Modified 5′-nucleotides resistant to 5′-nucleotidase: isolation of 3-(3-amino-3-carboxypropyl)uridine 5′-phosphate and N2,N2-dimethylguanosine 5′-phosphate from snake venom hydrolysates of transfer RNA

1976 ◽  
Vol 54 (5) ◽  
pp. 413-422 ◽  
Author(s):  
M. W. Gray
Keyword(s):  
Ribosomal Rna ◽  
Quantitative Measurement ◽  
Chinese Hamster ◽  
Assay Method ◽  
Wheat Embryo ◽  
E Coli ◽  
Yeast Trna ◽  
The Stability ◽  
Hydrolysis Conditions ◽  
Hydrolysis Of

A procedure for the quantitative measurement of the O2′-methylnucleoside constituents of RNA has recently been developed in this laboratory (Gray, M. W. Can. J. Biochem. 53,735–746 (1975)). This assay method is based on the resistance of O2′-methylnucleoside 5′-phosphates (pNm) (generated by phosphodiesterase hydrolysis of RNA) to subsequent dephosphorylation by venom 5′-nucleotidase (EC 3.1.3.5). In the present investigation, two base-modified 5′-nucleotides, each displaying an unusual resistance to 5′-nucleotidase, have been identified. These compounds have been characterized by a variety of techniques as N2,N2-dimethylguanosine 5′-phosphate [Formula: see text] and 3-(3-amino-3-carboxypropyl)uridine 5′-phosphate (p4abu3U). Because of their resistance to 5′-nucleotidase, [Formula: see text] and p4abu3U are isolated along with the pNm in the mononucleotide fraction of venom hydrolysates of transfer RNA.Under hydrolysis conditions, the stability of p4abu3U is comparable to that of a pNm, allowing quantitative assay of the nucleotide. The proportion (mean ± SD) of p4abu3U in venom hydrolysates of wheat embryo and Escherichia coli tRNA has been determined to be 0.35 ± 0.03 (n = 5) and 0.14 ± 0.02 (n = 4) mol%, respectively. The absence of p4abu3U in venom hydrolysates of yeast tRNA implies the absence of the corresponding nucleoside in yeast tRNA, in agreement with existing data. The variable recovery of [Formula: see text] from venom hydrolysates of wheat embryo and yeast tRNA indicates that under hydrolysis conditions, this base-modified nucleotide is only partially resistant to 5′-nucleotidase. The complete absence of[Formula: see text] in venom hydrolysates of E. coli tRNA is consistent with the known absence of N2,N2-dimethylguanosine in this RNA. These observations demonstrate that resistance to 5′-nucleotidase is a necessary but not sufficient criterion for concluding that a 5′-nucleotide is O2′-methylated.When applied to wheat embryo ribosomal RNA, the analytical methods described in this report failed to reveal any compound having the distinctive charge properties of p4abu3U. It therefore appears that 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, recently characterized as a constituent of the 18 S rRNA of Chinese hamster cells (Saponara, A. G. &Enger, M. D. Biochim. Biophys. Acta 349,61–77 (1974)), may not be present in wheat embryo ribosomal RNA.

Analysis of O2′-Methylnucleoside 5′-Phosphates in Snake Venom Hydrolysates of RNA: Identification of O2′-Methyl-5-Carboxymethyluridine as a Constituent of Yeast Transfer RNA

1975 ◽  
Vol 53 (7) ◽  
pp. 735-746 ◽  
Author(s):  
M. W. Gray
Keyword(s):  
Snake Venom ◽  
Deae Cellulose ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Assay Method ◽  
Blocking Group ◽  
Wheat Embryo ◽  
Yeast Trna ◽  
Snake Venom Phosphodiesterase ◽  
Hydrolysis Of

