The chain termini of polynucleotides formed by limited enzymic fragmentation of wheat embryo ribosomal RNA. Part 2. Studies of a snake venom ribonuclease and pancreas ribonuclease

1968 ◽  
Vol 46 (1) ◽  
pp. 93-107 ◽  
Author(s):  
B. D. McLennan ◽  
B. G. Lane
Keyword(s):  
Snake Venom ◽  
Ribosomal Rna ◽  
Mode Of Action ◽  
Ph Dependence ◽  
Wheat Embryo ◽  
Analytical Technique ◽  
Phosphodiester Bonds ◽  
Acetone Fractionation ◽  
High Degree ◽  
End Group

A snake venom ribonuclease, similar to the classical pancreas ribonuclease, can be freed of other known nucleolytic activities by acid treatment, or by acetone-fractionation of Russell viper venom. Utilizing an end-group analytical technique in conjunction with oligonucleotide analyses of both partial and complete digests of ribonucleates, it has been possible to broadly characterize the mode of action of the snake venom ribonuclease, even though it is present in such small amount in venom that extensive purification was not attempted. The venom ribonuclease parallels pancreas ribonuclease in its acid stability, pH dependence, and high degree of preferential specificity toward PypA bonds in ribonucleate chains. Studies of the limited fragmentation of ribosomal and soluble ribonucleates, as well as dinucleoside phosphates, have shown that there are subtle differences between the venom and pancreas ribonucleases. The results of the investigation suggest that the venom and pancreas ribonucleases can be useful as a means of introducing a limited number of preferential scissions into ribonucleate chains at PypA internucleoside phosphodiester bonds. Incidental to the principal investigations dealing with the mode of action of the venom and pancreas ribonucleases on ribonucleates, certain features pertaining to the nucleotide sequences in ribosomal and soluble ribonucleates from wheat embryo have been noted.

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 Cathepsin D. Purification of isoenzymes from human and chicken liver

1970 ◽  
Vol 117 (3) ◽  
pp. 601-607 ◽  
Author(s):  
A. J. Barrett
Keyword(s):  
Isoelectric Focusing ◽  
Cathepsin D ◽  
Ph Dependence ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Ph Optimum ◽  
Specific Activities ◽  
Acetone Fractionation ◽  
High Degree

1. The Barrett (1967) assay for cathepsin D was slightly modified. 2. The enzyme was purified from liver of man and chicken by a procedure involving autolysis, acetone fractionation, ion-exchange chromatography and isoelectric focusing. 3. Several isoenzymes of cathepsin D were resolved in the isoelectric-focusing step, and three major forms, α,β and γ, were distinguished for each species. 4. A modified analytical method of isoelectric focusing in polyacrylamide gel indicated a high degree of homogeneity of the purified β and γ isoenzymes from each species, and this was supported by their constant high specific activities. 5. Gel filtration of the isoenzymes in a calibrated column of Sephadex G-100 showed that each had a molecular weight of 45000. 6. Human cathepsin D had a pH optimum of 3.5, and that of chicken enzyme was 3.0, haemoglobin being used as substrate. In each species, the three isoenzymes have the same pH-dependence curve. 7. The purified cathepsin D samples showed very little action on acid-denatured albumin.

THE TERMINAL GROUPS OF RIBONUCLEATE CHAINS IN EACH OF THE 16S AND 23S COMPONENTS OF ESCHERICHIA COLI RIBOSOMAL RNA

1967 ◽  
Vol 45 (6) ◽  
pp. 937-948 ◽  
Author(s):  
J. L. Nichols ◽  
B. G. Lane
Keyword(s):  
Escherichia Coli ◽  
Chain Length ◽  
Ribosomal Rna ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Quantitative Estimates ◽  
16S Rna ◽  
The Mean ◽  
End Group

Ribosomal ribonucleates from Escherichia coli have been resolved into 16S and 28S components by sucrose density-gradient centrifugation, and the chain termini in each of the 16S and 23S RNA components have been analyzed by hydrolysis with alkali. The principal 5′-linked end group of 16S RNA was found to be adenosine, and the principal 5′-linked end group of 23S RNA was found to be uridine. The principal 3′-linked end group of 16S RNA was also found to be adenosine, whereas the principal 3′-linked end group of 23S RNA was found to be guanosine. Quantitative estimates of chain length based on analyses for 5′-iinked terminals indicate that the mean chain length for 16S RNA is about 1.3 × 103nucleotide residues and the mean chain length for 23S RNA is about 2.1 × 103nucleotide residues.

scholarly journals A novel view on an old drug, 5-fluorouracil: an unexpected RNA modifier with intriguing impact on cancer cell fate

