RESUMEN
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.
Asunto(s)
Venenos de Crotálidos/inmunología , Exonucleasas/inmunología , gammaglobulinas , Animales , Formación de Anticuerpos , Reacciones Antígeno-Anticuerpo , Estabilidad de Medicamentos , Exonucleasas/antagonistas & inhibidores , Sueros Inmunes , Inmunodifusión , Inmunoelectroforesis , Conejos/inmunología , gammaglobulinas/aislamiento & purificaciónRESUMEN
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.
Asunto(s)
Isoenzimas , Hidrolasas Diéster Fosfóricas , Aminoácidos/análisis , Animales , Carbohidratos/análisis , Lípidos/análisis , Metales/análisis , Fosfodiesterasa I , SerpientesRESUMEN
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.
Asunto(s)
Replicación del ADN/genética , ADN Superhelicoidal , Origen de Réplica/genética , Saccharomyces cerevisiae/genética , Venenos de Crotálidos/enzimología , ADN de Hongos , Electroforesis en Gel de Agar , Escherichia coli/genética , Hidrolasas Diéster Fosfóricas , Plásmidos/genética , Mapeo RestrictivoRESUMEN
The 5'-->3' exonuclease from beef spleen is a 160-kDa tetramer consisting of four subunits of two types. Partial reduction of the tetramer led to one stable intermediate state of the enzyme with Mr = 80 kDa. The aim of this paper was to attribute the exonucleolytic activity to one of the two monomers, to the dimer or to the tetramer. The different forms of the exonuclease were separated by SDS-polyacrylamide gel electrophoresis, transferred on an Immobilon-P membrane and subsequently renaturated. Antibodies monospecific against each of the two monomers as well as against the dimer were isolated and their inhibitory effect on the holoenzyme determined. It was found that after renaturation the two monomers did not possess any exonuclease activity while the 80-kDa dimer showed a lower recovery of the specific activity of the enzyme (20.8+/-0.23 nkat/nmol, (n = 5)) in comparison with the 160-kDa tetramer (64.8+/-0.75 nkat/nmol (n = 5)). It was demonstrated that the antibodies monospecific against the dimer caused 53% maximum inhibition of the 160-kDa exonuclease. The antibodies monospecific against 25- and 55-kDa monomers did not inhibit the activity of the holoenzyme. No single-strand endonuclease activity of the spleen exonuclease was observed when using supercoiled Bluescript KS+ plasmid DNA as a substrate. This data suggest the emergence of an 80 kDa active form of beef spleen exonuclease upon association of two monomers of the tetrameric enzyme.
Asunto(s)
Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/metabolismo , Bazo/enzimología , Animales , Sitios de Unión , Bovinos , Cromatografía de Afinidad , Exodesoxirribonucleasa V , Exodesoxirribonucleasas/aislamiento & purificación , Cinética , Sustancias Macromoleculares , Peso Molecular , Estructura Cuaternaria de ProteínaRESUMEN
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.
Asunto(s)
Venenos de Crotálidos/enzimología , ADN de Cadena Simple/metabolismo , Desoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Animales , Western Blotting , Venenos de Crotálidos/metabolismo , Exodesoxirribonucleasas/aislamiento & purificación , Hidrólisis , Peso Molecular , Especificidad por SustratoRESUMEN
The intrinsic fluorescence of the exonuclease isolated from Crotalus adamanteus venom, was studied. The position of its maximum at 335 nm and half-width of the emission band 55 nm (lambda exc. 295 nm) suggested the existence of at least two types of tryptophan residues in the enzyme molecule. Differential analysis of the fluorescence spectra obtained by excitation at 280 and 295 nm revealed about 12.5% contribution of the tyrosine fluorescence in the overall emission excited at 280 nm. The environment of the tryptophan residues in the exonuclease was studied by quenching of their fluorescence with various ionic (NO3-, NO2-, I-, Br- and Cs+) and non-ionic agents (acrylamide, chloroform-methanol). On this basis, fractions of inner (non-polar) and surface tryptophan residues located in charged and neutral regions of the enzyme molecule were evaluated. More than half of the residues (60%) was found in the inner part of the exonuclease while most of its surface tryptophans--in a neutral region(s).
Asunto(s)
Venenos de Crotálidos/análisis , Exonucleasas/análisis , Acrilamida , Acrilamidas/farmacología , Cesio/farmacología , Proteínas/análisis , Espectrometría de Fluorescencia/métodosRESUMEN
Some of the synthetic chromophoric substrates of various enzymes cannot be used for direct spectrophotometric recording of the reactions, when a difference between the pH optimum of the enzyme reaction and the pH of maximum absorption of the released chromophore exists. In the present paper we describe a new method for following the time course of the spleen exonuclease-catalyzed reaction with thymidine 3'-monophospho-p-nitrophenyl ester as a substrate, based on the difference obtained in the absorbency of the substrate and its products in the far UV (at 330 nm). This difference, not published before, permits direct spectrophotometric recording of the amount of the hydrolyzed chromophoric substrate in acidic pH, whereas the maximum absorption of the product as accepted in the literature, is in alkaline pH. The molar absorption coefficient of the measurement at pH 5.7 is determined to be epsilon = 522 M-1.mm-1.