Snake venom phosphodiesterase liberates the O2′-methylnucleoside (Nm) constituents of RNA as the corresponding 5′-nucleotides (pNm), which, in contrast to normal 5′-nucleotides (pN), are resistant to dephosphorylation by venom 5′-nucleotidase. This property provides the basis of a convenient and highly reproducible quantitative assay for Nm residues in RNA. The assay method involves: (1) hydrolysis of RNA with whole or partially-purified snake venom; (2) isolation of the pNm derivatives, as a group, by anion-exchange chromatography on DEAE-cellulose; (3) resolution of the individual pNm compounds by two-dimensional paper chromatography; (4) identification and quantitative measurement of pNm derivatives by ultraviolet absorption spectrophotometry. Using this procedure, the molar proportions of the Nm constituents of wheat embryo, yeast, and Escherichia coli tRNA have been determined. The close correspondence between the values measured by venom hydrolysis and those obtained by analysis of alkali-stable dinucleotide (Nm-Np) sequences attests to the validity of the venom assay, and further indicates that alkali-stable sequences larger than dinucleotides are not present in significant amounts in the tRNA of the above three organisms.During the present investigation, several ultraviolet-absorbing components, not immediately identifiable as ribose-methylated nucleotides, were isolated along with the expected O2′-methylnucleoside 5′-phosphates. Preliminary characterization of one of these compounds suggests that it is a derivative of a novel nucleoside, O2′-methyl-5-carboxymethyluridine (cm5Um). Venom hydrolysis of yeast tRNA liberates the 5′-nucleotide of cm5Um in the form of a carboxyl-blocked derivative (pU-2). During alkaline hydrolysis of yeast tRNA, the blocking group in U-2 is labilized and cm5Um is released as part of an alkali-stable dinucleotide, cm5Um-Ap. The proportion of pU-2 in venom hydrolysates of yeast tRNA (0.02 mol%, the same as the content of cm5Um-Ap in alkaline hydrolysates) suggests that O2′-methyl-5-carboxymethyluridine may be confined to a single isoaccepting species of tRNA in yeast.In an allied study, reinvestigation of the alkali-stable dinucleotide sequences of baker's yeast tRNA has confirmed previous results concerning the sequence distribution of O2′-methylribose in yeast tRNA (Gray, M. W. &Lane, B. G. (1967) Biochim. Biophys. Acta 134, 243–257).

Stability and Hydrolysis of Desomorphine-Glucuronide

2019 ◽  
Vol 43 (7) ◽  
pp. 536-542 ◽  
Author(s):  
Jessica Winborn ◽  
Sarah Kerrigan
Keyword(s):  
Glucuronic Acid ◽  
Helix Pomatia ◽  
Sample Treatment ◽  
E Coli ◽  
Hydrolysis Method ◽  
Flight Mass Spectrometry ◽  
The Stability ◽  
Hydrolysis Of ◽  
Additional Sample ◽  
Analgesic Potency

AbstractDesomorphine, the principal opioid in Krokodil, has an analgesic potency approximately ten-times that of morphine. Similar to other opioids, during phase II metabolism it undergoes conjugation with glucuronic acid to form desomorphine-glucuronide. Although hydrolysis of conjugated species is sometimes required prior to analysis, desomorphine-glucuronide has not been fully investigated. In this study, six hydrolysis procedures were optimized and evaluated. Deconjugation efficiencies using chemical and enzymatic hydrolysis were evaluated and stability in aqueous solution was assessed. Acid hydrolysis was compared with five β-glucuronidase sources (BGTurbo™, IMCSzyme™, Escherichia coli, Helix pomatia and Patella vulgata). At optimal conditions, each hydrolysis method produced complete hydrolysis (≥96%). However, under simulated challenging conditions, P. vulgata was the most efficient β-glucuronidase for the hydrolysis of desomorphine-glucuronide. Both BGTurbo™ and IMCSzyme™ offered fast hydrolysis with no need for sample cleanup prior to liquid chromatography-quadrupole/time of flight-mass spectrometry (LC-Q/TOF-MS) analysis. Hydrolysates using E. coli, H. pomatia and P. vulgata underwent additional sample treatment using β-Gone™ cartridges. Additionally, the stability of free and conjugated drug was evaluated at elevated temperature (60°C) in aqueous solutions between pH 4 and 10. No degradation was observed for either desomorphine or desomorphine-glucuronide under any of the conditions tested.

Wheat Embryo Ribonucleates. V. Generation of N2-Dimethylgnanylate When 'Fully Sequenced' Homogeneous Species of Transfer RNA are Used as Substrates for Wheat Embryo Methyltransferases

1975 ◽  
Vol 53 (6) ◽  
pp. 690-697 ◽  
Author(s):  
T. C. Kwong ◽  
B. G. Lane
Keyword(s):  
Molecular Basis ◽  
Transfer Rna ◽  
Target Site ◽  
Common Site ◽  
Tertiary Structures ◽  
Wheat Embryo ◽  
E Coli ◽  
Yeast Trna ◽  
The Subject ◽  
Random Distributions

1. When S-adenosyl[methyl-14C]methiomne and various species of transfer RNA are used as substrates for wheat embryo methyltransferases, the principal site of guanylate-N2 methylation can be shown to be a G-residue between the stems of the dihydrouridine and anticodon loops. This common site of guanylate-N2 methylation is referred to as the interstem target site.2. When the interstem target site is the non-terminal G-residue in a G-C-G-C sequence, as in the cases of Escherichia coli tRNA1Leu, tRNAIle, and tRNA3Ser, there is preponderant dimethylation to yield N2-dimethylguanylate.3. When the interstem target site is part of a U-C-G-U sequence, as in the case of E. coli tRNAtMet, there is diminished dimethylation and correspondingly increased monomethylation to yield N2-monomethylguanylate.4. When the interstem target site is the non-terminal G-residue in an A-U-G-G sequence, as in the case of yeast tRNAAsp, there is negligible dimethylation and almost exclusive monomethylation to yield N2-monomethylguanylate.5. The concerted way in which the primary, secondary, and tertiary structures of tRNA molecules might influence the efficacy of these methylations is the subject of a brief discussion. Attention is also focused on the evolutionary and molecular basis for the generally non-random distributions of methylated oligonucleotide sequences in ribosomal and transfer ribonucleates.

