Purification, characterization and complete amino acid sequence of nuclease C1 from Cunninghamella echinulata var. echinulata.
It is known, from the zymogram method of nuclease activity assay, that the crude extracts of Cunninghamella echinulata var. echinulata contained at least three distinct extracellular nucleases. Among them, the major form was 30 kDa in molecular mass and termed nuclease C1. In this report, nuclease C1 was purified to apparent homogeneity by chromatography on Cibacron blue-3GA, phenyl-Sepharose 4B and HiTrap Heparin. Nuclease C1 acquired enzymatic activity in the presence of Mn2+ or Mg2+ and was inhibited by EDTA. The activity was maximal at pH 7-8.5. The primary structure of nuclease C1 was completely determined using enzymatic digestion and gene cloning. The N-terminal 49 residues of nuclease C1 were first elucidated from a tryptic digest. Two degenerate upstream primers were subsequently designed to amplify the cDNA encoding nuclease C1. The resulting protein sequence of nuclease C1 was shown to be composed of 252 residues. It was intriguing to find that the protein sequence of nuclease C1 showed significant similarities with the sequences of the mitochondrial nucleases of Saccharomyces cerevisiae (44% identity) and Schizosaccharomyces pombe (42% identity). Residue His87 of nuclease C1 was postulated to be located at the active site from sequence similarity with secreted nuclease from Serratia marcescens.
Ho
H C
HC
Department of Biochemistry, School of Medicine, China Medical College, Taichung, Taiwan, Republic of China. hcho@mail.cmc.edu.tw
Shiau
P F
PF
Liu
F C
FC
Chung
J G
JG
Chen
L Y
LY
D000595:Amino Acid Sequence; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D003851:Deoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D009090:Mucorales / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry; D004718:Saccharomycetales / Q000201:enzymology; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D010446:Peptide Fragments; D003851:Deoxyribonucleases; C114723:nuclease C1