studies of a transfer of pre-assembled moieties. 相似文献
9.
Ron L. Batstone-Cunningham Robert E. Hardy Kilian Dill 《International journal of biological macromolecules》1983,5(5):314-317
Structural studies of homozygous glycophorin AM were undertaken by monitoring the 13C methyl resonances of 13C reductively methylated glycophorin AM (contains five Lys residues, and the N-terminal Ser residues) in various forms of glycosylation. The results indicate that removal of the α-d-NeuAc residues does not affect the structure about the N-terminal Ser residue. However, removal of the fifteen O-linked oligosaccharide units results in a structural effect about the N-terminal Ser residue. Partial methylation experiments performed on native glycophorin AM and deglycosylated glycophorin AM indicate that methylation of the lysine residue(s) may influence the structure about the N-terminal Ser residue, especially in the case of deglycosylated AM. 相似文献
10.
Rat adipocytes were incubated at 37°C with 2-deoxy-d-[1-14C]glucose ([14C]2dGlc) at various concentrations and the intracellular concentrations of [14C]2dGlc and deoxy[14C]glucose phosphate () were measured. Using 7 μM extracellular [14C]2dGlc, the intracellular [14C]2dGlc concentration approached the extracellular by 5 min in insulin-stimulated cells and by 60 min it exceeded the extracellular concentration by 50-fold. A maximum accumulation ratio of 3.5 was reached by 7 min using 1 mM and a ratio of 1.6 was reached by 1 to 3 min using 10 mM extracellular 2dGlc. The time at which the concentration of intracellular 2dGlc exceeded the extracellular was inversely related to the accumulation of . The rate of accumulation of total radioactivity ([14C]2dGlc plus decreased after 20 min using 7 μM extracellular [14C]2dGlc. This change occurred later at 22°C or in the absence of insulin and sooner at higher concentrations of 2dGlc. Experiments where uptake was stopped by dilution indicated that radioactivity appearing in the medium was [14C]2dGlc, but radioactivity disappearing from the cells was largerly . Addition of 10 mM unlabelled 2dGlc or glucose to cells preincubated with 7 μM [14C]2dGlc resulted in a more rapid loss of accumulated label from the cells, while addition of 10 mM , a non-metabolizeable sugar analogue with about the same affinity for the transport system as 2dGlc, was without effect. The results show that 2dGlc is accumulated against its concentration gradient. It is suggested that the mechanism involves first, dephosphorylation of and second, the presence of a diffusion barrier between the site of dephosphorylation and the transport site. 相似文献
11.
Robert E. Hardy Marsha E. Daman Anne M. Holbrooks Kilian Dill 《International journal of biological macromolecules》1984,6(2):103-107
13C nuclear magnetic resonance (n.m.r.) spectral data for 13C reductively methylated N-terminal tryptic glycopeptides and for 13C reductively methylated N-terminal glyco-octapeptides derived from homozygous glycophorins AM and AN are presented. Their 13C chemical shift data are compared with the previously published 13C n.m.r. data for 13C reductively methylated homozygous glycophorins AM and AN in order to investigate the means of display of the MN blood determinants by these species. The pH dependence of the 13C resonances of leucine of glyco-octapeptide AN and of serine of glyco-octapepti AM indicated that only a slight structural perturbation occurs at the N-terminus when a large portion of the glycoprotein molecule is removed. However, one structural ‘state’ of 13C reductively methylated glycophorin AM is lost when the glyco-octapeptide AM is produced. The 13C resonance of leucine of glycooctapeptide AN titrated with a of 7.7 (Hill coefficient ). The 13C resonance of serine, on the other hand, exhibited an unusual pH dependence, indicating the existence of some possible steric constraints or hydrogen bonding in this molecule. In comparison to the data obtained for 13C-labelled glycooctapeptide AM molecule, the pH dependence of the chemical shift of the 13C resonance of serine of tripeptide tri-L-serine is also presented. Circular dichroism (c.d.) spectra indicated that the reductive methylation technique does not cause a large perturbation of the glycophorin A molecule. 相似文献
12.
As part of our effort to understand the chromosomal organization of streptomycetes, we have developed a method for detecting large alterations in the DNA, in particular to visualize insertions, transpositions, and deletions. The method involves the labeling of the DNA of two strains with [3H]thymidine or [14C]thymidine, extraction and purification of the DNA, digestion with restriction endonucleases, and one-dimensional agarose gel electrophoresis of the two samples in the same slot. Following electrophoresis, the gel is cut into thin slices, and the ratio is measured in each slice. Deviations from the average standard ratio are caused by differences in the restriction site arrangement in the DNA of the two strains, which may be caused by rearrangements in the DNA. The method has a high resolution of one restriction fragment change. 相似文献
13.
