Purification and properties of a DNase inhibitor from Nicotiana tabacum cell cultures.
Extraction of Nicotiana tabacum cell cultures, chromatography on DEAE-cellulose and gel filtration resulted in a homogeneous protein (Mr = 14500), which strongly reduces the hydrolysis of Escherichia coli DNA by DNase I. DNA degradation by micrococcal nuclease is not inhibited. The inhibitor protein interacts with DNase I in the absence of DNA, as determined by the partial quenching of protein intrinsic fluorescence; a 1:1 stoichiometry is deduced. From the reduction of DNase I activity with increasing inhibitor concentration apparent equilibrium constants for the inhibitor X DNase-I complex have been calculated. This interaction is strongly temperature-dependent; at 20 degrees C and 26 degrees C dissociation constants of 5 nM and 110 nM, respectively, were determined. As a consequence a rather high enthalpy of interaction can be estimated.
D002478:Cells, Cultured; D003850:Deoxyribonuclease I; D003851:Deoxyribonucleases / Q000037:antagonists & inhibitors*; D004720:Endonucleases / Q000037:antagonists & inhibitors*; D007700:Kinetics; D008970:Molecular Weight; D010940:Plant Proteins / Q000302:isolation & purification / Q000494:pharmacology*; D010944:Plants / Q000032:analysis*; D010947:Plants, Toxic; D014026:Nicotiana / Q000032:analysis
D010940:Plant Proteins; D003851:Deoxyribonucleases; D004720:Endonucleases; D003850:Deoxyribonuclease I