Extracellular nuclease from Rhizopus stolonifer: purification and characteristics of - single strand preferential - deoxyribonuclease activity.
An extracellular nuclease from Rhizopus stolonifer (designated as nuclease Rsn) was purified to homogeneity by chromatography on DEAE-cellulose followed by Blue Sepharose. The M(r) of the purified enzyme determined by native PAGE was 67¿ omitted¿000 and it is a tetramer and each protomer consists of two unidentical subunits of M(r) 21¿ omitted¿000 and 13¿ omitted¿000. It is an acidic protein with a pI of 4.2 and is not a glycoprotein. The purified enzyme showed an obligate requirement of divalent cations like Mg(2+), Mn(2+) and Co(2+) for its activity but is not a metalloprotein. The optimum pH of the enzyme was 7.0 and was not influenced by the type of metal ion used. Although, the optimum temperature of the enzyme for single stranded (ss) DNA hydrolysis in presence of all three metal ions and for double stranded (ds) DNA hydrolysis in presence of Mg(2+) was 40 degrees C, it showed higher optimum temperature (45 degrees C) for dsDNA hydrolysis in presence of Mn(2+) and Co(2+). Nuclease Rsn was inhibited by divalent cations like Zn(2+), Cu(2+) and Hg(2+), inorganic phosphate and pyrophosphate, low concentrations of SDS, guanidine hydrochloride and urea, organic solvents like dimethyl sulphoxide, dimethyl formamide and formamide but not by 3'- or 5'-mononucleotides. The studies on mode and mechanism of action showed that nuclease Rsn is an endonuclease and cleaves dsDNA through a single hit mechanism. The end products of both ssDNA and dsDNA hydrolysis were predominantly oligonucleotides ending in 3'-hydroxyl and 5'-phosphoryl termini. Moreover, the type of metal ion used did not influence the mode and mechanism of action of the enzyme.
Rangarajan
S
S
Division of Biochemical Sciences, National Chemical Laboratory, Pune, India.
Shankar
V
V
D002413:Cations, Divalent; D004277:DNA, Single-Stranded / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000378:metabolism; D004720:Endonucleases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D004789:Enzyme Activation; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D012233:Rhizopus / Q000201:enzymology*; D012260:Ribonucleases / Q000737:chemistry; D012492:Salts; D012997:Solvents; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds; D013696:Temperature
D002413:Cations, Divalent; D004277:DNA, Single-Stranded; D012492:Salts; D012997:Solvents; D013438:Sulfhydryl Compounds; D004706:Endodeoxyribonucleases; D004720:Endonucleases; D012260:Ribonucleases