Luben B. Dolapchiev

The action of snake venom phosphodiesterase on liver ribosomal ribonucleic acids

Abstract The stepwise hydrolysis of rat-liver ribosomal RNAs (rRNA) with snake venom phosphodiesterase (EC 3.1.4.1) has been studied. The following results have been obtained: 1. 1. Hydrolysis of poly (U), poly (A) and equimolar mixtures of them proceeds to completion and yields pU and pA as the only major products of the reaction. Secondary structure in poly (U) · poly (A) has no detectable influence on the rate and the extent of hydrolysis. The substrate (Ap)nCp is resistant to the action of this enzyme, while (Ap)nC is rapidly degraded to give pA and pC. 2. 2. As shown by agar-gel electrophoresis, the liver rRNA studied is constituted of homogeneous 28-S and 18-S RNA molecules. They display secondary structure transitions typical for native rRNAs. The preparations studied are virtually free from endonucleolytic contaminants. 3. 3. Hydrolysis of rRNAs with venom phosphodiesterase goes to completion and yields pG, pC, pA and pU as the only major products. Analysis by determination of the nucleosides originating from chain terminals during hydrolysis, shows that the endonuclease contaminants in this system may account for no more than 1 to 2 internal breaks per RNA molecule. 4. 4. The molar ratio of the mononucleotides liberated on stepwise hydrolysis of liver rRNAs with venom phosphodiesterase varies with progress of enzymatic degradation. The product obtained at early stages of enzyme action has a higher content of pA and pU and a lower content of pG and pC. The same correlation is observed with 18-S rRNA and partly with 28-S rRNA. It is suggested that in rRNA the segment near the 3′-end of the chain contains more pA and pU and less pG and pC as compared with the segment near the 5′-end of the chain. 5. 5. Labeling in vivo of free uridine 5′-phosphates and cytidine 5′-phosphates with [6-14C]orotic acid shows with both nucleotides a maximum incorporation at 40 min. Ribosomal RNAs labeled for 90 min in vivo with [6-14C]orotic acid were analysed by partial degradation with venom phosphodiesterase and determination of the radioactivity of uridylic acid residues. The labeling of 18-S RNA is higher than of 28-S RNA. Enzymatic liberation of labeled uridylic acid from 18-S and 28-S RNA is non-uniform. With both 18-S and 28-S RNA, uridylic acid liberated at the initial stages of enzyme action is more highly labeled than uridylic acid from the remaining RNA fragments.

Venom exonuclease: II. Amino acid composition and carbohydrate, metal ion and lipid content in the Crotalus adamanteus venom exonuclease

Exonuclease from Crotalus adamanteus venom has only threonine as N-terminimal amono acid residue. It was examined for its amino acid composition, -SH and S-S groups. It has no free -SH groups and seven S-S bonds. The analysis of the carbohydrate residues in the enzyme proves that it is a glycoprotein. It contains neutral sugars (9.2%), amino sugars (1.9%) and ten sialic acid residues per molecule. The venom exonuclease is a metalloenzyme. This is proven by the existence of Mg2+, Zn2+ and Ca2+ and their specific role in the catalytic reactions. The enzyme contains also triacylglycerols (1.54%) and cholesterol esters (1.43%). The influence of the non-protein moieties of the exonuclease on its catalytic ability has been discussed.

Single-strand-specific DNase activity is an inherent property of the 140-kDa protein of the snake venom exonuclease 1

Polyclonal antibodies against the exonuclease from Crotalus adamanteus venom (the 140‐kDa protein) inhibit both the exonucleolytic and the single‐strand‐specific endonucleolytic activities, present in the exonuclease preparation. The antibodies also diminish the ability of the enzyme to split the negatively supercoiled Bluescript KS+ in the AT‐rich fragment near‐by the transcription termination site of the Ampicillin gene. Therefore the single‐strand‐specific endonucleolytic activity was attributed to the protein molecule of the exonuclease. The processivity of the exonucleolytic action was found to be less than 3 monomers as indicated by the heparin trapping method.

Pattern of [32P]orthophosphate incorporation in vitro into ribonucleic acid nucleotides of Ehrlich ascites tumor cells.

1. 1. Rapidly labelled RNA from whole Ehrlich ascites tumor cells had the same mononucleotide composition irrespective of conditions used for [32P]orthophosphate incorporation (in vivo or in vitro). It corresponded in each case to 30–40 % d-RNA (RNA similar in mononucleotide composition to DNA) and 60–70 % r-RNA (RNA similar in mononucleotide composition to ribosomal RNA). 2. RNA extracted with phenol-sodium dodecyl sulfate at 65° from nuclei of isolated ascites tumor cells showed three main peaks on agar gel electrophoresis, corresponding to ribosomal RNA (2 peaks) and soluble RNA. Polyvinyl sulfate was necessary to avoid partial degradation of RNA during its isolation and storage. 3. Nuclear RNA isolated from tumor cells labelled in vitro (30 min) with [32P]orthophosphate was hydrolyzed with 0.5 N KOH or with snake-venom phosphodiesterase (EC 3.1.4.1). The specific activities of the four nucleoside 2′(3′)-monophosphates were of the same order. The specific activity of GMP was on the average twelve-fold lower than that of the other three nucleoside 5′-monophosphates. Nucleotide incorporation into rapidly labelled nuclear RNA was statistically random. The mononucleotide composition of this RNA corresponds to 50 % d-RNA and 50 % r-RNA. 4. During the step-wise degradation of rapidly labelled nuclear RNA with snake-venom phosphodiesterase the specific activity of CMP increased, while that of AMP and UMP decreased.