Asunto(s)
Exonucleasas/metabolismo , Espectrofotometría/métodos , Bazo/enzimología , Compuestos Cromogénicos , Concentración de Iones de HidrógenoAsunto(s)
Monoéster Fosfórico Hidrolasas/metabolismo , Polinucleótidos/análisis , ARN/metabolismo , Ponzoñas/farmacología , Animales , Isótopos de Carbono , Electroforesis , Hígado/efectos de los fármacos , Masculino , Ácido Orótico/metabolismo , Ratas , Ribosomas/efectos de los fármacos , Serpientes , Espectrofotometría , TemperaturaAsunto(s)
Nucleótidos/biosíntesis , Fosfatos/metabolismo , Plasmacitoma/metabolismo , ARN Ribosómico/metabolismo , Adenosina Monofosfato/biosíntesis , Adenosina Monofosfato/aislamiento & purificación , Animales , Nucleótidos de Citosina/biosíntesis , Nucleótidos de Citosina/aislamiento & purificación , Electroforesis , Nucleótidos de Guanina/biosíntesis , Nucleótidos de Guanina/aislamiento & purificación , Hidrólisis , Métodos , Ratones , Ratones Endogámicos , Isótopos de Fósforo , ARN Ribosómico/aislamiento & purificación , Nucleótidos de Uracilo/biosíntesis , Nucleótidos de Uracilo/aislamiento & purificaciónRESUMEN
A method for purification of beef spleen exonuclease is described, leading to electrophoretically homogeneous enzyme preparation. The method consists of three step fractionation of crude enzyme (after ammonium sulfate precipitation) as follows - ion exchange chromatography on ECTEOLA-cellulose, affinity chromatography on Concanavalin A-Sepharose and molecular sieving. The enzyme thus obtained is practically free of any contaminating activities - endonuclease or phosphomonoesterase. The molecular weight of the exonuclease was determined (98 000 +/- 3 000 daltons) and some other parameters of the enzyme were calculated. The investigation of the pH and thermo-stabilities showed significantly narrow limits of the exonuclease activity. The effect of the urea on the enzyme activity has also been evaluated.
Asunto(s)
Exonucleasas/aislamiento & purificación , Bazo/enzimología , Animales , Bovinos , Cromatografía/métodos , Exonucleasas/metabolismo , Calor , Concentración de Iones de Hidrógeno , Punto Isoeléctrico , Peso Molecular , Desnaturalización Proteica/efectos de los fármacos , Urea/farmacologíaRESUMEN
In the presence of phosphomonoesterase contaminations the use of bis-p-nitrophenyl phosphate to measure phosphodiesterase activity gives inconclusive values because one of the products of the phosphodiesterase or nuclease reaction becomes a substrate of the contaminating enzyme. A direct determination of the hydrolyzed phosphodiesterase substrate in the UV range is possible at the isosbestic points of the transformation of the phosphomonoesterase substrate.
Asunto(s)
Hidrolasas Diéster Fosfóricas/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Espectrofotometría/métodos , Nitrofenoles , Especificidad por SustratoRESUMEN
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.
RESUMEN
A one-step purification of beef spleen exonuclease in the form of a DNA-enzyme complex is described. The purity of the exonuclease was verified by two-dimensional gel electrophoresis. It possesses molecular mass 160 kDa and pI 6.92. The one-dimensional sodium dodecyl sulfate gel after reduction with beta-mercaptoethanol suggests that the 160-kDa exonuclease consists of four polypeptide chains of two different types with molecular masses 55 and 25 kDa. The tetrameric structure of the exonuclease is supported by intermolecular disulfide bonds, and their partial reduction leads to the formation of one stable intermediate with molecular mass 80 kDa formed by binding one 55-kDa with one 25-kDa subunit into a dimer. During two-dimensional gel electrophoresis, the dimer showed pI 6.92 while monomers showed pI 6.78 for the 55-kDa and pI 6.29 for the 25-kDa. Two other intermediate states of two big and one small (135 kDa) and two small and one big subunit (105 kDa) were also visualized. They are unstable and easily dissociated into one 80-kDa dimer and either one 55-kDa or one 25-kDa monomer. The immunoblotting analysis with specific polyclonal antibodies against 160-kDa protein confirmed the subunit structure of the exonuclease. It was found that both monomers are glycosylated.