The chain termini of polynucleotides formed by limited enzymic fragmentation of wheat embryo ribosomal RNA. Part 1. Studies of snake venom phosphodiesterase

1968 ◽  
Vol 46 (1) ◽  
pp. 81-92 ◽  
Author(s):  
B. D. McLennan ◽  
B. G. Lane
Keyword(s):  
Snake Venom ◽  
Ribosomal Rna ◽  
Sedimentation Rate ◽  
Sharp Decrease ◽  
Degree Of Polymerization ◽  
Wheat Embryo ◽  
Snake Venom Phosphodiesterase ◽  
Endonucleolytic Cleavage ◽  
Limited Degree ◽  
Hydrolysis Of

Snake venom phosphodiesterase induces about fifteen exonucleolytic cleavages for each endonucleolytic cleavage during the first hour of hydrolysis of wheat embryo ribosomal RNA, under the conditions of hydrolysis used in this present investigation. The polynucleotide chains in the ribosomal RNA preparation have an average degree of polymerization in the neighborhood of 1300 nucleotide residues, and there is a mean of between 5 and 10 endonucleolytic breaks per chain during this first hour of phosphodiesterase-induced hydrolysis. The cleavages occur widely throughout most of the polynucleotide chains in the ribosomal RNA preparation, as judged by the sharp decrease in mean sedimentation rate which accompanies a limited degree (about 10%) of exonucleolysis of the RNA. Studies of phosphodiesterase-induced endonucleolysis of wheat embryo soluble RNA are reported, but because of the much lower initial degree of polymerization (about 80 nucleotide residues per polynucleotide chain), the results of endonucleolysis are less pronounced in terms of the proportional increment in chain termini and the proportional decrease of mean sedimentation rate. The endonucleolysis of RNA is discussed in terms of the minor nucleotide components in both ribosomal and soluble RNA, and particular reference is made to pseudouridylate which has been found in relatively high proportion among the chain termini after limited hydrolysis with venom phosphodiesterase.Purified venom phosphodiesterase preparations, devoid of ribonuclease or 5′-nucleotidase contamination, were found to convert nucleoside 2′(3′),5′-diphosphates to 5′-nucleotides under conditions which had virtually no effect on nucleoside 3′-phosphates or nucleoside 5′-phosphates. The possibility that this reaction may be catalyzed by the venom phosphodiesterase itself is discussed.

scholarly journals Purine quantification in digesta from ruminants by spectrophotometric and HPLC methods

1999 ◽  
Vol 81 (2) ◽  
pp. 107-112 ◽  
Author(s):  
H. P. S. Makkar ◽  
K. Becker
Keyword(s):  
Nucleic Acids ◽  
Accurate Determination ◽  
Internal Standard ◽  
Hplc Method ◽  
E Coli ◽  
Purine Bases ◽  
Microbial Preparation ◽  
Hydrolysis Conditions ◽  
Hydrolysis Of

The method of Zinn & Owens (1986;Canadian Journal of Animal Science66, 157–166), based on release of purine bases by HClO4followed by their precipitation with AgNO3, was used to study recovery of purines from lyophilized rumen microbial orEscherichia colipreparations added to matrices such as cellulose, starch and neutral-detergent fibre. The recovery of purines was poor (approximately 50 %). Under the hydrolysis conditions (12 M-HClO4, 90–95° for 1 h) used in the method of Zinn & Owens (1986), the recovery of purines from the rumen microbial preparations added to matrices measured using an HPLC method was 95–102 %, suggesting that the lower recovery of purines in the method of Zinn & Owens (1986) was not due to incomplete hydrolysis of nucleic acids. Using the HPLC method, adenine and allopurinol (an internal standard) were found to be heat-labile as substantial destruction was observed on heating at 121°. On the other hand, another commonly used internal standard, caffeine, was stable at 121°. A complete hydrolysis of nucleic acids from the rumen microbial preparation was observed with 2·5 ml 0·6 M-HClO4in a total volume of 3 ml (0·5 M-HClO4during hydrolysis) at 90–95° for 1 h, and under these conditions adenine, guanine, allopurinol and caffeine were stable. Moreover, under these milder hydrolysis conditions, the recovery of purine bases from the rumen microbial orE. colipreparations added to matrices ranged from 92 to 108 % using the method of Zinn & Owens (1986). Based on the results, changes in hydrolysis conditions have been proposed for accurate determination of purine bases using spectrophotometric or HPLC methods.

