Purification and characterization of fungal nuclease composed of heterogeneous subunits.
A nuclease was purified from the extract of wheat bran culture of Aspergillus sp. isolated from "Katsuobushi" by a series of column chromatographies. The purified nuclease showed a single protein band on nondenaturing polyacrylamide gel electrophoresis. The enzyme showed three protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Their molecular weights were estimated to be 80,000, 50,000, and 25,000. The molecular weight of the nuclease was estimated to be 125,000 by gel permeation chromatography. The enzyme showed maximum activity around pH 8.0 for DNA and RNA. The enzyme required Mg2+, Mn2+, or Co2+ for the appearance of activity. The enzyme was stable until 40 degrees C and in pH range of 5-9. The stability of the enzyme for temperature increased until 50 degrees C by Ca2+. The enzyme exonucleolytically degraded DNA and RNA by 3'-->5' mode to produce 5'-mononucleotides. The fungal nuclease acted on heat-denatured DNA and native DNA and RNA, but not bis(p-nitrophenyl)phosphate, p-nitrophenyl thymidine 5'-phosphate, and p-nitrophenyl thymidine 3'-phosphate. The enzyme did not show strict base specificity for DNA and RNA, while the affinity for substrate was affected by 3'-terminal bases. The enzyme preferentially degraded poly(C) and poly(U), but hardly degraded poly(A) and poly(G). The nuclease acted on closed circular double-stranded DNA to produce open circular DNA and then linear form DNA by single-strand scission. The nicking activity was intrinsic to the enzyme.
Ito
K
K
Department of Biology, Faculty of Science, Osaka City University, Japan.
Matsuura
Y
Y
Minamiura
N
N
D000818:Animals; D001230:Aspergillus / Q000201:enzymology*; D010584:Bacteriophage phi X 174 / Q000235:genetics; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D003035:Cobalt / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D004270:DNA, Circular / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005092:Exonucleases / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D009691:Nucleic Acid Denaturation; D009711:Nucleotides / Q000378:metabolism; D012331:RNA, Fungal / Q000378:metabolism; D012474:Salmon; D013696:Temperature
D004270:DNA, Circular; D004279:DNA, Viral; D009711:Nucleotides; D012331:RNA, Fungal; D003035:Cobalt; D008345:Manganese; D004247:DNA; D005092:Exonucleases; C086038:exonuclease, Aspergillus; D008274:Magnesium