NAR Cancer ◽  
2021 ◽  
Vol 3 (3) ◽  
Author(s):  
Mounira Chalabi-Dchar ◽  
Tanguy Fenouil ◽  
Christelle Machon ◽  
Anne Vincent ◽  
Frédéric Catez ◽  
...  
Keyword(s):  
Cell Fate ◽  
Ribosomal Rna ◽  
Mode Of Action ◽  
Rna Modifications ◽  
Cell Plasticity ◽  
Historical Knowledge ◽  
Cytotoxic Effects ◽  
Translational Activity ◽  
Pancreatic Cancers ◽  
Patients Experience

Abstract 5-Fluorouracil (5-FU) is a chemotherapeutic drug widely used to treat patients with solid tumours, such as colorectal and pancreatic cancers. Colorectal cancer (CRC) is the second leading cause of cancer-related death and half of patients experience tumour recurrence. Used for over 60 years, 5-FU was long thought to exert its cytotoxic effects by altering DNA metabolism. However, 5-FU mode of action is more complex than previously anticipated since 5-FU is an extrinsic source of RNA modifications through its ability to be incorporated into most classes of RNA. In particular, a recent report highlighted that, by its integration into the most abundant RNA, namely ribosomal RNA (rRNA), 5-FU creates fluorinated active ribosomes and induces translational reprogramming. Here, we review the historical knowledge of 5-FU mode of action and discuss progress in the field of 5-FU-induced RNA modifications. The case of rRNA, the essential component of ribosome and translational activity, and the plasticity of which was recently associated with cancer, is highlighted. We propose that translational reprogramming, induced by 5-FU integration in ribosomes, contributes to 5-FU-driven cell plasticity and ultimately to relapse.

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).

Hypermodified alkali-stable dinucleotide sequences in each of the high-molecular-weight (26S and 18S) ribosomal RNA species of wheat

1977 ◽  
Vol 55 (5) ◽  
pp. 582-586 ◽  
Author(s):  
M. W. Gray ◽  
R. S. Cunningham
Keyword(s):  
Molecular Weight ◽  
Ribosomal Rna ◽  
18S Rrna ◽  
28S Rrna ◽  
Animal Cells ◽  
Wheat Embryo ◽  
Rrna Species ◽  
Wheat Embryos ◽  
The Individual ◽  
26S Rrna

Two hypermodified, alkali-stable dinucleotide sequences, each containing abase modification in addition to sugar methylation, are known to be present in wheat embryo 26S + 18S rRNA (Gray, M. W. (1974) Biochemistry 13, 5453–5463). Quantitative analysis of unfractionated 26S + 18S rRNA had suggested that each of these sequences (Cm-ψp and ψm-Ap, where Cm = O2′-methylcytidine and ψm = O2′-methylpseudouridine) was present in either the 18S or the 26S rRNA species, but not in both, at a frequency of not more than once per chain. In the study reported here, the individual 32P-labeled 18S and 26S rRNA species were isolated from viable wheat embryos germinated in the presence of [32P]orthophosphate. From analyses of phosphodiesterase and alkaline hydrolysates of the separated [32P]RNAs, we conclude that ψm-Ap is confined to wheat cytosol 18S rRNA, whereas Cm-ψp is localized in wheat cytosol 26S rRNA. The presence of ψm in the 18S rRNA of wheat stands in contrast with the situation in animal cells, where this hypermodified nucleoside is located in the 28S rRNA (Khan, M. S. N. &Maden, B. E. H. (1976) J. Mol. Biol. 101, 235–254)

scholarly journals C. elegans pharyngeal pumping provides a whole organism bio-assay to investigate anti-cholinesterase intoxication and antidotes

2020 ◽  
Author(s):  
Patricia G. Izquierdo ◽  
Vincent O’Connor ◽  
Christopher Green ◽  
Lindy Holden-Dye ◽  
John Tattersall
Keyword(s):  
Mode Of Action ◽  
Neuromuscular Junctions ◽  
Acetylcholinesterase Activity ◽  
Cholinesterase Inhibition ◽  
Genetic Determinants ◽  
C Elegans ◽  
Functional Conservation ◽  
Wide Range ◽  
High Degree ◽  
Bio Assay

