Isolation, characterisation and crystallization of deoxyribonuclease I from bovine and rat parotid gland and its interaction with rabbit skeletal muscle actin.
A purification procedure is described yielding DNase I from bovine and rat parotid glands of high homogeneity. The apparent molecular masses of the DNases I isolated have been found by sodium dodecyl sulfate/polyacrylamide gel electrophoresis to be 34 and 32 kDa for bovine and rat parotid DNase I, respectively, and thus differ from the enzyme isolated from bovine pancreas (31 kDa). By a number of different criteria concerning their enzymic behaviour, the isolated enzymes could be clearly classified as DNases I, i.e. endonucleolytic activity preferentially on native double-stranded DNA yielding 5'-oligonucleotides, a pH optimum at about 8.0, the dependence of their enzymic activity on divalent metal ions, their inhibition by 2-nitro-5-thiocyanobenzoic acid and by skeletal muscle actin. Comparison of their primary structure by analysis of their amino acid composition and also two-dimensional fingerprints and isoelectric focusing indicate gross similarities between the enzymes isolated from bovine pancreas and parotid, but distinct species differences, i.e. between the enzymes isolated from bovine and rat parotid. All the DNases I are glycoproteins. From bovine parotid DNase I crystals suitable for X-ray structure analysis could be obtained. The DNases I from both parotid sources specifically interact with monomeric actin forming 1:1 stoichiometric complexes. Their binding constants to monomeric actin differ, being 2 X 10(8) M-1 and 5.5 X 10(6) M-1 for bovine and rat parotid DNase I, respectively. Only the enzyme isolated from bovine sources is able to depolymerize filamentous actin.
D000199:Actins / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D002417:Cattle; D003460:Crystallization; D003850:Deoxyribonuclease I; D004706:Endodeoxyribonucleases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D008297:Male; D009132:Muscles / Q000378:metabolism*; D010306:Parotid Gland / Q000201:enzymology*; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D051381:Rats; D013045:Species Specificity
D000199:Actins; D004706:Endodeoxyribonucleases; D003850:Deoxyribonuclease I