Prakash Bhuta Jiří Žemclička 《Biochimica et Biophysica Acta (BBA)/General Subjects》1985,841(2):145-150
The (1c) and (2c), obtained by chemical synthesis, are substrates for ribosomal peptidyltransferase from Escherichia coli. Nucleoside 1c, which mimics an anti conformation of antibiotic formycin, has 80% of the acceptor activity of puromycin at 5 · 10?4 M determined by the release of N-Ac-Phe residue from the 70 S complex. The reaction product, (1d), was characterized by paper electrophoresis before and after alkaline hydrolysis. By contrast, nucleoside 2c, which resembles a syn conformation of formycin, exhibited only 20% of the acceptor activity of puromycin at 5 · 10∮4 M and essentially none in the concentration region between 1 · 10?6 and 1 · 10?4 M. The results which are in accord with previous models have shown that a substrate with its base in an anti conformation is preferable for the acceptor site of peptidyltransferase than the corresponding syn counterpart, Nevertheless, it is possible that an intermediate conformation, for example, high anti (amphi-minus), is an optimal arrangement for acceptor site substrates. 相似文献
14.
Bernard Grandchamp Yves Nordmahn 《Biochemical and biophysical research communications》1977,74(3):1089-1095
Using []Coproporphyrinogen obtained from human red blood cells incubated with [] δ aminolevulinic acid, we measured lymphocytes Coproporphyrinogen III Oxidase activity in 17 subjects with hereditary coproporphyria. The mean activity was about 50 % of that in lymphocytes from normal subjects. This finding suggests that decreased coproporphyrinogen III oxidase activity reflects the primary genetic defect in Hereditary Coproporphyria. The technique described allows easy detection of asymptomatic carriers. 相似文献
15.
Edward L. Schwartz Anthony F. Hadfield Alfred E. Brown Alan C. Sartorelli 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1983,762(4):489-497
Two analogs of N-acetylmannosamine, (Ac4-NAcMan) and the 2-trifluoroacetamido derivative (Ac4F3-NAcMan), were synthesized as potential inhibitors of the formation of sialic acid-containing glycoconjugates and were examined for their ability to modify the incorporation of into cellular glycoconjugates of Friend murine erythroleukemia cells. Ac4F3-NAcMan and Ac4-NAcMan inhibited cellular replication in suspension culture at concentrations of 0.02 and 0.08 mM, respectively. The cytotoxicity of Ac4-NAcMan was relatively reversible, whereas that produced by Ac4F3-NAcMan was not, as judged by measurement of the cloning efficiencies of cells exposed to these agents. The analogs inhibited incorporation of into ethanol-soluble and -insoluble materials. Separation of ethanol-soluble metabolites by HPLC demonstrated that Ac4F3-NAcMan caused accumulation of radioactivity from in CMP-N-acetylneuraminic acid (CMP-NeuNAc) equal to the decrease in macromolecular-bound 3H caused by this agent. In contrast, similar exposure to Ac4-NAcMan produced a large increase in the amount of radioactivity in ethanol-soluble N-acetylneuraminic acid while decreasing the amount of label from in cellular CMP-NeuNAc, suggesting that the analogs differ in their biochemical sites of action. Treatment of cells with either analog increased the amount of neuraminidase-hydrolyzable sialic acid-like material on the cell surface; this appeared to be due to the incorporation of the analogs into cellular glycoconjugates, since incubation of cells with 3H-labeled analogs resulted in the appearance of radioactivity in cellular ethanol-insoluble and neuraminidase-hydrolyzable material. Incubation of cells with Ac4-NAcMan labeled with 14C in the 4-O-acetyl group further demonstrated that incorporation occurred with approx. 50% retention of this substituent. Thus, both the amount and the nature of the surface sialic acid constituents of treated cells were altered, suggesting that these or similar analogs could potentially be used to modify cellular membrane function. 相似文献
16.
Catherine Ronin Simone Bouchilloux 《Biochimica et Biophysica Acta (BBA)/General Subjects》1978,539(4):481-488
Pig thyroid rough microsomes catalyzed the transfer of glucose from UDP-[14C]Glc to glycolipids extractable with chloroform/methanol, glycolipids extractable with a water-saturated chloroform/methanol and to a residual material. Kinetics of labeling were compatible with a precursor-product relationship between the second type of glycolipid and residuals.The [14C] Glc-glycolipids soluble in CHCl3/CH3OH/H2O, 10 : 10 : 3, behaved on DEAE-cellulose mainly as pyrophospho derivatives, with some less acidic radioactivity, probably . Their saccharide moieties released by mild acid appeared polydisperse on paper chromatography, a part of them being estimated larger than a nonasaccharide marker GlcNAc-[Man]8. The 14C-labeled glucosylated glycoproteins have represented all the considerable polymeric label remaining after lipid extraction. Their pronase glycopeptides were submitted to a differential reductive alkaline hydrolysis and it was concluded that their [14C] glucose belongs mainly to N-glycosically linked units. On gel filtration, the released saccharides exhibited an average size of nine monosaccharide units (from six to twelve with a relatively high proportion of material containing more than nine sugars).In a [14C] Glc-microsomal extract, 29% of the non-lipid radioactivity was found immunoreactive with an antiserum to pig thyroglobulin. 相似文献
17.