AbstractInhibition of acetylcholinesterase by either organophosphates or carbamates causes anti-cholinesterase poisoning. This arises through a wide range of neurotoxic effects triggered by the overstimulation of the cholinergic receptors at synapses and neuromuscular junctions. Without intervention, this poisoning can lead to profound toxic effects, including death, and the incomplete efficacy of the current treatments, particularly for oxime-insensitive agents, provokes the need to find better antidotes. Here we show how the non-parasitic nematode Caenorhabditis elegans offers an excellent tool for investigating the acetylcholinesterase intoxication. The C. elegans neuromuscular junctions show a high degree of molecular and functional conservation with the cholinergic transmission that operates in the autonomic, central and neuromuscular synapses in mammals. In fact, the anti-cholinesterase intoxication of the worm’s body wall neuromuscular junction has been unprecedented in understanding molecular determinants of cholinergic function in nematodes and other organisms. We extend the use of the model organism’s feeding behaviour as a tool to investigate carbamate and organophosphate mode of action. We show that inhibition of the cholinergic-dependent rhythmic pumping of the pharyngeal muscle correlates with the inhibition of the acetylcholinesterase activity caused by aldicarb, paraoxons and DFP exposure. Further, this bio-assay allows one to address oxime dependent reversal of cholinesterase inhibition in the context of whole organism recovery. Interestingly, the recovery of the pharyngeal function after such anti-cholinesterase poisoning represents a sensitive and easily quantifiable phenotype that is indicative of the spontaneous recovery or irreversible modification of the worm acetylcholinesterase after inhibition. These observations highlight the pharynx of C. elegans as a new tractable approach to explore anti-cholinesterase intoxication and recovery with the potential to resolve critical genetic determinants of these neurotoxins’ mode of action.

Degranulation of Rabbit Platelets with PAF-Acether: A New Procedure for Unravelling the Mode of Action of Platelet-Activating Substances

1982 ◽  
Vol 48 (01) ◽  
pp. 067-071 ◽  
Author(s):  
B Boris Vargaftig ◽  
Danielel Joseph ◽  
Guy Marlas ◽  
L-G Chevance
Keyword(s):  
Snake Venom ◽  
Mode Of Action ◽  
Dense Body ◽  
Platelet Activating Factor ◽  
Thromboxane A2 ◽  
Venom Component ◽  
Rabbit Platelets ◽  
Body Components ◽  
Membrane Changes ◽  
Membrane Components

SummaryAggregation and secretion of ATP induced by thrombin, collagen, the snake venom component convulxin and platelet-activating factor (PAF-acether) were studied after the exposure of rabbit platelets to 1 μM of PAF-acether. This concentration, which is around 6 orders of magnitude above the concentration needed to induce full aggregation, was required to remove most of the releasable ATP from the platelets. The depleted platelets aggregated to PAF-acether, to thrombin and to convulxin under conditions where only very low amounts of ATP were secreted, confirming that these agents do not require the release of dense body components to trigger aggregation. Furthermore, when exposure to PAF-acether was associated to inactivation of platelet cyclooxygenase with aspirin, aggregation to thrombin persisted, validating the claim that thrombin induces aggregation by a third pathway unrelated to ADP and to thromboxane A2. Aggregation by collagen was markedly reduced by exposure of the platelets to PAF-acether or to aspirin; when both procedures were associated, aggregation was suppressed. Failure to desensitize the rabbit platelets to PAF-acether upon exposure to high amounts of it indicates the absence of irreversible membrane changes due to PAF-acether, and allows its use as a depleting procedure for the dense body materials, which does not affect platelet membrane components as is the case for thrombin.

END GROUP AND SEDIMENTATION DATA ON FRAGMENTED HIGH MOLECULAR WEIGHT RIBONUCLEATES

1963 ◽  
Vol 41 (1) ◽  
pp. 1927-1941 ◽  
Author(s):  
B. G. Lane ◽  
J. Diemer ◽  
C. A. Blashko
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Snake Venom ◽  
Group Analysis ◽  
Sedimentation Coefficient ◽  
Snake Venom Phosphodiesterase ◽  
Ehrlich Ascites ◽  
Spontaneous Hydrolysis ◽  
Hydrolysis Of ◽  
End Group

A method for end group analysis of ribonucleate preparations using purified snake venom phosphodiesterase is described. Unusual difficulties encountered with the method are discussed. The technique is useful for detection of end groups resulting from enzymic and chemical fragmentation of high molecular weight ribonucleates. Preliminary studies indicate that the method has limited usefulness because of a spontaneous hydrolysis of ribonucleates which occurs under the conditions which are optimal for hydrolysis with snake venom phosphodiesterase (pH 9, in the presence of magnesium). Physicochemical studies have shown that the pronounced dependence of sedimentation coefficient on ionic strength which has been reported by other investigators is also observed with fragmented high molecular weight ribonucleates and with 16S + 24S ribonucleates of Ehrlich ascites cells. The changes of sedimentation rate are associated with configurational and aggregation effects.

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