ABSTRACT
With the use of the strain-overproducer restriction endonuclease R.EcoRV was isolated and purified to homogeneity. The molecular mass of the enzyme was determined by gel filtration and polyacrylamide gel electrophoresis to be 25 000 daltons. According to the data of immunological tests R.EcoRV differs in its antigenic characteristics from restriction endonucleases R.EcoRI and R.EcoRII. Dependence of enzyme activity on pH, ionic strength, temperature, presence of divalent cations (Mn2+, Mg2+, Co2+, Zn2+, Ni2+ and Cd2+) and organic solvents (glycerol, dimethylsulfoxide, ethanol) has been studied. It was shown that under conditions of replacement of Mg2+ for Mn2+ or after addition of organic solvents relaxation of R.EcoRV specificity takes place. It was shown also that R.EcoRV is able to digest T-even bacteriophage DNAs with different types and extents of modification. DNA modified by the action of MR.EcoRV system in vivo is susceptible to R.EcoRV in vitro. Under conditions of relaxed specificity noncanonical sites are susceptible to R.EcoRV attack. The fragments resulted may be cloned in canonical pBR322 EcoRV site.
Subject(s)
DNA Restriction Enzymes/analysis , DNA, Bacterial/metabolism , DNA, Viral/metabolism , Deoxyribonucleases, Type II Site-Specific , Base Sequence , Chromatography, Gel , DNA Restriction Enzymes/immunology , DNA Restriction Enzymes/isolation & purification , Deoxyribonuclease EcoRI , Electrophoresis, Polyacrylamide Gel , Epitopes/analysis , Escherichia coli/genetics , Molecular Weight , T-Phages/geneticsSubject(s)
Dioxygenases , Oxygenases/analysis , Pseudomonas aeruginosa/enzymology , Xylenes/metabolism , Amino Acid Sequence , Catechol 1,2-Dioxygenase , Catechol 2,3-Dioxygenase , Culture Media , Kinetics , Mutation , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/growth & development , Substrate SpecificityABSTRACT
Molecular weight of native apotransketolase from baker's yeast is found to be 159000 +/- 6000 by means of sedimentation equilibrium and sedimentation-diffusion rate. The enzyme in a relatively low concentration reversibly dissociates into two subunits with molecular weight of about 80 000 at pH 7.6 and 20 degrees C. The equilibrium constant of the reaction monomer-dimer is 4.4 . 10(3) M-1. A decrease of the temperature stimulates the association of monomers into dimer, while the shift of pH 7.6 into acid or alkaline region stimulates the dissociation process. Dissociation becames irreversible at pH less than 5 and greater than 10.5.