Kinetics of hydrolysis of uridylic oligonucleotides with different degree of polymerisation by snake venom phosphodiesterase

Snake venom phosphodiesterase (orthophosphoric diester phosphohydrolase, EC 3.1.4.1) is an exonuclease, splitting successively 5’-mononucleotides from the polynucleotide end, bearing free 3’-hydroxyl group [l-3] . It has been reported that the reaction time course shows an atypical rate of liberation of the product from the polynucleotide substrate during the hydrolysis [3]. The molar concentration of the substrate remains constant for a long enough period of time, depending on the degree of polymerisation of the substrate molecule. Therefore, the increasing reaction rate with progress of the hydrolysis could be explained by the influence of the chain length of the substrate molecule. In the present study we followed the change of some kinetic parameters of the enzyme reaction catalyzed by snake venom phosphodiesterase as a function of the length of the substrate molecule.

Production of phosphomonoesterases by Nicotiana tabacum 1507 in an aqueous two-phase system.

Studies were conducted on Nicotiana tabacum 1507 cultivation in an aqueous two-phase system (PD(5)) formed by adding 4% PEG (MW 20,000) and 7.5% dextran (MW 70,000) to the medium. The time course of growth and changes in the phase volumes of the PD(5) system, as well as the biosynthesis, secretion, and partitioning of phosphomonoesterases during 11 days of cultivation, were followed. In comparison with N. tabacum 1507 cultivation as a free suspension, on day 8 of cultivation in the PD(5) system the yields of acid and alkaline extracellular phosphomonoesterases were 18 and 10 times higher, respectively. Partitioning took place mainly in the bottom phase with specific activity being 4.5 and 3.5 times higher, respectively. (c) 1996 John Wiley & Sons, Inc.

Venom exonuclease. III. Immunochemical characterization and modification by specific antibody.

The immunoelectrophoretic purity of the exonuclease preparation, isolated from Crotalus adamanteus venom according to a procedure previously published (Dolapchiev, L.B., Sulkowski, E. and Laskowski, M., Sr. (1974) Biochem. Biophys. Res. Commun. 61, 271-281), is reported. The enzyme showed one precipitin line against antibody prepared against partially purified venom. The exonuclease is unstable in dilute (1.25 microgram/ml and below) solutions. Bovine serum albumin stabilized the enzyme nonspecifically whereas the homologous antibody demonstrated a specific stabilizing effect under the same conditions. The binding of the anti-enzyme with the enzyme caused inhibition of both its activities--phosphodiesterase and pyrophosphatase. The inhibition of the exonuclease when attacking high molecular weight substrates is similar to the above and is of the same noncompetitive type. The thermal stability of venom exonuclease is reported.

Venom exonuclease — kinetics and specificity towards different nucleotide substrates

Abstract The steady-state kinetics of the reactions catalyzed by the exonuclease from Crotalus adamanteus venom were studied by the titrigraphic method. The kinetic parameters of the enzyme reactions were determined for the hydrolysis of a number of different substrates with phosphodiester and prophosphate bonds. When low molecular weeight substrates were hydrolysed, a relative base specificity was observed. The hydrolysis of polymeric substrates showed an increase in the reaction rate of the consecutive liberation of the mononucleotide product, parallel with the decrease of the substrate chain length. A strict correlation of the kinetic parameters of the hydrolysis of the first phosphodiester bond of oligomeric substrates with the degree of polymerization of these substrates was obtained. On the basis of the determined kinetic parameters of hydrolysis for the different substrates under study, some thermodynamic parameters of the enzyme reactions were calculated. A hypothetical model is put forward for the active site of the venom exonuclease.

Tobacco plant cell cultures with high content of extracellular phosphohydrolases

Abstract A new, fast, and easy assay based on the “cell squash” method for analysis of a large number of tobacco cell cultures which produces phosphohydrolytic enzymes is described. The time course of growth and changes in the amount of the secreted phosphohydrolytic enzymes, such as acid and alkaline phosphomonoesterases or acid and alkaline specific or nonspecific phosphodiesterases (exonucleases), in Nicotiana tabacum 1507, 1508, and 1509 cell cultures selected by this method were studied. The highest amounts of phosphohydrolytic enzymes were measured in the culture medium of N. tabacum 1507 at 2.8 μkat ml−1 for alkaline phosphodiesterase and 2.5 μkat ml−1 for the acid enzymes. The amount of 5′-phosphodiesterase and 3′-phosphodiesterase produced was 0.93 μkat ml−1 and 1.1 μkat ml−1, respectively. The culture line N. tabacum 1508 which was derived from the N. tabacum 1507 that secreted double the amount of extracellular alkaline phosphohydrolases produced the same amount of 5′-phosphodiesterase as N. tabacum 1507.

Supercoiling unwinds two-micrometer plasmid yeast DNA at the origin of replication.

All studied origins of replication of DNA in Saccharomyces cerevisiae contain DNA unwinding elements. The introduction of unrestrained negative supercoiling leads to melting of the two DNA strands in DNA unwinding elements. To understand the mechanism of DNA replication it is important to know whether the most unstable region of DNA coincides with the origin of replication. Two-micrometer plasmid DNA from S. cerevisiae inserted in pBR322 was investigated by cleaving with snake venom phosphodiesterase. Its single-strand endonucleolytic activity allows cutting of negatively supercoiled DNA in the DNA unwinding elements. The sites of the venom phosphodiesterase hydrolysis were mapped by restriction enzymes. This study shows that the unwinding of the two-micrometers plasmid DNA of S. cerevisiae takes place only in the origin of replication as a result of unrestrained negative supercoiling.