scholarly journals Assay method for Vibrio cholerae and Escherichia coli enterotoxins by automated counting of floating chinese hamster ovary cells in culture medium

1978 ◽  
Vol 7 (5) ◽  
pp. 479-485
Author(s):  
R T Nozawa ◽  
T Yokota ◽  
S Kuwahara
Keyword(s):  
Escherichia Coli ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Culture Medium ◽  
Culture Media ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Assay Method ◽  
Ovary Cells ◽  
E Coli

As Chinese hamster ovary (CHO) cells on plastic proliferate, many cells float off into the medium instead of piling up after they form a monolayer. Fewer cells were floating in the medium when CHO cells were incubated with cholera toxin at a concentration as low as 10 pg/ml. The toxin increased the adhesiveness of the cells forming confluent monolayers so that the floating cells accumulated on the adherent monolayers. On the basis of this finding, a simple, quantitative assay method for cholera and Escherichia coli enterotoxins was devised by cultivating CHO cells in a Linbro multidish and counting the cells in the medium with a Coulter Counter. The method was sensitive enough to detect toxins in 100- to 200-fold-diluted culture media of toxigenic E. coli strains. Little or no activity was detected by this method in the culture medium of nontoxigenic E. coli.

Visualisation of E. coli ribosomal RNA in situ by electron spectroscopic imaging and image averaging

1992 ◽  
Vol 50 (1) ◽  
pp. 466-467
Author(s):  
Daniel Beniac ◽  
George Harauz
Keyword(s):  
Ribosomal Rna ◽  
Three Dimensional ◽  
Spectroscopic Imaging ◽  
Electron Spectroscopic Imaging ◽  
E Coli ◽  
Microscopical Technique ◽  
Quantitative Image ◽  
Dimensional Reconstruction ◽  
Image Averaging ◽  
Protein Components

The structures of E. coli ribosomes have been extensively probed by electron microscopy of negatively stained and frozen hydrated preparations. Coupled with quantitative image analysis and three dimensional reconstruction, such approaches are worthwhile in defining size, shape, and quaternary organisation. The important question of how the nucleic acid and protein components are arranged with respect to each other remains difficult to answer, however. A microscopical technique that has been proposed to answer this query is electron spectroscopic imaging (ESI), in which scattered electrons with energy losses characteristic of inner shell ionisations are used to form specific elemental maps. Here, we report the use of image sorting and averaging techniques to determine the extent to which a phosphorus map of isolated ribosomal subunits can define the ribosomal RNA (rRNA) distribution within them.

A New Fluorogenic Substrate Method for Assay of Bacterial Endotoxins Using Limulus Hemocyte Lysate

1979 ◽  
Author(s):  
T Harada ◽  
M Ohki ◽  
M Niwa ◽  
S Iwanaga
Keyword(s):  
Antithrombin Iii ◽  
Assay Method ◽  
Sensitive Assay ◽  
Fluorogenic Substrate ◽  
Toxin Concentration ◽  
Amidase Activity ◽  
E Coli ◽  
Bacterial Endotoxins ◽  
Plasmin Inhibitor ◽  
Limulus Test

Limulus hemocyte lysate contains a proclotting enzyme, which is transformed to the active clotting enzyme in the presence of gram-negative bacterial endotoxins. The clotting enzyme coagulates a clottable protein, named coagulogen, contained also in the lysate. This gelation reaction of the lysate, named Limulus test, has been widely employed as a simple and very sensitive assay method for endotoxins. We developed a new fluorogenic substrate, Boc-Leu-Gly-Arg-4-methylcoumarin amide, for Limulus clotting enzyme and established an enzymatic assay method for endotoxins, using the substrate. Because the endotoxin mediates the activation of proclotting enzyme in the lysate, the measurement of amidase activity could be applicable for quantitation of the endotoxins. In fact, the amidase activity determined fluorometrically increased by increasing concentration of E. coli 0111: B4 endotoxin added to the lysate, and a linear relationship between the toxin concentration and the activity was observed in the range of 5X10-6to 5xl0-2 µg endotoxin. The method was a fifty times more sensitive than that of the Limulus test and was very reproducible. However, the method was not directly applicable for the assay of endotoxins in circulating blood, as the amidase activity was strongly inhibited by antithrombin III and α2-plasmin inhibitor. Thus, some pretreatment with heat or chloroform on plasma samples before the assay was required.

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