Liposomes prepared with cholesterol and dipalmitoyl phosphatidylcholine were incubated with a clone of normal rat kidney fibroblast of cells in culture. The cells took up [14C]cholesterol in proportion to the concentration of liposomes in the incubation medium, and the uptake increased with time over the four hours of study. Two cell membrane enzymes, adenylate cyclase and (, exhibited decreased activity after treatment with cholesterol-containing liposomes. The decrease in adenylate cyclase activity was directly proportional to the uptake of [14C]cholesterol. When a variety of subclones of NRK 5W were examined some were found to respond to cholesterol treatment and some did not. These data are consistent with the view that membrane cholesterol content plays a role in controlling the activity of some plasma membrane enzymes. 相似文献
18.
Reynold Spector 《Archives of biochemistry and biophysics》1980,205(1):85-93
In vitro, the accumulation and release of [methyl-3H]thymidine ([]thymidine) by the isolated choroid plexus, the anatomical locus of the blood-cerebrospinal fluid barrier, was studied. With concentrations of []thymidine in the medium of 1.0 μm (or greater), the choroid plexus accumulated []thymidine against a concentration gradient by a process that depended on intracellular energy production but did not depend on intracellular binding or metabolism of the []thymidine. This transport process was inhibited (although differentially) by various nucleosides and low temperatures but not by 2-deoxyribose or pyrimidine bases. With concentrations of less than 1.0 μm []thymidine in the medium, the choroid plexus accumulated []thymidine against a concentration gradient. However, the majority of the []thymidine within the choroid plexus was metabolized to []thymidine nucleotides at low extracellular []thymidine concentrations (3 nm). This accumulation process depended, in large part, on saturable intracellular phosphorylation. Thymidine was the principal form released from choroid plexuses that had been incubated for various times in media containing concentrations of thymidine from 3 to 1.0 mm. The release of thymidine from choroid plexus was depressed by cold temperatures and a very high (2.56 mmol/kg) intracellular thymidine concentration. 相似文献
19.
Flufenamate, a non-steroidal anti-inflammatory drug, is a powerful inhibitor of anion transport in the human erythrocyte (). The concentration dependence of the binding to ghosts reveals two saturable components. [14C]Flufenamate binds with high affinity () to 8.5·105 sites per cell (the same value as the number of band 3 protein per cell); it also binds, with lower affinity () to a second set of sites (4.6·107 per cell). Pretreatment of cells with 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS), a specific inhibitor of anion transport, prevents [14C]flufenamate binding only to high affinity sites. These results suggest that high affinity sites are located on the band 3 protein involved in anion transport. Extracellular chymotrypsin and pronase at low concentration cleave the 95 kDa band 3 into 60 kDa and 35 kDa fragments without affecting either anion transport or [14C]flufenamate binding. Splitting by trypsin at the inner membrane surface of the 60 kDa chymotryptic fragment into 17 kDa transmembrane fragment and 40 kDa water-soluble fragment does not affect [14C]flufenamate binding. In contrast degradation at the outer membrane surface of the 35 kDa fragment by high concentration of pronase or papain decreases both anion transport capacity and number of high affinity binding sites for [14C]flufenamate. Thus it appears that 35 kDa peptide is necessary for both anion transport and binding of the inhibitors and that the binding site is located in the membrane-associated domain of the band 3 protein. 相似文献
20.
Assembly of the vesicular stomatitis virus envelope: incorporation of viral polypeptides into the host plasma membrane 总被引:17,自引:0,他引:17
A E David 《Journal of molecular biology》1973,76(1):135-148
Incorporation of viral polypeptides into the host plasma membrane is an essential step in the formation of the lipoprotein envelope of vesicular stomatitis virus. A quantitative study of this process was carried out using a double-isotope labeling procedure. Infected cells were incubated for two hours with 14C-labeled amino acids, pulse-labeled with [3H]leucine and incubated for various times with an excess of non-radioactive leucine. The ratio was determined for each viral polypeptide in isolated plasma membranes and in the whole cell by polyacrylamide gel electrophoresis. It was found that [3H]leucine-labeled viral polypeptides could be detected in the plasma membranes immediately following a 30-second pulse but that the ratios of polypeptides in the plasma membrane did not reach the ratios in the whole cells until the end of a two-minute chase period. The addition of puromycin to the cultures at the end of the pulse period did not affect subsequent incorporation of [3H]leucine-labeled polypeptides into the plasma membrane. The incorporation of various amino acid analogs into the viral polypeptides did not affect the efficiency with which they were incorporated into the plasma membranes. It is proposed that viral polypeptides are selected for incorporation into the plasma membrane from a small interior pool of completed molecules. 相似文献